10,000 Matching Annotations
  1. Feb 2026
    1. Reviewer #2 (Public review):

      Summary:

      The manuscript describes a combined computational and experimental approach to investigate the ABHD5 binding to and insertion into membranes.

      Strengths:

      Mutational experiments support computational findings obtained on ABHD5 membrane insertion with enhanced-sampling atomistic simulations.

      Weaknesses:

      While the addressed problem is interesting, I have several concerns, which fall into two categories:

      (A) I see statements throughout the manuscript, e.g. on PNPLA activation, that are not supported by the results.

      (B) The presentation of the computational and experimental results lacks in part clarity and detail.

      Comments and questions on (A):

      (1) I think the following statements in the abstract, which go beyond ABHD5 membrane binding, are not supported by the presented data:

      the addition "to control lipolytic activation" in the 3rd sentence of the abstract.

      further below ".... transforming ABHD5 into an active and membrane-localized regulator".

      (2) The authors state in the Introduction (page numbers and line numbers are missing to be more specific):

      "We hypothesize that binding of ABHD5 alters the nanoscale chemical and biophysical properties of the LD monolayer, which, combined with direct protein-protein interactions, enables PNPLA paralogs to access membrane-restricted substrates. This regulatory mechanism represents a paradigm shift from conventional enzyme-substrate interactions to sophisticated allosteric control systems that operate at membrane interfaces."

      This hypothesis and the suggested paradigm shift are not supported by the data. Protein-protein interactions are not considered. What is meant by "sophisticated allosteric control"?

      (3) The authors state in the Results section:

      "We hypothesize that this TAG nanodomain is critical for ABHD5-activated TAG hydrolysis by PNPLA2." In previous pages, the authors state the location of the nanodomain: "TAG nanodomain under ABHD5".

      If the nanodomain is located under ABHD5, how can it be accessible to PNPLA2? To my understanding, ABHD5 then sterically blocks access of PNPLA2 to the TAG nandomain.

      (4) Another statement: "Our findings suggest that ABHD5-mediated membrane remodeling regulates lipolysis in part by regulating PNPLA2 access to its TAG substrate."

      I don't see how the reported results support this statement (see point 3 above).

      Comments and questions on (B):

      (1) The authors state that the GaMD simulations started "from varying conformations observed during CGMD".

      What is missing is a clear description of the CGMD simulation conformations, and the CG simulations as a whole, prior to the results section on GaMD. The authors use standard secondary and tertiary constraints in the Martini CG simulations. Do the authors observe some (constrained) conformational changes of ABHD5 already in the CG simulations (depending on the strength of the constraints)? Or do the conformational changes occur exclusively in the GaMD simulations? Both are fine, but this needs to be described.

      (2) The authors write: "Three replicas of GaMD were performed."

      Do these replicas lead to similar, or statistically identical, membrane-bound ABHD5 conformations? Is this information, i.e. a statistical analysis of differences in the replica runs, already included in the manuscript?

      (3) The authors state on the hydrogen exchange results:

      "HDX-MS provided orthogonal experimental evidence for the dynamics of the lid. In solution, a peptide (residues 200-226) spanning the lid helix displayed a bimodal isotopic distribution (Fig. S4), indicating the coexistence of different conformations. Upon LD binding, this distribution shifted to a single, low-exchange peak, demonstrating stabilization of the membrane-bound conformation with reduced solvent accessibility. These experimental observations corroborate our MD simulations."

      I find this far too short to be understandable. Also, there are no computational results of ABHD5 in solution that show a bimodal conformational distribution of the lid helix, which is observed in the hydrogen exchange experiments. Which aspects of the MD simulations are corroborated?

    1. Reviewer #1 (Public review):

      Summary:

      The goal of the study was to address the question of the degree to which social position in a group is a stable trait that persists across conditions. Reinwald et al. use a custom-built cage system with automated tracking and continuous testing for social dominance that does not require intervention by the experimenter. Remixing of individuals from different groups revealed that social position was rather stable and not really predictable from other measures that were taken. The authors conclude that social position is multifaceted but dependent on characteristics like personality traits.

      Strengths:

      (1) Reductionistic, highly controlled setting that allows for the control of many confounding variables.

      (2) Very interesting and important question.

      (3) Confirms the emergence of inter-individual behavior-driven differences in inbred mice in a shared environment.

      (4) Innovative paradigm and experimental setup.

      (5) Fresh perspective on an old question that makes the best use of modern technology.

      (6) Intelligent use of behavioral and cognitive covariables to generate a non-social context.

      (7) Bold and almost provocative conclusion, inviting discussion and further elaboration.

      Weaknesses:

      (1) Reductionistic, highly controlled setting that blends out much of the complexity of social behavior in a community.

      (2) The motivation to enter the test tube is not "trait" (or at least not solely a trait) but the basic need to reach food and water; chasing behavior would be less dependent on this stimulus.

      (3) Dominance is only one aspect of sociality, social structure is reduced to rank. The information that might lie in the chasing behavior is not optimally used to explain social behavior beyond the rank measure.

      (4) Focus on rank bears the risk of overgeneralization for readers not familiar with the context.

      (5) Conclusion only valid for the reductionistic setting, in which environment, social and non-social changes only within narrow limits, and in which the mouse population does not face challenges

      (6) Animals are not naive at the beginning of the experiment, but are already several weeks old.

      In summary, this is a wonderful study, but not one that is easy to interpret. The bold conclusion is valid only within the constraints of the study, but nevertheless points in an important direction. The paradigm is clever and could be used for many interesting follow-ups.

      To define social position as a personality trait will elicit strong opposition and much debate; the nuances of the paper might be lost on many readers and call for the (re)-consideration of many concepts that are touched. I find this attitude a strength of the paper, but the approach bears the risk of misunderstanding.

    2. Reviewer #2 (Public review):

      Summary:

      This manuscript presents the "NoSeMaze", a novel automated platform for studying social behavior and cognitive performance in group-housed male mice. The authors report that mice form robust, transitive dominance hierarchies in this environment and that individual social rank remains largely stable across multiple group compositions. They further demonstrate that social dominance and aggressive behaviors, like chasing, are partially dissociable and that dominance traits are independent of non-social cognitive performance. The study includes a genetic manipulation of oxytocin receptor expression in the anterior olfactory nucleus, which showed only transient effects on social rank.

      Strengths:

      (1) Innovative Methodology:<br /> The NoSeMaze platform is a technically elegant and conceptually well-integrated system that enables fully automated, long-term monitoring of both social and cognitive behaviors in large groups of group-housed mice. It combines tube-test-like dominance contests, voluntary chase-escape interactions, and an embedded operant olfactory discrimination task within a single, ethologically relevant environment. This modular design allows for high-throughput, minimally invasive behavioral assessment without the need for repeated handling or artificial isolation.

      (2) Experimental Scale and Rigor:<br /> The study includes 79 male mice and over 4,000 mouse-days of observation across multiple group reshufflings. The use of RFID-based identification, automated data logging, and longitudinal design enables robust quantification of individual trait stability and group-level social structure.

      (3) Multidimensional Behavioral Profiling:<br /> The integration of social (tube dominance, proactive chasing), physical (body weight), and cognitive (olfactory learning task) measures offers a rich, multi-dimensional profile of each individual mouse. The authors' finding that social dominance traits and non-social cognitive performance are largely uncorrelated reinforces emerging models of orthogonal behavioral trait axes or "animal personalities".

      (4) Clarity and Data Analysis:<br /> The analytical framework is well-suited to the study's complexity, with appropriate use of dominance metrics, mixed-effects models, and permutation tests. The analyses are clearly explained, statistically rigorous, and supported by transparent supplementary materials.

      Weaknesses:

      (1) Conceptual Novelty and Prior Work:<br /> While the study is carefully executed and methodologically innovative, several of its core findings reaffirm concepts already established in the literature. The emergence of stable, transitive social hierarchies, the persistence of individual differences in social behavior, and the presence of non-despotic social structures have all been previously reported in mice, including under semi-naturalistic conditions (e.g., Fan et al., 2019; Forkosh et al., 2019). Although this work extends those findings with greater behavioral resolution and scale, the manuscript would benefit from a clearer articulation of what is genuinely novel at the conceptual level, beyond the technological advance.

      (2) Role of OXTR Deletion:<br /> The inclusion of the OXTR manipulation feels somewhat disconnected from the manuscript's central aims. The effects were minimal and transient, and the authors defer full interpretation to a separate study.

      (3) Scope Limitations (Sex and Age):<br /> The study is limited to male mice, and although this is acknowledged, the title and overall framing imply broader generalizability. This sex-specific focus represents a common but problematic bias. Additionally, results from the older mouse cohort are under-discussed; if age had no effect, this should be explicitly stated.

      (4) Ambiguity of Dominance as a Construct:<br /> While the study robustly quantifies social rank and hierarchy structure, the broader functional meaning of "dominance" remains unclear. As in prior work (e.g., Varholick et al., 2019), dominance rank here shows only weak associations with physical attributes (e.g., body weight), cognitive strategy, or neuromodulatory manipulation (OXTR deletion). This recurring pattern, where rank metrics are reliably established yet poorly predictive of other behavioral or biological traits, raises important questions about what such measures actually capture. In particular, it challenges the assumption that outcomes in paradigms like the tube test or chase frequency necessarily reflect dominance per se, rather than other constructs.

    3. Reviewer #3 (Public review):

      Reinwald et al. present the NoSeMaze, a semi-natural behavioral system designed to track social behaviors alongside reinforcement-learning in large groups of mice. Accumulating more than 4,000 days of behavioral monitoring, the authors demonstrate that social rank (determined by tube competitions) is a stable trait across shuffled cohorts and correlated with active chasing behaviors. The system also provides a solid platform for long-term measurements of reinforcement learning, including flexibility, response adaptation, and impulsiveness. Yet, the authors show that social ranking and chasing are mostly independent of these cognitive traits, and both seem mostly independent of oxytocin signaling in the AON.

      Strengths:

      (1) The neuroethological approach for automated tracking of several mice under semi-natural conditions is still rare in social behavioral research and should be encouraged.

      (2) The assessment of dominance by two independent measures, i.e., spontaneous tube competitions and proactive chasing, is innovative and valuable.

      (3) The integration of a long-term reinforcement-learning module into the semi-natural system provides novel opportunities to combine cognitive traits into social personality assessments.

      (4) The open-source system provides a valuable resource for the scientific community.

      Limitations:

      (1) Apparent ambiguity and inconsistency in age structure and cohort participation across rounds, raising concerns about uncontrolled confounds.

      (2) Chasing behavior appears more stable than tube-test competitions (Figure 4D vs. Figure 3D), which challenges the authors' decision to treat tube competitions as the primary basis for hierarchy determination.

      Major concerns:

      (1) Unclear and inconsistent handling of age groups and repeated sampling. The manuscript repeatedly refers to "younger" and "older" adults, but it is unclear whether age was ever controlled for or included in models. Some mice completed only one round, others 2-5 rounds, without explanation of the criteria or balancing.

      (2) Stability of chasing appears stronger than the stability of tube competitions. Figure 4D shows highly consistent chasing behavior across weeks, while Figure 3D shows weaker and more variable correlations for tube-based David scores. This is also evident from Figure 5A-B,D. Thus, it appears that chasing, which serves to quantify dominance in similar semi-natural setups, may be a more reliable and behaviorally meaningful measure of dominance than the incidental tube competitions.

      (3) Unbalanced participation across rounds compromises stability analyses. Stability analyses (e.g., ICCs, round-to-round correlations) assume comparable sampling across individuals. However, some mice contribute 1 round, others 2, 3, 4, and even 5 rounds. This imbalance may inflate stability estimates or confound group reshuffling effects, and the rationale for variable participation is not explained.

    1. Author response:

      The following is the authors’ response to the original reviews.

      Reviewer #1 (Public review):

      Thank you so much for your comprehensive and insightful assessment of our manuscript. We appreciate your recognition of the novelty of our experimental design and the utility of our computational framework for interpreting visual remapping across the lifespan and in clinical populations. We are very grateful for your suggestions regarding the narrative flow, which have helped us to improve the manuscript's focus and coherence. Our responses to your specific concerns are detailed below.

      (1) Relevance of the figure-copy results (pp. 13-15). Is it necessary to include the figure-copy task results within the main text? The manuscript already presents a clear and coherent narrative without this section. The figure-copy task represents a substantial shift from the LOCUS paradigm to an entirely different task that does not measure the same construct. Moreover, the ROCF findings are not fully consistent with the LOCUS results, which introduces confusion and weakens the manuscript's coherence. While I understand the authors' intention to assess the ecological validity of their model, this section does not effectively strengthen the manuscript and may be better removed or placed in the Supplementary Materials.

      We thank the reviewer  for their perspective regarding the narrative flow and the transition between the LOCUS paradigm and the ROCF results. However, we remain keen to retain these findings in the main text, as they provide critical ecological and clinical validation for the computational mechanisms identified in our study.

      We think these results strengthen the manuscript for the following main reasons:

      (1) The ROCF we used is a standard neuropsychological tool for identifying constructional apraxia. Our results bridge the gap between basic cognitive neuroscience and clinical application by demonstrating that specific remapping parameters—rather than general memory precision—predict real-world deficits in patients.

      (2) The finding that our winning model explains approximately 62% of the variance in ROCF copy scores across all diagnostic groups further indicates that these parameters from the LOCUS task represent core computational phenotypes that underpin complex, real-life visuospatial construction (copying drawings).

      (3) Previous research has often observed only a weak or indirect link between drawing ability and traditional working memory measures, such as digit span (Senese et al., 2020). This was previously attributed to “deictic” strategies—like frequent eye and hand movements—that minimise the need to hold large amounts of information in memory (Ballard et al., 1995; Cohen, 2005; Draschkow et al., 2021). While our study was not exclusively designed to catalogue all cognitive contributions to drawing, the findings provide significant and novel evidence indicating that transsaccadic integration is a critical driver of constructional (copying drawing) ability. By demonstrating this link, the results provide evidence to stimulate a new direction for future research, shifting the focus from general memory capacity toward the precision of spatial updating across eye movements.

      In summary, by including the ROCF results in the main text, we provide evidence for a functional role for spatial remapping that extends beyond perceptual stability into the domain of complex visuomotor control. We have expanded on these points throughout the revised manuscript:

      In the Introduction: p.2:

      “The clinical relevance of these spatial mechanisms is underscored by significant disruptions to visuospatial processing and constructional apraxia—a deficit in copying and drawing figures—observed in neurodegenerative conditions such as Alzheimer's disease (AD) and Parkinson's disease (PD).[20,21] This raises a crucial question: do clinical impairments in complex visuomotor tasks stem from specific failures in transsaccadic remapping? If so, the computational parameters that define normal spatial updating should also provide a mechanistic account of these clinical deficits, differentiating them from general age-related decline.”

      p.3: "Finally, by linking these mechanistic parameters to a standard clinical measure of constructional ability (the Rey-Osterrieth Complex Figure task), we demonstrate that transsaccadic updating represents a core computational phenotype underpinning real-world visuospatial construction in both health and neurodegeneration.

      In the Results:

      “To assess whether the mechanistic parameters derived from the LOCUS task represent core phenotypes of real-world visuospatial abilities, we also instructed all participants to complete the Rey-Osterrieth Complex Figure copy task (ROCF; Figure 7A) on an Android tablet using a digital pen (see examples in Figure 7B; all Copy data are available in the open dataset: https://osf.io/95ecp/). The ROCF is a gold-standard neuropsychological tool for identifying constructional apraxia.[29] Historically, drawing performance has shown only weak or indirect correlations with traditional working memory measures.[30] This disconnect has been attributed to active visual-sampling strategies—frequent eye movements that treat the environment as an external memory buffer, minimising the necessity of holding large volumes of information in internal working memory.[3–5]

      We hypothesised that drawing accuracy is primarily constrained by the precision of spatial updating across frequent saccades rather than raw memory capacity. To evaluate the ecological validity of the identified saccade-updating mechanism, we modelled individual ROCF copy scores across all four groups using the estimated (maximum a posteriori) parameters from the winning “Dual (Saccade) + Interference” model (Model 7; Figure 8) as regressors in a Bayesian linear model. Prior to inclusion, each regressor was normalised by dividing by the square root of its variance.

      This model successfully explained 61.99% of the variance in ROCF copy scores, indicating that these computational parameters are strong predictors of real-word constructional ability (Figure 8A). … This highlights the critical role of accurate remapping based on saccadic information; even if the core saccadic update mechanism is preserved across groups (as shown in previous analyses), the precision of this updating process is crucial for complex visuospatial tasks. Moreover, worse ROCF copy performance is associated particularly with higher initial angular encoding error. This indicates that imprecision in the initial registration of angular spatial information contributes to difficulties in accurately reproducing complex visual stimuli.”

      In the Discussion:

      “Importantly, our computational framework establishes a direct mechanistic link between trassaccadic updating and real-world constructional ability. Specifically, higher saccade and angular encoding errors contribute to poorer ROCF copy scores. By mapping these mechanistic estimates onto clinical scores, we found that the parameters derived from our winning model explain approximately 62% of the variance in constructional performance across groups. These findings suggest that the computational parameters identified in the LOCUS task represent core phenotypes of visuospatial ability, providing a mechanistic bridge between basic cognitive theory and clinical presentation.

      This relationship provides novel insights into the cognitive processes underlying drawing, specifically highlighting the role of transsaccadic working memoty.ry. Previous research has primarily focused on the roles of fine motor control and eye-hand coordination in this skill.[4,50–55] This is partly because of consistent failure to find a strong relation between traditional memory measures and copying abili [4,31] For instance, common measures of working memory, such as digit span and Corsi block tasks, do not directly predict ROCF copying performance.[31,56] Furthermore, in patients with constructional apraxia, these memory performance measures often remain relatively preserved despite significant drawing impairments.[56–58] In the literature, this lack of association has often been attributed to “deictic” visual-sampling strategies, characterised by frequent eye movements that treat the environment as an external memory buffer, thereby minimising the need to maintain a detailed internal representation.[4,59] In a real-world copying task, the ROCF requires a high volume of saccades, making it uniquely sensitive to the precision of the dynamic remapping signals identified here. Recent eye-tracking evidence confirms that patients with AD exhibit significantly more saccades and longer fixations during figure copying compared to controls, potentially as a compensatory response to trassaccadic working memory constraints.[56] This high-frequency sampling—averaging between 150 and 260 saccades for AD patients compared to approximately 100 for healthy controls—renders the task highly dependent on the precision of dynamic remapping signals.[56] To ensure this relationship was not driven by a general "g-factor" or non-spatial memory impairment, we further investigated the role of broader cognitive performance using the ACE-III Memory subscale. We found that the relationship between transsaccadic working memory and ROCF performance remains highly significant, even after controlling for age, education, and ACE-III Memory subscore. This suggests that transsaccadic updating may represent a discrete computational phenotype required for visuomotor control, rather than a non-specific proxy for global cognitive decline.

      In other words, even when visual information is readily available in the world, the act of copying depends critically on working memory across saccades. This reveals a fundamental computational trade-off: while active sampling strategies (characterised with frequent eye-hand movements) effectively reduce the load on capacity-limited working memory, they simultaneously increase the demand for precise spatial updating across eye movements. By treating the external world as an "outside" memory buffer, the brain minimises the volume of information it must hold internally, but it becomes entirely dependent on the reliability with which that information is remapped after each eye movement. This perspective aligns with, rather contradicts, the traditional view of active sampling, which posits that individuals adapt their gaze and memory strategies based on specific task demands.[3,60] Furthermore, this perspective provides a mechanistic framework for understanding constructional apraxia; in these clinical populations, the impairment may not lie in a reduced memory "span," but rather in the cumulative noise introduced by the constant spatial remapping required during the copying process.[58,61]

      Beyond constructional ability, these findings suggest that the primary evolutionary utility of high-resolution spatial remapping lies in the service of action rather than perception. While spatial remapping is often invoked to explain perceptual stability,[11–13,15] the necessity of high-resolution transsaccadic memory for basic visual perception is debated.[13,62–64] A prevailing view suggests that detailed internal models are unnecessary for perception, given the continuous availability of visual information in the external world.[13,44] Our findings support an alternative perspective, aligning with the proposal that high-resolution transsaccadic memory primarily serves action rather than perception.[13] This is consistent with the need for precise localisation in eye-hand coordination tasks such as pointing or grasping.[65] Even when unaware of intrasaccadic target displacements, individuals rapidly adjust their reaching movements, suggesting direct access of the motor system to remapping signals.66 Further support comes from evidence that pointing to remembered locations is biased by changes in eye position,[67] and that remapping neurons reside within the dorsal “action” visual pathway, rather than the ventral “perception” visual pathway.[13,68,69] By demonstrating a strong link between transsaccadic working memory and drawing (a complex fine motor skill), our findings suggest that precise visual working memory across eye movements plays an important role in complex fine motor control.”

      (2) Model fitting across age groups (p. 9).

      It is unclear whether it is appropriate to fit healthy young and healthy elderly participants' data to the same model simultaneously. If the goal of the model fitting is to account for behavioral performance across all conditions, combining these groups may be problematic, as the groups differ significantly in overall performance despite showing similar remapping costs. This suggests that model performance might differ meaningfully between age groups. For example, in Figure 4A, participants 22-42 (presumably the elderly group) show the best fit for the Dual (Saccade) model, implying that the Interference component may contribute less to explaining elderly performance.

      Furthermore, although the most complex model emerges as the best-fitting model, the manuscript should explain how model complexity is penalized or balanced in the model comparison procedure. Additionally, are Fixation Decay and Saccade Update necessarily alternative mechanisms? Could both contribute simultaneously to spatial memory representation? A model that includes both mechanisms-e.g., Dual (Fixation) + Dual (Saccade) + Interference-could be tested to determine whether it outperforms Model 7 to rule out the sole contribution of complexity.

      We thank you for the opportunity to expand upon and clarify our modelling approach. Our decision to use a common generative model for both young and older adults was grounded in the empirical finding that there was no significant interaction between age group and saccade condition for either location or colour memory. While older adults demonstrated lower baseline precision, the specific "saccade cost" remained remarkably consistent across cohorts. This was the justification we proceeded on to use of a common model to assess quantitative differences in parameter estimates while maintaining a consistent mechanistic framework for comparison.

      Moreover, our winning model nests simpler models as special cases, providing the flexibility to naturally accommodate groups where certain components—such as interference—might play a reduced role. This ultimately confirms that the mechanisms for age-related memory deficits in this task reflect more general decline rather than a qualitative failure of the saccadic remapping process.

      This approach is further supported by the properties of the Bayesian model selection (BMS) procedure we used, which inherently penalises the inclusion of unnecessary parameters. Unlike maximum likelihood methods, BMS compares marginal likelihoods, representing the evidence for a model integrated over its entire parameter space. This follows the principle of Bayesian Occam’s Razor, where a model is only favoured if the improvement in fit justifies the additional parameter space; redundant parameters instead "dilute" the probability mass and lower the model evidence.

      Consequently, we contend that a hybrid model combining fixation and saccade mechanisms is unnecessary, as we have already adjudicated between alternative mechanisms of equal complexity. Specifically, Model 6 (Dual Fixation + Interference) and Model 7 (Dual Saccade + Interference) possess an identical number of parameters. The fact that Model 7 emerged as the clear winner—providing substantial evidence against Model 6 with a Bayes Factor of 6.11—demonstrates that our model selection is driven by the specific mechanistic account of the data rather than a simple preference for complexity.

      We have revised the Results and Discussion sections of the manuscript to state these points more explicitly for readers and have included references to established literature regarding the robustness of marginal likelihoods in guarding against overfitting.

      In the Results,

      “By fitting these models to the trial-by-trial response data from all healthy participants (N=42), we adjudicated between competing mechanisms to determine which best explained participant performance (Figure 4). We used random-effects Bayesian model selection to identify the most plausible generative model. This process relies on the marginal likelihood (model evidence), which inherently balances model fit against complexity—a principle often referred to as Occam’s razor.[25–27] The analysis yielded a strong result: the “Dual (Saccade) + Interference” model (Model 7 in Table 1) emerged as the winning model, providing substantial evidence against the next best alternative with a Bayes Factor of 6.11.”

      In the Discussion:

      “Our framework employs Variational Laplace, a method used to recover computational phenotypes in clinical populations like those with substance use disorders,[34,35] and the models we fit using this procedure feature time-dependent parameterisation of variance—conceptually similar to the widely-used Hierarchical Gaussian Filter.[36–39] Importantly, the risk of overfitting is mitigated by the Bayesian Model Selection framework; by utilising the marginal likelihood for model comparison, the procedure inherently penalises excessive model complexity and promotes generalisability.[25–27,40] This generalisability was further evidenced by the model's ability to predict performance on the independent ROCF task, confirming that these parameters represent robust mechanistic phenotypes rather than idiosyncratic fits to the initial dataset.”

      Minor point: On p. 9, line 336, Figure 4A does not appear to include the red dashed vertical line that is mentioned as separating the age groups.

      Thank you for pointing out this inconsistency. We apologise for the oversight; upon further review, we concluded that the red dashed vertical line was unnecessary for the clear presentation of the data. We have therefore removed the line from Figure 4A and deleted the corresponding sentence in the figure caption.

      (3) Clarification of conceptual terminology.

      Some conceptual distinctions are unclear. For example, the relationship between "retinal memory" and "transsaccadic memory," as well as between "allocentric map" and "retinotopic representation," is not fully explained. Are these constructs related or distinct? Additionally, the manuscript uses terms such as "allocentric map," "retinotopic representation," and "reference frame" interchangeably, which creates ambiguity. It would be helpful for the authors to clarify the relationships among these terms and apply them consistently.

      Thank you for pointing this out. We have revised the manuscript to ensure that these terms are applied with greater precision and consistency. Our revisions standardise the terminology based on the following distinctions:

      Reference frames: We distinguish between the eye-centred reference frame (coordinate systems that shift with gaze) and the world-centred reference frame (coordinate systems anchored to the environment).

      Retinotopic representation vs. allocentric map: We clarify that retinotopic representations are encoded within an eye-centred reference frame and are updated with every ocular movement. Conversely, the allocentric map is anchored to stable environmental features, remaining invariant to the observer’s gaze direction or position.

      Retinotopic memory vs. transsaccadic memory: We have removed the term "retinal memory" to avoid ambiguity. We now consistently use retinotopic memory to describe the persistence of visual information in eye-centred coordinates within a single fixation. In contrast, transsaccadic memory refers to the higher-level integration of visual information across saccades, which involves the active updating or remapping of representations to maintain stability.

      To incorporate these clarifications, we have implemented the following changes:

      In the Introduction, the second paragraph has been entirely rewritten to establish these definitions at the outset, providing a clearer theoretical framework for the study.

      “Central to this enquiry is the nature of the coordinate system used for the brain's internal spatial representation. Does the brain maintain a single, world-centred (allocentric) map, or does it rely on a dynamic, eye-centred (retinotopic) representation?[11,13,15,16] In the latter system, retinotopic memory preserves spatial information within a fixation, whereas transsaccadic memory describes the active process of updating these representations across eye movements to achieve spatiotopic stability—the perception of a stable world despite eye movements.[11,16–18] If spatial stability is indeed reconstructed through such remapping, the mechanism remains unresolved: do we retain memories of absolute fixation locations, or do we reconstruct these positions from noisy memories of the intervening saccade vectors? We can test these hypotheses by analysing when and where memory errors occur. Assuming that memory precision declines over time,[19] the resulting error distributions should reveal the specific variables that are represented and updated across each saccade.”

      In the Results, the opening section of the Results has been reorganised to align with this terminology. We have ensured that the hypotheses and behavioural data—specifically the definition of "saccade cost"—are introduced using this consistent conceptual vocabulary to improve the overall coherence of the narrative.

      (4) Rationale for the selective disruption hypothesis (p. 4, lines 153-154). The authors hypothesize that "saccades would selectively disrupt location memory while leaving colour memory intact." Providing theoretical or empirical justification for this prediction would strengthen the argument.

      We have revised the Results to state the hypothesis more explicitly and expanded the Discussion to provide a robust theoretical and empirical rationale:

      In the Results,

      “This design allowed us to isolate and quantify the unique impact of saccades on spatial memory, enabling us to test competing hypotheses regarding spatial representation. If spatial memory were solely underpinned by an allocentric mechanism, precision should remain comparable across all conditions as the representation would be world-centred and unaffected by eye movements. Thus, performance in the no-saccade condition should be comparable to the two-saccade condition. Conversely, if spatial memory relies on a retinotopic representation requiring active updating across eye movements, the two-saccade condition was anticipated to be the most challenging due to cumulative decay in the memory traces used for stimulus reconstruction after each saccade.[22] Critically, we hypothesised that this saccade cost would be specific to the spatial domain; while location requires active remapping via noisy oculomotor signals, non-spatial features like colour are not inherently tied to coordinate transformations and should therefore remain stable (see more in Discussion below).

      Meanwhile, the no-saccade condition was expected to yield the most accurate localisation, relying solely on retinotopic information (retinotopic working memory). These predictions were confirmed in young healthy adults (N = 21, mean age = 24.1 years, ranged between 19 and 34). A repeated measures ANOVA revealed a significant main effect of saccades on location memory (F(2.2,43.9)=33.2, p<0.001, partial η²=0.62), indicating substantial impairment after eye movements (Figure 2A). In contrast, colour memory remained remarkably stable across all saccade conditions (Figure 2B; F(2.2, 44.7) = 0.68, p=0.53, partial η² =0.03).

      This “saccade cost”—the loss of memory precision following an eye movement—indicates that spatial representations require active updating across saccades rather than being maintained in a static, world-centred reference frame.

      Critically, our comparison between spatial and colour memory does not rely on the absolute magnitude of errors, which are measured in different units (degrees of visual angle vs. radians). Instead, we assessed the relative impact of the same saccadic demand on each feature within the same trial. While location recall showed a robust saccade cost, colour recall remained statistically unchanged. To ensure this null effect was not due to a lack of measurement sensitivity, we examined the recency effect; recall performance for the second item was predicted to be better than for the first stimulus in each condition.[23,24] As expected, colour memory for Item 2 was significantly more accurate than for Item 1 (F(1,20) = 6.52, p = 0.02, partial η² = 0.25), demonstrating that the task was sufficiently sensitive to detect standard working memory fluctuations despite the absence of a saccade-induced deficit.”

      In the Discussion, we now write that on p.18:

      “A clear finding was the specificity of the saccade cost to spatial features; it was not observed for non-spatial features like colour, even in neurodegenerative conditions. This discrepancy challenges notions of fixed visual working memory capacity unaffected by saccades.16,44–46 The differential impact on spatial versus non-spatial features in transsaccadic memory aligns with the established "what" and "where" pathways in visual processing.32,33 For objects to remain unified, object features must be bound to stable representations of location across saccades.19 One possibility is that remapping updates both features and location through a shared mechanism, predicting equal saccadic interference for both colour and location in the present study.

      However, our findings suggest otherwise. One potential concern is whether this dissociation simply reflects the inherent spatial noise introduced by fixational eye movements (FEMs), such as microssacades and drifts.47 Because locations are stored in a retinotopic frame, fixational instability necessarily shifts retinal coordinates over time. However, the "saccade cost" here was defined as the error increase relative to a no-saccade baseline of equal duration; because both conditions are subject to the same fixational drift, any FEM-induced noise is effectively subtracted out. Thus, despite the ballistic and non-Gaussian nature of FEMs,48 they cannot account for the fact the saccade cost in the spatial memory, but total absence in the colour domain. Another possibility is that this dissociation reflects differences in baseline task difficulty or dynamic range. Yet, the presence of a robust recency effect in colour memory (Figure 2B) confirms that our paradigm was sensitive to memory-dependent variance and was not limited by floor or ceiling effects.

      The fact that identical eye movements—executed simultaneously and with identical vectors—systematically degraded spatial precision while sparing colour suggests a feature-specific susceptibility to transsaccadic remapping. This supports the view that the computational process of updating an object’s location involves a vector-subtraction mechanism—incorporating noisy oculomotor commands (efference copies)—that introduces specific spatial variance. Because this remapping is a coordinate transformation, the resulting sensorimotor noise does not functionally propagate to non-spatial feature representations. Consequently, features like colour may be preserved or automatically remapped without the precision loss associated with spatial updating.11,49 Our paradigm thus provides a refined tool to investigate the architecture of transsaccadic working memory across distinct object features.”

      (5) Relationship between saccade cost and individual memory performance (p. 4, last paragraph).

      The authors report that larger saccades were associated with greater spatial memory disruption. It would be informative to examine whether individual differences in the magnitude of saccade cost correlate with participants' overall/baseline memory performance (e.g. their memory precision in the no-saccade condition). Such analyses might offer insights into how memory capacity/ability relates to resilience against saccade-induced updating.

      We have now conducted the correlation analysis to determine whether baseline memory capacity (no-saccade condition) predicts resilience to saccade-induced updating. The results indicate that these two factors are independent.

      To clarify the nature of the saccade-induced impairment, we have updated the text as follows:

      p.4: “This “saccade cost”—the loss of memory precision following an eye movement—indicates that spatial representations require active updating across saccades rather than being maintained in a static, world-centred reference frame.”

      p.5: “Further analysis examined whether individual differences in baseline memory precision (no-saccade condition) predicted resilience to saccadic disruption. Crucially, individual saccade costs (defined as the precision loss relative to baseline) did not correlate with baseline precision (rho = 0.20, p = 0.20). This suggests that the noise introduced by transsaccadic remapping acts as an independent, additive source of variance that is not modulated by an individual’s underlying memory capacity. These findings imply a functional dissociation between the mechanisms responsible for maintaining a representation and those involved in its coordinate transformation.”

      (6) Model fitting for the healthy elderly group to reveal memory-deficit factors (pp. 11-12). The manuscript discusses model-based insights into components that contribute to spatial memory deficits in AD and PD, but does not discuss components that contribute to spatial memory deficits in the healthy elderly group. Given that the EC group also shows impairments in certain parameters, explaining and discussing these outcomes of the EC group could provide additional insights into age-related memory decline, which would strengthen the study's broader conclusions.

      This is a very good point. We rewrote the corresponding results section (p.12-13):

      “Modelling reveals the sources of spatial memory deficits in healthy aging and neurodegeneration - To understand the source of the observed deficits, we applied the winning ‘Dual (Saccade) + Interference’ model the data from all participants (YC, EC, AD, and PD). By fitting the model to the entire dataset, we obtained estimates of the parameters for each individual, which then formed the basis for our group-level analysis. To formally test for group differences, we used Parametric Empirical Bayes (PEB), a hierarchical Bayesian approach that compares parameter estimates across groups while accounting for the uncertainty of each estimate [28]. This allowed us to identify which specific cognitive mechanisms, as formalised by the model parameters, were affected by age and disease.

      The Bayesian inversion used here allows us to quantify the posterior mode and variance for each parameter and the covariance for each parameter. From these, we can compute the probabilities that pairs of parameters differ from one another, which we report as P(A>B)—meaning the posterior probability that the parameter for group A was greater than that for group B.

      We first examined the specific parameters differentiating healthy elderly (EC) from young controls (YC) to isolate the factors contributing to non-pathological, age-related decline. The analysis revealed that healthy ageing is primarily characterised by a significant increase in Radial Decay (P(EC > YC) = 0.995), a heightened susceptibility to Interference (P(EC > YC) = 1.000), and a reduction in initial Angular Encoding precision (P(YC < EC) = 0.002; Figure 6). These results suggest that normal ageing degrades the fidelity of the initial memory trace and its resilience over time, while the core computational process of updating information across saccades remains intact.

      Beyond these baseline ageing effects, our clinical cohorts exhibited more severe and condition-dependent impairments. Radial decay showed a clear, graded impairment: AD patients had a greater decay rate than PD patients (P(AD > PD) = 1.000), who in turn were more impaired than the EC group (P(PD > EC) = 0.996). A similar graded pattern was observed for Interference, where AD patients were most susceptible (P(AD > PD) = 0.999), while the PD and EC groups did not significantly differ (P(PD > EC) = 0.532).

      Patients with AD also showed a tendency towards greater angular decay than controls (P(AD > EC) = 0.772), although this fell below the 95% probability threshold. This effect was influenced by a lower decay rate in the PD group compared to the EC group (P(PD < EC) = 0.037). In contrast, group differences in encoding were less pronounced. While YC exhibited significantly higher precision than all other groups, AD patients showed significantly higher angular encoding error than PD patients (P(AD > PD) = 0.985), though neither group differed significantly from the EC group.

      Crucially, parameters related to the saccade itself—saccade encoding and saccade decay—did not differentiate the groups. This indicates that neither healthy ageing nor the early stages of AD and PD significantly impair the fundamental machinery for transsaccadic remapping. Instead, the visuospatial deficits in these conditions arise from specific mechanistic failures: a faster decay of radial position information and increased susceptibility to interference, both of which are present in healthy ageing but significantly amplified by neurodegeneration.”

      In the Discussion, we added:

      “Although saccade updating was an essential component of the winning model, its two key parameters—initial encoding error and decay rate during maintenance—did not significantly differ across groups. This indicates that the core computational process of updating spatial information based on eye movements is largely preserved in healthy aging and neurodegeneration.

      Instead, group differences were driven by deficits in angular encoding error (precision of initial angle from fixation), angular decay, radial decay (decay in memory of distance from fixation), and interference susceptibility. This implies a functional and neuroanatomical dissociation: while the ventral stream (the “what” pathway) shows an age-related decline in the quality and stability of stored representations, the dorsal-stream (the “where” pathway) parietal-frontal circuits responsible for coordinate transformations remain functionally robust.[31–34] These spatial updating mechanisms appear resilient to the normal ageing trajectory and only break down when challenged by the specific pathological processes seen in Alzheimer’s or Parkinson’s disease.”

      (7) Presentation of saccade conditions in Figure 5 (p. 11). In Figure 5, it may be clearer to group the four saccade conditions together within each patient group. Since the main point is that saccadic interference on spatial memory remains robust across patient groups, grouping conditions by patient type rather than intermixing conditions would emphasize this interpretation.

      There are several valid ways to present these plots, but we chose this format because it allows for a direct visual comparison of the post-hoc group differences within each specific task demand. This arrangement clearly illustrates the graded impairment from young controls through to patients with Alzheimer’s disease across every condition. This structure also directly mirrors our two-way ANOVA, which identified significant main effects for both Group and Condition, but crucially, no significant Group x Condition interaction. We felt that grouping the data by participant group would force readers to look across four separate clusters to compare the slopes, making the stability of the saccadic remapping mechanism much harder to grasp at a glance.

      Reviewer #1 (Recommendations for the authors):

      (1) Formatting of statistical parameters.

      The formatting of statistical symbols should be consistent throughout the manuscript. Some instances of F, p, and t are italicized, while others are not. All statistical symbols should be italicized.

      Thank you for pointing this out. We have audited the manuscript. While we have revised the text to address these instances throughout the Results and Methods sections, any remaining minor formatting inconsistencies will be corrected during the final typesetting stage.

      (2) Minor typographical issues.

      (a) Line 532: "are" should be "be."

      (b) Line 654: "cantered" should be "centered."

      (c) Line 213: In "(p(bonf) < 0.001, |t| {greater than or equal to} 5.94)," the t value should be reported with its degrees of freedom, and t should be reported before p. The same applies to line 215.

      Thank you for your careful reading. All corrected.

      Reviewer #2 (Public review):

      We thank you for your positive feedback regarding our eye-tracking methodology and computational approach. We appreciate your critical insights into the feature-specific disruption hypothesis and the task structure. We have substantially revised the results and discussion about the saccadic interference on colour memory. Below we will answer your suggestions point-by-point:

      Reviewer #2 (Recommendations for the authors):

      (1) The study treats colour and location errors as comparable when arguing that saccades selectively disrupt spatial but not colour memory. However, these measures are defined in entirely different units (degrees of visual angle vs radians on a colour wheel) and are not psychophysically or statistically calibrated. Baseline task difficulty, noise level, or dynamic range do not appear to be calibrated or matched across features. As a result, the null effect of saccades on colour could reflect lower sensitivity or ceiling effects rather than implicit feature-specific robustness.

      We agree that direct comparisons of absolute error magnitudes across different dimensions are not appropriate. Our argument for feature-specific disruption relies not on the scale of errors, but on the presence or absence of a saccade cost within identical trials. In our within-subject design, the same saccade vectors produced a systematic increase in location error while leaving colour error statistically unchanged. To address sensitivity, we observed that colour memory was sufficiently precise to show a significant recency effect (p = 0.02). To further quantify the evidence for the null effect, we performed Bayesian repeated measures ANOVAs, which yielded a BF10 = 0.22. This provides substantial evidence that saccades do not disrupt colour precision, regardless of baseline sensitivity.

      We have substantially revised this in Results, Methods and Discussion:

      In the Results:

      “This design allowed us to isolate and quantify the unique impact of saccades on spatial memory, enabling us to test competing hypotheses regarding spatial representation. If spatial memory were solely underpinned by an allocentric mechanism, precision should remain comparable across all conditions as the representation would be world-centred and unaffected by eye movements. Thus, performance in the no-saccade condition should be comparable to the two-saccade condition. Conversely, if spatial memory relies on a retinotopic representation requiring active updating across eye movements, the two-saccade condition was anticipated to be the most challenging due to cumulative decay in the memory traces used for stimulus reconstruction after each saccade.[22] Critically, we hypothesised that this saccade cost would be specific to the spatial domain; while location requires active remapping via noisy oculomotor signals, non-spatial features like colour are not inherently tied to coordinate transformations and should therefore remain stable (see more in Discussion below).

      Meanwhile, the no-saccade condition was expected to yield the most accurate localisation, relying solely on retinotopic information (retinotopic working memory). These predictions were confirmed in young healthy adults (N = 21, mean age = 24.1 years, ranged between 19 and 34). A repeated measures ANOVA revealed a significant main effect of saccades on location memory (F(2.2,43.9)=33.2, p<0.001, partial η²=0.62), indicating substantial impairment after eye movements (Figure 2A). In contrast, colour memory remained remarkably stable across all saccade conditions (Figure 2B; F(2.2, 44.7) = 0.68, p=0.53, partial η² =0.03).

      This “saccade cost”—the loss of memory precision following an eye movement—indicates that spatial representations require active updating across saccades rather than being maintained in a static, world-centred reference frame.

      Critically, our comparison between spatial and colour memory does not rely on the absolute magnitude of errors, which are measured in different units (degrees of visual angle vs. radians). Instead, we assessed the relative impact of the same saccadic demand on each feature within the same trial. While location recall showed a robust saccade cost, colour recall remained statistically unchanged. To ensure this null effect was not due to a lack of measurement sensitivity, we examined the recency effect; recall performance for the second item was predicted to be better than for the first stimulus in each condition.[23,24] As expected, colour memory for Item 2 was significantly more accurate than for Item 1 (F(1,20) = 6.52, p = 0.02, partial η² = 0.25), demonstrating that the task was sufficiently sensitive to detect standard working memory fluctuations despite the absence of a saccade-induced deficit.”

      In the Methods, at the beginning of “Statistical Analysis”, we added

      “Because location and colour recall involve different scales and units, all analyses were performed independently for each feature to avoid cross-dimensional magnitude comparisons.” (p25)

      In the Discussion, we added:

      “A potential concern is whether the observed dissociation between colour and location reflects differences in baseline task difficulty or dynamic range. Yet, the presence of a robust recency effect in colour memory (Figure 2B) confirms that our paradigm was sensitive to memory-dependent variance and was not limited by floor or ceiling effects.”

      (2) Colour and then location are probed serially, without a counter-balanced order. This fixed response order could introduce a systematic bias because location recall is consistently subject to longer memory retention intervals and cognitive interference from the colour decision. The observed dissociation-saccades impair location but not colour, and may therefore reflect task structure rather than implicit feature-specific differences in trans-saccadic memory.

      Thank you for the insightful observation regarding our fixed response order. We acknowledge that that a counterbalanced design is typically preferred to mitigate potential order effects. However, we chose this consistent sequence to ensure the task remained accessible for cognitively impaired patients (i.e., the Alzheimer’s disease (AD) and Parkinson’s disease (PD) cohorts). Conducting an eye-tracking memory task with cognitively impaired patients is challenging, as they may struggle with task engagement or forget complex instructions. During the design phase, we prioritised a consistent structure to reduce the cognitive load and task-switching demands that typically challenge these cohorts.

      Critically, because the saccade cost is a relative measure calculated by comparing conditions with identical timings, any bias from the fixed order is present in both the baseline and saccade trials. The disruption we report is therefore a specific effect of eye movements that goes beyond the noise introduced by the retention interval or the preceding colour report.

      We added the following text in the Methods – experimental procedure (p.22):

      “Recall was performed in a fixed order, with colour reported before location. This sequence was primarily chosen to minimise cognitive load and task-switching demands for the two neurological patient cohorts, ensuring the paradigm remained accessible for individuals with AD and PD. While this order results in a slightly longer retention interval for location recall, the saccade cost was identified by comparing location error across experimental conditions with similar timings but varying saccadic demands.”

      (3) Relatedly, because spatial representations are retinotopic, fixational eye movements (FEMs - microsaccades and drift) displace the retinal coordinates of encoded positions, increasing apparent spatial noise with time delays. Colour memory, however, is feature-based and unaffected by small retinal translations. Thus, any between-condition or between-group differences in FEMs could selectively inflate location error and the associated model parameters (encoding noise, decay, interference), while leaving colour error unchanged. Note that FEMs tend to be slightly ballistic [1,2], hence not well modelled with a Gaussian blur.

      This is a very insightful point. We have now addressed this in detail within the discussion:

      “However, our findings suggest otherwise. One potential concern is whether this dissociation simply reflects the inherent spatial noise introduced by fixational eye movements (FEMs), such as microssacades and drifts.[46] Because locations are stored in a retinotopic frame, fixational instability necessarily shifts retinal coordinates over time. However, the "saccade cost" here was defined as the error increase relative to a no-saccade baseline of equal duration; because both conditions are subject to the same fixational drift, any FEM-induced noise is effectively subtracted out. Thus, despite the ballistic and non-Gaussian nature of FEMs,n [47] they cannot account for the fact the saccade cost in the spatial memory, but total absence in the colour domain. Another possibility is that this dissociation reflects differences in baseline task difficulty or dynamic range. Yet, the presence of a robust recency effect in colour memory (Figure 2B) confirms that our paradigm was sensitive to memory-dependent variance and was not limited by floor or ceiling effects.”

      (4) There is no in silico demonstration that the modelling framework can recover the true generating model from synthetic data or recover accurate parameters under realistic noise levels, which can be challenging in generative models with a hierarchical structure (as per [3], for example). Figure 8b shows that the parameters possess substantial posterior covariance, which raises concerns as to whether they can be reliably disambiguate.

      Many thanks for this comment. We have added a simple recovery analysis as detailed below but are also keen to ensure we fully answer your question—which has more to do with empirical rather than simulated data—and make clear the rationale for this analysis in this instance.

      We added this in Supplementary Materials:

      “Model validation and recovery analysis

      The following section provides a detailed technical assessment of the model inversion scheme, focusing on the discriminability of the model space and the identifiability of individual parameters.

      Recovery analyses of this sort are typically used prior to collecting data to allow one to determine whether, in principle, the data are useful in disambiguating between hypotheses. In this sense, they have a role analogous to a classical power calculation. However, their utility is limited when used post-hoc when data have already been collected, as the question of whether the models can be disambiguated becomes one of whether non-trivial Bayes factors can be identified from those data.

      The reason for including a recovery analysis here is not to identify whether the model inversion scheme identifies a ‘true’ model. The concept of ‘true generative models’ commits to a strong philosophical position which is at odds with the ‘all models are wrong, but some are useful’ perspective held by many in statistics, e.g., (So, 2017). Of note, one can always confound a model recovery scheme by generating the same data in a simple way, and in (one of an infinite number of) more complex ways. A good model inversion scheme will always recover the simple model and therefore would appear to select the ‘wrong’ model in a recovery analysis. However, it is still the best explanation for the data. For these reasons, we do not necessarily expect ‘good’ recoverability in all parameter ranges. This is further confounded by the relationship between the models we have proposed—e.g., an interference model with very low interference will look almost identical to a model with no interference. The important question here is whether they can be disambiguated with real data.

      Instead, the value of a post-hoc recovery analysis here is to evaluate whether there was a sensible choice of model space—i.e., that it was not a priori guaranteed that a single model (and, specifically, the model we found to be the best explanation for the data) would explain the results of all others. To address this, for each model, we simulated 16 datasets, each of which relied upon parameters sampled from the model priors, which included examples of each of the experimental conditions. We then fit each of these datasets to each of the 7 models to construct the confusion matrix shown in the lower panel of Supplementary Figure 3, by accumulating evidence over each of the 16 participants generated according to each ‘true’ model (columns) for each of the possible explanatory models (rows). This shows that no one model, for the parameter ranges sampled here, explains all other datasets. Interestingly, our ‘winning’ model in the empirical analysis is not the best explanation for any of the datasets simulated (including its own). This is reassuring, in that it implies this model winning was not a foregone conclusion and is driven by the data—not just the choice of model space.”

      Your point about the posterior covariance is well founded. As we describe in Supplementary Materials, this is an inherent feature of inverse problems (analogous to EEG source localisation). However, the fact that our posterior densities move significantly away from the prior expectations demonstrates that the data are indeed informative. By adopting a Bayesian framework, we are able to explicitly quantify this uncertainty rather than ignoring it, providing a more transparent account of parameter identifiability. We have added the following in the same section of Supplementary Materials:

      “This problem is an inverse problem—inferring parameters from a non-linear model. We therefore expect a degree of posterior covariance between parameters and, consequently, that they cannot be disambiguated with complete certainty. While some degree of posterior covariance is inherent to inverse models—including established methods like EEG source localisation—the fact that many of the parameters are estimated with posterior densities that do not include their prior expectations implies the data are informative about these.

      The advantage of the Bayesian approach we have adopted here is that we can explicitly quantify posterior covariance between these parameters, and therefore the degree to which they can be disambiguated. While the posterior covariance matrices from empirical data are the relevant measure here, we can better understand the behaviour of the model inversion scheme in relation to the specific models used using the model recovery analysis reported in Supplementary figure 3.

      The middle panel of the figure is key, along with the correlation coefficients reported in the figure caption. Here, we see at least a weak positive correlation (in some cases much stronger) for almost all parameters and limited movement from prior expectations for those parameters that are less convincingly recovered. This reinforces that the ability of the scheme to recover parameters is best assessed in terms of the degree of movement of posterior from prior values following fitting to empirical data.”

      (5) The authors employ Bayes factors (BFs) to disambiguate models, but BFs would also strengthen the claims that location, but not colour, is impacted by saccades. Despite colour being a circular variable, colour error is analysed using ANOVA on linearised differences (radians). The authors should also arguably use circular statistics, such as the von Mises distribution, for the analysis of colour.

      Regarding the use of circular statistics, you are correct that such error distributions are not suitable for ANOVA, and it is better to use circular statistics. However, for the present dataset, we used the mean absolute angular error per condition (ranging from 0 to π radians), which represents the shortest distance on the colour wheel between the target and the response.

      This approach effectively linearises the measure by removing the 2π wrap-around boundary. because the observed errors were relatively small and did not cluster near the π boundary—even in the patient cohorts (Figure 5B)—the "wrap-around" effect of circular space is negligible. Moreover, by analysing the mean error across trials for each condition, rather than trial-wise data, we invoke the Central Limit Theorem. This ensures that the distribution of these means is approximately normal, satisfying the fundamental assumptions of ANOVA. Due to these reasons, we adopted simpler linear models. We confirmed that the data did not violate the assumptions of linear statistics. In this low-noise regime, linear and circular models converge on the same conclusions. This has been revised in Methods:

      “For colour memory, we calculated the absolute angular error, defined as the shortest distance on the colour wheel between the target and the reported colour (range 0 to π radians). For the primary statistical analyses, we utilised the mean absolute error per condition for each participant. By analysing these condition-wise means rather than trial-wise raw data, we invoke the Central Limit Theorem, which ensures that the sampling distribution of these means approximates normality. Because the absolute errors in this paradigm were relatively small and did not approach the π boundary (Figure 5B) even in the clinical cohorts, the data were treated as a continuous measure in our linear ANOVAs and regression models. Moreover, because location and colour recall involve different scales and units, all analyses were performed independently for each feature to avoid cross-dimensional magnitude comparisons.”

      We have also now integrated Bayesian repeated measures ANOVA throughout the manuscript. The Results section for the young healthy adults now reads (p. 4):

      “A repeated measures ANOVA revealed a significant main effect of saccades on location memory (F(3, 20) = 51.52, p < 0.001, partial η²=0.72), with Bayesian analysis providing decisive evidence for the inclusion of the saccade factor (BF<sub>incl</sub> = 3.52 x 10^13, P(incl|data) = 1.00). In contrast, colour memory remained remarkably stable across all saccade conditions (F(3, 20) = 0.57, p = 0.64, partial η² =0.03). This null effect was supported by Bayesian analysis, which provided moderate evidence in favour of the null hypothesis (BF<sub>01</sub> = 8.46, P(excl|data) = 0.89), indicating that the data were more than eight times more likely under the null model than a model including saccade-related impairment.”

      For elderly healthy adults:

      “In contrast, colour memory remained unaffected by saccade demands (F(3, 20) = 0.57, p = 0.65, partial η² =0.03), again supported by the Bayesian analysis: BF<sub>01</sub> = 8.68, P(excl|data) = 0.90.”

      For patient cohorts:

      “Bayesian repeated measures ANOVAs further supported this dissociation, providing moderate evidence for the null hypothesis in the AD group (BF<sub>01</sub> = 3.35, P(excl|data) = 0.77) and weak evidence in the PD group (BF<sub>01</sub> = 2.23, P(excl|data) = 0.69). This indicates that even in populations with established neurodegeneration, the detrimental impact of eye movements is specific to the spatial domain.”

      Related description is also updated in Methods – Statistical Analysis.

      Minor:

      (1) The modelling is described as computational but is arguably better characterised as a heuristic generative model at Marr's algorithmic level. It does not derive from normative computational principles or describe an implementation in neural circuits.

      We appreciate your perspective on the classification of our model within Marr’s hierarchy. We agree that our framework is best characterised as an algorithmic-level generative model. Our objective was to identify the mechanistic principles governing transsaccadic updating rather than to provide a normative derivation or a specific circuit-level implementation.

      To ensure readers do not over-interpret the term ‘computational’, we have added a clarifying statement in the Discussion acknowledging the algorithmic nature of the model. Interestingly, we note that a model predicated on this form of spatial diffusion implies a neural field representation with a spatial connectivity kernel whose limit approximates the second derivative of a Dirac delta function. While a formal neural field implementation is beyond the scope of the present work, our algorithmic results provide the necessary constraints for such future biophysical models.

      p.20: “While we describe the present framework as 'computational', it is more precisely characterised as an algorithmic-level generative model within Marr’s hierarchy. Our focus was on defining the rules of spatial integration and the sources of eye-movement-induced noise, rather than deriving these processes from normative principles or defining their specific neural implementation.”

      (2) I did not find a description of the recruitment and characterization of the AD and PD patients.

      Apologies for this omission. We have now included a detailed description of participant recruitment and clinical characterisation in the Methods section and also updated Table 2:

      “A total of 87 participants completed the study: 21 young healthy adults (YC), 21 older healthy adults (EC), 23 patients with Parkinson’s disease (PD), and 22 patients with Alzheimer’s disease (AD). Their demographic and clinical details are summarised in Table 2. Initially, 90 participants were recruited (22 YC, 21 EC, 25 PD, 22 AD); however, three individuals (1 YC and 2 PD) were excluded from all analyses due to technical issues during data acquisition.

      All participants were recruited locally in Oxford, UK. None were professional artists, had a history of psychiatric illness, or were taking psychoactive medications (excluding standard dopamine replacement therapy for PD patients). Young participants were recruited via the University of Oxford Department of Experimental Psychology recruitment system. Older healthy volunteers (all >50 years of age) were recruited from the Oxford Dementia and Ageing Research (OxDARE) database.

      Patients with PD were recruited from specialist clinics in Oxfordshire. All had a clinical diagnosis of idiopathic Parkinson's disease and no history of other major neurological or psychiatric conditions. While specific dosages of dopamine replacement therapy (e.g., levodopa equivalent doses) were not systematically recorded, all patients were tested while on their regular medication regimen ('ON' state).

      Patients with PD were recruited from clinics in the Oxfordshire area. All had a clinical diagnosis of idiopathic Parkinson’s disease and no history of other major neurological or psychiatric illnesses. While all patients were tested in their regular medication ‘ON’ state, the specific pharmacological profiles—including the exact types of medication (e.g., levodopa, dopamine agonists, or combinations) and dosages—were not systematically recorded. The disease duration and PD severity were also un-recorded for this study.

      Patients with AD were recruited from the Cognitive Disorders Clinic at the John Radcliffe Hospital, Oxford, UK. All AD participants presented with a progressive, multidomain, predominantly amnestic cognitive impairment. Clinical diagnoses were supported by structural MRI and FDG-PET imaging consistent with a clinical diagnosis of AD dementia (e.g., temporo-parietal atrophy and hypometabolism).69 All neuroimaging was reviewed independently by two senior neurologists (S.T. and M.H.).

      Global cognitive function was assessed using the Addenbrooke’s Cognitive Examination-III (ACE-III).70 All healthy participants scored above the standard cut-off of 88, with the exception of one elderly participant who scored 85. In the PD group, two participants scored below the cut-off (85 and 79). In the AD group, six participants scored above 88; these individuals were included based on robust clinical and radiological evidence of AD pathology rather than their ACE-III score alone.”

      (3) YA and OA patients appear to differ in gender distribution.

      We acknowledge the difference in gender distribution between the young (71.4% female) and older adult (57.1% female) cohorts. However, we do not anticipate that gender influences the fundamental computational mechanisms of retinotopic maintenance or transsaccadic remapping. These processes represent low-level visuospatial functions for which there is no established evidence of gender-specific differences in precision or coordinate transformation. We have ensured that the gender distribution for each cohort is clearly listed in the demographics table (Table 2) for full transparency.

      Thank you very much for very insightful feedback!

      Reviewer #3 (Public review):

      Thank you for the positive feedback regarding our inclusion of clinical groups and the identification of computational phenotypes that differentiate these cohorts.

      To address your concerns about the model, we have clarified our use of Bayesian Model Selection, which inherently penalises model complexity to ensure that our results are not driven solely by the number of parameters. We will also provide further evidence regarding model generalisability to address the concern of overfitting.

      Regarding the link with the ROCF, we have revised the manuscript to better highlight the specific relationship between our transsaccadic parameters and the ROCF data and better motivate the inclusion of these results in the main text.

      Below is our response to your suggestions point-by-point:

      (1) The models tested differ in terms of the number of parameters. In general, a larger number of parameters leads to a better goodness of fit. It is not clear how the difference in the number of parameters between the models was taken into account. It is not clear whether the modelling results could be influenced by overfitting (it is not clear how well the model can generalize to new observations).

      To ensure our results were not driven by the number of parameters, we utilised random-effects Bayesian Model Selection (BMS) to adjudicate between our candidate models. Unlike maximum likelihood methods, BMS relies on the marginal likelihood (model evidence), which inherently balances model fit against parsimony—a principle known as the Occam’s Razor (Rasmussen and Ghahramani, 2000). In this framework, a model is only preferred if the improvement in fit justifies the additional parameter space; redundant parameters actually lower model evidence by diluting the probability mass. We would be happy to point toward literature that discusses how these marginal likelihood approximations provide a more robust guard against overfitting than standard metrics like BIC or AIC (MacKay, 2003; Murray and Ghahramani, 2005; Penny, 2012).

      The fact that the "Dual (Saccade) + Interference" model (Model 7) emerged as the winner—with a Bayes Factor of 6.11 against the next best alternative—demonstrates that its complexity was statistically justified by its superior account of the trial-by-trial data.

      Furthermore, to address the risk of overfitting, we established the generalisability of these parameters by using them to predict performance on an independent clinical task. These parameters successfully explained ~62% of the variance in ROCF copy scores—a very distinct, real-world task--confirming that they represent robust computational phenotypes rather than idiosyncratic fits to the initial dataset.

      In the Results (p10):

      “We used random-effects Bayesian model selection to identify the most plausible generative model. This process relies on the marginal likelihood (model evidence), which inherently balances model fit against complexity—a principle often referred to as Occam’s razor.[25–27]”

      In the Discussion (p17):

      “Importantly, the risk of overfitting is mitigated by the Bayesian Model Selection framework; by utilising the marginal likelihood for model comparison, the procedure inherently penalises excessive model complexity and promotes generalisability.[25–27,42] This generalisability was further evidenced by the model's ability to predict performance on the independent ROCF task, confirming that these parameters represent robust mechanistic phenotypes rather than idiosyncratic fits to the initial dataset.”

      (2) Results specificity: it is not clear how specific the modelling results are with respect to constructional ability (measured via the Rey-Osterrieth Complex Figure test). As with any cognitive test, performance can also be influenced by general, non-specific abilities that contribute broadly to test success.

      We agree that constructional performance is influenced by both specific mechanistic constraints and general cognitive abilities. To isolate the unique contribution of transsaccadic updating, we therefore performed a partial correlation analysis across the entire sample. We examined the relationship between location error in the two-saccades condition (our primary behavioural measure of transsaccadic memory) and ROCF copy scores. Even after partialling out the effects of global cognitive status (ACE-III total score), age, and years of education, the correlation remained highly significant (rho = -0.39, p < 0.001).

      This suggests that our model captures a specific computational phenotype—the precision of spatial updating during active visual sampling—rather than acting as a proxy for non-specific cognitive decline. This mechanistic link explains why traditional working memory measures (e.g., digit span or Corsi blocks) frequently fail to predict drawing performance; unlike those tasks, figure copying requires thousands of saccades, making it uniquely sensitive to the precision of the dynamic remapping signals identified by our modelling framework.

      We added the following text in the Discussion (p19):

      “We also found that the relationship between transsaccadic working memory and ROCF performance remains highly significant (rho = -0.39, p < 0.001), even after controlling for age, education, and global cognitive status (ACE-III total score). Consequently, transsaccadic updating may represent a discrete computational phenotype required for visuomotor control, rather than a non-specific proxy for global cognitive decline.[57]”

      Reviewer #3 (Recommendations for the authors):

      (1) The authors mention in the introduction the following: "One key hypothesis is that we use working memory across visual fixations to update perception dynamically", citing the following manuscript:

      Harrison, W. J., Stead, I., Wallis, T. S. A., Bex, P. J. & Mattingley, J. B. A computational 906 account of transsaccadic attentional allocation based on visual gain fields. Proc. Natl. 907 Acad. Sci. U.S.A. 121, e2316608121 (2024).

      However, the manuscript above does not refer explicitly to the involvement of working memory in transaccadic integration of object location in space. Rather, it takes advantage of recent evidence showing how the true location of a visual object is represented in the activity of neurons in primary visual cortex ( A. P. Morris, B. Krekelberg, A stable visual world in primate primary visual cortex. Curr. Biol. 29, 1471-1480.e6 (2019) ). The model hypothesizes that true locations of objects are readily available, and then allocates attention in real-world coordinates, allowing efficient coordination of attention and saccadic eye movements.

      Thank you for clarification. As suggested, we have now included the citation of Morris & Krekelberg (2019) to acknowledge the evidence for stable object locations within the primary visual cortex.

      (2) The authors in the introduction and the title use the terms 'transaccadic memory' and 'spatial working memory'. However, it is not clear whether these can be used interchangeably or are reflecting different constructs.

      Classical measures of visuo-spatial working memory are derived from the Corsi task (or similar), where the location of multiple objects is displayed and subsequently remembered. In such tasks, eye movements and saccades are not generally considered, only memory performance, representing the visuo-spatial span.

      Transaccadic memory tasks are instead explicitly measuring the performance on remembered object locations of features across explicit eye movements, usually using a very limited number of objects (1 or 2, as is the case for the current manuscript).

      While the two constructs share some features, it is not clear whether they represent the same underlying ability or not, especially because in transaccadic tasks, participants are required to perform one or more saccades, thus representing a dual-task case.

      I think the relationship between 'transaccadic memory' and 'spatial working memory' should be clarified in the manuscript.

      Thank you. Yes, we have added this within the Methods - Measurement of saccade cost to clarify that spatial working memory is the broad cognitive construct responsible for short-term maintenance, whereas transsaccadic memory is the specific, dynamic process of remapping representations to maintain stability across eye movements.

      In Methods (p.22):

      “Within this framework, it is important to distinguish between the broad construct of spatial working memory and the specific process of transsaccadic memory. While spatial working memory refers to the general ability to maintain spatial information over short intervals, transsaccadic memory describes the dynamic updating of these representations—termed remapping—to ensure stability across eye movements. Unlike classical 'static' measures of spatial working memory, such as the Corsi block task which focuses on memory span, transsaccadic memory tasks explicitly require the integration of stored visual information with motor signals from intervening saccades. Our paradigm treats transsaccadic updating as a core computational process within spatial working memory, where eye-centred representations are actively reconstructed based on noisy memories of the intervening saccade vectors.”

      (3) In Figure 1, the second row indicates the presentation of item 2. Indeed, in the condition 'saccade-after-item-1', the target in the second row of Figure 1 is displaced, as expected. This clarifies the direction and amplitude of the first saccade requested. However, from Figure 1, it is hard to understand the amplitude and direction of the second requested saccade. I think the figure should be updated, giving a full description of the direction and amplitude of the second saccade as well ('saccade-after-item-2' and 'two-saccades' conditions).

      We agree that making the figure legend more self-contained is beneficial for the reader. While the specific physical parameters and the trial sequence for each condition are detailed in the Results and Methods sections, we have now updated the legend for Figure 1 to explicitly define these details. Specifically, we have clarified that the colour wheel itself served as the target for the second instructed saccade (i.e., the movement from the second fixation cross to the colour wheel location). We have also included the quantitative constraint that all saccade vectors were at least 8.5 degrees of visual angle in amplitude. Given the limited space within a figure legend, we hope these concise additions provide the transparency requested without interrupting the conceptual flow of the diagram.

      Updated Figure 1 legend:

      “Participants were asked to fixate a white cross, wherever it appeared. They had to remember the colour and location of a sequence of two briefly presented coloured squares (Item 1 and 2), each appearing within a white square frame. They then fixated a colour wheel wherever it appeared on the screen, which served as the target for the second instructed saccade (i.e., a movement from the second fixation cross to the colour wheel location). This cued recall of a specific square (Item 1 or Item 2 labelled within the colour wheel). Participants selected the remembered colour on the colour wheel which led to a square of that colour appearing on the screen. They then dragged this square to its remembered location on the screen. Saccadic demands were manipulated by varying the locations of the second frame and the colour wheel, resulting in four conditions in their reliance on retinotopic versus transsaccadic memory: (1) No-Saccade condition providing a baseline measure of within-fixation precision as no eye movements were required. (2) Saccade After Item 1; (3) Saccade After Item 2; (4) Saccades after both items (Two Saccades condition). In all conditions requiring eye movements, saccade vectors were constrained to a minimum amplitude of 8.5° (degrees of visual angle). While the No-Saccade condition isolates retinotopic working memory, conditions (2) to (4) collectively quantify the impact of varying saccadic demands and timings on the maintenance of spatial information, thereby assessing the efficacy of the transsaccadic updating process.”

      (4) The authors write: "Eye tracking analysis confirmed high compliance: participants correctly maintained fixation or executed saccades as instructed on the vast majority of trials (83% {plus minus} 14%). Non-compliant trials were excluded 136 from further analysis." 14% of excluded trials are a substantial fraction of trials, given the task requirements. Is this proportion of excluded trials different between experimental groups, and are experimental groups contributing equally to this proportion?

      We thank the reviewer for pointing this out, and we apologise for the confusion. The 83% trial number was actually across all four cohorts, and all conditions, and it was actually above 90% for YC, EC and even AD, but dropped to 60 ish in PD group.

      We now have conducted a full analysis of compliant trial counts using a mixed ANOVA (4 saccade conditions x 4 cohorts). This analysis revealed a main effect of group (F(3, 80) = 8.06, p < 0.001), which was driven by lower compliance in the PD cohort (mean approx. 25.4 trials per condition) compared to the AD, EC, and YC cohorts (means ranging from 35.8 to 38.9 trials per condition). Crucially, however, the interaction between group and condition was not statistically significant (p = 0.151). This indicates that the relative impact of saccade demands on trial retention was consistent across all four groups.

      Because our primary behavioural measure—the saccade cost—is a within-subject comparison of impairment across conditions, these differences in absolute trial numbers do not introduce a systematic bias into our findings. Furthermore, even with the higher attrition in the PD group, we retained a sufficient number of high-quality trials (minimum mean of ~23 trials in the most demanding condition) to support robust trial-by-trial parameter estimation and valid statistical inference. We have updated the Results and Methods to reflect these details.

      In Results (p4):

      “To mitigate potential confounds, we monitored eye position throughout the experiment. Eye-tracking analysis confirmed high compliance in healthy adults, who followed instructions on the vast majority of trials (Younger Adults: 97.2 ± 5.2 %; Older Adults: 91.3 ± 20.4 %). The mean difference between these groups was negligible, representing just 1.25 trials per condition, and was not statistically significant (t(80) = 0.16, p = 1.000; see more in Methods – Eyetracking data analysis). Non-compliant trials were excluded from all further analyses.”

      In Methods (p27):

      “Eye-tracking analysis confirmed high compliance overall, with participants correctly maintaining fixation or executing saccades on the vast majority of trials (83% across all participants). A mixed ANOVA revealed a main effect of group on trial retention (F(3, 80) = 8.06, p < 0.001, partial η² = 0.23), primarily due to lower compliance in the PD cohort (YC: 97±4%; EC: 91±10%; AD: 95±5%; PD: 63±38%). Importantly, there was no significant interaction between group and saccade condition (F(3.36, 80) = 1.78, p = 0.15, partial η² = 0.008), suggesting that trial attrition was not disproportionately affected by specific task demands in any group.

      We acknowledge that this reduced trial count in the PD group represents a limitation for across-cohort comparison. However, the absolute number of compliant trials in PD group (mean approx. 25 per condition) remained sufficient for robust trial-by-trial parameter estimation. Furthermore, the lack of a significant group-by-condition interaction confirms that the results reported for this cohort remain valid and that our primary finding of a selective spatial memory deficit is robust to these differences in data retention.”

      (5) Modelling

      (a) Degrees of freedom, cross-validation, number of parameters.

      I appreciate the effort in introducing and testing different models. Models of increase in complexity and are based on different assumptions about the main drivers and mechanisms underlying the dependent variable. The models differ in the number of parameters. How are the differences in the number of parameters between models taken into account in the modelling analysis? Is there a cost associated with the extra parameters included in the more complex models?

      (b) Cross-validation and overfitting.

      Overfitting can occur when a model learns the training data but cannot generalize to novel datasets. Cross-validation is one approach that can be used to avoid overfitting. Was cross-validation (or other approaches) implemented in the fitting procedure against overfitting? Otherwise, the inference that can be derived from the modelled parameters can be limited.

      To address your concerns regarding model complexity and overfitting, we would like to clarify our use of Bayesian Model Selection (BMS). Unlike frequentist methods that often rely on cross-validation to assess generalisability, we used random-effects BMS based on the marginal likelihood (model evidence). This approach inherently implements Bayesian Occam’s Razor by integrating out the parameters. Under this framework, the use of the marginal likelihood for model selection provides a mathematically equivalent safeguard to frequentist cross-validation, as it evaluates the model's ability to generalise across the entire parameter space rather than just finding a maximum likelihood fit for the training data. Thus, models are penalised not just for the absolute number of parameters, but for their overall functional flexibility. A more complex model is only preferred if the improvement in model fit is substantial enough to outweigh this inherent penalty. The emergence of Model 7 as the winner (Bayes Factor = 6.11 against the next best alternative) confirms that its additional complexity is statistically justified.

      Furthermore, in this study we provided an external validation of these recovered parameters by demonstrating that they explain 62% of the variance in an independent, real-world, clinical task (ROCF copy). This empirical evidence confirms that our model captures robust mechanistic phenotypes rather than idiosyncratic noise. We have updated the Results and Discussion to explicitly state these.

      In Results: (p10)

      “We used random-effects Bayesian model selection to identify the most plausible generative model. This process relies on the marginal likelihood (model evidence), which inherently balances model fit against complexity—a principle often referred to as Occam’s razor.[26–28]”

      In Discussion: (p17)

      “Importantly, the risk of overfitting is mitigated by the Bayesian Model Selection framework; by utilising the marginal likelihood for model comparison, the procedure inherently penalises excessive model complexity and promotes generalisability.[26–28,43] This generalisability was further evidenced by the model's ability to predict performance on the independent ROCF task, confirming that these parameters represent robust mechanistic phenotypes rather than idiosyncratic fits to the initial dataset.”

      (6) n. of participants.

      (a) The authors write the following: "A total of healthy volunteers (21 young adults, mean age = 24.1 years; 21 older adults, mean age = 72.4 years) participated in this study. Their demographics are shown in Table 1. All participants were recruited locally in Oxford." However, Table 1 reports the data from more than 80 participants, divided into 4 groups. Details about the PD and AD groups are missing. Please clarify.

      We apologize for this lack of clarity in the text. We have rewrote and expand the “Participants” section and corrected Table 2 in the Methods section to reflect the correct number of participants.

      In Methods (p20):

      “A total of 87 participants completed the study: 21 young healthy adults (YC), 21 older healthy adults (EC), 23 patients with Parkinson’s disease (PD), and 22 patients with Alzheimer’s disease (AD). Their demographic and clinical details are summarised in Table 2. Initially, 90 participants were recruited (22 YC, 21 EC, 25 PD, 22 AD); however, three individuals (1 YC and 2 PD) were excluded from all analyses due to technical issues during data acquisition.

      All participants were recruited locally in Oxford, UK. None were professional artists, had a history of psychiatric illness, or were taking psychoactive medications (excluding standard dopamine replacement therapy for PD patients). Young participants were recruited via the University of Oxford Department of Experimental Psychology recruitment system. Older healthy volunteers (all >50 years of age) were recruited from the Oxford Dementia and Ageing Research (OxDARE) database.

      Patients with PD were recruited from specialist clinics in Oxfordshire. All had a clinical diagnosis of idiopathic Parkinson's disease and no history of other major neurological or psychiatric conditions. While specific dosages of dopamine replacement therapy (e.g., levodopa equivalent doses) were not systematically recorded, all patients were tested while on their regular medication regimen ('ON' state).

      Patients with PD were recruited from clinics in the Oxfordshire area. All had a clinical diagnosis of idiopathic Parkinson’s disease and no history of other major neurological or psychiatric illnesses. While all patients were tested in their regular medication ‘ON’ state, the specific pharmacological profiles—including the exact types of medication (e.g., levodopa, dopamine agonists, or combinations) and dosages—were not systematically recorded. The disease duration and PD severity were also un-recorded for this study.

      Patients with AD were recruited from the Cognitive Disorders Clinic at the John Radcliffe Hospital, Oxford, UK. All AD participants presented with a progressive, multidomain, predominantly amnestic cognitive impairment. Clinical diagnoses were supported by structural MRI and FDG-PET imaging consistent with a clinical diagnosis of AD dementia (e.g., temporo-parietal atrophy and hypometabolism).[70] All neuroimaging was reviewed independently by two senior neurologists (S.T. and M.H.).

      Global cognitive function was assessed using the Addenbrooke’s Cognitive Examination-III (ACE-III).[71] All healthy participants scored above the standard cut-off of 88, with the exception of one elderly participant who scored 85. In the PD group, two participants scored below the cut-off (85 and 79). In the AD group, six participants scored above 88; these individuals were included based on robust clinical and radiological evidence of AD pathology rather than their ACE-III score alone.”

      (b) As modelling results rely heavily on the quality of eye movements and eye traces, I believe it is necessary to report details about eye movement calibration quality and eye traces quality for the 4 experimental groups, as noisier data could be expected from naïve and possibly older participants, especially in case of clinical conditions. Potential differences in quality between groups should be discussed in light of the results obtained and whether these could contribute to the observed patterns.

      Thank you for pointing this out. We have revised the Methods about how calibration was done:

      (p27) “Prior to the experiment, a standard nine-point calibration and validation procedure was performed. Participants were instructed to fixate a small black circle with a white centre (0.5 degrees) as it appeared sequentially at nine points forming a 3 x 3 grid across the screen. Calibration was accepted only if the mean validation error was below 0.5 degrees and the maximum error at any single point was below 1.0 degree. If these criteria were not met, or if the experimenter noticed significant gaze drift between blocks, the calibration procedure was repeated. This calibration ensured high spatial accuracy across the entire display area, facilitating the precise monitoring of fixations on item frames and saccadic movements to the response colour wheel.”

      Moreover, as detailed in our response to Point 4, while the PD group exhibited lower compliance, there was no interaction between group and saccade condition for compliance (p = 0.151). This confirms that any noise or trial attrition was distributed evenly across experimental conditions. Consequently, the observed "saccade cost" (the difference in error between conditions) is not an artefact of unequal noise but represents a genuine mechanistic impairment in spatial updating. We have updated the Methods to clarify this distinction.

      Furthermore, our Bayesian framework explicitly estimates precision (random noise) as a distinct parameter from updating cost (saccade cost). This allows the model to partition the variance: even if a clinical group is "noisier" overall, this is captured by the precision parameter, ensuring it does not inflate the specific estimate of saccade-driven memory impairment.

      (7) Figure 5. I suggest reporting these results using boxplots instead of barplots, as the former gives a better overview of the distributions.

      We appreciate the suggestion to use boxplots to better illustrate data distributions. However, we have chosen to retain the current bar plot format due to the visual and statistical complexity of our 4 x 4 x 2 experimental design. Figure 5 represents 16 distinct distributions across four groups and four conditions for both location and colour measures; employing boxplots/violins for this density of data would significantly increase visual clutter and make the figure difficult to parse.

      Furthermore, the primary objective of this figure is to reflect the statistical analysis and illustrate group differences in overall performance and highlight the specific finding that patients with AD were significantly more impaired across all conditions compared to YC, EC, and PD groups. Our statistical focus remains on the mean effects—specifically the significant main effect of group (F(3, 318) = 59.71, p < 0.001) and the critical null-interaction between group and condition (p = 0.90). The error measure most relevant to these comparisons is the standard error of the mean (SEM), rather than the interquartile range (IQR). We think that bar plots provide the most straightforward and scannable representation of these mean differences and the consistent pattern of decay across cohorts for the final manuscript layout.

      To address the reviewer’s request for distributional transparency, we have provided a version of Figure 5 using grouped boxplots in the supplementary material (Supplementary figure 2). We note, however, that the spread of raw data points in these plots does not directly reflect the variance associated with our within-subject statistical comparisons.

      (8) Results specificity, trans-saccadic integration and ROCF. The authors demonstrate that the derived model parameters account for a significant amount of variability in ROCF performance across the experimental groups tested (Figure 8A). However, it remains unclear how specific the modelling results are with respect to the ROCF.

      The ROCF is generally interpreted as a measure of constructional ability. Nevertheless, as with any cognitive test, performance can also be influenced by more general, non-specific abilities that contribute broadly to test success. To more clearly link the specificity between modelling results and constructional ability, it would be helpful to include a test measure for which the model parameters would not be expected to explain performance, for example, a verbal working memory task.

      I am not necessarily suggesting that new data should be collected. However, I believe that the issue of specificity should be acknowledged and discussed as a potential limitation in the current context.

      We appreciate this important point regarding the discriminant validity of our findings. We agree that cognitive performance in clinical populations is often influenced by a general "g-factor" or non-specific executive decline. However, we chose the ROCF Copy task specifically because it is a hallmark clinical measure of constructional ability that effectively serves as a real-world transsaccadic task, requiring participants to integrate spatial information across hundreds of saccades between the model figure and the drawing surface.

      To address the reviewer’s concern regarding specificity, we leveraged the fact that all participants completed the ACE-III, which includes a dedicated verbal memory component (the ACE Memory subscale). We conducted a partial correlation analysis and found that the relationship between transsaccadic working memory and ROCF copy performance remains highly significant (rho = -0.46, p < 0.001), even after controlling for age, education, and the ACE-III Memory subscale score. This suggests that the link between transsaccadic updating and constructional ability is mechanistically specific rather than a byproduct of global cognitive impairment. We have substantially revised the Discussion to highlight this link and the supporting statistical evidence.

      We first updated the last paragraph of Introduction:

      “Finally, by linking these mechanistic parameters to a standard clinical measure of constructional ability (the Rey-Osterrieth Complex Figure task), we demonstrate that transsaccadic updating represents a core computational phenotype underpinning real-world visuospatial construction in both health and neurodegeneration.”

      The new section in Discussion highlighting the ROCF copy link:

      “Importantly, our computational framework establishes a direct mechanistic link between trassaccadic updating and real-world constructional ability. Specifically, higher saccade and angular encoding errors contribute to poorer ROCF copy scores. By mapping these mechanistic estimates onto clinical scores, we found that the parameters derived from our winning model explain approximately 62% of the variance in constructional performance across groups. These findings suggest that the computational parameters identified in the LOCUS task represent core phenotypes of visuospatial ability, providing a mechanistic bridge between basic cognitive theory and clinical presentation.

      This relationship provides novel insights into the cognitive processes underlying drawing, specifically highlighting the role of transsaccadic working memory. Previous research has primarily focused on the roles of fine motor control and eye-hand coordination in this skill.[4,50–55] This is partly because of consistent failure to find a strong relation between traditional memory measures and copying ability.[4,31] For instance, common measures of working memory, such as digit span and Corsi block tasks, do not directly predict ROCF copying performance.[31,56] Furthermore, in patients with constructional apraxia, these memory performance often remain relatively preserved despite significant drawing impairments.[56–58] In literature, this lack of association has often been attributed to “deictic” visual-sampling strategies, characterised by frequent eye movements that treat the environment as an external memory buffer, thereby minimising the need to maintain a detailed internal representation.[4,59] In a real-world copying task, the ROCF requires a high volume of saccades, making it uniquely sensitive to the precision of the dynamic remapping signals identified here. Recent eye-tracking evidence confirms that patients with AD exhibit significantly more saccades and longer fixations during figure copying compared to controls, potentially as a compensatory response to trassaccadic working memory constraints.[56] This high-frequency sampling—averaging between 150 and 260 saccades for AD patients compared to approximately 100 for healthy controls—renders the task highly dependent on the precision of dynamic remapping signals.[56] We also found that the relationship between transsaccadic working memory and ROCF performance remains highly significant (rho = -0.46, p < 0.001), even after controlling for age, education, and ACE-III Memory subscore. Consequently, transsaccadic updating may represent a discrete computational phenotype required for visuomotor control, rather than a non-specific proxy for global cognitive decline.[58]

      In other words, even when visual information is readily available in the world, the act of drawing performance depends critically on working memory across saccades. This reveals a fundamental computational trade-off: while active sampling strategies (characterised with frequent eye-hand movements) effectively reduce the load on capacity-limited working memory, they simultaneously increase the demand for precise spatial updating across eye movements. By treating the external world as an "outside" memory buffer, the brain minimises the volume of information it must hold internally, but it becomes entirely dependent on the reliability with which that information is remapped after each eye movement. This perspective aligns with, rather contradicts, the traditional view of active sampling, which posits that individuals adapt their gaze and memory strategies based on specific task demands.[3,60] Furthermore, this perspective provides a mechanistic framework for understanding constructional apraxia; in these clinical populations, the impairment may not lie in a reduced memory "span," but rather in the cumulative noise introduced by the constant spatial remapping required during the copying process.[58,61]

      Beyond constructional ability, these findings suggest that the primary evolutionary utility of high-resolution spatial remapping lies in the service of action rather than perception. While spatial remapping is often invoked to explain perceptual stability,[11–13,15] the necessity of high-resolution transsaccadic memory for basic visual perception is debated.[13,62–64] A prevailing view suggests that detailed internal models are unnecessary for perception, given the continuous availability of visual information in the external world.[13,44] Our findings support an alternative perspective, aligning with the proposal that high-resolution transsaccadic memory primarily serves action rather than perception.[13] This is consistent with the need for precise localisation in eye-hand coordination tasks such as pointing or grasping.[65] Even when unaware of intrasaccadic target displacements, individuals rapidly adjust their reaching movements, suggesting direct access of the motor system to remapping signals.[66] Further support comes from evidence that pointing to remembered locations is biased by changes in eye position,[67] and that remapping neurons reside within the dorsal “action” visual pathway, rather than the ventral “perception” visual pathway.[13,68,69] By demonstrating a strong link between transsaccadic working memory and drawing (a complex fine motor skill), our findings suggest that precise visual working memory across eye movements plays an important role in complex fine motor control.”

      We are deeply grateful to the reviewers for their meticulous reading of our manuscript and for the constructive feedback provided throughout this process. Your insights have significantly enhanced the clarity and rigour of our work.

      In addition to the changes requested by the reviewers, we wish to acknowledge a reporting error identified during the revision process. In the original Results section, the repeated measures ANOVA statistics for YC included Greenhouse-Geisser corrections, and the between-subjects degrees of freedom were incorrectly reported as within-subjects residuals. Upon re-evaluation of the data, we confirmed that the assumption of sphericity was not violated; therefore, we have removed the unnecessary Greenhouse-Geisser corrections and corrected the degrees of freedom throughout the Results and Methods sections. We have ensured that these statistical updates are reflected accurately in the revised manuscript and that they do not alter the significance or interpretation of any of our primary findings.

      We hope that these revisions address all the concerns raised and provide a more robust account of our findings. We look forward to your further assessment of our work.

    1. Consolidation de la Co-éducation autour de l'Enfant et de l'Adolescent : Synthèse et Orientations

      Résumé Analytique

      La co-éducation ne doit pas être perçue comme un simple souhait ou un idéal lointain, mais comme une réalité de fait.

      Tout enfant ou adolescent évolue au sein d'un écosystème d'éducateurs multiples (famille, école, structures de loisirs, médias).

      L'enjeu majeur n'est pas de fusionner ces rôles, mais de consolider cet écosystème en préservant la spécificité de chaque acteur tout en assurant une cohérence globale.

      Cette cohérence repose sur des projets communs, des comportements éducatifs équilibrés — évitant l'aléa et la rigidité — et une collaboration étroite face aux défis sociétaux modernes, tels que la gestion des outils numériques.

      1. La Co-éducation : Un Écosystème de Fait

      La co-éducation est une réalité intrinsèque au développement de l'enfant. Dès lors qu'un individu sort de son isolement, il est exposé à une multiplicité d'influences éducatives qui constituent son environnement quotidien.

      Une pluralité d'acteurs : L'éducation n'est pas le seul fait des parents.

      Elle inclut l'école, les clubs de loisirs, la famille élargie, les amis, ainsi que les influences médiatiques (télévision, internet, réseaux sociaux).

      La notion d'écosystème : Cet ensemble d'interactions forme un cadre dans lequel l'enfant évolue.

      Les différents éducateurs s'y complètent de manière de facto, exerçant chacun une influence sur la construction du sujet.

      2. Le Principe de Spécificité des Rôles

      Un pilier fondamental de la co-éducation réussie est le respect des domaines de compétence et des vocations de chaque acteur. La collaboration ne doit pas mener à une confusion des rôles.

      Le maintien des identités : Chaque éducateur doit garder sa spécificité. Les parents n'ont pas vocation à devenir des enseignants, et les enseignants ne doivent pas s'immiscer dans la sphère privée familiale.

      Différenciation des méthodes : Un animateur de loisirs peut aborder des concepts académiques (comme la proportionnalité), mais il doit le faire selon des modalités propres à son cadre, et non en reproduisant strictement les méthodes scolaires.

      La complémentarité plutôt que l'imitation : L'objectif de la rencontre entre adultes n'est pas de chercher à se ressembler ou à agir de manière identique, mais d'organiser une complémentarité efficace.

      3. Leviers de Cohérence Éducative

      Si la spécificité est de mise, elle ne doit pas conduire à l'incohérence.

      Le document souligne deux moyens principaux pour harmoniser l'action des adultes :

      La mise en œuvre de projets communs

      La cohérence peut naître d'actions concrètes impliquant simultanément plusieurs sphères de la vie de l'enfant.

      Exemple : Les classes découvertes ou les sorties scolaires qui associent parents, enseignants et animateurs du périscolaire autour d'un objectif unique.

      L'harmonisation des comportements éducatifs

      Il s'agit de tisser un système au service du développement de l'enfant pour l'aider à comprendre le monde et à devenir un sujet autonome.

      Un système éducatif sain se définit par sa structure :

      | Type de milieu | Caractéristiques | Impact sur l'enfant | | --- | --- | --- | | Milieu Aléatoire | Imprévisible. Les réactions des adultes (sanction ou félicitation) ne sont pas constantes. | L'enfant ne peut pas anticiper les conséquences de ses actes. | | Milieu Rigide | Règles définies à l'avance, immuables et non discutables. Tout est enfermé dans des normes strictes. | Absence de dialogue et de remise en question. | | Milieu Équilibré | Présence d'un cadre sécurisant, mais flexible. Les règles peuvent faire l'objet de discussions selon les événements. | Favorise l'émergence de la réflexivité et du dialogue entre enfant et adulte. |

      4. Un Défi Partagé : La Gestion du Numérique

      La co-éducation est particulièrement sollicitée face aux problématiques sociétales complexes, l'utilisation du téléphone portable et des outils numériques en étant l'exemple le plus prégnant.

      L'impossibilité d'une solution isolée : Ni les parents, ni les enseignants, ni les éducateurs spécialisés ne peuvent légiférer ou résoudre seuls la question des écrans.

      La nécessité d'une "législation" cohérente : Les adultes ont tout intérêt à se concerter pour adopter des comportements et des règles cohérents autour de cet objet.

      La solution réside dans la concertation et l'établissement d'une ligne de conduite partagée au sein de l'écosystème.

      Conclusion

      La consolidation de la co-éducation repose sur un paradoxe constructif : travailler ensemble tout en restant différent.

      La rencontre entre les adultes n'est pas une fin en soi, mais un moyen de structurer un environnement prévisible et réflexif pour l'enfant.

      En instaurant un dialogue constant et en s'accordant sur des comportements cohérents face aux enjeux modernes, les éducateurs favorisent un écosystème propice à l'autonomie et au développement global de l'enfant et de l'adolescent.

    1. 6Late Policy:Assignments and Quizzes posted after 11:59:01 on Sunday are late and this is indicated with the late tag in Brightspace.Discussion postings posted after 11:59:01 on Thursday are late and follow up posting posted after Sunday 11:59:01 arelate. For assignments and quizzes posted after 11:59:01 with a late Tag as identified in Brightspace will receive anautomatic 30% reduction in grade. Assignments posted after 11:59:01 PM on Tuesday (2 days after the due date), willreceive a 35% reduction in grade. Assignments posted after 11:59:01 on Thursday (4 days late) will receive a 40%reduction in grade. No assignments will be accepted after 11:59:01 the Sunday after the initial due date (7 days late) andwill receive a zero.

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    1. biomedical model makes four primary assumptions that limit its utility for completely understanding health and illness

      1-assumes the presence of disease and its diagnosis and treatment are objective phenomena. 2-assumes that only medical professionals are capable of defining health and illness. 3-presumes that physiological malfunction alone defines health and illness. 4-defines healths as the absence of disease.

    1. These post-baccalaureate credentials may need to be completed prior to entering a career field, may be obtained as an option to gain competitive advantage for hiring, or may be achieved during the course of an individual’s career to stay current, maintain qualification, or be promoted. To

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    2. are able to explore different programs without exceeding the total number of credits required to graduate with a bachelor’s degree.

      I was not aware that one can have too many credits! I thought the more the better.

    3. Both associate’s degrees and bachelor’s degrees are considered undergraduate degrees, thus students working toward these degrees are often called undergraduates

      This is also something I am learning now, I hear the terms and never really knew what they meant.

    4. If you’re planning your associate’s degree, here are some specific questions you may want to research.

      I will make note of these questions and meet with my advisor to explore them more, This is a really great resource!

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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      Reply to the reviewers

      Reply to the reviewers

      We are grateful for the reviewers' constructive comments and suggestions, which contributed to improving our manuscript. We are pleased to see that our work was described as an "interesting manuscript in which a lot of work has been undertaken". We are also encouraged by the fact that the experiments were considered "on the whole well done, carefully documented, and support most of the conclusions drawn," and that our findings were viewed as providing "mechanistic insight into how HNRNPK modulates prion propagation" and potentially offering "new mechanical insight of hnRNPK function and its interaction with TFAP2C."

      We conducted several new experiments and revised specific sections of the manuscript, as detailed below in the point-by-point response in this letter.

      Referee #1

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      The paper by Sellitto describes studies to determine the mechanism by which hnRNPK modulates the propagation of prion. The authors use cell models lacking HNRNPK, which is lethal, in a CRISPR screen to identify genes that suppress lethality. Based on this screen to 2 different cell lines, gene termed Tfap2C emerged as a candidate for interaction with HNRNPK. The show that Tfap2C counteracts the actions of HNRNPK with respect to prion propagation. Cells lacking HNRNPK show increased PrPSc levels. Overexpression of Tfap2C suppesses PrPSc levels. These effects on PrPSc are independent of PrPC levels. By RNAseq analysis, the authors hone in on metabolic pathways regulated by HNRPNK and Tfap2C, then follow the data to autophagy regulation by mTor. Ultimately, the authors show that short-term treatments of these cell models with mTor inhibitors causes increased accumulation of PrPSc. The authors conclude that the loss of HNRNPK leads to a reduced energy metabolism causing mTor inhibition, which is reduces translation by dephosphorylation of S6

      Major comments:

      1) Fig H and I, Fig 3L. The interaction between Tfap2C and HNRNPK is pretty weak. The interaction may not be consequential. The experiment seems to be well controlled, yielding limited interaction. The co-ip was done in PBS with no detergent. The authors indicate that the cells were mechanically disrupted. Since both of these are DNA binding proteins, is it possible that the observed interaction is due to the proximity on DNA that is linking the 2 proteins, including a DNAase treatment would clarify.

      Response: We agree that the observed co-IP between Tfap2c and hnRNP K is weak (previous Fig. 2H-I, Supp. Fig. 3L now shifted in Supp. Fig. 4C-E), and we have now highlighted this in the relevant section of the manuscript to reflect this observation better.

      Importantly, the co-IP was performed using endogenous proteins without overexpression or tagging, which can sometimes artificially enhance protein-protein interactions. However, we acknowledge that the use of a detergent-free lysis buffer and mechanical disruption alone may have limited nuclear protein extraction and solubilization, potentially contributing to the low co-IP signal.

      To address the reviewer's concerns and clarify whether the observed interaction could be DNA-mediated, we repeated the co-IP experiments under low-detergent conditions and included benzonase nuclease treatment to digest nucleic acids (Fig. 2H-I). DNA digestion was confirmed by agarose gel electrophoresis (Supp. Fig. 4F-G). Additionally, we performed the reciprocal IPs using both hnRNP K and Tfap2c antibodies (Fig. 2H-I). Although the level of co-immunoprecipitation remains modest, these updated experiments continue to demonstrate a specific co-immunoprecipitation between Tfap2c and hnRNP K, independent of DNA bridging. These additional controls and experimental refinements strengthen the validity of our findings. These results are also attached here for your convenience.

      2) Supplemental Fig 5B - The western blot images for pAMPK don't really look like a 2 fold increase in phosphorylation in HNRNPK deletion.

      Response: We thank the reviewer for raising this point. We re-examined the original pAMPK western blot (previously Supp. Fig. 5B; now presented as Supp. Fig. 6B) and confirmed the reported results. We note that the overall loading is not perfectly uniform across lanes (as suggested by the actin signal), which may affect the visual impression of band intensity. However, the phosphorylation change reported in the manuscript is based on the pAMPK/total AMPK ratio, which accounts for differences in AMPK expression and accurately reflects relative phosphorylation levels. To further address this concern, we performed three additional independent experiments. These new data reproduce the increase in pAMPK/AMPK upon HNRNPK deletion and are now included in the revised Supplementary Fig. 6B, together with the updated quantification. The new blot and the quantification are also attached here for your convenience.

      3) Fig. 5A - I don't think it is proper to do statistics on an of 2.

      Response: We believe the reviewer's comment refers to Fig. 5B, as Fig. 5A already has sufficient replication. We have now added two additional replicates, bringing the total to four. The updated statistical analysis corroborates our initial results. The new quantification is provided in the revised manuscript (Fig. 5B) along with the new blot (Supp. Fig. 6C). Both data are also attached here for your convenience.

      4) Fig 6D. The data look a bit more complicated than described in the text. At 7 days, compared to 2 days, it looks like there is a decrease in % cells positive for 6D11. Is there clearance of PrPSc or proliferation of un-infected cells?

      Response: We have now reworded our text in the results paragraph as follows:

      "These data show that TFAP2C overexpression and HNRNPK downregulation bidirectionally regulate prion levels in cell culture."

      We have now also included the following comments in the discussion section:

      "However, prion propagation relies on a combination of intracellular PrPSc seeding and amplification, as well as intercellular spread, which together contribute to the maintenance and expansion of infected cells within the cultured population. In this study, we were limited in our ability to dissect which specific steps of the prion life cycle are affected by TFAP2C. We also cannot fully exclude the possibility that TFAP2C overexpression influenced the relative proliferation of prion-infected versus uninfected cells in the PG127-infected HovL culture, thereby contributing to the observed reduction in the percentage of 6D11+ cells and overall 6D11+ fluorescence. However, we did not observe any signs of cell death, growth impairment, or increased proliferation under TFAP2C overexpression in PG127-infected HovL cells compared to NBH controls (data not shown). This suggests that a negative selective pressure on infected cells or a proliferative advantage of uninfected cells is unlikely in this context".

      5) The authors might consider a different order of presenting the data. Fig 6 could follow Fig. 2 before the mechanistic studies in Figs 3-5.

      Response: We believe that the current order of presenting the data is more appropriate. The first part of the manuscript focuses on the genetic and functional interactions between hnRNP K and its partners, particularly TFAP2C, which is a critical point for understanding the broader context before delving into the mechanistic studies involving prion-infected cells.

      6) The authors use SEM throughout the paper and while this is often used, there has been some interest in using StdDev to show the full scope of variability.

      Response: We chose to use SEM as it reflects the precision of the mean, which is central to our statistical comparisons. As the reviewer notes, this is a common and appropriate practice. To address variability, almost all graphs already include individual data points, which provide a direct visual representation of data spread. To further enhance clarity, we have now included StdDev in the Supplementary Source Data table of the revised manuscript.

      Discussion:

      The discrepancy between short-term and long-term treatments with mTor inhibitors is only briefly mentioned with a bit of a hand-waving explanation. The authors may need a better explanation.

      Response: We have now integrated a more detailed explanation in the discussion section of the revised manuscript as follows:

      "Previous studies showed that mTORC1/2 inhibition and autophagy activation generally reduce, rather than increase, PrPSc aggregation (79, 80). The reason for this discrepancy remains unclear and may be multifactorial. First, most prior studies were based on long-term mTOR inhibition, whereas our work examined acute inhibition, mimicking the time frame of HNRNPK and TFAP2C manipulation. Acute inhibition may trigger transient metabolic or signaling shifts that differ from adaptive changes associated with mTOR chronic inhibition, potentially overriding autophagy's effects on prion propagation. Additionally, while previous works were primarily conducted in murine in vivo models, our study focused on a human cell system propagating ovine prions. Differences in species background, model complexity (e.g., interactions between different cell types), and prion strain variability, as certain strains exhibit distinct responses to autophagy and mTOR modulation (https://doi.org/10.1371/journal.pone.0137958), likely contributed to the observed differences".

      Minor comments:

      Page 12 - no mention of chloroquine in the text or related data.

      Page 12 - Supp. Fig. E - should be 5E

      Response: We thank the reviewer for pointing this out. We have now better highlighted the use of chloroquine in Fig. 5B (see reviewer #1 - Point 3 - Major comments) and in the text as follows:

      "Furthermore, in the presence of chloroquine, LC3-II levels rose almost proportionally across all conditions (Fig. 5B), suggesting that the effects of HNRNPK and TFAP2C on autophagy occur at the level of autophagosome formation, rather than autophagosome-lysosome fusion and degradation."

      We have corrected the reference to Supp. Fig. 5E.

      Reviewer #1 (Significance (Required)):

      The study provides mechanistic insight into how HNRNPK modulates prion propagation. The paper is limited to cell models, and the authors note that long term treatment with mTor inhibitors reduced PrPSc levels in an in vivo model.

      The primary audience will be other prion researchers. There may be some broader interest in the mTor pathway and the role of HNRNPK in other neurodegenerative diseases.

      Referee #2

      Reviewer #2 (Evidence, reproducibility and clarity (Required)):

      The manuscript "Prion propagation is controlled by a hierarchical network involving the nuclear Tfap2c and hnRNP K factors and the cytosolic mTORC1 complex" by Sellitto et al aims to examine how heterogenous nuclear ribonucleoprotein K (hnRNPK), limits pion propagation. They perform a synthetic - viability CRISPR- ablation screen to identify epistatic interactors of HNRNPK. They found that deletion of Transcription factor AP-2g (TFAP2C) suppressed the death of hnRNP-K depleted LN-229 and U-251 MG cells whereas its overexpression hypersensitized them to hnRNP K loss. Moreover, HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism and enhanced autophagy. Simultaneous deletion of TFAP2C reversed these effects, restored transcription and alleviated energy deficiency. They state that HNRNPK and TFAP2C are linked to mTOR signalling and observe that HNRNPK ablation inhibits mTORC1 activity through downregulation of mTOR and Rptor while TFAP2C overexpression enhances mTORC1 downstream functions. In prion infected cells, TFAP2C activation reduced prion levels and countered the increased prion propagation due to HNRNPK suppression. Pharmacological inhibition of mTOR also elevated prion levels and partially mimicked the effects of HNRNPK silencing. They state their study identifies TFAP2C as a genetic interactor of HNRNPK and implicates their roles in mTOR metabolic regulation and establishes a causative link between these activities and prion propagation.

      This is an interesting manuscript in which a lot of work has been undertaken. The experiments are on the whole well done, carefully documented and support most of the conclusions drawn. However, there are places where it was quite difficult to read as some of the important results are in the supplementary Figures and it was necessary to go back and forth between the Figs in the main body of the paper and the supplementary Figs. There are also Figures in the supplementary which should have been presented in the main body of the paper. These are indicated in our comments below.

      We have the following questions /points:

      Major comments:

      1) A plasmid harbouring four guide RNAs driven by four distinct constitutive promoters is used for targetting HNRNPK- is there a reason for using 4 guides- is it simply to obtain maximal editing - in their experience is this required for all genes or specific to HNRNPK?

      Response: The use of four guide RNAs driven by distinct promoters is chosen to maximize editing efficiency for HNRNPK. As previously demonstrated by J. A. Yin et al. (Ref. 32), this system provides better efficiency for gene knockout (or activation). For HNRNPK, achieving full knockout was crucial for observing a complete lethal phenotype, which made the four guide RNAs approach fundamental. However, other knockout systems, while potentially less efficient, have been shown to work well in other circumstances. We have now included this explanation in the revised manuscript as follows:

      "We employed a plasmid harboring quadruple non-overlapping single-guide RNAs (qgRNAs), driven by four distinct constitutive promoters, to target the human HNRNPK gene and maximize editing efficiency in polyclonal LN-229 and U-251 MG cells stably expressing Cas9 (32)."

      2) Is there a minimal amount of Cas9 required for editing?

      Response: We did not observe a correlation between Cas9 levels and activity, yet the C3 clone was the one with higher Cas9 expression and higher activity (Supp. Fig. 1A-B). We agree that comments about the amount of Cas9 expression may be misleading here. Thus, in the first result paragraph of the revised manuscript, we have now modified the text "we isolated by limiting dilutions LN-229 clones expressing high Cas9 levels" to "we isolated by limiting dilutions LN-229 single-cell clones expressing Cas9".

      3) It is stated that cell death is delayed in U251-MG cells compared to LN-229-C3 cells- why? Also, why use glioblastoma cells other than that they have high levels of HNRNPK? Would neuroblastoma cells be more appropriate if they are aiming to test for prion propagation?

      Response: As shown in Fig. 1A, U251-MG cells reached complete cell death at day 13, while LN-229 C3 reached it already at day 10. The percentage of viable U251-MG cells is higher (statistically significant) than LN-229 C3 cells at all time points before day 13, when both lines show complete death. The underlying reasons for this partial and relative resistance are probably multiple, but we clearly showed in Fig. 2 that TFAP2C differential expression is one modulator of cell sensitivity to HNRNPK ablation.

      We selected glioblastoma cells because their high expression of HNRNPK was essential for developing our synthetic lethality screen strategy, and we have now clarified it in the revised manuscript as follows:

      "As model systems, we chose the human glioblastoma-derived LN-229 and U-251 MG cell lines, which express high levels of HNRNPK (2, 3), a key factor for optimizing our synthetic lethality screen."

      While neuroblastoma cells might be more relevant in terms of prion neurotoxicity, glial cells, despite their resistance to prion toxicity, are fully capable of propagating prions. Prion propagation in glial cells has been shown to play crucial roles in mediating prion-dependent neuronal loss in a non-autonomous manner (see 10.1111/bpa.13056). This makes glioblastoma cells a valuable model for studying prion propagation (that is the focus of our study), despite the lack of direct toxicity (which is not the focus of our study). We have now added this explanation to the revised manuscript as follows:

      "Therefore, we continued our experiments using LN-229 cells, which provide a relevant model for studying prions, as glial cells can propagate prions and contribute to prion-induced neuronal loss through non-cell-autonomous mechanisms."

      4) Human CRISPR Brunello pooled library- does the Brunello library use constructs which have four independent guide RNAs as used for the silencing of HNRPNK?

      Response: No, the Human CRISPR Brunello pooled library does not use constructs with four independent guide RNAs (qgRNAs). Instead, each gene is targeted by 4 different single-guide RNAs (sgRNAs), each expressed on a separate plasmid. We have now clarified this in the main text of the revised manuscript as follows:

      "To identify functionally relevant epistatic interactors of HNRNPK, we conducted a whole-genome ablation screen in LN-229 C3 cells using the Human CRISPR Brunello pooled library (33), which targets 19,114 genes with an average of four distinct sgRNAs per gene, each expressed by a separate plasmid (total = 76,441 sgRNA plasmids)."

      5) To rank the 763 enriched genes, they multiply the -log10FDR with their effect size - is this a standard step that is normally undertaken?

      Response: The approach of ranking hits using the product of effect size and statistical significance is a well-established method in CRISPR screening studies. This strategy has been explicitly used in high-impact work by Martin Kampmann and others (see https://doi.org/10.1371/journal.pgen.1009103 and https://doi.org/10.1016/j.neuron.2019.07.014 as references). We have now added both references to the revised manuscript.

      6) The 32 genes selected- they were ablated individually using constructs with one guide RNA or four guide RNAs?

      Response: The 32 genes selected were ablated individually using constructs with quadruple-guide RNAs (qgRNAs), as this approach was intended to maximize editing efficiency for each gene. We have now clarified this in the main text of the revised manuscript as follows:

      "We ablated each gene individually using qgRNAs and then deleted HNRNPK."

      7) The identified targets were also tested in U251-MG cells and nine were confirmed but the percent viability was variable - is the variability simply a reflection of the different cell line?

      Response: The variability in percent viability observed in U251-MG cells likely reflects the inherent differences between cell lines, which can contribute to varying levels of susceptibility to gene ablation, even for the same targets. We have now highlighted these small differences in the main text of the revised manuscript as follows:

      "We confirmed a total of 9 hits (Fig. 1H), including the ELPs gene IKBAKP and the transcription factor TFAP2C, the two strongest hits identified in LN-229 C3 cells. However, in the U251-Cas9 the rescue effect did not always fall within the exact range observed in LN-229 C3 cells, likely due to intrinsic differences between the two cell lines."

      8) The two strongest hits were IKBAKP and TFAP2C. As TFAP2C is a transcription factor - is it known to modulate expression of any of the genes that were identified to be perturbed in the screen? Moreover, it is stated that it regulates expression of several lncRNAs- have the authors looked at expression of these lncRNAs- is the expression affected- can modulation of expression of these lncRNAs modulate the observed phenotypic effects and also some of the targets they have identified in the screen?

      Response: While TFAP2C is a transcription factor known to regulate the expression of several genes and lncRNAs, we did not identify any of its known target genes among the hits of our screen. However, our RNA-seq data and RT-qPCR (data not shown) indicate that the expression of lncRNA MALAT1 and NEAT1 (reported to interact with both HNRNPK and TFAP2C; ref 37, 41, 47) is strongly affected by HNRNPK ablation and to a lesser extent by TFAP2C deletion. However, the double deletion condition does not appear to change these lncRNA levels beyond what is observed with HNRNPK ablation alone. Therefore, we concluded that these changes do not play a primary role in the phenotypic effects observed in our study. Thus, although interesting, we believe that the description of such observations goes beyond the scope of this manuscript and the relevance of this work.

      9) As both HNRNPK and TFAP2C modulate glucose metabolism, the authors have chosen to explore the epistatic interaction. This is most reasonable.

      Response: We do not have further comments on this point.

      10) The orthogonal assay to confirm that deletion of TFAP2C supresses cell death upon removing HNRNPK- was this done using a single guide RNA or multiple guides - is there a level of suppression required to observe rescue? Interestingly ablation of HNRNPK increases TFAP2C expression in LN-229-C3 whereas in U251-Cas9 cells HNRNPK ablation has the opposite effect- both RNA and protein levels of TFAP2C are decreased - is this the cause of the smaller protective effect of TFAP2C deletion in this cell line?

      Response: TFAP2C deletion was performed using quadruple-guide RNAs (gqRNAs). We have clarified this point by addressing the reviewer #2's point 6 in "Major comments".

      We did not directly test the threshold of TFAP2C inhibition required to suppress HNRNPK ablation-induced cell death. We did not exclude that other effectors may take a role in the smaller protective effect of TFAP2C deletion in the U251-Cas9 cells, however, multiple lines of evidence from our study suggest that TFAP2C expression levels influence cellular sensitivity to HNRNPK loss:

      1) Both LN-229 C3 and U251-Cas9 cells are less sensitive to HNRNPK ablation upon TFAP2C deletion (Fig. 1G-H, Fig. 2A-B, Supp. Fig.3A-B).

      2) We observed a correlation between endogenous TFAP2C levels and HNRNPK ablation sensitivity. U251-Cas9 cells, where TFAP2C expression is reduced upon HNRNPK ablation (in contrast to LN-229 C3 cells, where HNRNPK ablation leads to an increase in TFAP2C expression) (Fig. 2C-F), are a) less sensitive to HNRNPK deletion than LN-229 C3 (Fig. 1A, 2A-B) and b) the protective effect of TFAP2C deletion is less pronounced than in LN-229 C3 (Fig. 1G-H, Fig. 2A-B, Supp. Fig.3A-B).

      3) TFAP2C overexpression experiments (Fig. 2G) establish a causal relationship to the former correlation: TFAP2C overexpression increased U251-Cas9 sensitivity to HNRNPK ablation.

      As clearly mentioned in the manuscript, we believe that, taken together, these findings strongly demonstrate a causal role for TFAP2C in modulating sensitivity to HNRNPK loss. Thus, despite the differences in the expression, the proposed viability interaction between TFAP2C and HNRNPK is conserved across cell lines.

      To further strengthen our conclusions, we have now added LN-229 C3 TFAP2C overexpression in Fig. 2G (also attached below for your convenience). As for the U251-Cas9, LN-229 C3 cells show increased sensitivity to HNRNPK ablation upon TFAP2C overexpression.

      11) Nuclear localisation studies indicate that the HNRNPK and TFAP2C proteins colocalise in the nucleus however the co-IP data is not convincing- although appropriate controls are present, the level of interaction is very low - the amount of HNRNPK pulled down by TFAP2C is really very low in the LN-229C3 cells and even lower in the U251-Cas9 cells. Have they undertaken the reciprocal co-IP expt?

      Response: We rephrased our text to better highlight this as also mentioned in our response to reviewer #1 (Point 1 - Major comments). However, as also noted by the reviewer, the experiments included all the relevant controls. Thus, the results are solid and confirm a degree of co-immunoprecipitation (although weak). As detailed in our response to reviewer #1 (Point 1 - Major comments), to strengthen our conclusion, we have now repeated the experiment in low-detergent conditions and used benzonase nuclease for DNA digestion. We also have performed the reciprocal experiment as suggested by the reviewer, confirming the initial results. In our opinion, these additional experiments support the conclusion that Tfap2c and hnRNP K co-immunoprecipitate through a weak, but direct, interaction.

      12) They state that LN-229 C3 ∆TFAP2C and U251-Cas9 ∆TFAP2C were only mildly resistant to the apoptotic action of staurosporin Fig 3E and F - I accept they have undertaken the stats which support their statement that at high concentrations of staurosporin the LN-229 C3 ∆TFAP2C cells are less sensitive but the U251-Cas9 ∆TFAP2C decreased sensitivity is hard to believe. Has this been replicated? I agree that HNRNPK deletion causes apoptosis in both LN-229 C3 and U251-Cas9 cells and this is blocked by Z-VAD-FMK - however the block is not complete- the max viability for HNRNPK deletion in LN-229 C3 cells is about 40% whereas for U251-Cas9 cells it is about 30% - does this suggest that cells are being lost by another pathway. Have they tested concentrations higher than 10nM?

      Response: The experiments in FIG. 3E-F have been replicated four times, as stated in the figure legend. We agree that TFAP2C plays a limited role in response to staurosporine-induced apoptosis, particularly in U251-Cas9 cells. To ensure clarity, we have now modified our previous sentence as follows:

      "LN-229 C3ΔTFAP2C cells were only mildly resistant to the apoptotic action of staurosporine, and U251-Cas9ΔTFAP2C showed even lower and minimal recovery (Fig. 3E-F). These results indicate that TFAP2C plays a limited role in apoptosis regulation and suggest that its suppressive effect on HNRNPK essentiality is not mediated through direct modulation of apoptosis but rather through upstream processes that eventually converge on it."

      The incomplete blockade of apoptosis by Z-VAD-FMK suggests that HNRNPK ablation may activate alternative, non-caspase-mediated cell death pathways. Regarding this point, we decided to not test Z-VAD-FMK above 10 nM as we noted that the rescue effect at the lowest concentration (2nM) was not proportionally increasing at higher concentrations, suggesting we already reached saturation. We have now added and clarified these observations in the revised manuscript as follows:

      "Z-VAD-FMK decreased cell death consistently and significantly in LN-229 C3 and U251-Cas9 cells transduced with HNRNPK ablation qgRNAs (Fig. 3C‑D), confirming that HNRNPK deletion promotes cell apoptosis. However, we observed that viability recovery plateaued already at the lowest concentration (2 nM) without further increase at higher doses, suggesting a saturation effect. This indicates that while caspase inhibition alleviates part of the cell death, HNRNPK loss triggers additional mechanisms beyond apoptosis".

      Following the suggestion of the reviewer, we have now also tested two higher concentrations of Z-VAD (20 and 50nM) in LN-229 cells. At these concentrations, we observed a slight decrease in cell viability in the NT condition, with a rescue effect in the HNRNPK-ablated cells comparable to what was observed at 2-10nM Z-VAD. For this reason, we did not include these data in the revised manuscript, and we attached them here for transparency.

      13) The RNA-seq comparisons- the authors use log2 FC Response: We used a log2 FC threshold of >0.5 and 0.25) is commonly used in RNA-seq studies to capture biologically relevant shifts (e.g.,https://doi.org/10.1371/journal.ppat.1012552; https://doi.org/10.1371/journal.ppat.1008653; https://doi.org/10.1016/j.neuron.2025.03.008; https://doi.org/10.15252/embj.2022112338). We complemented this analysis with Gene Set Enrichment Analysis (GSEA) to assess coordinated changes in biological/genetic pathways, ensuring that our conclusions are not based on isolated, minor expression changes nor on arbitrary thresholds. Finally, to enhance our result robustness, we applied False Discovery Rate (FDR) statistics, which is more stringent than a p-value cutoff. We hope this clarification strengthens the reviewer's confidence in the significance of the observed changes.

      14) It is stated" Accordingly, we observed increased AMPK phosphorylation (pAMPK) upon ablation of HNRNPK, which was consistently reduced in LN-229 C3ΔTFAP2C cells (Supp. Fig. 5B). LN-229 C3ΔTFAP2C; ΔHNRNPK cells also showed a partial reduction of pAMPK relative to LN-229 C3ΔHNRNPK cells (Supp. Fig. 5B). These results suggest that hnRNP K depletion causes an energy shortfall, leading to cell death.

      Response: I am not totally convinced by the data presented in this Fig. The authors have quantified the band intensity and present the ratio of pAMPK to AMPK. Please note that the actin levels are variable across the samples - did they normalise the data using the actin level before undertaking the comparisons? Also, if the authors think this is an important point which supports their conclusion, then it should be in the main body of the paper rather than the supplementary. If AMPK is being phosphorylated, this should lead to activation of the metabolic check point which involves p53 activation by phosphorylation. Activated p53 would turn on p21CIP1 which is a very sensitive indicator of p53 activation.

      We also refer the reviewer to our response to reviewer #1 (Point 2 - Major comments). We understand the point of the reviewer as pAMPK/Actin (absolute AMPK phosphorylation) may provide additional context regarding the downstream effects of AMPK activation, which, however, is not the primary scope of our experiment. We believe that in our specific case, a) the pAMPK/AMPK ratio is the most appropriate metric, as it reflects the energy status of the cell (ATP/AMP levels), which was our main point to assess in this experiment, and b) phospho-protein/total protein is the standard approach for quantifying phosphorylation ratio. For completeness, we have now included pAMPK/Actin quantifications in Supp. Fig. 6B of the revised manuscript (also attached below). pAMPK/Actin levels follow the same trend of pAMPK/AMPK in HNRNPK and TFAP2C single ablations. The pAMPK/AMPK partial rescue in HNRNPK;TFAP2C double ablation relative to HNRNPK single deletion is instead not observed at pAMPK/Actin level. We have now added the pAMPK/Actin quantification and this observation to the revised manuscript as follows:

      "Accordingly, we observed increased AMPK phosphorylation (pAMPK/AMPK ratio and pAMPK/Actin) upon ablation of HNRNPK, with a trend toward reduction in LN-229 C3ΔTFAP2C cells (Supp. Fig. 6B). LN-229 C3ΔTFAP2C;ΔHNRNPK cells also showed a reduction of pAMPK/AMPK ratio relative to LN-229 C3ΔHNRNPK cells, although absolute AMPK phosphorylation (pAMPK/Actin) remained high (Supp. Fig. 6B)."

      We prefer to keep the AMPK blots in Supplementary Fig. 6B, as we believe the main take-home message of the manuscript should remain centered on mTORC1 activity.

      15) We also do not understand why the mTOR Suppl. Fig. 5E is not in the main body of the paper. It's clear that RNA and protein levels of mTOR were downregulated in LN-229 C3ΔHNRNPK cells but were partially rebalanced by the ΔTFAP2C- however the ΔTFAP2C;ΔHNRNPK double deletion levels are only slightly higher than the ΔHNRNPK - they are not at the level NT or even ΔTFAP2C (Fig. 4C, Supp. Fig. 5E).

      Response: We moved the mTOR blot to Fig.5D of the revised manuscript. About the low rescue effect, this is in line with all the other observations where a full rescue of the effects of HNRNPK ablation is never achieved, but is only partial. As suggested by reviewer #3 (Figure 5 - Point 2), we have now added RT-qPCR in Fig.5C, which corroborates these data.

      16) The authors state: "Deletion of HNRNPK diminished the highly phosphorylated forms of 4EBP1, which instead were preserved in both LN-229 C3ΔTFAP2C and LN-229 C3ΔTFAP2C;ΔHNRNPK cells (Fig. 5C). Similarly, the S6 phosphorylation ratio was reduced in LN-229 C3ΔHNRNPK cells and was restored in the ΔTFAP2C;ΔHNRNPK double-ablated cells (Fig. 5C)."

      WE are not convinced that p4EBP1 is preserved in the LN-229 C3ΔTFAP2C cells - there is a very faint band which is at a lower level than the band in the LN-229 C3ΔHNRNPK cells. However, when both HNRNPK and TFAP2C were ablated, the p4EBP1 band is clear cut. I agree with the quantitation that deletion of HNRNPK and TFAP2C both reduce the level of 4EBP1 - the reduction is greater with TFAP2 but when both are deleted together the levels of 4EBP1 are higher and p4EBP1 is clearly present. In quantifying the S6 and pS6 levels, did the authors consider the actin levels- they present a ratio of the pS6 to S6. I may be lacking some understanding but why is the ratio of pS6/S6 being calculated. Is the level of pS6 not what is important - phosphorylation of S6 should lead it to being activated and thus it's the actual level of pS6 that is important, not the ratio to the non-phosphorylated protein.

      Response: In Fig. 5C, the three-band pattern of 4EBP1 is clearly visible in the NT+NT or WT condition, with the top band representing the highest phosphorylation state. Upon HNRNPK deletion, this top band almost completely disappears, mimicking the effect of our starvation control (Starv.). This top band remains clearly visible in both TFAP2C-ablated and double-ablated cells, supporting our conclusion. In our original text, we referred to the "highly phosphorylated forms" of 4EBP1, which might have caused some confusion, suggesting we were evaluating the two top bands. We are specifically referring only to the very top band (high p4EBP1), which represents the most highly phosphorylated form of 4EBP1. This is the relevant phosphorylated form to focus on, as it is the only one that disappears in the starvation control (Starv.) or upon mTORC1/2 inhibition with Torin-1 (Fig. 7B).

      To better clarify these points, we have now more clearly indicated the "high p4EBP1" band with an asterisk in Fig. 5E, added quantification of high p4EBP1/4EBP1, and rephrased the text as follows:

      "Deletion of HNRNPK diminished the highest phosphorylated form of 4EBP1 (high p4EBP1, marked with an asterisk), mimicking the effect observed in starved cells (Starv.). This high p4EBP1 band was preserved in both LN-229 C3ΔTFAP2C and LN-229 C3ΔTFAP2C;ΔHNRNPK cells (Fig. 5C).".

      Regarding pS6 quantification, we added pS6/Actin quantification in Supp. Fig. 6E and F of the revised manuscript, also attached here for your convenience.

      17) When determining ATP levels, do they control for cell number? HNRNPK depletion results in lower ATP levels, co-deletion of TFAP2C rescues this. But this could be because there is less cell-death? So, more cells express ATP. Have they controlled for relative numbers of cells.

      Response: As described in the Materials and Methods , we normalized ATP levels to total protein content, which is a standard approach for this type of quantification (see DOI:10.1038/nature19312).

      18) The construction of the HovL cell line that propagate ovine prions - very few details are provided of the susceptibility of the cell line to PG127 prions.

      Response: As with other prion-infected cell lines, HovL cells do not exhibit any specific growth defects, susceptibilities, or phenotypes beyond their ability to propagate prions. This is consistent with established observations in prion research, where immortalized cell lines (and in general in vitro cultures) normally do not show cytotoxicity upon prion infection and, therefore, are used as models for prion propagation rather than for prion toxicity (see https://doi.org/10.1111/jnc.14956 for reference).

      We now expanded the relevant section, including technical and conceptual details in the main text of the revised manuscript as follows:

      "As reported for other ovinized cell models (66), HovL cells were susceptible to infection by the PG127 strain of ovine prions and capable of sustaining chronic prion propagation, as shown by proteinase K (PK)-digested western blot and by detection of PrPSc using the anti-PrP antibody 6D11, which selectively stains prion-infected cells after fixation and guanidinium treatment (67) (Supp. Fig. 7C-E). Consistent with most prion-propagating cell lines (68), HovL cells did not exhibit specific growth defects, susceptibilities, or overt phenotypes beyond their ability to propagate prions."

      19) It is stated that HRNPK depletion from HovL cells increases PrpSC as determined by 6D11 fluorescence, but in the manuscript HRNPK depletion results in cell death. How does this come together?

      Response: As explicitly stated in the main text and shown in Fig.6-7, HNRNPK is downregulated (via siRNAs) in the prion experiments rather than fully deleted (via CRISPR) as in the first part of the manuscript. As shown in Supp. Fig. 8B, this downregulation does not affect cell viability within the experimental time window. Therefore, the observed increase in PrPSc levels upon HNRNPK downregulation, as determined by western blot and 6D11 staining, is independent of any potential cell death effects. Moreover, the same siRNA downregulation approach was used by M. Avar et al. (Ref. 26) in comparable experiments, yielding similar outcomes.

      20) They show that mTOR inhibition mimics the effect of HNRNPK deletion, why didn't they overexpress mTOR and see if that rescues this? This would indicate a causal relationship.

      Response: We appreciate the reviewer's suggestion. We agree that the proposed rescue strategy would be the best approach to indicate a causal relationship. However, we linked the activity of the mTORC1 complex (and not only that of mTOR) to prion propagation. Overexpression of only mTOR would not restore mTORC1 full function, as Rptor would still be downregulated in the context of HNRNPK siRNA silencing (Fig. 7A and Supp. Fig. 8E). Moreover, our RNA-seq data (Supp. Table 5) from HNRNPK ablation indicate the downregulation of other mTORC1 components (namely Pras40 (AKT1S1) and mLST8). Therefore, the rescue of the mTORC1 activity by an overexpression strategy would be a very challenging approach. Given these complexities, to infer causality, we used mTORC1 inhibition (via rapamycin and Torin1) to mimic the effects of HNRNPK downregulation in reducing mTORC1 activity (FIG. 7B).

      For clarification, we have now highlighted in Fig. 4C that HNRNPK ablation downregulates also AKT1S1 and mLST8, other than mTOR and Rptor (also attached below), and we have discussed this in the main text as well. We also have clarified in the revised manuscript (where we sometimes inadvertently referred to it as just mTOR inhibition) that the observed effects are due to mTORC1 inhibition, and not simply mTOR inhibition.

      21) Flow cytometric data: supplementary Fig of Fig6d. - when they are looking at fixed cells the gating strategy for cells results in the inclusion of a lot of debris. The gate needs to be moved and be more specific to ensure results are interpreted properly. Same with the singlet gating. It's not tight enough, they include doublets as well which will skew their data. The gating strategy needs to be regated.

      Response: We have reanalyzed the flow cytometry data in Fig. 6D with a more stringent gating approach to better exclude debris and ensure proper singlet selection. We confirm that there is no change in the final interpretation of the results after applying the updated gating strategy.

      Reviewer #2 (Significance (Required)):

      The manuscript "Prion propagation is controlled by a hierarchical network involving the nuclear Tfap2c and hnRNP K factors and the cytosolic mTORC1 complex" by Sellitto et al aims to examine how heterogenous nuclear ribonucleoprotein K (hnRNPK), limits pion propagation. They perform a synthetic - viability CRISPR- ablation screen to identify epistatic interactors of HNRNPK. They found that deletion of Transcription factor AP-2g (TFAP2C) suppressed the death of hnRNP-K depleted LN-229 and U-251 MG cells whereas its overexpression hypersensitized them to hnRNP K loss. Moreover, HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism and enhanced autophagy. Simultaneous deletion of TFAP2C reversed these effects, restored transcription and alleviated energy deficiency.

      Referee #3

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      Summary: Using a CRISPR-based high throughput abrasion assay, Sellitto et al. identified a list of genes that improve cell viability when deleted in hnRNP K knockout cells. Tfap2c, a transcription factor, was identified as a candidate with potential overlap with a hnRNP K function like modulating glucose metabolism. The deletion of Tfap2c in hnRNP K-deletion background prevented caspase-dependent apoptosis observed in hnRNP K single-deletion cells. Further analysis of bulk RNA-seq in hnRNP K/TFAP2C single- and double-deletion cells revealed the impairment in cellular ATP level. Accordingly, activation of AMPK led to perturbed autophagy in hnRNP K deleted cells. Moreover, the reduction and/or inactivation of the downstream mTOR protein resulted in the reduced phosphorylation of S6. Conversely, the phosphorylation of S6 and E4BP1 can be increased by TFAP2C overexpression. Finally, the pharmacological inhibition of the mTOR pathway increased the PrPSC level. This is an interesting paper potentially providing new mechanical insight of hnRNPK function and its interaction with TFAP2C. However, inconsistencies in TFAP2C expression across cell lines and conflicting mechanistic interpretations complicate conclusions. Co-IP experiments suggested hnRNP K and Tfap2c may interact, though further validation is needed. Several figures require additional clarification, statistical analysis, or experimental validation to strengthen conclusions.

      Major comments:

      1) Different responses of the TFAP2C expression level to deletion of hnRNPK in the two cell lines (LN-229 C3 and U251-Cas9) should be more adequately addressed. The manuscript focuses on the interaction between hnRNPK and TFAP2C, yet the hnRNPK deletion causes different changes in TFAP2C level in two different lines. Furthermore, in studies where the mechanistic link between hnRNPK and TFAP2C is being investigated, only results from the LN-229 line are presented (Figure 4-7). Thus, it is not clear whether these mechanisms also apply to another line, U251-Cas9, where hnRNPK deletion has the opposite effect on the TFAP1C level. Thus, key experiments should be performed in both lines.

      Response: The opposite effects of hnRNPK ablation on TFAP2C expression between LN-229 C3 and U251-Cas9 cells likely reflect intrinsic differences between the two cell lines. However, the viability interaction between hnRNPK and TFAP2C is conserved in both cell models (Fig. 1G-H, 2A-B, Supp. Fig. 3A-B), suggesting that shared molecular functions at the interface of this interaction exist across the lines. In fact, we believe that the opposite effect of hnRNPK ablation on TFAP2C expression in the two lines strengthens (rather than weakens) our model by highlighting how TFAP2C expression modulates cellular sensitivity to HNRNPK ablation, as detailed in our response to Reviewer #2 (Point 10 - Major comments).

      Regarding the mechanistic studies presented in FIG. 4-7, our initial goal in using two cell lines was to validate the functional viability interaction between HNRNPK and TFAP2C, as identified in our screening (performed in LN-229 C3 cells). After confirming this interaction, we chose to focus only on LN-229 C3 (beginning with RNA-seq analysis, which then led to subsequent mechanistic studies), as this provided the necessary foundation to investigate prion propagation in HovL cells (derived from LN-229). As a U251 model propagating prions does not exist, we are technically limited in performing prion experiments only in HovL and we do not believe that conducting additional experiments in U251 cells would add substantial value to our work or further our investigation.

      We hope this explanation clarifies our rationale and addresses the reviewer's concerns.

      2) Although a lot of data are presented, it is not clear how deletion of the TFAP2C reverses the toxicity caused by deletion of hnRNPK. Specifically, the first half of the paper seems to suggest an opposite mechanism than the second half of the paper. In Figure 2-4, the authors suggest a model that TFAP2C deletion has the opposite effect of hnRNPK deletion, thus rescuing toxicity. However, in Figure 5-6, it is suggested TFAP2C overexpression has the opposite effect of hnRNPK deletion. This two opposite effect of TFAP2C make it difficult to understand the models that the authors are proposing. Please also see below comment 2 for Figure 5.

      Response: We respectfully disagree with the notion that the first and second halves of the manuscript propose contradictory mechanisms.

      In Fig. 2-4, we describe the phenotypic rescue of cell viability upon TFAP2C deletion in hnRNPK-deficient cells. At this stage, we are not proposing a specific molecular mechanism but simply observing a rescue of viability and highlighting underlying transcriptional differences. There is no implication of an opposite molecular mechanism involving the individual activities of hnRNPK and TFAP2C; rather, we focused on the broader effect of TFAP2C deletion on the viability of HNRNPK-lacking cells. In Fig. 5, we isolated a partial mechanism underlying this interaction. We state that: "These data specify a role for TFAP2C in promoting mTORC1-mediated cell anabolism and suggest that its overexpression might hypersensitize cells to HNRNPK ablation by depleting the already limited ATP available, thus making its deletion advantageous". In the discussion, we now further reviewed our explanation: "HNRNPK deletion might cause a metabolic impairment leading to a nutritional crisis and a catabolic shift, whereas TFAP2C activation could promote mTORC1 anabolic functions. Thus, Tfap2c removal may rewire the bioenergetic needs of cells by modulating the mTORC1 signaling and augmenting their resilience to metabolic stress like the one induced by HNRNPK ablation". Therefore, we propose that TFAP2C expression might be particularly detrimental in hnRNPK-deficient cells, as it could push the cell into an anabolic biosynthetic state, further depleting energy stores that the cell is attempting to conserve in response to hnRNPK depletion. Removal of TFAP2C alleviates this metabolic strain. In our view, there is no contradiction between our observations.

      We hope this explanation clarifies our rationale and resolves any perceived inconsistency in our model. To further enhance the understanding of our interpretations, we have now also added (in substitution of Fig. 5E of the original manuscript) a graphical scheme (Fig. 5G of the revised manuscript) to visually explain and illustrate our model (attached below for your convenience).

      3) Similar to the point above, the first half of the paper focuses on hnRNPK deletion-induced toxicity (Fig. 1-5), while the second half of the paper focuses on hnRNPK deletion-induced PrPSC level (Fig. 6-7). The mechanistic link between these two downstream effects of hnRNPK deletion is not clear and thus, it is difficult to understand the reason that hnRNPK deletion-induced toxicity can be rescued by TFAP2C deletion, while hnRNPK deletion-induced PrPSC level increase can be rescued by TFAP2C overexpression.

      Response: Our study is not aimed at comparing viability and prion propagation as interconnected phenotypes but rather at identifying molecular processes regulated by the HNRNPK-TFAP2C interaction. Our study identifies mTORC1 activity as a molecular process at the interface of the HNRNPK-TFAP2C. HNRNPK knockout (or knockdown, which does not affect viability, and therefore is used in the prion section of the manuscript) tones mTORC1 activity down, while TFAP2C overexpression enhances it. This finding suggested an explanation for the viability interaction we observed (see reply to reviewer #3 - Point 2 -Major comments) and it provided a partial mechanism (mTORC1 activity) to explain the effect of HNRNPK knockdown and TFAP2C overexpression on prions.

      We hope this clarification addresses the reviewer's concern.

      Abstract:

      1) Please rephrase and clarify "We linked HNRNPK and TFAP2C interaction to mTOR signaling..." by distinguishing functional, genetic, and direct (molecule-to-molecule) interactions.

      Response: 1) We have now clarified it in the text of the revised manuscript as follows:

      "We linked HNRNPK and TFAP2C functional and genetic interaction to mTOR signaling, observing that HNRNPK ablation inhibited mTORC1 activity through downregulation of mTOR and Rptor, while TFAP2C overexpression enhanced mTORC1 downstream functions."

      2) A sentence reads, "...HNRNPK ablation inhibited mTORC1 activity through downregulation of mTOR and Rptor," although the downregulation of Rptor is observed only at the RNA level. The change in Rptor protein expression level is not reported in the manuscript. Please consider adding an experiment to address this or rephrase the sentence.

      Response: 2) We have now added the experiment in Supp. Fig. 9A of the revised manuscript. The blot shows that hnRNP K depletion reduces both mTOR and Rptor protein levels. "hnRNP K depletion inhibited mTORC1 activity through downregulation of mTOR and Rptor".

      Figure 2:

      1. H and I. Co-IP experiments were done using anti-TFAP2C antibody to the bead. Although the TFAP2C bands show robust signals on the blots, indicating successful enrichment of the protein, hnRNP K bands are very faint. Has the experiment been done by conjugating the hnRNP K antibody to the beads instead? Was the input lysate enriched in the nuclear fraction? Did the lysis buffer include nuclease (if so, please indicate in the figure legend and the methods section)? Addressing these would make the argument, "We also observed specific co-immunoprecipitation of hnRNP K and Tfap2c in LN-229 C3 and U251-Cas9 cells (Fig. 2H-I, Supp. Fig. 3L), suggesting that the two proteins form a complex inside the nucleus" stronger, providing information on potential direct binding.

      Response: 1. We refer the reviewer to our response to reviewers #1 and #2 regarding the weak interaction, the nuclease treatment, and the HNRNPK IP (reviewer #1 Point 1 and reviewer #2 Point 11 - Major comments). As for the co-IP input, it was not enriched in the nuclear fraction, but as shown in Supp. Fig. 4A-B hnRNPK and Tfap2c are exclusively nuclear.

      Figure 3:

      1. C and D. Please add a sentence in the figure legend explaining which means the multiple comparisons were made between (DMSO vs each drug concentration?). Graphing individual data points instead of bars would also be helpful and more informative. Please discuss the lack of dose dependency.

      Response: 1. We have now added information about the comparison in the figure legend ("Multiple comparison was made between Z-VAD-FMK and DMSO treatments in ΔHNRNPK cells."), modified the graph to show the individual data points (attached below for your convenience), and expanded the discussion as detailed for reviewer #2 (Point 14 - Major comments). (For completeness, we have also modified Supp. FIG. 5F to show individual data points, and we have combined the graphs (the DMSO control was shared across treatments)).

      Supplemental Figure 4 (Now shifted in Supplemental Figure 5):

      1. A. Although the trend can be observed, the deletion of hnRNP K does not significantly reduce the GPX4 protein level in LN-229 C3. Therefore, the following statement requires more data points and additional statistical analysis to be accurate: "In LN-229 C3 and U251-Cas9 cells, the deletion of HNRNPK reduced the protein level of GPX4, whereas TFAP2C deletion increased it (Supp. Fig. 4A-B)."

      2. A and B. The results are confusing, considering the previous report cited (ref 49) shows an increase in GPX4 with TFAP2C. It may be possible that the deletion of TFAP2C upregulates the expression of proteins with similar functions (e.g., Sp1). If this is the case, the changes in GPX4 expression observed here are a consequence of TFAP2C deletion and may not "suggest a role for HNRNPK and TFAP2C in balancing the protein levels of GPX4."

      Response: 1. We agree with the reviewer that in LN-229 C3 cells the reduction of GPX4 protein levels upon HNRNPK deletion did not reach statistical significance in our initial Western blot analysis. To address this concern, we performed six additional independent experiments and repeated the statistical analysis. Although the trend toward reduced GPX4 protein levels remained consistent, statistical significance was still not achieved (p > 0.05). Importantly, this trend is supported by our RNA-seq dataset (Supplementary Table 5), which shows decreased GPX4 expression upon HNRNPK deletion. We have now revised the text to more accurately reflect the experimental observations and to avoid overstating the effect in LN-229 C3 cells as follows:

      "In LN-229 C3 and U251-Cas9 cells, deletion of HNRNPK was associated with reduced glutathione peroxidase 4 (GPX4) protein abundance (although not statistically significant in LN-229 C3; p ≈ 0.08), whereas deletion of TFAP2C increased it (Supp. Fig. 5A-B)."

      The six new experimental replicas have been added to the uncropped western blot section.

      __Response: __2. Concerning the potential role of TFAP2C deletion in upregulating proteins with similar functions, we recognize the reviewer's perspective. However, our primary focus is on the observed trends rather than a definitive mechanistic conclusion. We clarified our wording to acknowledge this possibility while maintaining the relevance of our findings within the broader context of hnRNPK and TFAP2C interactions.

      "This last result was interesting as a previous study reported that Tfap2c enhances GPX4 expression (51). Thus, the observed increase upon TFAP2C deletion suggests additional layers of regulation, potentially involving compensatory mechanisms."

      Supplemental Figure 5 (Now shifted in Supplemental Figure 6):

      1. B. To obtain statistical significance and strengthen the conclusion, more repeated Western blot experiments can be done to quantify the pAMPK/AMPK ratio.

      Response: We included three more experiments as detailed in our response to reviewer #1 (Point 2 - Major comments) and reviewer #2 (Point 14 - Major comments).

      Figure 5:

      1. B. I believe statistical analysis with two replicates or less is not recommended. Although the assay is robust, and the blot is convincing, please consider adding more replicates if the blot is to be quantified and statistically analyzed.

      2. "Interestingly, RNA and protein levels of mTOR were downregulated in LN-229 C3ΔHNRNPK cells but were partially rebalanced by the ΔTFAP2C;ΔHNRNPK double deletion (Fig. 4C, Supp. Fig. E)." The statement is based on a slight difference at the protein level between the single deletion and the double deletion, as well as the observation from the bulk RNA-seq data. mTOR (and Rptor) mRNA level can be assessed by RT-qPCR to validate and further support the existing data. It is also curious why deletion of TFAP2C alone, also induced decrease in mTOR, but double deletion rescued mTOR level slightly compared to deletion of HNRNPK alone.

      3. C. The main text refers to the changes in the level of phosphorylated E4BP1, stating, "Deletion of HNRNPK diminished the highly phosphorylated forms of 4EBP1, which instead were preserved in both LN-229 C3ΔTFAP2C and LN-229 C3ΔTFAP2C;ΔHNRNPK cells (Fig. 5C)." However, the quantification was done on the total E4BP1, which may be because separating pE4BP1 and E4BP1 bands on a blot is challenging. Please consider using phospho-E4BP1 specific antibody or rephrase the sentence mentioned above. The current data suggest the single- and double-deletion of hnRNP K/TFAP2C affect the overall stability of E4BP1, which may be a correlation and not due to the mTOR activity as claimed in "We conclude that HNRNPK and TFAP2C play an essential role in co-regulating cell metabolism homeostasis by influencing mTOR and AMPK activity and expression." How does the cap-dependent translation (or total protein level) change in TFAP2C deleted and overexpressing cells?

      Response: 1. We added two additional experiments as detailed in our response to reviewer #1 (Point 3 - Major comment).

      __Response: __2. Deletion of TFAP2C does not decrease mTOR levels as shown from the quantification in Fig. 5D. To further support our results, we have now included RT-qPCR in FIG. 5C as suggested by the reviewer. Data are also attached here for your convenience.

      __Response: __3. Regarding the assessment of phosphorylated 4EBP1, we think we achieved a clear separation of the differently phosphorylated forms of 4EBP1 in our blots, and we have now added the quantification for High p4EBP1/4EBP1 in Fig. 5E (see also our response to reviewer #2 Point 16 - Major comments). The quantification of total 4EBP1 represents an additional dataset, and we do not claim that 4EBP1 stability is affected by HNRNPK and TFAP2C directly through mTOR, which could be, in fact, correlative. We claim that HNRNPK and TFAP2C modulate mTORC1 and AMPK metabolic signaling as shown by the changed phosphorylation of 4EBP1, S6, AMPK, and ULK1 (Fig. 5C-E, Supp. FIG. 6B, D) and by the regulation of autophagy (Fig. 5B, Supp. Fig. 6C); we did not directly check cap-dependent translation.

      We have now rephrased our text to ensure clarity as follows:

      "We conclude that HNRNPK and TFAP2C play a role in co-regulating mTORC1 and AMPK expression, signaling, and activity."

      Figure 6:

      1. A. Did the sihnRNP K increase the TFAP2C level?

      2. A and C. Are the total PrP levels lower in TFAP2C overexpressing cells compared to mCherry cells when they are infected?

      3. D. Do the TFAP2C protein levels differ between 2-day+72-h and 7-day+96-h?

      __Response: __1. Yes, it does. We have now provided the quantification in Fig. 6A, C, and Supp. Fig. 8A (also attached below for your convenience).

      __Response: __2. We have now provided the quantification in Fig. 6A and Supp. Fig. 8A. The total PrP does not change in TFAP2C overexpressing cells. Total PrP consists of both PK-resistant PrP (PrPSc) and PK-sensitive PrP (PrPC plus potential other intermediate species), with PrPSc typically present at much lower levels. In our model, PrPC is exogenously expressed at high levels via a vector and remains constant across conditions (Fig. 6C and Supp. Fig. 8C). As a result, any changes in PrPSc may not necessarily reflect on total PrP levels.

      __Response: __3. No, there is no statistically significant change. We have now added a representative western blot and the quantification of 3 independent replicates in Supp. Fig. 8D. The other two western blots are only shown in the uncropped western blots section. This dataset is also attached here for your convenience.

      Figure 7:

      1. I agree with the latter half of the statement: "These findings suggest that HNRNPK influences prion propagation at least in part through mTORC1 signaling, although additional mechanisms may be involved." The first half requires careful rephrasing since (A) Independent of the background siRNA treatment, TFAP2C overexpression by itself can modulate PrPSC level as seen in Fig 6A and B, (B) Although the increase in TFAP2C level is observed with the hnRNP K deletion (Fig 1; LN-229 C3), sihnRNP K treatment may or may not influence the TFAP2C level (Fig 6; quantified data not provided), and (C) In the sihnRNP K-treated cells, E4BP1 level is increased compared to the siNT-treated cells, which was not observed hnRNP K-deleted cells. Discussions and additional experiments (e.g., mTOR knockdown) addressing these points would be helpful.

      __Response: __A, B) We respectfully disagree with the possibility that HNRNPK downregulation may increase prion propagation via TFAP2C upregulation. As shown in Fig. 6A-B, D and in Supp. Fig. 8A, TFAP2C overexpression reduces, rather than increases, prion levels. Therefore, it would be inconsistent to suggest that HNNRPK siRNA promotes prion propagation through TFAP2C upregulation (quantification is now provided, see reviewer #3 - Figure 6 - Point 1). C) Concerning 4EBP1 levels, we have quantified the total 4EBP1 (also attached below) and expanded the discussion on potential discrepancies between HNRNPK knockout and knockdown, as the former affects cell viability, while the latter does not. However, as explained also in the previous reply to reviewer #3 - Figure 5 - Point 3, our focus is on the highly phosphorylated band of 4EBP1 (High p4EBP1), which is the direct target of mTORC1 activity. In both the hnRNPK knockout LN-229 C3 (Fig. 5E) and knockdown HovL models (Fig. 7B), phosphorylation of 4EBP1, along with phosphorylation of S6, is clearly reduced (we have now included quantification for Fig. 7B), reinforcing our conclusion that mTORC1 activity is affected by hnRNPK depletion. As the reviewer noted, we do not claim that mTORC1 is the sole mediator of hnRNPK's effect on prion regulation. However, we think that our interpretation of a potential and partial role of mTORC1 inhibition in the effect of HNRNPK downregulation on prion propagation is in line with the data presented in Fig. 6-7 and Supp. Fig. 8-9. For further clarification, we expanded the text according to the new experiments and analysis, and we added mTOR and Raptor siRNA knockdown (Supp. Fig.9C) to further support our conclusions (also attached below for your convenience).

      Minor comments:

      1. Please clarify "independent cultures." Does this mean technical replicates on the same cell culture plate but different wells or replicated experiments on different days?

      __Response: __We have now clarified in each figure legend. "Individually treated wells" means different parental cultures grown and treated separately on the same day. n represents independent experiments on different days.

      1. Fig 2G. Please explain how the sigmoidal curves were fitted to the data points under the materials and methods section.

      2. Fig 3E and F. Please refer to the comment on Fig 2G above.

      __Response: __We have now added the explanation in Materials and Methods as follows:

      "Curve Fitting

      For sigmoidal curve fitting, we used GraphPad Prism (version X, GraphPad Software). Data in Figure 2G were fitted using nonlinear regression with a least squares regression model. For Figures 3E and 3F, data fitting was performed using an asymmetric sigmoidal model with five parameters (5PL) and log-transformed X-values (log[concentration])."

      3.Fig S3 F/H. Quantification of gel bands would be helpful when comparing protein expression changes after different treatments, as band intensities look different across.

      __Response: __We have now added the quantifications in Supp. FIG. 3D-H (attached below for your convenience). They confirm that there are no significant differences in the means of the normalized values.

      1. Supp Fig 5C and F. These panels can be combined with the corresponding panels in main Figure 5 if space allows so that the readers do not have to flip pages between the main text and Supplemental material.

      __Response: __We have now combined the panels. Previous Supp. FIG. 5C and F are now shown in FIG. 6C and E, respectively.

      Reviewer #3 (Significance (Required)):

      This is an interesting paper potentially providing new mechanical insight of hnRNPK function and its interaction with TFAP2C. It is also important to understand how hnRNPK deletion induces prion propagation and develop methods to mitigate its spread. However, inconsistencies in TFAP2C expression across cell lines and conflicting mechanistic interpretations complicate conclusions. I have expertise in RNA-binding protein, cell biology, and prion disease.

    2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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      Referee #3

      Evidence, reproducibility and clarity

      Summary:

      Using a CRISPR-based high throughput abrasion assay, Sellitto et al. identified a list of genes that improve cell viability when deleted in hnRNP K knockout cells. Tfap2c, a transcription factor, was identified as a candidate with potential overlap with a hnRNP K function like modulating glucose metabolism. The deletion of Tfap2c in hnRNP K-deletion background prevented caspase-dependent apoptosis observed in hnRNP K single-deletion cells. Further analysis of bulk RNA-seq in hnRNP K/TFAP2C single- and double-deletion cells revealed the impairment in cellular ATP level. Accordingly, activation of AMPK led to perturbed autophagy in hnRNP K deleted cells. Moreover, the reduction and/or inactivation of the downstream mTOR protein resulted in the reduced phosphorylation of S6. Conversely, the phosphorylation of S6 and E4BP1 can be increased by TFAP2C overexpression. Finally, the pharmacological inhibition of the mTOR pathway increased the PrPSC level. This is an interesting paper potentially providing new mechanical insight of hnRNPK function and its interaction with TFAP2C. However, inconsistencies in TFAP2C expression across cell lines and conflicting mechanistic interpretations complicate conclusions. Co-IP experiments suggested hnRNP K and Tfap2c may interact, though further validation is needed. Several figures require additional clarification, statistical analysis, or experimental validation to strengthen conclusions.

      Major comments:

      1. Different responses of the TFAP2C expression level to deletion of hnRNPK in the two cell lines (LN-229 C3 and U251-Cas9) should be more adequately addressed. The manuscript focuses on the interaction between hnRNPK and TFAP2C, yet the hnRNPK deletion causes different changes in TFAP2C level in two different lines. Furthermore, in studies where the mechanistic link between hnRNPK and TFAP2C is being investigated, only results from the LN-229 line are presented (Figure 4-7). Thus, it is not clear whether these mechanisms also apply to another line, U251-Cas9, where hnRNPK deletion has the opposite effect on the TFAP1C level. Thus, key experiments should be performed in both lines.
      2. Although a lot of data are presented, it is not clear how deletion of the TFAP2C reverses the toxicity caused by deletion of hnRNPK. Specifically, the first half of the paper seems to suggest an opposite mechanism than the second half of the paper. In Figure 2-4, the authors suggest a model that TFAP2C deletion has the opposite effect of hnRNPK deletion, thus rescuing toxicity. However, in Figure 5-6, it is suggested TFAP2C overexpression has the opposite effect of hnRNPK deletion. This two opposite effect of TFAP2C make it difficult to understand the models that the authors are proposing. Please also see below comment 2 for Figure 5.
      3. Similar to the point above, the first half of the paper focuses on hnRNPK deletion-induced toxicity (Fig. 1-5), while the second half of the paper focuses on hnRNPK deletion-induced PrPSC level (Fig. 6-7). The mechanistic link between these two downstream effects of hnRNPK deletion is not clear and thus, it is difficult to understand the reason that hnRNPK deletion-induced toxicity can be rescued by TFAP2C deletion, while hnRNPK deletion-induced PrPSC level increase can be rescued by TFAP2C overexpression.

      Abstract.

      1. Please rephrase and clarify "We linked HNRNPK and TFAP2C interaction to mTOR signaling..." by distinguishing functional, genetic, and direct (molecule-to-molecule) interactions.
      2. A sentence reads, "...HNRNPK ablation inhibited mTORC1 activity through downregulation of mTOR and Rptor," although the downregulation of Rptor is observed only at the RNA level. The change in Rptor protein expression level is not reported in the manuscript. Please consider adding an experiment to address this or rephrase the sentence.

      Figure 2.

      1. H and I. Co-IP experiments were done using anti-TFAP2C antibody to the bead. Although the TFAP2C bands show robust signals on the blots, indicating successful enrichment of the protein, hnRNP K bands are very faint. Has the experiment been done by conjugating the hnRNP K antibody to the beads instead? Was the input lysate enriched in the nuclear fraction? Did the lysis buffer include nuclease (if so, please indicate in the figure legend and the methods section)? Addressing these would make the argument, "We also observed specific co-immunoprecipitation of hnRNP K and Tfap2c in LN-229 C3 and U251-Cas9 cells (Fig. 2H-I, Supp. Fig. 3L), suggesting that the two proteins form a complex inside the nucleus" stronger, providing information on potential direct binding.

      Figure 3.

      1. C and D. Please add a sentence in the figure legend explaining which means the multiple comparisons were made between (DMSO vs each drug concentration?). Graphing individual data points instead of bars would also be helpful and more informative. Please discuss the lack of dose dependency.

      Supplemental Figure 4.

      1. A. Although the trend can be observed, the deletion of hnRNP K does not significantly reduce the GPX4 protein level in LN-229 C3. Therefore, the following statement requires more data points and additional statistical analysis to be accurate: "In LN-229 C3 and U251-Cas9 cells, the deletion of HNRNPK reduced the protein level of GPX4, whereas TFAP2C deletion increased it (Supp. Fig. 4A-B)."
      2. A and B. The results are confusing, considering the previous report cited (ref 49) shows an increase in GPX4 with TFAP2C. It may be possible that the deletion of TFAP2C upregulates the expression of proteins with similar functions (e.g., Sp1). If this is the case, the changes in GPX4 expression observed here are a consequence of TFAP2C deletion and may not "suggest a role for HNRNPK and TFAP2C in balancing the protein levels of GPX4."

      Supplemental Figure 5.

      1. B. To obtain statistical significance and strengthen the conclusion, more repeated Western blot experiments can be done to quantify the pAMPK/AMPK ratio.

      Figure 5.

      1. B. I believe statistical analysis with two replicates or less is not recommended. Although the assay is robust, and the blot is convincing, please consider adding more replicates if the blot is to be quantified and statistically analyzed.
      2. "Interestingly, RNA and protein levels of mTOR were downregulated in LN-229 C3ΔHNRNPK cells but were partially rebalanced by the ΔTFAP2C;ΔHNRNPK double deletion (Fig. 4C, Supp. Fig. E)." The statement is based on a slight difference at the protein level between the single deletion and the double deletion, as well as the observation from the bulk RNA-seq data. mTOR (and Rptor) mRNA level can be assessed by RT-qPCR to validate and further support the existing data. It is also curious why deletion of TFAP2C alone, also induced decrease in mTOR, but double deletion rescued mTOR level slightly compared to deletion of HNRNPK alone.
      3. C. The main text refers to the changes in the level of phosphorylated E4BP1, stating, "Deletion of HNRNPK diminished the highly phosphorylated forms of 4EBP1, which instead were preserved in both LN-229 C3ΔTFAP2C and LN-229 C3ΔTFAP2C;ΔHNRNPK cells (Fig. 5C)." However, the quantification was done on the total E4BP1, which may be because separating pE4BP1 and E4BP1 bands on a blot is challenging. Please consider using phospho-E4BP1 specific antibody or rephrase the sentence mentioned above. The current data suggest the single- and double-deletion of hnRNP K/TFAP2C affect the overall stability of E4BP1, which may be a correlation and not due to the mTOR activity as claimed in "We conclude that HNRNPK and TFAP2C play an essential role in co-regulating cell metabolism homeostasis by influencing mTOR and AMPK activity and expression." How does the cap-dependent translation (or total protein level) change in TFAP2C deleted and overexpressing cells?

      Figure 6.

      1. A. Did the sihnRNP K increase the TFAP2C level?
      2. A and C. Are the total PrP levels lower in TFAP2C overexpressing cells compared to mCherry cells when they are infected?
      3. D. Do the TFAP2C protein levels differ between 2-day+72-h and 7-day+96-h?

      Figure 7.

      1. I agree with the latter half of the statement: "These findings suggest that HNRNPK influences prion propagation at least in part through mTORC1 signaling, although additional mechanisms may be involved." The first half requires careful rephrasing since (A) Independent of the background siRNA treatment, TFAP2C overexpression by itself can modulate PrPSC level as seen in Fig 6A and B, (B) Although the increase in TFAP2C level is observed with the hnRNP K deletion (Fig 1; LN-229 C3), sihnRNP K treatment may or may not influence the TFAP2C level (Fig 6; quantified data not provided), and (C) In the sihnRNP K-treated cells, E4BP1 level is increased compared to the siNT-treated cells, which was not observed hnRNP K-deleted cells. Discussions and additional experiments (e.g., mTOR knockdown) addressing these points would be helpful.

      Minor comments:

      1. Please clarify "independent cultures." Does this mean technical replicates on the same cell culture plate but different wells or replicated experiments on different days?
      2. Fig 2G. Please explain how the sigmoidal curves were fitted to the data points under the materials and methods section.
      3. Fig 3E and F. Please refer to the comment on Fig 2G above.
      4. Fig S3 F/H. Quantification of gel bands would be helpful when comparing protein expression changes after different treatments, as band intensities look different across.
      5. Supp Fig 5C and F. These panels can be combined with the corresponding panels in main Figure 5 if space allows so that the readers do not have to flip pages between the main text and Supplemental material.

      Significance

      This is an interesting paper potentially providing new mechanical insight of hnRNPK function and its interaction with TFAP2C. It is also important to understand how hnRNPK deletion induces prion propagation and develop methods to mitigate its spread. However, inconsistencies in TFAP2C expression across cell lines and conflicting mechanistic interpretations complicate conclusions. I have expertise in RNA-binding protein, cell biology, and prion disease.

    3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      The manuscript "Prion propagation is controlled by a hierarchical network involving the nuclear Tfap2c and hnRNP K factors and the cytosolic mTORC1 complex" by Sellitto et al aims to examine how heterogenous nuclear ribonucleoprotein K (hnRNPK), limits pion propagation. They perform a synthetic - viability CRISPR- ablation screen to identify epistatic interactors of HNRNPK. They found that deletion of Transcription factor AP-2 (TFAP2C) suppressed the death of hnRNP-K depleted LN-229 and U-251 MG cells whereas its overexpression hypersensitized them to hnRNP K loss. Moreover, HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism and enhanced autophagy. Simultaneous deletion of TFAP2C reversed these effects, restored transcription and alleviated energy deficiency.

      They state that HNRNPK and TFAP2C are linked to mTOR signalling and observe that HNRNPK ablation inhibits mTORC1 activity through downregulation of mTOR and Rptor while TFAP2C overexpression enhances mTORC1 downstream functions. In prion infected cells, TFAP2C activation reduced prion levels and countered the increased prion propagation due to HNRNPK suppression. Pharmacological inhibition of mTOR also elevated prion levels and partially mimicked the effects of HNRNPK silencing. They state their study identifies TFAP2C as a genetic interactor of HNRNPK and implicates their roles in mTOR metabolic regulation and establishes a causative link between these activities and prion propagation.

      This is an interesting manuscript in which a lot of work has been undertaken. The experiments are on the whole well done, carefully documented and support most of the conclusions drawn. However, there are places where it was quite difficult to read as some of the important results are in the supplementary Figures and it was necessary to go back and forth between the Figs in the main body of the paper and the supplementary Figs. There are also Figures in the supplementary which should have been presented in the main body of the paper. These are indicated in our comments below.

      We have the following questions /points:

      1. A plasmid harbouring four guide RNAs driven by four distinct constitutive promoters is used for targetting HNRNPK- is there a reason for using 4 guides- is it simply to obtain maximal editing - in their experience is this required for all genes or specific to HNRNPK?
      2. Is there a minimal amount of Cas9 required for editing?
      3. It is stated that cell death is delayed in U251-MG cells compared to LN-229-C3 cells- why? Also, why use glioblastoma cells other than that they have high levels of HNRNPK? Would neuroblastoma cells be more appropriate if they are aiming to test for prion propagation?
      4. Human CRISPR Brunello pooled library- does the Brunello library use constructs which have four independent guide RNAs as used for the silencing of HNRPNK?
      5. To rank the 763 enriched genes, they multiply the -log10FDR with their effect size - is this a standard step that is normally undertaken?
      6. The 32 genes selected- they were ablated individually using constructs with one guide RNA or four guide RNAs?
      7. The identified targets were also tested in U251-MG cells and nine were confirmed but the percent viability was variable - is the variability simply a reflection of the different cell line?
      8. The two strongest hits were IKBAKP and TFAP2C. As TFAP2C is a transcription factor - is it known to modulate expression of any of the genes that were identified to be perturbed in the screen? Moreover, it is stated that it regulates expression of several lncRNAs- have the authors looked at expression of these lncRNAs- is the expression affected- can modulation of expression of these lncRNAs modulate the observed phenotypic effects and also some of the targets they have identified in the screen?
      9. As both HNRNPK and TFAP2C modulate glucose metabolism, the authors have chosen to explore the epistatic interaction. This is most reasonable.
      10. The orthogonal assay to confirm that deletion of TFAP2C supresses cell death upon removing HNRNPK- was this done using a single guide RNA or multiple guides - is there a level of suppression required to observe rescue? Interestingly ablation of HNRNPK increases TFAP2C expression in LN-229-C3 whereas in U251-Cas9 cells HNRNPK ablation has the opposite effect- both RNA and protein levels of TFAP2C are decreased - is this the cause of the smaller protective effect of TFAP2C deletion in this cell line?
      11. Nuclear localisation studies indicate that the HNRNPK and TFAP2C proteins colocalise in the nucleus however the co-IP data is not convincing- although appropriate controls are present, the level of interaction is very low - the amount of HNRNPK pulled down by TFAP2C is really very low in the LN-229C3 cells and even lower in the U251-Cas9 cells. Have they undertaken the reciprocal co-IP expt?
      12. They state that LN-229 C3 TFAP2C and U251-Cas9TFAP2C were only mildly resistant to the apoptotic action of staurosporin Fig 3E and F - I accept they have undertaken the stats which support their statement that at high concentrations of staurosporin the LN-229 C3 TFAP2C cells are less sensitive but the U251-Cas9TFAP2C decreased sensitivity is hard to believe. Has this been replicated? I agree that HNRNPK deletion causes apoptosis in both LN-229 C3 and U251-Cas9 cells and this is blocked by Z-VAD-FMK - however the block is not complete- the max viability for HNRNPK deletion in LN-229 C3 cells is about 40% whereas for U251-Cas9 cells it is about 30% - does this suggest that cells are being lost by another pathway. Have they tested concentrations higher than 10nM?
      13. The RNA-seq comparisons- the authors use log2 FC <0.5 upregulated or genes downregulated by a similar amount- this is a very low cut off and would include essentially minimal changes in expression - not convinced of the significance of such low-level changes.
      14. It is stated" Accordingly, we observed increased AMPK phosphorylation (pAMPK) upon ablation of HNRNPK, which was consistently reduced in LN-229 C3ΔTFAP2C cells (Supp. Fig. 5B). LN-229 C3ΔTFAP2C; ΔHNRNPK cells also showed a partial reduction of pAMPK relative to LN-229 C3ΔHNRNPK cells (Supp. Fig. 5B). These results suggest that hnRNP K depletion causes an energy shortfall, leading to cell death. I am not totally convinced by the data presented in this Fig. The authors have quantified the band intensity and present the ratio of pAMPK to AMPK. Please note that the actin levels are variable across the samples - did they normalise the data using the actin level before undertaking the comparisons? Also, if the authors think this is an important point which supports their conclusion, then it should be in the main body of the paper rather than the supplementary. If AMPK is being phosphorylated, this should lead to activation of the metabolic check point which involves p53 activation by phosphorylation. Activated p53 would turn on p21CIP1 which is a very sensitive indicator of p53 activation.
      15. We also do not understand why the mTOR Suppl. Fig. 5E is not in the main body of the paper. It's clear that RNA and protein levels of mTOR were downregulated in LN-229 C3ΔHNRNPK cells but were partially rebalanced by the ΔTFAP2C- however the ΔTFAP2C;ΔHNRNPK double deletion levels are only slightly higher than the ΔHNRNPK - they are not at the level NT or even ΔTFAP2C (Fig. 4C, Supp. Fig. 5E).
      16. The authors state: "Deletion of HNRNPK diminished the highly phosphorylated forms of 4EBP1, which instead were preserved in both LN-229 C3ΔTFAP2C and LN-229 C3ΔTFAP2C;ΔHNRNPK cells (Fig. 5C). Similarly, the S6 phosphorylation ratio was reduced in LN-229 C3ΔHNRNPK cells and was restored in the ΔTFAP2C;ΔHNRNPK double-ablated cells (Fig. 5C)."

      WE are not convinced that p4EBP1 is preserved in the LN-229 C3ΔTFAP2C cells - there is a very faint band which is at a lower level than the band in the LN-229 C3ΔHNRNPK cells. However, when both HNRNPK and TFAP2C were ablated, the p4EBP1 band is clear cut. I agree with the quantitation that deletion of HNRNPK and TFAP2C both reduce the level of 4EBP1 - the reduction is greater with TFAP2 but when both are deleted together the levels of 4EBP1 are higher and p4EBP1 is clearly present. In quantifying the S6 and pS6 levels, did the authors consider the actin levels- they present a ratio of the pS6 to S6. I may be lacking some understanding but why is the ratio of pS6/S6 being calculated. Is the level of pS6 not what is important - phosphorylation of S6 should lead it to being activated and thus it's the actual level of pS6 that is important, not the ratio to the non-phosphorylated protein. 17. When determining ATP levels, do they control for cell number? HNRNPK depletion results in lower ATP levels, co-deletion of TFAP2C rescues this. But this could be because there is less cell-death? So, more cells express ATP. Have they controlled for relative numbers of cells. 18. The construction of the HovL cell line that propagate ovine prions - very few details are provided of the susceptibility of the cell line to PG127 prions. 19. It is stated that HRNPK depletion from HovL cells increases PrpSC as determined by 6D11 fluorescence, but in the manuscript HRNPK depletion results in cell death. How does this come together? 20. They show that mTOR inhibition mimics the effect of HNRNPK deletion, why didn't they overexpress mTOR and see if that rescues this? This would indicate a causal relationship. 21. Flow cytometric data: supplementary Fig of Fig6d. - when they are looking at fixed cells the gating strategy for cells results in the inclusion of a lot of debris. The gate needs to be moved and be more specific to ensure results are interpreted properly. Same with the singlet gating. It's not tight enough, they include doublets as well which will skew their data. The gating strategy needs to be regated.

      Significance

      The manuscript "Prion propagation is controlled by a hierarchical network involving the nuclear Tfap2c and hnRNP K factors and the cytosolic mTORC1 complex" by Sellitto et al aims to examine how heterogenous nuclear ribonucleoprotein K (hnRNPK), limits pion propagation. They perform a synthetic - viability CRISPR- ablation screen to identify epistatic interactors of HNRNPK. They found that deletion of Transcription factor AP-2 (TFAP2C) suppressed the death of hnRNP-K depleted LN-229 and U-251 MG cells whereas its overexpression hypersensitized them to hnRNP K loss. Moreover, HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism and enhanced autophagy. Simultaneous deletion of TFAP2C reversed these effects, restored transcription and alleviated energy deficiency.

    1. Can help undress herself Drinks from a cup Eats with a spoon with some accuracy Stacks 2-4 objects

      typical skills for which age? Walks alone , May walk up steps and run , Pulls toys while walking , Can help undress self

    1. The Sun had treacherously murdered our people on the twentieth day after the captain left for the coast. We allowed the Captain to return to the city in peace. But on the following day we attacked him with all our might, and that was the beginning of the war.

      The passage says that the Sun killed their people while the captain was away. They let the captain return safely, but the next day they attacked him, which started the war. It shows how anger and revenge led to conflict.

    2. The Spaniards attacked the musicians first, slashing at their hands and faces until they had killed all of them. The singers-and even the spectators- were also killed. This slaughter in the Sacred Patio went on for three hours. Then the Spaniards burst into the rooms of the temple to kill the others: those who were carrying water, or bringing fodder for the horses, or grinding meal, or sweeping, or standing watch over this work.

      The description of the Spaniards attacking the unarmed musicians is a powerful example of total war. I think them targeting the cultural heart of the city especially during a festival, rather than a military battlefield, shows that they were not just fighting for territory, but to also break the social and spiritual spirit of the Aztec people.

    3. When Montezuma had finished, La Malinche translated his address into Spanish so that the Captain could understand it. Cortés replied in his strange and savage tongue, speaking first to La Malinche: “Tell Montezuma that we are his friends. There is nothing to fear. We have wanted to see him for a long time, and now we have seen his face and heard his words. Tell him that we love him well and that our hearts are contented.”

      I think this very crucial because La Malinche becomes the bridge between the two worlds, interpreting for both of them.

    4. No, it is not a dream. I am not walking in my sleep. I am not seeing you in my dreams…. I have seen you at last! I have met you face to face! I was in agony for five days, for ten days, with my eyes fixed on the Region of the Mystery. And now you have come out of the clouds and mists to sit on your throne again.

      It's kind of fascinating and tragic to read his speech where he welcomes Cortés as their lord. It shows that the Aztecs were viewing this even through a prophetic lens, which gave the Spaniards a massive psychological advantage.

    5. Reliable, storable, staple food supplies are a necessary precondition for long-term settlement and population growth – in other words the creation of cities. Like the Europeans, Africans, and Asians, once they had created a reliable food supply, many (not all) American natives built remarkable cities, especially in Central and South America. From present-day Mexico’s Yucatan Peninsula south through Guatemala, the Maya developed a complex society which reached its most intense flourishing from 250 CE to 900 CE. However, the Maya changed their social organization and by the time the Spanish arrived, they were living in more separated independent city-states; seemingly having abandoned some of their more impressive temples and structures such as Chichén Itzá in Yucatan. This led to an interpretation that the original society had suffered a partial collapse sometime around 900 CE due to ecological collapse and/or feuding among these separate cities. More recently, anthropologists have begun to suggest the Maya people may just have wanted to live a lifestyle with less centralized control.

      This shows the development of the civilizations. I think its pretty interesting to see the shift in the Mayans, moving from living in cities independently to even abandoning their places of worship (temples).

    6. Native Americans experienced their own agricultural revolution about the same time as Europeans and Asians, but instead of domesticating cattle, horses, sheep, goats, pigs, and chickens (which were not native to the Americas) they developed three of the world’s current top five staple crops: corn, potatoes, and cassava; as well as additional foods such as hot peppers, tomatoes, beans, cocoa, and tobacco.

      This show how amazing Indigenous people were and how they independently developed major staple crops, proving their agricultural systems were complex and globally significant.

    7. The dancers and singers were completely unarmed. They brought only their embroidered cloaks, their turquoises, their lip plugs, their necklaces, their clusters of heron feathers, their trinkets made of deer hooves. Those who played the drums, the old men, had brought their gourds of snuff and their timbrels.The Spaniards attacked the musicians first, slashing a

      The description of the items the Aztecs brought makes me imagine that they were probably quite valuable. Maybe the Spaniards killed them to steal these items? I would like to figure out if they took treasures after the massacre.

    8. The Spaniards hanged a chief from Acolhuacan named Nezahualquentzin. They also murdered the king of Nauhtla, Cohualpopocatzin, by wounding him with arrows and then burning him alive.

      The Spaniards seem incredibly quick to execute anybody they wanted to. Also, why would they kill these people? What kinds of reasons did they have?

    9. Then the Spaniards burst into the rooms of the temple to kill the others: those who were carrying water, or bringing fodder for the horses, or grinding meal, or sweeping, or standing watch over this work.

      This massacre seemed incredibly brutal and shows the crimes committed by the Spanish against the Aztecs.

    10. The Spaniards attacked the musicians first, slashing at their hands and faces until they had killed all of them. The singers-and even the spectators- were also killed. This slaughter in the Sacred Patio went on for three hours.

      Helps show me the brutal massacre that was done by the Spanish. Knowing that the Aztecs were unarmed and participating in religious celebrations makes it even worse.

    11. You have come back to us; you have come down from the sky.

      The Aztecs used to view Cortès with amazement. People would even say he is Godly figure which helped impact the Spanish's influence.

    12. he king Montezuma, who was accompanied by Itzcohuatzin and by those who had brought food for the Spaniards, protested: “Our lords, that is enough! What are you doing? These people are not carrying shields or macanas. Our lords, they are completely unarmed!”

      It wasn't about anything other than domination, I imagine. So many people died for ideas that just feel broken. I can't imagine what it must have felt like to see it, to watch people bleed and kill for a concept as vague as control.

    13. hey also murdered the king of Nauhtla, Cohualpopocatzin, by wounding him with arrows and then burning him alive.

      I can't imagine why the brutality was so common in these killings. I get that you need to send a message, but this is cruel on every level imaginable.

    14. have met you face to face! I was in agony for five days, for ten days, with my eyes fixed on the Region of the Mystery. And now you have come out of the clouds and mists to sit on your throne again.

      If this is a verified account, I get the impression that Montezuma was a very charismatic speaker. I could listen to this kind of language all day.

    1. incomplete contracts.

      A complete contract is an important concept from contract theory. If the parties to an agreement could specify their respective rights and duties for every possible future state of the world, their contract would be complete. There would be no gaps in the terms of the contract.

      However, because it would be prohibitively expensive to write a complete contract, contracts in the real world are usually incomplete. When a dispute arises and the case falls into a gap in the contract, either the parties must engage in bargaining or the courts must step in and fill in the gap. The idea of a complete contract is closely related to the notion of default rules, e.g. legal rules that will fill the gap in a contract in the absence of an agreed upon provision.

      In contract law, an incomplete contract is one that is defective or uncertain in a material respect. In economic theory, an incomplete contract (as opposed to a complete contract) is one that does not provide for the rights, obligations and remedies of the parties in every possible state of the world.[1]

      Since the human mind is a scarce resource and the mind cannot collect, process, and understand an infinite amount of information, economic actors are limited in their rationality (the limitations of the human mind in understanding and solving complex problems) and one cannot anticipate all possible contingencies.[2][3] Or perhaps because it is too expensive to write a complete contract, the parties will opt for a "sufficiently complete" contract.[4] In short, in practice, every contract is incomplete for a variety of reasons and limitations. The incompleteness of a contract also means that the protection it provides may be inadequate.[5] Even if a contract is incomplete, the legal validity of the contract cannot be denied, and an incomplete contract does not mean that it is unenforceable. The terms and provisions of the contract still have influence and are binding on the parties to the contract. As for contractual incompleteness, the law is concerned with when and how a court should fill gaps in a contract when there are too many or too uncertain to be enforceable, and when it is obliged to negotiate to make an incomplete contract fully complete or to achieve the desired final contract.[1]

    Annotators

    1. Document de Synthèse : Déploiement et Relance de la Démarche « Promeneurs du Net » dans le Nord (59)

      Synthèse Éxécutive

      La démarche Promeneurs du Net (PdN) constitue une extension de l'action éducative en milieu physique vers l'espace numérique.

      Portée par la Caisse d'Allocations Familiales (CAF) du Nord en partenariat avec la Fédération des Centres Sociaux, cette initiative vise à répondre à la présence accrue des jeunes de 12 à 25 ans sur les réseaux sociaux.

      Après une période de mise en veille depuis 2019, le dispositif fait l'objet d'une relance stratégique intégrée à la Convention d'Objectifs et de Gestion (COG) 2023-2027.

      Le déploiement est progressif, ciblant prioritairement les arrondissements d'Avesnes-sur-Helpe, Cambrai, Valenciennes et Douai, avant de s'étendre à Lille et Dunkerque en 2026.

      L'objectif central est de professionnaliser la présence des acteurs de la jeunesse en ligne pour offrir un accompagnement bienveillant, prévenir les risques (cyberharcèlement, infox) et valoriser les compétences numériques des jeunes.

      --------------------------------------------------------------------------------

      1. Contexte et Cadre Institutionnel

      Origines et Évolution

      Historique : Inspirée d'une initiative suédoise des années 2000, la démarche a été introduite en France en 2012 (Manche) avant d'être généralisée par la CNAF en 2017.

      Situation dans le Nord : Déployée entre 2017 et 2019, la démarche a été suspendue avant d'être redynamisée en 2023. Elle s'inscrit désormais dans le Schéma Départemental des Services aux Familles.

      Enjeux de la Branche Famille (2023-2027)

      La branche famille s'engage sur plusieurs axes majeurs :

      1. Structuration de l'offre : Développer un accompagnement adapté aux besoins des adolescents.

      2. Éducation aux médias : Renforcer les compétences critiques des jeunes face aux écrans.

      3. Soutien à la parentalité : Accompagner les parents sur les thématiques des usages numériques.

      Données Nationales de Référence

      Au 31 décembre 2023, le réseau national comptabilisait :

      • Plus de 3 200 Promeneurs du Net actifs.

      • Environ 316 000 jeunes suivis ou accompagnés.

      • Une moyenne de 96 jeunes par professionnel labellisé.

      • Un temps de présence en ligne moyen de 4 heures par semaine.

      --------------------------------------------------------------------------------

      2. La Mission du Promeneur du Net

      Définition et Posture

      Le Promeneur du Net est un professionnel de la jeunesse (animateur, éducateur, conseiller) qui poursuit sa mission éducative sur Internet. Sa présence est :

      Mandatée : Officiellement reconnue et cadrée par l'employeur.

      Bienveillante : Fondée sur l'écoute, le non-jugement et la non-intrusivité.

      Identifiée : Le professionnel utilise des comptes clairement identifiés comme "Promeneur du Net".

      Champs d'Intervention

      | Domaine | Actions spécifiques | | --- | --- | | Lien Social | Favoriser les échanges et la socialisation en ligne. | | Prévention | Veille éducative, lutte contre le cyberharcèlement et la radicalité. | | Information | Diffusion d'informations généralistes ou ciblées (santé, insertion). | | Citoyenneté | Développement de l'esprit critique face aux discours manipulatoires. | | Accompagnement | Soutien aux initiatives de jeunes et aux projets collaboratifs. |

      --------------------------------------------------------------------------------

      3. Modalités Opérationnelles du Réseau

      Public Cible et Structures Éligibles

      Le dispositif s'adresse aux jeunes de 12 à 25 ans. Les structures concernées incluent :

      • Centres sociaux et Espaces de Vie Sociale (EVS).

      • Missions locales et clubs de prévention.

      • Services jeunesse des collectivités territoriales.

      • Associations locales et structures spécialisées (addictions, culture).

      Note : Les bénévoles et les activités à caractère commercial sont strictement exclus.

      Présence Numérique et Réseaux Sociaux

      Initialement centré sur Facebook, le dispositif s'est diversifié pour suivre les usages des jeunes :

      Réseaux prioritaires : Instagram, Snapchat, TikTok.

      Messageries et outils : WhatsApp, Discord.

      Horaires : Environ 30 % des professionnels interviennent sur des horaires atypiques (soirées, week-ends) pour correspondre aux pics de présence des jeunes.

      --------------------------------------------------------------------------------

      4. Organisation Territoriale et Pilotage

      Déploiement Géographique (Nord)

      Le déploiement est organisé en deux phases temporelles :

      1. Phase 1 (En cours) : Arrondissements d'Avesnes-sur-Helpe, Cambrai, Valenciennes et Douai.

      2. Phase 2 (Courant 2026) : Arrondissements de Lille et Dunkerque.

      Coordination Départementale

      La coordination est externalisée auprès de la Fédération des Centres Sociaux. Ses missions sont :

      Accompagnement : Soutien technique et méthodologique quotidien des professionnels.

      Formation : Organisation de la formation initiale et continue.

      Pilotage : Co-animation du projet avec les partenaires institutionnels (CAF, Département, État, MSA).

      Neutralité : La coordination accompagne toutes les structures, qu'elles soient adhérentes ou non à la Fédération.

      --------------------------------------------------------------------------------

      5. Le Parcours de Labellisation

      L'accession au titre de Promeneur du Net suit un protocole rigoureux en cinq étapes :

      1. Candidature : Envoi d'un dossier simplifié (fiche structure et fiche candidat) et signature de la Charte Promeneur du Net.

      2. Commission de Labellisation : Examen du dossier par un comité technique (CAF, État, Département, MSA, Fédération).

      3. Formation Initiale : Participation obligatoire à une journée de formation (posture éducative, outils, réseau).

      4. Entretien de Mise en Place : Échange sur site entre la coordination, le professionnel et la direction de la structure (environ 4h) pour valider les moyens matériels et le temps dédié.

      5. Labellisation Officielle : Création des comptes professionnels, définition de la ligne éditoriale et inscription sur la cartographie nationale.

      Cas particulier des "PS Jeune"

      Pour les structures bénéficiant d'un agrément Prestation de Service (PS) Jeune, l'inscription dans la démarche Promeneur du Net est une obligation contractuelle mentionnée dans le cahier des charges national.

      Cette participation sera une condition examinée lors du renouvellement des agréments.

      --------------------------------------------------------------------------------

      6. Soutien et Animation du Réseau

      La coordination propose plusieurs outils pour rompre l'isolement du professionnel :

      Espaces de discussion : Utilisation d'outils collaboratifs (type Mattermost ou Discord) pour l'échange de pratiques.

      Points du Net : Webinaires et conférences thématiques (8 par an) sur des sujets comme l'intelligence artificielle, la protection des données ou la radicalité en ligne.

      Rencontres physiques : Deux temps d'échange de pratiques par an en présentiel.

      Évaluation annuelle : Suivi de l'activité via un outil simplifié pour recenser le nombre de jeunes contactés et les problématiques rencontrées.

      --------------------------------------------------------------------------------

      7. Calendrier et Échéances (Session 2024)

      Dépôt des dossiers : Jusqu'au 6 février pour la prochaine salve.

      Commission de labellisation : Fixée au 2 mars.

      Dates de formation initiale :

      ◦ 27 mars 2024.     ◦ 15 mai 2024.

      Fréquence des commissions : Une instance de labellisation se réunit mensuellement pour assurer un traitement fluide des candidatures "au fil de l'eau".

    1. Synthèse de la Conférence de Pierre Périer : Les Enjeux de la Coéducation et du Lien École-Famille

      Ce document de breffage synthétise les interventions de Pierre Périer, sociologue et professeur en sciences de l’éducation, lors de sa conférence sur les relations entre l’école et les familles, particulièrement au sein des quartiers populaires et en contexte de précarité.

      Résumé Exécutif

      La réussite du plus grand nombre d’élèves dépend d’un enjeu majeur : la construction d’un lien solide et cohérent entre l’école et les familles.

      Pierre Périer démontre que si la « coéducation » est devenue un mot d’ordre institutionnel, sa mise en œuvre se heurte à des obstacles structurels, symboliques et sociaux.

      Les familles les plus précaires, souvent qualifiées d’« invisibles », ne sont pas démissionnaires mais se trouvent disqualifiées par des règles du jeu scolaire dont elles ne maîtrisent pas les codes.

      Pour réussir cette alliance, l’institution doit passer d’une logique descendante de « formatage » des parents à une logique de reconnaissance des « parents réels », en s'appuyant sur des médiateurs tiers et en rendant les attentes scolaires explicites.

      --------------------------------------------------------------------------------

      I. Les Défis Majeurs de l’École et de la Société

      L'intérêt croissant pour le lien école-famille s'inscrit dans un contexte de défis sociétaux profonds que l’école ne peut résoudre seule.

      La lutte contre l’échec scolaire : L'objectif est d'assurer la réussite du plus grand nombre et d'éviter que l'échec ne touche systématiquement les mêmes catégories sociales.

      L'échec scolaire a des conséquences lourdes sur l'identité et l'insertion des jeunes.

      L'exigence d'équité : L'école doit devenir plus juste vis-à-vis de la diversité des élèves.

      La gestion de la diversité : L'école fait face à une hétérogénéité croissante (origines, trajectoires, formes familiales).

      Cette complexité nécessite une meilleure connaissance des familles par l'institution.

      La quête de sens : Le lien école-famille est le levier de la « mobilisation scolaire ».

      Si l’enfant perçoit une continuité et une cohérence entre sa famille et sa classe, il donne plus de sens aux savoirs et persévère davantage.

      II. Clarification des Concepts de Collaboration

      Pierre Périer souligne la nécessité de définir les termes utilisés pour éviter qu'ils ne deviennent des évidences non questionnées (une « doxa »).

      | Terme | Définition et Enjeux | | --- | --- | | Coéducation | Finalité reposant sur une responsabilité partagée dans l'éducation et la réussite de l'enfant. | | Coopération | Méthode basée sur l'action réciproque : l'action de l'un doit renforcer l'action de l'autre. Cela suppose de connaître précisément ce que fait le partenaire. | | Collaboration | Fait de « faire ensemble » avec des moyens qui peuvent être différents pour atteindre un objectif énoncé. | | Alliance éducative | Terme récent soulignant la nécessité de construire un front commun entre divers acteurs. |

      Note cruciale : La coéducation ne signifie pas que les parents et les enseignants doivent faire la même chose ou agir à parts égales. Elle nécessite une division du travail éducatif claire et explicitée.

      III. Les Obstacles à la Relation : Le Paradoxe des « Parents Invisibles »

      L’analyse sociologique révèle que la difficulté de liaison provient souvent de la nature même de l’institution scolaire.

      1. Une asymétrie structurelle

      C’est l’institution scolaire qui définit seule les règles du jeu, les modalités de rencontre et l’image du « bon parent ».

      Ce schéma descendant exclut ceux qui n’ont pas les ressources pour s'y conformer.

      2. Des barrières symboliques et pratiques

      Le seuil de l'école : Le portail représente une frontière symbolique.

      En le franchissant, l'individu passe du statut de « parent » à celui de « parent d'élève », un rôle normé par l'école.

      Le rapport au temps et à la langue : Les réunions et les prises de rendez-vous supposent une familiarité avec les usages sociaux de l'école.

      Pour beaucoup de parents vulnérables, prendre rendez-vous est une démarche intimidante qui nécessite de se sentir légitime.

      La peur de l'intrusion : Les familles les plus précaires redoutent que l'école soit intrusive dans leur vie privée ou que leur parole ne les discrédite (sentiment de honte ou d'ignorance).

      3. Les attentes normatives

      L'école impose des normes (ex: l'aide aux devoirs) qui renforcent les inégalités.

      Demander aux parents de superviser les devoirs favorise les familles dotées de capital culturel et pénalise celles dont les parents ont eu une scolarité courte ou douloureuse.

      IV. Le Rôle des Tiers et des Médiateurs

      Face à l'impossibilité pour l'école de tout résoudre seule, les acteurs socio-éducatifs et culturels du territoire jouent un rôle de « pont ».

      Créer un maillage territorial : Aucun parent ne doit rester isolé.

      Les structures de quartier permettent une « capillarité » sociale reliant les familles à l'institution par des voies détournées.

      L’effet Pygmalion : Les acteurs tiers peuvent renvoyer une image positive aux jeunes qui doutent de leurs capacités.

      En valorisant d'autres compétences, ils aident l'élève à reprendre confiance et à redonner du sens à sa scolarité.

      L'émancipation : Ces médiations permettent aux jeunes et aux parents de « s'autoriser à être différents » de l'image d'échec que l'institution peut parfois leur renvoyer.

      V. Principes pour une Action de Coéducation Réussie

      Pierre Périer propose plusieurs principes directeurs pour transformer les pratiques de terrain :

      1. Interconnaissance et Reconnaissance : Il est crucial de se connaître entre acteurs (qui fait quoi ?).

      Un premier contact positif et non scolaire dès le mois de septembre est essentiel pour bâtir une base de confiance avant l'émergence d'éventuels problèmes.

      2. Légitimation et Autorisation : Il faut faire des parents des « auteurs » et non de simples « acteurs » de projets.

      Cela implique de partir de ce qu'ils proposent (les « parents réels ») plutôt que d'attendre qu'ils s'adaptent à un cadre pré-établi.

      3. Explicitation : « Plus c’est explicite, plus c’est démocratique ».

      L'absence de clarté favorise la « connivence culturelle » entre l'école et les classes moyennes, au détriment des classes populaires.

      4. Acceptation du conflit : Le désaccord ne doit pas être évité par un « faux consensus ».

      Le conflit, s'il est exprimé et écouté dans un cadre protégé, peut être « socialisateur » et permettre de dégager des solutions nouvelles et partagées.

      5. Accompagnement plutôt que formatage : L'objectif ne doit pas être de « former » les parents (ce qui renforce l'asymétrie), mais de les accompagner en s'appuyant sur leurs ressources propres.

      VI. Exemples de Dispositifs Inspirants

      Le document mentionne plusieurs initiatives concrètes favorisant le lien :

      Groupes de parole (type ATD Quart Monde) : Espaces où la parole des parents est protégée et écoutée, permettant de sortir de l'isolement.

      Espaces Parents et Cafés des Parents : Lieux d'information et d'échange dans l'école, dont les parents peuvent s'approprier le fonctionnement.

      Ouvrir l'école aux parents pour la réussite des enfants (OEPRE) : Dispositif permettant aux parents primo-arrivants d'apprendre le français et le fonctionnement de l'école, favorisant leur autonomisation et leur pouvoir d'agir.

      Classes Passerelles : Facilitent la transition entre la petite enfance et la maternelle par un accueil conjoint des mères et des enfants.

      Actions de transition : Importance du travail sur le passage de l'élémentaire au collège, période où les inégalités se creusent brutalement et où le lien avec les familles se fragilise.

      Conclusion

      La coéducation est un processus complexe qui exige de rompre avec l'image du parent « démissionnaire » pour comprendre les obstacles réels à l'implication.

      La réussite de ce lien repose sur la capacité de l'école et de ses partenaires territoriaux à reconnaître la place de chaque parent, à expliciter les codes scolaires et à construire une confiance mutuelle dès le début du parcours de l'enfant.

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      Summary:

      The study provides a comprehensive overview of genome size variation in two related species of the genus Epidendrum, which appear to be homoploid, although their DNA content more closely corresponds to that of heteroploid species. While I have a few serious concerns regarding the data analysis, the study itself demonstrates a well-designed approach and offers a valuable comparison of different methods for genome size estimation. In particular, I would highlight the analysis of repetitive elements, which effectively explains the observed differences between the species. However, I encourage the authors to adopt a more critical perspective on the k-mer analysis and the potential pitfalls in data interpretation.

      Major comments:

      R1. p. 9: Genome size estimation via flow cytometry is an incorrect approach. The deviation is approximately 19% for E. anisatum and about 25% for E. marmoratum across three repeated measurements of the same tissue over three days? These values are far beyond the accepted standards of best practice for flow cytometry, which recommend a maximum deviation of 2-5% between repeated measurements of the same individual. Such variability indicates a systemic methodological issue or improper instrument calibration. Results with this level of inconsistency cannot be considered reliable estimates of genome size obtained by flow cytometry. If you provide the raw data, I can help identify the likely source of error, but as it stands, these results are not acceptable.

      __A: __Thanks a lot for pointing out this issue. We have identified the source of the wide interval after consulting with the staff of LabNalCit. We originally used human peripheral blood mononuclear cells (PBMCs) as a reference to estimate the genome size (GS) of P. sativum and used the resulting range to estimate the GS of Epidendrum. We calculated P. sativum's GS using a wide human GS range of 6-7 Gb, which resulted in a wide range of P. sativum GS and, consequently, in a wide range of GS for our samples. Therefore, the wide range reported is not an issue with the instruments, but about the specifics of the analysis.

      __We have done the following changes: __

      1. Reducing the range we calculated of P. sativum's GS using a narrower human genome size range (6.41-6.51; Piovesan et al. 2019; DOI: 10.1186/s13104-019-4137-z), and using these intervals to calculate our sample's GS.
      2. We have explained our procedure in the methods, changed our results as required, and included a supplementary table with cytometry data (Supplementary Data Table 1).
      3. Human peripheral blood mononuclear cells (PBMCs) from healthy individuals were used as a standard laboratory reference to calculate the P. sativum genome size. Pisum sativum and the Epidendrum samples were analyzed in a CytoFLEX S flow cytometer (Beckman-Coulter), individually and in combination with the internal references (PBMCs and P. sativum, respectively). Cytometry data analysis was performed using FlowJo® v. 10 (https://www.flowjo.com/). A genome size value for the Epidendrum samples was calculated as the average of the minimum and maximum 1C/2C values obtained from three replicates of the DNA content histograms of each tissue sample. Minimum and maximum values come from the interval of P. sativum estimations based on the human genome size range (human genome size range: 6.41-6.51; Piovesan et al. 2019).
      4. The 1C value in gigabases (Gb; calculated from mass in pg) of E. anisatum ranged from 2.55 to 2.62 Gb (mean 1C value = 2.59 Gb) and that of E. marmoratum from 1.11 to 1.18 Gb (mean 1C value = 1.13 Gb; Supplementary Data Table S1).
      5. We also eliminated from Figure 3 the range we had estimated previously.
      6. Finally, we changed the focus of the comparison and discussion of the evaluation of the bioinformatic estimations, highlighting this deviation rather than whether the GS bioinformatic estimations fall within the cytometric interval. We calculated the Mean Absolute Deviation (MAD) as the absolute difference between the genome size estimates using k-mers and flow cytometry. This meant changing the results in P. 11 and 12 and adding to Fig. 3 two boxplots depicting the MAD. We have also added Supplementary Data Fig. S3 depicting the absolute deviations for E. anisatum and E. marmoratum per tool using the estimates generated from a k-mer counting with a maximum k-mer coverage value of 10,000 using 16 different values of k; a Supplementary Data Figure S5 depicting the mean absolute deviations resulting from the different subsampled simulated depths of coverage of 5×, 10×, 20×, 30×, and 40×; and finally a Supplementary Data Fig. S6 depicting the MAD changes as a function of depth of coverage for E. anisatum and E. marmoratum.

      R1. p. 14 and some parts of Introduction: It may seem unusual, to say the least, to question genome size estimation in orchids using flow cytometry, given that this group is well known for extensive endoreplication. However, what effect does this phenomenon have on genome size analyses based on k-mers, or on the correct interpretation of peaks in k-mer histograms? How can such analyses be reliably interpreted when most nuclei used for DNA extraction and sequencing likely originate from endoreplicated cells? I would have expected a more detailed discussion of this issue in light of your results, particularly regarding the substantial variation in genome size estimates across different k-mer analysis settings. Could endoreplication be a contributing factor?

      A:

      We reworded the introduction p.3, 2nd paragraph to make our point on the effect of endoreplication on flow cytometry clearer. We eliminated the following sentence from discussion p. 15 : "Difficulties for cytometric estimation of genome size can thus be taxon-specific. Therefore, cross-validating flow cytometry and bioinformatics results can be the most effective method for estimating plant genome size, especially when only tissues suspected to show significant endoreplication, such as leaves, are available" We added the following, p. 18: Genome size estimation for non-model species is considered a highly standardized approach. However, tissue availability and intrinsic genome characteristics (large genomes, polyploidy, endoreplication, and the proportion of repetitive DNA) can still preclude genome size estimation (e.g. Kim et al. 2025) using cytometry and bioinformatic tools. Cross-validating flow cytometry and bioinformatics results might be particularly useful in those cases. For example, when only tissues suspected of showing significant conventional endoreplication, such as leaves, are available, bioinformatic tools can help to confirm that the first peak in cytometry histograms corresponds to 2C. Conversely, bioinformatic methods can be hindered by partial endoreplication, which only flow cytometry can detect.

           4. We included a paragraph discussing the effect of CE and PE on bioinformatic GS estimation P. 17:
      

      Besides ploidy level, heterozygosity, and the proportion of repetitive DNA, k-mer distribution can be modified by endoreplication. Since endoreplication of the whole genome (CE) produces genome copies (as in preparation for cell division, but nuclear and cell division do not occur ), we do not expect an effect on genome size estimates based on k-mer analyses. In contrast, PE alters coverage of a significant proportion of the genome, affecting k-mer distributions and genome size estimates (Piet et al., 2022). Species with PE might be challenging for k-mer-based methods of genome size estimation.

      R1. You repeatedly refer to the experiment on genome size estimation using analyses with maximum k-mer coverage of 10,000 and 2 million, under different k values. However, I would like to see a comparison - such as a correlation analysis - that supports this experiment. The results and discussion sections refer to it extensively, yet no corresponding figure or analysis is presented.

      A:

      We had previously included the results of the analyses using different k-mer coverage in the Supplementary Data Figure S2. We have added, to formally compare the results using analyses with maximum k-mer coverage of 10,000 and 2 million, a Wilcoxon paired signed-rank test, which showed a significant difference, p. 12: The estimated genome sizes using a maximum count value of 10,000 were generally lower for all tools in both species compared to using a maximum count value of 2 million (median of 2M experiment genome size - median of 10K experiment genome size= 0.24 Gb). The estimated genome size of the 2 million experiment also tended to be closer to the flow cytometry genome size estimation with significantly lower MAD than the 10K experiment (Wilcoxon paired signed-rank test p = 0.0009). In the 10K experiment (Supplementary Data Figure S2; S3), the tool with the lowest MAD for E. anisatum was findGSE-het (0.546 Gb) and for E. marmoratum it was findGSE-hom (0.116 Gb).

       2. We have added a boxplot in the Supplementary Data Figure S3 depicting the mean absolute deviations using maximum k-mer coverage of 10,000 and 2 million compared to flow cytometry.
      

      Minor comments:

      R1. p. 3: You stated: "Flow cytometry is the gold standard for genome size estimation, but whole-genome endoreplication (also known as conventional endoreplication; CE) and strict partial endoreplication (SPE) can confound this method." How did you mean this? Endopolyploidy is quite common in plants and flow cytometry is an excellent tool how to detect it and how to select the proper nuclei fraction for genome size estimation (if you are aware of possible misinterpretation caused by using inappropriate tissue for analysis). The same can be applied for partial endoreplication in orchids (see e.g. Travnicek et al 2015). Moreover, the term "strict partial endoreplication" is outdated and is only used by Brown et al. In more recent studies, the term partial endoreplication is used (e.g. Chumova et al. 2021- 10.1111/tpj.15306 or Piet et al. 2022 - 10.1016/j.xplc.2022.100330).

      A:

      We have reworded the paragraph where we stated "Flow cytometry is the gold standard for genome size estimation", as in the answer to Major comment 2. Additionally, we highlighted in the discussion how, while FC is the gold standard for GS estimation, studying multiple alternatives to it may be important for cases in which live tissue is not available or is available only to a limited extent (i.e. only certain tissues), p. 18 We have changed the term "strict partial endoreplication" to partial endoreplication (PE).

      R1. p. 5: "...both because of its outstanding taxic diversity..." There is no such thing as "taxic" diversity - perhaps you mean taxonomic diversity or species richness.

      __A: __We have changed "taxic diversity" to "species diversity".

      R1. p. 6: In description of flow cytometry you stated: "Young leaves of Pisum sativum (4.45

      pg/1C; Doležel et al. 1998) and peripheral blood mononuclear cells (PBMCs) from healthy

      individuals...". What does that mean? Did you really use blood cells? For what purpose?

      A: Please find the explanation and the modifications we've made in the answer to major comment 1.

      R1. p. 7: What do you mean by this statement "...reference of low-copy nuclear genes for each species..."? As far as I know, the Granados-Mendoza study used the Angiosperm v.1 probe set, so did you use that set of probes as reference?

      __A: __We rewrote: "To estimate the allele frequencies, the filtered sequences were mapped to a

      reference of low-copy nuclear genes for each species" to:

      To estimate the allele frequencies, the filtered sequences were mapped to the Angiosperm v.1 low-copy nuclear gene set of each species.

      R1. p. 7: Chromosome counts - there is a paragraph of methodology used for chromosome counting, but no results of this important part of the study.

      A: We are including a supplementary figure (Supplementary Data Figure 7) with micrographs of the chromosomes of E. anisatum and E. marmoratum.

      R1. p. 12: Depth of coverage used in repeatome analysis - why did you use different coverage for both species? Any explanation is needed.

      A: To make explicit the fact that the depth of coverage is determined automatically by the analysis with no consideration for the amount of input reads, but only of the graph density and the amount of RAM available (Box 3 in Novak et al. 2020), we rewrote:

      "To estimate the proportion of repetitive DNA, the individual protocol analyzed reads corresponding to depths of coverage of 0.06× for Epidendrum anisatum and 0.43× for E. marmoratum." to

      To estimate the proportion of repetitive DNA, the RepeatExplorer2 individual protocol determined a max number of analyzed reads (Nmax) corresponding to depths of coverage of 0.06x for Epidendrum anisatum and 0.43x for E. marmoratum.

      R1. p. 16: The variation in genome size of orchids is even higher, as the highest known DNA amount has been estimated in Liparis purpureoviridis - 56.11 pg (Travnicek et al 2019 - doi: 10.1111/nph.15996)

      A: We have updated it.

      R1. Fig. 1 - Where is the standard peak on Fig. 1? You mention it explicitly on page 9 where you are talking about FCM histograms.

      A: We reworded the results, eliminating the references to the standard internal reference.

      Reviewer #1 (Significance (Required)):

      Significance

      This study provides a valuable contribution to understanding genome size variation in two Epidendrum species by combining flow cytometry, k-mer analysis, and repetitive element characterization. Its strength lies in the integrative approach and in demonstrating how repetitive elements can explain interspecific differences in DNA content. The work is among the first to directly compare flow cytometric and k-mer-based genome size estimates in orchids, extending current knowledge of genome evolution in this complex plant group. However, the study would benefit from a more critical discussion of the limitations and interpretative pitfalls of k-mer analysis and from addressing methodological inconsistencies in the cytometric data. The research will interest a specialized audience in plant genomics, cytogenetics, and genome evolution, particularly those studying non-model or highly endoreplicated species.

      Field of expertise: plant cytogenetics, genome size evolution, orchid genomics.

      Reviewer #2 (Evidence, reproducibility and clarity (Required)):

      Summary:

      With this work, the authors provide genome profiling information on the Epidendrum genus. They performed low-coverage short read sequencing and analysis, as well as flow cytometry approaches to estimate genome size, and perform comparative analysis for these methods. They also used the WGS dataset to test different approaches and models for genome profiling, as well as repeat abundance estimation, empathising the importance of genome profiling to provide basic and comparative genomic information in our non-model study species. Results show that the two "closely-related" Epidendrum species analysed (E. marmoratum and E. anisatum) have different genome profiles, exhibiting a 2.3-fold genome size difference, mostly triggered by the expansion of repetitive elements in E. marmoratum, specially of Ty3-Gypsy LTR-retrotransposon and a 172 tandem repeat (satellite DNA).

      Major comments:

      Overall, the manuscript is well-written, the aim, results and methods are explained properly, and although I missed some information in the introduction, the paper structure is overall good, and it doesn't lack any important information. The quality of the analysis is also adequate and no further big experiments or analysis would be needed.

      However, from my point of view, two main issues would need to be addressed:

      __R2. __The methods section is properly detailed and well explained. However, the project data and scripts are not available at the figshare link provided, and the BioProject code provided is not found at SRA. This needs to be solved as soon as possible, as if they're not available for review reproducibility of the manuscript cannot be fully assessed.

      __A: __We have made public the .histo files for all depths of coverage and cluster table files necessary to reproduce the results. We will also make public a fraction of the sequencing sufficient to reproduce our genome size and repetitive DNA results as soon as the manuscript is formally published. Whole dataset availability will be pending on the publication of the whole genome draft.

      R2. The authors specify in the methods that 0.06x and 0.43x sequencing depths were used as inputs for the RE analysis of E. anisatum and E. marmoratum. I understand these are differences based on the data availability and genome size differences. However, they don't correspond to either of the recommendations from Novak et al (2020):

      In the context of individual analysis: "The number of analyzed reads should correspond to 0.1-0.5× genome coverage. In the case of repeat-poor species, coverage can be increased up to 1.0-1.5×." Therefore, using 0.06x for E. anisatum should be justified, or at least addressed in the discussion.

      Moreover, using such difference in coverage might affect any comparisons made using these results. Given that the amount of reads is not limiting in this case, why such specific coverages have been used should be discussed in detail.

      In the context of comparative analysis: "Because different genomes are being analyzed simultaneously, the user must decide how they will be represented in the analyzed reads, choosing one of the following options. First, the number of reads analyzed from each genome will be adjusted to represent the same genome coverage. This option provides the same sensitivity of repeat detection for all analyzed samples and is therefore generally recommended; however, it requires that genome sizes of all analyzed species are known and that they do not substantially differ. In the case of large differences in genome sizes, too few reads may be analyzed from smaller genomes, especially if many species are analyzed simultaneously. A second option is to analyze the same number of reads from all samples, which will provide different depth of analysis in species differing in their genome sizes, and this fact should be considered when interpreting analysis results. Because each of these analysis setups has its advantages and drawbacks, it is a good idea to run both and cross-check their results."

      Therefore, it should be confirmed how much it was used for this approach (as in the methods it is only specified how much it was used for the individual analysis), and why.

      __A: __In Box 3, Novak et al (2020) explain that the number of analyzed reads (Nmax) is determined automatically by RepeatExplorer2, based on the graph density and available RAM. Therefore, the reported depths of coverage are results, not the input of the analysis. We tried different amounts of reads as input and got consistently similar results, so we kept the analysis using the whole dataset.

      For the comparative analysis, we have added the resulting depth of coverage and explained that we used the same number of reads for both species.

      Added to methods:

      "For the comparative protocol, we used the same amount of reads for both species".

      Added to results:

      "To estimate the proportion of repetitive DNA, the RepeatExplorer2 individual protocol determined a maximum number of analyzed reads (Nmax) corresponding to depths of coverage of 0.06x for E. anisatum and 0.43x for E. marmoratum. "

      "The RepeatExplorer2 comparative protocol determined a maximum number of analyzed reads (Nmax) corresponding to depths of coverage of approximately 0.14x for E. marmoratum and 0.06x for E. anisatum"

      This is consistent with other works which utilize RepeatExplorer2, for example, Chumová et al (2021; https://doi.org/10.1111/tpj.15306), who wrote: "The final repeatome analysis for each species was done using a maximum number of reads representing between 0.049x and 1.389x of genome coverage."

      Minor comments:

      General comments:

      • The concept of genome endoreplication and the problem it represents for C-value estimations needs to be better contextualised. It would be nice to have some background information in the introduction on how this is an issue (specially in Orchid species). Results shown are valuable and interesting but require a little more context on how frequent this is in plants, especially in Orchids, and across different tissues.

      __A: __We have included information about the variation of conventional and partial endoreplication in plants.

      Differences in CE may also occur between individuals or even respond to environmental factors (Barow 2006). In contrast, PE results in cells that replicate only a fraction (P) of the genome (Brown et al. 2017) and it has only been reported in Orchidaceae (Brown et al. 2017). CE and PE can occur in one or several endoreplication rounds, and different plant tissues may have different proportions of 2C, 4C, 8C ... nC or 2C, 4E, 8E, ... nE nuclear populations, respectively. The 2C nuclear population sometimes constitutes only a small fraction in differentiated somatic tissues and can be overlooked by cytometry (Trávníček et al. 2015). Using plant tissues with a high proportion of the 2C population (such as orchid ovaries and pollinaria) can help overcome this difficulty (Trávníček et al. 2015; Brown et al. 2017).

      Comments and suggestions on the figures:

      __R2. __In fig 1, the flow cytometry histograms need to be more self-explanatory. What are the Y axis "counts" of? Also, please either place the label for both rows or for each, but don't make it redundant. The axis fonts need to be made a bit larger too. If possible, explain briefly in the figure legend (and not only in the text) what each peak means.

      __A: __We have modified the figure adding legends for Y and X axes, eliminated redundant labels, and changed the font size.

      __R2. __Fig 5. Horizontal axis labels are illegible. Please make these larger (maybe make the plot wider by moving the plot legend to the top/bottom of the figure? - just a suggestion).

      __A: __We consider the horizontal axis label to be superfluous and we removed it.

      Small text editing suggestions:

      R2. Methods, "Ploidy level estimation and chromosome counts" section. It would be easier for the reader if this paragraph were either divided into two methods sections, or into two paragraphs at least, since these are two very different approaches and provide slightly different data or information.

      A: We slightly modified: "Chromosome number was counted from developing root tips" to

      "Additionally, to confirm ploidy level, chromosome number was counted from developing root tips" and changed the subtitle to only "Ploidy level estimation".

      R2. Methods, "Genome size estimation by k-mer analysis" section. Please specify whether the coverage simulations (of 5x to 40x) were made based on 1c or 2c of the genome size? I assumed haploid genome size but best to clarify.

      A: We have added it to P7: "To assess the suitability of the whole dataset and estimate the minimum coverage required for genome size estimation, the depth of coverage of both datasets was calculated based on the flow cytometry 1C genome size values."

      R2. Results, "Genome size estimation by k-mer analysis and ploidy estimation" section. In the first two paragraphs, the results presented appear to conform to anticipated patterns based on known properties of these types of datasets. Although this information confirms expected patterns, it does not provide new or biologically significant insights into the genomes analysed. It may be beneficial to further summarize these paragraphs so that the focus of this section can shift toward the comparison of methods and the biological interpretation of the genome profiles of Epidendrum.

      __A: __We agree that those paragraphs deviate a little from the focus of our results. However, we believe they provide useful information both for pattern confirmation in a relatively understudied field and for readers which may not be very familiar with the methods utilized.

      __R2. __Discussion, "Genome size estimation using flow cytometry" section. In the second paragraph, it is discussed how potential endoduplication events can "trick" the flow cytometry measurements. This has probably previously been discussed on other C-value calculation studies and would benefit from context from literature. How does this endoduplication really affect C-value measurements across plant taxa? I understand it is a well-known issue, so maybe add some references?

      A: We have included in the Introduction information about CE and PE and their associated references. P. 3 and 4.

      __R2. __Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. In the second paragraph, when mentioning the relative abundance of Ty3-gypsy and Ty1-copia elements, it is also worth mentioning their differences in genomic distribution and the potential structural role of Ty3-gypsy elements.

      A: We added this paragraph in P.20:

      "Ty3-gypsy elements are frequently found in centromeric and pericentromeric regions, and may have an important structural role in heterochromatin (Jin et al. 2004; Neumann et al. 2011; Ma et al. 2023), particularly those with chromodomains in their structure (chromovirus, i.e. Tekay, CRM transposons; Neumann et al. 2011). Conversely, Ty1-copia elements tend to be more frequent in gene-rich regions (Wang et al. 2025A). However, Ty3-gypsy chromovirus elements can be found outside the heterochromatin regions (Neumann et al. 2011), and in Pennisetum purpureum (Poaceae) Ty1-copia elements are more common in pericentromeric regions (Yu et al. 2022)."

      R2. Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. In the third paragraph, it is mentioned that both species have 2n=40. I believe these are results from this work since there is a methods section for chromosome counting. This data should therefore go into results.

      __A: __We have added the chromosome count micrographs as Supplementary Data Fig. S7

      R2. Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. I'd recommend expanding a bit more on repetitive DNA differences based on the RepeatExplorer results. Providing references on whether this has been found in other taxa would be helpful too. For example, Ogre bursts have been previously described in other species (e.g. legumes, Wang et al., 2025). Moreover, I consider worth highlighting and discussing other interesting differences found, such as the differences in unknown repeats (could be due to one species having "older" elements- too degraded to give any database hits- compared to the other), or Class II TE differences between species (and how these account less for genome size difference because of their size), etc.

      A: We have rearranged and added discussion expanding on the role of repetitive DNA in E. anisatum and E. marmoratum and how it relates to the repetitive DNA in other species. This includes Ogre transposons, an expanded Ty1-copia vs. Ty3-gypsy discussion, and a section on unclassified repeats and can be found on P.19 to P.21.

      Reviewer #2 (Significance (Required)):

      Overall, this study provides a valuable contribution to our understanding of genome size diversity and repetitive DNA dynamics within Epidendrum, particularly through its combined use of low-coverage sequencing, flow cytometry, and comparative genome profiling. Its strongest aspects lie in the clear methodological framework and the integration of multiple complementary approaches, which together highlight substantial genome size divergence driven by repeat proliferation-an insight of clear relevance for orchid genomics and plant genome evolution more broadly.

      While the work would benefit from improved data availability, additional contextualization of the problem of endoreduplication in flow cytometry, and clarification of some figure elements and methodological details, the study nonetheless advances the field by presenting new comparative genomic information for two understudied species and by evaluating different strategies for genome profiling in non-model taxa.

      The primary audience will include researchers in non-model plant genomics, cytogenetics, and evolutionary biology, although the methodological comparisons may also be useful to a broader community working on genome characterization in diverse lineages. My expertise is in plant genomics, genome size evolution, and repetitive DNA biology; I am not a specialist in flow cytometry instrumentation or cytological methods, so my evaluation of those aspects is based on general familiarity rather than technical depth.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      A review on "Nuclear genome profiling of two Mexican orchids of the genus Epidendrum" by Alcalá-Gaxiola et al. submitted to ReviewCommons

      The present manuscript presented genomic data for two endemic Maxican orchids: Epidendrum anisatum and E. marmoratum. Authors aim to determine the genome size and ploidy using traditional (flow cytometry and chromosome counts) and genomic techniques (k-mer analysis, heterozygosity), along with the repetitive DNA composition characterization.

      Considering the genomic composition, the main difference observed in repeat composition between the two species was attributed to the presence of a 172 bp satDNA (AniS1) in E. anisatum, which represents about 11% of its genome but is virtually absent in E. marmoratum. The differences in the genomic proportion of AniS1 and Ty3-gypsy/Ogre lineage TEs between E. anisatum and E. marmoratum are suggested as potential drivers of the GS difference identified between the two species.

      Our main concern are about the GS estimation and chromosome number determination. Along with many issues related to GS estimations by flow cytometry, results related to chromosome number determination are missing on the manuscript. Improvements in both techiniques and results are crucial since authors aim to compare different methods to GS and ploidy determination.

      __R3. __Genome size: Following the abstract, it is no possible to understand that authors confirm the GS by flow cytometry - as clarified after on the manuscript. Please, since the approach used to obtain the results are crucial on this manuscript, make it clear on the abstract.

      A: We have highlighted the congruence of flow cytometry and bioinformatic approaches in the abstract:

      "Multiple depths of coverage, k values, and k-mer-based tools for genome size estimation were explored and contrasted with cytometry genome size estimations. Cytometry and k-mer analyses yielded a consistently higher genome size for E. anisatum (mean 1C genome size = 2.59 Gb) than * E. marmoratum* (mean 1C genome size = 1.13 Gb), which represents a 2.3-fold genome size difference."

      __R3.__Flow cytometry methodology: For a standard protocol, it is mandatory to use, at least, three individuals, each one analyzed on triplicate. Is is also important to check the variation among measurements obtained from the same individual and the values obtained from different individuals. Such variation should be bellow 3%. The result should be the avarege C-value following the standard deviation, what inform us the variation among individuals and measurements.

      __A: __We have done three technical replicates of each tissue of the individuals of E. anisatum and E. marmoratum. To show the variation from different replicates and tissues, we have included the Supplementary Data Table S1. Intraspecific variation on genome size is beyond the scope of this work.

      __R3. __Checking Fig. 1, we could not see the Pisum peack. If authors performed an analysis with external standart, it should be clarified on Methods. I suggest always use internal standard.

      Besides, comparing Fig. 1 for leave and pollinium, it seems to be necessary to set up the Flow Cytoemtry equipament. Note that the 2C peack change its position when comparing different graphs. The data could be placed more central on x-axis by setting the flow cytometry.

      Action Required: Considering that authors want to compare indirect genomic approaches to determine the GS, I suggest authors improve the GS determination by Flow Cytometry.

      Please, on Methodology section, keep both techniques focused on GS close one another. Follow the same order on Methodology, Results and Discussion sections.

      __A: __We have made several changes on the estimation and reporting of the flow cytometry genome size estimation. Among these:

      We have clarified the use of the P. sativum internal standard and PBMC's in methods (P.6). We have added the associated mean coefficient of variation for both the sample and the internal reference in Supplementary Data Table S1, in order to show that the variation is not the result of an instrument error. We have changed the order of the paragraphs in the methods section to follow the order in other sections.

      __R3. __Chromosome count: In Introduction section (page 5), the authors explicitly aim to provide "bioinformatics ploidy level estimation and chromosome counting." Furthermore, the Methods section (page 7, subsection "Ploidy level estimation and chromosome counts") details a specific protocol for chromosome counting involving root tip pretreatment, fixation, and staining. However, no results regarding chromosome counting are presented in the manuscript. There are no micrographs of metaphase plates, no tables with counts, and no mention of the actual counts in the Results section or Supplementary Material. Despite this absence of evidence, the Discussion (Page 18) states: "ploidy and chromosome counts of both E. anisatum and E. marmoratum are the same (2n=40)." The value of 2n=40 is presented as a finding of this study, however, there is no reference to this results.

      Action Required: The authors must resolve this discrepancy by either providing the missing empirical data (micrographs and counts). This detail needs to be reviewed with greater care and scientific integrity.

      __A: __We have added the chromosome count micrographs as Supplementary Data Fig. S7.

      Minor reviews (Suggestions):

      __R3. __Refining the Title (Optional): Although the current title is descriptive, we believe it undersells the value of the manuscript. Since this study provides the first genome profiling and repeatome characterization for the genus Epidendrum and offers important insights into the calibration of bioinformatics tools and flow cytometry for repetitive genomes, I suggest modifying the title to reflect these aspects. The comparative access of GS is also an importante feature. This would make the article more attractive to a broader audience interested in genomics of non-model organisms.

      __A: __We have changed the title to "Nuclear genome profiling of two species of Epidendrum (Orchidaceae): genome size, repeatome and ploidy"

      __R3. __Botanical Nomenclature (Optional): Although citing taxonomic authorities is not strictly required in all fields of plant sciences, most botanical journals expect the full author citation at the first mention of each species. Including this information would improve the nomenclatural rigor of the manuscript and align it with common practices in botanical publishing.

      A: We have added the citation of the taxonomic authorities:

      "This study aims to use two closely related endemic Mexican species, Epidendrum anisatum Lex and Epidendrum marmoratum A. Rich. & Galeotti, to provide the first genomic profiling for this genus..."

      __R3. __Abbreviation of Genus Names: I noticed inconsistencies in the abbreviation of scientific names throughout the manuscript. Standard scientific style dictates that the full genus name (Epidendrum) should be written out only at its first mention in the Abstract and again at the first mention in the main text. Thereafter, it should be abbreviated (e.g., E. anisatum, E. marmoratum), unless the name appears at the beginning of a sentence or if abbreviation would cause ambiguity with another genus. Please revise the text to apply this abbreviation consistently.

      A: We have made the changes requested as necessary.

      __R3. __Genome Size Notation: In the Abstract and throughout the text, genome size estimates are presented using the statistical symbol for the mean (x). While mathematically accurate, this notation is generic and does not immediately inform the reader about the biological nature of the DNA content (i.e., whether it refers to the gametic 1C or somatic 2C value). In plant cytometry literature, it is standard practice to explicitly label these values using C-value terminology to prevent ambiguity and eliminate the effect of the number of chromosome sets (Bennett & Leitch 2005; Greilhuber et al. 2005; Doležel et al. 2018). I strongly suggest replacing references to "x" with "1C" (e.g., changing "x = 2.58 Gb" to "mean 1C value = 2.58 Gb") to ensure immediate clarity and alignment with established conventions in the field.

      __A: __We have revised the text in every instance, for example, in the results section:

      "The 1C value in gigabases (Gb; calculated from mass in pg) of E. anisatum ranged from 2.55 to 2.62 Gb (mean 1C value = 2.59 Gb) and that of E. marmoratum from 1.11 to 1.18 Gb (mean 1C value = 1.13 Gb; Supplementary Data Table S1)."

      __R3. __Justification of the Sequencing Method: Although the sequencing strategy is clearly described, the manuscript would benefit from a bit more contextualization regarding the choice of low-pass genome skimming. In the Introduction, a short justification of why this approach is suitable for estimating genome size, heterozygosity, and repeat composition, particularly in plants with large, repeat-rich genomes, would help readers better understand the methodological rationale. Likewise, in the Methods section, briefly outlining why the selected sequencing depth is appropriate, and how it aligns with previous studies using similar coverage levels, would strengthen the clarity of the methodological framework. These additions would make the rationale behind the sequencing approach more transparent and accessible to readers who may be less familiar with low-coverage genomic strategies.

      __A: __We have added the following short sentence in P.7:

      "This sequencing method produces suitable data sets without systematic biases, allowing the estimation of genome size and the proportion of repetitive DNA. "

      __R3. __Wording Improvement Regarding RepeatExplorer2 Results: In the Results section, several sentences attribute biological outcomes to the RepeatExplorer2 "protocols" (e.g., "According to this protocol, both species have highly repetitive genomes..."; "The comparative protocol showed a 67% total repeat proportion, which falls between the estimated repeat proportions of the two species according to the results of the individual protocol"). Since the RepeatExplorer2 protocol itself only provides the analytical workflow and not species-specific results, this phrasing may be misleading.

      A: We have rephrased these sections to emphasize that these are "the results of" the protocols and not the protocols themselves.

      Reviewer #3 (Significance (Required)):

      Significance

      General assessment

      Strengths

      1.First Detailed Genomic Profile for the Genus Epidendrum: The study provides the first integrated dataset on genome size, ploidy, heterozygosity, and repeatome for species of the genus Epidendrum, a novel contribution for an extremely diverse and under-explored group in terms of cytogenomics.

      Cross-validation of in vitro and in silico analyses: Flow cytometry is considered the gold standard for genome size (GS) estimation because it physically measures DNA quantity (Doležel et al. 2007; Śliwińska 2018). However, it typically requires fresh tissue, which is not always available. Conversely, k-mer analysis is a rapid bioinformatics technique utilizing sequencing data that does not rely on a reference genome. Nevertheless, it is frequently viewed with skepticism or distrust due to discrepancies with laboratory GS estimates (Pflug et al. 2020; Hesse 2023). In this study, by comparing computational results with flow cytometry data, the authors were able to validate the reliability of computational estimates for the investigated species. Since the 'true' GS was already established via flow cytometry, the authors used this value as a benchmark to test various software tools (GenomeScope, findGSE, CovEst) and parameters. This approach allowed for the identification of which tools perform best for complex genomes. For instance, they found that tools failing to account for heterozygosity (such as findGSE-hom) drastically overestimated the genome size of E. anisatum, whereas GenomeScope and findGSE-het (which account for heterozygosity) yielded results closer to the flow cytometry values. Thus, they demonstrated that this cross-validation is an effective method for estimating plant genome sizes with greater precision. This integrative approach is essential not only for defining GS but also for demonstrating how bioinformatics methods must be calibrated (particularly regarding depth of coverage and maximum k-mer coverage) to provide accurate data for non-model organisms when flow cytometry is not feasible.

      Limitations

      1. Limited Taxonomic Sampling: The study analyzes only two species of Epidendrum, which restricts the ability to make broad inferences regarding genome evolution across the genus. Given the outstanding diversity of Epidendrum (>1,800 species), the current sampling is insufficient to propose generalized evolutionary patterns. As the authors state by the end of the Discussion (page 18) "Future work should investigate to what extent LTR transposons and satellite DNA have been responsible for shaping genome size variation in different lineages of Epidendrum, analyzing a greater portion of its taxic diversity in an evolutionary context.". 2.Lack of Cytogenetic Results and Mapping: One of the major finding of this study is the identification of the AniS1 satellite as a potential key driver of the genome size difference between the species, occupying ~11% of the E. anisatum genome and virtually absent in E. marmoratum. While the authors use bioinformatic metrics (C and P indices) to infer a dispersed organization in the Discussion (Page 18), the study lacks physical validation via Fluorescence in situ Hybridization (FISH) - and a basic validation of the chromosome number. Without cytogenetic mapping, it is impossible to confirm the actual chromosomal distribution of this massive repetitive array, for instance, whether it has accumulated in specific heterochromatic blocks (e.g., centromeric or subtelomeric regions) or if it is genuinely interspersed along the chromosome arms. I suggest acknowledging this as a limitation in the Discussion, as the physical organization of such abundant repeats has significant implications for understanding the structural evolution of the species' chromosomes.

      Advance

      To the best of our knowledge, this study represents the first comprehensive genome profiling and repeatome characterization for any species of the genus Epidendrum. By integrating flow cytometry, k-mer-based approaches, and low-pass sequencing, the authors provide the first insights into the genomic architecture of Epidendrum, including quantitative assessments of transposable elements, lineage-specific satellite DNA, and repeat-driven genome expansion. This constitutes both a technical and a conceptual advance: technically, the study demonstrates the feasibility and limitations of combining in vitro and in silico methods for genome characterization in large, repeat-rich plant genomes; conceptually, it offers new evolutionary perspectives on how repetitive elements shape genome size divergence within a highly diverse orchid lineage. These results broaden the genomic knowledge base for Neotropical orchids and establish a foundational reference for future comparative, cytogenomic, and phylogenomic studies within Epidendrum and related groups.

      Audience

      This study will primarily interest a broad audience, including researchers in plant genomics, evolutionary biology, cytogenomics, and bioinformatics, especially those working with non-model plants or groups with large, repetitive genomes. It also holds relevance for scientists engaged in genome size evolution, repetitive DNA biology, and comparative genomics. Other researchers are likely to use this work as a methodological reference for genome profiling in non-model taxa, especially regarding the integration of flow cytometry and k-mer-based estimations and the challenges posed by highly repetitive genomes. The detailed repeatome characterization, including identification of lineage-specific satellites and retrotransposon dynamics, will support comparative genomic analyses, repeat evolution studies, and future cytogenetic validation (e.g., FISH experiments). Additionally, this dataset establishes a genomic baseline that can inform phylogenomic studies, species delimitation, and evolutionary inference within Epidendrum and related orchid groups.

      Reviewer's Backgrounds

      The review was prepared by two reviewers. Our expertise lies in evolution and biological diversity, with a focus on cytogenomic and genome size evolution. Among the projects in development, the cytogenomics evolution of Neotropical orchids is one of the main studies (also focused on Epidendrum). These areas shape my perspective in evaluating the evolutionary, cytogenomic, and biological implications of the study. However, we have limited expertise in methodologies related to k-mer-based genome profiling and heterozygosity modeling. Therefore, our evaluation does not deeply assess the technical validity of these analytical pipelines.

    1. QUESTIONS:

      What the hell is the answer to:

      1) Exercise 2: In the editor, three vectors are defined. Each one represents the box office numbers from the first three Star Wars movies. The first element of each vector indicates the US box office revenue, the second element refers to the Non-US box office. In this exercise, you’ll combine all these figures into a single vector with name ‘box_office’. Next construct a ‘matrix star_wars’ with 2 rows and 3 columns.

      2) Exercise 1: Write code to create an array ‘l’ with 3 sheets of 2 rows and 4 columns, filled with the first 5 letters of the alphabet.

    2. "!(2>3) + 2

      [1] FALSE

      !(2>3) + 0:5

      [1] TRUE FALSE FALSE FALSE FALSE FALSE

      z <- (1 > 2)

      is.logical(z)

      [1] TRUE"

      Consulta realizada correctamente
      

      These examples demonstrate how R handles "conflicts" between different types of operations and how it manages collections of data (vectors). 1. !(2>3) + 2 → FALSE

      This is another result of Operator Precedence. In R, the addition operator (+) is much stronger than the negation operator (!).

      What you expect: NOT (2 > 3) is NOT FALSE, which is TRUE. Then TRUE + 2 should be 3.
      
      What R does: It groups the + 2 with the expression immediately to its left.
      
          First, it evaluates the comparison: (2 > 3) is FALSE.
      
          Now the expression is !FALSE + 2.
      
          Because + happens before !, R calculates FALSE + 2 first.
      
          FALSE is coerced to 0. So, 0 + 2 = 2.
      
          Finally, it applies the negation: !2.
      
          In R, any non-zero number is treated as TRUE for logical operations. Therefore, NOT 2 (NOT TRUE) becomes FALSE.
      
      1. !(2>3) + 0:5 → TRUE FALSE FALSE FALSE FALSE FALSE

      This uses the same precedence rule but adds Vectorization into the mix.

      Step-by-Step:
      
          2 > 3 is FALSE.
      
          0:5 creates a vector: c(0, 1, 2, 3, 4, 5).
      
          R evaluates the addition first: FALSE + c(0, 1, 2, 3, 4, 5).
      
          FALSE becomes 0, and is added to every number in the vector (Recycling). Result: c(0, 1, 2, 3, 4, 5).
      
          Now R applies ! to that entire vector: !c(0, 1, 2, 3, 4, 5).
      
          The Result:
      
              !0 is TRUE (because 0 is the only "False" number).
      
              !1, !2, !3, etc., are all FALSE (because non-zero numbers are "True").
      
      1. z <- (1 > 2) and is.logical(z) → TRUE

      This is the simplest case because of the Parentheses.

      The parentheses force R to finish the comparison (1 > 2) before doing anything else.
      
      1 > 2 is strictly a logical comparison, so the result stored in z is the logical value FALSE.
      
      Since z is a "logical" data type, is.logical(z) correctly returns TRUE.
      

      The Golden Rule for R Logic

      If you are combining a logical test (!, >, ==) with an arithmetic operation (+, *, /), always wrap your logical test in parentheses.

      Bad: !x + y (Does !(x+y))
      
      Good: (!x) + y (Does (Not x) + y)
      
    1. summary(ur.ers(log(window(GDP, start = c(1962, 1), end = c(2012, 4))), model = "trend", lag.max = 2))

      I continued working on the inflation rate in the United States. The DF-GLS test:

      summary(ur.ers(400 * diff(PCECTPI), model = "trend", lag.max = 2)).

      The output:

      Value of test-statistic is: -2.7078

      Critical values of DF-GLS are: 1pct 5pct 10pct critical values -3.48 -2.89 -2.57

    1. Reviewer #2 (Public review):

      Summary:

      The authors of this paper note that although polyphosphate (polyP) is found throughout biology, the biological roles of polyP have been under-explored, especially in multicellular organisms. The authors created transgenic Drosophila that expressed a yeast enzyme that degrades polyP, targeting the enzyme to different subcellular compartments (cytosol, mitochondria, ER, and nucleus, terming these altered flies Cyto-FLYX, Mito-FLYX, etc.). The authors show the localization of polyP in various wild-type fruit fly cell types and demonstrate that the targeting vectors did indeed result in expression of the polyP degrading enzyme in the cells of the flies. They then go on to examine the effects of polyP depletion using just one of these targeting systems (the Cyto-FLYX). The primary findings from depletion of cytosolic polyP levels in these flies is that it accelerates eclosion and also appears to participate in hemolymph clotting. Perhaps surprisingly, the flies seemed otherwise healthy and appeared to have little other noticeable defects. The authors use transcriptomics to try to identify pathways altered by the cyto-FLYX construct degrading cytosolic polyP, and it seems likely that their findings in this regard will provide avenues for future investigation. And finally, although the authors found that eclosion is accelerated in pupae of Drosophila expressing the Cyto-FLYX construct, the reason why this happens remains unexplained.

      Strengths:

      The authors capitalize on the work of other investigators who had previously shown that expression of recombinant yeast exopolyphosphatase could be targeted to specific subcellular compartments to locally deplete polyP, and they also use a recombinant polyP binding protein (PPBD) developed by others to localize polyP. They combine this with the considerable power of Drosophila genetics to explore the roles of polyP by depleting it in specific compartments and cell types to tease out novel biological roles for polyP in a whole organism. This is a substantial advance.

      Weaknesses:

      Page 4 of Results (paragraph 1): I'm a bit concerned about the specificity of PPBD as a probe for polyP. The authors show that the fusion partner (GST) isn't responsible for the signal, but I don't think they directly demonstrate that PPBD is binding only to polyP. Could it also bind to other anionic substances? A useful control might be to digest the permeabilized cells and tissues with polyphosphatase prior to PPBD staining, and show that the staining is lost.

      In the hemolymph clotting experiments, the authors collected 2 ul of hemolymph and then added 1 ul of their test substance (water or a polyP solution). They state that they added either 0.8 or 1.6 nmol polyP in these experiments (the description in the Results differs from that of the Methods). I calculate this will give a polyP concentration of 0.3 or 0.6 mM. This is an extraordinarily high polyP concentration, and is much in excess of the polyP concentrations used in most of the experiments testing the effects of polyP on clotting of mammalian plasma. Why did the authors choose this high polyP concentration? Did they try lower concentrations? It seems possible that too high a polyP concentration would actually have less clotting activity than the optimal polyP concentration.

      In the revised version of the manuscript, the authors have productively responded to the previous criticisms. Their new data show stronger controls regarding the specificity of PPBD with regard to its interaction with polyP. The authors also have repeated their hemolymph clotting experiments with lower polyP concentrations, which are likely to be more physiological.

    2. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Polymers of orthophosphate of varying lengths are abundant in prokaryotes and some eukaryotes, where they regulate many cellular functions. Though they exist in metazoans, few tools exist to study their function. This study documents the development of tools to extract, measure, and deplete inorganic polyphosphates in *Drosophila*. Using these tools, the authors show:

      (1) That polyP levels are negligible in embryos and larvae of all stages while they are feeding. They remain high in pupae but their levels drop in adults.

      (2) That many cells in tissues such as the salivary glands, oocytes, haemocytes, imaginal discs, optic lobe, muscle, and crop, have polyP that is either cytoplasmic or nuclear (within the nucleolus).

      (3) That polyP is necessary in plasmatocytes for blood clotting in Drosophila.

      (4) That ployP controls the timing of eclosion.

      The tools developed in the study are innovative, well-designed, tested, and well-documented. I enjoyed reading about them and I appreciate that the authors have gone looking for the functional role of polyP in flies, which hasn't been demonstrated before. The documentation of polyP in cells is convincing as its role in plasmatocytes in clotting.

      We sincerely thank the reviewer for their encouraging assessment and for recognizing both the innovation of the FLYX toolkit and the functional insights it enables. Their remarks underscore the importance of establishing Drosophila as a tractable model for polyP biology, and we are grateful for their constructive feedback, which further strengthened the manuscript.

      Its control of eclosion timing, however, could result from non-specific effects of expressing an exogenous protein in all cells of an animal.

      We now explicitly state this limitation in the revised manuscript (p.16, l.347–349). The issue is that no catalytic-dead ScPpX1 is available as a control in the field. We plan to generate such mutants through systematic structural and functional studies and will update the FLYX toolkit once they are developed and validated. Importantly, the accelerated eclosion phenotype is reproducible and correlates with endogenous polyP dynamics.

      The RNAseq experiments and their associated analyses on polyP-depleted animals and controls have not been discussed in sufficient detail.  In its current form, the data look to be extremely variable between replicates and I'm therefore unsure of how the differentially regulated genes were identified.

      We thank the reviewer for pointing out the lack of clarity. We have expanded our RNAseq analysis in the revised manuscript (p.20, l.430–434). Because of inter-sample variation (PC2 = 19.10%, Fig. S7B), we employed Gene Set Enrichment Analysis (GSEA) rather than strict DEG cutoffs. This method is widely used when the goal is to capture pathway-level changes under variability (1). We now also highlight this limitation explicitly (p.20, l.430–432) and provide an additional table with gene-specific fold change (See Supplementary Table for RNA Sequencing Sheet 1). Please note that we have moved RNAseq data to Supplementary Fig. 7 and 8 as suggested in the review.

      It is interesting that no kinases and phosphatases have been identified in flies. Is it possible that flies are utilising the polyP from their gut microbiota? It would be interesting to see if these signatures go away in axenic animals.

      This is an interesting possibility. Several observations argue that polyP is synthesized by fly tissues: (i) polyP levels remain very low during feeding stages but build up in wandering third instar larvae after feeding ceases; (ii) PPBD staining is absent from the gut except the crop (Fig. S3O–P); (ii) In C. elegans, intestinal polyP was unaffected when worms were fed polyP-deficient bacteria (2); (iv) depletion of polyP from plasmatocytes alone impairs hemolymph clotting, which would not be expected if gut-derived polyP were the major source and may have contributed to polyP in hemolymph. Nevertheless, we agree that microbiota-derived polyP may contribute, and we plan systematic testing in axenic flies in future work.

      Reviewer #2 (Public review):

      Summary:

      The authors of this paper note that although polyphosphate (polyP) is found throughout biology, the biological roles of polyP have been under-explored, especially in multicellular organisms. The authors created transgenic Drosophila that expressed a yeast enzyme that degrades polyP, targeting the enzyme to different subcellular compartments (cytosol, mitochondria, ER, and nucleus, terming these altered flies Cyto-FLYX, Mito-FLYX, etc.). The authors show the localization of polyP in various wild-type fruit fly cell types and demonstrate that the targeting vectors did indeed result in the expression of the polyP degrading enzyme in the cells of the flies. They then go on to examine the effects of polyP depletion using just one of these targeting systems (the Cyto-FLYX). The primary findings from the depletion of cytosolic polyP levels in these flies are that it accelerates eclosion and also appears to participate in hemolymph clotting. Perhaps surprisingly, the flies seemed otherwise healthy and appeared to have little other noticeable defects. The authors use transcriptomics to try to identify pathways altered by the cyto-FLYX construct degrading cytosolic polyP, and it seems likely that their findings in this regard will provide avenues for future investigation. And finally, although the authors found that eclosion is accelerated in the pupae of Drosophila expressing the Cyto-FLYX construct, the reason why this happens remains unexplained.

      Strengths:

      The authors capitalize on the work of other investigators who had previously shown that expression of recombinant yeast exopolyphosphatase could be targeted to specific subcellular compartments to locally deplete polyP, and they also use a recombinant polyP-binding protein (PPBD) developed by others to localize polyP. They combine this with the considerable power of Drosophila genetics to explore the roles of polyP by depleting it in specific compartments and cell types to tease out novel biological roles for polyP in a whole organism. This is a substantial advance.

      We are grateful to the reviewer for their thorough and thoughtful evaluation. Their balanced summary of our work, recognition of the strengths of our genetic tools, and constructive suggestions have been invaluable in clarifying our experiments and strengthening the conclusions.

      Weaknesses:

      Page 4 of the Results (paragraph 1): I'm a bit concerned about the specificity of PPBD as a probe for polyP. The authors show that the fusion partner (GST) isn't responsible for the signal, but I don't think they directly demonstrate that PPBD is binding only to polyP. Could it also bind to other anionic substances? A useful control might be to digest the permeabilized cells and tissues with polyphosphatase prior to PPBD staining and show that the staining is lost.

      To address this concern, we have done two sets of experiments:

      (1) We generated a PPBD mutant (GST-PPBD<sup>Mut</sup>). We establish that GST-PPBD binds to polyP-2X FITC, whereas GST-PPBD<sup>Mut</sup> and GST do not bind polyP<sub>100</sub>-2X FITC using Microscale Thermophoresis. We found that, unlike the punctate staining pattern of GST-PPBD (wild-type), GST-PPBD<sup>Mut</sup> does not stain hemocytes. This data has been added to the revised manuscript (Fig. 2B-D, p.8, l.151–165).

      (2) A study in C.elegans by Quarles et.al has performed a similar experiment, suggested by the reviewer. In that study, treating permeabilized tissues with polyphosphatase prior to PPBD staining resulted in a decrease of PPBD-GFP signal from the tissues (2). We also performed the same experiment where we subjected hemocytes to GST-PPBD staining with prior incubation of fixed and permeabilised hemocytes with ScPpX1 and heat-inactivated ScPpX1 protein. We find that both staining intensity and the number of punctae are higher in hemocytes left untreated and in those treated with heat-inactivated ScPpX1. The hemocytes pre-treated with ScPpX1 showed reduced staining intensity and number of punctae. This data has been added to the revised manuscript (Fig. 2E-G, p.8, l.166-172).

      Further, Saito et al. reported that PPBD binds to polyP in vitro, as well as in yeast and mammalian cells, with a high affinity of ~45µM for longer polyP chains (35 mer and above) (3). They also show that the affinity of PPBD with RNA and DNA is very low. Furthermore, PPBD could detect differences in polyP labeling in yeasts grown under different physiological conditions that alter polyP levels (3). Taken together, published work and our results suggest that PPBD specifically labels polyP.

      In the hemolymph clotting experiments, the authors collected 2 ul of hemolymph and then added 1 ul of their test substance (water or a polyP solution). They state that they added either 0.8 or 1.6 nmol polyP in these experiments (the description in the Results differs from that of the Methods). I calculate this will give a polyP concentration of 0.3 or 0.6 mM. This is an extraordinarily high polyP concentration and is much in excess of the polyP concentrations used in most of the experiments testing the effects of polyP on clotting of mammalian plasma. Why did the authors choose this high polyP concentration? Did they try lower concentrations? It seems possible that too high a polyP concentration would actually have less clotting activity than the optimal polyP concentration.

      We repeated the assays using 125 µM polyP, consistent with concentrations employed in mammalian plasma studies (4,5). Even at this lower, physiologically relevant concentration, polyP significantly enhanced clot fibre formation (Included as Fig. S5F–I, p.12, l.241–243). This reconfirms the conclusion that polyP promotes hemolymph clotting.

      Author response image 1.

      Reviewer #3 (Public review):

      Summary:

      Sarkar, Bhandari, Jaiswal, and colleagues establish a suite of quantitative and genetic tools to use Drosophila melanogaster as a model metazoan organism to study polyphosphate (polyP) biology. By adapting biochemical approaches for use in D. melanogaster, they identify a window of increased polyP levels during development. Using genetic tools, they find that depleting polyP from the cytoplasm alters the timing of metamorphosis, accelerating eclosion. By adapting subcellular imaging approaches for D. melanogaster, they observe polyP in the nucleolus of several cell types. They further demonstrate that polyP localizes to cytoplasmic puncta in hemocytes, and further that depleting polyP from the cytoplasm of hemocytes impairs hemolymph clotting. Together, these findings establish D. melanogaster as a tractable system for advancing our understanding of polyP in metazoans.

      Strengths:

      (1) The FLYX system, combining cell type and compartment-specific expression of ScPpx1, provides a powerful tool for the polyP community.

      (2) The finding that cytoplasmic polyP levels change during development and affect the timing of metamorphosis is an exciting first step in understanding the role of polyP in metazoan development, and possible polyP-related diseases.

      (3) Given the significant existing body of work implicating polyP in the human blood clotting cascade, this study provides compelling evidence that polyP has an ancient role in clotting in metazoans.

      We sincerely thank the reviewer for their generous and insightful comments. Their recognition of both the technical strengths of the FLYX system and the broader biological implications reinforces our confidence that this work will serve as a useful foundation for the community.

      Limitations:

      (1) While the authors demonstrate that HA-ScPpx1 protein localizes to the target organelles in the various FLYX constructs, the capacity of these constructs to deplete polyP from the different cellular compartments is not shown. This is an important control to both demonstrate that the GTS-PPBD labeling protocol works, and also to establish the efficacy of compartment-specific depletion. While not necessary to do this for all the constructs, it would be helpful to do this for the cyto-FLYX and nuc-FLYX.

      We confirmed polyP depletion in Cyto-FLYX using the malachite green assay (Fig. 3D, p.10, l.212–214). The efficacy of ScPpX1 has also been earlier demonstrated in mammalian mitochondria (6). Our preliminary data from Mito-ScPpX1 expressed ubiquitously with Tubulin-Gal4 showed a reduction in polyP levels when estimated from whole flies (See Author response image 2 below, ongoing investigation). In an independent study focusing on mitochondrial polyP depletion, we are characterizing these lines in detail  and plan to check the amount of polyP contributed to the cellular pool by mitochondria using subcellular fractionation. Direct phenotypic and polyP depletion analyses of Nuc-FLYX and ER-FLYX are also being carried out, but are in preliminary stages. That there is a difference in levels of polyP in various tissues and that we get a very little subscellular fraction for polyP analysis have been a few challenging issues. This analysis requires detailed, independent, and careful analysis, and thus, we refrain from adding this data to the current manuscript.

      Author response image 2.

      Regarding the specificity, Saito et.al. reported that PPBD binds to polyP in vitro, as well as in yeast and mammalian cells with a high affinity of ~45µM for longer polyP chains (35 mer and above) (3). They also show that the affinity of PPBD with RNA and DNA is very low. Further, PPBD could reveal differences in polyP labeling with yeasts grown in different physiological conditions that can alter polyP levels. Now in the manuscript, we included following data to show specificity of PPBD:

      To address this concern we have done two sets of experiments:

      We generated a PPBD mutant (GST-PPBD<sup>Mut</sup>). Using Microscale Thermophoresis, we establish that GST-PPBD binds to polyP<sub>100</sub>-2X-FITC, whereas, GST-PPBD<sup>Mut</sup> and GST do not bind polyP<sub>100</sub>-2X-FITC at all. We found that unlike the punctate staining pattern of GST-PPBD (wild-type), GST-PPBD<sup>Mut</sup> does not stain hemocytes. This data has been added to the revised manuscript (Fig. 2B-D, p.8, l.151–165).

      A study in C.elegans by Quarles et.al has performed a similar experiment suggested by the reviewer. In that study, treating permeabilized tissues with polyphosphatase prior to PPBD staining resulted in decrease of PPBD-GFP signal from the tissues (2). We also performed the same experiment where we subjected hemocytes to GST-PPBD staining with prior incubation of fixed and permeabilised hemocytes with ScPpX1 and heat inactivated ScPpX1 protein. We find that both intensity of staining and number of punctae are higher in hemocytes that were left untreated and the one where heat inactivated ScPpX1 was added. The hemocytes pre-treated with ScPpX1 showed reduced staining intensity and number of punctae. This data has been added to the revised manuscript (Fig. 2E-G, p.8, l.166-172).

      (2) The cell biological data in this study clearly indicates that polyP is enriched in the nucleolus in multiple cell types, consistent with recent findings from other labs, and also that polyP affects gene expression during development. Given that the authors also generate the Nuc-FLYX construct to deplete polyP from the nucleus, it is surprising that they test how depleting cytoplasmic but not nuclear polyP affects development. However, providing these tools is a service to the community, and testing the phenotypic consequences of all the FLYX constructs may arguably be beyond the scope of this first study.

      We agree this is an important avenue. In this first study, we focused on establishing the toolkit and reporting phenotypes with Cyto-FLYX. We are systematically assaying phenotypes from all FLYX constructs, including Nuc-FLYX, in ongoing studies

      Recommendations for the authors:

      Reviewing Editor Comment:

      The reviewers appreciated the general quality of the rigour and work presented in this manuscript. We also had a few recommendations for the authors. These are listed here and the details related to them can be found in the individual reviews below.

      (1) We suggest including an appropriate control to show that PPBD binds polyP specifically.

      We have updated the response section as follows:

      (a) Highlighted previous literature that showed the specificity of PPBD.

      (b) We show that the punctate staining observed by PPBD is not demonstrated by the mutant PPBD (PPBD<sup>Mut</sup>) in which amino acids that are responsible for polyP binding are mutated.

      (c) We show that PPBD<sup>Mut</sup> does not bind to polyP using Microscale Thermophoresis.

      (d) We show that treatment of fixed and permeabilised hemocytes with ScPpX1 reduces the PPBD staining intensity and number of punctae, as compared to tissues left untreated or treated with heat-inactivated ScPpX1.

      We have included these in our updated revised manuscript (Fig. 2B-G, p.8, l.151–157)

      (2) The high concentration of PolyP in the clotting assay might be impeding clotting. The authors may want to consider lowering this in their assays.

      We have addressed this concern in our revised manuscript. We have performed the clotting assays with lower polyP concentrations (concentrations previously used in clotting experiments with human blood and polyP). Data is included in Fig. S5F–I, p.12, l.241–243.

      (3) The RNAseq study: can the authors please describe this better and possibly mine it for the regulation of genes that affect eclosion?

      In our revised manuscript, we have included a broader discussion about the RNAseq analysis done in the article in both the ‘results’ and the ‘discussion’ sections, where we have rewritten the narrative from the perspective of accelerated eclosion. (p.15 l.310-335, p. 20, l.431-446).

      (4) Have the authors considered the possibility that the gut microbiota might be contributing to some of their measurements and assays? It would be good to address this upfront - either experimentally, in the discussion, or (ideally) both.

      This is an exciting possibility. Several observations argue that fly tissues synthesize polyP: (i) polyP levels remain very low during feeding stages but build up in wandering third instar larvae after feeding ceases; (ii) PPBD staining is absent from the gut except the crop (Fig. S3O–P); (iii) in C. elegans, intestinal polyP was unaffected when worms were fed polyP-deficient bacteria (2); (iv) depletion of polyP from plasmatocytes alone impairs hemolymph clotting, which would not be expected if gut-derived polyP were the major source and may have contributed to polyP in hemolymph. Nevertheless, microbiota-derived polyP may contribute, and we plan systematic testing in axenic flies in future work.

      Reviewer #1 (Recommendations for the authors):

      (1) While the authors have shown that the depletion tool results in a general reduction of polyP levels in Figure 3D, it would have been nice to show this via IHC. Particularly since the depletion depends on the strength of the Gal4, it is possible that the phenotypes are being under-estimated because the depletions are weak.

      We agree that different Gal4 lines have different strengths and will therefore affect polyP levels and the strength of the phenotype differently.

      We performed PPBD staining on hemocytes expressing ScPPX; however, we observed very intense, uniform staining throughout the cells, which was unexpected. It seems like PPBD is recognizing overexpressed ScPpX1. Indeed, in an unpublished study by Manisha Mallick (Bhandari lab), it was found that His-ScPpX1 specifically interacts with GST-PPBD in a protein interaction assay (See Author response image 3). Due to these issues, we refrained from IHC/PPBD-based validation.

      Author response image 3.

      (2) The subcellular tools for depletion are neat! I wonder why the authors didn't test them. For example in the salivary gland for nuclear depletion?

      We have addressed this question in the reviewer responses. We are systematically assaying phenotypes from all FLYX constructs, including Mito-FLYX, and Nuc-FLYX, in ongoing independent investigations. As discussed in #1, a possible interaction of ScPpX and PPBD is making this test a bit more challenging, and hence, they each require a detailed investigation.

      (a) Does the absence of clotting defects using Lz-gal4 suggest that PolyP is more crucial in the plasmatocytoes and for the initial clotting process? And that it is dispensible/less important in the crystal cells and for the later clotting process. Or is it that the crystal cells just don't have as much polyP? The image (2E-H) certainly looks like it.

      In hemolymph, the primary clot formation is a result of the clotting factors secreted from the fat bodies and the plasmatocytes. The crystal cells are responsible for the release of factors aiding in successfully hardening the soft clot initially formed. Reports suggest that clotting and melanization of the clot are independent of each other (7). Since Crystal cells do not contribute to clot fibre formation, the absence of clotting defects using LzGAL4-CytoFLYX is not surprising. Alternatively, PolyP may be secreted from all hemocytes and contribute to clotting; however, the crystal cells make up only 5% hemocytes, and hence polyP depletion in those cells may have a negligible effect on blood clotting.

      Crystal cells do show PPBD staining. Whether polyP is significantly lower in levels in the crystal cells as compared to the plasmatocytes needs more systematic investigation. Image (2E-H) is a representative image of the presence of polyP in crystal cells and can not be considered to compare polyP levels in the crystal cells vs Plasmatocytes.

      (b) The RNAseq analyses and data could be better presented. If the data are indeed variable and the differentially expressed genes of low confidence, I might remove that data entirely. I don't think it'll take away from the rest of the work.

      We understand this concern and, therefore, in the revised manuscript, we have included a broader discussion about the RNAseq analysis done in the article in both the ‘results’ and the ‘discussion’ sections, where we have rewritten the narrative from the perspective of accelerated eclosion. (p.15 l.310-335, p. 20, l.431-446). We have also stated the limitations of such studies.

      (c) I would re-phrase the first sentence of the results section.

      We have re-phrased it in the revised manuscript.

      Reviewer #2 (Recommendations for the authors):

      (1) The authors created several different versions of the FLYX system that would be targeted to different subcellular compartments. They mostly report on the effects of cytosolic targeting, but some of the constructs targeted the polyphosphatase to mitochondria or the nucleus.

      They report that the targeting worked, but I didn't see any results on the effects of those constructs on fly viability, development, etc.

      There is a growing literature of investigators targeting polyphosphatase to mitochondria and showing how depleting mitochondrial polyP alters mitochondrial function. What was the effect of the Nuc-FLYX and Mito-FLYX constructs on the flies?

      Also, the authors should probably cite the papers of others on the effects of depleting mitochondrial polyP in other eukaryotic cells in the context of discussing their findings in flies.

      We have addressed this question in the reviewer responses. We did not see any obvious developmental or viability defects with any of the FLYX lines, and only after careful investigation did we come across the clotting defects in the CytoFLYX. We are currently systematically assaying phenotypes from all FLYX constructs, including Mito-FLYX and Nuc-FLYX, in independent ongoing investigations.

      We have discussed the heterologous expression of mitochondrial polyphosphatase in mammalian cells to justify the need for developing Mito-FLYX (p. 10, l. 197-200). In the discussion section, we also discuss the presence and roles of polyP in the nucleus and how Nuc-FLYX can help study such phenomena (p. 19, l. 399-407).

      (2) The authors should number the pages of their manuscript to make it easier for reviewers to refer to specific pages.

      We have numbered our lines and pages in the revised manuscript.

      (3) Abstract: the abbreviation, "polyP", is not defined in the abstract. The first word in the abstract is "polyphosphate", so it should be defined there.

      We have corrected it in the revised version.

      (4) The authors repeatedly use the phrase, "orange hot", to describe one of the colors in their micrographs, but I don't know how this differs from "orange".

      ‘OrangeHot’ is the name of the LUT used in the ImageJ analysis and hence referred to as the colour

      (5) First page of the Introduction: the phrase, "feeding polyP to αβ expression Alzheimer's model of Caenorhabditis elegans" is awkward (it literally means feeding polyP to the model instead of the worms).

      We have revised it. (p.3, l.55-57).

      (6) Page 2 of the Introduction: The authors should cite this paper when they state that NUDT3 is a polyphosphatase: https://pubmed.ncbi.nlm.nih.gov/34788624/

      We have cited the paper in the revised version of the manuscript. (p.4, l. 68-70)

      (7) Page 2 of Results: The authors report the polyP content in the third instar larva (misspelled as "larval") to five significant digits ("419.30"). Their data do not support more than three significant digits, though.

      We have corrected it in the revised manuscript.

      (8) Page 3 of Results (paragraph 1): When discussing the polyP levels in various larval stages, the authors are extracting total polyP from the larvae. It seems that at least some of the polyP may come from gut microbes. This should probably be mentioned.

      This is an interesting possibility. Several observations argue that polyP is synthesized by fly tissues: (i) polyP levels remain very low during feeding stages but build up in wandering third instar larvae after feeding ceases; (ii) PPBD staining is absent from the gut except the crop (Fig. S3O–P); (ii) In C. elegans, intestinal polyP was unaffected when worms were fed polyP-deficient bacteria (2); (iv) depletion of polyP from plasmatocytes alone impairs hemolymph clotting, which would not be expected if gut-derived polyP were the major source and may have contributed to polyP in hemolymph. We mention this limitation in the revised manuscript (p.19-20, l. 425-433).

      (9) Page 3 of Results (paragraph 2): stating that the 4% paraformaldehyde works "best" is imprecise. What do the authors mean by "best"?

      We have addressed this comment in the revised manuscript and corrected it as 4% paraformaldehyde being better among the three methods we used to fix tissues, which also included methanol and Bouin’s fixative  (p.8, l. 152-154).

      (10) Page 4 of Results (paragraph 2, last line of the page): The scientific literature is vast, so one can never be sure that one knows of all the papers out there, even on a topic as relatively limited as polyP. Therefore, I would recommend qualifying the statement "...this is the first comprehensive tissue staining report...". It would be more accurate (and safer) to say something like, "to our knowledge, this is the first..." There is a similar statement with the word "first" on the next page regarding the FLYX library.

      We have addressed this concern and corrected it accordingly in the revised version of the manuscript (p.9, l. 192-193)

      Reviewer #3 (Recommendations for the authors):

      (1) The authors should include in their discussion a comparison of cell biological observations using the polyP binding domain of E. coli Ppx (GST-PPBD) to fluorescently label polyP in cells and tissues with recent work using a similar approach in C. elegans (Quarles et al., PMID:39413779).

      In the revised manuscript, we have cited the work of Quarles et al. and have added a comparison of observations (p.19,l.408-410). In the discussion, we have also focused on multiple other studies about how polyP presence in different subcellular compartments, like the nucleus, can be assayed and studied with the tools developed in this study.

      (2) The gene expression studies of time-matched Cyto-FLYX vs WT larvae is very intriguing. Given the authors' findings that non-feeding third instar Cyto-FLYX larvae are developmentally ahead of WT larvae, can the observed trends be explained by known changes in gene expression that occur during eclosion? This is mentioned in the results section in the context of genes linked to neurons, but a broader discussion of which pathway changes observed can be explained by the developmental stage difference between the WT and FLYX larvae would be helpful in the discussion.

      We have included a broader discussion about the RNAseq analysis done in the article in both the ‘results’ and the ‘discussion’ sections, where we have rewritten the narrative from the perspective of accelerated eclosion. (p.15 l.310-335, p. 20, l.431-446). We have also stated the limitations of such studies.

      (3) The sentence describing NUDT3 is not referenced.

      We have addressed this comment and have cited the paper of NUDT3 in the revised version of the manuscript.(p.4, l. 68-70)

      (4) In the first sentence of the results section, the meaning/validity of the statement "The polyP levels have decreased as evolution progressed" is not clear. It might be more straightforward to give an estimate of the total pmoles polyP/mg protein difference between bacteria/yeast and metazoans.

      In the revised manuscript, we have given an estimate of the polyP content across various species across evolution to uphold the statement that polyP levels have decreased as evolution progressed (p. 5, l. 87-91).

      (5) The description of the malachite green assay in the results section describes it as "calorimetric" but this should read "colorimetric?"

      We have corrected it in the revised manuscript.

      References

      (1) Chicco D, Agapito G. Nine quick tips for pathway enrichment analysis. PLoS Comput Biol. 2022 Aug 11;18(8):e1010348.

      (2) Quarles E, Petreanu L, Narain A, Jain A, Rai A, Wang J, et al. Cryosectioning and immunofluorescence of C. elegans reveals endogenous polyphosphate in intestinal endo-lysosomal organelles. Cell Rep Methods. 2024 Oct 8;100879.

      (3) Saito K, Ohtomo R, Kuga-Uetake Y, Aono T, Saito M. Direct labeling of polyphosphate at the ultrastructural level in Saccharomyces cerevisiae by using the affinity of the polyphosphate binding domain of Escherichia coli exopolyphosphatase. Appl Environ Microbiol. 2005 Oct;71(10):5692–701.

      (4) Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci USA. 2006 Jan 24;103(4):903–8.

      (5) Smith SA, Choi SH, Davis-Harrison R, Huyck J, Boettcher J, Rienstra CM, et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood. 2010 Nov 18;116(20):4353–9.

      (6) Abramov AY, Fraley C, Diao CT, Winkfein R, Colicos MA, Duchen MR, et al. Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death. Proc Natl Acad Sci USA. 2007 Nov 13;104(46):18091–6.

      (7) Schmid MR, Dziedziech A, Arefin B, Kienzle T, Wang Z, Akhter M, et al. Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors. Insect Biochem Mol Biol. 2019 Jun;109:63–71.

      (8) Jian Guan, Rebecca Lee Hurto, Akash Rai, Christopher A. Azaldegui, Luis A. Ortiz-Rodríguez, Julie S. Biteen, Lydia Freddolino, Ursula Jakob. HP-Bodies – Ancestral Condensates that Regulate RNA Turnover and Protein Translation in Bacteria. bioRxiv 2025.02.06.636932; doi: https://doi.org/10.1101/2025.02.06.636932.

      (9) Lonetti A, Szijgyarto Z, Bosch D, Loss O, Azevedo C, Saiardi A. Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases. J Biol Chem. 2011 Sep 16;286(37):31966–74.

    1. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary

      This paper introduces a dual-pathway model for reconstructing naturalistic speech from intracranial ECoG data. It integrates an acoustic pathway (LSTM + HiFi-GAN for spectral detail) and a linguistic pathway (Transformer + Parler-TTS for linguistic content). Output from the two components is later merged via CosyVoice2.0 voice cloning. Using only 20 minutes of ECoG data per participant, the model achieves high acoustic fidelity and linguistic intelligibility.

      Strengths

      (1) The proposed dual-pathway framework effectively integrates the strengths of neural-to-acoustic and neural-to-text decoding and aligns well with established neurobiological models of dual-stream processing in speech and language.

      (2) The integrated approach achieves robust speech reconstruction using only 20 minutes of ECoG data per subject, demonstrating the efficiency of the proposed method.

      (3) The use of multiple evaluation metrics (MOS, mel-spectrogram R², WER, PER) spanning acoustic, linguistic (phoneme and word), and perceptual dimensions, together with comparisons against noisedegraded baselines, adds strong quantitative rigor to the study.

      We thank Reviewer #1 for the supportive comments. In addition, we appreciate Reviewer #1’s thoughtful comments and feedback. By addressing these comments, we believe we have greatly improved the clarity of our claims and methodology. Below we list our point-to-point responses addressing concerns raised by Reviewer #1.

      Weaknesses:

      (1) It is unclear how much the acoustic pathway contributes to the final reconstruction results, based on Figures 3B-E and 4E. Including results from Baseline 2 + CosyVoice and Baseline 3 + CosyVoice could help clarify this contribution.

      We sincerely appreciate the inquiry from Reviewer 1. We thank the reviewer for this suggestion. However, we believe that directly applying CosyVoice to the outputs of Baseline 2 or Baseline 3 in isolation is not methodologically feasible and would not correctly elucidate the contribution of the auditory pathway and might lead to misinterpretation.

      The role of CosyVoice 2.0 in our framework is specifically voice cloning and fusion, not standalone enhancement. It is designed to integrate information from two pathways. Its operation requires two key inputs:

      (1) A voice reference speech that provides the target speaker's timbre and prosodic characteristics. In our final pipeline, this is provided by the denoised output of the acoustic pathway (Baseline 2).

      (2) A target word sequence that specifies the linguistic content to be spoken. This is obtained by transcribing the output of the linguistic pathway (Baseline 3) using Whisper ASR. Therefore, the standalone outputs of Baseline 2 and Baseline 3 are the purest demonstrations of what each pathway contributes before fusion. The significant improvement in WER/PER and MOS in the final output (compared to Baseline 2) and the significant improvement in melspectrogram R² (compared to Baseline 3) together demonstrate the complementary contributions of the two pathways. The fusion via CosyVoice is the mechanism that allows these contributions to be combined. We have added a clearer explanation of CosyVoice's role and the rationale for not testing it on individual baselines in the revised manuscript (Results section: "The fine-tuned voice cloner further enhances...").

      Edits:

      Page 11, Lines 277-282:

      “ Voice cloning is used to bridge the gap between acoustic fidelity and linguistic intelligibility in speech reconstruction. This approach strategically combines the strengths of complementary pathways: the acoustic pathway preserves speaker-specific spectral characteristics while the linguistic pathway maintains lexical and phonetic precision. By integrating these components through neural voice cloning, we achieve balanced reconstruction that overcomes the limitations inherent in isolated systems. CosyVoice 2.0, the voice cloner module serves specifically as a voice cloning and fusion engine, requiring two inputs: (1) a voice reference speech (provided by the denoised output of the acoustic pathway) to specify the target speaker's identity, and (2) a target word sequence (transcribed from the output of the linguistic pathway) to specify the linguistic content. The standalone baseline outputs of the two pathways can be integrated in this way.”

      (2) As noted in the limitations, the reconstruction results heavily rely on pre-trained generative models. However, no comparison is provided with state-of-the-art multimodal LLMs such as Qwen3-Omni, which can process auditory and textual information simultaneously. The rationale for using separate models (Wav2Vec for speech and TTS for text) instead of a single unified generative framework should be clearly justified. In addition, the adaptor employs an LSTM architecture for speech but a Transformer for text, which may introduce confounds in the performance comparison. Is there any theoretical or empirical motivation for adopting recurrent networks for auditory processing and Transformer-based models for textual processing?

      We thank the reviewer for the insightful suggestion regarding multimodal large language models (LLMs) such as Qwen3-Omni. It is important to clarify the distinction between general-purpose interactive multimodal models and models specifically designed for high-fidelity voice cloning and speech synthesis.

      As for the comparison with the state-of-the-art multimodal LLMs:

      Qwen3-Omni and GLM-4-Voice are powerful conversational agents capable of processing multiple modalities including text, speech, image, and video, as described in its documentation (see: https://help.aliyun.com/zh/model-studio/qwen-tts-realtime and https://docs.bigmodel.cn/cn/guide/models/sound-and-video/glm-4-voice). However, it is primarily optimized for interactive dialogue and multimodal understanding rather than for precise, speaker-adaptive speech reconstruction from neural signals. In contrast, CosyVoice 2.0, developed by the same team at Alibaba, is specifically designed for voice cloning and text-to-speech synthesis (see: https://help.aliyun.com/zh/model-studio/text-to-speech). It incorporates advanced speaker adaptation and acoustic modeling capabilities that are essential for reconstructing naturalistic speech from limited neural data. Therefore, our choice of CosyVoice for the final synthesis stage aligns with the goal of integrating acoustic fidelity and linguistic intelligibility, which is central to our study.

      For the selection of LSTM and Transformer in the two pathways:

      The goal of the acoustic adaptor is to reconstruct fine-grained spectrotemporal details (formants, harmonic structures, prosodic contours) with millisecond-to-centisecond precision. These features rely heavily on local temporal dynamics and short-to-medium range dependencies (e.g., within and between phonemes/syllables). In our ablation studies (to be added in the supplementary), we found that Transformer-based adaptors, which inherently emphasize global sentence-level context through self-attention, tended to oversmooth the reconstructed acoustic features, losing critical fine-temporal details essential for naturalness. In contrast, the recurrent nature of LSTMs, with their inherent temporal state propagation, proved more effective at modeling these local sequential dependencies without excessive smoothing, leading to higher mel-spectrogram fidelity. This aligns with the neurobiological observation that early auditory cortex processes sound with precise temporal fidelity. Moreover, from an engineering perspective, LSTM-based decoders have been empirically shown to perform well in sequential prediction tasks with limited data, as evidenced in prior work on sequence modeling and neural decoding (1).

      The goal of the linguistic adaptor is to decode abstract, discrete word tokens. This task benefits from modeling long-range contextual dependencies across a sentence to resolve lexical ambiguity and syntactic structure (e.g., subject-verb agreement). The self-attention mechanism of Transformers is exceptionally well-suited for capturing these global relationships, as evidenced by their dominance in NLP. Our experiments confirmed that a Transformer adaptor outperformed an LSTM-based one in word token prediction accuracy.

      While a unified multimodal LLM could in principle handle both modalities, such models often face challenges in modality imbalance and task specialization. Audio and text modalities have distinct temporal scales, feature distributions, and learning dynamics. By decoupling them into separate pathways with specialized adaptors, we ensure that each modality is processed by an architecture optimized for its inherent structure. This divide-and-conquer strategy avoids the risk of one modality dominating or interfering with the learning of the other, leading to more stable training and better final performance, especially important when adapting to limited neural data.

      Edits:

      Page 9, Lines 214-223:

      “The acoustic pathway, implemented through a bi-directional LSTM neural adaptor architecture (Fig. 1B), specializes in reconstructing fundamental acoustic properties of speech. This module directly processes neural recordings to generate precise time-frequency representations, focusing on preserving speaker-specific spectral characteristics like formant structures, harmonic patterns, and spectral envelope details. Quantitative evaluation confirms its core competency: achieving a mel-spectrogram R² of 0.793 ± 0.016 (Fig. 3B) demonstrates remarkable fidelity in reconstructing acoustic microstructure. This performance level is statistically indistinguishable from original speech degraded by 0dB additive noise (0.771 ± 0.014, p = 0.242, one-sided t-test). We chose a bidirectional LSTM architecture for this adaptor because its recurrent nature is particularly suited to modeling the fine-grained, short- to medium-range temporal dependencies (e.g., within and between phonemes and syllables) that are critical for acoustic fidelity. An ablation study comparing LSTM against Transformerbased adaptors for this task confirmed that LSTMs yielded superior mel-spectrogram reconstruction fidelity (higher R²), as detailed in Table S1, likely by avoiding the oversmoothing of spectrotemporal details sometimes induced by the strong global context modeling of Transformers”.

      “To confirm that the acoustic pathway’s output is causally dependent on the neural signal rather than the generative prior of the HiFi-GAN, we performed a control analysis in which portions of the input ECoG recording were replaced with Gaussian noise. When either the first half, second half, or the entirety of the neural input was replaced by noise, the melspectrogram R² of the reconstructed speech dropped markedly, corresponding to the corrupted segment (Fig. S5). This demonstrates that the reconstruction is temporally locked to the specific neural input and that the model does not ‘hallucinate’ spectrotemporal structure from noise. These results validate that the acoustic pathway performs genuine, input-sensitive neural decoding”.

      Edits:

      Page 10, Lines 272-277:

      “We employed a Transformer-based Seq2Seq architecture for this adaptor to effectively capture the long-range contextual dependencies across a sentence, which are essential for resolving lexical ambiguity and syntactic structure during word token decoding. This choice was validated by an ablation study (Table S2), indicating that the Transformer adaptor outperformed an LSTM-based counterpart in word prediction accuracy”

      (3) The model is trained on approximately 20 minutes of data per participant, which raises concerns about potential overfitting. It would be helpful if the authors could analyze whether test sentences with higher or lower reconstruction performance include words that were also present in the training set.

      Thank you for raising the important concern regarding potential overfitting given the limited size of our training dataset (~20 minutes per participant). To address this point directly, we performed a detailed lexical overlap analysis between the training and test sets.

      The test set contains 219 unique words. Among these:

      127 words (58.0%) appeared in the training set (primarily high-frequency, common words).

      92 words (42.0%) were entirely novel and did not appear in the training set. We further examined whether trials with the best reconstruction (WER = 0) relied more on training vocabulary. Among these top-performing trials, 55.0% of words appeared in the training set. In contrast, the worst-performing trials showed 51.9% overlap in words in the training set. No significant difference was observed, suggesting that performance is not driven by simple lexical memorization.

      The presence of a substantial proportion of novel words (42%) in the test set, combined with the lack of performance advantage for overlapping content, provides strong evidence that our model is generalizing linguistic and acoustic patterns rather than merely memorizing the training vocabulary. High reconstruction performance on unseen words would be improbable under severe overfitting.

      Therefore, we conclude that while some lexical overlap exists (as expected in natural language), the model’s performance is driven by its ability to decode generalized neural representations, effectively mitigating the overfitting risk highlighted by the reviewer.

      (4) The phoneme confusion matrix in Figure 4A does not appear to align with human phoneme confusion patterns. For instance, /s/ and /z/ differ only in voicing, yet the model does not seem to confuse these phonemes. Does this imply that the model and the human brain operate differently at the mechanistic level?

      We thank the reviewer for this detailed observation regarding the difference between our model's phoneme confusion patterns and typical human perceptual confusions (e.g., the lack of /s/-/z/ confusion).

      The reviewer is correct in inferring a mechanistic difference. This divergence is primarily attributable to the Parler-TTS model acting as a powerful linguistic prior. Our linguistic pathway decodes word tokens, which Parler-TTS then converts to speech. Trained on massive corpora to produce canonical pronunciations, Parler-TTS effectively performs an implicit "error correction." For instance, if the neural decoding is ambiguous between the words "sip" and "zip," the TTS model's strong prior for lexical and syntactic context will likely resolve it to the correct word, thereby suppressing purely acoustic confusions like voicing.

      This has important implications for interpreting our model's errors and its relationship to brain function. The phoneme errors in our final output reflect a combination of neural decoding errors and the generative biases of the TTS model, which is optimized for intelligibility rather than mimicking raw human misperception. This does imply our model operates differently from the human auditory periphery. The human brain may first generate a percept with acoustic confusions, which higher-level language regions then disambiguate. Our model effectively bypasses the "confused percept" stage by directly leveraging a pre-trained, high-level language model for disambiguation. This is a design feature contributing to its high intelligibility, not necessarily a flaw. This observation raises a fascinating question: Could a model that more faithfully simulates the hierarchical processing of the human brain (including early acoustic confusions) provide a better fit to neural data at different processing stages? Future work could further address this question.

      Edits:

      add another paragraph in Discussion (Page 14, Lines 397-398):

      “The phoneme confusion pattern observed in our model output (Fig. 4A) differs from classic human auditory confusion matrices. We attribute this divergence primarily to the influence of the Parler-TTS model, which serves as a strong linguistic prior in our pipeline. This component is trained to generate canonical speech from text tokens. When the upstream neural decoding produces an ambiguous or erroneous token sequence, the TTS model’s internal language model likely performs an implicit ‘error correction,’ favoring linguistically probable words and pronunciations. This underscores that our model’s errors arise from a complex interaction between neural decoding fidelity and the generative biases of the synthesis stage”

      (5) In general, is the motivation for adopting the dual-pathway model to better align with the organization of the human brain, or to achieve improved engineering performance? If the goal is primarily engineeringoriented, the authors should compare their approach with a pretrained multimodal LLM rather than relying on the dual-pathway architecture. Conversely, if the design aims to mirror human brain function, additional analysis, such as detailed comparisons of phoneme confusion matrices, should be included to demonstrate that the model exhibits brain-like performance patterns.

      Our primary motivation is engineering improvement, to overcome the fundamental trade-off between acoustic fidelity and linguistic intelligibility that has limited previous neural speech decoding work. The design is inspired by the related works of the convergent representation of speech and language perception (2). However, we do not claim that our LSTM and Transformer adaptors precisely simulate the specific neural computations of the human ventral and dorsal streams. The goal was to build a high-performance, data-efficient decoder. We will clarify this point in the Introduction and Discussion, stating that while the architecture is loosely inspired by previous neuroscience results, its primary validation is its engineering performance in achieving state-of-the-art reconstruction quality with minimal data.

      Edits:

      Page 14, Line 358-373:

      “In this study, we present a dual-path framework that synergistically decodes both acoustic and linguistic speech representations from ECoG signals, followed by a fine-tuned zero-shot text-to-speech network to re-synthesize natural speech with unprecedented fidelity and intelligibility. Crucially, by integrating large pre-trained generative models into our acoustic reconstruction pipeline and applying voice cloning technology, our approach preserves acoustic richness while significantly enhancing linguistic intelligibility beyond conventional methods. Our dual-pathway architecture, while inspired by converging neuroscience insights on speech and language perception, was principally designed and validated as an engineering solution. The primary goal to build a practical decoder that achieves state-of-theart reconstruction quality with minimal data. The framework's success is therefore ultimately judged by its performance metrics, high intelligibility (WER, PER), acoustic fidelity (melspectrogram R²), and perceptual quality (MOS), which directly address the core engineering challenge we set out to solve. Using merely 20 minutes of ECoG recordings, our model achieved superior performance with a WER of 18.9% ± 3.3% and PER of 12.0% ± 2.5% (Fig. 2D, E). This integrated architecture, combining pre-trained acoustic (Wav2Vec2.0 and HiFiGAN) and linguistic (Parler-TTS) models through lightweight neural adaptors, enables efficient mapping of ECoG signals to dual latent spaces. Such methodology substantially reduces the need for extensive neural training data while achieving breakthrough word clarity under severe data constraints. The results demonstrate the feasibility of transferring the knowledge embedded in speech-data pre-trained artificial intelligence (AI) models into neural signal decoding, paving the way for more advanced brain-computer interfaces and neuroprosthetics”.

      Reviewer #2 (Public review):

      Summary:

      The study by Li et al. proposes a dual-path framework that concurrently decodes acoustic and linguistic representations from ECoG recordings. By integrating advanced pre-trained AI models, the approach preserves both acoustic richness and linguistic intelligibility, and achieves a WER of 18.9% with a short (~20-minute) recording.

      Overall, the study offers an advanced and promising framework for speech decoding. The method appears sound, and the results are clear and convincing. My main concerns are the need for additional control analyses and for more comparisons with existing models.

      Strengths:

      (1) This speech-decoding framework employs several advanced pre-trained DNN models, reaching superior performance (WER of 18.9%) with relatively short (~20-minute) neural recording.

      (2) The dual-pathway design is elegant, and the study clearly demonstrates its necessity: The acoustic pathway enhances spectral fidelity while the linguistic pathway improves linguistic intelligibility.

      We thank Reviewer #2 for supportive comments. In addition, we appreciate Reviewer #2’s thoughtful comments and feedback. By addressing these comments, we believe we have greatly improved the clarity of our claims and methodology. Below we list our point-to-point responses addressing concerns raised by Reviewer #2.

      Weaknesses:

      The DNNs used were pre-trained on large corpora, including TIMIT, which is also the source of the experimental stimuli. More generally, as DNNs are powerful at generating speech, additional evidence is needed to show that decoding performance is driven by neural signals rather than by the DNNs' generative capacity.

      Thank you for raising this crucial point regarding the potential for pre-trained DNNs to generate speech independently of the neural input. We fully agree that it is essential to disentangle the contribution of the neural signals from the generative priors of the models. To address this directly, we have conducted two targeted control analyses, as you suggested, and have integrated the results into the revised manuscript (see Fig. S5 and the corresponding description in the Results section):

      (1) Random noise input: We fed Gaussian noise (matched in dimensionality and temporal structure to real ECoG recordings) into the trained adaptors. The outputs were acoustically unstructured and linguistically incoherent, confirming that the generative models alone cannot produce meaningful speech without valid neural input.

      (2) Partial sentence input (real + noise): For the acoustic pathway, we systematically replaced portions of the ECoG input with noise. The reconstruction quality (mel-spectrogram R²) dropped significantly in the corrupted segments, demonstrating that the decoding is temporally locked to the neural signal and does not “hallucinate” speech from noise.

      These results provide strong evidence that our model’s performance is causally dependent on and sensitive to the specific neural input, validating that it performs genuine neural decoding rather than merely leveraging the generative capacity of the pre-trained DNNs.

      The detailed edits are in the “recommendations” below. (See recommendations (1) and (2))

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) Clarify the results shown in Figure 4E. The integrated approach appears to perform comparably to Baseline 3 in phoneme class clarity. However, Baseline 3 represents the output of the linguistic pathway alone, which is expected to encode information primarily at the word level.

      We appreciate the reviewer's observation and agree that clarification is needed. The phoneme class clarity (PCC) metric shown in Figure 4E measures whether mis-decoded phonemes are more likely to be confused within their own class (vowel-vowel or consonantconsonant) rather than across classes (vowel-consonant). A higher PCC indicates that the model's errors tend to be phonologically similar sounds (e.g., one vowel mistaken for another), which is a reasonable property for intelligibility.

      We would like to clarify the nature of Baseline 3. As stated in the manuscript (Results section: "The linguistic pathway reconstructs high-intelligibility, higher-level linguistic information"), Baseline 3 is the output of our linguistic pathway. This pathway operates as follows: the ECoG signals are mapped to word tokens via the Transformer adaptor, and these tokens are then synthesized into speech by the frozen Parler-TTS model. Crucially, the input to Parler-TTS is a sequence of word tokens.

      It is important to distinguish between the levels of performance measured: Word Error Rate (WER) reflects accuracy at the lexical level (whole words). The linguistic pathway achieves a low WER by design, as it directly decodes word sequences. Phoneme Error Rate (PER) reflects accuracy at the sublexical phonetic level (phonemes). A low WER generally implies a low PER, because robust word recognition requires reliable phoneme-level representations within the TTS model's prior. This explains why Baseline 3 also exhibits a low PER. However, acoustic fidelity (captured by metrics like mel-spectrogram R²) requires the preservation of fine-grained spectrotemporal details such as pitch, timbre, prosody, and formant structures, information that is not directly encoded at the lexical level and is therefore not a strength of the purely linguistic pathway.

      While Parler-TTS internally models sub-word/phonetic information to generate the acoustic waveform, the primary linguistic information driving the synthesis is at the lexical (word) level. The generated speech from Baseline 3 therefore contains reconstructed phonemic sequences derived from the decoded word tokens, not from direct phoneme-level decoding of ECoG.

      Therefore, the comparable PCC between our final integrated model and Baseline 3 (linguistic pathway) suggests that the phoneme-level error patterns (i.e., the tendency to confuse within-class phonemes) in our final output are largely inherited from the high-quality linguistic prior embedded in the pre-trained TTS model (Parler-TTS). The integrated framework successfully preserves this desirable property from the linguistic pathway while augmenting it with speaker-specific acoustic details from the acoustic pathway, thereby achieving both high intelligibility (low WER/PER) and high acoustic fidelity (high melspectrogram R²).

      We will revise the caption of Figure 4E and the corresponding text in the Results section to make this interpretation explicit.

      Edits:

      Page 12, Lines 317-322:

      “In addition to the confusion matrices, we categorized the phonemes into vowels and consonants to assess the phoneme class clarity. We defined "phoneme class clarity" (PCC) as the proportion of errors where a phoneme was misclassified within the same class versus being misclassified into a different class. The purpose of introducing PCC is to demonstrate that most of the misidentified phonemes belong to the same category (confusion between vowels or consonants), rather than directly comparing the absolute accuracy of phoneme recognition. For instance, a vowel being mistaken for another vowel would be considered a within-class error, whereas a vowel being mistaken for a consonant would be classified as a between-class error” 

      (2) Add results from Baseline 2 + CosyVoice and Baseline 3 + CosyVoice to clarify the contribution of the auditory pathway.

      Thank you for the suggestion. We appreciate the opportunity to clarify the role of CosyVoice in our framework.

      As explained in our response to point (1), CosyVoice 2.0 is designed as a fusion module that requires two inputs: 1) a voice reference (from the acoustic pathway) to specify speaker identity, and 2) a word sequence (from the linguistic pathway) to specify linguistic content. Because it is not a standalone enhancer, applying CosyVoice to a single pathway output (e.g., Baseline 2 or 3 alone) is not quite feasible and would not reflect its intended function and could lead to misinterpretation of each pathway’s contribution.

      Instead, we have evaluated the contribution of each pathway by comparing the final integrated output against each standalone pathway output (Baseline 2 and 3). The significant improvements in both acoustic fidelity and linguistic intelligibility demonstrate the complementary roles of the two pathways, which are effectively fused through CosyVoice.

      (3) Justify your choice of using LSTM and Transformer architecture for the auditory and linguistic neural adaptors, respectively, and how your methods could compare to using a unified generative multimodal LLM for both pathways.

      Thank you for revisiting this important point. We appreciate your interest in the architectural choices and their relationship to state-of-the-art multimodal models.

      As detailed in our response to point (2), our choice of LSTM for the acoustic pathway and Transformer for the linguistic pathway is driven by task-specific requirements, supported by ablation studies (Supplementary Tables 1–2). The acoustic pathway benefits from LSTM’s ability to model fine-grained, local temporal dependencies without over-smoothing. The linguistic pathway benefits from Transformer’s ability to capture long-range semantic and syntactic context.

      Regarding comparison with unified multimodal LLMs (e.g., Qwen3-Omni), we clarified that such models are optimized for interactive dialogue and multimodal understanding, while our framework relies on specialist models (CosyVoice 2.0, Parler-TTS) that are explicitly designed for high-fidelity, speaker-adaptive speech synthesis, a requirement central to our decoding task.

      We have incorporated these justifications into the revised manuscript (Results and Discussion sections) and appreciate the opportunity to further emphasize these points.

      Edits:

      Page 9, Lines 214-223:

      “The acoustic pathway, implemented through a bi-directional LSTM neural adaptor architecture (Fig. 1B), specializes in reconstructing fundamental acoustic properties of speech. This module directly processes neural recordings to generate precise time-frequency representations, focusing on preserving speaker-specific spectral characteristics like formant structures, harmonic patterns, and spectral envelope details. Quantitative evaluation confirms its core competency: achieving a mel-spectrogram R² of 0.793 ± 0.016 (Fig. 3B) demonstrates remarkable fidelity in reconstructing acoustic microstructure. This performance level is statistically indistinguishable from original speech degraded by 0dB additive noise (0.771 ± 0.014, p = 0.242, one-sided t-test). We chose a bidirectional LSTM architecture for this adaptor because its recurrent nature is particularly suited to modeling the fine-grained, short- to medium-range temporal dependencies (e.g., within and between phonemes and syllables) that are critical for acoustic fidelity. An ablation study comparing LSTM against Transformerbased adaptors for this task confirmed that LSTMs yielded superior mel-spectrogram reconstruction fidelity (higher R²), as detailed in Table S1, likely by avoiding the oversmoothing of spectrotemporal details sometimes induced by the strong global context modeling of Transformers”.

      “To confirm that the acoustic pathway’s output is causally dependent on the neural signal rather than the generative prior of the HiFi-GAN, we performed a control analysis in which portions of the input ECoG recording were replaced with Gaussian noise. When either the first half, second half, or the entirety of the neural input was replaced by noise, the melspectrogram R² of the reconstructed speech dropped markedly, corresponding to the corrupted segment (Fig. S5). This demonstrates that the reconstruction is temporally locked to the specific neural input and that the model does not ‘hallucinate’ spectrotemporal structure from noise. These results validate that the acoustic pathway performs genuine, input-sensitive neural decoding”.

      Page 10, Lines 272-277:

      “We employed a Transformer-based Seq2Seq architecture for this adaptor to effectively capture the long-range contextual dependencies across a sentence, which are essential for resolving lexical ambiguity and syntactic structure during word token decoding. This choice was validated by an ablation study (Table S2), indicating that the Transformer adaptor outperformed an LSTM-based counterpart in word prediction accuracy”.

      (4) Discuss the differences between the model's phoneme confusion matrix in Figure 4A and human phoneme confusion patterns. In addition, please clarify whether the adoption of the dual-pathway architecture is primarily intended to simulate the organization of the human brain or to achieve engineering improvements.

      The observed difference between our model's phoneme confusion matrix and typical human perceptual confusion patterns (e.g., the noted lack of confusion between /s/ and /z/) is, as the reviewer astutely infers, likely attributable to the TTS model (Parler-TTS) acting as a powerful linguistic prior. The linguistic pathway decodes word tokens, and Parler-TTS converts these tokens into speech. Parler-TTS is trained on massive text and speech corpora to produce canonical, clean pronunciations. It effectively performs a form of "error correction" or "canonicalization" based on its internal language model. For example, if the neural decoding is ambiguous between "sip" and "zip", the TTS model's strong prior for lexical and syntactic context may robustly resolve it to the correct word, suppressing purely acoustic confusions like voicing. Therefore, the phoneme errors in our final output reflect a combination of neural decoding errors and the TTS model's generation biases, which are optimized for intelligibility rather than mimicking human misperception. We will add this explanation to the paragraph discussing Figure 4A.

      Our primary motivation is engineering improvement, to overcome the fundamental tradeoff between acoustic fidelity and linguistic intelligibility that has limited previous neural speech decoding work. The design is inspired by the convergent representation of speech and language perception (1). However, we do not claim that our LSTM and Transformer adaptors precisely simulate the specific neural computations of the human ventral and dorsal streams. The goal was to build a high-performance, data-efficient decoder. We will clarify this point in the Introduction and Discussion, stating that while the architecture is loosely inspired by previous neuroscience results, its primary validation is its engineering performance in achieving state-of-the-art reconstruction quality with minimal data.

      Edits:

      Pages 2-3, Lines 74-85:

      “Here, we propose a unified and efficient dual-pathway decoding framework that integrates the complementary strengths of both paradigms to enhance the performance of re-synthesized natural speech from the engineering performance. Our method maps intracranial electrocorticography (ECoG) signals into the latent spaces of pre-trained speech and language models via two lightweight neural adaptors: an acoustic pathway, which captures low-level spectral features for naturalistic speech synthesis, and a linguistic pathway, which extracts high-level linguistic tokens for semantic intelligibility. These pathways are fused using a finetuned text-to-speech (TTS) generator with voice cloning, producing re-synthesized speech that retains both the acoustic spectrotemporal details, such as the speaker’s timbre and prosody, and the message linguistic content. The adaptors rely on near-linear mappings and require only 20 minutes of neural data per participant for training, while the generative modules are pre-trained on large unlabeled corpora and require no neural supervision”.

      Page 14, Lines 358-373:

      “In this study, we present a dual-path framework that synergistically decodes both acoustic and linguistic speech representations from ECoG signals, followed by a fine-tuned zero-shot text-to-speech network to re-synthesize natural speech with unprecedented fidelity and intelligibility. Crucially, by integrating large pre-trained generative models into our acoustic reconstruction pipeline and applying voice cloning technology, our approach preserves acoustic richness while significantly enhancing linguistic intelligibility beyond conventional methods. Our dual-pathway architecture, while inspired by converging neuroscience insights on speech and language perception, was principally designed and validated as an engineering solution. The primary goal to build a practical decoder that achieves state-of-the-art reconstruction quality with minimal data. The framework's success is therefore ultimately judged by its performance metrics, high intelligibility (WER, PER), acoustic fidelity (mel-spectrogram R²), and perceptual quality (MOS), which directly address the core engineering challenge we set out to solve. Using merely 20 minutes of ECoG recordings, our model achieved superior performance with a WER of 18.9% ± 3.3% and PER of 12.0% ± 2.5% (Fig. 2D, E). This integrated architecture, combining pre-trained acoustic (Wav2Vec2.0 and HiFi-GAN) and linguistic (Parler-TTS) models through lightweight neural adaptors, enables efficient mapping of ECoG signals to dual latent spaces. Such methodology substantially reduces the need for extensive neural training data while achieving breakthrough word clarity under severe data constraints. The results demonstrate the feasibility of transferring the knowledge embedded in speech-data pre-trained artificial intelligence (AI) models into neural signal decoding, paving the way for more advanced brain-computer interfaces and neuroprosthetics”.

      Reviewer #2 (Recommendations for the authors):

      (1) My main question is whether any experimental stimuli overlap with the data used to pre-train the models. The authors might consider using pre-trained models trained on other corpora and training their own model without the TIMIT corpus. Additionally, as pretrained models were used, it might be helpful to evaluate to what extent the decoding is sensitive to the input neural recording or whether the model always outputs meaningful speech. The authors might consider two control analyses: a) whether the model still generates speech-like output if the input is random noise; b) whether the model can decode a complete sentence if the first half recording of a sentence is real but the second half is replaced with noise.

      We thank the reviewer for raising this crucial point regarding potential data leakage and the sensitivity of decoding to neural input.

      We confirm that the pre-training phase of our core models (Wav2Vec2.0 encoder, HiFiGAN decoder) was conducted exclusively on the LibriSpeech corpus (960 hours), which is entirely separate from the TIMIT corpus used for our ECoG experiments. The subsequent fine-tuning of the CosyVoice 2.0 voice cloner for speaker adaptation was performed on the training set portion of the entire TIMIT corpus. Importantly, the test set for all neural decoding evaluations was strictly held out and never used during any fine-tuning stage. This data separation is now explicitly stated in the " Methods" sections for the Speech Autoencoder and the CosyVoice fine-tuning.

      Regarding the potential of training on other corpora, we agree it is a valuable robustness check. Previous work has demonstrated that self-supervised speech models like Wav2Vec2.0 learn generalizable representations that transfer well across domains (e.g., Millet et al., NeurIPS 2022). We believe our use of LibriSpeech, a large and diverse corpus, provides a strong, general-purpose acoustic prior.

      We agree with the reviewer that control analyses are essential to demonstrate that the decoded output is driven by neural signals and not merely the generative prior of the models. We have conducted the following analyses and will include them in the revised manuscript (likely in a new Supplementary Figure or Results subsection):

      (a) Random Noise Input: We fed Gaussian noise (matched in dimensionality and temporal length to the real ECoG input) into the trained acoustic and linguistic adaptors. The outputs were evaluated. The acoustic pathway generated unstructured, noisy spectrograms with no discernible phonetic structure, and the linguistic pathway produced either highly incoherent word sequences or failed to generate meaningful tokens. The fusion via CosyVoice produced unintelligible babble. This confirms that the generative models alone cannot produce structured speech without meaningful neural input.

      (b) Partial Sentence Input (Real + Noise): In the acoustic pathway, we replaced the first half, the second half, and all the ECoG recording for test sentences with Gaussian noise. The melspectrogram R<sup>2</sup> showed a clear degradation in the reconstructed speech corresponding to the noisy segment. We did not do similar experiments in the linguistic pathway because the TTS generator is pre-trained by HuggingFace. We did not train any parameters of Parler-TTS. These results strongly indicate that our model's performance is contingent on and sensitive to the specific neural input, validating that it is performing genuine neural decoding.

      Edits:

      Page 19, Lines 533-538:

      “The parameters in Wav2Vec2.0 were frozen within this training phase. The parameters in HiFi-GAN were optimized using the Adam optimizer with a fixed learning rate of 10<sub>-5</sub>, 𝛽<sub>!</sub> = 0.9, 𝛽<sub>2</sub> = 0.999. We trained this Autoencoder in LibriSpeech, a 960-hour English speech corpus with a sampling rate of 16kHz, which is entirely separate from the TIMIT corpus used for our ECoG experiments. We spent 12 days in parallel training on 6 Nvidia GeForce RTX3090 GPUs. The maximum training epoch was 2000. The optimization did not stop until the validation loss no longer decreased”.

      Edits:

      Page9, Lines214-223:

      “The acoustic pathway, implemented through a bi-directional LSTM neural adaptor architecture (Fig. 1B), specializes in reconstructing fundamental acoustic properties of speech. This module directly processes neural recordings to generate precise time-frequency representations, focusing on preserving speaker-specific spectral characteristics like formant structures, harmonic patterns, and spectral envelope details. Quantitative evaluation confirms its core competency: achieving a mel-spectrogram R² of 0.793 ± 0.016 (Fig. 3B) demonstrates remarkable fidelity in reconstructing acoustic microstructure. This performance level is statistically indistinguishable from original speech degraded by 0dB additive noise (0.771 ± 0.014, p = 0.242, one-sided t-test). We chose a bidirectional LSTM architecture for this adaptor because its recurrent nature is particularly suited to modeling the fine-grained, short- to medium-range temporal dependencies (e.g., within and between phonemes and syllables) that are critical for acoustic fidelity. An ablation study comparing LSTM against Transformer-based adaptors for this task confirmed that LSTMs yielded superior mel-spectrogram reconstruction fidelity (higher R²), as detailed in Table S1, likely by avoiding the oversmoothing of spectrotemporal details sometimes induced by the strong global context modeling of Transformers”.

      “To confirm that the acoustic pathway’s output is causally dependent on the neural signal rather than the generative prior of the HiFi-GAN, we performed a control analysis in which portions of the input ECoG recording were replaced with Gaussian noise. When either the first half, second half, or the entirety of the neural input was replaced by noise, the melspectrogram R² of the reconstructed speech dropped markedly, corresponding to the corrupted segment (Fig. S5). This demonstrates that the reconstruction is temporally locked to the specific neural input and that the model does not ‘hallucinate’ spectrotemporal structure from noise. These results validate that the acoustic pathway performs genuine, input-sensitive neural decoding”

      (2) For BCI applications, the decoding speed matters. Please report the model's inference speed. Additionally, the authors might also consider reporting cross-participant generalization and how the accuracy changes with recording duration.

      We thank the reviewer for these practical and important suggestions. 

      Inference Speed: You are absolutely right. On our hardware (single NVIDIA GeForce RTX 3090 GPU), the current pipeline has an inference time that is longer than the duration of the target speech segment. The primary bottlenecks are the sequential processing in the autoregressive linguistic adaptor and the high-resolution waveform generation in CosyVoice 2.0. This latency currently limits real-time application. We have now added this in the Discussion acknowledging this limitation and stating that future work must focus on architectural optimizations (e.g., non-autoregressive models, lighter vocoders) and potential hardware acceleration to achieve real-time performance, which is critical for a practical BCI.

      Cross-Participant Generalization: We agree that this is a key question for scalability. Our framework already addresses part of the cross-participant generalization challenge through the use of pre-trained generative modules (HiFi-GAN, Parler-TTS, CosyVoice 2.0), which are pretrained on large corpora and shared across all participants. Only a small fraction of the model, the lightweight neural adaptors, is subject-specific and requires a small amount of supervised fine-tuning (~20 minutes per participant). This design significantly reduces the per-subject calibration burden. As the reviewer implies, the ultimate goal would be pure zero-shot generalization. A promising future direction is to further improve cross-participant alignment by learning a shared neural feature encoder (e.g., using contrastive or self-supervised learning on aggregated ECoG data) before the personalized adaptors. We have added a paragraph in the Discussion outlining this as a major next step to enhance the framework’s practicality and further reduce calibration time.

      Accuracy vs. Recording Duration: Thank you for this insightful suggestion. To systematically evaluate the impact of training data volume on performance, we have conducted additional experiments using progressively smaller subsets of the full training set (i.e., 25%, 50%, and 75%). When we used more than 50% of the training data, performance degrades gracefully rather than catastrophically with less data, which is promising for potential clinical scenarios where data collection may be limited. We add another figure (Fig. S4) to demonstrate this.

      Edits:

      Pages 15-16, Lines 427-452:

      “There are several limitations in our study. The quality of the re-synthesized speech heavily relies on the performance of the generative model, indicating that future work should focus on refining and enhancing these models. Currently, our study utilized English speech sentences as input stimuli, and the performance of the system in other languages remains to be evaluated. Regarding signal modality and experimental methods, the clinical setting restricts us to collecting data during brief periods of awake neurosurgeries, which limits the amount of usable neural activity recordings. Overcoming this time constraint could facilitate the acquisition of larger datasets, thereby contributing to the re-synthesis of higher-quality natural speech. Furthermore, the inference speed of the current pipeline presents a challenge for real-time applications. On our hardware (a single NVIDIA GeForce RTX 3090 GPU), synthesizing speech from neural data takes approximately two to three times longer than the duration of the target speech segment itself. This latency is primarily attributed to the sequential processing in the autoregressive linguistic adaptor and the computationally intensive high-fidelity waveform generation in the vocoder (CosyVoice 2.0). While the current study focuses on offline reconstruction accuracy, achieving real-time or faster-than-real-time inference is a critical engineering goal for viable speech BCI prosthetics. Future work must therefore prioritize architectural optimizations, such as exploring non-autoregressive decoding strategies and more efficient neural vocoders, alongside potential hardware acceleration. Additionally, exploring non-invasive methods represents another frontier; with the accumulation of more data and the development of more powerful generative models, it may become feasible to achieve effective non-invasive neural decoding for speech resynthesis. Moreover, while our framework adopts specialized architectures (LSTM and Transformer) for distinct decoding tasks, an alternative approach is to employ a unified multimodal large language model (LLM) capable of joint acoustic-linguistic processing. Finally, the current framework requires training participant-specific adaptors, which limits its immediate applicability for new users. A critical next step is to develop methods that learn a shared, cross-participant neural feature encoder, for instance, by applying contrastive or selfsupervised learning techniques to larger aggregated ECoG datasets. Such an encoder could extract subject-invariant neural representations of speech, serving as a robust initialization before lightweight, personalized fine-tuning. This approach would dramatically reduce the amount of per-subject calibration data and time required, enhancing the practicality and scalability of the decoding framework for real-world BCI applications”

      “In summary, our dual-path framework achieves high speech reconstruction quality by strategically integrating language models for lexical precision and voice cloning for vocal identity preservation, yielding a 37.4% improvement in MOS scores over conventional methods. This approach enables high-fidelity, sentence-level speech synthesis directly from cortical recordings while maintaining speaker-specific vocal characteristics. Despite current constraints in generative model dependency and intraoperative data collection, our work establishes a new foundation for neural decoding development. Future efforts should prioritize: (1) refining few-shot adaptation techniques, (2) developing non-invasive implementations, (3) expanding to dynamic dialogue contexts, and (4) cross-subject applications. The convergence of neurophysiological data with multimodal foundation models promises transformative advances, not only revolutionizing speech BCIs but potentially extending to cognitive prosthetics for memory augmentation and emotional communication. Ultimately, this paradigm will deepen our understanding of neural speech processing while creating clinically viable communication solutions for those with severe speech impairments”

      Edits: 

      add another section in Methods: Page 22, Line 681:

      “Ablation study on training data volume”.

      “To assess the impact of training data quantity on decoding performance, we conducted an additional ablation experiment. For each participant, we created subsets of the full training set corresponding to 25%, 50%, and 75% of the original data by random sampling while preserving the temporal continuity of speech segments. Personalized acoustic and linguistic adaptors were then independently trained from scratch on each subset, following the identical architecture and optimization procedures described above. All other components of the pipeline, including the frozen pre-trained generators (HiFi-GAN, Parler-TTS) and the CosyVoice 2.0 voice cloner, remained unchanged. Performance metrics (mel-spectrogram R², WER, PER) were evaluated on the same held-out test set for all data conditions. The results (Fig. S4) demonstrate that when more than 50% of the training data is utilized, performance degrades gracefully rather than catastrophically, which is a promising indicator for clinical applications with limited data collection time”.

      (3) I appreciate that the author compared their model with the MLP, but more comparisons with previous models could be beneficial. Even simply summarizing some measures of earlier models, such as neural recording duration, WER, PER, etc., is ok.

      Thank you for this suggestion. We agree that a broader comparison contextualizes our contribution. We also acknowledge that given the differences in tasks, signal modality, and amount of data, it’s hard to draw a direct comparison. The main goal of this table is to summarize major studies, their methods and results for reference. We have now added a new Supplementary Table that summarizes key metrics from several recent and relevant studies in neural speech decoding. The table includes:

      - Neural modality (e.g., ECoG, sEEG, Utah array)

      - Approximate amount of neural data used per subject for decoder training

      - Primary task (perception vs. production)

      -Decoding framework

      -Reported Word Error Rate (WER) or similar intelligibility metrics (e.g., Character Error Rate)

      -Reported acoustic fidelity metrics (if available, e.g., spectral correlation)

      This table includes works such as Anumanchipalli et al., Nature 2019; Akbari et al., Sci Rep 2019; Willett et al., Nature 2023; and other contemporary studies. The table clearly shows that our dual-path framework achieves a highly competitive WER (~18.9%) using an exceptionally short neural recording duration (~20 minutes), highlighting its data efficiency. We will refer to this table in the revised manuscript.

      Edits:

      Page 14, Lines 374-376:

      “Our framework establishes a framework for speech decoding by outperforming prior acousticonly or linguistic-only approaches (Table S3) through integrated pretraining-powered acoustic and linguistic decoding”

      Minor:

      (1) Some processes might be described earlier, for example, the electrodes were selected, and the model was trained separately for each participant. That information was only described in the Method section now.

      Thank you for catching these. We have revised the manuscript accordingly.

      Edits:

      Page4, Lines 89-95:

      “Our proposed framework for reconstructing speech from intracranial neural recordings is designed around two complementary decoding pathways: an acoustic pathway focused on preserving low-level spectral and prosodic detail, and a linguistic pathway focused on decoding high-level textual and semantic content. For every participant, our adaptor is independently trained, and we select speech-responsive electrodes (selection details are provided in the Methods section) to tailor the model to individual neural patterns. These two streams are ultimately fused to synthesize speech that is both natural-sounding and intelligible, capturing the full richness of spoken language. Fig. 1 provides a schematic overview of this dual-pathway architecture”

      (2) Line 224-228 Figure 2 should be Figure 3

      Thank you for catching these. We have revised the manuscript accordingly. The information about participant-specific training and electrode selection is now briefly mentioned in the "Results" overview (section: "The acoustic and linguistic performance..."), with details still in the Methods. The figure reference error has been corrected.

      Edits:

      Page7, Lines 224-228:

      “However, exclusive reliance on acoustic reconstruction reveals fundamental limitations. Despite excellent spectral fidelity, the pathway produces critically impaired linguistic intelligibility. At the word level, intelligibility remains unacceptably low (WER = 74.6 ± 5.5%, Fig. 3D), while MOS and phoneme-level precision fares only marginally better (MOS = 2.878 ± 0.205, Fig. 3C; PER = 28.1 ± 2.2%, Fig. 3E)”.

      (3) For Figure 3C, why does the MOS seem to be higher for baseline 3 than for ground truth? Is this significant?

      This is a detailed observation. Baseline 3 achieves a mean opinion score of 4.822 ± 0.086 (Fig. 3C), significantly surpassing even the original human speech (4.234 ± 0.097, p = 6.674×10⁻33). We believe this trend arises because the TIMIT corpus, recorded decades ago, contains inherent acoustic noise and relatively lower fidelity compared to modern speech corpus. In contrast, the Parler-TTS model used in Baseline 3 is trained on massive, highquality, clean speech datasets. Therefore, it synthesizes speech that listeners may subjectively perceive as "cleaner" or more pleasant, even if it lacks the original speaker's voice. Crucially, as the reviewer implies, our final integrated output does not aim to maximize MOS at the cost of speaker identity; it successfully balances this subjective quality with high intelligibility and restored acoustic fidelity. We will add a brief note explaining this possible reason in the caption of Figure 3C.

      Edits:

      Page9, Lines 235-245:

      “The linguistic pathway reconstructs high-intelligibility, higher-level linguistic information”

      “The linguistic pathway, instantiated through a pre-trained TTS generator (Fig. 1B), excels in reconstructing abstract linguistic representations. This module operates at the phonological and lexical levels, converting discrete word tokens into continuous speech signals while preserving prosodic contours, syllable boundaries, and phonetic sequences. It achieves a mean opinion score of 4.822 ± 0.086 (Fig. 3C) - significantly surpassing even the original human speech (4.234 ± 0.097, p = 6.674×10⁻33) in that the TIMIT corpus, recorded decades ago, contains inherent acoustic noise and relatively lower fidelity compared to modern speech corpus.  Complementing this perceptual quality, objective intelligibility metrics confirm outstanding performance: WER reaches 17.7 ± 3.2%, with PER at 11.0 ± 2.3%”.

      Reference

      (1) Chen M X, Firat O, Bapna A, et al. The best of both worlds: Combining recent advances in neural machine translation[C]//Proceedings of the 56th annual meeting of the association for computational linguistics (Volume 1: Long papers). 2018: 76-86

      (2) P. Chen et al. Do Self-Supervised Speech and Language Models Extract Similar Representations as Human Brain? 2024 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP 2024). 2225–2229 (2024).

      (3) H. Akbari, B. Khalighinejad, J. L. Herrero, A. D. Mehta, N. Mesgarani, Towards reconstructing intelligible speech from the human auditory cortex. Scientific reports 9, 874 (2019).

      (4) S. Komeiji et al., Transformer-Based Estimation of Spoken Sentences Using Electrocorticography. Int Conf Acoust Spee, 1311-1315 (2022).

      (5) L. Bellier et al., Music can be reconstructed from human auditory cortex activity using nonlinear decoding models. Plos Biology 21,  (2023).

      (6) F. R. Willett et al., A high-performance speech neuroprosthesis. Nature 620,  (2023).

      (7) S. L. Metzger et al., A high-performance neuroprosthesis for speech decoding and avatar control. Nature 620, 1037-1046 (2023).

      (8) J. W. Li et al., Neural2speech: A Transfer Learning Framework for NeuralDriven Speech Reconstruction. Int Conf Acoust Spee, 2200-2204 (2024).

      (9) X. P. Chen et al., A neural speech decoding framework leveraging deep learning and speech synthesis. Nat Mach Intell 6,  (2024).

      (10) M. Wairagkar et al., An instantaneous voice-synthesis neuroprosthesis. Nature,  (2025).

    1. Author response:

      The following is the authors’ response to the original reviews.

      Reviewer #1

      Chen et al. engineered and characterized a suite of next-generation GECIs for the Drosophila NMJ that allow for the visualization of calcium dynamics within the presynaptic compartment, at presynaptic active zones, and in the postsynaptic compartment. These GECIs include ratiometric presynaptic Scar8m (targeted to synaptic vesicles), ratiometric active zone localized Bar8f (targeted to the scaffold molecule BRP), and postsynaptic SynapGCaMP8m. The authors demonstrate that these new indicators are a large improvement on the widely used GCaMP6 and GCaMP7 series GECIs, with increased speed and sensitivity. They show that presynaptic Scar8m accurately captures presynaptic calcium dynamics with superior sensitivity to the GCaMP6 and GCaMP7 series and with similar kinetics to chemical dyes. The active-zone targeted Bar8f sensor was assessed for the ability to detect release-site-specific nanodomain changes, but the authors concluded that this sensor is still too slow to accurately do so. Lastly, the use of postsynaptic SynapGCaMP8m was shown to enable the detection of quantal events with similar resolution to electrophysiological recordings. Finally, the authors developed a Python-based analysis software, CaFire, that enables automated quantification of evoked and spontaneous calcium signals. These tools will greatly expand our ability to detect activity at individual synapses without the need for chemical dyes or electrophysiology.

      We thank this Reviewer for the overall positive assessment of our manuscript and for the incisive comments.

      (1) The role of Excel in the pipeline could be more clearly explained. Lines 182-187 could be better worded to indicate that CaFire provides analysis downstream of intensity detection in ImageJ. Moreover, the data type of the exported data, such as .csv or .xlsx, should be indicated instead of 'export to graphical program such as Microsoft Excel'.

      We thank the Reviewer for these comments, many of which were shared by the other reviewers. In response, we have now 1) more clearly explained the role of Excel in the CaFire pipeline (lines 677-681), 2) revised the wording in lines 676-679 to indicate that CaFire provides analysis downsteam of intensity detection in ImageJ, and 3) Clarified the exported data type to Excel (lines 677-681). These efforts have improved the clarity and readability of the CaFire analysis pipeline.

      (2) In Figure 2A, the 'Excel' step should either be deleted or included as 'data validation' as ImageJ exports don't require MS Excel or any specific software to be analysed. (Also, the graphic used to depict Excel software in Figure 2A is confusing.)

      We thank the reviewer for this helpful suggestion. In the Fig. 2A, we have changed the Excel portion and clarified the processing steps in the revised methods. Specifically, we now indicate that ROIs are first selected in Fiji/ImageJ and analyzed to obtain time-series data containing both the time information and the corresponding imaging mean intensity values. These data are then exported to a spreadsheet file (e.g., Excel), which is used to organize the output before being imported into CaFire for subsequent analysis. These changes can be found in the Fig. 2A and methods (lines 676-681).

      (3) Figure 2B should include the 'Partition Specification' window (as shown on the GitHub) as well as the threshold selection to give the readers a better understanding of how the tool works.

      We absolutely agree with this comment, and have made the suggested changes to the Fig. 2B. In particular, we have replaced the software interface panels and now include windows illustrating the Load File, Peak Detection, and Partition functions. These updated screenshots provide a clearer view of how CaFire is used to load the data, detect events, and perform partition specification for subsequent analysis. We agree these changes will give the readers a better understanding of how the tool works, and we thank the reviewer for this comment.

      (4) The presentation of data is well organized throughout the paper. However, in Figure 6C, it is unclear how the heatmaps represent the spatiotemporal fluorescence dynamics of each indicator. Does the signal correspond to a line drawn across the ROI shown in Figure 6B? If so, this should be indicated.

      We apologize that the heatmaps were unclear in Fig panel 6C (Fig. 7C in the Current revision). Each heatmap is derived from a one-pixel-wide vertical line within a miniature-event ROI. These heatmaps correspond to the fluorescence change in the indicated SynapGCaMP variant of individual quantal events and their traces shown in Fig. 7C, with a representative image of the baseline and peak fluorescence shown in Fig. 7B. Specifically, we have added the following to the revised Fig. 7C legend:

      The corresponding heatmaps below were generated from a single vertical line extracted from a representative miniature-event ROI, and visualize the spatiotemporal fluorescence dynamics (ΔF/F) along that line over time.

      (5) In Figure 6D, the addition of non-matched electrophysiology recordings is confusing. Maybe add "at different time points" to the end of the 6D legend, or consider removing the electrophysiology trace from Figure 6D and referring the reader to the traces in Figure 7A for comparison (considering the same point is made more rigorously in Figure 7).

      This is a good point, one shared with another reviewer. We apologize this was not clear, and have now revised this part of the figure to remove the electrophysiological traces in what is now Fig. 7 while keeping the paired ones still in what is now Fig. 8A as suggested by the reviewer. We agree this helps to clarify the quantal calcium transients.

      (6) In GitHub, an example ImageJ Script for analyzing the images and creating the inputs for CaFire would be helpful to ensure formatting compatibility, especially given potential variability when exporting intensity information for two channels. In the Usage Guide, more information would be helpful, such as how to select ∆R/R, ideally with screenshots of the application being used to analyze example data for both single-channel and two-channel images.

      We agree that additional details added to the GitHub would be helpful for users of CaFire. In response, we have now added the following improvements to the GitHub site: 

      - ImageJ operation screenshots

      Step-by-step illustrations of ROI drawing and Multi Measure extraction.

      - Example Excel file with time and intensity values

      Demonstrates the required data format for CaFire import, including proper headers.

      - CaFire loading screenshots for single-channel and dual-channel imaging

      Shows how to import GCaMP into Channel 1 and mScarlet into Channel 2.

      - Peak Detection and Partition setting screenshots

      Visual examples of automatic peak detection, manual correction, and trace partitioning.

      - Instructions for ROI Extraction and CaFire Analysis

      A written guide describing the full workflow from ROI selection to CaFire data export.

      These changes have improved the usability and accessibility of CaFire, and we thank the reviewer for these points.

      Reviewer #2

      Calcium ions play a key role in synaptic transmission and plasticity. To improve calcium measurements at synaptic terminals, previous studies have targeted genetically encoded calcium indicators (GECIs) to pre- and postsynaptic locations. Here, Chen et al. improve these constructs by incorporating the latest GCaMP8 sensors and a stable red fluorescent protein to enable ratiometric measurements. In addition, they develop a new analysis platform, 'CaFire', to facilitate automated quantification. Using these tools, the authors demonstrate favorable properties of their sensors relative to earlier constructs. Impressively, by positioning postsynaptic GCaMP8m near glutamate receptors, they show that their sensors can report miniature synaptic events with speed and sensitivity approaching that of intracellular electrophysiological recordings. These new sensors and the analysis platform provide a valuable tool for resolving synaptic events using all-optical methods.

      We thank the Reviewer for their overall positive evaluation and comments.

      Major comments:

      (1) While the authors rigorously compared the response amplitude, rise, and decay kinetics of several sensors, key parameters like brightness and photobleaching rates are not reported. I feel that including this information is important as synaptically tethered sensors, compared to freely diffusible cytosolic indicators, can be especially prone to photobleaching, particularly under the high-intensity illumination and high-magnification conditions required for synaptic imaging. Quantifying baseline brightness and photobleaching rates would add valuable information for researchers intending to adopt these tools, especially in the context of prolonged or high-speed imaging experiments.

      This is a good point made by the reviewer, and one we agree will be useful for researchers to be aware. First, it is important to note that the photobleaching and brightness of the sensors will vary depending on the nature of the user’s imaging equipment, which can vary significantly between widefield microscopes (with various LED or halogen light sources for illumination), laser scanning systems (e.g., line scans with confocal systems), or area scanning systems using resonant scanners (as we use in our current study). Under the same imaging settings, GCaMP8f and 8m exhibit comparable baseline fluorescence, whereas GCaMP6f and 6s are noticeably dimmer; because our aim is to assess each reagent’s potential under optimal conditions, we routinely adjust excitation/camera parameters before acquisition to place baseline fluorescence in an appropriate dynamic range. As an important addition to this study, motivated by the reviewer’s comments above, we now directly compare neuronal cytosolic GCaMP8m expression with our Scar8m sensor, showing higher sensitivity with Scar8m (now shown in the new Fig. 3F-H).

      Regarding photobleaching, GCaMP signals are generally stable, while mScarlet is more prone to bleaching: in presynaptic area scanned confocal recordings, the mScarlet channel drops by ~15% over 15 secs, whereas GCaMP6s/8f/8m show no obvious bleaching over the same window (lines 549-553). In contrast, presynaptic widefield imaging using an LED system (CCD), GCaMP8f shows ~8% loss over 15 secs (lines 610-611). Similarly, for postsynaptic SynapGCaMP6f/8f/8m, confocal resonant area scans show no obvious bleaching over 60 secs, while widefield shows ~2–5% bleaching over 60 secs (lines 634-638). Finally, in active-zone/BRP calcium imaging (confocal), mScarlet again bleaches by ~15% over 15 s, while GCaMP8f/8m show no obvious bleaching. The mScarlet-channel bleaching can be corrected in Huygens SVI (Bleaching correction or via the Deconvolution Wizard), whereas we avoid applying bleaching correction to the green GCaMP channel when no clear decay is present to prevent introducing artifacts. This information is now added to the methods (lines 548-553).

      (2) In several places, the authors compare the performance of their sensors with synthetic calcium dyes, but these comparisons are based on literature values rather than on side-by-side measurements in the same preparation. Given differences in imaging conditions across studies (e.g., illumination, camera sensitivity, and noise), parameters like indicator brightness, SNR, and photobleaching are difficult to compare meaningfully. Additionally, the limited frame rate used in the present study may preclude accurate assessment of rise times relative to fast chemical dyes. These issues weaken the claim made in the abstract that "...a ratiometric presynaptic GCaMP8m sensor accurately captures .. Ca²⁺ changes with superior sensitivity and similar kinetics compared to chemical dyes." The authors should clearly acknowledge these limitations and soften their conclusions. A direct comparison in the same system, if feasible, would greatly strengthen the manuscript.

      We absolutely agree with these points made the reviewer, and have made a concerted effort to address them through the following:

      We have now directly compared presynaptic calcium responses on the same imaging system using the chemical dye Oregon Green Bapta-1 (OGB-1), one of the primary synthetic calcium indicators used in our field. These experiments reveal that Scar8f exhibits markedly faster kinetics and an improved signal-to-noise ratio compared to OGB-1, with higher peak fluorescence responses (Scar8f: 0.32, OGB-1: 0.23). The rise time constants of the two indicators are comparable (both ~3 msecs), whereas the decay of Scar8f is faster than that of OGB-1 (Scar8f: ~40, OGB-1: ~60), indicating more rapid signal recovery. These results now directly demonstrate the superiority of the new GCaMP8 sensors we have engineered over conventional synthetic dyes, and are now presented in the new Fig. 3A-E of the manuscript.

      We agree with the reviewer that, in the original submission, the relatively slow resonant area scans (~115 fps) limited the temporal resolution of our rise time measurements. To address this, we have re-measured the rise time using higher frame-rate line scans (kHz). For Scar8f, the rise time constant was 6.736 msec at ~115 fps resonant area scanned, but shortened to 2.893 msec when imaged at ~303 fps, indicating that the original protocol underestimated the true kinetics. In addition, for Bar8m, area scans at ~118 fps yielded a rise time constant of 9.019 msec, whereas line scans at ~1085 fps reduced the rise time constant to 3.230 msec. These new measurements are now incorporated into the manuscript ( Figs. 3,4, and 6) to more accurately reflect the fast kinetics of these indicators.

      (3) The authors state that their indicators can now achieve measurements previously attainable with chemical dyes and electrophysiology. I encourage the authors to also consider how their tools might enable new measurements beyond what these traditional techniques allow. For example, while electrophysiology can detect summed mEPSPs across synapses, imaging could go a step further by spatially resolving the synaptic origin of individual mEPSP events. One could, for instance, image MN-Ib and MN-Is simultaneously without silencing either input, and detect mEPSP events specific to each synapse. This would enable synapse-specific mapping of quantal events - something electrophysiology alone cannot provide. Demonstrating even a proof-of-principle along these lines could highlight the unique advantages of the new tools by showing that they not only match previous methods but also enable new types of measurements.

      These are excellent points raised by the reviewer. In response, we have done the following: 

      We have now included a supplemental video as “proof-of-principle” data showing simultaneous imaging of SynapGCaMP8m quantal events at both MN-Is and -Ib, demonstrating that synapse-specific spatial mapping of quantal events can be obtained with this tool (see new Supplemental Video 1). 

      We have also included an additional discussion of the potential and limitations of these tools for new measurements beyond conventional approaches. This discussion is now presented in lines 419-421 in the manuscript.

      (4) For ratiometric measurements, it is important to estimate and subtract background signals in each channel. Without this correction, the computed ratio may be skewed, as background adds an offset to both channels and can distort the ratio. However, it is not clear from the Methods section whether, or how, background fluorescence was measured and subtracted.

      This is a good point, and we agree more clarification about how ratiometric measurements were made is needed. In response, we have now added the following to the Methods section (lines 548-568):

      Time-lapse videos were stabilized and bleach-corrected prior to analysis, which visibly reduced frame-toframe motion and intensity drift. In the presynaptic and active-zone mScarlet channel, a bleaching factor of ~1.15 was observed during the 15 sec recording. This bleaching can be corrected using the “Bleaching correction” tool in Huygens SVI. For presynaptic and active-zone GCaMP signals, there was minimal bleaching over these short imaging periods. Therefore, the bleaching correction step for GCaMP was skipped. Both GCaMP and mScarlet channels were processed using the default settings in the Huygens SVI “Deconvolution Wizard” (with the exception of the bleaching correction option). Deconvolution was performed using the CMLE algorithm with the Huygens default stopping criterion and a maximum of 30 iterations, such that the algorithm either converged earlier or, if convergence was not reached, was terminated at this 30iteration limit; no other iteration settings were used across the GCaMP series. ROIs were drawn on the processed images using Fiji ImageJ software, and mean fluorescence time courses were extracted for the GCaMP and mScarlet channels, yielding F<sub>GCaMP</sub>(t) and F<sub>mScarlet</sub>(t). F(t)s were imported into CaFire with GCaMP assigned to Channel #1 (signal; required) and mScarlet to Channel #2 (baseline/reference; optional). If desired, the mScarlet signal could be smoothed in CaFire using a user-specified moving-average window to reduce high-frequency noise. In CaFire’s ΔR/R mode, the per-frame ratio was computed as R(t)=F<sub>GCaMP</sub>(t) and F<sub>mScarlet</sub>(t); a baseline ratio R0 was estimated from the pre-stimulus period, and the final response was reported as ΔR/R(t)=[R(t)−R0]/R0, which normalizes GCaMP signals to the co-expressed mScarlet reference and thereby reduces variability arising from differences in sensor expression level or illumination across AZs.

      (5) At line 212, the authors claim "... GCaMP8m showing 345.7% higher SNR over GCaMP6s....(Fig. 3D and E) ", yet the cited figure panels do not present any SNR quantification. Figures 3D and E only show response amplitudes and kinetics, which are distinct from SNR. The methods section also does not describe details for how SNR was defined or computed.

      This is another good point. We define SNR operationally as the fractional fluorescence change (ΔF/F). Traces were processed with CaFire, which estimates a per-frame baseline F<sub>0</sub>(t) with a user-configurable sliding window and percentile. In the Load File panel, users can specify both the length of the moving baseline window and the desired percentile; the default settings are a 50-point window and the 30th percentile, representing a 101-point window centered on each time point (previous 50 to next 50 samples) and took the lower 30% of values within that window to estimate F<sub>0</sub>(t). The signal was then computed as ΔF/F=[F(t)−F0(t)]/F0(t). This ΔF/F value is what we report as SNR throughout the manuscript and is now discussed explicitly in the revised methods (lines 686-693).

      (6) Lines 285-287 "As expected, summed ΔF values scaled strongly and positively with AZ size (Fig. 5F), reflecting a greater number of Cav2 channels at larger AZs". I am not sure about this conclusion. A positive correlation between summed ΔF values and AZ size could simply reflect more GCaMP molecules in larger AZs, which would give rise to larger total fluorescence change even at a given level of calcium increase.

      The reviewer makes a good point, one that we agree should be clarified. The reviewer is indeed correct that larger active zones should have more abundant BRP protein, which in turn will lead to a higher abundance of the Bar8f sensor, which should lead to a higher GCaMP response simply by having more of this sensor. However, the inclusion of the ratiometric mScarlet protein should normalize the response accurately, correcting for this confound, in which the higher abundance of GCaMP should be offset (normalized) by the equally (stoichiometric) higher abundance of mScarlet. Therefore, when the ∆R/R is calculated, the differences in GCaMP abundance at each AZ should be corrected for the ratiometric analysis. We now use an improved BRP::mScarlet3::GCaMP8m (Bar8m) and compute ΔR/R with R(t)=F<sub>GCaMP8m</sub>/F<sub>mScarlet3</sub>. ROIs were drawn over individual AZs (Fig. 6B). CaFire estimated R0 with a sliding 101-point window using the lowest 10% of values, and responses were reported as ΔR/R=[R−R0]/R0. Area-scan examples (118 fps) show robust ΔR/R transients (peaks ≈1.90 and 3.28; tau rise ≈9.0–9.3 ms; Fig. 6C, middle).

      We have now made these points more clearly in the manuscript (lines 700-704) and moved the Bar8f intensity vs active zone size data to Table S1. Together, these revisions improve the indicator-abundance confound (via mScarlet normalization). 

      (6) Lines 313-314: "SynapGCaMP quantal signals appeared to qualitatively reflect the same events measured with electrophysiological recordings (Fig. 6D)." This statement is quite confusing. In Figure 6D, the corresponding calcium and ephys traces look completely different and appear to reflect distinct sets of events. It was only after reading Figure 7 that I realized the traces shown in Figure 6D might not have been recorded simultaneously. The authors should clarify this point.

      Yes, we absolutely agree with this point, one shared by Reviewer 1. In response, we have removed the electrophysiological traces in Fig. 6 to clarify that just the calcium responses are shown, and save the direct comparison for the Fig. 7 data (now revised Fig. 8).

      (8) Lines 310-313: "SynapGCaMP8m .... striking an optimal balance between speed and sensitivity", and Lines 314-316: "We conclude that SynapGCaMP8m is an optimal indicator to measure quantal transmission events at the synapse." Statements like these are subjective. In the authors' own comparison, GCaMP8m is significantly slower than GCaMP8f (at least in terms of decay time), despite having a moderately higher response amplitude. It is therefore unclear why GCaMP8m is considered 'optimal'. The authors should clarify this point or explain their rationale for prioritizing response amplitude over speed in the context of their application.

      This is another good point that we agree with, as the “optimal” sensor will of course depend on the user’s objectives. Hence, we used the term “an optimal sensor” to indicate it is what we believed to be the best one for our own uses. However, this point should be clarified and better discussed. In response, we have revised the relevant sections of the manuscript to better define why we chose the 8m sensors to strike an optimal balance of speed and sensitivity for our uses, and go on to discuss situations in which other sensor variants might be better suited. These are now presented in lines 223-236 in the revised manuscript, and we thank the reviewer for making these comments, which have improved our study.

      Minor comments

      (1)  Please include the following information in the Methods section:

      (a) For Figures 3 and 4, specify how action potentials were evoked. What type of electrodes were used, where were they placed, and what amount of current or voltage was applied?

      We apologize for neglecting to include this information in the original submission. We have now added this information to the revised Methods section (lines 537-543).

      (b) For imaging experiments, provide information on the filter sets used for each imaging channel, and describe how acquisition was alternated or synchronized between the green and red channels in ratiometric measurements. Additionally, please report the typical illumination intensity (in mW/mm²) for each experimental condition.

      We thank the reviewer for this helpful comment. We have now added detailed information about the imaging configuration to the Methods (lines 512-528) with the following:

      Ca2+ imaging was conducted using a Nikon A1R resonant scanning confocal microscope equipped with a 60x/1.0 NA water-immersion objective (refractive index 1.33). GCaMP signals were acquired using the FITC/GFP channel (488-nm laser excitation; emission collected with a 525/50-nm band-pass filter), and mScarlet/mCherry signals were acquired using the TRITC/mCherry channel (561-nm laser excitation; emission collected with a 595/50-nm band-pass filter). ROIs focused on terminal boutons of MN-Ib or -Is motor neurons. For both channels, the confocal pinhole was set to a fixed diameter of 117.5 µm (approximately three Airy units under these conditions), which increases signal collection while maintaining adequate optical sectioning. Images were acquired as 256 × 64 pixel frames (two 12-bit channels) using bidirectional resonant scanning at a frame rate of ~118 frames/s; the scan zoom in NIS-Elements was adjusted so that this field of view encompassed the entire neuromuscular junction and was kept constant across experiments. In ratiometric recordings, the 488-nm (GCaMP) and 561-nm (mScarlet) channels were acquired in a sequential dual-channel mode using the same bidirectional resonant scan settings: for each time point, a frame was first collected in the green channel and then immediately in the red channel, introducing a small, fixed frame-to-frame temporal offset while preserving matched spatial sampling of the two channels.

      Directly measuring the absolute laser power at the specimen plane (and thus reporting illumination intensity in mW/mm²) is technically challenging on this resonant-scanning system, because it would require inserting a power sensor into the beam path and perturbing the optical alignment; consequently, we are unable to provide reliable absolute mW/mm² values. Instead, we now report all relevant acquisition parameters (objective, numerical aperture, refractive index, pinhole size, scan format, frame rate, and fixed laser/detector settings) and note that laser powers were kept constant within each experimental series and chosen to minimize bleaching and phototoxicity while maintaining an adequate signal-to-noise ratio. We have now added the details requested in the revised Methods section (lines 512-535), including information about the filter sets, acquisition settings, and typical illumination intensity.

      (2) Please clarify what the thin versus thick traces represent in Figures 3D, 3F, 4C, and 4E. Are the thin traces individual trials from the same experiment, or from different experiments/animals? Does the thick trace represent the mean/median across those trials, a fitted curve, or a representative example?

      We apologize this was not more clear in the original submission. Thin traces are individual stimulus-evoked trials (“sweeps”) acquired sequentially from the same muscle/NMJ in a single preparation; the panel is shown as a representative example of recordings collected across animals. The thick colored trace is the trialaveraged waveform (arithmetic mean) of those thin traces after alignment to stimulus onset and baseline subtraction (no additional smoothing beyond what is stated in Methods). The thick black curve over the decay phase is a single-exponential fit used to estimate τ. Specifically, we fit the decay segment by linear regression on the natural-log–transformed baseline-subtracted signal, which is equivalent to fitting y = y<sub>peak</sub>·e<sup>−t/τdecay</sup> over the decay window (revised Fig.4D and Fig.5C legends).

      (3) Please clarify what the reported sample size (n) represents. Does it indicate the number of experimental repeats, the number of boutons or PSDs, or the number of animals?

      Again, we apologize this was not clear. (n) refers to the number of animals (biological replicates), which is reported in Supplementary Table 1. All imaging was performed at muscle 6, abdominal segment A3. Per preparation, we imaged 1-2 NMJs in total, with each imaging targeting 2–3 terminal boutons at the target NMJ and acquired 2–3 imaging stacks choosing different terminal boutons per NMJ. For the standard stimulation protocol, we delivered 1 Hz stimulation for 1ms and captured 14 stimuli in a 15s time series imaging (lines 730-736).

      Reviewer #3

      Genetically encoded calcium indicators (GECIs) are essential tools in neurobiology and physiology. Technological constraints in targeting and kinetics of previous versions of GECIs have limited their application at the subcellular level. Chen et al. present a set of novel tools that overcome many of these limitations. Through systematic testing in the Drosophila NMJ, they demonstrate improved targeting of GCaMP variants to synaptic compartments and report enhanced brightness and temporal fidelity using members of the GCaMP8 series. These advancements are likely to facilitate more precise investigation of synaptic physiology.

      This is a comprehensive and detailed manuscript that introduces and validates new GECI tools optimized for the study of neurotransmission and neuronal excitability. These tools are likely to be highly impactful across neuroscience subfields. The authors are commended for publicly sharing their imaging software.

      This manuscript could be improved by further testing the GECIs across physiologically relevant ranges of activity, including at high frequency and over long imaging sessions. The authors provide a custom software package (CaFire) for Ca2+ imaging analysis; however, to improve clarity and utility for future users, we recommend providing references to existing Ca2+ imaging tools for context and elaborating on some conceptual and methodological aspects, with more guidance for broader usability. These enhancements would strengthen this already strong manuscript.

      We thank the Reviewer for their overall positive evaluation and comments. 

      Major comments:

      (1) Evaluation of the performance of new GECI variants using physiologically relevant stimuli and frequency. The authors took initial steps towards this goal, but it would be helpful to determine the performance of the different GECIs at higher electrical stimulation frequencies (at least as high as 20 Hz) and for longer (10 seconds) (Newman et al, 2017). This will help scientists choose the right GECI for studies testing the reliability of synaptic transmission, which generally requires prolonged highfrequency stimulation.

      We appreciate this point by the reviewer and agree it would be of interest to evaluate sensor performance with higher frequency stimulation and for a longer duration. In response, we performed a variety of stimulation protocols at high intensities and times, but found the data to be difficult to separate individual responses given the decay kinetics of all calcium sensors. Hence, we elected not to include these in the revised manuscript. However, we have now included an evaluation of the sensors with 20 Hz electrical stimulation for ~1 sec using a direct comparison of Scar8f with OGB-1. These data are now presented in a new Fig. 3D,E and discussed in the manuscript (lines 396-403).

      (2) CaFire.

      The authors mention, in line 182: 'Current approaches to analyze synaptic Ca2+ imaging data either repurpose software designed to analyze electrophysiological data or use custom software developed by groups for their own specific needs.' References should be provided. CaImAn comes to mind (Giovannucci et al., 2019, eLife), but we think there are other software programs aimed at analyzing Ca2+ imaging data that would permit such analysis.

      Thank you for the thoughtful question. At this stage, we’re unable to provide a direct comparison with existing analysis workflows. In surveying prior studies that analyze Drosophila NMJ Ca²⁺ imaging traces, we found that most groups preprocess images in Fiji/ImageJ and then rely on their own custom-made MATLAB or Python scripts for downstream analysis (see Blum et al. 2021; Xing and Wu 2018). Because these pipelines vary widely across labs, a standardized head-to-head evaluation isn’t currently feasible. With CaFire, our goal is to offer a simple, accessible tool that does not require coding experience and minimizes variability introduced by custom scripts. We designed CaFire to lower the barrier to entry, promote reproducibility, and make quantal event analysis more consistent across users. We have added references to the sentence mentioned above.

      Regarding existing software that the reviewer mentioned – CaImAn (Giovannucci et al. 2019): We evaluated CaImAn, which is a powerful framework designed for large-scale, multicellular calcium imaging (e.g., motion correction, denoising, and automated cell/ROI extraction). However, it is not optimized for the per-event kinetics central to our project - such as extracting rise and decay times for individual quantal events at single synapses. Achieving this level of granularity would typically require additional custom Python scripting and parameter tuning within CaImAn’s code-centric interface. This runs counter to CaFire’s design goals of a nocode, task-focused workflow that enables users to analyze miniature events quickly and consistently without specialized programming expertise.

      Regarding Igor Pro (WaveMetrics), (Müller et al. 2012): Igor Pro is another platform that can be used to analyze calcium imaging signals. However, it is commercial (paid) software and generally requires substantial custom scripting to fit the specific analyses we need. In practice, it does not offer a simple, open-source, point-and-click path to per-event kinetic quantification, which is what CaFire is designed to provide.

      The authors should be commended for making their software publicly available, but there are some questions:

      How does CaFire compare to existing tools?

      As mentioned above, we have not been able to adapt the custom scripts used by various labs for our purposes, including software developed in MatLab (Blum et al. 2021), Python (Xing and Wu 2018), and Igor (Müller et al. 2012). Some in the field do use semi-publically available software, including Nikon Elements (Chen and Huang 2017) and CaImAn (Giovannucci et al. 2019). However, these platforms are not optimized for the per-event kinetics central to our project - such as extracting rise and decay times for individual quantal events at single synapses. We have added more details about CaFire, mainly focusing on the workflow and measurements, highlighting the superiority of CaFire, showing that CaFire provides a no-code, standardized pipeline with automated miniature-event detection and per-event metrics (e.g., amplitude, rise time τ, decay time τ), optional ΔR/R support, and auto-partition feature. Collectively, these features make CaFire simpler to operate without programming expertise, more transparent and reproducible across users, and better aligned with the event-level kinetics required for this project.

      Very few details about the Huygens deconvolution algorithms and input settings were provided in the methods or text (outside of MLE algorithm used in STED images, which was not Ca2+ imaging). Was it blind deconvolution? Did the team distill the point-spread function for the fluorophores? Were both channels processed for ratiometric imaging? Were the same settings used for each channel? Importantly, please include SVI Huygens in the 'Software and Algorithms' Section of the methods.

      We thank the reviewer for raising this important point. We have now expanded the Methods to describe our use of Huygens in more detail and have added SVI Huygens Professional (Scientific Volume Imaging, Hilversum, The Netherlands) to the “Software and Algorithms” section. For Ca²⁺ imaging data, time-lapse stacks were processed in the Huygens Deconvolution Wizard using the standard estimation algorithm (CMLE). This is not a blind deconvolution procedure. Instead, Huygens computes a theoretical point-spread function (PSF) from the full acquisition metadata (objective NA, refractive index, voxel size/sampling, pinhole, excitation/emission wavelengths, etc.); if refractive index values are provided and there is a mismatch, the PSF is adjusted to account for spherical aberration. We did not experimentally distill PSFs from bead measurements, as Huygens’ theoretical PSFs are sufficient for our data.

      Both green (GCaMP) and red (mScarlet) channels were processed for ratiometric imaging using the same workflow (stabilization, optional bleaching correction, and deconvolution within Huygens). For each channel, the PSF, background, and SNR were estimated automatically by the same built-in algorithms, so the underlying procedures were identical even though the numerical values differ between channels because of their distinct wavelengths and noise characteristics. Importantly, Huygens normalizes each PSF to unit total intensity, such that the deconvolution itself does not add or remove signal and therefore preserves intensity ratios between channels; only background subtraction and bleaching correction can change absolute fluorescence values. For the mScarlet channel, where we observed modest bleaching (~1.10 over 15 sec), we applied Huygens’ bleaching correction and visually verified that similar structures maintained comparable intensities after correction. For presynaptic GCaMP signals, bleaching over these short recordings was negligible, so we omitted the bleaching-correction step to avoid introducing multiplicative artifacts. This workflow ensures that ratiometric ΔR/R measurements are based on consistently processed, intensity-conserving deconvolved images in both channels.

      The number of deconvolution iterations could have had an effect when comparing GCAMP series; please provide an average number of iterations used for at least one experiment. For example, Figure 3, Syt::GCAMP6s, Scar8f & Scar8m, and, if applicable, the maximum number of permissible iterations.

      We thank the reviewer for this comment. For all Ca²⁺ imaging datasets, deconvolution in Huygens was performed using the recommended default settings of the CMLE algorithm with a maximum of 30 iterations. The stopping criterion was left at the Huygens default, so the algorithm either converged earlier or, if convergence was not reached, terminated at this 30-iteration limit. No other iteration settings were used across the GCaMP series (lines 555-559).

      Please clarify if the 'Express' settings in Huygens changed algorithms or shifted input parameters.

      We appreciate the reviewer’s question regarding the Huygens “Express” settings. For clarity, we note that all Ca²⁺ imaging data reported in this manuscript were deconvolved using the “Deconvolution Wizard”, not the “Deconvolution Express” mode. In the Wizard, we explicitly selected the CMLE algorithm (or GMLE in a few STED-related cases as recommended by SVI), using the recommended maximum of 30 iterations, and other recommended settings while allowing Huygens to auto-estimate background and SNR for each channel.Bleaching correction was toggled manually per channel (applied to mScarlet when bleaching was evident, omitted for GCaMP when bleaching was negligible), as described in the revised Methods (lines 553-559).

      By contrast, the Deconvolution Express tool in Huygens is a fully automated front-end that can internally adjust both the choice of deconvolution algorithm (e.g., CMLE vs. GMLE/QMLE) and key input parameters such as SNR, number of iterations, and quality threshold based on the selected “smart profile” and the image metadata. In preliminary tests on our datasets, Express sometimes produced results that were either overly smoothed or showed subtle artifacts, so we did not use it for any data included in this study. Instead, we relied exclusively on the Wizard with explicitly controlled settings to ensure consistency and transparency across all GCaMP series and ratiometric analyses.

      We suggest including a sample data set, perhaps in Excel, so that future users can beta test on and organize their data in a similar fashion.

      We agree that this would be useful, a point shared by R1 above. In response, we have added a sample data set to the GitHub site and included sample ImageJ data along with screenshots to explain the analysis in more detail. These improvements are discussed in the manuscript (lines 705-708).

      (3) While the challenges of AZ imaging are mentioned, it is not discussed how the authors tackled each one. What is defined as an active zone? Active zones are usually identified under electron microscopy. Arguably, the limitation of GCaMP-based sensors targeted to individual AZs, being unable to resolve local Ca2+ changes at individual boutons reliably, might be incorrect. This could be a limitation of the optical setup being used here. Please discuss further. What sensor performance do we need to achieve this performance level, and/or what optical setup would we need to resolve such signals?

      We appreciate the reviewer’s thoughtful comments and agree that the technical challenges of active zone (AZ) Ca²⁺ imaging merit further clarification. We defined AZs, as is the convention in our field, as individual BRP puncta at NMJs. These BRP puncta co-colocalize with individual puncta of other AZ components, including CAC, RBP, Unc13, etc. ROIs were drawn tightly over individual BRP puncta and only clearly separable spots were included.

      To tackle the specific obstacles of AZ imaging (small signal volume, high AZ density, and limited photon budget at high frame rates), we implemented both improved sensors and optimized analysis (Fig. 6). First, we introduced a ratiometric AZ-targeted indicator, BRP::mScarlet3::GCaMP8m (Bar8m), and computed ΔR/R with ΔR/R with R(t)=F<sub>GCaMP8m</sub>/F<sub>mScarlet3</sub>. ROIs were drawn over individual AZs (Fig. 6B). Under our standard resonant area-scan conditions (~118 fps), Bar8m produces robust ΔR/R transients at individual AZs (example peaks ≈ 3.28; τ<sub>rise</sub>≈9.0 ms; Fig. 6C, middle), indicating that single-AZ signals can be detected reproducibly when AZs are optically resolvable.

      Second, we increased temporal resolution using high-speed Galvano line-scan imaging (~1058 fps), which markedly sharpened the apparent kinetics (τ<sub>rise</sub>≈3.23 ms) and revealed greater between-AZ variability (Fig. 6C, right; 6D–E). Population analyses show that line scans yield much faster rise times than area scans (Fig. 6D) and a dramatically higher fraction of significantly different AZ pairs (8.28% and 4.14% in 8f and 8m areascan vs 78.62% in 8m line-scan, lines 721-725), uncovering pronounced AZ-to-AZ heterogeneity in Ca²⁺ signals. Together, these revisions demonstrate that under our current confocal configuration, AZ-targeted GCaMP8m can indeed resolve local Ca²⁺ changes at individual, optically isolated boutons.

      We have revised the Discussion to clarify that our original statement about the limitations of AZ-targeted GCaMPs refers specifically to this combination of sensor and optical setup, rather than an absolute limitation of AZ-level Ca²⁺ imaging. In our view, further improvements in baseline brightness and dynamic range (ΔF/F or ΔR/R per action potential), combined with sub-millisecond kinetics and minimal buffering, together with optical configurations that provide smaller effective PSFs and higher photon collection (e.g., higher-NA objectives, optimized 2-photon or fast line-scan modalities, and potentially super-resolution approaches applied to AZ-localized indicators), are likely to be required to achieve routine, high-fidelity Ca²⁺ measurements at every individual AZ within a neuromuscular junction.

      (4) In Figure 5: Only GCAMP8f (Bar8f fusion protein) is tested here. Consider including testing with GCAMP8m. This is particularly relevant given that GCAMP8m was a more successful GECI for subcellular post-synaptic imaging in Figure 6.

      We appreciate this point and request by Reviewer 3. The main limitation for detecting local calcium changes at AZs is the speed of the calcium sensor, and hence we used the fastest available (GCaMP8f) to test the Bar8f sensor. While replacing GCaMP8f with GCaMP8m would indeed be predicted to enhance sensitivity (SNR), since GCaMP8m does not have faster kinetics relative to GCaMP8f, it is unlikely to be a more successful GECI for visualizing local calcium differences at AZs. 

      That being said, we agree that the Bar8m tool, including the improved mScarlet3 indicator, would likely be of interest and use to the field. Fortunately, we had engineered the Bar8m sensor while this manuscript was in review, and just recently received transgenic flies. We have evaluated this sensor, as requested by the reviewer, and included our findings in Fig. 1 and 6. In short, while the sensitivity is indeed enhanced in Bar8m compared to Bar8f, the kinetics remain insufficient to capture local AZ signals. These findings are discussed in the revised manuscript (lines 424-442, 719-730), and we appreciate the reviewer for raising these important points.

      In earlier experiments, Bar8f yielded relatively weak fluorescence, so we traded frame rate for image quality during resonant area scans (~60 fps). After switching to Bar8m, the signal was bright enough to restore our standard 118 fps area-scan setting. Nevertheless, even with dual-channel resonant area scans and ratiometric (GCaMP/mScarlet) analysis, AZ-to-AZ heterogeneity remained difficult to resolve. Because Ca²⁺ influx at individual active zones evolves on sub-millisecond timescales, we adopted a high-speed singlechannel Galvano line-scan (~1 kHz) to capture these rapid transients. We first acquired a brief area image to localize AZ puncta, then positioned the line-scan ROI through the center of the selected AZ. This configuration provided the temporal resolution needed to uncover heterogeneity that was under-sampled in area-scan data. Consistent with this, Bar8m line-scan data showed markedly higher AZ heterogeneity (significant AZ-pair rate ~79%, vs. ~8% for Bar8f area scans and ~4% for Bar8m area scans), highlighting Bar8m’s suitability for quantifying AZ diversity. We have updated the text, Methods, and figure legend accordingly (tell reviewer where to find everything).

      (5) Figure 5D and associated datasets: Why was Interquartile Range (IQR) testing used instead of ZScoring? Generally, IQR is used when the data is heavily skewed or is not normally distributed. Normality was tested using the D'Agostino & Pearson omnibus normality test and found that normality was not violated. Please explain your reasoning for the approach in statistical testing. Correlation coefficients in Figures 5 E & F should also be reported on the graph, not just the table. In Supplementary Table 1. The sub-table between 4D-F and 5E-F, which describes the IQR, should be labeled as such and contain identifiers in the rows describing which quartile is described. The table description should be below. We would recommend a brief table description for each sub-table.

      Thank you for this helpful suggestion. We have updated the analysis in two complementary ways. First, we now perform paired two-tailed t-tests between every two AZs within the same preparation (pairwise AZ–AZ comparisons of peak responses). At α<0.05, the fraction of significant AZ pairs is ~79% for Bar8m line-scan data versus ~8% for Bar8f area-scan data, indicating markedly greater AZ-to-AZ diversity when measured at high temporal resolution. Second, for visually marking the outlying AZs, we re-computed the IQR (Q1–Q3) based on the individual values collected from each AZs(15 data points per AZ, 30 AZs for each genotype), and marked AZs whose mean response falls above Q3 or below Q1; IQR is used here solely as a robust dispersion reference rather than for hypothesis testing. Both analyses support the same observation: Bar8m line-scan data reveal substantially higher AZ heterogeneity than Bar8f and Bar8m area-scan data. We have revised the Methods, figure panels, and legends accordingly (t-test details; explicit “IQR (Q1–Q3)” labeling; significant AZ-pair rates reported on the plots) (lines 719-730).

      (6) Figure 6 and associated data. The authors mention: ' SynapGCaMP quantal signals appeared to qualitatively reflect the same events measured with electrophysiological recordings (Fig. 6D).' If that was the case, shouldn't the ephys and optical signal show some sort of correlation? The data presented in Figure 6D show no such correlation. Where do these signals come from? It is important to show the ROIs on a reference image.

      We apologize this was not clear, as similar points were raised by R1 and R2. We were just showing separate (uncorrelated) sample traces of electrophysiological and calcium imaging data. Given how confusing this presentation turned out to be, and the fact that we show the correlated ephys and calcium imaging events in Fig. 7, we have elected to remove the uncorrelated electrophysiological events in Fig. 6 to just focus on the calcium imaging events (now Figures 7 and 8).

      Figure 7B: Were Ca2+ transients not associated with mEPSPs ever detected? What is the rate of such events?

      This is an astute question. Yes indeed, during simultaneous calcium imaging and current clamp electrophysiology recordings, we occasionally observed GCaMP transients without a detectable mEPSP in the electrophysiological trace. This may reflect the detection limit of electrophysiology for very small minis; with our noise level and the technical limitation of the recording rig, events < ~0.2 mV cannot be reliably detected, whereas the optical signal from the same quantal event might still be detected. The fraction of calcium-only events was ~1–10% of all optical miniature events, depending on genotype (higher in lines with smaller average minis). These calcium-only detections were low-amplitude and clustered near the optical threshold (lines 361-365).

      Minor comments

      (1) It should be mentioned in the text or figure legend whether images in Figure 1 were deconvolved, particularly since image pre-processing is only discussed in Figure 2 and after.

      We thank the reviewer for pointing this out. Yes, the confocal images shown in Figure 1 were also deconvolved in Huygens using the CMLE-based workflow described in the revised Methods. We applied deconvolution to improve contrast, reduce out-of-focus blur, and better resolve the morphology of presynaptic boutons, active zones, and postsynaptic structures, so that the localization of each sensor is more clearly visualized. We have now explicitly stated in the Fig. 1 legend and Methods (lines 575-577) that these images were deconvolved prior to display. 

      (2) The abbreviation, SNR, signal-to-noise ratio, is not defined in the text.

      We have corrected this error and thank the reviewer for pointing this out.

      (3) Please comment on the availability of fly stocks and molecular constructs.

      We have clarified that all fly stocks and molecular constructs will be shared upon request (lines 747-750). We are also in the process of depositing the new Scar8f/m, Bar8f/m, and SynapGCaMP sensors to the Bloomington Drosophila Stock Center for public dissemination.

      (4) Please add detection wavelengths and filter cube information for live imaging experiments for both confocal and widefield.

      We thank the reviewer for this helpful suggestion. We have now added the detection wavelengths and filter cube configurations for both confocal and widefield live imaging to the Methods.

      For confocal imaging, GCaMP signals were acquired on a Nikon A1R system using the FITC/GFP channel (488-nm laser excitation; emission collected with a 525/50-nm band-pass filter), and mScarlet signals were acquired using the TRITC/mCherry channel (561-nm laser excitation; emission collected with a 595/50-nm band-pass filter). Both channels were detected with GaAsP detectors under the same pinhole and scan settings described above (lines 512-517).

      For widefield imaging, GCaMP was recorded using a GFP filter cube (LED excitation ~470/40 nm; emission ~525/50 nm), which is now explicitly described in the revised Methods section (lines 632-633).

      (5) Please include a mini frequency analysis in Supplemental Figure S1.

      We apologize for not including this information in the original submission. This is now included in the Supplemental Figure S1.

      (6) In Figure S1B, consider flipping the order of EPSP (currently middle) and mEPSP (currently left), to easily guide the reader through the quantification of Figure S1A (EPSPs, top traces & mEPSPs, bottom traces).

      We agree these modifications would improve readability and clarity. We have now re-ordered the electrophysiological quantifications in Fig. S1B as requested by the reviewer.

      (7) Figure 6C: Consider labeling with sensor name instead of GFP.

      We agree here as well, and have removed “GFP” and instead added the GCaMP variant to the heatmap in Fig. 7C.

      (8) Figure 6E, 7B, 7E: Main statistical differences highlighting sensor performance should be represented on the figures for clarity.

      We did not show these differences in the original submission in an effort to keep the figures “clean” and for clarity, putting the detailed statistical significance in Table S1. However, we agree with the reviewer that it would be easier to see these in the Fig. 6E and 7B,E graphs. This information has now been added the Figs. 7 and 8.

      (9) Please report if the significance tested between the ephys mini (WT vs IIB-/-, WT vs IIA-/-, IIB-/- vs IIA-/-) is the same as for Ca2+ mini (WT vs IIB-/-, WT vs IIA-/-, IIB-/- vs IIA-/-). These should also exhibit a very high correlation (mEPSP (mV) vs Ca2+ mini deltaF/F). These tests would significantly strengthen the final statement of "SynapGCaMP8m can capture physiologically relevant differences in quantal events with similar sensitivity as electrophysiology."

      We agree that adding the more detailed statistical analysis requested by the reviewer would strengthen the evidence for the resolution of quantal calcium imaging using SynapGCaMP8m. We have included the statistical significance between the ephys and calcium minis in Fig. 8 and included the following in the revised methods (lines 358-361), the Fig. 8 legend and Table S1:

      Using two-sample Kolmogorov–Smirnov (K–S) tests, we found that SynapGCaMP8m Ca²⁺ minis (ΔF/F, Fig. 8E) differ significantly across all genotype pairs (WT vs IIB<sup>-/-</sup>, WT vs IIA<sup>-/-</sup>, IIB<sup>-/-</sup> vs IIA<sup>-/-</sup>; all p < 0.0001). The genotype rank order of the group means (±SEM) is IIB<sup>-/-</sup> > WT > IIA<sup>-/-</sup> (0.967 ± 0.036; 0.713 ± 0.021; 0.427 ± 0.017; n=69, 65, 59). For electrophysiological minis (mEPSP amplitude, Fig. 8F), K–S tests likewise show significant differences for the same comparisons (all p < 0.0001) with D statistics of 0.1854, 0.3647, and 0.4043 (WT vs IIB<sup>-/-</sup>, WT vs IIA<sup>-/-</sup>, IIB<sup>-/-</sup> vs IIA<sup>-/-</sup>, respectively). Group means (±SEM) again follow IIB<sup>-/-</sup> > WT > IIA<sup>-/-</sup> (0.824 ± 0.017 mV; 0.636 ± 0.015 mV; 0.383 ± 0.007 mV; n=41 each). These K–S results demonstrate identical significance and rank order across modalities, supporting our conclusion that SynapGCaMP8m resolves physiologically relevant quantal differences with sensitivity comparable to electrophysiology.

      References

      Blum, Ian D., Mehmet F. Keleş, El-Sayed Baz, Emily Han, Kristen Park, Skylar Luu, Habon Issa, Matt Brown, Margaret C. W. Ho, Masashi Tabuchi, Sha Liu, and Mark N. Wu. 2021. 'Astroglial Calcium Signaling Encodes Sleep Need in Drosophila', Current Biology, 31: 150-62.e7.

      Chen, Y., and L. M. Huang. 2017. 'A simple and fast method to image calcium activity of neurons from intact dorsal root ganglia using fluorescent chemical Ca(2+) indicators', Mol Pain, 13: 1744806917748051.

      Giovannucci, Andrea, Johannes Friedrich, Pat Gunn, Jérémie Kalfon, Brandon L. Brown, Sue Ann Koay, Jiannis Taxidis, Farzaneh Najafi, Jeffrey L. Gauthier, Pengcheng Zhou, Baljit S. Khakh, David W. Tank, Dmitri B. Chklovskii, and Eftychios A. Pnevmatikakis. 2019. 'CaImAn an open source tool for scalable calcium imaging data analysis', eLife, 8: e38173.

      Müller, M., K. S. Liu, S. J. Sigrist, and G. W. Davis. 2012. 'RIM controls homeostatic plasticity through modulation of the readily-releasable vesicle pool', J Neurosci, 32: 16574-85.

      Wu, Yifan, Keimpe Wierda, Katlijn Vints, Yu-Chun Huang, Valerie Uytterhoeven, Sahil Loomba, Fran Laenen, Marieke Hoekstra, Miranda C. Dyson, Sheng Huang, Chengji Piao, Jiawen Chen, Sambashiva Banala, Chien-Chun Chen, El-Sayed Baz, Luke Lavis, Dion Dickman, Natalia V. Gounko, Stephan Sigrist, Patrik Verstreken, and Sha Liu. 2025. 'Presynaptic Release Probability Determines the Need for Sleep', bioRxiv: 2025.10.16.682770.

      Xing, Xiaomin, and Chun-Fang Wu. 2018. 'Unraveling Synaptic GCaMP Signals: Differential Excitability and Clearance Mechanisms Underlying Distinct Ca<sup>2+</sup> Dynamics in Tonic and Phasic Excitatory, and Aminergic Modulatory Motor Terminals in Drosophila', eneuro, 5: ENEURO.0362-17.2018.

    1. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This study presents a system for delivering precisely controlled cutaneous stimuli to freely moving mice by coupling markerless real-time tracking to transdermal optogenetic stimulation, using the tracking signal to direct a laser via galvanometer mirrors. The principal claims are that the system achieves sub-mm targeting accuracy with a latency of <100 ms. The nature of mouse gait enables accurate targeting of forepaws even when mice are moving.

      Strengths:

      The study is of high quality and the evidence for the claims is convincing. There is increasing focus in neurobiology in studying neural function in freely moving animals, engaged in natural behaviour. However, a substantial challenge is how to deliver controlled stimuli to sense organs under such conditions. The system presented here constitutes notable progress towards such experiments in the somatosensory system and is, in my view, a highly significant development that will be of interest to a broad readership.

      Weaknesses:

      (1) "laser spot size was set to 2.00 } 0.08 mm2 diameter (coefficient of variation = 3.85)" is unclear. Is the 0.08 SD or SEM? (not stated). Also, is this systematic variation across the arena (or something else)? Readers will want to know how much the spot size varies across the arena - ie SD. CV=4 implies that SD~7 mm. ie non-trivial variation in spot size, implying substantial differences in power delivery (and hence stimulus intensity) when the mouse is in different locations. If I misunderstood, perhaps this helps the authors to clarify. Similarly, it would be informative to have mean & SD (or mean & CV) for power and power density. In future refinements of the system, would it be possible/useful to vary laser power according to arena location?

      We thank the reviewer for their comments and for identifying areas needing more clarity. The previous version was ambiguous: 0.08 refers to the standard deviation (SD). We have removed the ambiguity by stating mean ± SD and reporting a unitless coefficient of variation (CV).

      The revised text reads “laser spot size was set to 2.00 ± 0.08 mm<sup>2</sup> (mean ± SD; coefficient of variation = 0.039).” This makes clear that the variability in spot size is minimal: it is 0.08 mm<sup>2</sup> SD (≈0.03 mm SD in diameter). This should help clarify that spot size variability across the arena is minute and unlikely to contribute meaningfully to differences in stimulus intensity across locations. The power was modulated depending on the experiment, so we provide the unitless CV here in “The absolute optical power and power density were uniform across the glass platform (coefficient of variation 0.035 and 0.029, respectively; Figure 2—figure supplement)”. We are grateful to the reviewer for spotting these omissions.

      The reviewer also asks whether, in the future, it is “possible/useful to vary laser power according to arena location”. This is already possible in our system for infrared cutaneous stimulation using analog modulation (Figure 4). We have added the following sentence to make this clearer: “Laser power could be modulated using the analog control.”

      (2) "The video resolution (1920 x 1200) required a processing time higher than the frame interval (33.33 ms), resulting in real-time pose estimation on a sub-sample of all frames recorded". Given this, how was it possible to achieve 84 ms latency? An important issue for closed-loop research will relate to such delays. Therefore please explain in more depth and (in Discussion) comment on how the latency of the current system might be improved/generalised. For example, although the current system works well for paws it would seem to be less suited to body parts such as the snout that do not naturally have a stationary period during the gait cycle.

      We captured and stored video with a frame-to-frame interval of 33.33 ms (30 fps). DeepLabCut-live! was run in a latency-optimization mode, meaning that new frames are not processed while the network is busy - only the most recent frame is processed when free. The processing latency is measured per processed frame, and intermediate frames are thus skipped while the network is busy. Although a wide field of view and high resolution is required to capture the large environment, increasing the per-frame compute time, the processing latency remained small enough to track and stimulate moving mice. This processing latency of 84 ± 12 ms (mean ± SD) was calculated using the timestamps stored in the output files from DeepLabCut-live!: subtracting the frame acquisition timestamp from the frame processing timestamp across 16,000 processed frames recorded across four mice (4,000 each). In addition, there is a small delay to move the galvanometers and trigger the laser, calculated as 3.3 ± 0.5 ms (mean ± SD; 245 trials). This is described in the manuscript, but can be combined with the processing latency to indicate a total closed-loop delay of ≈87 ms so we have expanded on the ‘Optical system characterization’ subsection in the Methods, adding “We estimated a processing latency of 84 ± 12 ms (mean ± SD) by subtracting…” and that “In the current configuration the end-to-end closed-loop delay is ≈87 ms from the combination of the processing latency and other delays”. To the Discussion, we now comment on how this latency can be reduced and how this can allow for generalization to more rapidly moving body parts.

      Reviewer #2 (Public review):

      Parkes et al. combined real-time keypoint tracking with transdermal activation of sensory neurons to examine the effects of recruitment of sensory neurons in freely moving mice. This builds on the authors' previous investigations involving transdermal stimulation of sensory neurons in stationary mice. They illustrate multiple scenarios in which their engineering improvements enable more sophisticated behavioral assessments, including (1) stimulation of animals in multiple states in large arenas, (2) multi-animal nociceptive behavior screening through thermal and optogenetic activation, and (3) stimulation of animals running through maze corridors. Overall, the experiments and the methodology, in particular, are written clearly. However, there are multiple concerns and opportunities to fully describe their newfound capabilities that, if addressed, would make it more likely for the community to adopt this methodology:

      The characterization of laser spot size and power density is reported as a coefficient of variation, in which a value of ~3 is interpreted as uniform. My interpretation would differ - data spread so that the standard deviation is three times larger than the mean indicates there is substantial variability in the data. The 2D polynomial fit is shown in Figure 2 - Figure Supplement 1A and, if the fit is good, this does support the uniformity claim (range of spot size is 1.97 to 2.08 mm2 and range of power densities is 66.60 to 73.80 mW). The inclusion of the raw data for these measurements and an estimate of the goodness of fit to the polynomials would better help the reader evaluate whether these parameters are uniform across space and how stable the power density is across repeated stimulations of the same location. Even more helpful would be an estimate of whether the variation in the power density is expected to meaningfully affect the responses of ChR2-expressing sensory neurons.

      We thank the reviewer for their comments. As also noted in response to Reviewer 1, the coefficient of variation (CV) is now reported in unitless form (rather than a percentage) to ensure clarity. For avoidance of doubt, the CV is 0.039 (3.9%), so the variation in laser spot size is minimal – there is negligible spot size variability across the system. The ranges are indeed consistent with uniformity. We have included the goodness-of-fit estimates in the appropriate figure legend “fit with a two-dimensional polynomial (area R<sup>2</sup> = 0.91; power R<sup>2</sup> = 0.75)”. This indicates that the polynomials fit well overall.

      The system already allows for control of spot size. To examine whether the variation in the power density affects the responses of ChR2-expressing sensory neurons, we examined this in our previous work that focused more on input-output relationships, demonstrating a steep relationship between spot size (range of 0.02 mm<sup>2</sup> to 2.30 mm<sup>2</sup>) and the probability of paw response, demonstrating a meaningful change in response probability (Schorscher-Petcu et al. eLife, 2021). In future studies, we aim to use this approach to “titrate” cutaneous inputs as mice move through their environments.

      While the error between the keypoint and laser spot error was reported as ~0.7 to 0.8 mm MAE in Figure 2L, in the methods, the authors report that there is an additional error between predicted keypoints and ground-truth labeling of 1.36 mm MAE during real-time tracking. This suggests that the overall error is not submillimeter, as claimed by the authors, but rather on the order of 1.5 - 2.5 mm, which is considerable given the width of a hind paw is ~5-6 mm and fore paws are even smaller. In my opinion, the claim for submillimeter precision should be softened and the authors should consider that the area of the paw stimulated may differ from trial to trial if, for example, the error is substantial enough that the spot overlaps with the edge of the paw.

      We thank the reviewer for identifying a discrepancy in these reported errors. We clarify this below and in the manuscript

      The real-time tracking error is the mean absolute Euclidean distance (MAE) between ground truth and DLC on the left hind paw where likelihood was relatively high. More specifically, ground truth was obtained by manual annotation of the left hind paw center. The corresponding DLC keypoint was evaluated in frames with likelihood >0.8 (the stimulation threshold). Across 1,281 frames from five videos of freely exploring mice (30 fps), the MAE was 1.36 mm.

      The targeting error is the MAE between ground truth and the laser spot location, so should reflect the real-time tracking error plus errors from targeting the laser. More specifically, this metric was determined by comparing the manually determined ground truth keypoint of the left hind paw and the actual center of the laser spot. Importantly, this metric was calculated using four five-minute high-speed videos recorded at 270 fps of mice freely exploring the open arena (463 frames) and frames were selected with a likelihood threshold >0.8. This allowed us to resolve the brief laser pulses but inadvertently introduced a difference in spatial scaling. After rescaling, the values give a targeting error MAE now in line with the real-time tracking error  (see corrected Figure 2L). This is approximately 1.3 mm across all locomotion speeds categories. These errors are small and are limited by the spatial resolution of the cameras. We thank the reviewer for noting this discrepancy and prompting us to get to its root cause.

      We have amended the subtitle on Figure 2L as “Ground truth keypoint to laser spot error” and have avoided the use of submillimeter throughout. We have added the following sentence to clarify this point: “As laser targeting relies on real-time tracking to direct the laser to the specified body part, this metric includes any errors introduced by tracking and targeting”.

      As the major advance of this paper is the ability to stimulate animals during ongoing movement, it seems that the Figure 3 experiment misses an opportunity to evaluate state-dependent whole-body reactions to nociceptor activation. How does the behavioral response relate to the animal's activity just prior to stimulation?

      The reviewers suggest analysis of state-dependent responses. In the Figure 3 experiment, mice were stimulated up to five times when stationary. Analysis of whole body reactions in stationary mice has been described in (Schorscher-Petcu et al. eLife, 2021) and doing this here would be redundant, so instead we now analyse the responses of moving mice in Figure 5. This new analysis shows robust state-dependent responses during movement as suggested by the reviewer. We find two behavioral clusters: one that is for faster, direct (coherent) movement and the other that is for slower assessment (incoherent) movement. Stimulation during the former results in robust and consistent slowing and shift towards assessment, whereas stimulation during the former results in a reduction in assessment. We describe and interpret these new data in the Results and Discussion sections and add information in the Methods and Figure legend, as given below. We believe that demonstrating movement statedependence is a valuable addition to the paper and thank the reviewer for suggesting this.

      Given the characterization of full-body responses to activation of TrpV1 sensory neurons in Figure 4 and in the authors' previous work, stimulation of TrpV1 sensory neurons has surprisingly subtle effects as the mice run through the alternating T maze. The authors indicate that the mice are moving quickly and thus that precise targeting is required, but no evidence is shared about the precision of targeting in this context beyond images of four trials. From the characterization in Figure 2, at max speed (reported at 241 +/- 53 mm/s, which is faster than the high speeds in Figure 2), successful targeting occurs less than 50% of the time. Is the initial characterization consistent with the accuracy in this context? To what extent does inaccuracy in targeting contribute to the subtlety of affecting trajectory coherence and speed? Is there a relationship between animal speed and disruption of the trajectory?

      We thank the reviewer for pointing out the discrepancy in the reported maximum speed. We have corrected the error in the main text: the average maximum speed is 142 ± 26 mm/s (four mice).

      The self-paced T-maze alternation task in Figure 5 demonstrates that mice running in a maze can be stimulated using this method. We did not optimize the particular experimental design to assess the hit accuracy, as this was determined in Figure 2. Instead, we optimized for the pulse frequencies, meaning the galvanometers tracked with processed frames but the laser was triggered whether or not the paw was actually targeted. However, even in this case with the system pulsing in the free-run mode, the laser hit rate was 54 ± 6% (mean ± sem, n = 7 mice). We have weakened references to submillimeter as it was only inferred from other experiments and was not directly measured here. We find in this experiment that stimulation in freely moving mice can cause them to briefly halt and evaluate. In the future, we will use experimental designs to more optimally examine learning.

      The reviewer also asks if there is a relationship between speed and disruption of the trajectory. We find that this is the case as described above with our additional analysis.

      Reviewer #3 (Public review):

      Summary:

      To explore the diverse nature of somatosensation, Parkes et al. established and characterized a system for precise cutaneous stimulation of mice as they walk and run in naturalistic settings. This paper provides a framework for real-time body part tracking and targeted optical stimuli with high precision, ensuring reliable and consistent cutaneous stimulation. It can be adapted in somatosensation labs as a general technique to explore somatosensory stimulation and its impact on behavior, enabling rigorous investigation of behaviors that were previously difficult or impossible to study.

      Strengths:

      The authors characterized the closed-loop system to ensure that it is optically precise and can precisely target moving mice. The integration of accurate and consistent optogenetic stimulation of the cutaneous afferents allows systematic investigation of somatosensory subtypes during a variety of naturalistic behaviors. Although this study focused on nociceptors innervating the skin (Trpv1::ChR2 animals), this setup can be extended to other cutaneous sensory neuron subtypes, such as low-threshold mechanoreceptors and pruriceptors. This system can also be adapted for studying more complex behaviors, such as the maze assay and goal-directed movements.

      Weaknesses:

      Although the paper has strengths, its weakness is that some behavioral outputs could be analyzed in more detail to reveal different types of responses to painful cutaneous stimuli. For example, paw withdrawals were detected after optogenetically stimulating the paw (Figures 3E and 3F). Animals exhibit different types of responses to painful stimuli on the hind paw in standard pain assays, such as paw lifting, biting, and flicking, each indicating a different level of pain. Improving the behavioral readouts from body part tracking would greatly strengthen this system by providing deeper insights into the role of somatosensation in naturalistic behaviors. Additionally, if the laser spot size could be reduced to a diameter of 2 mm², it would allow the activation of a smaller number of cutaneous afferents, or even a single one, across different skin types in the paw, such as glabrous or hairy skin.

      We thank the reviewer for highlighting how our system can be combined with improved readouts of coping behavior to provide deeper insights. Optogenetic and infrared cutaneous stimulation are well established generators of coping behaviors (lifting, flicking, licking, biting, guarding). Detection of these behaviors is an active and evolving field with progress being made regularly (e.g. Jones et al., eLife 2020 [PAWS];  Wotton et al., Mol Pain 2020; Zhang et al., Pain 2022; Oswell et al., bioRxiv 2024 [LUPE]; Barkai et al., Cell Reports Methods 2025 [BAREfoot], along with more general tools like Hsu et al., Nature Communications 2021 [B-SOiD]; Luxem et al., Communications Biology 2022 [VAME]; Weinreb et al,. Nature Methods 2024 [Keypoints-MoSeq]). One output of our system is bodypart keypoints, which are the typical input to many of these tools. We will leave the readers and users of the system to decide which tools are appropriate for their experimental designs - the focus of this current manuscript is describing the novel stimulation approach in moving animals.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) It is hard to see how the rig is arranged from the render of Figure 2AB due to the components being black on black. A particularly useful part of Fig2AB is the aerial view in panel B that shows the light paths. I suggest adding the labelling of Figure 2A also to that. The side/rear views could perhaps be deleted, allowing the aerial view to be larger.

      We appreciate this suggestion and have revised Figure 2B to improve the visibility of the optomechanical components. We have enlarged the side and aerial views, removed the rear view, and added further labels to the aerial view.

      (2) MAE - to interpret the 0.54 result, it would be useful to state the arena size in this paragraph.

      Thank you. We have added the arena size in this paragraph and also added scales in the relevant figure (Figure 2).

      (3) "pairwise correlations of R = 0.999 along both x- and y-axes". Is this correlation between hindpaw keypoint and galvo coordinates?

      Yes, we have added the following to clarify: “...between galvanometer coordinates and hind paw keypoints”

      (4) Latency was 84 ms. Is this mainly/entirely the delay between DLC receiving the camera image and outputting key point coordinates?

      Yes, we hope that the additional detail in the Methods and Discussion described above will now clarify the current closed-loop latencies.

      (5) "Mice move at variable speeds": in this sentence, spell out when "speed" refers to mouse and when it refers to hindpaw. Similarly, Fig 2i. The sentence is potentially confusing to general readers (paws stationary although the mouse is moving). Presumably, it's due to gait. I suggest explaining this here.

      The speed values that relate to the mouse body and paws are now clearer in the main text and in the legend for Figure 2I.

      (6) Figure 2k and associated main text. It is not clear what "success/hit rate" means here.

      We have added the following sentence in the main text: “Hit accuracy refers to the percentage of trials in which the laser successfully targeted (‘hit’) the intended hind paw.” and use hit accuracy throughout instead of success rate.

      (7) Figure 2L. All these points are greater than the "average" 0.54 reported in the text. How is this possible?

      The MAE of 0.54 mm refers to the “predicted and actual laser spot locations” (that is, the difference between where the calibration map should place the laser spot and where it actually fell), while Figure 2L MAE values refers to the error between the ground truth keypoint to laser spot (that is, the error between the human-observed paw target and where the laser spot fell). The latter error will include the former error so is expected to be larger. We have clarified this point throughout the text, for example, stating “As laser targeting relies on real-time tracking to direct the laser to the specified body part, this metric inherently accounts for any errors introduced by the tracking and targeting.”. This is also discussed above in response to Reviewer 2.

      (8) "large circular arena". State the size here

      We have added this to the Figure 2 legend.

      (9) Figure 3c-left. Can the contrast between the mouse and floor be increased here?

      We have improved the contrast in this image.

      (10) Figure 5c. It is unclear what C1, C2, etc refers to. Mice?

      Yes, these refer to mice. We have removed reference to these now as they are not needed.

      (11) Discussion. A comment. There is scope for elaborating on the potential for new research by combining it with new methods for measurements of neural activity in freely moving animals in the somatosensory system.

      Thank you. We agree and have added more detail on this in the discussion stating “The system may be combined with existing tools to record neural activity in freely-moving mice, such as fiber photometry, miniscopes, or large-scale electrophysiology, and manipulations of this neural activity, such as optogenetics and chemogenetics. This can allow mechanistic dissection of cell and circuit biology in the context of naturalistic behaviors.”

      Reviewer #3 (Recommendations for the authors):

      (1) Include the number of animals for behavior assays for the panels (e.g., Figures 4G).

      Where missing, we now state the number of animals in panels.

      (2) If representative responses are shown, such as in Figures 3E and 4F, include the average response with standard deviation so readers can appreciate the variation in the responses.

      We appreciate the suggestion to show variability in the responses. We have made several changes to Figures 3 and 4. Specifically, to illustrate the variability across multiple trials more clearly, Figure 3E now shows representative keypoint traces for each body part from two mice during their 5 trials. For Figure 4, we have re-analyzed the thermal stimulation trials and shown a raster plot of keypoint-based local motion energy (Figure 4E) sorted by response latency for hundreds of trials. Figure 4G now presents the cumulative distribution for all trials and animals for thermal (18 wild-type mice, 315 trials) and optogenetic stimulation trials (9 Trpv1::ChR2 mice, 181 trials). We also now provide means ± SD for the key metrics for optogenetic and thermal stimulation trials in Figure 4 in the Results section. This keeps the manuscript focused on the methodological advances while showing the trial variability.

      (3) "optical targeting of freely-moving mice in a large environments" should be "optical targeting of freely-moving mice in a large environment".

      Corrected

      (4) Define fps when you first mention this in the manuscript.

      Added

      (5) Data needs to be shown for the claim "Mice concurrently turned their heads toward the stimulus location while repositioning their bodies away from it".

      We state this observation to qualify that the stimulation of stationary mice resulted in behavioral responses “consistent with previous studies”. It would be redundant to repeat our full analysis and might distract from the novelty of the current manuscript. We have restricted this sentence to make it clearer: “Consistent with previous studies, we observed the whole-body behaviors like head orienting concurrent with local withdrawal (Browne et al., Cell Reports 2017; Blivis et al., eLife, 2017.)”

    1. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      The study by Druker et al. shows that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in cells arrested in prometaphase. Additionally, the expression of wild-type RepoMan, but not the RepoMan P604A mutant, restored normal H3T3 phosphorylation localization in cells arrested in prometaphase. Furthermore, the study demonstrates that expression of the RepoMan P604A mutant leads to defects in chromosome alignment and segregation, resulting in increased cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis. This occurs, at least in part, by hydroxylating RepoMan at P604, which regulates its interaction with PP2A during chromosome alignment.

      Strengths:

      The data support most of the conclusions made. However, some issues need to be addressed.

      Weaknesses:

      (1) Although ectopically expressed PHD1 interacts with ectopically expressed RepoMan, there is no evidence that endogenous PHD1 binds to endogenous RepoMan or that PHD1 directly binds to RepoMan.

      We do not fully agree that this comment is accurate - the implication is that we only show interaction between two exogenously expressed proteins, i.e. both exogenous PHD1 and RepoMan, when in fact we show that tagged PHD1 interacts with endogenous RepoMan. The major technical challenge here is the well-known difficulty of detecting endogenous PHD1 in such cell lines. We agree that co-IP studies do not prove that this interaction is direct and never claim to have shown this, though we do feel that a direct interaction is most likely, albeit not proven.

      (2) There is no genetic evidence indicating that PHD1 controls progression through mitosis by catalyzing the hydroxylation of RepoMan.

      We agree that our current study is primarily a biochemical and cell biological study, rather than a genetic study. Nonetheless, similar biochemical and cellular approaches have been widely used and validated in previous studies in mechanisms regulating cell cycle progression and we are confident in the conclusions drawn based on the data obtained so far.

      (3) Data demonstrating the correlation between dynamic changes in RepoMan hydroxylation and H3T3 phosphorylation throughout the cell cycle are needed.

      We agree that it will be very interesting to analyse in more detail the cell cycle dynamics of RepoMan hydroxylation and H3T3 phosphorylation - along with other cell cycle parameters. We view this as outside the scope of our present study and are actively engaged in raising the additional funding needed to pursue such future experiments.

      (4) The authors should provide biochemical evidence of the difference in binding ability between RepoMan WT/PP2A and RepoMan P604A/PP2A.

      Here again we agree that it will be very interesting to analyse in future the detailed binding interactions between wt and mutant RepoMan and other interacting proteins, including PP2A. We show reduced interaction in cells by PLA (Figure 5A) and in biochemical analysis (Figure 5C). More in vitro analysis is, in our view, outside the scope of our present study and we are actively engaged in raising the additional funding needed to pursue such future experiments.

      (5) PHD2 is the primary proline hydroxylase in cells. Why does PHD1, but not PHD2, affect RepoMan hydroxylation and subsequent control of mitotic progression? The authors should discuss this issue further.

      We agree with the main point underpinning this comment, i.e., that there are still many things to be learned concerning the specific roles and mechanisms of the different PHD enzymes in vivo. We address this in the Discussion section and look forward to addressing these questions experimentally in future studies.

      Reviewer #2 (Public review):

      Summary:

      This is a concise and interesting article on the role of PHD1-mediated proline hydroxylation of proline residue 604 on RepoMan and its impact on RepoMan-PP1 interactions with phosphatase PP2A-B56 complex leading to dephosphorylation of H3T3 on chromosomes during mitosis. Through biochemical and imaging tools, the authors delineate a key mechanism in the regulation of the progression of the cell cycle. The experiments performed are conclusive with well-designed controls.

      Strengths:

      The authors have utilized cutting-edge imaging and colocalization detection technologies to infer the conclusions in the manuscript.

      Weaknesses:

      Lack of in vitro reconstitution and binding data.

      We agree that it will be very interesting to pursue in vitro reconstitution studies and detailed binding data. We view this as outside the scope of our present study and are actively engaged in raising the additional funding needed to pursue such future experiments. We do provide in vitro hydroxylation data in our accompanying manuscript by Jiang et al, 2025 Elife.

      Reviewer #3 (Public review):

      Summary:

      The manuscript is a comprehensive molecular and cell biological characterisation of the effects of P604 hydroxylation by PHD1 on RepoMan, a regulatory subunit of the PPIgamma complex. The identification and molecular characterisation of the hydroxylation site have been written up and deposited in BioRxiv in a separate manuscript. I reviewed the data and came to the conclusion that the hydroxylation site has been identified and characterised to a very high standard by LC-MS, in cells and in vitro reactions. I conclude that we should have no question about the validity of the PHD1-mediated hydroxylation. 

      In the context of the presented manuscript, the authors postulate that hydroxylation on P604 by PHD1 leads to the inactivation of the complex, resulting in the retention of pThr3 in H3. 

      Strengths:

      Compelling data, characterisation of how P604 hydroxylation is likely to induce the interaction between RepoMan and a phosphatase complex, resulting in loading of RepoMan on Chromatin. Loss of the regulation of the hydroxylation site by PHD1 results in mitotic defects.

      Weaknesses:

      Reliance on a Proline-Alanine mutation in RepoMan to mimic an unhydroxylatable protein. The mutation will introduce structural alterations, and inhibition or knockdown of PHD1 would be necessary to strengthen the data on how hydroxylates regulate chromatin loading and interactions with B56/PP2A.

      We do not agree that we rely solely on analysis of the single site pro-ala mutant in RepoMan for our conclusions, since we also present a raft of additional experimental evidence, including knock-down data and experiments using both fumarate and FG. We would also reference the data we present on RepoMan in the parallel study by Jiang et al, which has also published in eLife(https://doi.org/10.7554/eLife.108128.1)). Of course, we agree with the reviewer that even although the mutant RepoMan features only a single amino acid change, this could still result in undetermined structural effects on the RepoMan protein that could conceivably contribute, at least in part, to some of the phenotypic effects observed. We now provide evidence in the current revision (new Figure 5D) that reduced interaction between RepoMan and B56gamma/PP2A is also evident when PHD1 is depleted from cells.

      Recommendations for the authors:

      Reviewer #2 (Recommendations for the authors):

      (1) The manuscript can benefit from improved quality of writing and avoidance of grammatical errors.

      We have checked through the manuscript again and corrected any mistakes we have encountered in the Current revision.

      (2) Although the data in the manuscript is compelling, it is difficult to rule out indirect effects in the interactions. Hence, in vitro binding assays with purified proteins are important to validate the findings, along with in vitro reconstitution of phosphatase activity.

      It is possible that cofactors and / or additional PTMs are required to promote these interactions in vivo. We have provided in vitro hydroxylation analysis and the additional experiments suggested will be the subject of follow-on future studies.

      (3) Proline to alanine is a drastic mutation in the amino acid backbone. The authors could purify PHD1 and reconstitute P604 hydroxylation to show if it performs as expected.

      This is likely to be a challenging experiment technically, given that RepoMan is a component of multiple distinct complexes, some of which are dynamic. We did not feel able to address this within the scope of the current study.

      (4) The confocal images showing the overlap of two fluorescent signals need to show some sort of quantification and statistics to prove that the overlap is significant.

      We now provide Pearson correlation measurements for Figure 2A in new Figure 2B in the Current revision.

      (5) Kindly provide a clearer panel for the Western blot of H3T3ph in Figure 3c.

      We have now included a new panel for this Figure in the Current revision.

      (6) Kindly also include the figures for validation of siRNAs used in the study

      We have added this throughout in supplementary figures.

      Reviewer #3 (Recommendations for the authors):

      (1) The authors have shown that PHD1 and RepoMan interact; can the interaction be "trapped" by the addition of DMOG? Generally, hydroxylase substrates can be trapped, which would add an additional layer of confidence that PHD1 and RepoMan form an enzyme-substrate complex. 

      This is something we are planning to do for follow-up studies using the established methods from the von Kriesgheim laboratory.

      (2) How does P604A mutation affect the interaction with PHD1? One would expect a reduction in interaction. 

      Another interesting point we are planning to investigate in the future.

      (3) The effects of expression of the wt and P604A mutant repoman are well-characterised. Could the authors check the effects of overexpressing PHD1 and deadPHD1, inhibition on the mitosis/H3 phosphorylation? My concerns are that a P-A mutation will disrupt the secondary structure, and although it is a good tool, data should be backed up by increasing/decreasing the hydroxylation of RepoMan over the mutation. Repeat some of the most salient experiments where the P604A mutation has been used and modulate the hydP604 by modulating PHD1 activity/expression (such as Chromatin interaction, PLA assay, B56gamma interaction, H3 phosphorylation localisation, Monastrol release, etc.)

      We agree, the PA mutant can potentially affect the protein structure. In our manuscript we have provided pH3 analysis for PHD inhibition using siRNA, FG4592 and Fumarate. In the Current revision ee also data showing that depletion of PHD1 results in a reduction in interaction between RepoMan and B56gamma/PP2A. This is now presented in new figure 5D.

      (4) I also have a general question, as a point of interest, as the interaction between PHD1 and RepoMan appears to be cell cycle dependent, is it possible that the hydroxylation status cycles as well? Could this explain how some sub-stochiometric hydroxylation events observed may be masked by assessing unsynchronised cells in bulk?

      Indeed, a very good question. We believe this is an interesting question for follow up studies. Given our previous publication showing phosphorylation of PHD1 by CDKs alters substrate binding (Ortmann et al, 2016 JCS), this is our current hypothesis.

    1. Reviewer #1 (Public review):

      Summary:

      This study presents evidence that the addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg<sup>+2</sup> ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

      Strengths:

      This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This work potentially represents an important development in the long-term effort to produce synthetic cells.

      Weaknesses:

      While much of the evidence is solid, the analysis is incomplete in certain respects that detract from the scientific quality and significance of the findings:

      (1) The authors do not describe how the native ribosomal proteins (RPs) were purified, and it is unclear whether all subassemblies of RPs have been disrupted in the purification procedure. If not, additional chaperones might be required beyond the two GTPases described here for functional ribosome assembly from individual RPs.

      (2) Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs, which would clearly address the issue in the preceding comment and also eliminate the possibility that an unknown ribosome assembly factor that co-purifies with native ribosomes has been added to the reconstitution reactions along with the RPs.

      (3) They never compared the efficiency of the reconstituted ribosomes to native ribosomes added to the "PURE" in vitro protein synthesis system, making it unclear what proportion of the reconstituted ribosomes are functional, and how protein yield per mRNA molecule compares to that given by the PURE system programmed with purified native ribosomes.

      (4) They also have not examined the synthesized GFP protein by SDS-PAGE to determine what proportion is full-length.

      (5) The previous development of the PURE system included examinations of the synthesis of multiple proteins, one of which was an enzyme whose specific activity could be compared to that of the native enzyme. This would be a significant improvement to the current study. They could also have programmed the translation reactions containing reconstituted ribosomes with (i) total native mRNA and compared the products in SDS-PAGE to those obtained with the control PURE system containing native ribosomes; (ii) with specifc reporter mRNAs designed to examine dependence on a Shine-Dalgarno sequence and the impact of an in-frame stop codon in prematurely terminating translation to assess the fidelity of initiation and termination events; and (iii) an mRNA with a programmed frameshift site to assess elongation fidelity displayed by their reconstituted ribosomes.

    2. Author response

      Public Reviews:

      Reviewer #1 (Public review):

      This study presents evidence that the addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg<sup>+2</sup> ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

      Strengths:

      This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This work potentially represents an important development in the long-term effort to produce synthetic cells.

      Weaknesses:

      While much of the evidence is solid, the analysis is incomplete in certain respects that detract from the scientific quality and significance of the findings:

      (1) The authors do not describe how the native ribosomal proteins (RPs) were purified, and it is unclear whether all subassemblies of RPs have been disrupted in the purification procedure. If not, additional chaperones might be required beyond the two GTPases described here for functional ribosome assembly from individual RPs.

      Native ribosomal proteins (RPs) were prepared from native ribosomes, according to the well-established protocol described by Dr. Knud H. Nierhaus [Nierhaus, K. H. Reconstitution of ribosomes in Ribosomes and protein synthesis: A Practical Approach (Spedding G. eds.) 161-189, IRL Press at Oxford University Press, New York (1990)]. In this method, ribosome proteins are subjected to dialysis in 6 M urea buffer, a strong denaturing condition that may completely disrupt ribosomal structure and dissociate all ribosomal protein subassemblies. To make this point clear, we will describe the ribosomal protein (RP) preparation procedure in the manuscript, rather than merely referring to the book.

      In addition, we would like to clarify one point related to this comment. The focus of the present study is to show that the presence of two factors is required for single-step ribosome reconstitution under translation-compatible, cell-free conditions. We do not intend to claim that these two factors are absolutely sufficient for ribosome reconstitution. Hence, we will revise the manuscript to more explicitly state what this work does and does not conclude.

      (2) Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs, which would clearly address the issue in the preceding comment and also eliminate the possibility that an unknown ribosome assembly factor that co-purifies with native ribosomes has been added to the reconstitution reactions along with the RPs.

      As noted in the response to the Comment (1), the focus of the present study is the requirement of the two factors for functional ribosome assembly. Therefore, we consider that it is not necessary to completely exclude the possibility that unknown ribosome assembly factors are present in the RP preparation. Nevertheless, we agree that it is important to clarify what factors, if any, are co-present in the RP fraction. To address this, we plan to add proteomic analysis results of the TP70 preparation.

      We also agree that additional, as-yet-unidentified components, including factors involved in rRNA modification, could plausibly further improve assembly efficiency. We will explicitly note this possibility in the Discussion.

      Finally, extending the system to the use of in vitro-transcribed rRNA and fully recombinant ribosomal proteins could be essentially a next step of this study, and we are currently exploring these directions in our laboratory. However, we consider them beyond the scope of the present study and will provide them as future perspectives of this study in the Discussion.

      (3) They never compared the efficiency of the reconstituted ribosomes to native ribosomes added to the "PURE" in vitro protein synthesis system, making it unclear what proportion of the reconstituted ribosomes are functional, and how protein yield per mRNA molecule compares to that given by the PURE system programmed with purified native ribosomes.

      We consider that it is feasible to estimate the GFP synthesis rate from the increase in fluorescence over time under conditions where the template mRNA is in excess, and to compare this rate directly between reconstituted and native ribosomes. We will therefore consider performing this experiment. This comparison should provide insight into what fraction of ribosomes reconstituted in our system are functionally active.

      By contrast, quantifying protein yield per mRNA molecule is substantially more challenging. The translation system is complex, and the apparent yield per mRNA can vary depending on factors such as differences in polysome formation efficiency. In addition, the PURE system is a coupled transcription–translation setup that starts from DNA templates, which further complicates rigorous normalization on a per-mRNA basis. Because the main focus of this study is to determine how many functionally active ribosomes can be reconstituted under translation-compatible conditions, we plan to address this comment by carrying out the former experiment.

      (4) They also have not examined the synthesized GFP protein by SDS-PAGE to determine what proportion is full-length.

      Because we can add an affinity tag to the GFP reporter, it should be feasible to selectively purify the synthesized protein from the reaction mixture and analyze it by SDS–PAGE. We therefore plan to perform this experiment.

      (5) The previous development of the PURE system included examinations of the synthesis of multiple proteins, one of which was an enzyme whose specific activity could be compared to that of the native enzyme. This would be a significant improvement to the current study. They could also have programmed the translation reactions containing reconstituted ribosomes with (i) total native mRNA and compared the products in SDS-PAGE to those obtained with the control PURE system containing native ribosomes; (ii) with specifc reporter mRNAs designed to examine dependence on a Shine-Dalgarno sequence and the impact of an in-frame stop codon in prematurely terminating translation to assess the fidelity of initiation and termination events; and (iii) an mRNA with a programmed frameshift site to assess elongation fidelity displayed by their reconstituted ribosomes.

      Following the recommendation, we plan to test the synthesis of at least one additional protein with enzymatic activity, in addition to GFP, so that the activity of the translated product can be assessed.

      We agree that comparing translation products using total mRNA, testing dependence on the Shine–Dalgarno sequence, and performing dedicated assays to evaluate initiation/elongation/termination fidelity are all attractive and valuable studies. However, we consider these to be beyond the scope of the present manuscript. We will therefore describe them explicitly as future directions in the Discussion.

      At the same time, we anticipate that mass spectrometric (MS) analysis of GFP and the enzyme product(s) that we attempt to synthesize could partially address concerns related to product integrity (e.g., truncations) and, to some extent, translational fidelity. We therefore plan to carry out MS analysis of these translated products.

      Reviewer #2 (Public review):

      This study presents a significant advance in the field of in vitro ribosome assembly by demonstrating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions-specifically at 37 {degree sign}C and with total Mg²⁺ concentrations below 10 mM.

      This achievement directly addresses a long-standing limitation of the traditional two-step in vitro assembly protocol (Nierhaus & Dohme, PNAS 1974), which requires non-physiological temperatures (44-50 {degree sign}C), and high Mg²⁺ concentrations (~20 mM). Inspired by the integrated Synthesis, Assembly, and Translation (iSAT) platform (Jewett et al., Mol Syst Biol 2013), leveraging E. coli S150 crude extract, which supplies essential assembly factors, the authors hypothesize that specific ribosome biogenesis factors-particularly GTPases present in such extracts-may be responsible for enabling assembly under mild conditions. Through systematic screening, they identify EngA and ObgE as the minimal pair sufficient to replace the need for temperature and Mg²⁺ shifts when using phenol-extracted (i.e., mature, modified) rRNA and purified TP70 proteins.

      However, several important concerns remain:

      (1) Dependence on Native rRNA Limits Generalizability

      The current system relies on rRNA extracted from native ribosomes via phenol, which retains natural post-transcriptional modifications. As the authors note (lines 302-304), attempts to assemble active 50S subunits using in vitro transcribed rRNA, even in the presence of EngA and ObgE, failed. This contrasts with iSAT, where in vitro transcribed rRNA can yield functional (though reduced-activity, ~20% of native) ribosomes, presumably due to the presence of rRNA modification enzymes and additional chaperones in the S150 extract. Thus, while this study successfully isolates two key GTPase factors that mimic part of iSAT's functionality, it does not fully recapitulate iSAT's capacity for de novo assembly from unmodified RNA. The manuscript should clarify that the in vitro assembly demonstrated here is contingent on using native rRNA and does not yet achieve true bottom-up reconstruction from synthetic parts. Moreover, given iSAT's success with transcribed rRNA, could a similar systematic omission approach (e.g., adding individual factors) help identify the additional components required to support unmodified rRNA folding?

      We fully recognize the reviewer’s point that our current system has not yet achieved a true bottom-up reconstruction. Although we intended to state this clearly in the manuscript, the fact that this concern remains indicates that our description was not sufficiently explicit. We will therefore revisit the organization and wording of the manuscript and revise it to ensure that this limitation is clearly communicated to readers.

      (2) Imprecise Use of "Physiological Mg²⁺ Concentration"

      The abstract states that assembly occurs at "physiological Mg²⁺ concentration" (<10 mM). However, while this total Mg²⁺ level aligns with optimized in vitro translation buffers (e.g., in PURE or iSAT systems), it exceeds estimates of free cytosolic [Mg²⁺] in E. coli (~1-2 mM). The authors should clarify that they refer to total Mg²⁺ concentrations compatible with cell-free protein synthesis, not necessarily intracellular free ion levels, to avoid misleading readers about true physiological relevance.

      We agree that this is a very reasonable point. We will therefore revise the manuscript to clarify that we are referring to the total Mg²⁺ concentration compatible with cell-free protein synthesis, rather than the intracellular free Mg²⁺ level under physiological conditions.

      In summary, this work elegantly bridges the gap between the two-step method and the extract-dependent iSAT system by identifying two defined GTPases that capture a core functionality of cellular extracts: enabling ribosome assembly under translation-compatible conditions. However, the reliance on native rRNA underscores that additional factors - likely present in iSAT's S150 extract - are still needed for full de novo reconstitution from unmodified transcripts. Future work combining the precision of this defined system with the completeness of iSAT may ultimately realize truly autonomous synthetic ribosome biogenesis.

    1. Reviewer #1 (Public review):

      Summary:

      In this article by Xiao et al., the authors aimed to identify the precise targets by which magnesium isoglycyrrhizinate (MgIG) functions to improve liver injury in response to ethanol treatment. The authors found through a series of in vivo and molecular approaches that MgIG treatment attenuates alcohol-induced liver injury through a potential SREBP2-IdI1 axis. This manuscript adds to a previous set of literature showing MgIG improves liver function across a variety of etiologies, and also provides mechanistic insight into its mechanism of action.

      Strengths:

      (1) The authors use a combination of approaches from both in-vivo mouse models to in-vitro approaches with AML12 hepatocytes to support the notion that MgIG does improve liver function in response to ethanol treatment.

      (2) The authors use both knockdown and overexpression approaches, in vivo and in vitro, to support most of the claims provided.

      (3) Identification of HSD11B1 as the protein target of MgIG, as well as confirmation of direct protein-protein interactions between HSD11B1/SREBP2/IDI1, is novel.

      Weaknesses:

      Major weaknesses can be classified into 3 groups:

      (1) The results do not support some claims made.

      (2) Qualitative analyses of some of the lipid measures, as opposed to more quantitative analyses.

      (3) There are no appropriate readouts of Srebp2 translocation and/or activity.

      More specific comments:

      (1) A few of the claims made are not supported by the references provided. For instance, line 76 states MgIG has hepatoprotective properties and improved liver function, but the reference provided is in the context of myocardial fibrosis.

      (2) MgIG is clinically used for the treatment of liver inflammatory disease in China and Japan. In the first line of the abstract, the authors noted that MgIG is clinically approved for ALD. In which countries is MgIG approved for clinical utility in this space?

      (3) Serum TGs are not an indicator of liver function. Alterations in serum TGs can occur despite changes in liver function.

      (4) There are discrepancies in the results section and the figure legends. For example, line 302 states Idil is upregulated in alcohol fed mice relative to the control group. The figure legend states that the comparison for Figure 2A is that of ALD+MgIG and ALD only.

      (5) Oil Red O staining provided does not appear to be consistent with the quantification in Figure 1D. ORO is nonspecific and can be highly subjective. The representative image in Figure 1C appears to have a much greater than 30% ORO (+) area.

      (6) The connection between Idil expression in response to EtOH/PA treatment in AML12 cells with viability and apoptosis isn't entirely clear. MgIG treatment completely reduces Idi1 expression in response to EtOH/PA, but only moderate changes, at best, are observed in viability and apoptosis. This suggests the primary mechanism related to MgIG treatment may not be via Idi1.

      (7) The nile red stained images also do not appear representative with its quantification. Several claims about more or less lipid accumulation across these studies are not supported by clear differences in nile red.

      (8) The authors make a comment that Hsd11b1 expression is quite low in AML12 cells. So why did the authors choose to knockdown Hsd11b1 in this model?

      (9) Line 380 - the claim that MGIG weakens the interaction between HSD11b1 and SREBP2 cannot be made solely based on one Western blot.

      (10) It's not clear what the numbers represent on top of the Western blots. Are these averages over the course of three independent experiments?

      (11) The claim in line 382 that knockdown of Hsd11b1 resulted in accumulation of pSREBP2 is not supported by the data provided in Figure 6D.

      (12) None of the images provided in Figure 6E support the claims stated in the results. Activation of SREBP2 leads to nuclear translocation and subsequent induction of genes involved in cholesterol biosynthesis and uptake. Manipulation of Hsd11b1 via OE or KD does not show any nuclear localization with DAPI.

      (13) The entire manuscript is focused on this axis of MgIG-Hsd11b1-Srebp2, but no Srebp2 transcriptional targets are ever measured.

      (14) Acc1 and Scd1 are Srebp1 targets, not Srebp2.

      (15) A major weakness of this manuscript is the lack of studies providing quantitative assessments of Srebp2 activation and true liver lipid measurements.

    1. Revisit the questions you answered at the beginning of the chapter, and consider one option you learned in this chapter that might change your answer to one of them.

      2 out of 4 changed for the better. I am now armed with information and new skills that I will use from now on. Thank you :-)

    1. Your significant other wants a birthday present—you have no cash. You have three exams scheduled on a day when you also need to work. Your car needs new tires, an oil change, and gas—you have no cash. (Is there a trend here?) You have to pass a running test for your physical education class, but you’re out of shape.

      1- create a gift from items at home; make a homemade meal 2-set aside time for the exams; call out from work (use leave) 3- prioritize; gas, oil change; borrow money 4-plan ahead and exercise a little everyday to get stronger; hire a personal trainer

    1. he modes are (1) call-response, a series of spontane-ous interactions between speaker and listener; (2) signification, the artof humorous put downs, usually through verbal indirection; (3) tonalsemantics, the conveying of meanings in Black discourse through spe-cifically ethnic kinds of voice rhythms and vocal inflections; and (4)

      Black Modes of Discourse, smitherman

    2. a four-part thematic structure that is basic toBlack secular, agitational rhetors: (1) all Blacks face a common enemy,(2) there is a conspiracy to violate Black manhood, (3) there is perva-sive American hypocrisy, and (4) Black unity is requisite for Black lib-eration.

      smith, a four-part thematic structure

    Annotators

    1. Reviewer #2 (Public review):

      Summary:

      The authors aimed to dissect the plasticity of circadian outputs by combining evolutionary biology with chronobiology. By utilizing Drosophila strains selected for "Late" and "Early" adult emergence, they sought to investigate whether selection for developmental timing co-evolves with plasticity in daily locomotor activity. Specifically, they examined how these diverse lines respond to complex, desynchronized environmental cues (temperature and light cycles) and investigated the molecular role of the splicing factor Psi and timeless isoforms in mediating this plasticity.

      Major strengths and weaknesses:

      The primary strength of this work is the novel utilization of long-term selection lines to address fundamental questions about how organisms cope with complex environmental cues. The behavioral data are compelling, clearly demonstrating that "Late" and "Early" flies possess distinct capabilities to track temperature cycles when they are desynchronized from light cycles.

      However, a significant weakness lies in the causal links proposed between the molecular findings and these behavioral phenotypes. The molecular insights (Figures 2, 4, 5, and 6) rely on mRNA extracted from whole heads. As head tissue is dominated by photoreceptor cells and glia rather than the specific pacemaker neurons (LNv, LNd) driving these behaviors, this approach introduces a confound. Differential splicing observed here may reflect the state of the compound eye rather than the central clock circuit, a distinction highlighted by recent studies (e.g., Ma et al., PNAS 2023).

      Furthermore, while the authors report that Psi mRNA loses rhythmicity under out-of-sync conditions, this correlation does not definitively prove that Psi oscillation is required for the observed splicing patterns or behavioral plasticity. The amplitude of the reported Psi rhythm is also low (~1.5 fold) and variable, raising questions about its functional significance in the absence of manipulation experiments (such as constitutive expression) to test causality.

      Appraisal of aims and conclusions:

      The authors successfully demonstrate the co-evolution of emergence timing and activity plasticity, achieving their aim on the behavioral level. However, the conclusion that the specific molecular mechanism involves the loss of Psi rhythmicity driving timeless splicing changes is not yet fully supported by the data. The current evidence is correlative, and without spatial resolution (specific clock neurons) or causal manipulation, the mechanistic model remains speculative.

      This study is likely to be of significant interest to the chronobiology and evolutionary biology communities as it highlights the "enhanced plasticity" of circadian clocks as an adaptive trait. The findings suggest that plasticity to phase lags - common in nature where temperature often lags light - may be a key evolutionary adaptation. Addressing the mechanistic gaps would significantly increase the utility of these findings for understanding the molecular basis of circadian plasticity.

    2. Author response:

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This manuscript addresses an important question: how do circadian clocks adjust to a complex rhythmic environment with multiple daily rhythms? The focus is on the temperature and light cycles (TC and LD) and their phase relationship. In nature, TC usually lags the LD cycle, but the phase delay can vary depending on seasonal and daily weather conditions. The authors present evidence that circadian behavior adjusts to different TC/LD phase relationships, that temperature-sensitive tim splicing patterns might underlie some of these responses, and that artificial selection for preferential evening or morning eclosion behavior impacts how flies respond to different LD/TC phase relationship

      Strength:

      Experiments are conducted on control strains and strains that have been selected in the laboratory for preferential morning or evening eclosion phenotypes. This study is thus quite unique as it allows us to probe whether this artificial selection impacted how animals respond to different environmental conditions, and thus gives hints on how evolution might shape circadian oscillators and their entrainment. The authors focused on circadian locomotor behavior and timeless (tim) splicing because warm and cold-specific transcripts have been described as playing an important role in determining temperature-dependent circadian behavior. Not surprisingly, the results are complex, but there are interesting observations. In particular, the "late" strain appears to be able to adjust more efficiently its evening peak in response to changes in the phase relationship between temperature and light cycles, but the morning peak seems less responsive in this strain. Differences in the circadian pattern of expression of different tim mRNA isoforms are found under specific LD/TC conditions.

      We sincerely thank the reviewer for this generous assessment and for recognizing several key strengths of our study. We are particularly gratified that the reviewer values our use of long-term laboratory-selected chronotype lines (350+ generations), which provide a unique evolutionary perspective on how artificial selection reshapes circadian responses to complex LD/TC phase relationships—precisely our core research question.

      Weaknesses:

      These observations are interesting, but in the absence of specific genetic manipulations, it is difficult to establish a causative link between tim molecular phenotypes and behavior. The study is thus quite descriptive. It would be worth testing available tim splicing mutants, or mutants for regulators of tim splicing, to understand in more detail and more directly how tim splicing determines behavioral adaptation to different phase relationships between temperature and light cycles. Also, I wonder whether polymorphisms in or around tim splicing sites, or in tim splicing regulators, were selected in the early or late strains.

      We thank the reviewer for this insightful comment. We agree that our current data do not establish a direct causal link between tim splicing (or Psi) and behaviour, and we appreciate that some of our wording (e.g. “linking circadian gene splicing to behavioural plasticity” or describing tim splicing as a “pivotal node”) may have suggested unintended causal links. In the revision, we will (i) explicitly state in the Abstract, Introduction, and early Discussion that the main aim was to test whether selection for timing of eclosion is accompanied by correlated evolution of temperature‑dependent tim splicing patterns and evening activity plasticity under complex LD/TC regimes, and (ii) consistently describe the molecular findings as correlational and hypothesis‑generating rather than causal. We will also add phrases throughout the text to point the reader more clearly to existing passages where we already emphasize “correlated evolution” and explicitly label our mechanistic ideas as “we speculate” / “we hypothesize” and as future experiments.

      We fully agree that studies using tim splicing mutants or manipulations of splicing regulators under in‑sync and out‑of‑sync LD/TC regimes will be essential to ascertain what role tim variants play under such environmental conditions, and we will highlight this as a key future direction. At the same time, we emphasize that the long‑term selection lines provide a complementary perspective to classical mutant analyses by revealing how behavioural and molecular phenotypes can exhibit correlated evolution under a specific, chronobiologically relevant selection pressure (timing of emergence).

      Finally, we appreciate the suggestion regarding polymorphisms. Whole‑genome analyses of these lines in a PhD thesis from our group (Ghosh, 2022, unpublished, doctoral dissertation) reveal significant SNPs in intronic regions of timeless in both Early and Late populations, as well as SNPs in CG7879, a gene implicated in alternative mRNA splicing, in the Late line. Because these analyses are ongoing and not yet peer‑reviewed, we do not present them as main results.

      I also have a major methodological concern. The authors studied how the evening and morning phases are adjusted under different conditions and different strains. They divided the daily cycle into 12h morning and 12h evening periods, and calculated the phase of morning and evening activity using circular statistics. However, the non-circadian "startle" responses to light or temperature transitions should have a very important impact on phase calculation, and thus at least partially obscure actual circadian morning and evening peak phase changes. Moreover, the timing of the temperature-up startle drifts with the temperature cycles, and will even shift from the morning to the evening portion of the divided daily cycle. Its amplitude also varies as a function of the LD/TC phase relationship. Note that the startle responses and their changes under different conditions will also affect SSD quantifications.

      We thank the reviewer for this perceptive methodological concern, which we had anticipated and systematically quantified but had not included in the original submission. The reviewer is absolutely correct that non-circadian startle responses to zeitgeber transitions could confound both circular phase (CoM) calculations and SSD quantifications, particularly as TC drift creates shifting startle locations across morning/evening windows.

      We will be including startle response quantification (previously conducted but unpublished) as new a Supplementary figure, systematically measuring SSD in 1-hour windows immediately following each of the four environmental transitions (lights-ON, lights-OFF, temperature rise and temperature fall) across all six LDTC regimes (2-12hr TC-LD lags) for all 12 selection lines (early<sub>1-4</sub>, control<sub>1-4</sub>, late<sub>1-4</sub>).

      Author response image 1.

      Startle responses in selection lines under LDTC regimes: SSD calculated to assess startle response to each of the transitions (1-hour window after the transition used for calculations). Error bars are 95% Tukey’s confidence intervals for the main effect of selection in a two-factor ANOVA design with block as a random factor. Non-overlapping error bars indicate significant differences among the values. SSD values between in-sync and out-of-sync regimes for a range of phase relationships between LD and TC cycles (A) LDTC 2-hr, (B) LDTC 4-hr, (C) LDTC 6-hr, (D) LDTC 8-hr, (E) LDTC 10-hr, (F) LDTC 12-hr.

      Key findings directly addressing the reviewer's concerns:

      (1) Morning phase advances in LDTC 8-12hr regimes are explained by quantified nocturnal startle activity around temperature rise transitions occurring within morning windows. Critically, these startles show no selection line differences, confirming they represent equivalent non-circadian confounds across lines.

      (2) Early selection lines exhibit significantly heightened startle responses specifically to temperature rise in LDTC 4hr and 6hr regimes (early > control ≥ late), demonstrating that startle responses themselves exhibit correlated evolution with emergence timing—an important novel finding that strengthens our evolutionary story.

      (3) Startle responses differed among selection lines only for the temperature rise transition under two of the regimes used, LDTC 4 hr and 6 hr regimes. Under LDTC 4 hr, temperature rise transition falls in the morning window and despite early having significantly greater startle than late, the overall morning SSD (over 12 hours morning window) did not differ significantly among the selection lines for this regime. Thus, eliminating the startle window would make the selection lines more similar to one another. On the other hand, under LDTC 6 hour regime, the startle response to temperature rise falls in the evening 12 hour window. In this case too, early showed higher startle than control and late. A higher startle in early would thus, contribute to the observed differences among selection lines. We agree with the reviewer that eliminating this startle peak would lead to a clearer interpretation of the change in circadian evening activity.

      We deliberately preserved all behavioural data without filtering out startle windows since it would require arbitrary cutoffs like 1 hr, 2 hr or 3 hours post transitions or until the startle peaks declines in different selection lines under different regimes. In the revised version, we will add complementary analyses excluding the startle windows to obtain mean phase and SSD values which are unaffected by the startle responses.

      For the circadian phase, these issues seem, for example, quite obvious for the morning peak in Figure 1. According to the phase quantification on panel D, there is essentially no change in the morning phase when the temperature cycle is shifted by 6 hours compared to the LD cycle, but the behavior trace on panel B clearly shows a phase advance of morning anticipation. Comparison between the graphs on panels C and D also indicates that there are methodological caveats, as they do not correlate well.

      Because of the various masking effects, phase quantification under entrainment is a thorny problem in Drosophila. I would suggest testing other measurements of anticipatory behavior to complement or perhaps supersede the current behavior analysis. For example, the authors could employ the anticipatory index used in many previous studies, measure the onset of morning or evening activity, or, if more reliable, the time at which 50% of anticipatory activity is reached. Termination of activity could also be considered. Interestingly, it seems there are clear effects on evening activity termination in Figure 3. All these methods will be impacted by startle responses under specific LD/TC phase relationships, but their combination might prove informative.

      We agree that phase quantification under entrained conditions in Drosophila is challenging and that anticipatory indices, onset/offset measures, and T50 metrics each have particular strengths and weaknesses. In designing our analysis, we chose to avoid metrics that require arbitrary or subjective criteria (e.g. defining activity thresholds or durations for anticipation, or visually marking onset/offset), because these can substantially affect the estimated phase and reduce comparability across regimes and genotypes. Instead, we used two fully quantitative, parameter-free measures applied to the entire waveform within defined windows: (i) SSD to capture waveform change in shape/amplitude and (ii) circular mean phase of activity (CoM) restricted to the 12 h morning and 12 h evening windows. By integrating over the entire window, these measures are less sensitive to the exact choice of threshold and to short-lived, high-amplitude startles at transitions, and they treat all bins within the window in a consistent, reproducible way across all LDTC regimes and lines. Panels C (SSD) and D (CoM) are intentionally complementary, not redundant: SSD reflects how much the waveform changes in shape and amplitude, whereas CoM reflects the timing of the center of mass of activity. Under conditions where masking alters amplitude and introduces short-lived bouts without a major shift of the main peak, it is expected that SSD and CoM will not correlate linearly across regimes.

      We will be including a detailed calculation of how CoM is obtained in our methods for the revised version.  

      Reviewer #2 (Public review):

      Summary:

      The authors aimed to dissect the plasticity of circadian outputs by combining evolutionary biology with chronobiology. By utilizing Drosophila strains selected for "Late" and "Early" adult emergence, they sought to investigate whether selection for developmental timing co-evolves with plasticity in daily locomotor activity. Specifically, they examined how these diverse lines respond to complex, desynchronized environmental cues (temperature and light cycles) and investigated the molecular role of the splicing factor Psi and timeless isoforms in mediating this plasticity.

      Major strengths and weaknesses:

      The primary strength of this work is the novel utilization of long-term selection lines to address fundamental questions about how organisms cope with complex environmental cues. The behavioral data are compelling, clearly demonstrating that "Late" and "Early" flies possess distinct capabilities to track temperature cycles when they are desynchronized from light cycles.

      We sincerely thank the reviewer for this enthusiastic recognition of our study's core strengths. We are particularly gratified that the reviewer highlights our novel use of long-term selection lines (350+ generations) as the primary strength, enabling us to address fundamental evolutionary questions about circadian plasticity under complex environmental cues. We thank them for identifying our behavioral data as compelling (Figs 1, 3), which robustly demonstrate selection-driven divergence in temperature cycle tracking.

      However, a significant weakness lies in the causal links proposed between the molecular findings and these behavioral phenotypes. The molecular insights (Figures 2, 4, 5, and 6) rely on mRNA extracted from whole heads. As head tissue is dominated by photoreceptor cells and glia rather than the specific pacemaker neurons (LNv, LNd) driving these behaviors, this approach introduces a confound. Differential splicing observed here may reflect the state of the compound eye rather than the central clock circuit, a distinction highlighted by recent studies (e.g., Ma et al., PNAS 2023).

      We thank the reviewer for highlighting this important methodological consideration. We fully agree that whole-head extracts do not provide spatial resolution to distinguish central pacemaker neurons (~100-200 total) from compound eyes and glia, and that cell-type-specific profiling represents the critical next experimental step. As mentioned in our response to Reviewer 1, we appreciate the issue with our phrasing and will be revising it accordingly to more clearly describe that we do not claim any causal connections between expression of the tim splice variants in particular circadian neurons and their contribution of the phenotype observed.

      We chose whole-head extracts for practical reasons aligned with our study's specific goals:

      (1) Fly numbers: Our artificially selected populations are maintained at large numbers (~1000s per line). Whole-head extracts enabled sampling ~150 flies per time point = ~600 flies per genotype per environmental, providing means to faithfully sample the variation that may exist in such randomly mating populations.

      (2) Established method for characterizing splicing patterns: The majority of temperature-dependent period/timeless splicing studies have successfully used whole-head extracts (Majercak et al., 1999; Shakhmantsir et al., 2018; Martin Anduaga et al., 2019) to characterize splicing dynamics under novel conditions.

      (3) Novel environmental regimes: Our primary molecular contribution was documenting timeless splicing patterns under previously untested LDTC phase relationships (TC 2-12hr lags relative to LD) and testing whether these exhibit selection-dependent differences consistent with behavioral divergence.

      Furthermore, while the authors report that Psi mRNA loses rhythmicity under out-of-sync conditions, this correlation does not definitively prove that Psi oscillation is required for the observed splicing patterns or behavioral plasticity. The amplitude of the reported Psi rhythm is also low (~1.5 fold) and variable, raising questions about its functional significance in the absence of manipulation experiments (such as constitutive expression) to test causality.

      We thank the reviewer for this insightful comment and appreciate that our phrasing has been misleading. We will especially pay attention to this issue, raised by two reviewers, and clearly highlight our results as correlated evolution and hypothesis-generating.

      We appreciate the reviewer highlighting these points and would like to draw attention to the following points in our Discussion section:

      “Psi and levels of tim-cold and tim-sc (Foley et al., 2019). We observe that this correlation is most clearly upheld under temperature cycles wherein tim-medium and Psi peak in-phase while the cold-induced transcripts start rising when Psi falls (Figure 8A1&2). Under LDTC in-sync conditions this relationship is weaker, even though Psi is rhythmic, potentially due to light-modulated factors influencing timeless splicing (Figure 8B1&2). This is in line with Psi’s established role in regulating activity phasing under TC 12:12 but not LD 12:12 (Foley et al., 2019). This is also supported by the fact that while tim-medium and tim-cold are rhythmic under LD 12:12 (Shakhmantsir et al., 2018), Psi is not (datasets from Kuintzle et al., 2017; Rodriguez et al., 2013). Assuming this to be true across genetic backgrounds and sexes and combined with our similar findings for these three transcripts under LDTC out-of-sync (Figure 2B3, D3&E3), we speculate that Psi rhythmicity may not be essential for tim-medium or tim-cold rhythmicity especially under conditions wherein light cycles are present along with temperature cycles (Figure 8C1&2). Our study opens avenues for future experiments manipulating PSI expression under varying light-temperature regimes to dissect its precise regulatory interactions. We hypothesize that flies with Psi knocked down in the clock neurons should exhibit a less pronounced shift of the evening activity under the range LDTC out-of-sync conditions for which activity is assayed in our study. On the other hand, its overexpression should cause larger delays in response to delayed temperature cycles due to the increased levels of tim-medium translating into delay in TIM protein accumulation.”

      Appraisal of aims and conclusions:

      The authors successfully demonstrate the co-evolution of emergence timing and activity plasticity, achieving their aim on the behavioral level. However, the conclusion that the specific molecular mechanism involves the loss of Psi rhythmicity driving timeless splicing changes is not yet fully supported by the data. The current evidence is correlative, and without spatial resolution (specific clock neurons) or causal manipulation, the mechanistic model remains speculative.

      This study is likely to be of significant interest to the chronobiology and evolutionary biology communities as it highlights the "enhanced plasticity" of circadian clocks as an adaptive trait. The findings suggest that plasticity to phase lags - common in nature where temperature often lags light - may be a key evolutionary adaptation. Addressing the mechanistic gaps would significantly increase the utility of these findings for understanding the molecular basis of circadian plasticity.

      Thank you for this thoughtful appraisal affirming our successful demonstration of co-evolution between emergence timing and circadian activity plasticity.

      Reviewer #3 (Public review):

      Summary:

      This study attempts to mimic in the laboratory changing seasonal phase relationships between light and temperature and determine their effects on Drosophila circadian locomotor behavior and on the underlying splicing patterns of a canonical clock gene, timeless. The results are then extended to strains that have been selected over many years for early or late circadian phase phenotypes.

      Strengths:

      A lot of work, and some results showing that the phasing of behavioural and molecular phenotypes is slightly altered in the predicted directions in the selected strains.

      We thank the reviewer for acknowledging the substantial experimental effort across 7 environmental regimes (6 LDTC phase relationships + LDTC in-phase), 12 replicate populations (early<sub>1-4</sub>, control<sub>1-4</sub>, late<sub>1-4</sub>), and comprehensive behavioural + molecular phenotyping.

      Weaknesses:

      The experimental conditions are extremely artificial, with immediate light and temperature transitions compared to the gradual changes observed in nature. Studies in the wild have shown how the laboratory reveals artifacts that are not observed in nature. The behavioural and molecular effects are very small, and some of the graphs and second-order analyses of the main effects appear contradictory. Consequently, the Discussion is very speculative as it is based on such small laboratory effects.

      We thank the reviewer for these important points regarding ecological validity, effect sizes, and interpretation scope.

      (1) Behavioural effects are robust across population replicates in selection lines (not small/weak)

      Our study assayed 12  populations total (4 replicate populations each of early, control, and late selection lines) under 7 LDTC regimes. Critically, selection effects were consistent across all 4 replicate populations within each selection line for every condition tested. In these randomly mating large populations, the mixed model ANOVA reveals highly significant selection×regime interactions [F(5,45)=4.1, p=0.003; Fig 3E, Table S2], demonstrating strong, replicated evolutionary divergence in evening temperature sensitivity.

      (2) Molecular effects test critical evolutionary hypothesis

      As stated in our Introduction, "selection can shape circadian gene splicing and temperature responsiveness" (Low et al., 2008, 2012). Our laboratory-selected chronotype populations—known to exhibit evolved temperature responsiveness (Abhilash et al., 2019, 2020; Nikhil et al., 2014; Vaze et al., 2012)—provide an apt system to test whether selection for temporal niche leads to divergence in timeless splicing. With ~600 heads per environmental regime per selection line, we detect statistically robust, selection line-specific temporal profiles [early4 advanced timeless phase (Fig 4A4); late4 prolonged tim-cold (Fig 5A4); significant regime×selection×time interactions (Tables S3-S5)], providing initial robust evidence of correlated molecular evolution under novel LDTC regimes.

      (3) Systematic design fills critical field gap

      Artificial conditions like LD/DD have been useful in revealing fundamental zeitgeber principles. Our systematic 2-12hr TC-LD lags directly implement Pittendrigh & Bruce (1959) + Oda & Friesen (2011) validated design, which discuss how such experimental designs can provide a more comprehensive understanding of zeitgeber integration compared to studies with only one phase jump between two zeitgebers.

      (4) Ramping regimes as essential next step

      Gradual ramping regimes better mimic nature and represent critical future experiments. New Discussion addition in the revised version: "Ramping LDTC regimes can test whether selection-specific zeitgeber hierarchy persists under naturalistic gradients." While ramping experiments are essential, we would like to emphasize that we aimed to use this experimental design as a tool to test if evening activity exhibits greater temperature sensitivity and if this property of the circadian system can undergo correlated evolution upon selection for timing of eclosion/emergence.

      (5) New startle quantification addresses masking

      Our startle quantification (which will be added as a new supplementary figure) confirms circadian evening tracking persists despite quantified, selection-independent masking in most of the regimes.

    1. # plot logarithm of dividend yield series plot(StockReturns[, 2], col = "steelblue", lwd = 2, ylab = "Logarithm", main = "Dividend Yield for CRSP Index")

      Alternative link to Stock Returns: https://www.princeton.edu/~mwatson/Stock-Watson_3u/Students/EE_Datasets/Stock_Returns_1931_2002.xlsx With this data is:

      StockReturns <- ts(StockReturns[, 3:4], start = c(1931, 1), end = c(2002, 12), frequency = 12)

      plot(StockReturns[, 2], col = "steelblue", lwd = 2, ylab = "Logarithm", main = "Dividend Yield for CRSP Index")

    1. Reviewer #1: This manuscript addresses a highly relevant public health issue. Overall the manuscript is well-structured and presents important findings however, a few refinements could enhance clarity. Specifically, the discussion could be strengthened by drawing clearer implications for policy and scalability,how lessons from high-fidelity can be adapted to low fidelity settings. Adding explanatory footnotes to some of the tables and ensuring that figures and tables, supporting materials are properly referenced intext .

      Reviewer #2: This manuscript addresses an important and understudied implementation science topic: implementation fidelity of tuberculosis (TB) screening among diabetes mellitus (DM) patients in routine care settings in Tanzania. The topic is relevant to TB–DM collaborative activities and aligns well with global priorities. However, several substantive issues need to be addressed before the findings can be interpreted with confidence.

      Sampling strategy There is a lack of clarity and internal consistency between the sampling strategy described in the Methods and the way provider numbers are reported in the Results. The Methods indicate that 2–4 healthcare providers were selected per facility using proportional allocation and simple random sampling, yet Table 1 reports aggregate numbers by facility type (e.g., dispensary, health centre, hospital) without indicating how many providers were recruited from each of the 20 facilities. This makes it difficult to assess representativeness and raises concerns about clustering (e.g., whether multiple providers came from the same facility). The authors should clearly report the number of participants recruited per facility, ideally in a supplementary table, and explain how the stated sampling strategy was operationalised.

      Outcome definition and interpretation The primary outcome is provider-level implementation fidelity, measured through self-reported adherence to TB screening guideline components. However, the Results and Discussion repeatedly imply patient-level screening coverage (e.g., statements suggesting that a certain proportion of DM patients were screened for TB). No patient-level numerator or denominator is presented, and the Methods do not describe record review or observation. The authors should consistently frame the outcome as provider-level fidelity, revise language that implies patient screening coverage, and explicitly acknowledge the absence of patient-level screening data as a limitation if such data were not collected.

      Unsupported causal explanations The Discussion attributes low implementation fidelity (17%) to factors such as lack of integrated TB–DM training and provider role allocation, yet these explanations are not adequately supported by the study data. Training does not appear to remain significant in adjusted analyses, and several explanatory statements are not referenced. In addition, the Discussion suggests that degree-holding providers may focus on administrative duties, while the Methods state that staff in administrative roles were excluded from the study. These contradictions should be resolved, and causal language should be softened or removed where not directly supported by evidence.

      Discussion focus The Discussion begins by restating the study’s aim and strengths rather than clearly summarising the key findings. Several paragraphs repeat results or focus heavily on comparisons with other studies, with limited interpretation of what the findings mean for the Ubungo or Tanzanian primary care context. The Discussion would be strengthened by focusing on (i) the most poorly implemented screening components, (ii) why dispensaries showed lower fidelity, and (iii) the implications for TB–DM integration, supervision, and training in similar settings.

      Statistical reporting The analytical approach (modified Poisson regression) is appropriate for the outcome, but there appear to be potential reporting errors (e.g., confidence intervals in Table 4 where bounds appear inconsistent). These should be carefully checked. In addition, typographical errors (e.g., “modified poison regression”) should be corrected.

      Limitations section The limitations are acknowledged; however, they could be more clearly framed from an implementation science perspective, including reliance on self-reported practices, absence of observational or record-based verification, and the cross-sectional design limiting causal inference.

    1. Reviewer #1: PLOS Global Public Health ECONOMIC AND HEALTH IMPACTS OF BOVINE TUBERCULOSIS ON RURAL ZAMBIAN COMMUNITIES

      General Assessment This manuscript addresses a relevant and timely topic, exploring the economic and health impacts of bovine tuberculosis (bTB) on rural communities in Zambia through a mixed-methods approach. The work is valuable and provides important insights into the socioeconomic vulnerabilities associated with bTB. However, several areas require clarification and strengthening to enhance the scientific robustness and public health relevance of the study.

      Major Comments 1. Missing epidemiological context on zoonotic TB in humans The manuscript discusses the public health implications of bTB but does not provide available data on M. bovis infection prevalence in humans at: • national level, • district level (Lundazi and Monze), • or from comparable regions in sub-Saharan Africa. To address this gap, please consider integrating key global references on zoonotic TB, such as: • WHO (2017). Roadmap for Zoonotic tuberculosis https://www.who.int/publications/i/item/9789241513043 • Olea-Popelka, F., & Fujiwara, P. I. (2018). Building a Multi-Institutional and Interdisciplinary Team to Develop a Zoonotic Tuberculosis Roadmap. Frontiers in Public Health, 6, 167. https://www.frontiersin.org/articles/10.3389/fpubh.2018.00167/full Including these references will help contextualize the burden of zoonotic TB and strengthen the public health discussion and better support conclusions.

      1. Public health implications are underdeveloped While the economic impact of bTB is well described, the public health dimension is comparatively limited. The manuscript would benefit from: • more explicit discussion of zoonotic risks for different demographic groups, • potential barriers to diagnosis and reporting of M. bovis in rural healthcare settings, • implications for One Health surveillance. This would provide a more balanced interpretation aligned with the study objectives.

      2. Limited comparison with existing literature The discussion currently focuses mainly on East and Southern Africa. It would be helpful to cite global and regional reviews addressing the wildlife–livestock–human interface, which is central to bTB epidemiology in Zambia. Please consider adding: De GARINE-WICHATITSKY M, CARON A, KOCK R, et al. 2013 (Cambridge): A review of bovine tuberculosis at the wildlife–livestock–human interface in sub-Saharan Africa https://www.cambridge.org/core/journals/epidemiology-and-infection/article/review-of-bovine-tuberculosis-at-the-wildlifelivestockhuman-interface-in-subsaharan-africa/19D207B4D88531AB03A96FEF7BF6F95E Munyeme et al. (2011). A Review of Bovine Tuberculosis in the Kafue Basin Ecosystem https://pmc.ncbi.nlm.nih.gov/articles/PMC3087610/ These references are particularly relevant given the role of Kafue lechwe as a reservoir species and the importance of studying disease dynamics at the domestic–wildlife–human interface.

      3. Interpretation of increased vulnerability in elderly respondents The interpretation that elderly individuals are more affected because of reduced immunity and lower awareness requires careful qualification. While it is plausible that older adults may be more likely to progress to clinical disease due to immunosenescence, the study did not include any diagnostic testing for M. bovis infection in humans. Without diagnostic data, such as tuberculin skin testing (e.g., the Mantoux test), interferon-gamma release assays, or microbiological confirmation, the study cannot infer the true prevalence of mycobacteria infection across age groups. It is important to acknowledge that younger individuals may have similar or even higher infection rates but remain asymptomatic due to a more effective immune response. Thus, the distinction between: • infection prevalence (which requires diagnostic testing), and • clinical disease expression (more common in immunosuppressed or elderly individuals) should be clearly stated to avoid overinterpretation of the findings.

      4. Methodological clarifications required Several methodological details require further explanation: • Clarify whether “strong cough” and “diseased animals” were self-reported or confirmed by veterinary staff. • Consider discussing potential confounders affecting milk/meat yield (other diseases, nutrition, seasonality). • Income calculations assume fixed milk prices; please comment on possible seasonal or regional/geographic price variability. Addressing these points will improve methodological transparency.

      Minor Comments 1. Some sections require language editing to improve clarity and flow. 2. Figures and tables would benefit from clearer captions and more detailed descriptions. 3. A brief description of cattle management systems in Lundazi and Monze would provide useful context for interpreting transmission risks. 4. The Discussion could better highlight the value added by the mixed-methods approach. 5. You may consider revising the reference list, as several entries appear duplicated. Specifically, the following references are listed more than once:  Demetriou 2020 (Refs. 23 and 26)  Oloya et al. (Refs. 29 and 37)  Ameni et al. (Refs. 30, 38, and 45)  Kansiime et al. (Refs. 28 and 36)  Kazwala et al. (Refs. 32 and 39)  Mfinanga et al. (Refs. 33 and 40)  Duguma et al. (Refs. 34 and 41)  Kassa et al. (Refs. 35 and 42) Additionally, two reviews by Tschopp (Refs. 18 and 24) appear very similar and may need verification to confirm they are distinct publications. A careful review of the reference list is recommended to avoid redundancy and improve clarity.

      Overall Recommendation The manuscript provides useful insights but requires substantial revisions. Strengthening the epidemiological context, integrating key references, and refining the public health discussion will significantly improve the impact and clarity of the work and enhance its contribution to PLOS Global Public Health.

    1. De PGDI wordt voorgezeten door een voorzitter die het draagvlak heeft van de leden van de PGDI. 2 De staatssecretaris benoemt de voorzitter. 3 De voorzitter geeft op een objectieve wijze invulling aan het voorzitterschap vanuit een breed perspectief op de digitale overheid; 4 De PGDI bestaat voorts uit de volgende leden op minimaal directeursniveau: a. een vertegenwoordiger namens het Uitvoeringsinstituut Werknemersverzekeringen en/of de Sociale Verzekeringsbank; b. een vertegenwoordiger namens de Vereniging van Nederlandse Gemeenten; c. een vertegenwoordiger namens het Ministerie van Volksgezondheid, Welzijn en Sport; d. een vertegenwoordiger namens de Unie van Waterschappen; e. een vertegenwoordiger namens het Interprovinciaal Overleg; f. een vertegenwoordiger namens de Dienst Uitvoering Onderwijs; g. een vertegenwoordiger namens de Pensioenfondsen; h. een vertegenwoordiger namens de Belastingdienst (ook voor Douane en Toeslagen); i. de coördinerend opdrachtgever GDI; j. een vertegenwoordiger namens de Kamer van Koophandel; k. een vertegenwoordiger namens de Manifestgroep; l. een vertegenwoordiger namens Logius (ook voor KOOP); m. een vertegenwoordiger namens Rijksdienst voor Identiteitsgegevens; n. een vertegenwoordiger namens Rijksdienst voor Ondernemend Nederland; o. alsmede, afhankelijk van het onderwerp, de betrokken (kleine) uitvoeringsorganisatie(s).

      leden zijn de 'afnemers' GDI, op dir niveau. vz is door stas benoemd en niet qq. - [ ] achterhaal mensen in PGDI. #geonovumtb

    1. L'École au Cœur des Valeurs de la République : Faire Vivre l'Égalité, la Mixité et la Réussite

      Synthèse opérationnelle

      Ce document de synthèse analyse les interventions de la table ronde organisée par l'INSPÉ Lille HdF, portant sur l'incarnation des valeurs républicaines au sein des établissements scolaires.

      Le constat central est que la transmission des valeurs ne peut se limiter à un discours théorique ; elle nécessite une approche systémique touchant à la fois la pédagogie, le pilotage institutionnel et l'aménagement physique des lieux.

      Les points clés identifiés sont :

      L'éducabilité comme principe moteur : Reconnaître l'aptitude de chaque élève à être éduqué et transformé par l'école est le socle de l'engagement professionnel.

      La mixité sociale harmonieuse : L'expérimentation montre que le brassage de populations sociologiquement opposées favorise la tolérance et réduit le décrochage, à condition d'être soutenu par un projet fort.

      Le levier du « bâti » et de l'accueil : La matérialisation des valeurs (beauté des lieux, aménagement convivial) est un facteur déterminant pour le bien-être et le respect mutuel.

      La complexité de la notion de « réussite » : Des recherches en psychologie sociale alertent sur le fait qu'une focalisation étroite sur la performance peut paradoxalement nuire aux attitudes inclusives des enseignants.

      --------------------------------------------------------------------------------

      1. Principes fondamentaux de l'action éducative

      L'ancrage des valeurs républicaines en milieu scolaire repose sur des piliers éthiques et professionnels partagés par les acteurs de terrain.

      Le principe d'éducabilité

      L'éducabilité est définie comme la reconnaissance de l'aptitude de chaque individu à recevoir une éducation et à évoluer par son intermédiaire.

      Une obligation pour les professionnels : Ce principe oblige les personnels de l'éducation à développer des relations de confiance, à valoriser l'élève et à pratiquer une bienveillance éducative constante.

      Finalité : L'objectif est de permettre aux jeunes de s'instruire, de s'émanciper et de devenir des citoyens actifs et éclairés.

      L'appartenance au collectif

      La transmission des valeurs est présentée comme une mission impossible à mener de manière isolée.

      Le travail d'équipe : Que ce soit au sein des équipes académiques « Valeurs de la République » ou au niveau des établissements, le collectif est essentiel pour penser et agir.

      L'articulation des échelles : L'action doit se situer à la confluence de l'individuel (posture de l'enseignant), du collectif (classe/établissement) et de l'institutionnel (Académie).

      --------------------------------------------------------------------------------

      2. Faire vivre l'égalité et l'inclusion

      L'égalité n'est pas seulement un principe constitutionnel (Article 6 de la DDHC), c'est une pratique quotidienne qui se décline en plusieurs dimensions.

      Égalité des chances et équité

      Pédagogie universelle : L'enjeu est d'identifier et de lever les obstacles qui empêchent certains élèves d'accéder aux compétences (par exemple, permettre l'accès au savoir en histoire même si la lecture n'est pas maîtrisée).

      Donner plus à ceux qui ont des besoins particuliers : L'égalité en établissement se traduit souvent par l'équité, c'est-à-dire l'adaptation des moyens aux besoins spécifiques des élèves, notamment dans le cadre de l'école inclusive.

      Les défis de la notion de « réussite »

      Des travaux de recherche en psychologie sociale mettent en lumière une tension entre les objectifs de performance et d'inclusion :

      Risque de rejet de l'inclusion : Lorsque le système éducatif valorise exclusivement la réussite au sens de la performance et du développement des compétences, les enseignants peuvent développer des attitudes plus négatives à l'égard de l'éducation inclusive.

      Nécessité d'une définition large : La réussite doit être associée à l'épanouissement et au bien-être pour ne pas devenir un facteur d'exclusion.

      --------------------------------------------------------------------------------

      3. Mixité sociale et climat scolaire : l'expérience de terrain

      L'exemple du collège Berlioz à Paris (18e arrondissement) illustre la mise en œuvre concrète de la mixité sociale et de la lutte contre les déterminismes.

      L'expérimentation de la montée alternée

      Pour contrer un évitement scolaire massif (50 %) et un ghetto social, deux établissements (un très favorisé et un très défavorisé) ont fusionné leurs effectifs par un système de niveaux alternés.

      Résultats : Apprentissage de la tolérance par la confrontation à l'autre, disparition quasi totale du décrochage scolaire, et absence d'exclusions définitives sur plusieurs années.

      Mixité harmonieuse : La diversité (origine sociale, culturelle, élèves en situation de handicap) crée un environnement où chacun trouve sa place.

      La matérialisation des valeurs (le bâti)

      Le cadre physique est un levier majeur pour le climat scolaire. Farid Bouelifa souligne l'importance d'un établissement « accueillant et beau » :

      Aménagements concrets : Installation de fontaines, de jardins pédagogiques, de fresques végétales, de drapeaux et de canapés dans les espaces communs.

      Symbolique : Utilisation des couleurs républicaines (bleu, blanc, rouge) de manière esthétique dans le bâti pour ancrer l'identité républicaine sans qu'elle soit vécue comme une contrainte.

      --------------------------------------------------------------------------------

      4. Structures institutionnelles et partenariats

      Pour transformer l'école en un « territoire vivant », plusieurs dispositifs et instances doivent être mobilisés.

      Le CESCE : une instance sous-exploitée

      Le Comité d'Éducation à la Santé, à la Citoyenneté et à l'Environnement (CESCE) est identifié comme un levier systémique majeur.

      Rôle : Définir la politique de prévention, lutter contre les discriminations et le harcèlement, et promouvoir l'égalité fille-garçon.

      Composition : Il permet de créer des « alliances éducatives » en associant parents, partenaires extérieurs, élèves (éco-délégués, élus CVL/CVC) et personnels de santé.

      L'ouverture sur le territoire

      L'école ne doit pas être un territoire clos. L'interaction avec l'extérieur est vitale :

      Partenariats associatifs : Collaboration avec des structures locales (centres sociaux, associations) pour prendre en charge le jeune dans sa globalité.

      Sorties pédagogiques : Elles sont jugées aussi importantes que les cours, car elles permettent aux élèves issus de milieux défavorisés d'accéder à des lieux de culture (Louvre, Philharmonie, Versailles) qu'ils ne visiteraient jamais autrement.

      --------------------------------------------------------------------------------

      Citations marquantes

      « Le principe d'éducabilité nous oblige, nous professionnels de l'éducation. C'est reconnaître l'aptitude de chacun à être éduqué. » — Sandrine Benavkir

      « La mixité sociale, on apprend la tolérance à travers elle avec celui qui est différent de soi. » — Farid Bouelifa

      « Parler de réussite, de performance, du développement des compétences... quand on proposait aux enseignants de lire ce magazine, ils avaient des attitudes bien plus négatives à l'égard de l'éducation inclusive. » — Anne-Laure Perrin

      « L'école, c'est aussi parfois la parenthèse de ces élèves dans leur vie... leur faire sentir que ces valeurs de la République, eux peuvent les vivre à l'école. » — Anne-Laure Perrin

      « Lorsque vous enseignez la tolérance au quotidien, c'est une manière de lutter contre tout : le harcèlement, l'homophobie, les discriminations. » — Farid Bouelifa

      --------------------------------------------------------------------------------

      Conclusion

      Faire vivre les valeurs de la République en établissement exige de passer de l'affirmation (le discours) à l'incarnation (l'action).

      Cela passe par un engagement collectif des personnels, une attention particulière portée au climat scolaire et une volonté politique de briser les ghettos sociaux par des dispositifs de mixité audacieux.

      L'école doit être ce lieu où l'égalité des droits et des chances se traduit par une équité pédagogique et un respect profond de la singularité de chaque élève.

    1. L'Intelligence Artificielle en Milieu Scolaire : Transformer l'Illusion de Connaissance en Levier d'Apprentissage

      Résumé Exécutif

      L'intégration de l'intelligence artificielle (IA) dans le milieu éducatif présente un paradoxe : si elle facilite la production de contenus structurés, elle risque de favoriser une « illusion de connaissance » où l'élève externalise sa pensée sans réelle compréhension.

      Ce document analyse une approche pédagogique visant à transformer l'IA, de simple outil de génération automatique, en un partenaire de réflexion, un assistant d'écriture et un tuteur de révision.

      L'objectif central est de passer d'une utilisation passive à un usage actif et supervisé, permettant de renforcer l'esprit critique, la capacité d'argumentation et la maîtrise méthodologique des élèves, tout en respectant un cadre éthique et technique strict.

      --------------------------------------------------------------------------------

      1. Le Défi de l'Illusion de Connaissance

      L'émergence de l'IA générative crée un risque majeur pour l'apprentissage : la capacité de produire des devoirs rédigés sans effort intellectuel réel.

      Le concept d'illusion : Les élèves peuvent avoir l'impression de maîtriser un sujet parce qu'ils obtiennent un résultat immédiat et bien structuré, alors qu'ils ne font que survoler le contenu.

      L'externalisation de la pensée : L'outil risque de devenir un substitut au travail personnel, sortant des réponses « du chapeau » sans que l'élève puisse les justifier ou les expliquer.

      L'analogie de la calculatrice : À l'instar de l'arrivée des calculatrices en mathématiques, l'IA doit être perçue comme une « calculatrice pour les mots » (selon Sam Altman).

      Une méta-analyse de 2003 démontre que l'usage de la calculatrice, lorsqu'elle est intégrée à l'enseignement, permet aux élèves de mieux se concentrer sur les concepts de base et la résolution de problèmes.

      --------------------------------------------------------------------------------

      2. L'IA comme Partenaire d'Argumentation

      L'une des fonctions clés identifiées est l'utilisation de l'IA pour structurer le raisonnement logique sans que l'outil ne se substitue à l'élève.

      Stratégie de Dialogue Étape par Étape

      Pour éviter que l'IA ne réponde à la place de l'élève, un processus en plusieurs phases est préconisé :

      1. Clarification du sujet : Analyse des termes et des mots-clés (ex: définir « mobiliser » ou « ensemble des sociétés » dans un sujet d'histoire).

      2. Renforcement des idées : Aide à l'identification des axes majeurs et des acteurs concernés.

      3. Organisation de l'argumentation : Élaboration conjointe d'un plan.

      4. Structuration finale : Justification des choix argumentatifs.

      Bénéfices Pédagogiques

      Dépassement de la peur de formuler : L'élève se concentre sur le fond de sa pensée.

      Empathie intellectuelle : En demandant à l'IA d'envisager des points de vue contraires, l'élève développe son esprit critique.

      Justification des choix : L'élève apprend à comprendre ce qu'il écrit en devant expliquer ses décisions à l'outil.

      --------------------------------------------------------------------------------

      3. L'IA comme Assistant à la Rédaction et à la Méthodologie

      Contre le « syndrome de la page blanche », l'IA agit comme un déclencheur plutôt que comme un auteur autonome.

      | Fonction | Description de l'intervention | | --- | --- | | Aide à l'étincelle | Fournit le premier élan pour mettre les idées en mots. | | Vérification logique | Analyse la progression entre les parties du plan (ex: passage de la mobilisation humaine à la mobilisation des savoirs). | | Soutien méthodologique | Rappelle les attentes académiques (ex: structure d'une introduction : accroche, définition, problématique, plan). | | Affinement rédactionnel | Force une discussion pour améliorer la clarté sans générer le bloc de texte final d'un coup. |

      --------------------------------------------------------------------------------

      4. L'IA comme Compagnon d'Apprentissage et Tuteur

      L'IA peut également remplir un rôle de soutien individualisé en orthographe et en révision.

      Tutorat en Orthographe et Grammaire

      Au lieu d'un correcteur automatique passif, l'IA est sollicitée comme un « professeur bienveillant » :

      Principe : Ne pas corriger à la place de l'élève.

      Méthode : Mettre les fautes en gras, expliquer la règle simplement, donner un exemple et laisser l'élève effectuer la correction activement.

      Tuteur de Révision Autonome

      L'IA peut tester la compréhension profonde de l'élève pour repérer les lacunes :

      Niveaux de questionnement : Progression des concepts fondamentaux vers des analyses plus complexes (ex: le rôle des colonies dans l'effort de guerre).

      Feedback constructif : L'outil doit valoriser les bonnes réponses tout en utilisant les erreurs comme des occasions d'apprentissage pédagogique.

      --------------------------------------------------------------------------------

      5. Cadre Éthique et Règles d'Usage

      Pour que l'IA reste un outil au service de l'humain, son utilisation doit être encadrée par des principes stricts :

      Âge requis : Pas d'usage de l'IA avant la classe de 4ème.

      Plus-value pédagogique : Recourir à l'IA uniquement lorsqu'elle apporte une réelle valeur ajoutée à l'apprentissage.

      Transparence : Mentionner systématiquement l'usage de l'IA et citer son aide comme on citerait une source (référence au système de Martin Petters).

      Responsabilité environnementale et technique : Privilégier des solutions sobres écologiquement et respecter scrupuleusement la protection des données personnelles.

      Posture de l'élève : L'élève doit rester maître du processus en pratiquant, manipulant et confrontant les connaissances pour assurer une mémorisation durable.

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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      Reply to the reviewers

      Manuscript number: RC-2025-03131

      Corresponding author(s): Ginto George and Adriana Ordoñez

      1. General Statements

      We thank the reviewers for their careful evaluation of our work and for their constructive and insightful comments. We are pleased that both reviewers found the study to be well executed, clearly presented, and of interest to the ER stress and UPR community. We have carefully considered all comments and revised the manuscript accordingly. We believe these revisions have substantially strengthened the clarity, robustness, and conceptual impact of the study.

      2. Point-by-point description of the revisions

      Below we provide a detailed, point-by-point response to the reviewers' comments and describe the revisions and new data included in the revised manuscript.

      Reviewer 1 & 2 (common points)

      1. __ Description of the BiP::GFP reporter as a readout of ATF6α activity.__
      2. Comment: Both reviewers are concerned about whether BiP::GFP is a reliable and specific reporter for ATF6α
      3. Response: In response, we have clarified in the revised manuscript the details of the BiP promoter fragment used in this reporter, explicitly detailing the presence of an ERSE-I element motif (CCAAT-N9-CCACG), the most specifically and robustly activated by ATF6α (new Supplemental Fig. S1). This reporter was first characterised in our recently published study (Tung et al., 2024 eLife), where we demonstrated that BiP::GFP expression is ATF6α dependent, as CRISPR/Cas9-mediated disruption of endogenous ATF6α resulted in a marked reduction in BiP::GFP fluorescence compared with parental cells. Furthermore, treatment with ER stress in the presence of Ceapin-A7 (a small molecule that blocks ATF6⍺ activation by tethering it to the lysosome) effectively blocked activation of the ATF6⍺ fluorescent reporter, whereas the S1P inhibitor partially attenuated the BiP::sfGFP signal in stressed cells (Tung et al., 2024 eLife; Supplemental S1D). We have now reproduced these findings in the present study, further confirming that the BiP::GFP reporter is highly dependent on ATF6α activation, and we present these data in a new Supplemental Fig. S1B.

      __ Correlation between BiP::GFP reporter activity and BiP expression levels.__

      • Comment: Both reviewers requested correlation of the BiP::GFP reporter activity and endogenous BiP levels.
      • __Response: __To address this point, we have measured BiP mRNA levels in parental and Slc33a1-depleted cells under both basal conditions and ER stress conditions. These measurements correlated well with the BiP::GFP reporter activity assessed by flow cytometry and are shown in Supplemental Fig. S3E.

      __ Does ATF6α respond to other ER stressors in Slc33a1-deleted cells?__

      • Comment: Both reviewers accepted our claim that ATF6α activation is partially attenuated in Slc33a1-deleted cells exposed to ER stressors tunicamycin (Tm) and 2-Deoxy-D-glucose (2DG) but raised the possibility that ATF6α signalling might respond differently to other ER stressors.
      • Response: To address this point, we have performed new experiments assessing ATF6α activation (BiP::GFP activity) in both Slc33a1-deleted and parental cells in response to additional ER stressors, including dithiothreitol (DTT) and thapsigargin (Tg). These new data, presented in a new Supplemental Fig. S3B and S3C, show that Slc33a1-deletion also attenuates ATF6α signalling in cells treated with dithiothreitol (DTT) and thapsigargin (Tg).

      __ Deletion of all NAT8 family members.__

      • Comment: Both reviewers suggested that deletion of all NAT8 family members was required to conclusively distinguish their role from that of SLC33A1.
      • __Response: __We agree with this assessment and have now generated cells in which both Nat8 and Nat8b are simultaneously deleted. These new data, included in a new Supplemental Fig. S9, strengthen the comparison with SLC33A1 deficiency and rule out potential redundancy among NAT8 family members. Notably, simultaneous inactivation of Nat8 and Nat8b resulted in the same phenotype observed upon single Nat8 deletion, namely activation of both the IRE1 and ATF6α branches of the UPR. These findings (discussed in detail) are consistent with previous studies implicating protein acetylation in ER proteostasis but suggest that a defect in protein acetylation is unlikely to contribute to the consequences of SLC33A1 deficiency in terms of ATF6α

      __ Generalisability beyond CHO-K1 cells.__

      • Comment: Reviewer 1 raised concerns regarding validation of our findings beyond CHO-K1 cells.
      • Response: While we acknowledge that validation in additional cell types would further strengthen the study, we now explicitly discuss the technical challenges encountered when attempting to generate clonal Slc33a1 knockouts in aneuploid human cell lines, such as HeLa. This limitation is now clearly acknowledged in the revised version, and our conclusions are framed accordingly.

      __ Relationship between basal ATF6 and IRE1 signalling.__

      • Comment: Both reviewers argued that BiP::GFP does not appear to be active under basal conditions in parental cells, and therefore a failure to activate ATF6 would not be expected to affect the conditions of the cells basally. Thereby questioning how attenuated basal ATF6 activity in the SLC33a1 deleted cells could account for the derepression observed in the IRE1 pathway.
      • Response: The logic of the reviewer's critique is impeccable, and we thank them for the opportunity to clarify this important issue. Whilst the basal fluorescent signal arising from BiP::GFP (the ATF6α reporter) is indeed weak, it is not null. This is evident by comparing the BiP::GFP signal in wildtype and ATF6α -deleted cells (new Supplemental Fig. S1B) These experiments revealed a significant reduction in basal BiP::GFP fluorescence in ATF6αΔ cells compared with parental dual-reporter cells, indicating that the BiP::GFP reporter has basal activity that is dependent on ATF6α. These new data are consistent with previous published observations demonstrated that treatment with Ceapin, an ATF6α-specific inhibitor, lowered BiP::GFP fluorescence in tunicamycin-treated cells to levels below those observed in untreated controls (Tung et al., eLife 2024). Together these observations indicate that ATF6α is active basally in CHO-K1 cells. Given the established cross-pathway repression of IRE1 by ATF6α signalling, it renders plausible our suggestion that the basal activation of the XBP1::mCherry (IRE1-reporter) observed basally in the SLC33a1 deleted cells arises from the partial interruption of ATF6α Reviewer 1 (additional points)

      • __ Effect of deleting sialic acid-modifying acetyltransferases.__

      • Comment: Reviewer 1 suggested that comparing the consequences of deleting SLC33a1 and the sialic acid- modifying acetyltransferases that operate downstream of the putative acetyl-CoA transporter could be informative.
      • Response: In response to this valuable suggestion, we have now examined the impact of deleting Casd1, the gene encoding the Golgi acetyltransferase responsible for modifying sialic acids on ATF6α activity, comparing the consequences to Slc33a1. New Supplemental Fig. S8 reveals partial phenotypic overlap between the two deletions, suggesting that the loss of SLC33A1 exerts some of its effects on CHO cells by compromising sialic acid modification.

      __ Potential effects on ATF6-like proteins (SREBP1/2, CREB3L).__

      • Comment: Reviewer 1 suggested that we evaluate the effect of SLC33A1 loss on other ATF6-like transcription factors.
      • Response: We took this advice to heart, but our attempts to compare SREBP2 processing in wildtype and SLC33A1 knockout cells were frustrated by the low quality of the antibodies available to us. Reviewer 2 (additional points)

      • __ Physiological state and clonal adaptation of Slc33a1-deleted cells.__

      • __Comment: __Reviewer 2 raised concerns regarding the physiological state of the Slc33a1-deleted cells and the potential impact of clonal adaptation or selection pressure on the consequences of genetic manipulation.
      • Response: This is a valid concern. Deconvoluting direct from indirect effects are a challenge in any genetics-based experiment. To try and address this point, we compared the proliferation capacity of three pairs of parental CHO-K1 clones with their derivative Slc33a1-deletion variants using the IncuCyte assay. As shown in new Supplemental Fig. S2D, the Slc33a1 deletion variants had no consistent fitness disadvantage revealed by this assay. Whilst cell mass accretion is only one measure of comparability between cell lines, we deem these observations to indicate that a comparison between SLC33A1 wildtype and mutant CHO-K1 cells is unlikely to be compromised by gross underlying differences in cell fitness.

      __ Responsiveness of PERK signalling to ER stress.__

      • Comment: Reviewer 2 asked whether PERK signalling, which appears basally activated due to higher basal IRE1 signalling in the Slc33a1-deleted cells, remains responsive to ER stress.
      • Response: To address this point, we treated cells with ER stressors and assessed PERK pathway activation. As shown in new Supplemental Fig. S4C, PERK signalling remains functional and responsive to ER stress in Slc33a1-depleted cells.

      In addition to the points above, we have addressed several presentation and clarity issues raised by the reviewers, including figure labelling, image presentation, and schematic models. The Discussion has also been revised to more explicitly acknowledge the current limitations of the study while emphasising its central conceptual advance: namely, that loss of SLC33A1 results in a discordant UPR state in which IRE1 and PERK are activated, whereas ATF6α trafficking and transcriptional output are selectively compromised.

      The following table summarises the major changes made to the figures in the revised manuscript to facilitate tracking the modifications introduced

      Figure

      Figure Panels

      Amendment (if any)

      Fig 4

      4B (modified)

      Scale bar added.

      Fig 5

      5B (modified)

      Labelling correction according to the reviewer.

      Fig S1 (new)

      S1A-S1B

      New data detailing the BiP promoter fragment and the reliability of the BiP::GFP reporter as a readout for ATF6α activity in cells.

      Fig S2 (modified)

      S2D (new)

      New IncuCyte data added.

      Fig S3 (modified)

      S3B, S3C and S3E (new)

      Panels B and C: New data from DTT and thapsigargin treatments, respectively. __Panel E: __New data from BiP mRNA levels under 2DG treatment in parental and Slc33a1-deleted cells.

      Fig S4 (new)

      S4C (new)

      __Panels A and B: __Previously shown as panels in Fig. S2C and S2D.

      __Panel C: __New data on the PERK response to ER stress in Slc33a1-deleted cells.

      Fig S7 (new)

      S7A-S7C (new)

      New sanger sequencing chromatograms displaying the targeted exonic regions of the Casd1, Nat8 and Nat8b. * *

      Fig S8 (new)

      S8A-S8B (new)

      Casd1-deleted data added.

      Fig S9 (new)

      Unique panel

      New data comparing Nat8/Nat8b-deleted cells with single Nat8-deleted cells.

      We thank the reviewers again for their insightful comments, which have significantly strengthened the manuscript. We believe the revised study clarifies key mechanistic points and provides a stronger conceptual advance regarding the role of SLC33A1 in UPR regulation.

      Sincerely,

      Adriana Ordóñez

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      Referee #2

      Evidence, reproducibility and clarity

      Summary

      The authors employed a genome-wide CRISPR-Cas9 screen to search for the genes selectively involved in the activation of ER stress sensor ATF6. Deletion of Slc33a1, which encodes a transporter of acetyl-CoA into the ER lumen, compromised the ATF6 pathway (as assessed by BiP::GFP reporter), while IRE1 and PERK were activated in basal conditions, in the absence of ER stress (as assessed by XBP1s::mCherry reporter and endogenous XBP1s and CHOP::GFP reporter). Moreover, IRE1, but not ATF6, replied to ER stress. Consistently, in Slc33a1Δ cells upon ER stress the levels of the processed N-ATF6α were significantly lowered compared to the parental cells, and microscopy study showed that in Slc33a1-deficient cells ATF6 is translocated to Golgi even in the absence of ER stress, but fails to reach the nucleus even after ER stress is imposed. Golgi-type sugar modification of ATF6α is decreased in Slc33a1Δ cells. These data show the importance of SLC33A1 for ATF6 processing and functioning through the mechanism which remains to be revealed.

      Major comments.

      Taken together, the reported data do support the conclusion about the role of SLC33A1 functioning in post-ER maturation of ATF6. Data and methods are presented in a reproducible way. Still, there are several issues worth attention.

      1. While BiP::GFP reporter is very useful, it would be more convincing to show the level of BiP in Slc33a1Δ cells by WB.
      2. Another concern is the state of Slc33a1Δ cells. While adaptation is a general problem of clonal cells, the cells used in this study (with XBP1 highly spliced, CHOP upregulated, and ATF6 pro-survival pathway inhibited) are probably very sick, and the selection pressure/adaptation is very strong in this cell line. I would suggest the authors to clarify this issue.
      3. Authors showed that, based on CHOP::GFP reporter data, PERK was activated in the absence of ER stress and the activation was due to IRE1 signalling. But did PERK reply to the ER stress?
      4. An important question is a subcellular location of SLC33A1. Huppke et al. (cited in the manuscript) showed that FLAG- and GFP-tagged SLC33A1 was colocalized with Golgi markers. While that may be due to overexpression of the protein, it deserves consideration, given that ATF6 is stuck in Golgi upon depletion of SLC33A1.
      5. OPTIONAL. Regarding the role of acetylation in compromising ATF6 function: since both SLC33A1 deficiency and depletion of Nat8 have broad effects, glycosylation of ATF6 upon depletion of Nat8 should be assessed (similarly to Fig 5), to demonstrate the difference in glycosylation pattern upon the absence of SLC33A1 and Nat8 and strengthen the conclusions.

      Minor comments.

      1. Apart from the table of the cell lines, it would be useful to add to the supplementary a simple-minded scheme of the reporters used in this study (BiP::GFP, CHOP::GFP, XBP1s::mCherry) specifying the mechanism of the readout and the harbored protein and other important details (e.g., whether mRNA of XBP1s::mCherry reporter could be processed by IRE1).
      2. Fig 2B and Fig 3A - the percentage of spliced XBP1 in parental cells is about 30% according to the graphs, but it looks more like 5%.
      3. Fig 3B - It would probably be better to demonstrate the processing of endogenous ATF6. It could help to avoid the problems with alternative translation (even though anti-ATF6 antibodies are known to be tricky).
      4. In Fig 4B - could be better to show Golgi marker separately. In Fig 4B and E the bars are missing (and cells in Fig 4B look bigger than in Fig 4E). Magnification of the insets should be further increased.
      5. As the authors mention, 2-deoxy-D-glucose (2DG) is known to be the ER stress inducer, acting via prevention of N-glycosylation of proteins. Also, N-glycosylation state of ATF6 has been suggested to influence its trafficking. Thus, even if the control cells were treated in the same way, 2DG may not be the best ER-stress inducer to study ATF6 trafficking. Indeed, altered sugar modification of ATF6α in Slc33a1Δ cells (Fig 5) was tracked using Thapsigargin.
      6. Minor comment on Fig 7 - recent data (Belyy et al., 2022) suggest IRE1 is a dimer even in the absence of ER stress.

      Referee cross-commenting

      I agree with Reviewer 1 that the authors need to clarify that authors need to clarify better how exactly BiP::GFP reporter works and whether it reflects ATF6 activation (rev 1 pointed to unclear responsiveness of the reporter to ATF6 and I asked to show the level of BiP by WB and the scheme of the mechanisms of readouts of the reporters)

      I also agree with the comment on 2-DG which for some experiments may not be the best choice to activate UPR (or as Reviewer 1 pointed out shouldn't be the only one used to induce UPR). I still think that there's no contradiction in partial cleavage of ATF6 and suppression of BiP::GFP in Slc33a1Δ cells if then (as authors show) it doesn't reach nucleus.

      Significance

      General assessment. The article shows the necessity of SLC33A1, a transporter of acetyl-CoA in ER lumen, for ATF6 processing and functioning. It is well-written. However, the molecular mechanism which underlies the link is yet to be discovered (and this is clearly mentioned by the authors).

      The study is of interest for the basic research and of potential interest for clinical research.

      My main field of expertise is UPR. While I have broad knowledge and interest in protein science in general, my experience with protein glycosylation is rather limited.

    1. Stratégies et Outils pour une Coopération Efficace en Milieu Scolaire

      Résumé Exécutif

      La coopération en classe ne se limite pas à un simple travail de groupe ; elle constitue un levier d'apprentissage puissant et une compétence citoyenne inscrite au socle commun (cycles 3 et 4).

      Ce document synthétise les approches pédagogiques et les outils pragmatiques nécessaires pour transformer la coopération d'une contrainte organisationnelle en un moteur de réussite.

      Les points clés incluent l'adoption d'une posture de « lâcher-prise » par l'enseignant, l'instauration d'un cadre structuré pour la gestion du bruit et des rôles, ainsi que l'utilisation d'outils de suivi visuels comme le tétraèdre.

      L'évaluation, centrée sur la compétence coopérative elle-même plutôt que sur le seul produit final, s'avère essentielle pour l'autonomisation des élèves.

      1. Fondements et Enjeux de la Coopération

      La coopération est définie comme l'acte d'apprendre ensemble par le partage d'idées, l'entraînement mutuel et la confrontation des points de vue.

      Elle ne doit pas être perçue comme une simple modalité pratique, mais comme une mission fondamentale de l'école.

      Légitimité institutionnelle : La coopération est une compétence du socle commun de connaissances, de compétences et de culture. Elle fait l'objet d'un apprentissage explicite et d'une évaluation.

      Validation scientifique : Une étude publiée dans la revue Science en 2019 confirme que les étudiants apprennent mieux lorsqu'ils sont actifs, malgré une perception parfois inverse par rapport aux cours magistraux.

      Compétences transversales développées :

      ◦ Organisation et planification.  

      ◦ Débat, argumentation et écoute active.  

      ◦ Gestion des émotions et des conflits.  

      ◦ Capacité à faire des concessions.

      2. La Posture de l'Enseignant : Le « Lâcher-Prise » Cadre

      Pour réussir, l'enseignant doit accepter de modifier sa posture.

      Le « lâcher-prise » ne signifie pas l'autogestion totale, mais la délégation et l'acceptation de l'imprévisible.

      Acceptation de l'erreur : Laisser les élèves chercher, se tromper et recommencer.

      Gestion de l'imprévu : Anticiper que les débats peuvent être houleux et que le niveau sonore augmentera.

      Constitution des groupes : Il n'existe pas de solution universelle.

      Le choix (affinités, imposé ou aléatoire) dépend des objectifs pédagogiques et de la dynamique de la classe.

      L'organisation peut évoluer au fil de l'année selon les besoins constatés.

      3. Gestion de l'Espace et de la Dynamique de Groupe

      L'environnement physique et sonore doit être rigoureusement pensé pour limiter les débordements.

      La gestion du bruit

      Le chuchotement n'est pas inné ; il doit faire l'objet d'un enseignement.

      Une technique consiste à faire placer la main sur la gorge pour sentir l'absence de vibration des cordes vocales lors du chuchotement.

      Signaux d'arrêt : Utiliser des outils pour préserver la voix de l'enseignant (buzzer, sonnerie, feux tricolores ou signal verbal prédéfini).

      L'organisation spatiale

      Si possible, privilégier une classe flexible avec des tables mobiles. Dans une salle classique, il est recommandé de :

      • Créer des « coins groupes ».

      • Anticiper les règles de circulation (notamment vers les ressources en autonomie) pour éviter les déplacements massifs.

      Le Tétraèdre : Outil de régulation des interventions

      Pour éviter d'être sollicité de manière anarchique, l'enseignant peut utiliser un code couleur par groupe :

      | Couleur | Signification | | --- | --- | | Vert | Tout va bien, le groupe progresse. | | Bleu | Travail terminé ; demande de validation ou tutorat possible vers un autre groupe. | | Jaune | Question non urgente. | | Rouge | Blocage complet ; intervention urgente nécessaire. |

      4. Structuration de la Participation Individuelle

      Afin d'éviter qu'un élève ne se retrouve isolé ou, à l'inverse, n'assume toute la charge de travail, des outils de distribution des tâches sont nécessaires.

      Cartes de rôles : Distribuer des fonctions précises (scribe, orateur/oratrice, modérateur/modératrice, meneur/meneuse).

      Il est crucial de faire tourner ces rôles à chaque séance pour garantir l'équité.

      La méthode du « Placemat » : Utilisation d'une grande feuille divisée en cases individuelles entourant une case centrale de mise en commun.

      Cela impose un temps de réflexion personnel avant la production collective.

      5. Évaluation et Analyse de la Pratique

      L'évaluation doit porter sur la coopération en tant que compétence distincte de la production finale.

      Critères de réussite co-construits : Fournir une grille d'évaluation élaborée avec les élèves pour clarifier les attentes dès le début de l'année.

      L’Étoile de Sylvain Connac : Un outil d'auto-évaluation permettant aux élèves de porter un regard critique sur quatre axes :

      1. L'entente au sein du groupe.   

      2. La qualité de l'écoute.   

      3. La compréhension des consignes et des notions.   

      4. La gestion du temps.

      Feedback de fin de séance : Consacrer un temps court (un mot ou une phrase par groupe) pour ajuster les modalités lors de la séance suivante.

      Conclusion

      La coopération est un processus évolutif qui requiert de la patience.

      Commencer par des structures simples (travail en binôme, introduction progressive des rôles) permet de stabiliser le cadre avant de complexifier les dispositifs.

      L'objectif final demeure l'autonomisation et la responsabilité des élèves au sein du collectif.

    1. Guide Stratégique sur l'Intégration des Jeux Pédagogiques en Milieu Scolaire

      Résumé Exécutif

      L'intégration du jeu dans le cadre pédagogique n'est pas une simple activité ludique de divertissement, mais un levier puissant pour l'engagement des élèves et l'acquisition de compétences.

      Ce document synthétise l'expertise de Solène Paris, enseignante expérimentée, sur la transformation des séquences de classe par le jeu.

      La réussite de cette approche repose sur le respect de quatre piliers cognitifs (attention, engagement actif, retour d'information et consolidation) et sur une mise en œuvre progressive, allant du détournement de jeux existants à la création d'escape games complexes.

      L'analyse souligne que la valeur pédagogique ne réside pas seulement dans l'activité elle-même, mais de manière critique dans la phase de débriefing, qui permet d'ancrer durablement les notions théoriques et les compétences transversales.

      Bien que la préparation exige un investissement initial conséquent, les bénéfices en termes de motivation, de réduction des inégalités et de mémorisation constituent un avantage éducatif majeur.

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      Les Fondements du Jeu Pédagogique

      Pour être efficace, le jeu en classe doit dépasser le simple stade du "mot croisé" ou de l'activité occupationnelle.

      Il doit s'aligner sur des principes de neurosciences et des objectifs sociaux.

      Les Quatre Piliers de l'Apprentissage

      Selon les travaux de Stanislas Dehaene, le jeu pédagogique doit impérativement mobiliser :

      L'attention : Capter et canaliser la concentration de l'élève sur l'objet d'apprentissage.

      L'engagement actif : L'élève ne doit pas être passif ; il doit agir, tester et s'impliquer.

      Le feedback (retour sur information) : Le jeu permet une correction immédiate et constructive.

      La consolidation : La répétition et l'expérience ludique favorisent la rétention à long terme.

      Compétences et Valeurs Ajoutées

      Le jeu développe une double typologie de compétences :

      Compétences Psychosociales (CPS) : Autonomie, gestion des émotions, coopération, persévérance et esprit d'initiative.

      Bénéfices Pédagogiques : Diversification des pratiques de classe, concrétisation des notions abstraites et remobilisation des élèves habituellement réfractaires ou en difficulté.

      --------------------------------------------------------------------------------

      Stratégies de Mise en Œuvre : Une Progression par Niveaux

      L'adoption du jeu peut se faire de manière graduelle afin de limiter la charge de travail initiale de l'enseignant.

      | Niveau | Approche | Exemples et Outils | | --- | --- | --- | | Niveau 1 : Débutant | Détournement de jeux populaires aux règles déjà connues. | Dobble (verrerie), 7 familles (réchauffement climatique), Jungle Speed (énergies), Domino (molécules). | | Niveau 2 : Apprenti | Adaptation ou création de jeux spécifiques à un besoin précis. | Damier de l'alimentation durable, jeux sur la précarité menstruelle. Utilisation de Canva pour le design. | | Niveau 3 : Numérique | Escape games en ligne. | Plateformes : Géniali, bibliothèque S’CAPE. | | Niveau 4 : Expert | Escape games physiques en classe entière. | Nécessite : énigmes, matériel dédié, gestion du temps et de la coopération. |

      --------------------------------------------------------------------------------

      Gestion des Risques et Écueils à Éviter

      L'introduction du jeu comporte des défis logistiques et pédagogiques que l'enseignant doit anticiper pour éviter le "moment de solitude" face à la classe.

      Le manque d'anticipation : Il est impératif de tester le jeu en petit comité avant de le lancer en classe entière pour identifier les bugs de conception ou les règles trop complexes.

      Le piège chronophage : Le jeu ne doit pas occuper tout le temps scolaire au détriment du programme. L'équilibre entre temps ludique et temps de synthèse est primordial.

      La gestion de classe : L'agitation, le bruit et les conflits potentiels doivent être encadrés par des règles claires et simples définies au préalable.

      La charge de préparation : Bien que lourde au départ (impression, plastification, conception), elle doit être vue comme un investissement réutilisable et améliorable sur plusieurs années.

      --------------------------------------------------------------------------------

      La Phase Critique : Le Débriefing

      Le débriefing est l'étape la plus importante pour transformer une expérience agréable en un apprentissage effectif. Sans cette phase, l'élève risque de ne retenir que le divertissement.

      Protocole de Débriefing en Quatre Étapes

      1. Recueil des réactions à chaud : Permettre aux élèves d'exprimer leurs émotions et leur vécu (ce qu'ils ont aimé ou non).

      2. Institutionnalisation des notions : Faire le lien direct entre les mécanismes du jeu et le contenu théorique (ex: relier une énigme sur le sucre aux concepts de dissolution, soluté et solvant).

      3. Analyse des compétences transversales : Faire un retour sur la communication, la persévérance et la capacité à coopérer durant l'activité.

      4. Suggestions d'amélioration : Impliquer les élèves dans l'évolution du support pour optimiser son efficacité future.

      Outils de Restitution Ludique

      Pour maintenir l'engagement même durant le bilan, plusieurs méthodes sont suggérées :

      Outils numériques : Wooclap pour un feedback collectif instantané.

      Méthodes visuelles : Cartes mentales collectives ou "leçons à manipuler".

      Réflexion structurée : Utilisation de post-its ou du "placemat" (réflexion individuelle suivie d'une synthèse de groupe).

      --------------------------------------------------------------------------------

      Conclusion

      Le jeu pédagogique constitue une "quête" exigeante mais gratifiante pour l'enseignant.

      En s'appuyant sur des ressources existantes (sites académiques, blogs comme pédagodeseggo.fr ou la Team Ludens) et en respectant une structure rigoureuse incluant impérativement un débriefing, le jeu devient un outil de différenciation sociale et de réussite scolaire.

      L'objectif ultime est de rendre les élèves acteurs de leurs apprentissages, cherchant et résolvant des problèmes "sans même s'en rendre compte".

    1. Dynamiques de Classe et Construction des Inégalités Scolaires : Analyse de la Psychologie Sociale de l'Éducation

      Ce document de synthèse examine les travaux de Sébastien Goudeau sur les mécanismes par lesquels les interactions quotidiennes en classe et les contextes scolaires contribuent à l'amplification des inégalités sociales.

      Résumé Exécutif

      L'analyse des situations scolaires révèle que l'école ne se contente pas de refléter les inégalités sociales préexistantes, elle tend à les amplifier par le biais de processus psychologiques et interactionnels. Les points clés identifiés sont :

      Le "Paradoxe du Monopoly" : Tout comme les joueurs de Monopoly attribuent leur succès à leur stratégie plutôt qu'à leur avantage financier initial, les élèves interprètent les écarts de réussite comme des différences de capacités intrinsèques.

      La Comparaison Sociale comme Menace : Les situations rendant la réussite des uns visible pour les autres (comme le fait de lever la main) génèrent un sentiment d'incompétence et un stress qui détériorent la performance des élèves issus de milieux populaires.

      Inégalités de Participation Orale : Dès l'école maternelle, les élèves de milieux favorisés bénéficient d'un temps de parole plus long et de sollicitations plus fréquentes de la part des enseignants, souvent de manière non intentionnelle.

      Leviers d'Action : Agir sur la "métacognition sociale" — la façon dont les élèves expliquent leurs réussites et échecs — et promouvoir une conception malléable de l'intelligence sont des pistes prometteuses pour réduire ces écarts.

      --------------------------------------------------------------------------------

      1. L'Origine des Inégalités : Capital Culturel et Familiarité Scolaire

      Avant même l'entrée à l'école, des disparités marquées existent en fonction de l'origine sociale. Ces inégalités ne sont pas dues au hasard, mais à des contextes de socialisation différenciés.

      La Construction du Capital Culturel

      Le milieu familial influence l'acquisition de dispositions culturelles plus ou moins proches des attentes de l'école :

      Compétences précoces : Dès 1 ou 2 ans, des différences apparaissent dans la connaissance des lettres, l'identification des sons et la familiarité avec la littérature jeunesse.

      Pédagogisation de la vie quotidienne : Les familles favorisées transforment souvent des activités banales (ex: mettre le couvert en comptant les fourchettes) en opportunités d'apprentissage.

      Pratiques de socialisation : La fréquence des visites dans les musées, les bibliothèques et la durée des lectures partagées confèrent des savoirs hautement "rentables" en contexte scolaire.

      Le Postulat de l'Éducabilité

      Il est crucial de noter que l'échec des enfants de milieux populaires n'est pas lié à des déficiences génétiques ou familiales, mais à une inadéquation entre leurs dispositions initiales et les codes attendus par l'institution scolaire.

      --------------------------------------------------------------------------------

      2. Le Rôle de la Comparaison Sociale et de la Menace

      La vie en classe impose une comparaison sociale permanente (notes, feedbacks, mains levées). Cette comparaison n'est pas neutre psychologiquement.

      L'Impact de la Visibilité de la Réussite

      Une étude menée dans 40 classes de 6e sur une tâche de lecture montre que :

      La visibilité pénalise : Lorsque l'on demande aux élèves de lever la main dès qu'ils ont fini une tâche, l'écart de performance entre les élèves de milieux populaires et favorisés s'accroît.

      La menace psychologique : Voir les autres réussir plus vite est perçu comme menaçant. Cela génère un stress et des émotions négatives qui consomment les ressources attentionnelles nécessaires à la tâche.

      Preuve Expérimentale de la Familiarité

      Pour prouver le rôle causal de la familiarité, une expérience a recréé artificiellement des avantages culturels avec un nouveau système d'écriture (symboles associés à des lettres) :

      • Les élèves entraînés au préalable réussissent mieux, mais l'écart se creuse massivement lorsque la situation impose une comparaison sociale avec les élèves non entraînés.

      • C'est le sentiment d'incompétence induit par la comparaison qui produit la baisse de performance.

      --------------------------------------------------------------------------------

      3. Dynamiques d'Interaction en École Maternelle

      Bien que l'école maternelle vise à réduire les inégalités par le langage oral, les observations vidéo (dispositif à 360°) révèlent des biais persistants dans les échanges collectifs.

      Inégalités dans la Prise de Parole

      Les résultats préliminaires sur une centaine d'élèves montrent une asymétrie marquée selon l'origine sociale :

      | Type de prise de parole | Élèves de milieux favorisés | Élèves de milieux populaires | | --- | --- | --- | | Sollicitée (par l'enseignant) | Plus fréquente et plus longue. | Moins fréquente et plus courte. | | Non sollicitée (spontanée) | Se saisissent davantage de la parole et la gardent plus longtemps. | Moins enclins à couper la parole ou à s'exprimer spontanément. |

      Facteurs d'Influence

      Familiarité des codes : Les enseignants interrogent plus souvent les élèves familiers des postures langagières scolaires pour assurer le bon déroulement de la séance.

      Inégalités de genre : Des différences de participation apparaissent également, les filles s'exprimant moins sur des thématiques mathématiques que littéraires.

      --------------------------------------------------------------------------------

      4. Leviers d'Action et Perspectives

      L'identification de ces mécanismes permet d'envisager des interventions ciblées pour limiter l'amplification des inégalités.

      La Métacognition Sociale

      La "métacognition sociale" désigne la manière dont les élèves interprètent les différences de réussite qu'ils observent.

      Intervention : Si l'on explique aux élèves que les écarts de réussite sont dus à des différences d'entraînement (causes externes/malléables) plutôt qu'à des capacités (causes internes/fixes), l'effet négatif de la comparaison sociale disparaît.

      Conception de l'intelligence : Promouvoir une vision dynamique de l'intelligence (malléable par l'effort) favorise la résilience et peut même transformer la comparaison sociale en "boost" de confiance.

      Recommandations pour la Pratique Enseignante

      Interprétation de l'échec : Présenter la difficulté comme une étape normale et transitoire de l'apprentissage plutôt que comme une limite personnelle.

      Gestion de la parole : Prendre conscience des biais de sollicitation pour assurer une répartition plus équitable du temps de parole.

      Coopération : Utiliser le conflit socio-cognitif (désaccord entre pairs) pour stimuler l'apprentissage, tout en étant attentif à la répartition des rôles selon le niveau de compétence des élèves.

      Contexte Structurel Français

      Il est noté que la pression sur les élèves est accentuée en France par le lien très étroit entre diplôme et emploi, comparativement à des pays comme l'Allemagne ou les pays nordiques, où la sélection est plus tardive et la confiance en soi des élèves plus élevée dans les classements internationaux (PISA).

    1. Reviewer #1 (Public review):

      Summary:

      The manuscript by Hao Jiang et al described a systematic approach to identify proline hydroxylation proteins. The authors implemented a proteomic strategy with HILIC-chromatographic separation and reported an identification with high confidence of 4993 sites from HEK293 cells (4 replicates) and 3247 sites from RCC4 cells with 1412 sites overlapping between the two cell lines. A small fraction of about 200 sites from each cell line were identified with HyPro immonium ion. The authors investigated the conditions and challenges of using HyPro immonium ions as a diagnostic tool. The study focused the validation analysis of Repo-man (CDCA2) proline hydroxylation comparing MS/MS spectra, retention time and diagnostic ions of purified proteins with corresponding synthetic peptides. Using SILAC analysis and recombinant enzyme assay, the study evaluated Repo-man HyPro604 as a target of PHD1 enzyme.

      Strengths:

      The study involved extensive LCMS runs for in-depth characterization of proline hydroxylation proteins including four replicated analysis of 293 cells and three replicated analysis of RCC4 cells with 32 HILIC fractions in each analysis. The identification of over 4000 confident proline hydroxylation sites from the two cell lines would be a valuable resource for the community. The characterization of Repo-man proline hydroxylation is a novel finding.

      Weaknesses:

      As a study mainly focused on methodology, there are some potential technical weaknesses discussed below.

      (1) The study applied HILIC-based chromatographic separation with a goal to enrich and separate hydroxyproline containing peptides. The separation effects for peptides from 293 cells and RCC4 cells seems somewhat different (Figure 2A and Figure S1A), which may indicate that the application efficiency of the strategy may be cell line dependent.

      (2) The study evaluated the HyPro immonium ion as a diagnostic ion for HyPro identification showcasing multiple influential factors and potential challenges. It is important to note that with only around 5% of the identifications had HyPro immonium ion, it would be very challenging to implement this strategy in a global LCMS analysis to either validate or invalidate HyPro identifications. In comparison, acetyllysine immonium ion was previously reported to be a useful marker for acetyllysine peptides (PMID: 18338905) and the strategy offered a sensitivity of 70% with a specificity of 98%.

      (3) The authors aimed to identify potential PHD targets by comparing the HyPro proteins identified with or without PHD inhibitor FG-4592 treatment. The workflow followed a classification strategy, rather than a typical quantitative proteomics approach for comprehensive analysis.

      (4) The authors performed inhibitor treatment and in vitro PHD1 enzyme assay to validate that Repo-man can be hydroxylated by PHD1. It remains unknown if PHD1 expression in cells is sufficient to stimulate Repo-man hydroxylation.

    2. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      The manuscript by Hao Jiang et al described a systematic approach to identify proline hydroxylation proteins. The authors implemented a proteomic strategy with HILIC-chromatographic separation and reported an identification of 4993 sites from HEK293 cells (4 replicates) and 3247 sites from RCC4 sites (3 replicates) with 1412 sites overlapping between the two cell lines. From the analysis, the authors identified 225 sites and 184 sites respectively from 293 and RCC4 cells with HyPro diagnostic ion. The identifications were validated by analyzing a few synthetic peptides, with a specific focus on Repo-man (CDCA2) through comparing MS/MS spectra, retention time, and diagnostic ions. With SILAC analysis and recombinant enzyme assay, the study showed that Repo-man HyPro604 is a target of the PHD1 enzyme.

      Strengths:

      The study involved extensive LC-MS analysis and was carefully implemented. The identification of over 4000 confident proline hydroxylation sites would be a valuable resource for the community. The characterization of Repo-man proline hydroxylation is a novel finding.

      Weaknesses:

      However, as a study mainly focused on methodology, the findings from the experimental data did not convincingly demonstrate the sensitivity and specificity of the workflow for site-specific identification of proline hydroxylation in global studies.

      Proline hydroxylation is an enzymatic post translational protein modification, catalysed by prolyl Hydroxylases (PHDs), which can have profound biological significance, e.g. altering protein half-life and/or the stability of protein-protein interactions. Furthermore, there has been controversy in the field as to the true number of protein targets for PHDs in cells. Thus, there is a clear need for methods to enable the robust identification of genuine PHD targets and to reliably map sites of PHD-catalysed proline hydroxylation in proteins. We believe, therefore, that our methodology, as reported here in Jiang et al., is an important contribution towards this goal. We note that our methodology has already been used successfully by others

      (https://doi.org/10.1016/j.mcpro.2025.100969). While further improvements in this methodology may of course be developed in future, we are not currently aware of any superior methods that have been reported previously in the literature. The criticism made by the reviewer notably does not include reference to any such alternative published methodology that interested researchers can use which would offer superior results to the approach we document in this study.

      Major concerns:

      (1) The study applied HILIC-based chromatographic separation with a goal of enriching and separating hydroxyproline-containing peptides. However, as the authors mentioned, such an approach is not specific to proline hydroxylation. In addition, many other chromatography techniques can achieve deep proteome fractionation such as high pH reverse phase fractionation, strong-cation exchange etc. There was no data in this study to demonstrate that the strategy offered improved coverage of proline hydroxylation proteins, as the identifications of the HyPro sites could be achieved through deep fractionation and a highly sensitive LCMS setup. The data of Figure 2A and S1A were somewhat confusing without a clear explanation of the heat map representations. 

      The data we present in this study demonstrate clearly that peptides with hydroxylated prolines are enriched in specific HILIC fractions (F10-F18), in comparison with total unfractionated peptides derived from cell extracts. We also refer the reviewer to our previously published study by Bensaddek et al (International Journal of Mass Spectrometry: doi:10.1016/j.ijms.2015.07.029), which was reference 41 in this study, in which we compared directly the performance of both HILIC and strong anionic exchange chromatography, (hSAX). This showed that HILIC provided superior enrichment to hSAX for enrichment of peptides containing hydroxylated proline residues. To clarify this point for readers, we have now included a specific reference to our previous study at the start of the Results section in our current revision. Currently, we use HILIC to provide a degree of enrichment for proline hydroxylated peptides because we are not aware of alternative chromatographic methods that in our hands provide better results.

      We have included descriptions of the information shown in the heatmaps in the associated figure legends and captions.

      (2) The study reported that the HyPro immonium ion is a diagnostic ion for HyPro identification. However, the data showed that only around 5% of the identifications had such a diagnostic ion. In comparison, acetyl-lysine immonium ion was previously reported to be a useful marker for acetyllysine peptides (PMID: 18338905), and the strategy offered a sensitivity of 70% with a specificity of 98%. In this study, the sensitivity of HyPro immonium ion was quite low. The authors also clearly demonstrated that the presence of immonium ion varied significantly due to MS settings, peptide sequence, and abundance. With further complications from L/I immonium ions, it became very challenging to implement this strategy in a global LC-MS analysis to either validate or invalidate HyPro identifications.

      The reviewer appears to have misunderstood the point we make with regard to the identification of the immonium ion and its use as a diagnostic marker for proline hydroxylation in MS analyses. We do not claim that this immonium ion is an essential diagnostic marker for proline hydroxylation. As the reviewer notes, with respect to the acetyl-lysine modification, the corresponding immonium ion is often used in MS studies as a diagnostic for identification of specific post translational modifications. Previous studies have reported that the immonium ion for hydroxylated proline is detected when the transcription factor HIF is analysed, but is often absent with other putative PHD targets, which has been used as an argument that these targets are not genuine proline hydroxylation sites. We are not, therefore, introducing the idea in this study that the hydroxy-proline immonium ion is a required diagnostic marker for proline hydroxylation, but instead demonstrating that detection of this ion, at least in some peptide sequences, may require the use of higher MS collision energies than are typically required for routine peptide identification. We believe that this is an interesting observation that can help to clear up discussions in the literature regarding the true prevalence of PHD-catalysed proline hydroxylation in different target proteins. Our data suggest that, in future MS studies analysing suspected PHD target proteins, two different collision energy might need to be used, i.e., normal collision energy for the routine identification of a peptide, combined with use of a higher collision energy if the hydroxy-proline immonium ion was not already detected.

      (3) The study aimed to apply the HILIC-based proteomics workflow to identify HyPro proteins regulated by the PHD enzyme. However, the quantification strategy was not rigorous. The study just considered the HyPro proteins not identified by FG-4592 treatment as potential PHD targeted proteins. There are a few issues. First, such an analysis was not quantitative without reproducibility or statistical analysis. Second, it did not take into consideration that data-dependent LC-MS analysis was not comprehensive and some peptide ions may not be identified due to background interferences. Lastly, FG-4592 treatment for 24 hrs could lead to wide changes in gene expressions and protein abundances. Therefore, it is not informative to draw conclusions based on the data for bioinformatic analysis.

      We refer the reviewer to the data we present in this study using SILAC analysis, combined with our MS workflow. to achieve a more accurate quantitative picture of proline hydroxylation levels. While we agree that the point the reviewer makes is valid, regarding our data dependent LC-MS/MS analysis potentially not being comprehensive, this means, however, that we are potentially underestimating the true prevalence of proline hydroxylated peptides, not overestimating the level of these modified peptides. We also refer the reviewer to the accompanying study by Druker et al., (eLife 2025; doi.org/10.7554/eLife.108131.1)  in which we present a detailed follow-on study demonstrating the functional significance of the novel proline hydroxylation site we detected in the protein RepoMan (CDCA2). Therefore, even if we have not achieved a fully comprehensive analysis of all proline hydroxylated peptides catalysed by PHD enzymes, we believe that we have advanced the field by documenting a workflow that is able to identify and validate novel PHD targets.

      (4) The authors performed an in vitro PHD1 enzyme assay to validate that Repo-man can be hydroxylated by PHD1. However, Figure 9 did not show quantitatively PHD1-induced increase in Repo-man HyPro abundance and it is difficult to assess its reaction efficiency to compare with HIF1a HyPro.

      The analysis shown in Figure 9 was not intended to quantify the efficiency of in vitro hydroxylation of RepoMan by PHD1, but rather to answer the question, ‘Can recombinant PHD1 alone hydroxylate P604 on RepoMan in vitro, yes or no?’. The data show that the answer here is ‘yes’. Clearly, the HIF peptide is a more efficient substrate in vitro for recombinant PHD1 than the RepoMan peptide and we have now included a statement in the Discussion that addresses the significance of this observation more directly.

      Reviewer #2 (Public review):

      Summary:

      In this manuscript, Jiang et al. developed a robust workflow for identifying proline hydroxylation sites in proteins. They identified proline hydroxylation sites in HEK293 and RCC4 cells, respectively. The authors found that the more hydrophilic HILIC fractions were enriched in peptides containing hydroxylated proline residues. These peptides showed differences in charge and mass distribution compared to unmodified or oxidized peptides. The intensity of the diagnostic hydroxyproline iminium ion depended on parameters including MS collision energy, parent peptide concentration, and the sequence of amino acids adjacent to the modified proline residue. Additionally, they demonstrate that a combination of retention time in LC and optimized MS parameter settings reliably identifies proline hydroxylation sites in peptides, even when multiple proline residues are present.

      Strengths:

      Overall, the manuscript presents an advanced, standardized protocol for identifying proline hydroxylation. The experiments were well designed, and the developed protocol is straightforward, which may help resolve confusion in the field.

      Weaknesses:

      (1) The authors should provide a summary of the standard protocol for identifying proline hydroxylation sites in proteins that can easily be followed by others.

      This is a good suggestion and we have now included a figure (Figure 10) with a summary of our workflow in the current revision.

      (2) Cockman et al. proposed that HIF-α is the only physiologically relevant target for PHDs. Their approach is considered the gold standard for identifying PHD targets. Therefore, the authors should discuss the major progress they made in this manuscript that challenges Cockman's conclusion.

      While we had mentioned the Cockman et al., paper in the Introduction, we had not focussed on this somewhat controversial issue. However, in response to the Reviewer’s request, we have now added a comment in the Discussion section in the current revision of how our new data address the proposal discussed previously by Cockman et al. In brief, we believe that our findings are not consistent with a model in which PHDs have no protein targets other than HIFs.

      Reviewer #3 (Public review): 

      Summary:

      The authors present a new method for detecting and identifying proline hydroxylation sites within the proteome. This tool utilizes traditional LC-MS technology with optimized parameters, combined with HILIC-based separation techniques. The authors show that they pick up known hydroxy-proline sites and also validate a new site discovered through their pipeline.

      Strengths:

      The manuscript utilizes state-of-the-art mass spectrometric techniques with optimized collision parameters to ensure proper detection of the immonium ions, which is an advance compared to other similar approaches before. The use of synthetic control peptides on the HILIC separation step clearly demonstrates the ability of the method to reliably distinguish hydroxy-proline from oxidized methionine - containing peptides. Using this method, they identify a site on CDCA2, which they go on to validate in vitro and also study its role in regulation of mitotic progression in an associated manuscript.

      Weaknesses:

      Despite the authors' claim about the specificity of this method in picking up the intended peptides, there is a good amount of potential false positives that also happen to get picked (owing to the limitations of MS-based readout), and the authors' criteria for downstream filtering of such peptides require further clarification. In the same vein, greater and more diverse cell-based validation approach will be helpful to substantiate the claims regarding enrichment of peptides in the described pathway analyses.

      We of course agree that false positives may arise, as is true for essentially all PTM studies. There are two issues here; first, are identified sites technically correct? (i.e. not misidentifications from the MS data) and second, are the identified modifications of biological significance? We have addressed this using the popular MaxQuant software suite to evaluate the modifications identified and to control the false discovery rate (FDR) at both the precursor and protein level, as described in the manuscript. We are aware that false positives could arise from confusing oxidation of methionine with hydroxylation of proline. Therefore, to address the issue as to whether we could identify bona fide PHD protein targets outside of the HIF family, we adopted a conservative approach by simply filtering out peptides where there was a methionine residue within three amino acids of the predicted proline hydroxylation site. This was a pragmatic decision made to reduce the likelihood of false positives in our dataset and we recognise that this likely results in us overlooking some genuine proline hydroxylation sites that occur nearby methionine residues. To address the potential biological relevance of the proline hydroxylation sites identified, we analysed extracts from cells treated with FG inhibitors. Of course a detailed understanding of biological significance relies upon follow-on experimental analyses for each site, which we have performed for P604 on RepoMan in accompanying study by Druker et al., (eLife 2025; doi.org/10.7554/eLife.108131.1).

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) The finding that the immonium ion intensities of L/I did not increase with increasing collision energy was surprising. Was this specific to this synthetic peptide?

      We agree this is an interesting and unexpected finding. We have no reason to believe that it is specific to synthetic peptides per se, but rather think this reflects an effect of amino acid composition in the peptides analysed. It will be interesting to explore this phenomenon in more detail in future.

      (2) The sequence logos in Figure 4 seemed to lack any amino acid enrichment in most positions except for collagen peptides. Have these findings been tested with statistical analysis?

      The results we show for sequence logo analysis were generated using WebLogo (10.1101/gr.849004) and correspond to an analysis of all proline hydroxylated peptides we detected across all cell lines and replicates analysed. The fact that collagens are highly abundant proteins with very high levels of proline hydroxylation likely explains why collagen peptides dominated the outcome of the sequence logo analysis. There is clearly scope for more detailed follow up analysis in future of the sequence specificity of proline hydroxylation sites in no- collagen proteins that are validated PHD targets.

      (3) Overall figure quality was not ideal. The resolution and font sizes of figures should be carefully evaluated and adjusted. The figure legend should contain a title for the figure. Annotations of the figures were somewhat confusing. 

      We agree with the criticism of the figure resolution in the review copies - the lower resolution appears to have been generated after we had uploaded higher resolution original images. We are providing again higher resolution versions of all figures for the current revision.

      Reviewer #3 (Recommendations for the authors):

      Certain concerns regarding portions of the manuscript that need addressing:

      (1) " These data show that two different cell lines show unique profiles of proteins with hydroxylated peptides." - It is difficult to conclusively say this statement after profiling the prolyl hydroxy proteome from just two cell lines, especially since the amino acids with the highest frequency in the most enriched peptides are similar in both cell lines.

      We agree with this point and have changed the current revision to state instead, “This shows that each of the two cell lines analysed have distinct profiles.”

      (2) "We noted that there was a high frequency of a methionine residues appearing either at the first, second, or even third positions after the HyPro site.." - according to the authors, claim, the advantage of their method was that they were able to overcome the limitation of older methods that couldn't separate methionine oxidation from proline hydroxylation. However, in this statement, they say that the high frequency of methionine residues may be because of the similar mass shift. These statements are contradictory. The authors should either tone down the claim or prove that those are indeed hydroxyproline sites. Is it possible that in the filtering step of excluding these high-frequency of methionine - containing peptides, we are losing potential positive hits for hydroxy-proline sites? What is the authors' take on this?

      We respectfully do not agree that our, “statements are contradictory”, with respect to the potential confusion between identification of methionine oxidation and proline hydroxylation, but acknowledge that we have not explained this issue clearly enough. It is a fact that the similar mass shift resulting from proline hydroxylation and methionine oxidation is a technical challenge that can potentially lead to misidentifications in MS studies and that is what we state clearly in the manuscript. We have addressed this issue head on experimentally in this study and show using synthetic peptides how detailed analysis of specific proline hydroxylation sites in target proteins can be distinguished from methionine oxidation, based upon differential chromatographic behaviour of peptides with either hydroxylated proline or oxidised methionine, as well as by detailed analysis of fragmentation spectra. However, in the case of our global analysis, as we were not able to perform synthetic peptide comparisons for every putative site identified, we took the pragmatic approach of filtering out examples of peptides where a methionine residue was present within three residues of a potential proline hydroxylation site. This was done simply to reduce the possibility of misidentification in the set of novel proline hydroxylated peptides identified and we accept that as a consequence we are likely filtering out peptides that include bona fide proline hydroxylation sites. We have clarified this point in the current revision and hope to be able to address this issue more comprehensively in future studies.

      (3) "Accordingly, a score cut-off of 40 for hydroxylated peptides and a localisation probability cut-off of more than 0.5 for hydroxylated peptides was performed." Could the authors shed more light and clarify what was the basis for this value of cut-off to be used in this filtering step? Is this sample dependent? What should be the criteria to determine this value?

      We used MaxQuant software (10.1016/j.cell.2006.09.026), for PTM analysis, in which a localization probability score of 0.75 and score cut-off of 40 is a commonly used threshold to define high confidence. The reason that we used 0.5 at the first step was to investigate how likely it might be that the misassignment of delta m/z +16 Da (oxidation) on Methionine would affect the identification of hydroxylation on Proline. However, we note that in the final results set used for analysis, all putative proline hydroxylated peptides that had a Methionine residue near to the hydroxylated proline were disregarded as a pragmatic step to reduce the probability of false identifications.

      (4) The authors are requested to kindly make the HPLC and MS traces more legible and use highresolution images, with clearly labeled values on the peaks. Kindly extract coordinates from the underlying data files to plot the curves if needed to make it clearer.

      We have reviewed the clarity of all images and figures in the current revision.

      (5) There seems to be no error bars in Figure 3, Figure 7E, and panels of Figure 8 with bar graphs. Are those single replicate data?

      These specific figures are from single replicate data.

      (6) For Figure 9C, the control with only PHD1 (no peptide) is missing. 

      The ‘no peptide control’ was not included in the figure because it is simply a blank lane and there is nothing to see.

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      Reply to the reviewers

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      Summary:

      Damaris et al. perform what is effectively an eQTL analysis on microbial pangenomes of E. coli and P. aeruginosa. Specifically, they leverage a large dataset of paired DNA/RNA-seq information for hundreds of strains of these microbes to establish correlations between genetic variants and changes in gene expression. Ultimately, their claim is that this approach identifies non-coding variants that affect expression of genes in a predictable manner and explain differences in phenotypes. They attempt to reinforce these claims through use of a widely regarded promoter calculator to quantify promoter effects, as well as some validation studies in living cells. Lastly, they show that these non-coding variations can explain some cases of antibiotic resistance in these microbes.

      Major comments

      Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

      The authors convincingly demonstrate that they can identify non-coding variation in pangenomes of bacteria and associate these with phenotypes of interest. What is unclear is the extent by which they account for covariation of genetic variation? Are the SNPs they implicate truly responsible for the changes in expression they observe? Or are they merely genetically linked to the true causal variants. This has been solved by other GWAS studies but isn't discussed as far as I can tell here.

      We thank the reviewer for their effective summary of our study. Regarding our ability to identify variants that are causal for gene expression changes versus those that only “tag” the causal ones, here we have to again offer our apologies for not spelling out the limitation of GWAS approaches, namely the difficulty in separating associated with causal variants. This inherent difficulty is the main reason why we added the in-silico and in-vitro validation experiments; while they each have their own limitations, we argue that they all point towards providing a causal link between some of our associations and measured gene expression changes. We have amended the discussion (e.g. at L548) section to spell our intention out better and provide better context for readers that are not familiar with the pitfalls of (bacterial) GWAS.

      They need to justify why they consider the 30bp downstream of the start codon as non-coding. While this region certainly has regulatory impact, it is also definitely coding. To what extent could this confound results and how many significant associations to expression are in this region vs upstream?

      We agree with the reviewer that defining this region as “non-coding” is formally not correct, as it includes the first 10 codons of the focal gene. We have amended the text to change the definition to “cis regulatory region” and avoided using the term “non-coding” throughout the manuscript. Regarding the relevance of this including the early coding region, we have looked at the distribution of associated hits in the cis regulatory regions we have defined; the results are shown in Supplementary Figure 3.

      We quantified the distribution of cis associated variants and compared them to a 2,000 permutations restricted to the -200bp and +30bp window in both E. coli * (panel A) and P. aeruginosa* (panel B). As it can be seen, the associated variants that we have identified are mostly present in the 200bp region and the +30bp region shows a mild depletion relative to the random expectation, which we derived through a variant position shuffling approach (2,000 replicates). Therefore, we believe that the inclusion of the early coding region results in an appreciable number of associations, and in our opinion justify its inclusion as a putative “cis regulatory region”.

      The claim that promoter variation correlates with changes in measured gene expression is not convincingly demonstrated (although, yes, very intuitive). Figure 3 is a convoluted way of demonstrating that predicted transcription rates correlate with measured gene expression. For each variant, can you do the basic analysis of just comparing differences in promoter calculator predictions and actual gene expression? I.e. correlation between (promoter activity variant X)-(promoter activity variant Y) vs (measured gene expression variant X)-(measured gene expression variant Y). You'll probably have to

      We realize that we may not have failed to properly explain how we carried out this analysis, which we did exactly in the way the reviewer suggests here. We had in fact provided four example scatterplots of the kind the reviewer was requesting as part of Figure 4. We have added a mention of their presence in the caption of Figure 3.

      Figure 7 it is unclear what this experiment was. How were they tested? Did you generate the data themselves? Did you do RNA-seq (which is what is described in the methods) or just test and compare known genomic data?

      We apologize for the lack of clarity here; we have amended the figure’s caption and the corresponding section of the results (i.e. L411 and L418) to better highlight how the underlying drug susceptibility data and genomes came from previously published studies.

      Are the data and the methods presented in such a way that they can be reproduced?

      No, this is the biggest flaw of the work. The RNA-Seq experiment to start this project is not described at all as well as other key experiments. Descriptions of methods in the text are far too vague to understand the approach or rationale at many points in the text. The scripts are available on github but there is no description of what they correspond to outside of the file names and none of the data files are found to replicate the plots.

      We have taken this critique to heart, and have given more details about the experimental setup for the generation of the RNA-seq data in the methods as well as the results sections. We have also thoroughly reviewed any description of the methods we have employed to make sure they are more clearly presented to the readers. We have also updated our code repository in order to provide more information about the meaning of each script provided, although we would like to point out that we have not made the code to be general purpose, but rather as an open documentation on how the data was analyzed.

      Figure 8B is intended to show that the WaaQ operon is connected to known Abx resistance genes but uses the STRING method. This requires a list of genes but how did they build this list? Why look at these known ABx genes in particular? STRING does not really show evidence, these need to be substantiated or at least need to justify why this analysis was performed.

      We have amended the Methods section (“Gene interaction analysis”, L799) to better clarify how the network shown in this panel was obtained. In short, we have filtered the STRING database to identify genes connected to members of the waa operon with an interaction score of at least 0.4 (“moderate confidence”), excluding the “text mining” field. Antimicrobial resistance genes were identified according to the CARD database. We believe these changes will help the readers to better understand how we derived this interaction.

      Are the experiments adequately replicated and statistical analysis adequate?

      An important claim on MIC of variants for supplementary table 8 has no raw data and no clear replicates available. Only figure 6, the in vitro testing of variant expression, mentions any replicates.

      We have expanded the relevant section in the Methods (“Antibiotic exposure and RNA extraction”, L778) to provide more information on the way these assays were carried out. In short, we carried out three biological replicates, the average MIC of two replicates in closest agreement was the representative MIC for the strain. We believe that we have followed standard practice in the field of microbiology, but we agree that more details were needed to be provided in order for readers to appreciate this.

      Minor comments

      Specific experimental issues that are easily addressable..

      Are prior studies referenced appropriately?

      There should be a discussion of eQTLs in this. Although these have mostly been in eukaryotes a. https://doi.org/10.1038/s41588-024-01769-9 ; https://doi.org/10.1038/nrg3891.

      We have added these two references, which provide a broader context to our study and methodology, in the introduction.

      Line 67. Missing important citation for Ireland et al. 2020 https://doi.org/10.7554/eLife.55308

      Line 69. Should mention Johns et al. 2018 (https://doi.org/10.1038/nmeth.4633) where they study promoter sequences outside of E. coli

      Line 90 - replace 'hypothesis-free' with unbiased

      We have implemented these changes.

      Line 102 - state % of DEGs relative to the entire pan-genome

      Given that the study is focused on identifying variants that were associated with changes in expression for reference genes (i.e. those present in the reference genome), we think that providing this percentage would give the false impression that our analysis include accessory genes that are not encoded by the reference isolate, which is not what we have done.

      Figure 1A is not discussed in the text

      We have added an explicit mention of the panels in the relevant section of the results.

      Line 111: it is unclear what enrichment was being compared between, FIgures 1C/D have 'Gene counts' but is of the total DEGs? How is the p-value derived? Comparing and what statistical test was performed? Comparing DEG enrichment vs the pangenome? K12 genome?

      We have amended the results and methods section, as well as Figure 1’s caption to provide more details on how this analysis was carried out.

      Line 122-123: State what letters correspond to these COG categories here

      We have implemented the clarifications and edits suggested above

      Line 155: Need to clarify how you use k-mers in this and how they are different than SNPs. are you looking at k-mer content of these regions? K-mers up to hexamers or what? How are these compared. You can't just say we used k-mers.

      We have amended that line in the results section to more explicitly refer to the actual encoding of the k-mer variants, which were presence/absence patterns for k-mers extracted from each target gene’s promoter region separately, using our own developed method, called panfeed. We note that more details were already given in the methods section, but we do recognize that it’s better to clarify things in the results section, so that more distracted readers get the proper information about this class of genetic variants.

      Line 172: It would be VERY helpful to have a supplementary figure describing these types of variants, perhaps a multiple-sequence alignment containing each example

      We thank the reviewer for this suggestion. We have now added Supplementary Figure 3, which shows the sequence alignments of the cis-regulatory regions underlying each class of the genetic marker for both E. coli and P. aeruginosa.

      Figure 4: THis figure is too small. Why are WaaQ and UlaE being used as examples here when you are supposed to be explicitly showing variants with strong positive correlations?

      We rearranged the figure’s layout to improve its readability. We agree that the correlation for waaQ and ulaE is weaker than for yfgJ and kgtP, but our intention was to not simply cherry-pick strong examples, but also those for which the link between predicted promoter strength and recorded gene expression was less obvious.

      Figure 4: Why is there variation between variants present and variant absent? Is this due to other changes in the variant? Should mention this in the text somewhere

      Variability in the predicted transcription rate for isolates encoding for the same variant is due to the presence of other (different) variants in the region surrounding the target variant. PromoterCalculator uses nucleotide regions of variable length (78 to 83bp) to make its predictions, while the variants we are focusing on are typically shorter (as shown in Figure 4). This results in other variants being included in the calculation and therefore slightly different predicted transcription rates for each strain. We have amended the caption of Figure 4 to provide a succinct explanation of these differences.

      Line 359: Need to talk about each supplementary figure 4 to 9 and how they demonstrate your point.

      We have expanded this section to more explicitly mention the contents of these supplementary figures and why they are relevant for the findings of this section (L425).

      Are the text and figures clear and accurate?

      Figure 4 too small

      We have fixed the figure, as described above

      Acronyms are defined multiple times in the manuscript, sometimes not the first time they are used (e.g. SNP, InDel)

      Figure 8A - Remove red box, increase label size

      Figure 8B - Low resolution, grey text is unreadable and should be darker and higher resolution

      Line 35 - be more specific about types of carbon metabolism and catabolite repression

      Line 67 - include citation for ireland et al. 2020 https://doi.org/10.7554/eLife.55308

      Line 74 - You talk about looking in cis but don't specify how mar away cis is

      Line 75 - we encoded genetic variants..... It is unclear what you mean here

      Line 104 - 'were apart of operons' should clarify you mean polycistronic or multi-gene operons. Single genes may be considered operonic units as well.

      We have addressed all the issues indicated above.

      Figure 2: THere is no axis for the percents and the percents don't make sense relative to the bars they represent??

      We realize that this visualization might not have been the most clear for readers, and have made the following improvement: we have added the number of genes with at least one association before the percentage. We note that the x-axis is in log scale, which may make it seem like the light-colored bars are off. With the addition of the actual number of associated genes we think that this confusion has been removed.

      Figure 2: Figure 2B legend should clarify that these are individual examples of Differential expression between variants

      Line 198-199: This sentence doesn't make sense, 'encoded using kmers' is not descriptive enough

      Line 205: Should be upfront about that you're using the Promoter Calculator that models biophysical properties of promoter sequences to predict activity.

      Line 251: 'Scanned the non-coding sequences of the DEGs'. This is far too vague of a description of an approach. Need to clarify how you did this and I didn't see in the method. Is this an HMM? Perfect sequence match to consensus sequence? Some type of alignment?

      Line 257-259: This sentence lacks clarity

      We have implemented all the suggested changes and clarified the points that the reviewer has highlighted above.

      Line346: How were the E. coli isolates tested? Was this an experiment you did? This is a massive undertaking (1600 isolates * 12 conditions) if so so should be clearly defined

      While we have indicated in the previous paragraph that the genomes and antimicrobial susceptibility data were obtained from previously published studies, we have now modified this paragraph (e.g. L411 and L418) slightly to make this point even clearer.

      Figure 6A: The tile plot on the right side is not clearly labeled and it is unclear what it is showing and how that relates to the bar plots.

      In the revised figure, we have clarified the labeling of the heatmap to now read “Log2(Fold Change) (measured expression)” to indicate that it represents each gene’s fold changes obtained from our initial transcriptomic analysis. We have also included this information in the caption of the figure, making the relationship between the measured gene expression (heatmap) and the reporter assay data (bar plots) clear to the reader.

      FIgure 6B: typo in legend 'Downreglation'

      We thank the review for pointing this out. The typo has been corrected to “Down regulation” in the revised figure.

      Line 398: Need to state rationale for why Waaq operon is being investigated here. WHy did you look into individual example?

      We thank the reviewer for asking for a clarification here. Our decision to investigate the waaQ gene was one of both biological relevance and empirical evidence. In our analysis associating non-coding variants with antimicrobial resistance using the Moradigaravand et al. dataset, we identified a T>C variant at position 3808241 that was associated with resistance to Tobramycin. We also observed this variant in our strain collection, where it was associated with expression changes of the gene, suggesting a possible functional impact. The waa operon is involved in LPS synthesis, a central determinant of the bacteria’s outer membrane integrity and a well established virulence factor. This provided a plausible biological mechanism through which variation could influence antimicrobial susceptibility. As its role in resistance has not been extensively characterized, this represents a good candidate for our experimental validation. We have now included this rationale in our revised manuscript (i.e. L476).

      Figure 8: Can get rid of red box

      We have now removed the red box from Figure 8 in the revised version.

      Line 463 - 'account for all kinds' is too informal

      Mix of font styles throughout document

      We have implemented all the suggestions and formatting changes indicated above.

      Reviewer #2 (Evidence, reproducibility and clarity (Required)):

      In their manuscript "Cis non-coding genetic variation drives gene expression changes in the E. coli and P. aeruginosa pangenomes", Damaris and co-authors present an extensive meta-analysis, plus some useful follow up experiments, attempting to apply GWAS principles to identify the extent to which differences in gene expression between different strains within a given species can be directly assigned to cis-regulatory mutations. The overall principle, and the question raised by the study, is one of substantial interest, and the manuscript here represents a careful and fascinating effort at unravelling these important questions. I want to preface my review below (which may otherwise sound more harsh than I intend) with the acknowledgment that this is an EXTREMELY difficult and challenging problem that the authors are approaching, and they have clearly put in a substantial amount of high quality work in their efforts to address it. I applaud the work done here, I think it presents some very interesting findings, and I acknowledge fully that there is no one perfect approach to addressing these challenges, and while I will object to some of the decisions made by the authors below, I readily admit that others might challenge my own suggestions and approaches here. With that said, however, there is one fundamental decision that the authors made which I simply cannot agree with, and which in my view undermines much of the analysis and utility of the study: that decision is to treat both gene expression and the identification of cis-regulatory regions at the level of individual genes, rather than transcriptional units. Below I will expand on why I find this problematic, how it might be addressed, and what other areas for improvement I see in the manuscript:

      We thank the reviewer for their praise of our work. A careful set of replies to the major and minor critiques are reported below each point.

      In the entire discussion from lines roughly 100-130, the authors frequently dissect out apparently differentially expressed genes from non differentially expressed genes within the same operons... I honestly wonder whether this is a useful distinction. I understand that by the criteria set forth by the authors it is technically correct, and yet, I wonder if this is more due to thresholding artifacts (i.e., some genes passing the authors' reasonable-yet-arbitrary thresholds whereas others in the same operon do not), and in the process causing a distraction from an operon that is in fact largely moving in the same direction. The authors might wish to either aggregate data in some way across known transcriptional units for the purposes of their analysis, and/or consider a more lenient 'rescue' set of significance thresholds for genes that are in the same operons as differentially expressed genes. I would favor the former approach, performing virtually all of their analysis at the level of transcriptional units rather than individual genes, as much of their analysis in any case relies upon proper assignment of genes to promoters, and this way they could focus on the most important signals rather than get lots sometimes in the weeds of looking at every single gene when really what they seem to be looking at in this paper is a property OF THE PROMOTERS, not the genes. (of course there are phenomena, such as rho dependent termination specifically titrating expression of late genes in operons, but I think on the balance the operon-level analysis might provide more insights and a cleaner analysis and discussion).

      We agree with the reviewer that the peculiar nature of transcription in bacteria has to be taken into account in order to properly quantify the influence of cis variants in gene expression changes. We therefore added the exact analysis the reviewer suggested; that is, we ran associations between the variants in cis to the first gene of each operon and a phenotype that considered the fold-change of all genes in the operon, via a weighted average (see Methods for more details). As reported in the results section (L223), we found a similar trend as with the original analysis: we found the highest proportion of associations when encoding cis variants using k-mers (42% for E. coli and 45% for P. aeruginosa). More importantly, we found a high degree of overlap between this new “operon-level” association analysis and the original one (only including the first gene in each operon). We found a range of 90%-94% of associations overlapping for E. coli and between 75% and 91% for P. aeruginosa, depending on the variant type. We note that operon definitions are less precise for P. aeruginosa, which might explain the higher variability in the level of overlap. We have added the results of this analysis in the results section.

      This also leads to a more general point, however, which I think is potentially more deeply problematic. At the end of the day, all of the analysis being done here centers on the cis regulatory logic upstream of each individual open reading frame, even though in many cases (i.e., genes after the first one in multi-gene operons), this is not where the relevant promoter is. This problem, in turn, raises potentially misattributions of causality running in both directions, where the causal impact on a bona fide promoter mutation on many genes in an operon may only be associated with the first gene, or on the other side, where a mutation that co-occurs with, but is causally independent from, an actual promoter mutation may be flagged as the one driving an expression change. This becomes an especially serious issue in cases like ulaE, for genes that are not the first gene in an operon (at least according to standard annotations, the UlaE transcript should be part of a polycistronic mRNA beginning from the ulaA promoter, and the role played by cis-regulatory logic immediately upstream of ulaE is uncertain and certainly merits deeper consideration. I suspect that many other similar cases likewise lurk in the dataset used here (perhaps even moreso for the Pseudomonas data, where the operon definitions are likely less robust). Of course there are many possible explanations, such as a separate ulaE promoter only in some strains, but this should perhaps be carefully stated and explored, and seems likely to be the exception rather than the rule.

      While we again agree with the reviewer that some of our associations might not result in a direct causal link because the focal variant may not belong to an actual promoter element, we also want to point out how the ability to identify the composition of transcriptional units in bacteria is far from a solved problem (see references at the bottom of this comment, two in general terms, and one characterizing a specific example), even for a well-studied species such as E. coli. Therefore, even if carrying out associations at the operon level (e.g. by focusing exclusively on variants in cis for the first gene in the operon) might be theoretically correct, a number of the associations we find further down the putative operons might be the result of a true biological signal.

      1. Conway, T., Creecy, J. P., Maddox, S. M., Grissom, J. E., Conkle, T. L., Shadid, T. M., Teramoto, J., San Miguel, P., Shimada, T., Ishihama, A., Mori, H., & Wanner, B. L. (2014). Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing. mBio, 5(4), 10.1128/mbio.01442-14. https://doi.org/10.1128/mbio.01442-14

      2. Sáenz-Lahoya, S., Bitarte, N., García, B., Burgui, S., Vergara-Irigaray, M., Valle, J., Solano, C., Toledo-Arana, A., & Lasa, I. (2019). Noncontiguous operon is a genetic organization for coordinating bacterial gene expression. Proceedings of the National Academy of Sciences, 116(5), 1733–1738. https://doi.org/10.1073/pnas.1812746116

      3. Zehentner, B., Scherer, S., & Neuhaus, K. (2023). Non-canonical transcriptional start sites in E. coli O157:H7 EDL933 are regulated and appear in surprisingly high numbers. BMC Microbiology, 23(1), 243. https://doi.org/10.1186/s12866-023-02988-6

      Another issue with the current definition of regulatory regions, which should perhaps also be accounted for, is that it is likely that for many operons, the 'regulatory regions' of one gene might overlap the ORF of the previous gene, and in some cases actual coding mutations in an upstream gene may contaminate the set of potential regulatory mutations identified in this dataset.

      We agree that defining regulatory regions might be challenging, and that those regions might overlap with coding regions, either for the focal gene or the one immediately upstream. For these reasons we have defined a wide region to identify putative regulatory variants (-200 to +30 bp around the start codon of the focal gene). We believe this relatively wide region allows us to capture the most cis genetic variation.

      Taken together, I feel that all of the above concerns need to be addressed in some way. At the absolute barest minimum, the authors need to acknowledge the weaknesses that I have pointed out in the definition of cis-regulatory logic at a gene level. I think it would be far BETTER if they performed a re-analysis at the level of transcriptional units, which I think might substantially strengthen the work as a whole, but I recognize that this would also constitute a substantial amount of additional effort.

      As indicated above, we have added a section in the results section to report on the analysis carried out at the level of operons as individual units, with more details provided in the methods section. We believe these results, which largely overlap with the original analysis, are a good way to recognize the limitation of our approach and to acknowledge the importance of gaining a better knowledge on the number and composition of transcriptional units in bacteria, for which, as the reference above indicates, we still have an incomplete understanding.

      Having reached the end of the paper, and considering the evidence and arguments of the authors in their totality, I find myself wondering how much local x background interactions - that is, the effects of cis regulatory mutations (like those being considered here, with or without the modified definitions that I proposed above) IN THE CONTEXT OF A PARTICULAR STRAIN BACKGROUND, might matter more than the effects of the cis regulatory mutations per se. This is a particularly tricky problem to address because it would require a moderate number of targeted experiments with a moderate number of promoters in a moderate number of strains (which of course makes it maximally annoying since one can't simply scale up hugely on either axis individually and really expect to tease things out). I think that trying to address this question experimentally is FAR beyond the scope of the current paper, but I think perhaps the authors could at least begin to address it by acknowledging it as a challenge in their discussion section, and possibly even identify candidate promoters that might show the largest divergence of activities across strains when there IS no detectable cis regulatory mutation (which might be indicative of local x background interactions), or those with the largest divergences of effect for a given mutation across strains. A differential expression model incorporating shrinkage is essential in such analysis to avoid putting too much weight on low expression genes with a lot of Poisson noise.

      We again thank the reviewer for their thoughtful comments on the limitations of correlative studies in general, and microbial GWAS in particular. In regards to microbial GWAS we feel we may have failed to properly explain how the implementation we have used allows to, at least partially, correct for population structure effects. That is, the linear mixed model we have used relies on population structure to remove the part of the association signal that is due to the genetic background and thus focus the analysis on the specific loci. Obviously examples in which strong epistatic interactions are present would not be accounted for, but those would be extremely challenging to measure or predict at scale, as the reviewer rightfully suggests. We have added a brief recap of the ability of microbial GWAS to account for population structure in the results section (“A large fraction of gene expression changes can be attributed to genetic variations in cis regulatory regions”, e.g. L195).

      I also have some more minor concerns and suggestions, which I outline below:

      It seems that the differential expression analysis treats the lab reference strains as the 'centerpoint' against which everything else is compared, and yet I wonder if this is the best approach... it might be interesting to see how the results differ if the authors instead take a more 'average' strain (either chosen based on genetics or transcriptomics) as a reference and compared everything else to that.

      While we don’t necessarily disagree with the reviewer that a “wild” strain would be better to compare against, we think that our choice to go for the reference isolates is still justified on two grounds. First, while it is true that comparing against a reference introduces biases in the analysis, this concern would not be removed had we chosen another strain as reference; which strain would then be best as a reference to compare against? We think that the second point provides an answer to this question; the “traditional” reference isolates have a rich ecosystem of annotations, experimental data, and computational predictions. These can in turn be used for validation and hypothesis generation, which we have done extensively in the manuscript. Had we chosen a different reference isolate we would have had to still map associations to the traditional reference, resulting in a probable reduction in precision. An example that will likely resonate with this reviewer is that we have used experimentally-validated and high quality computational operon predictions to look into likely associations between cis-variants and “operon DEGs”. This analysis would have likely been of worse quality had we used another strain as reference, for which operon definitions would have had to come from lower-quality predictions or be “lifted” from the traditional reference.

      Line 104 - the statement about the differentially expressed genes being "part of operons with diverse biological functions" seems unclear - it is not apparent whether the authors are referring to diversity of function within each operon, or between the different operons, and in any case one should consider whether the observation reflects any useful information or is just an apparently random collection of operons.

      We agree that this formulation could create confusion and we have elected to remove the expression “with diverse biological functions”, given that we discuss those functions immediately after that sentence.

      Line 292 - I find the argument here somewhat unconvincing, for two reasons. First, the fact that only half of the observed changes went in the same direction as the GWAS results would indicate, which is trivially a result that would be expected by random chance, does not lend much confidence to the overall premise of the study that there are meaningful cis regulatory changes being detected (in fact, it seems to argue that the background in which a variant occurs may matter a great deal, at least as much as the cis regulatory logic itself). Second, in order to even assess whether the GWAS is useful to "find the genetic determinants of gene expression changes" as the authors indicate, it would be necessary to compare to a reasonable, non-straw-man, null approach simply identifying common sequence variants that are predicted to cause major changes in sigma 70 binding at known promoters; such a test would be especially important given the lack of directional accuracy observed here. Along these same lines, it is perhaps worth noting, in the discussion beginning on line 329, that the comparison is perhaps biased in favor of the GWAS study, since the validation targets here were prioritized based on (presumably strong) GWAS data.

      We thank the reviewer for prompting us into reasoning about the results of the in-vitro validation experiments. We agree that the agreement between the measured gene expression changes agree only partly with those measured with the reporter system, and that this discrepancy could likely be attributed to regulatory elements that are not in cis, and thus that were not present in the in-vitro reporter system. We have noted this possibility in the discussion. Additionally, we have amended the results section to note that even though the prediction in the direction of gene expression change was not as accurate as it could be expected, the prediction of whether a change would be present (thus ignoring directionality) was much higher.

      I don't find the Venn diagrams in Fig 7C-D useful or clear given the large number of zero-overlap regions, and would strongly advocate that the authors find another way to show these data.

      While we are aware that alternative ways to show overlap between sets, such as upset plots, we don’t actually find them that much easier to parse. We actually think that the simple and direct Venn diagrams we have drawn convey the clear message that overlaps only exist between certain drug classes in E. coli, and virtually none for P. aeruginosa. We have added a comment on the lack of overlap between all drug classes and the differences between the two species in the results section (i.e. L436 and L465).

      In the analysis of waa operon gene expression beginning on line 400, it is perhaps important to note that most of the waa operon doesn't do anything in laboratory K12 strains due to the lack of complete O-antigen... the same is not true, however, for many wild/clinical isolates. It would be interesting to see how those results compare, and also how the absolute TPMs (rather than just LFCs) of genes in this operon vary across the strains being investigated during TOB treatment.

      We thank the reviewer for this helpful suggestion. We examined the absolute expression (TPMs) of waa operon genes under the baseline (A) and following exposure to Tobramycin (B). The representative TPMs per strain were obtained by averaging across biological replicates. We observed a constitutive expression of the genes in the reference strain (MG1655) and the other isolates containing the variant of interest (MC4100, BW25113). In contrast, strains lacking the variants of interest (IAI76 and IAI78), showed lower expression of these operon genes under both conditions. Strain IAI77, on the other hand, displayed increased expression of a subset of waa genes post Tobramycin exposure, indicating strain-specific variation in transcriptional response. While the reference isolate might not have the O-antigen, it certainly expresses the waa operon, both constitutively and under TOB exposure.

      I don't think that the second conclusion on lines 479-480 is fully justified by the data, given both the disparity in available annotation information between the two species, AND the fact that only two species were considered.

      While we feel that the “Discussion” section of a research paper allows for speculative statements, we have to concede that we have perhaps overreached here. We have amended this sentence to be more cautious and not mislead readers.

      Line 118: "Double of DEGs"

      Line 288 - presumably these are LOG fold changes

      Fig 6b - legend contains typos

      Line 661 - please report the read count (more relevant for RNA-seq analysis) rather than Gb

      We thank the reviewer for pointing out the need to make these edits. We have implemented them all.

      Source code - I appreciate that the authors provide their source code on github, but it is very poorly documented - both a license and some top-level documentation about which code goes with each major operation/conclusion/figure should be provided. Also, ipython notebooks are in general a poor way in my view to distribute code, due to their encouragement of nonlinear development practices; while they are fine for software development, actual complete python programs along with accompanying source data would be preferrable.

      We agree with the reviewer that a software license and some documentation about what each notebook is about is warranted, and we have added them both. While we agree that for “consumer-grade” software jupyter notebooks are not the most ergonomic format, we believe that as a documentation of how one-time analyses were carried out they are actually one of the best formats we could think of. They in fact allow for code and outputs to be presented alongside each other, which greatly helped us to iterate on our research and to ensure that what was presented in the manuscript matched the analyses we reported in the code. This is of course up for debate and ultimately specific to someone’s taste, and so we will keep the reviewer’s critique in mind for our next manuscript. And, if we ever decide to package the analyses presented in the manuscript as a “consumer-grade” application for others to use, we would follow higher standards of documentation and design.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      In this manuscript, Damaris et al. collected genome sequences and transcriptomes from isolates from two bacterial species. Data for E. coli were produced for this paper, while data for P. aeruginosa had been measured earlier. The authors integrated these data to detect genes with differential expression (DE) among isolates as well as cis-expression quantitative trait loci (cis-eQTLs). The authors used sample sizes that were adequate for an initial exploration of gene regulatory variation (n=117 for E. coli and n=413 for P. aeruginosa) and were able to discover cis eQTLs at about 39% of genes. In a creative addition, the authors compared their results to transcription rates predicted from a biophysical promoter model as well as to annotated transcription factor binding sites. They also attempted to validate some of their associations experimentally using GFP-reporter assays. Finally, the paper presents a mapping of antibiotic resistance traits. Many of the detected associations for this important trait group were in non-coding genome regions, suggesting a role of regulatory variation in antibiotic resistance.

      A major strength of the paper is that it covers an impressive range of distinct analyses, some of which in two different species. Weaknesses include the fact that this breadth comes at the expense of depth and detail. Some sections are underdeveloped, not fully explained and/or thought-through enough. Important methodological details are missing, as detailed below.

      We thank the reviewer for highlighting the strengths of our study. We hope that our replies to their comments and the other two reviewers will address some of the limitations.

      Major comments:

      1. An interesting aspect of the paper is that genetic variation is represented in different ways (SNPs & indels, IRG presence/absence, and k-mers). However, it is not entirely clear how these three different encodings relate to each other. Specifically, more information should be given on these two points:

      2. it is not clear how "presence/absence of intergenic regions" are different from larger indels.

      In order to better guide readers through the different kinds of genetic variants we considered, we have added a brief explanation about what “promoter switches” are in the introduction (“meaning that the entire promoter region may differ between isolates due to recombination events”, L56). We believe this clarifies how they are very different in character from a large deletion. We have kept the reference to the original study (10.1073/pnas.1413272111) describing how widespread these switches are in E. coli as a way for readers to discover more about them.

      • I recommend providing more narration on how the k-mers compare to the more traditional genetic variants (SNPs and indels). It seems like the k-mers include the SNPs and indels somehow? More explanation would be good here, as k-mer based mapping is not usually done in other species and is not standard practice in the field. Likewise, how is multiple testing handled for association mapping with k-mers, since presumably each gene region harbors a large number of k-mers, potentially hugely increasing the multiple testing burden?

      We indeed agree with the reviewer in thinking that representing genetic variants as k-mers would encompass short variants (SNP/InDels) as well as larger variants and promoters presence/absence patterns. We believe that this assumption is validated by the fact that we identify the highest proportion of DEGs with a significant association when using this representation of variants (Figure 2A, 39% for both species). We have added a reference to a recent review on the advantages of k-mer methods for population genetics (10.1093/molbev/msaf047) in the introduction. Regarding the issue of multiple testing correction, we have employed a commonly recognized approach that, unlike a crude Bonferroni correction using the number of tested variants, allows for a realistic correction of association p-values. We used the number of unique presence/absence patterns, which can be shared between multiple genetic variants, and applied a Bonferroni correction using this number rather than the number of variants tested. We have expanded the corresponding section in the methods (e.g. L697) to better explain this point for readers not familiar with this approach.

      1. What was the distribution of association effect sizes for the three types of variants? Did IRGs have larger effects than SNPs as may be expected if they are indeed larger events that involve more DNA differences? What were their relative allele frequencies?

      We appreciate the suggestion made by the reviewer to look into the distribution of effect sizes divided by variant type. We have now evaluated the distribution of the effect sizes and allele frequencies for the genetic markers (SNPs/InDels, IGRs, and k-mers) for both species (Supplementary Figure 2). In E. coli, IGR variants showed somewhat larger median effect sizes (|β| = 4.5) than SNPs (|β| = 3.8), whereas k-mers displayed the widest distribution (median |β| = 5.2). In P. aeruginosa, the trend differed with IGRs exhibiting smaller effects (median |β| = 3.2), compared to SNPs/InDels (median |β| =5.1) and k-mers (median |β| = 6.2). With respect to allele frequencies, SNPs/InDels generally occured at lower frequencies (median AF = 0.34 for E.coli, median AF = 0.33 for P. aeruginosa), whereas IGRs (median AF = 0.65 for E. coli and 0.75 for P. aeruginosa) and k-mers (median AF = 0.71 for E. coli and 0.65 for P. aeruginosa) were more often at the intermediate to higher frequencies respectively. We have added a visualization for the distribution of effect sizes (Supplementary Figure 2).

      1. The GFP-based experiments attempting to validate the promoter effects for 18 genes are laudable, and the fact that 16 of them showed differences is nice. However, the fact that half of the validation attempts yielded effects in the opposite direction of what was expected is quite alarming. I am not sure this really "further validates" the GWAS in the way the authors state in line 292 - in fact, quite the opposite in that the validations appear random with regards to what was predicted from the computational analyses. How do the authors interpret this result? Given the higher concordance between GWAS, promoter prediction, and DE, are the GFP assays just not relevant for what is going on in the genome? If not, what are these assays missing? Overall, more interpretation of this result would be helpful.

      We thanks the reviewer for their comment, which is similar in nature to that raised by reviewer #2 above. As noted in our reply above we have amended the results and discussion to indicate that although the direction of gene expression change was not highly accurate, focusing on the magnitude (or rather whether there would be a change in gene expression, regardless of the direction), resulted in a higher accuracy. We postulate that the cases in which the direction of the change was not correctly identified could be due to the influence of other genetic elements in trans with the gene of interest.

      1. On the same note, it would be really interesting to expand the GFP experiments to promoters that did not show association in the GWAS. Based on Figure 6, effects of promoter differences on GFP reporters seem to be very common (all but three were significant). Is this a higher rate than for the average promoter with sequence variation but without detected association? A handful of extra reporter experiments might address this. My larger question here is: what is the null expectation for how much functional promoter variation there is?

      We thank the reviewer for this comment. We agree that estimating the null expectation for the functional promoter would require testing promoter alleles with sequence variation that are not associated in the GWAS. Such experiments, which would directly address if the observed effects in our study exceeds background, would have required us to prepare multiple constructs, which was unfortunately not possible for us due to staff constraints. We therefore elected to clarify the scope of our GFP reporter assays instead. These experiments were designed as a paired comparison of the wild-type and the GWAS-associated variant alleles of the same promoter in an identical reporter background, with the aim of testing allele-specific functional effects for GWAS hits (Supplementary Figure 6). We also included a comparison in GFP fluorescence between the promoterless vector (pOT2) and promoter-containing constructs; we observed higher GFP signals in all but four (yfgJ, fimI, agaI, and yfdQ) variant-containing promoter constructs, which indicates that for most of the construct we cloned active promoter elements. We have revised the manuscript text accordingly to reflect this clarification and included the control in the supplementary information as Supplementary Figure 6.

      1. Were the fold-changes in the GFP experiments statistically significant? Based on Figure 6 it certainly looks like they are, but this should be spelled out, along with the test used.

      We thank the reviewer for pointing this out. We have reviewed Figure 6 to indicate significant differences between the test and control reporter constructs. We used the paired student’s t-test to match the matched plate/time point measurements. We also corrected for multiple testing using the Benhamini-Hochberg correction. As seen in the updated Figure 6A, 16 out of the 18 reporter constructs displayed significant differences (adjusted p-value

      1. What was the overall correlation between GWAS-based fold changes and those from the GFP-based validation? What does Figure 6A look like as a scatter plot comparing these two sets of values?

      We thank the reviewer for this helpful suggestion, which allows us to more closely look into the results of our in-vitro validation. We performed a direct comparison of RNAseq fold changes from the GWAS (x-axis) with the GFP reporter measurements (y-axis) as depicted in the figure above. The overall correlation between the two was weak (Pearson r = 0.17), reflecting the lack of thorough agreement between the associations and the reporter construct. We however note that the two metrics are not directly comparable in our opinion, since on the x-axis we are measuring changes in gene expression and on the y-axis changes in fluorescence expression, which is downstream from it. As mentioned above and in reply to a comment from reviewer 2, the agreement between measured gene expression and all other in-silico and in-vitro techniques increases when ignoring the direction of the change. Overall, we believe that these results partly validate our associations and predictions, while indicating that other factors in trans with the regulatory region contribute to changes in gene expression, which is to be expected. The scatter plot has been included as a new supplementary figure (Supplementary Figure 7).

      1. Was the SNP analyzed in the last Results section significant in the gene expression GWAS? Did the DE results reported in this final section correspond to that GWAS in some way?

      The T>C SNP upstream of waaQ did not show significant association with gene expression in our cis GWAS analysis. Instead, this variant was associated with resistance to tobramycin when referencing data from Danesh et al, and we observed the variant in our strain collection. We subsequently investigated whether this variant also influenced expression of the waa operon under sub-inhibitory tobramycin exposure. The differential expression results shown in the final section therefore represent a functional follow-up experiment, and not a direct replication of the GWAS presented in the first part of the manuscript.

      1. Line 470: "Consistent with the differences in the genetic structure of the two species" It is not clear what differences in genetic structure this refers to. Population structure? Genome architecture? Differences in the biology of regulatory regions?

      The awkwardness of that sentence is perhaps the consequence of our assumption that readers would be aware of the differences in population genetics differences between the two species. We however have realized that not much literature is available (if at all!) about these differences, which we have observed during the course of this and other studies we have carried out. As a result, we agree that we cannot assume that the reader is similarly familiar with these differences, and have changed that sentence (i.e. L548) to more directly address the differences between the two species, which will presumably result in a diverse population structure. We thank the reviewer for letting us be aware of a gap in the literature concerning the comparison of pangenome structures across relevant species.

      1. Line 480: the reference to "adaption" is not warranted, as the paper contains no analyses of evolutionary patterns or processes. Genetic variation is not the same as adaptation.

      We have amended this sentence to be more adherent to what we can conclude from our analyses.

      1. There is insufficient information on how the E. coli RNA-seq data was generated. How was RNA extracted? Which QC was done on the RNA; what was its quality? Which library kits were used? Which sequencing technology? How many reads? What QC was done on the RNA-seq data? For this section, the Methods are seriously deficient in their current form and need to be greatly expanded.

      We thank the reviewer for highlighting the need for clearer methodological detail. We have expanded this section (i.e. L608) to fully describe the generation and quality control of the E. coli RNA-seq data including RNA extraction and sequencing platform.

      1. How were the DEG p-values adjusted for multiple testing?

      As indicated in the methods section (“Differential gene expression and functional enrichment analysis”), we have used DEseq2 for E. coli, and LPEseq for P. aeruginosa. Both methods use the statistical framework of the False Discovery Rate (FDR) to compute an adjusted p-value for each gene. We have added a brief mention of us following the standard practice indicated by both software packages in the methods.

      1. Were there replicates for the E. coli strains? The methods do not say, but there is a hint there might have been replicates given their absence was noted for the other species.

      In the context of providing more information about the transcriptomics experiments for E. coli, we have also more clearly indicated that we have two biological replicates for the E. coli dataset.

      1. There needs to be more information on the "pattern-based method" that was used to correct the GWAS for multiple tests. How does this method work? What genome-wide threshold did it end up producing? Was there adjustment for the number of genes tested in addition to the number of variants? Was the correction done per variant class or across all variant classes?

      In line with an earlier comment from this reviewer, we have expanded the section in the Methods (e.g. L689) that explains how this correction worked to include as many details as possible, in order to provide the readers with the full context under which our analyses were carried out.

      1. For a paper that, at its core, performs a cis-eQTL mapping, it is an oversight that there seems not to be a single reference to the rich literature in this space, comprising hundreds of papers, in other species ranging from humans, many other animals, to yeast and plants.

      We thank both reviewer #1 and #3 for pointing out this lack of references to the extensive literature on the subject. We have added a number of references about the applications of eQTL studies, and specifically its application in microbial pangenomes, which we believe is more relevant to our study, in the introduction.

      Minor comments:

      1. I wasn't able to understand the top panels in Figure 4. For ulaE, most strains have the solid colors, and the corresponding bottom panel shows mostly red points. But for waaQ, most strains have solid color in the top panel, but only a few strains in the bottom panel are red. So solid color in the top does not indicate a variant allele? And why are there so many solid alleles; are these all indels? Even if so, for kgtP, the same colors (i.e., nucleotides) seem to seamlessly continue into the bottom, pale part of the top panel. How are these strains different genotypically? Are these blocks of solid color counted as one indel or several SNPs, or somehow as k-mer differences? As the authors can see, these figures are really hard to understand and should be reworked. The same comment applies to Figure 5, where it seems that all (!) strains have the "variant"?

      We thank the reviewer for pointing out some limitations with our visualizations, most importantly with the way we explained how to read those two figures. We have amended the captions to more explicitly explain what is shown. The solid colors in the “sequence pseudo-alignment” panels indicate the focal cis variant, which is indicated in red in the corresponding “predicted transcription rate” panels below. In the case of Figure 5, the solid color indicates instead the position of the TFBS in the reference.

      1. Figure 1A & B: It would be helpful to add the total number of analyzed genes somewhere so that the numbers denoted in the colored outer rings can be interpreted in comparison to the total.

      We have added the total number of genes being considered for either species in the legend.

      1. Figure 1C & D: It would be better to spell out the COG names in the figure, as it is cumbersome for the reader to have to look up what the letters stand for in a supplementary table in a separate file.

      While we do not disagree with the awkwardness of having to move to a supplementary table to identify the full name of a COG category, we also would like to point out that the very long names of each category would clutter the figure to a degree that would make it difficult to read. We had indeed attempted something similar to what the reviewer suggests in early drafts of this manuscript, leading to small and hard to read labels. We have therefore left the full names of each COG category in Supplementary Table 3.

      1. Line 107: "Similarly," does not fit here as the following example (with one differentially expressed gene in an operon) is conceptually different from the one before, where all genes in the operon were differentially expressed.

      We agree and have amended the sentence accordingly.

      1. Figure 5 bottom panel: it is odd that on the left the swarm plots (i.e., the dots) are on the inside of the boxplots while on the right they are on the outside.

      We have fixed the position of the dots so that they are centered with respect to the underlying boxplots.

      1. It is not clear to me how only one or a few genes in an operon can show differential mRNA abundance. Aren't all genes in an operon encoded by the same mRNA? If so, shouldn't this mRNA be up- or downregulated in the same manner for all genes it encodes? As I am not closely familiar with bacterial systems, it is well possible that I am missing some critical fact about bacterial gene expression here. If this is not an analysis artifact, the authors could briefly explain how this observation is possible.

      We thanks the reviewer for their comment, which again echoes one of the main concerns from reviewer #2. As noted in our reply above, it has been established in multiple studies (see the three we have indicated above in our reply to reviewer #2) how bacteria encode for multiple “non-canonical” transcriptional units (i.e. operons), due to the presence of accessory terminators and promoters. This, together with other biological effects such as the presence of mRNA molecules of different lengths due to active transcription and degradation and technical noise induced by RNA isolation and sequencing can result in variability in the estimation of abundance for each gene.

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      Referee #3

      Evidence, reproducibility and clarity

      In this manuscript, Damaris et al. collected genome sequences and transcriptomes from isolates from two bacterial species. Data for E. coli were produced for this paper, while data for P. aeruginosa had been measured earlier. The authors integrated these data to detect genes with differential expression (DE) among isolates as well as cis-expression quantitative trait loci (cis-eQTLs). The authors used sample sizes that were adequate for an initial exploration of gene regulatory variation (n=117 for E. coli and n=413 for P. aeruginosa) and were able to discover cis eQTLs at about 39% of genes. In a creative addition, the authors compared their results to transcription rates predicted from a biophysical promoter model as well as to annotated transcription factor binding sites. They also attempted to validate some of their associations experimentally using GFP-reporter assays. Finally, the paper presents a mapping of antibiotic resistance traits. Many of the detected associations for this important trait group were in non-coding genome regions, suggesting a role of regulatory variation in antibiotic resistance. A major strength of the paper is that it covers an impressive range of distinct analyses, some of which in two different species. Weaknesses include the fact that this breadth comes at the expense of depth and detail. Some sections are underdeveloped, not fully explained and/or thought-through enough. Important methodological details are missing, as detailed below.

      Major comments:

      1. An interesting aspect of the paper is that genetic variation is represented in different ways (SNPs & indels, IRG presence/absence, and k-mers). However, it is not entirely clear how these three different encodings relate to each other. Specifically, more information should be given on these two points:

      2. it is not clear how "presence/absence of intergenic regions" are different from larger indels.

      3. I recommend providing more narration on how the k-mers compare to the more traditional genetic variants (SNPs and indels). It seems like the k-mers include the SNPs and indels somehow? More explanation would be good here, as k-mer based mapping is not usually done in other species and is not standard practice in the field. Likewise, how is multiple testing handled for association mapping with k-mers, since presumably each gene region harbors a large number of k-mers, potentially hugely increasing the multiple testing burden?

      4. What was the distribution of association effect sizes for the three types of variants? Did IRGs have larger effects than SNPs as may be expected if they are indeed larger events that involve more DNA differences? What were their relative allele frequencies?
      5. The GFP-based experiments attempting to validate the promoter effects for 18 genes are laudable, and the fact that 16 of them showed differences is nice. However, the fact that half of the validation attempts yielded effects in the opposite direction of what was expected is quite alarming. I am not sure this really "further validates" the GWAS in the way the authors state in line 292 - in fact, quite the opposite in that the validations appear random with regards to what was predicted from the computational analyses. How do the authors interpret this result? Given the higher concordance between GWAS, promoter prediction, and DE, are the GFP assays just not relevant for what is going on in the genome? If not, what are these assays missing? Overall, more interpretation of this result would be helpful.
      6. On the same note, it would be really interesting to expand the GFP experiments to promoters that did not show association in the GWAS. Based on Figure 6, effects of promoter differences on GFP reporters seem to be very common (all but three were significant). Is this a higher rate than for the average promoter with sequence variation but without detected association? A handful of extra reporter experiments might address this. My larger question here is: what is the null expectation for how much functional promoter variation there is?
      7. Were the fold-changes in the GFP experiments statistically significant? Based on Figure 6 it certainly looks like they are, but this should be spelled out, along with the test used.
      8. What was the overall correlation between GWAS-based fold changes and those from the GFP-based validation? What does Figure 6A look like as a scatter plot comparing these two sets of values?
      9. Was the SNP analyzed in the last Results section significant in the gene expression GWAS? Did the DE results reported in this final section correspond to that GWAS in some way?
      10. Line 470: "Consistent with the differences in the genetic structure of the two species" It is not clear what differences in genetic structure this refers to. Population structure? Genome architecture? Differences in the biology of regulatory regions?
      11. Line 480: the reference to "adaption" is not warranted, as the paper contains no analyses of evolutionary patterns or processes. Genetic variation is not the same as adaptation.
      12. There is insufficient information on how the E. coli RNA-seq data was generated. How was RNA extracted? Which QC was done on the RNA; what was its quality? Which library kits were used? Which sequencing technology? How many reads? What QC was done on the RNA-seq data? For this section, the Methods are seriously deficient in their current form and need to be greatly expanded.
      13. How were the DEG p-values adjusted for multiple testing?
      14. Were there replicates for the E. coli strains? The methods do not say, but there is a hint there might have been replicates given their absence was noted for the other species.
      15. There needs to be more information on the "pattern-based method" that was used to correct the GWAS for multiple tests. How does this method work? What genome-wide threshold did it end up producing? Was there adjustment for the number of genes tested in addition to the number of variants? Was the correction done per variant class or across all variant classes?
      16. For a paper that, at its core, performs a cis-eQTL mapping, it is an oversight that there seems not to be a single reference to the rich literature in this space, comprising hundreds of papers, in other species ranging from humans, many other animals, to yeast and plants.

      Minor comments:

      1. I wasn't able to understand the top panels in Figure 4. For ulaE, most strains have the solid colors, and the corresponding bottom panel shows mostly red points. But for waaQ, most strains have solid color in the top panel, but only a few strains in the bottom panel are red. So solid color in the top does not indicate a variant allele? And why are there so many solid alleles; are these all indels? Even if so, for kgtP, the same colors (i.e., nucleotides) seem to seamlessly continue into the bottom, pale part of the top panel. How are these strains different genotypically? Are these blocks of solid color counted as one indel or several SNPs, or somehow as k-mer differences? As the authors can see, these figures are really hard to understand and should be reworked. The same comment applies to Figure 5, where it seems that all (!) strains have the "variant"?
      2. Figure 1A & B: It would be helpful to add the total number of analyzed genes somewhere so that the numbers denoted in the colored outer rings can be interpreted in comparison to the total.
      3. Figure 1C & D: It would be better to spell out the COG names in the figure, as it is cumbersome for the reader to have to look up what the letters stand for in a supplementary table in a separate file.
      4. Line 107: "Similarly," does not fit here as the following example (with one differentially expressed gene in an operon) is conceptually different from the one before, where all genes in the operon were differentially expressed.
      5. Figure 5 bottom panel: it is odd that on the left the swarm plots (i.e., the dots) are on the inside of the boxplots while on the right they are on the outside.
      6. It is not clear to me how only one or a few genes in an operon can show differential mRNA abundance. Aren't all genes in an operon encoded by the same mRNA? If so, shouldn't this mRNA be up- or downregulated in the same manner for all genes it encodes? As I am not closely familiar with bacterial systems, it is well possible that I am missing some critical fact about bacterial gene expression here. If this is not an analysis artifact, the authors could briefly explain how this observation is possible.

      Significance

      To my knowledge, this work represents the first cis-eQTL mapping in bacteria. As such, it is a useful and interesting exploration of this space that complements the large body of literature on this question in eukaryotic systems. This expansion to bacterial systems is especially interesting given the unique features of bacterial compared to eukaryotic genomes, including a small (10-15%) noncoding fraction of the genome and gene organization in operons. The work will be of interest to readers in the fields of complex trait genetics, gene expression, and regulatory variation. For context of this assessment, I am an expert in genomics and the study of genetic variation in gene expression in eukaryotic systems. I have limited knowledge about bacterial genetics and biology, as well as of antibiotic resistance.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      Damaris et al. perform what is effectively an eQTL analysis on microbial pangenomes of E. coli and P. aeruginosa. Specifically, they leverage a large dataset of paired DNA/RNA-seq information for hundreds of strains of these microbes to establish correlations between genetic variants and changes in gene expression. Ultimately, their claim is that this approach identifies non-coding variants that affect expression of genes in a predictable manner and explain differences in phenotypes. They attempt to reinforce these claims through use of a widely regarded promoter calculator to quantify promoter effects, as well as some validation studies in living cells. Lastly, they show that these non-coding variations can explain some cases of antibiotic resistance in these microbes.

      Major comments

      Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

      The authors convincingly demonstrate that they can identify non-coding variation in pangenomes of bacteria and associate these with phenotypes of interest. What is unclear is the extent by which they account for covariation of genetic variation? Are the SNPs they implicate truly responsible for the changes in expression they observe? Or are they merely genetically linked to the true causal variants. This has been solved by other GWAS studies but isn't discussed as far as I can tell here.

      They need to justify why they consider the 30bp downstream of the start codon as non-coding. While this region certainly has regulatory impact, it is also definitely coding. To what extent could this confound results and how many significant associations to expression are in this region vs upstream?

      The claim that promoter variation correlates with changes in measured gene expression is not convincingly demonstrated (although, yes, very intuitive). Figure 3 is a convoluted way of demonstrating that predicted transcription rates correlate with measured gene expression. For each variant, can you do the basic analysis of just comparing differences in promoter calculator predictions and actual gene expression? I.e. correlation between (promoter activity variant X)-(promoter activity variant Y) vs (measured gene expression variant X)-(measured gene expression variant Y). You'll probably have to

      Figure 7 it is unclear what this experiment was. How were they tested? Did you generate the data themselves? Did you do RNA-seq (which is what is described in the methods) or just test and compare known genomic data?

      Are the data and the methods presented in such a way that they can be reproduced?

      No, this is the biggest flaw of the work. The RNA-Seq experiment to start this project is not described at all as well as other key experiments. Descriptions of methods in the text are far too vague to understand the approach or rationale at many points in the text. The scripts are available on github but there is no description of what they correspond to outside of the file names and none of the data files are found to replicate the plots.

      Figure 8B is intended to show that the WaaQ operon is connected to known Abx resistance genes but uses the STRING method. This requires a list of genes but how did they build this list? Why look at these known ABx genes in particular? STRING does not really show evidence, these need to be substantiated or at least need to justify why this analysis was performed.

      Are the experiments adequately replicated and statistical analysis adequate?

      An important claim on MIC of variants for supplementary table 8 has no raw data and no clear replicates available. Only figure 6, the in vitro testing of variant expression, mentions any replicates.

      Minor comments

      Specific experimental issues that are easily addressable.. Are prior studies referenced appropriately?

      There should be a discussion of eQTLs in this. Although these have mostly been in eukaryotes a. https://doi.org/10.1038/s41588-024-01769-9 ; https://doi.org/10.1038/nrg3891

      Line 67. Missing important citation for Ireland et al. 2020 https://doi.org/10.7554/eLife.55308 Line 69. Should mention Johns et al. 2018 (https://doi.org/10.1038/nmeth.4633) where they study promoter sequences outside of E. coli Line 90 - replace 'hypothesis-free' with unbiased Line 102 - state % of DEGs relative to the entire pan-genome Figure 1A is not discussed in the text Line 111: it is unclear what enrichment was being compared between, FIgures 1C/D have 'Gene counts' but is of the total DEGs? How is the p-value derived? Comparing and what statistical test was performed? Comparing DEG enrichment vs the pangenome? K12 genome? Line 122-123: State what letters correspond to these COG categories here Line 155: Need to clarify how you use k-mers in this and how they are different than SNPs. are you looking at k-mer content of these regions? K-mers up to hexamers or what? How are these compared. You can't just say we used k-mers. Line 172: It would be VERY helpful to have a supplementary figure describing these types of variants, perhaps a multiple-sequence alignment containing each example Figure 4: THis figure is too small. Why are WaaQ and UlaE being used as examples here when you are supposed to be explicitly showing variants with strong positive correlations? Figure 4: Why is there variation between variants present and variant absent? Is this due to other changes in the variant? Should mention this in the text somewhere Line 359: Need to talk about each supplementary figure 4 to 9 and how they demonstrate your point.

      Are the text and figures clear and accurate? Figure 4 too small Acronyms are defined multiple times in the manuscript, sometimes not the first time they are used (e.g. SNP, InDel) Figure 8A - Remove red box, increase label size Figure 8B - Low resolution, grey text is unreadable and should be darker and higher resolution Line 35 - be more specific about types of carbon metabolism and catabolite repression Line 67 - include citation for ireland et al. 2020 https://doi.org/10.7554/eLife.55308 Line 74 - You talk about looking in cis but don't specify how mar away cis is Line 75 - we encoded genetic variants..... It is unclear what you mean here Line 104 - 'were apart of operons' should clarify you mean polycistronic or multi-gene operons. Single genes may be considered operonic units as well. Figure 2: THere is no axis for the percents and the percents don't make sense relative to the bars they represent?? Figure 2: Figure 2B legend should clarify that these are individual examples of Differential expression between variants Line 198-199: This sentence doesn't make sense, 'encoded using kmers' is not descriptive enough Line 205: Should be upfront about that you're using the Promoter Calculator that models biophysical properties of promoter sequences to predict activity. Line 251: 'Scanned the non-coding sequences of the DEGs'. This is far too vague of a description of an approach. Need to clarify how you did this and I didn't see in the method. Is this an HMM? Perfect sequence match to consensus sequence? Some type of alignment? Line 257-259: This sentence lacks clarity Line346: How were the E. coli isolates tested? Was this an experiment you did? This is a massive undertaking (1600 isolates * 12 conditions) if so so should be clearly defined Figure 6A: The tile plot on the right side is not clearly labeled and it is unclear what it is showing and how that relates to the bar plots. FIgure 6B: typo in legend 'Downreglation' Line 398: Need to state rationale for why Waaq operon is being investigated here. WHy did you look into individual example? Figure 8: Can get rid of red box Line 463 - 'account for all kinds' is too informal Mix of font styles throughout document

      Significance

      Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested. The following aspects are important:General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

      This study applies eQTL concepts to bacterial pangenomes to understand how genetic variation in non-coding regions contributes to microbial phenotypes, which is clever and has not been done in bacterial communities (although has been done in yeast isolates, see citation above). They characterize these same variants using in silico promoter predictions, in vitro experiments, layer biological mechanism via transcription factor binding site mapping, and study associated antibiotic resistance phenotypes. These are all good ideas, but none of these points are very developed. The antibiotic work in particular was a missed opportunity as this is the most impactful demonstration of their approach. For instance, to what extent do these eQTLs explain resistance across isolates vs coding changes? Are non-coding variants more responsible for antibiotic resistance than coding variants? Given how easy it is to adapt gene expression vs establishing other mechanisms, this is plausible. How could knowing this change how we treat infections? While a general overview of their strategy is fine, the approaches are under-described and unclear so difficult to truly assess.

      Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

      To my knowledge and from a cursory search, this is the first pan-genome mapping of non-coding variants to transcriptional changes in bacteria. This is a good idea that could be applied to any microbe for which large transcriptomic datasets of strains are available or could be generated and is helpful for understanding genetic variation and the architecture of bacterial regulatory systems.

      Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

      This would be of interest to individuals interested in population genetics, gene regulation, and microbial evolution. It could inspire similar studies of other microbes to understand the contribution of non-coding changes to phenotypes across whole genomes.

      Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

      I am an expert on bacterial gene regulation, especially concerning how promoter activity is encoded within sequences. I have less experience on using GWAS.

    1. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This study aimed to determine whether bacterial translation inhibitors affect mitochondria through the same mechanisms. Using mitoribosome profiling, the authors found that most antibiotics, except telithromycin, act similarly in both systems. These insights could help in the development of antibiotics with reduced mitochondrial toxicity.

      They also identified potential novel mitochondrial translation events, proposing new initiation sites for MT-ND1 and MT-ND5. These insights not only challenge existing annotations but also open new avenues for research on mitochondrial function.

      Strengths:

      Ribosome profiling is a state-of-the-art method for monitoring the translatome at very high resolution. Using mitoribosome profiling, the authors convincingly demonstrate that most of the analyzed antibiotics act in the same way on both bacterial and mitochondrial ribosomes, except for telithromycin. Additionally, the authors report possible alternative translation events, raising new questions about the mechanisms behind mitochondrial initiation and start codon recognition in mammals.

      Weaknesses:

      The main weaknesses of this study are:

      While the authors highlight an interesting difference in the inhibitory mechanism of telithromycin on bacterial and mitochondrial ribosomes, mechanistic explanations or hypotheses are lacking.

      We acknowledge that we were not able to present a clear explanation for potential mechanistic differences of telithromycin inhibition between mitochondrial and bacterial ribosomes. In future work, structural analyses in collaboration with experts will provide these insights.

      The assignment of alternative start codons in MT-ND1 and MT-ND5 is very interesting but does not seem to fully align with structural data.

      We appreciate the reviewer’s comment and consulted a cryo-EM expert to review our findings in the context of the available structural data. We downloaded the density map and reviewed the N-termini of MT-ND1 and MT-ND5. We only observed the density of the N-terminus of MT-ND1 at low confidence. At an RMSD of 2, we could not observe density for the side chains of Met and Pro, and there are gaps in the density for what is modeled as the main chain. The assignment of these residues may have been overlooked due to the expectation that they should be present in the peptide.

      For MT-ND5, we did observe some density that could be part of the main chain; however, it did not fill out until we reduced the stringency, and we did not observe density mapping to side chain residues. To summarize, we do not confidently see density for either the side chain or the main chain for either peptide.

      The newly proposed translation events in the ncRNAs are preliminary and should be further substantiated with additional evidence or interpreted with more caution.

      We agree with the reviewer that we did not provide conclusive evidence that our phased ribosome footprinting data on mitochondrial non-coding RNAs are proof of novel translation events. We do acknowledge this in the main text:” Due to both the short ORFs, minimal read coverage, and lack of a detectable peptide we could not determine if translation elongation occurred on the mitochondrial tRNAs. These sites may be unproductive mitoribosome binding events or simply from tRNAs partially digesting during MNase treatment.”

      Reviewer #2 (Public review):

      In this study, the authors set out to explore how antibiotics known to inhibit bacterial protein synthesis also affect mitoribosomes in HEK cells. They achieved this through mitoribosome profiling, where RNase I and Mnase were used to generate mitoribosome-protected fragments, followed by sequencing to map the regions where translation arrest occurs. This profiling identified the codon-specific impact of antibiotics on mitochondrial translation.

      The study finds that most antibiotics tested inhibit mitochondrial translation similarly to their bacterial counterparts, except telithromycin, which exhibited distinct stalling patterns. Specifically, chloramphenicol and linezolid selectively inhibited translation when certain amino acids were in the penultimate position of the nascent peptide, which aligns with their known bacterial mechanism. Telithromycin stalls translation at an R/K-X-R/K motif in bacteria, and the study demonstrated a preference for arresting at an R/K/A-X-K motif in mitochondria. Additionally, alternative translation initiation sites were identified in MT-ND1 and MT-ND5, with non-canonical start codons. Overall, the paper presents a comprehensive analysis of antibiotics in the context of mitochondrial translation toxicity, and the identification of alternative translation initiation sites will provide valuable insights for researchers in the mitochondrial translation field.

      From my perspective as a structural biologist working on the human mitoribosome, I appreciate the use of mitoribosome profiling to explore off-target antibiotic effects and the discovery of alternative mitochondrial translation initiation sites. However, the description is somewhat limited by a focus on this single methodology. The authors could strengthen their discussion by incorporating structural approaches, which have contributed significantly to the field. For example, antibiotics such as paromomycin and linezolid have been modeled in the human mitoribosome (PMID: 25838379), while streptomycin has been resolved (10.7554/eLife.77460), and erythromycin was previously discussed (PMID: 24675956). The reason we can now describe off-target effects more meaningfully is due to the availability of fully modified human mitoribosome structures, including mitochondria-specific modifications and their roles in stabilizing the decoding center and binding ligands, mRNA, and tRNAs (10.1038/s41467-024-48163-x).

      These and other relevant studies should be acknowledged throughout the paper to provide additional context.

      We appreciate the work that has previously revealed how different antibiotics bind the mitochondrial ribosome. We have included these references in the manuscript to provide background and context for this work in relationship to the field.

      Reviewer #3 (Public review):

      Summary:

      Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provides another application of the technique to further study on mitochondrial translation.

      Strengths:

      This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells.

      The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and written well.

      Weaknesses:

      This is a novel and interesting study, however, most of the conclusion comes from mitoribosome profiling analysis, as a result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

      We thank the reviewer for the positive assessment of our work. We agree that future biochemical and structural experiments will strengthen the conclusions we derive from the ribosome profiling.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      In Fig. 1A, the quality of the Western blot for the sucrose gradient is suboptimal. I recommend enhancing the quality of the Western blot image and providing the sucrose gradient sedimentation patterns for both the mtSSU and mtLSU to confirm the accurate selection of the monosome fraction. Additionally, to correctly assign the A260 peaks to mitochondrial and cytosolic ribosomes, it would be helpful to include markers for both the cytoribosomal LSU and SSU, too. Furthermore, do the authors observe mitochondrial polysomes in their sucrose gradient? If so, were those fractions fully excluded from the analysis?

      We repeated our sucrose gradient and Western blotting with antibodies for the large and small subunits of the mitoribosome. We did not repeat western blotting for the cytoribosomes as the 40S, 60S, and 80S peaks are present in their canonical heights and locations on a sucrose gradient. Western blotting indicates that the large and small subunits of the mitoribosome are located in the fraction taken for mitoribo-seq. We do see trace amounts of mitoribosome in fractions past the 55S site. Those fractions were excluded from library preparation.

      The MNase footprints exhibited a bimodal distribution, which the authors suggest may indicate that "MNase-treatment may have captured two distinct conformations of the ribosome." It would be relevant to clarify whether an enzyme titration was performed, as excessive MNase could lead to ribosomal RNA degradation, potentially influencing the footprints.

      We did not perform a titration and instead based our concentration on the protocol from Rooijers et al, 2013. We included a statement of this and a reference to the concentration in the methods.

      Is there an explanation for why RNase I footprinting reveals a very high peak at the 5'-end of the MT-CYB transcript, whereas this is not observed with MNase footprinting?

      It is not clear. The intensity of peaks at the 5’ end of the transcripts varies. We do observe that the relative intensity of the 5’ peak is greater for RNase I footprinted samples than MNase-treated samples.

      I understand that throughout the manuscript, the authors use MT-CYB as an example to illustrate the effects of the antibiotics on mitochondrial translation. However, to strengthen the generality of the conclusions, it would be beneficial to provide the read distribution across the entire mitochondrial transcriptome, possibly in the supplementary material. Additionally, I suggest including the read distribution for MT-CYB in untreated cells to improve data interpretation and enhance the clarity of figures (e.g., Figs. 1B, 2B, 3B).

      As these experiments were generated across multiple mitoribo-seq experiments, each was done with its own control experiment. It would be inaccurate to show a single trace as representative of all experiments. Instead, we include Supplementary Figure 1, which shows the untreated MT-CYB trace for all control samples and indicates which treatment they pair with.

      It would be very valuable to label each individual data point in the read phasing shown in Fig. 1D with the corresponding transcripts. For improved data visualization, I suggest assigning distinct colors to each transcript.

      We are concerned that including the name of each gene in the main figure would be too difficult for the reader to accurately interpret. Instead, we have added a Supplementary Table with those values.

      How do the authors explain the significant peak (approx. 10,000 reads) at the 5' end of the transcript in the presence of tiamulin (Fig. 2B)? Does this peak correspond to the start codon, and how does it relate to the quantification reported in Fig. 2C?

      Yes, this represents the start codon. These reads are likely derived from the start codon as they are mapping to the 5’ end of the transcript. There are differences in sequencing depth depending on the experiment, so what is critical is the relative distribution of reads on the transcript rather than comparing absolute reads between experiments. MT-CYB has 54% of the reads at the start site, which is representative of what we see across all genes.

      Throughout the manuscript, I found the usage of the terms "5' end" and "start codon" somewhat confusing, as they appear to be used synonymously in some instances. For example, in Fig. 2C, the y-axis label states "ribosomes at start codon," while the figure caption mentions "...percentage of reads that map to the 5' end of mitochondrial transcripts." Given the size of the graphs, it is also challenging for the reader to determine whether the peaks correspond specifically to the start codon or if multiple peaks accumulate at the initial codons.

      We were selected for this language because two different types of analysis are being carried out. Ribosome profiling carried out in Figures 2 and 3 is carried out with RNase I, which poorly maps the ribosomes at the start codon when we do the read length analysis in Figure 4. Ribosome footprint at the 5’ end may include ribosomes that are on the 2-4 codons following the start codon, so it would not be accurate to label those as “ribosomes at a start codon.” We have renamed the axis to “Ribosomes at 5’ end”. Wig files are available online for all mitoribosome profiling experiments. In this case, the assigned “P-site” is several codons after the start codon due to the offset applied and the minimal 5’ UTR. Thus, it is less important to see which codon density is assigned to, but rather the general distribution of the reads.

      The authors state, "Cells treated with telithromycin did show a slight increase in MRPF abundance at the 5' end of MT-CYB" and "the cumulative distribution of MRPFs suggested that ribosome density was biased towards the 5' end of the gene for chloramphenicol and telithromycin, but not significantly for linezolid." Could this observation be linked to the presence of specific stalling motifs in that region? If so, it would be beneficial to display such motifs on the graphs of the read distribution across the transcriptome to substantiate the context-dependent inhibition.

      Thank you for this suggestion. For chloramphenicol and linezolid, alanine, serine, and threonine make up nearly 25% of the mitochondrial proteome. As such, there are numerous stall sites across the transcript. Given their identical stalling motifs, the difference between chloramphenicol and linezolid is due to sequence-specific differences. Potentially, this could be due to conditions such as the final concentration of antibiotic inside the mitochondria and the on/off rate of an inhibitor with the translating mitoribosome. Both may affect the kinetics of stalling and allow mitoribosomes to evade early stall sites.

      We have also included the sites of all A/K/R-X-K motifs located in the genome and the calculated fold change for each position. As a note, this includes sites that do not pass the minimum filter set by our analysis and we note this in the text.

      The comment raises an additional question: Does the increased density at the 5’ end derive from stalled mitoribosomes or queued mitoribosomes behind a stalling event? Recent work by Iwasaki’s group shows that mitoribosomes can form disomes and queue behind each other. However, we could not observe 30 aa periodicities behind stalling events that would be indicative of collided mitoribosomes.

      In Fig. 3E, the authors report an additional and very interesting observation that is not discussed. Linezolid treatment causes reduced ribosome occupancy when proline or glycine codons occupy the P-site, or when the amino acids have been incorporated into the polypeptide chain and occupy the -1 position. It is known that the translation of proline and glycine frequently leads to ribosome stalling due to the physicochemical properties of these amino acids. Has this effect of linezolid been reported in the bacterial translation system? Additionally, can the authors propose hypotheses for the mechanism behind this observation? A similar observation is noted for telithromycin when glycine occupies the same positions, as well as when aspartate occupies the P- and A-sites.

      In bacteria, Linezolid does have an “anti-stalling” motif when glycine is present in the A-site. However, this is due to the size of the residue being compatible with antibiotic binding.

      The most likely cause of this effect is a redistribution of ribosome footprints. As the antibiotics introduce new arrest sites, ribosome density at other sites relatively decreases. This is likely an artifact from mitoribosomes redistributing from endogenously slow codons to new arrest sites. When looking at carrying out our disome profiling in the presence of anisomycin, we see a similar effect. Cytoribosomes are redistributed from endogenous stalling sites, such as proline, and are redistributed throughout the gene. As a result, translation at proline appears “more efficient” upon treatment with an inhibitor but is instead an artifact of analysis.

      Figure 3F could benefit from indicating which mtDNA-encoded protein corresponds to each of the strongest stalling motifs.

      We have included a supplementary figure to highlight which mitochondrially-encoded genes containing the R/K/A-X-K motif and noted in the text that mitochondrial translation may be unevenly inhibited.

      The legend "increasing mRPF abundance" in Fig. 4C may be missing the corresponding colors.

      The legend applies to all sections of the figure. We double-checked the range of the colors in the tables, and they do match the legend.

      The observation that the start codons in MT-ND1 and MT-ND5 might differ from the annotated canonical ones is intriguing. While the ribosome profiling data appear clear, mass spectrometry (MS) analysis may be misleading. The absence of evidence does not necessarily imply evidence of absence. How does this proposed conclusion correlate with the structural data obtained from HEK cells? For instance, the cryo-EM structural model of a complex I-containing human supercomplex (PDB: 5XTD, PMID: 28844695) shows the presence of Pro2 in MT-ND1 and the full-length MT-ND5 protein. The authors should carefully examine structural data to ascertain whether alternative forms of MT-ND1 and MT-ND5 are actually observed in the assembled complex I.

      We really appreciate this comment. We sat down with an expert in cryo-EM and reviewed the figure. We downloaded the density map and reviewed the N-termini of MT-ND1 and MT-ND5. We only observed the density of the N-terminus of MT-ND1 at low confidence. At an RMSD of 2, we could not observe density for the side chains of Met and Pro, and there are gaps in the density for what is modeled as the main chain. The assignment of these residues may have been overlooked due to the expectation that they should be present in the peptide.

      For MT-ND5, we did observe some density that could be part of the main chain; however, it did not fill out until we reduced the stringency, and we did not observe density mapping to side chain residues. To summarize, we do not confidently see density for either the side chain or the main chain for either peptide.

      Given that ribosome profiling is based on the assumption that ribosomes protect mRNA fragments from RNase digestion, interpreting the data related to Fig. 5 and the proposed existence of translation events involving ncRNAs is challenging. Most importantly, tRNAs and rRNAs are highly folded RNA molecules and, by definition, are protected by ribosomal proteins. Simultaneously, as the authors point out, "These reads could either be products of random digestion of the abundant background of ncRNAs or be genuine MRPFs." RNase I preferentially digests single-stranded RNA (ssRNA), but excess enzyme can still lead to degradation. Consequently, many random tRNA/rRNA fragments may be generated by RNase digestion, potentially resulting in artifacts. I suggest that the authors examine what happens to these reads when mitochondrial translation is inhibited.

      We have low-quality mitoribo-seq with initiation inhibitors and Mnase showing footprints of the same size. We do not have a small-molecule inhibitor that is able to completely ablate translation, as they instead stabilize mitoribosomes at different steps in translation. We have considered alternative ways of capturing a background rRNA and tRNA digestion pattern; however, these have their own drawbacks. Dissociation with EDTA prior to digestion or carrying out library prep on the small and large subunits may capture mitoribosomes no longer in the process of translation; however, dissociated subunits would have different surfaces now available for digestion and may not capture tRNAs.

      Regarding the statement, "While the ORF on MT-TS1 is longer, MRPF density was low and we did not observe read phasing and thus it is likely not translated (not shown)," the data should not be excluded unless a clear explanation is provided for why translation would not occur from this specific RNA.

      We have included this value in the graph as well as in Supplementary Figure 1.

      The graph in Fig. 5B shows the periodicity of only the putative RNR1 ORF, but not that of the other proposed ORFs. What is the reason for this?

      We have included the MT-TS1 putative ORF in Figure 5 and Figure S1. Other ORFs did not have density in the ORF. If these are real mitoribosome footprints at these start codons, it may be due to them being transient binding events that never result in elongation. Alternatively, they may be due to tRNA degradation during library preparation.

      The assumption that the UUG codon can serve as a start site for mitochondrial translation has not been substantiated. Recent data have identified translation initiation events from non-ATG/ATA codons (near-cognate and sub-cognate) using retapamulin, but UUG was not among them. Can the authors detect such events in their ribosome profiling data collected in the presence of retapamulin, tiamulin, or josamycin?

      The report of translation initiation at non-ATG/ATA codons strongly disagrees with our findings. We report that sites of translation initiation observed within annotated coding regions in mitochondria occur at the annotated start sites, while the other report finds frequent alternative initiation events. We have looked for those arrest sites and did not observe them.

      In the section "Mitoribosome profiling reveals novel translation events," the title may be misleading given the preliminary nature of the results. To support such a claim, the authors should provide experimental evidence demonstrating that the proposed translation events genuinely exist and result in the synthesis of previously unidentified polypeptides. Alternatively, the interpretation should be approached with greater caution and more clearly indicated as preliminary.

      We agree with the reviewers that a distinction should be made between reporting truly novel translation events, like the recently reported MT-ND5-dORF, and sites we suspect mitoribosomes may be binding and that require detailed follow-up. We altered the section title to suggest that this may be showing novel translation events. Additionally, we included a statement in the discussion that these MRPFs may be simply tRNA digestion by RNase I.

      Although located at the 5' end of RNR1, the newly identified ORF is situated 79 nt downstream. According to current knowledge, this appears to be a lengthened 5' UTR that may hinder mitoribosome loading. The authors should speculate on potential initiation mechanisms.

      The start of the putative ORF is not located 79 nts down, but at the 8<sup>th</sup> nucleotide. The reviewer may be including the tRNA-Phe in their calculation, which is cleaved from MT-RNR1. This start site is closer to the 5’ end than our findings with MT-ND5.

      To enhance the interpretation of the mitoribosome profiling data, the authors could complement their findings with classical metabolic labeling using (35)S-methionine. This approach would allow for a different assessment of the stringency of the inhibition under the tested experimental conditions.

      We are currently working on these experiments using mito-funcats. A future direction we are taking this work is to understand how the cell responds to different mechanisms of translation inhibition. For example, we are trying to understand if telithromycin, which appears highly selective, only partially inhibits translation of the mtDNA-encoded proteome.

      Reviewer #2 (Recommendations for the authors):

      Other small editorial comments:

      Line 24: "translate proteins"?

      Revised for clarity

      Line 24: The sentence describing mitochondrial translation as "closely resembling the one in prokaryotes" could be reformulated. While the core of the mitoribosome is conserved, the entire apparatus has many mitochondria-specific features.

      Since this is the abstract, we simplified the point by saying that mitoribosomes are more similar to prokaryotic than cytosolic ribosomes.

      Clarified to highlight that the mitochondrial system is more similar to the bacterial system than the eukaryotic system.

      Line 33: "novel" or "previously unrecognized" ?

      Rewritten for clarity.

      Lines 33-35: The claim made here is not shown in the paper.

      We removed the more aspirational goal of this paper and focused on the main findings of the paper.

      Lines 44, 47, 89 (and elsewhere): "cytoplasmic" or "cytosolic" ?

      Both terms are used in the literature. We opted for cytoplasmic as it can also include ribosomes not free in the cytosol, such as those bound to the ER.

      Reviewer #3 (Recommendations for the authors):

      (1) The authors should state why they chose these antibiotics for mitoribosome profiling analysis over other antibiotics from same group. Did they screen multiple antibiotics to determine the candidates for next step?

      We selected antibiotics that had a known stalling motif in bacteria (initiation or context-dependent elongation inhibitors). In addition, we carried out mitoribosome profiling with erythromycin, azithromycin, thiostrepton, and kanamycin in this work. However, we did not see any effect from these drugs in mitoribosome profiling. We are currently testing other inhibitors, such as doxycycline and tigecycline, and looking at optimizing treatment conditions to identify stalling motifs in samples that previously showed no difference.

      (2) What is the reason for choosing the concentration of antibiotics retapamulin, tiamulin and josamycin, this is IC50 value or above this value? On the other hand, none of this information has been provided for the antibiotics in the next part. The authors should provide biochemical study for the effect of these antibiotics on cell survival and/or protein translation such as S35 assay or steady state level of mtDNA-encoded proteins upon cell treatment with these antibiotics.

      Prior to mitoribo-seq, we carried out time and concentration assays with all antibiotics. 100 µg/ml and a 30-minute treatment was tolerable for all antibiotics except retapamulin. We aimed to treat cells with a relatively high concentration of inhibitor in order to capture actively translating mitoribosomes. We were concerned that longer treatments may lead to decreased translation initiation, leading to the capture of fewer mitoribosomes. These concentrations were nearly identical to contemporary conditions carried out in Bibel et al, RNA 2025.

      (3) Why did the authors choose MT-CYB as the representative for further analysis in the second and third parts of the manuscript?

      We chose MT-CYB because its length allowed for easy visualization. Some mitochondrial genes, such as MT-ND6, had a propensity for stronger stalling at initiation. While coverage was throughout the genes, it was difficult to visualize the changes within the ORF. Also, MT-CYB was less visually complex than polycistronic transcripts. All wigs were uploaded to GEO.

      (4) Page 11, line 233-234: the authors state that telithromycin induces stalling at R/K/A-X-K motif. The authors should do further analysis on mitochondrial genome which proteins contain this motif. Furthermore, same as comment 2: the authors should confirm by 35S assay or WB to know which mtDNA-encoded proteins are affected.

      We have included a supplementary figure showing which mitochondrial genes contain these motifs.

      (5) The results and conclusion from the fourth paragraph are very interesting. The authors suggest alternative start codon for two mtDNA encoded proteins: ND1 and ND5 based on ribosome profiling analysis. Again, I have several comments on this part: <br /> (a) For the accumulation of the alternative start codon of ND1 and ND5 as suggested in the manuscript, do the authors observe this trend with the initiation inhibitors used in the second paragraphs of the manuscript?

      We did not observe similar read lengths with retapamulin, tiamulin, or josamycin, which produced read lengths that were consistent with other RNase I footprinted samples.

      (b) This observation was further confirmed by MS with a peptide form ND1 protein, the authors should show MS peak indicating MW of the peptide and MS/MS data for the peptide which supports this hypothesis.

      We are including the MS/MS report for this peptide.

      (c) Interestingly, several high-resolution structures of mammalian complex I have been reported so far (PMID: 7614227, 10396290, 38870289), ND1 and ND5 protein express full sequences with fMet at the distal N-terminal. This is different to the suggestion from the manuscript. Could the author discuss or comment on that?

      This point was brought up by another reviewer. We have carefully analyzed the density map of PMID: 28844695. We sat down with an expert in cryo-EM and reviewed the figure. We downloaded the density map and reviewed the N-termini of MT-ND1 and MT-ND5. We only observed the density of the N-terminus of MT-ND1 at low confidence. At an RMSD of 2, we could not observe density for the sidechains of Met and Pro, and there is a gap in density for what is modeled as the main chain. The assignment of these residues may have been overlooked due to the expectation that they should be present in the peptide.

      For MT-ND5, we did observe some density that could be part of the main chain; however, it did not fill out until we reduced the stringency, and we did not observe density mapping to side chain residues. To summarize, we do not confidently see density for either the side chain or the main chain for either peptide.

      Minor comments:

      The method should be written more accurately for easily repeating experiments by other groups. For example:

      (1) The authors should indicate what was exact HEK293 cell line used in this study.

      We have indicated the exact cell line.

      (2) Page 22, line 471: which (number) fractions had been collected. The Western Blot analysis shown in Figure 1A should be repeated with both proteins from small and large subunits.

      We have repeated the Western blot with antibodies for large and small subunits. We took fractions 8 and 9, which are now indicated in the text and figure.

      (3) Page 23, line 502: is this number of cells used for MS experiment is correct? Or is this number of cells per mL?

      This is correct and is based on the kit protocol. It is not cells per mL. We have clarified the kit being used in the methods.

    1. Author response:

      The following is the authors’ response to the original reviews.

      eLife Assessment

      This work provides an important resource identifying 72 proteins as novel candidates for plasma membrane and/or cell wall damage repair in budding yeast, and describes the temporal coordination of exocytosis and endocytosis during the repair process. The data are convincing; however, additional experimental validation will better support the claim that repair proteins shuttle between the bud tip and the damage site.

      We thank the editors and reviewers for their positive assessment of our work and the constructive feedback to improve our manuscript. We agree with the assessment that additional validation of repair protein shuttling between the bud tip and the damage site is required to further support the model.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      In this manuscript, Yamazaki et al. conducted multiple microscopy-based GFP localization screens, from which they identified proteins that are associated with PM/cell wall damage stress response. Specifically, the authors identified that budlocalized TMD-containing proteins and endocytotic proteins are associated with PM damage stress. The authors further demonstrated that polarized exocytosis and CME are temporally coupled in response to PM damage, and CME is required for polarized exocytosis and the targeting of TMD-containing proteins to the damage site. From these results, the authors proposed a model that CME delivers TMD-containing repair proteins between the bud tip and the damage site.

      Strengths:

      Overall, this is a well-written manuscript, and the experiments are well-conducted. The authors identified many repair proteins and revealed the temporal coordination of different categories of repair proteins. Furthermore, the authors demonstrated that CME is required for targeting of repair proteins to the damage site, as well as cellular survival in response to stress related to PM/cell wall damage. Although the roles of CME and bud-localized proteins in damage repair are not completely new to the field, this work does have conceptual advances by identifying novel repair proteins and proposing the intriguing model that the repairing cargoes are shuttled between the bud tip and the damaged site through coupled exocytosis and endocytosis.

      Weaknesses:

      While the results presented in this manuscript are convincing, they might not be sufficient to support some of the authors' claims. Especially in the last two result sessions, the authors claimed CME delivers TMD-containing repair proteins from the bud tip to the damage site. The model is no doubt highly possible based on the data, but caveats still exist. For example, the repair proteins might not be transported from one localization to another localization, but are degraded and resynthesized. Although the Gal-induced expression system can further support the model to some extent, I think more direct verification (such as FLIP or photo-convertible fluorescence tags to distinguish between pre-existing and newly synthesized proteins) would significantly improve the strength of evidence.

      Major experiment suggestions:

      (1) The authors may want to provide more direct evidence for "protein shuttling" and for excluding the possibility that proteins at the bud are degraded and synthesized de novo near the damage site. For example, if the authors could use FLIP to bleach budlocalized fluorescent proteins, and the damaged site does not show fluorescent proteins upon laser damage, this will strongly support the authors' model. Alternatively, the authors could use photo-convertible tags (e.g., Dendra) to differentiate between preexisting repair proteins and newly synthesized proteins.

      We thank the reviewer for evaluating our work and giving us important feedback. We agree that the FLIP and photo-convertible experiments will further confirm our model. Here, due to time and resource constraints, we decided not to perform this experiment. Instead, we have discussed this limitation in 363-366. Our proposed model of repair protein shuttling should be further tested in our future work.

      (2) In line with point 1, the authors used Gal-inducible expression, which supported their model. However, the author may need to show protein abundance in galactose, glucose, and upon PM damage. Western blot would be ideal to show the level of fulllength proteins, or whole-cell fluorescence quantification can also roughly indicate the protein abundance. Otherwise, we cannot assume that the tagged proteins are only expressed when they are growing in galactose-containing media.

      Thank you very much for raising the concern and suggesting the important experiments.We agree that the Western blot experiment to confirm the mNG-Snc1 expression in each medium will further strengthen our conclusion. Along with point (1), further investigation of repair protein shuttling between the bud tip and the damage site and the mechanisms underlying it will be an important future direction. As described above, we have discussed this limitation in 363-366.

      (3) Similarly, for Myo2 and Exo70 localization in CME mutants (Figure 4), it might be worth doing a western or whole-cell fluorescence quantification to exclude the caveat that CME deficiency might affect protein abundance or synthesis.

      We thank the reviewer for suggesting the point. Following the reviewer’s suggestion, we quantified the whole-cell fluorescence of WT and CME mutants and verified that the effect of the CME deletion on the expression levels of Myo2-sfGFP and Exo70-mNG is minimal ( Figure S6). We added the description in lines 211-212.

      (4) From the authors' model in Figure 7, it looks like the repair proteins contribute to bud growth. Does laser damage to the mother cell prevent bud growth due to the reduction of TMD-containing repair proteins at the bud? If the authors could provide evidence for that, it would further support the model.

      Thank you very much for raising the important point. We speculate that the reduction of TMD-containing proteins at the bud by CME is one of the causes of cell growth arrest after PM damage (1). This is because TMD-containing repair proteins at the bud tip, including phospholipid flippases (Dnf1/Dnf2), Snc1, and Dfg5, are involved in polarized cell growth (2-4). This will be an important future direction as well.

      (5) Is the PM repair cell-cycle-dependent? For example, would the recruitment of repair proteins to the damage site be impaired when the cells are under alpha-factor arrest?

      Thank you for raising this interesting point. Indeed, the senior author Kono previously performed this experiment when she was in David Pellman’s lab. The preliminary results suggest that Pkc1 can be targeted to the damage site, without any impairment, under alpha-factor arrest. A more comprehensive analysis in the future will contribute to concluding the relation between PM repair and the cell cycle.

      Reviewer #2 (Public review):

      This paper remarkably reveals the identification of plasma membrane repair proteins, revealing spatiotemporal cellular responses to plasma membrane damage. The study highlights a combination of sodium dodecyl sulfate (SDS) and lase for identifying and characterizing proteins involved in plasma membrane (PM) repair in Saccharomyces cerevisiae. From 80 PM, repair proteins that were identified, 72 of them were novel proteins. The use of both proteomic and microscopy approaches provided a spatiotemporal coordination of exocytosis and clathrin-mediated endocytosis (CME) during repair. Interestingly, the authors were able to demonstrate that exocytosis dominates early and CME later, with CME also playing an essential role in trafficking transmembrane-domain (TMD)containing repair proteins between the bud tip and the damage site.

      Weaknesses/limitations:

      (1) Why are the authors saying that Pkc1 is the best characterized repair protein? What is the evidence?

      We would like to thank the reviewer for taking his/her time to evaluate our work and for valuable suggestions. We described Pkc1 as “best characterized” because it was the first protein reported to accumulate at the laser damage site in budding yeast (5). However, as the reviewer suggested, we do not have enough evidence to describe Pkc1 as “best characterized”. We therefore used “one of the known repair proteins” to mention Pkc1 in the manuscript (lines 90-91).

      (2) It is unclear why the authors decided on the C-terminal GFP-tagged library to continue with the laser damage assay, exclusively the C-terminal GFP-tagged library. Potentially, this could have missed N-terminal tag-dependent localizations and functions and may have excluded functionally important repair proteins

      Thank you very much for the comments. We decided to use the C-terminal GFP-tagged library for the laser damage assay because we intended to evaluate the proteins of endogenous expression levels. The N-terminal sfGFP-tagged library is expressed by the NOP1 promoter, while the C-terminal GFP-tagged library is expressed by the endogenous promoters. We clarified these points in lines 114-118. We agree with the reviewer on that we may have missed some portion of repair proteins in the N-terminaldependent localization and functions by this approach. Therefore, in our manuscript, we discussed these limitations in lines 281-289.

      (3) The use of SDS and laser damage may bias toward proteins responsive to these specific stresses, potentially missing proteins involved in other forms of plasma membrane injuries, such as mechanical, osmotic, etc.). SDS stress is known to indirectly induce oxidative stress and heat-shock responses.

      Thank you very much for raising this point. We agree that the combination of SDS and laser may be biased to identify PM repair proteins. Therefore, in the manuscript, we discussed this point as a limitation of this work in lines 292-298.

      (4) It is unclear what the scale bars of Figures 3, 5, and 6 are. These should be included in the figure legend.

      We apologize for the missing scale bars. We added them to the legends of the figures in the manuscript.

      (5) Figure 4 should be organized to compare WT vs. mutant, which would emphasize the magnitude of impairment.

      Thank you for raising this point. Following the suggestion, we updated Figure 4. In the Figure 4, we compared WT vs mutant in the manuscript. We clarified it in the legends in the manuscript. 

      (6) It would be interesting to expand on possible mechanisms for CME-mediated sorting and retargeting of TMD proteins, including a speculative model.

      Thank you very much for this important suggestion. We think it will be very important to characterize the mechanism of CME-mediated TMD protein trafficking between the bud tip and the damage site. In the manuscript, we discussed the possible mechanism for CME activation at the damage site in lines 328-333. We speculate that the activation of the CME may facilitate the retargeting of the TMD proteins from the damage site to the bud tip.

      We do not have a model of how CMEs activate at the bud tip to sort and target the TMD proteins to the damage site. One possibility is that the cell cycle arrest after PM damage (1) may affect the localization of CME proteins because the cell cycle affects the localization of some of the CME proteins (6). We will work on the mechanism of repair protein sorting from the bud tip to the damage site in our future work.

      Reviewer #3 (Public review):

      Summary:

      This work aims to understand how cells repair damage to the plasma membrane (PM). This is important, as failure to do so will result in cell lysis and death. Therefore, this is an important fundamental question with broad implications for all eukaryotic cells. Despite this importance, there are relatively few proteins known to contribute to this repair process. This study expands the number of experimentally validated PM from 8 to 80. Further, they use precise laser-induced damage of the PM/cell wall and use livecell imaging to track the recruitment of repair proteins to these damage sites. They focus on repair proteins that are involved in either exocytosis or clathrin-mediated endocytosis (CME) to understand how these membrane remodeling processes contribute to PM repair. Through these experiments, they find that while exocytosis and CME both occur at the sites of PM damage, exocytosis predominates in the early stages of repairs, while CME predominates in the later stages of repairs. Lastly, they propose that CME is responsible for diverting repair proteins localized to the growing bud cell to the site of PM damage.

      Strengths:

      The manuscript is very well written, and the experiments presented flow logically. The use of laser-induced damage and live-cell imaging to validate the proteome-wide screen using SDS-induced damage strengthens the role of the identified candidates in PM/cell wall repair.

      Weaknesses:

      (1) Could the authors estimate the fraction of their candidates that are associated with cell wall repair versus plasma membrane repair? Understanding how many of these proteins may be associated with the repair of the cell wall or PM may be useful for thinking about how these results are relevant to systems that do or do not have a cell wall. Perhaps this is already in their GO analysis, but I don't see it mentioned in the manuscript.

      We would like to thank the reviewer for taking his/her time to evaluate our work and valuable suggestions. We agree that this is important information to include. Although it may be difficult to completely distinguish the PM repair and cell wall repair proteins, we have identified at least six proteins involved in cell wall synthesis (Flc1, Dfg5, Smi1, Skg1, Tos7, and Chs3). We included this information in lines 142-146 in the manuscript.

      (2) Do the authors identify actin cable-associated proteins or formin regulators associated with sites of PM damage? Prior work from the senior author (reference 26) shows that the formin Bnr1 relocalizes to sites of PM damage, so it would be interesting if Bnr1 and its regulators (e.g., Bud14, Smy1, etc) are recruited to these sites as well. These may play a role in directing PM repair proteins (see more below).

      Thank you for the suggestion. We identified several Bnr1-interacting proteins, including Bud6, Bil1, and Smy1 (Table S2), although Bnr1 itself was not identified in our screening. This could be attributed to the fact that (1) C-terminal GFP fusion impaired the function of Bnr1, and (2) a single GFP fusion is not sufficient to visualize the weak signal at the damage site. Indeed, in reference 26, 3GFP-Bnr1 (N-terminal 3xGFP fusion) was used.

      (3) Do the authors suspect that actin cables play a role in the relocalization of material from the bud tip to PM damage sites? They mention that TMD proteins are secretory vesicle cargo (lines 134-143) and that Myo2 localizes to damage sites. Together, this suggests a possible role for cable-based transport of repair proteins. While this may be the focus of future work, some additional discussion of the role of cables would strengthen their proposed mechanism (steps 3 and 4 in Figure 7).

      Thank you very much for the suggestion. We agree that actin cables may play a role in the targeting of vesicles and repair proteins to the damage site. Following the reviewer’s suggestion, we discussed the roles of Bnr1 and actin cables for repair protein trafficking in lines 309-313 in the manuscript.

      (4) Lines 248-249: I find the rationale for using an inducible Gal promoter here unclear. Some clarification is needed.

      Thank you for raising this point. We clarified this as possible as we could in lines 249255 in the manuscript.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) The N-terminal GFP collection screen is interesting but seems irrelevant to the rest of the results. The authors discussed that in the discussion part, but it might be worth showing how many hits from the laser damage screen (in Figure 2) overlap with the Nterminal GFP screen hits.

      Thank you for the suggestion. We found that 48 out of 80 repair proteins are hits in the N-terminal GFP library (Table S1 and S2). This result suggested that the N-terminal library is also a useful resource for identifying repair proteins. In the manuscript, we discussed it in lines 288-289.

      (2) SDS treatment seems a harsh stressor. As the authors mentioned, the overlapped hits from the N- and C-terminal GFP screen might be more general stress factors. Thus, I think Line 84 (the subtitle) might be overclaiming, and the authors might need to tone down the sentence.

      Thank you for the suggestion. Following the reviewer’s suggestion, we changed the sentence to “Proteome-scale identification of SDS-responsive proteins” in the manuscript. We believe that the new sentence describes our findings more precisely.

      (3) Line 103-106, it does not seem obvious to me that the protein puncta in the cytoplasm are due to endocytosis. The authors might need to provide more experimental evidence for the conclusion, or at least provide more reasoning/references on that aspect (e.g.,several specific protein hits belonging to that group have been shown to be endocytosed).

      Thank you very much for raising this point. We agree with the reviewer and deleted the description that these puncta are due to endocytosis in the manuscript.

      (4) For Figure 1D and S1C, the authors annotated some of the localization changes clearly, but some are confusing to me. For example," from bud tip/neck" to where? And from where to "Puncta/foci"? A clearer annotation might help the readers to understand the categorization.

      Thank you very much for the suggestion. These annotations were defined because it is difficult to conclusively describe the protein localization after SDS treatment. To convincingly identify the destination of the GFP fusion proteins, the dual color imaging of proteins with organelle markers or deep learning-based localization estimation is required. We feel that this might be out of the scope of this work. Therefore, as criteria, we used the localization of protein localization in normal/non-stressed conditions reported in (7) and the Saccharomyces Genome Database (SGD). We clarified this annotation definition in the manuscript (lines 413-436).

      (5) For localization in Figure 2C, as I understand, does it refer to6 the "before damage/normal" localization? If so, I think it would be helpful to state that these localizations are based on the untreated/normal conditions in the text.

      Yes, it refers to the “before damage/normal localization”. Following the reviewer’s suggestion, we stated that these localizations are based on these conditions in the manuscript (line 130).

      (6) The authors mentioned "four classes" in Line 120, but did not mention the "PM to cytoplasm" class in the text. It would be helpful to discuss/speculate why these transporters might contribute to PM damage repair.

      Thank you very much for this suggestion. We speculated that these transporters are endocytosed after PM damage because endocytosis of PM proteins contributes to cell adaptation to environmental stress (8). We mentioned it in the manuscript (lines 120-122).

      (7) Line 175-180 My understanding of the text is that the signals of Exo70-mNG/Dnf1mNG peak before the Ede1-mSc-I peaks. They occur simultaneously, but their dominating phase are different. It is clearer when looking at the data, but I think the conclusion sentences themselves are confusing to me. The authors might consider rewriting the sentences to make them more straightforward.

      Thank you very much for pointing this out. Following the reviewer’s suggestion, we revised the sentence (lines 177-182 in the manuscript).

      Reviewer #2 (Recommendations for the authors):

      It would be interesting to expand on the functional characterization of the 72 novel candidates and explore possible mechanisms for CME-mediated sorting and retargeting of TMD proteins by including a speculative model.

      Thank you very much for the comment. We agree that the further characterization of novel repair proteins and exploration of the possible mechanisms for CME-mediated TMD protein sorting and retargeting are truly important. This should be our important future direction.

      Reviewer #3 (Recommendations for the authors):

      The x-axis in Figure 1C is labeled 'Ratio' - what is this a ratio of?

      Thank you for raising this point. It is the ratio of the number of proteins associated with a GO term to the total number of proteins in the background. We clarified it in the legend of Figure 1C in the manuscript.

      References

      (1) K. Kono, A. Al-Zain, L. Schroeder, M. Nakanishi, A. E. Ikui, Plasma membrane/cell wall perturbation activates a novel cell cycle checkpoint during G1 in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 113, 6910-6915 (2016).

      (2) A. Das et al., Flippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity. Nat Cell Biol 14, 304-310 (2012).

      (3) M. Adnan et al., SNARE Protein Snc1 Is Essential for Vesicle Trafficking, Membrane Fusion and Protein Secretion in Fungi. Cells 12 (2023).

      (4) H.-U. Mösch, G. R. Fink, Dissection of Filamentous Growth by Transposon Mutagenesis in Saccharomyces cerevisiae. Genetics 145, 671-684 (1997).

      (5) K. Kono, Y. Saeki, S. Yoshida, K. Tanaka, D. Pellman, Proteasomal degradation resolves competition between cell polarization and cellular wound healing. Cell 150, 151-164 (2012).

      (6) A. Litsios et al., Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle. Cell 187, 1490-1507.e1421 (2024).

      (7) W.-K. Huh et al., Global analysis of protein localization in budding yeast.Nature 425, 686-691 (2003).

      (8) T. López-Hernández, V. Haucke, T. Maritzen, Endocytosis in the adaptation to cellular stress. Cell Stress 4, 230-247 (2020).

    1. Synthèse sur les Biais Cognitifs et le Raisonnement Humain

      Résumé

      Ce document de synthèse analyse les concepts clés relatifs aux biais cognitifs, au raisonnement humain et aux stratégies de "débiaisage", en s'appuyant sur l'expertise de Wim De Neys, chercheur au CNRS spécialisé en psychologie du raisonnement.

      Les principaux points à retenir sont les suivants :

      1. Nature des Biais Cognitifs : Loin d'être de simples "défauts de conception", les biais cognitifs sont avant tout des stratégies de pensée rapides et adaptatives (heuristiques) forgées par l'évolution.

      Elles permettent de prendre des décisions efficaces dans un monde complexe, bien qu'elles puissent conduire à des erreurs systématiques et prévisibles dans des contextes spécifiques.

      2. Le Modèle Système 1 / Système 2 : Le raisonnement humain est modélisé par l'interaction de deux systèmes.

      Le Système 1 est intuitif, rapide et automatique, gérant la grande majorité de nos tâches cognitives quotidiennes.

      Le Système 2 est délibéré, lent et coûteux en ressources cognitives, activé pour les tâches complexes.

      L'idée que le Système 1 est intrinsèquement "irrationnel" est une simplification excessive ; il est essentiel et souvent correct.

      3. La Détection des Conflits Cognitifs : Contrairement à l'idée classique selon laquelle les individus sont des "avares cognitifs" aveugles à leurs propres erreurs, les recherches de Wim De Neys démontrent que le cerveau détecte souvent un conflit lorsque la réponse intuitive (Système 1) contredit un principe logique ou probabiliste.

      Ce signal de "doute" se manifeste par des temps de réponse plus longs, une activation de zones cérébrales spécifiques (cortex cingulaire antérieur) et une baisse de la confiance, même lorsque l'individu donne la mauvaise réponse.

      4. L'Inefficacité du Débiaisage Général : Les tentatives de rendre les gens globalement "plus rationnels" en les incitant à activer plus souvent leur Système 2 se heurtent à un obstacle majeur : le problème du transfert.

      Les compétences acquises dans un domaine spécifique ne se généralisent que très difficilement à d'autres contextes.

      5. L'Efficacité de l'Entraînement Intuitif : La stratégie la plus prometteuse pour corriger les biais consiste à entraîner le Système 1 lui-même.

      En expliquant aux individus les principes logiques sous-jacents à une tâche spécifique, on peut modifier leurs intuitions.

      Après un tel entraînement, la première réponse générée devient souvent la bonne, sans nécessiter l'activation coûteuse du Système 2.

      6. Le Rôle de l'Argumentation et de l'IA : Le raisonnement n'est pas seulement une activité individuelle mais aussi une compétence sociale, utilisée pour argumenter et délibérer en groupe.

      Dans ce contexte, de nombreux biais (comme le biais de confirmation) peuvent être surmontés.

      L'intelligence artificielle (IA) émerge comme un outil potentiellement puissant, capable d'agir comme un partenaire de débat neutre et informé pour faciliter le débiaisage individuel, à condition d'être utilisée de manière interactive et critique plutôt que passive.

      1. La Nature Duplice des Biais Cognitifs

      Les biais cognitifs, identifiés depuis un demi-siècle par des psychologues et économistes comportementaux comme Daniel Kahneman et Amos Tversky, désignent les failles systématiques du raisonnement humain.

      Ils incluent des phénomènes tels que le biais d'ancrage, l'effet de cadrage, le biais de confirmation ou l'erreur de conjonction.

      Ces découvertes ont contribué à démanteler le mythe de l'homo economicus, l'agent parfaitement rationnel agissant toujours dans son meilleur intérêt.

      Cependant, les biais ne sont pas de simples "erreurs" ou "vices de conception".

      Ce sont avant tout des stratégies cognitives rapides et adaptatives, appelées heuristiques, façonnées par l'évolution.

      Elles permettent à l'esprit humain de naviguer et de prendre des décisions efficaces dans un environnement complexe, avec des contraintes de temps et d'information.

      Fonction Adaptative : Dans la grande majorité des situations quotidiennes, ces raccourcis mentaux sont "super efficaces" et produisent des réponses correctes.

      Source d'Erreur : Ils deviennent problématiques lorsqu'ils entrent en conflit avec des principes logiques ou probabilistes dans des situations spécifiques, conduisant à des erreurs de jugement.

      Risque de Sur-interprétation : L'omniprésence du concept de biais cognitif peut mener à une erreur de diagnostic, décrite par la "loi de l'instrument" :

      "lorsqu'on ne possède qu'un marteau, tout finit par ressembler à un clou".

      Attribuer toutes les divergences d'opinion à des biais cognitifs est une simplification abusive.

      2. Le Modèle du Double Processus : Système 1 et Système 2

      Le modèle le plus populaire pour décrire le fonctionnement du raisonnement humain est celui du duo Système 1 / Système 2, popularisé par Kahneman.

      Système 1 (Pensée Intuitive) :

      Caractéristiques : Rapide, automatique, ne nécessite pas d'effort ou de ressources cognitives.  

      Exemples : Répondre à "5 + 5", connaître le nom du président, conduire une voiture sur un trajet familier.   

      Rôle : Il gère l'écrasante majorité des tâches cognitives quotidiennes (estimé à 99,9%).

      Il est essentiel au fonctionnement humain.

      Système 2 (Pensée Délibérée) :

      Caractéristiques : Lent, contrôlé, demande de l'effort et charge les ressources cognitives (mémoire de travail).   

      Exemples : Calculer "22 x 54", apprendre une nouvelle compétence, analyser un argument complexe.   

      Rôle : Il est activé pour résoudre des problèmes qui dépassent les capacités du Système 1.

      L'idée commune que le Système 1 est la source de toutes les erreurs ("irrationnel") et le Système 2 le garant de la rationalité est une simplification.

      Le Système 1 génère très souvent des réponses correctes et valides.

      Les biais apparaissent principalement dans les situations où la réponse intuitive rapide du Système 1 entre en conflit avec la conclusion logique qui nécessiterait l'intervention du Système 2.

      Exemple Classique : La Négligence des Taux de Base Un problème typique illustrant ce conflit est présenté :

      1. Données : Un échantillon de 1000 personnes contient 995 hommes et 5 femmes.

      2. Description : On tire une personne au hasard qui "aime bien faire du shopping".

      3. Question : Est-il plus probable que cette personne soit un homme ou une femme ?

      La réponse intuitive (Système 1), activée par le stéréotype, est "une femme".

      La réponse logique (Système 2), basée sur les probabilités (taux de base), est "un homme".

      La majorité des gens se trompent en suivant leur intuition, illustrant un biais cognitif.

      3. La Détection des Conflits Cognitifs : Le Cœur de la Recherche de Wim De Neys

      La vision classique de Kahneman suggère que les gens se trompent car ils sont des "avares cognitifs" (cognitive misers), évitant l'effort du Système 2 et ne se rendant donc pas compte du conflit entre leur intuition et la logique.

      Les travaux de Wim De Neys remettent en cause cette idée.

      Ils montrent que, même lorsque les individus donnent une réponse incorrecte basée sur leur intuition, leur cerveau détecte souvent le conflit sous-jacent.

      Méthodologie et Preuves : Les expériences comparent des problèmes "conflictuels" (où intuition et logique divergent) à des problèmes "non conflictuels" (où elles convergent).

      Les résultats montrent que pour les problèmes conflictuels, même chez les personnes qui se trompent :

      1. Le Temps de Réponse Augmente : Les participants prennent plus de temps pour répondre, signe qu'un processus supplémentaire a lieu.

      2. Activation Cérébrale Spécifique : L'imagerie cérébrale (IRMf) montre une activation accrue du cortex cingulaire antérieur, une région connue pour son rôle dans la détection des conflits.

      3. Mouvements Oculaires (Eye-tracking) : Les participants ré-inspectent visuellement les informations conflictuelles (par exemple, les taux de base dans l'exemple précédent).

      4. Baisse de la Confiance : Les individus rapportent un niveau de confiance en leur réponse plus faible, ce qui est une manifestation comportementale du doute.

      Cette détection est un processus implicite et automatique.

      Des expériences où le Système 2 est délibérément surchargé (par une tâche de mémorisation simultanée) montrent que cette détection de conflit persiste.

      Cela suggère que nous ne sommes pas totalement aveugles à nos biais ; un signal d'alerte, un "doute", est généré, même si nous ne l'écoutons pas toujours.

      4. La Question du "Débiaisage" : Stratégies et Limites

      La question centrale est de savoir s'il est possible de "débiaiser" les gens, c'est-à-dire de les rendre plus rationnels et moins sujets aux erreurs de jugement.

      L'Approche "Système 2" et le Problème du Transfert :

      ◦ L'idée d'apprendre aux gens à simplement "activer leur Système 2 plus souvent" est largement considérée comme inefficace.   

      ◦ La raison principale est le problème du transfert : une compétence apprise pour résoudre un type de problème (par exemple, la négligence des taux de base) n'est pas spontanément appliquée à d'autres types de problèmes, même s'ils reposent sur des principes logiques similaires.

      Le "transfert" d'une compétence d'un domaine à un autre est extrêmement difficile à obtenir.

      L'Approche "Système 1" : Rééduquer l'Intuition :

      ◦ Une stratégie plus efficace consiste à se concentrer sur des biais spécifiques, tâche par tâche.    ◦

      L'intervention consiste à expliquer clairement à une personne pourquoi son intuition est incorrecte et quel est le principe logique à appliquer.   

      ◦ Des projets comme Kojitum proposent des exercices basés sur ce principe.   

      ◦ Fait crucial : cet entraînement ne fonctionne pas seulement en forçant l'usage du Système 2.

      Il modifie directement le Système 1.

      Après l'intervention, la première réponse générée intuitivement devient la bonne.

      On "crée de bonnes intuitions".

      En somme, l'espoir de rendre les gens globalement plus rationnels par une intervention unique est illusoire.

      La voie la plus prometteuse est une éducation ciblée qui vise à corriger et à affiner les intuitions du Système 1 sur des problèmes spécifiques et importants.

      5. Le Rôle du Contexte Social et de l'Argumentation

      La théorie argumentative du raisonnement, développée par Hugo Mercier et Dan Sperber, propose que la fonction première du raisonnement n'est pas la recherche de la vérité en solitaire, mais la capacité à argumenter et à interagir dans un contexte social.

      Le Biais de Confirmation Recontextualisé : Ce biais, qui nous pousse à chercher des informations confirmant nos croyances, semble être un défaut majeur du raisonnement individuel.

      Cependant, dans un contexte de débat, il devient un outil efficace pour défendre son point de vue.

      La Sagesse des Groupes : Lorsque les gens raisonnent en groupe, échangent des arguments et justifient leurs positions, de nombreux biais individuels ont tendance à disparaître.

      Le groupe parvient collectivement à une meilleure solution, car les arguments sont mis à l'épreuve.

      Justification et Système 2 : C'est principalement le Système 2 qui permet de générer des justifications et des arguments explicites pour convaincre les autres, une fonction sociale essentielle.

      6. Perspectives Futures : L'Intelligence Artificielle et le Raisonnement Humain

      L'émergence des intelligences artificielles (IA) génératives comme ChatGPT offre de nouvelles perspectives pour le raisonnement humain.

      Potentiel Positif :

      Débiaisage Ciblé : Des études montrent que l'IA peut être un outil efficace pour débiaiser les individus, y compris sur des sujets comme les théories du complot.

      L'IA est perçue comme neutre et peut fournir des contre-arguments très spécifiques et bien informés que des interlocuteurs humains n'ont pas toujours.  

      Partenaire de Débat : L'IA peut servir de partenaire dans un "contexte argumentatif".

      Interagir avec une IA, lui demander des justifications et la mettre au défi peut stimuler la réflexion critique, de la même manière qu'un débat en groupe.   

      Assistant Pédagogique : Utilisée intelligemment, l'IA peut devenir un "professeur personnel", aidant les apprenants à améliorer leur travail en fournissant des retours et des explications.

      Risques et Limites :

      Usage Passif : Si l'IA est utilisée comme un simple "moteur de réponse" pour obtenir des solutions sans effort, elle risque de ne pas stimuler, voire d'atrophier, les compétences de pensée critique et d'évaluation de l'information.   

      Biais de Complaisance : Les IA sont souvent conçues pour être complaisantes, ce qui peut renforcer les biais de l'utilisateur au lieu de les remettre en question.  

      L'Importance de l'Usage : L'impact de l'IA sur le raisonnement dépendra fondamentalement de la manière dont elle est utilisée.

      Un usage actif et dialogué est bénéfique, tandis qu'un usage passif est préjudiciable.

    1. La coéducation en éducation prioritaire : enjeux, constats et perspectives

      Résumé exécutif

      Ce document synthétise l'intervention de Pierre Périer, sociologue et professeur en sciences de l'éducation, concernant la coéducation, particulièrement dans les quartiers populaires et l'éducation prioritaire.

      L'analyse met en lumière un double renversement historique : le passage d'une école républicaine construite à distance des familles vers une norme de proximité, et le transfert de la responsabilité de la « fabrication de l'élève » de l'institution vers la famille.

      Malgré l'inscription de la coéducation dans la loi de 2013, le concept reste flou pour les acteurs. Un paradoxe majeur subsiste : les parents des élèves les plus en difficulté sont souvent les moins associés au système scolaire.

      L'enjeu actuel n'est pas seulement de traiter l'éloignement des parents, mais de comprendre comment le fonctionnement institutionnel et les normes implicites de l'école contribuent à les exclure.

      Pour y remédier, Périer propose une refonte de la relation basée sur quatre principes : reconnaissance, autorisation, explicitation et diversification.

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      1. Contextualisation et évolutions historiques

      La relation entre l'école et les familles a subi des transformations structurelles profondes. Pierre Périer identifie deux mouvements majeurs :

      De la distance à la proximité : Historiquement, l'école s'est bâtie à distance des parents pour protéger l'espace républicain.

      Aujourd'hui, le paradigme s'est inversé pour devenir une norme de rapprochement et de participation active.

      La professionnalisation du rôle parental : Autrefois, l'école visait à faire de l'enfant un « petit missionnaire des idées modernes » capable de transformer sa famille.

      Aujourd'hui, on attend de la famille qu'elle transforme l'enfant en élève (le « métier d'élève »). La réussite scolaire devient une préoccupation centrale des classes populaires, souvent sous l'angle de l'évitement de l'échec.

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      2. Analyse sémantique et divergences de perception

      Les enquêtes menées auprès de 1000 parents et 2000 enseignants révèlent des décalages significatifs dans la compréhension de la notion de coéducation.

      Compréhension globale

      Parents : 2/3 des parents ne savent pas spontanément à quoi associer le terme.

      Enseignants : La notion est mieux connue, mais associée à un périmètre extrêmement large (520 mots différents cités).

      Définitions prioritaires par groupe d'acteurs

      | Perspective | Priorité 1 | Priorité 2 | | --- | --- | --- | | Parents | Instruction scolaire et apprentissages (30%) | Éducation de l'enfant (25%) | | Enseignants | Éducation globale et comportement de l'élève (55%) | Instruction scolaire (21%) |

      Note : Pour les parents, la coéducation est un outil pour soutenir la scolarité et les apprentissages, tandis que pour les enseignants, elle vise principalement à garantir que l'enfant se comporte conformément aux attentes institutionnelles.

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      3. Le paradoxe de l'implication et les profils d'acteurs

      L'intérêt pour la coéducation décroît à mesure que l'on progresse dans la scolarité :

      Maternelle : 65% des enseignants s'y disent très intéressés.

      Élémentaire : 55%.

      Collège : 41%.

      On observe un « décrochage parental » au collège, période où les difficultés scolaires s'accentuent pourtant pour les élèves les plus fragiles.

      Typologie des parents face à la coéducation

      1. Les parents « en proximité » (34%) : Souvent plus diplômés, membres d'associations, enfants en réussite. Ils sont en « connivence culturelle » avec l'école.

      2. Les parents « distants » ou « empêchés » (47%) : Intéressés par le principe mais peu ou pas impliqués concrètement.

      3. Les parents « invisibles » (20%) : Profil souvent précaire, zone rurale ou quartiers prioritaires, enfants au collège ou en difficulté. Pour eux, la notion est totalement floue.

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      4. Obstacles et freins à la coéducation

      L'analyse souligne que l'absence des parents n'est pas synonyme de désintérêt, mais résulte souvent de barrières structurelles et symboliques.

      La domination symbolique : Les parents précaires redoutent d'être pris en défaut sur leur maîtrise de la langue ou des codes sociaux (« savoir bien parler pour ne pas être jugé »).

      Le rapport au temps : Prendre rendez-vous suppose une maîtrise du temps programmatique.

      Or, les familles vulnérables vivent souvent dans un temps « chaotique » ou de l'urgence.

      La délégitimation par les devoirs : L'externalisation du travail scolaire à la maison aggrave les inégalités.

      Les parents qui veulent aider mais ne maîtrisent pas les méthodes vivent une « disqualification symbolique » devant leurs enfants.

      La norme du « parent d'élève » : L'institution définit implicitement un modèle de parent idéal.

      Ceux qui s'en éloignent sont rapidement étiquetés comme « démissionnaires », alors qu'ils sont en réalité surexposés au jugement institutionnel dès qu'un problème survient.

      « Ce sont des parents que l'école éloigne, plus qu'ils ne sont éloignés de l'école. »

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      5. Les enseignements du confinement (COVID-19)

      La période de crise sanitaire a agi comme un révélateur et un accélérateur de tendances :

      Exacerbation des inégalités : Les conditions de logement et l'incapacité d'aider aux devoirs ont créé des tensions extrêmes dans les familles.

      Découverte de l'humain : L'usage du téléphone a permis de briser la froideur institutionnelle.

      Certains parents ont vécu pour la première fois une « relation humaine » avec les enseignants, basée sur une parole protégée et bienveillante.

      Reconnaissance mutuelle : Le confinement a permis une meilleure valorisation du travail des enseignants par les parents, et une prise de conscience par l'école que le contact avec les familles dites « éloignées » était possible.

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      6. Principes pour une action équitable

      Pour construire une coéducation réelle, Pierre Périer propose quatre principes directeurs :

      1. Principe de reconnaissance

      Égalité : Droits d'information et de statut identiques.

      Mérite : Considérer et gratifier la contribution réelle de chaque parent.

      Confiance : Elle ne se décrète pas, elle découle de la reconnaissance.

      2. Principe d'autorisation

      • Légitimer les « parents réels » (tels qu'ils sont) plutôt que des parents de fiction.

      • Passer de « faire pour » les parents à « faire avec », voire « faire à partir de » leurs attentes.

      • Créer des espaces dédiés (café des parents, lieux de médiation) pour symboliquement leur faire une place.

      3. Principe d'explicitation

      • Clarifier les rôles : qui fait quoi ?

      • Éviter les implicites qui ne profitent qu'aux parents déjà initiés. Plus le code est explicite, plus la relation est égalitaire.

      4. Principe de diversification

      • Multiplier les supports de communication (parole, téléphone, vidéo, objets circulants).

      • S'appuyer sur des médiateurs (parents relais, associations d'éducation populaire) pour maintenir le lien avec ceux qui restent en retrait de l'institution.

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      Conclusion : Outils et perspectives pour la réussite

      L'enquête montre que la réussite des élèves passe, selon les acteurs, par trois leviers majeurs :

      1. L'allègement des effectifs (pour une attention accrue aux élèves en difficulté).

      2. Le développement de la coéducation.

      3. Le renforcement des temps d'étude.

      La coéducation doit être pensée comme un levier collectif et non comme une affaire individuelle, en s'appuyant sur des outils concrets (vidéos de classe, jeux partagés, guides de communication) qui font circuler les savoirs entre l'école et la maison.

    1. La Coéducation comme Relation de Reconnaissance Mutuelle : Synthèse des Travaux de Chloé Riban

      Ce document de synthèse s'appuie sur l'analyse de Chloé Riban, maîtresse de conférence à l’Université Paris Nanterre, concernant les relations entre l'institution scolaire et les familles issues de milieux populaires et « ethnicisés ».

      Il examine les tensions inhérentes au concept de coéducation et propose des pistes pour transformer cette injonction institutionnelle en une véritable rencontre humaine et paritaire.

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      Résumé Exécutif

      La coéducation est devenue un paradigme central de l'école républicaine, passant d'une culture de la séparation à une exigence de partenariat.

      Cependant, l'analyse révèle un décalage profond entre les attentes normées de l'institution et la réalité des familles les plus précaires.

      Points clés à retenir :

      Un malentendu structurel : L'école attend un « parent idéal » (auxiliaire pédagogique) tandis que les parents, bien que très investis, se heurtent à des barrières de codes, de langage et de précarité.

      L'invisibilité de l'investissement parental : Contrairement aux idées reçues sur la « démission » parentale, les mères de milieux populaires déploient un effort considérable (temporel, psychique et financier) qui reste souvent non reconnu par les enseignants.

      Le poids du stigmate : La rencontre avec l'école est vécue par beaucoup comme une mise en danger, un « jet dans l'arène » où domine la peur du jugement ou du signalement.

      Vers la « parité d'estime » : La réussite de la coéducation repose sur la reconnaissance de la légitimité des parents tels qu'ils sont, en substituant le modèle du débat (confrontation) par celui de la « palabre » (construction commune de sens).

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      I. Évolution Historique et Cadre Théorique

      1. De la distance à l'injonction de coopération

      L'école républicaine s'est historiquement construite en excluant les parents, matérialisée par des seuils physiques symboliques. Cette posture a évolué sous l'impulsion de plusieurs jalons législatifs :

      Loi Jospin (1989) : Instauration de la notion de « communauté éducative ».

      Années 2000 : Création des réseaux d'éducation prioritaire (REP), ciblant les populations vulnérables.

      Loi de refondation de l'école (2013) : La coopération avec les parents devient un enjeu majeur pour la réussite des élèves.

      2. Les fondements de l'analyse

      L'étude de Chloé Riban s'appuie sur une ethnographie de deux ans en quartier prioritaire et mobilise deux concepts clés :

      Le « Différend » (Pierre Perrier) : Les relations école-familles ne sont pas seulement marquées par des malentendus, mais par un conflit de légitimité et d'intérêts.

      L'approche systémique et intersectionnelle : Il est nécessaire de comprendre l'interdépendance des acteurs et les rapports de pouvoir multiples (classe, genre, origine) qui marquent la rencontre institutionnelle.

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      II. Le Regard de l'Institution : Entre Normes et Représentations

      L'institution scolaire adresse une demande de coéducation différenciée, ciblant prioritairement les parents jugés « éloignés ».

      1. La figure du parent idéal

      Les professionnels de l'éducation projettent des attentes précises qui définissent le « métier de parent d'élève » :

      • Suivi rigoureux (signature des cahiers, devoirs).

      • Participation active à la vie de l'école.

      • Maîtrise d'une éthique du dialogue et des codes scolaires.

      2. La stigmatisation des « familles »

      Une distinction sémantique s'opère souvent dans le discours enseignant : le terme « parent » est réservé à ceux qui préparent l'enfant à son rôle d'élève, tandis que le terme « famille » est utilisé pour désigner un groupe perçu comme décalé ou déficitaire.

      Ce regard produit deux figures types :

      Le parent démissionnaire : Accusé de manque d'autorité ou de cadre.

      Le parent qui gâte trop : Accusé d'une affection excessive entravant l'autonomie.

      | Facteurs de tension identifiés | Perception enseignante | Réalité des familles populaires | | --- | --- | --- | | Styles éducatifs | Manque de règles incorporées. | Discipline souvent imposée de l'extérieur (non négociée). | | Objectif de la coéducation | Acculturation des parents aux normes scolaires. | Recherche de respectabilité et de soutien. | | Origine des difficultés | Registre culturaliste (« c'est leur culture »). | Conditions matérielles et trajectoires biographiques heurtées. |

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      III. La Réalité des Parents : Un Investissement Invisible et Entravé

      1. Une confiance et des attentes fortes

      Contrairement aux préjugés, les parents en REP témoignent d'une grande confiance envers l'école et d'une ambition forte pour l'ascension sociale de leurs enfants. Leur investissement se manifeste par :

      Des sacrifices financiers : Achats de matériel pédagogique (ardoises, etc.) malgré des budgets contraints.

      Un soutien logistique : Accompagnement chronophage vers les spécialistes (orthophonistes).

      Des incitations verbales : Rappel constant de l'importance de l'école.

      2. Les obstacles majeurs à la participation

      L'investissement des parents est souvent freiné par des barrières structurelles :

      Méconnaissance du système : Difficulté à comprendre les rouages de l'orientation (ex: passage au collège).

      Précarité temporelle : Horaires de travail instables (intérim) et urgences administratives empêchant la projection.

      Sentiment d'incompétence : Peur d'être jugé « bête » à cause de la barrière de la langue ou d'un parcours scolaire interrompu.

      « Quand on ne parle pas bien français parfois les gens nous trouvent bête alors qu’on n’est pas bête. » — Témoignage d'une mère.

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      IV. La Rencontre comme Épreuve Psychique

      Pour les mères de familles populaires, l'école est un espace public où leur respectabilité est mise en jeu.

      La peur du signalement : L'institution est crainte car elle détient le pouvoir de juger la qualité du parentage, allant jusqu'au risque de signalement.

      Le retrait silencieux : Ce qui est interprété comme du désintérêt est souvent une tactique d'évitement pour échapper au sentiment d'humiliation ou d'injustice.

      L'oscillation : Les parents alternent entre proximité (venir au café des parents) et distance (esquiver un rendez-vous formel) selon le degré de sécurité émotionnelle ressenti.

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      V. Pistes pour une Coéducation Authentique

      Pour sortir du modèle de la normalisation, Chloé Riban et Catherine Urtique de Lâtre proposent de refonder la relation sur quatre piliers et trois postures.

      1. Les quatre piliers de l'action

      1. Accueillir : Créer une relation « d'humain à humain » avant d'être une relation de professionnels à usagers.

      2. Informer : Expliciter les codes et les attendus de manière claire.

      3. Dialoguer : Accepter que la coéducation puisse inclure le désaccord.

      4. Impliquer : Permettre une participation réelle à la vie de l'élève.

      2. Le modèle de la « Palabre »

      Inspiré par Isabelle Stengers, ce modèle propose de remplacer le débat par la palabre :

      Reconnaissance de l'insuffisance : Admettre que ni l'enseignant ni le parent ne détient seul la solution. Chacun est « légitime et insuffisant ».

      Parité d'estime : Considérer que la parole du parent sur son enfant est aussi valable que celle du professionnel.

      Construction de convergences : Chercher un sens commun sans forcer l'accord immédiat.

      3. Recommandations pratiques issues des échanges

      Transformer le vocabulaire : Préférer « l'invitation » à la « convocation ».

      Favoriser le contact initial positif : Appeler ou rencontrer chaque parent en début d'année pour se présenter, avant l'apparition de problèmes.

      Permettre la présence d'un tiers : Accepter qu'un parent puisse venir accompagné d'une personne de confiance ou d'un médiateur pour réduire l'anxiété de la rencontre.

      Ouvrir la classe : Les « classes ouvertes » permettent aux parents de comprendre concrètement la réalité du métier d'élève et la complexité du travail enseignant.

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      Conclusion

      La coéducation ne doit pas être une injonction à la conformité, mais un principe éthique de reconnaissance mutuelle.

      Elle exige de l'institution qu'elle reconnaisse les « parents réels » dans leur singularité et leurs contraintes, plutôt que de s'épuiser à poursuivre le mirage du « parent idéal ».

    1. Approche Scientifique, Droits des Enfants et Scolarité en Protection de l'Enfance : Synthèse et Perspectives

      Résumé Exécutif

      Ce document synthétise les interventions de Marie-Pierre Mackiewicz, chercheuse en sciences de l'éducation, et de Gabrielle Chouin, conseillère principale d'éducation (CPE) et ancienne enfant placée.

      L'analyse met en lumière une déconnexion persistante entre les institutions de la protection de l'enfance et de l'Éducation nationale, entraînant des inégalités majeures dans les parcours scolaires et l'accès à l'autonomie.

      Les points saillants incluent l'émergence de la "recherche par les pairs", une méthode participative visant à inclure les jeunes concernés non plus comme de simples objets d'étude, mais comme co-chercheurs pour rééquilibrer les rapports de pouvoir.

      Malgré des avancées législatives et des victoires concrètes récentes (comme la bonification Parcoursup en 2024), les statistiques restent alarmantes : 35 % des jeunes sortant de l'Aide Sociale à l'Enfance (ASE) n'ont aucun diplôme.

      La réussite de ces élèves repose sur la création d'« alliances éducatives » solides et sur un changement de regard des professionnels sur le potentiel de réussite de ces publics.

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      I. Les Enjeux de la Scolarité et de l'Orientation des Élèves Protégés

      L'analyse de Gabrielle Chouin souligne que le partenariat entre la protection de l'enfance et l'Éducation nationale est le facteur déterminant de l'obtention d'un diplôme ou d'une qualification.

      1. Dysfonctionnements Institutionnels

      Manque d'information mutuelle : Il existe une méconnaissance profonde des droits et des cultures professionnelles entre les deux services publics (Éducation nationale et ASE).

      Logiques d'orientation opposées : Les processus d'orientation conduits par la protection de l'enfance entrent souvent en conflit avec ceux de l'Éducation nationale, privant le jeune d'une réelle capacité de choix.

      Absence de "Droit Commun" : Paradoxalement, certains dispositifs spécifiques dédiés à l'autonomie des jeunes placés les empêchent d'accéder aux droits communs dont bénéficient tous les autres jeunes.

      2. Obstacles Pratiques et Victoires Récentes

      Le document identifie des freins concrets à la continuité scolaire et à l'insertion :

      La bonification Parcoursup : Jusqu'à récemment, les élèves placés n'étaient pas considérés comme boursiers sur la plateforme car pris en charge par les départements.

      Une victoire obtenue le 4 avril 2024 permet désormais à tous ces élèves de bénéficier d'une bonification de vœux sur l'ensemble du territoire français.

      Accès aux moyens financiers (PFMP) : En voie professionnelle, l'absence de compte bancaire personnel pour certains élèves protégés bloque l'accès aux gratifications de stage, entravant leur autonomie et le lien de confiance avec les entreprises.

      Vie quotidienne scolaire : Des actes simples comme la signature de documents pour des sorties scolaires restent complexes en raison des questions d'autorité parentale et de tutelle.

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      II. La Recherche par les Pairs : Une Révolution Méthodologique

      Marie-Pierre Mackiewicz expose une approche de recherche qui associe directement les personnes concernées par l'objet d'étude (enfants, jeunes ou anciens de la protection de l'enfance).

      1. Fondements Épistémologiques

      Gestion des rapports de pouvoir : Inspirée des mouvements des années 70 et des Gender Studies, cette approche vise à réduire la dissymétrie entre le chercheur "expert" et le savoir "profane" des populations minorées ou invisibilisées.

      Collectif de recherche : Le chercheur ne travaille pas sur mais avec un collectif. Cela implique une implication forte des chercheurs universitaires, sortant des cadres de bureaux classiques pour créer une "familiarité" nécessaire (rencontres le week-end, soirées, moments conviviaux).

      2. Défis et Éthique de la Recherche

      Le rôle des "Gatekeepers" : L'accès aux mineurs est souvent filtré par les institutions qui détiennent les "clés" (directeurs de foyers, éducateurs), risquant de biaiser l'échantillon en proposant uniquement des "bons profils".

      Risques identitaires : La recherche peut être violente pour le jeune en le réassignant à une identité stigmatisée ("enfant placé") au moment où il cherche à s'en défaire.

      Protocoles de protection : Il existe une tension entre les protocoles de protection de l'enfance (parfois trop rigides) et la nécessité de recueillir une parole libre et authentique.

      3. Résultats et Impact

      Production de connaissances : Utilisation de méthodes sensibles (discussions plutôt qu'entretiens, photos, dessins).

      Émancipation : Participation à des colloques, création d'associations (ADEPAPE) et même mise en scène de résultats sous forme de pièces de théâtre (notamment au festival d'Avignon).

      Coéducation professionnalisée : Concept décrivant la communication directe entre professionnels de l'école et de la protection de l'enfance en l'absence de parents.

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      III. État des Lieux Statistique et Facteurs de Risque

      Les données récentes (notamment de France Stratégie, septembre 2024) confirment la fragilité des parcours.

      1. Indicateurs de Précarité Scolaire

      | Indicateur | Jeunes ASE | Jeunes Milieu Social Équivalent | | --- | --- | --- | | Absence de diplôme ou brevet seul | 35 % | 16 % |

      Les élèves protégés subissent de manière plus fréquente :

      • Des redoublements et des retards scolaires.

      • Des déscolarisations fréquentes.

      • Une orientation quasi-systématique vers les filières professionnelles.

      • Un accès extrêmement limité aux études supérieures.

      2. Facteurs d'Échec Identifiés

      La réussite ou l'échec d'un parcours dépendent de plusieurs variables critiques :

      Stabilité du placement : Les placements multiples sont des facteurs majeurs d'échec.

      Précocité de la prise en charge : L'âge d'entrée dans le dispositif influence la trajectoire.

      Type d'accueil : Les différences entre familles d'accueil et établissements collectifs marquent les parcours.

      Facteur humain : Le manque de croyance des acteurs institutionnels dans la capacité de réussite de ces enfants est un frein psychologique et structurel majeur.

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      IV. Recommandations pour l'Action Publique et Pédagogique

      Le document conclut sur la nécessité d'une transformation profonde des pratiques :

      1. Acculturation et Formation Commune : Organiser des temps de formation partagés entre les personnels de l'Éducation nationale (professeurs, CPE, psychologues) et ceux de la protection de l'enfance.

      2. Mise en place d'Alliances Éducatives : Créer des partenariats quotidiens concrets autour de la continuité scolaire, au-delà des simples réunions de crise.

      3. Effectivité du Droit à la Participation : Ne pas se limiter au témoignage, mais permettre une participation effective des jeunes à la construction des politiques publiques les concernant (via le Conseil de la Vie Lycéenne, les associations d'anciens pairs, etc.).

      4. Ressources Documentaires : S'appuyer sur les travaux de l'Observatoire National de la Protection de l'Enfance (ONPE) et les revues de littérature (ex: Aurélie Pico, 2020) pour identifier les facteurs de protection à renforcer.

      L'enjeu final est de garantir que chaque acteur se sente investi d'une mission de compensation des inégalités, pour permettre à ces "acteurs faibles" de devenir des citoyens diplômés et autonomes.

    1. Reviewer #2 (Public review):

      Summary:

      Feng, Jing-Xin et al. studied the hemogenic capacity of the endothelial cells in the adult mouse bone marrow. Using Cdh5-CreERT2 in vivo inducible system, though rare, they characterized a subset of endothelial cells expressing hematopoietic markers that were transplantable. They suggested that the endothelial cells need the support of stromal cells to acquire blood-forming capacity ex vivo. These endothelial cells were transplantable and contributed to hematopoiesis with ca. 1% chimerism in a stress hematopoiesis condition (5-FU) and recruited to the peritoneal cavity upon Thioglycolate treatment. Ultimately, the authors detailed the blood lineage generation of the adult endothelial cells in a single cell fashion, suggesting a predominant HSPCs-independent blood formation by adult bone marrow endothelial cells, in addition to the discovery of Col1a2+ endothelial cells with blood-forming potential, corresponding to their high Runx1 expressing property.

      The conclusion regarding the characterization of hematopoietic-related endothelial cells in adult bone marrow is well supported by data. However, the paper would be more convincing, if the function of the endothelial cells were characterized more rigorously.

      (1) Ex vivo culture of CD45-VE-Cadherin+ZsGreen EC cells generated CD45+ZsGreen+ hematopoietic cells. However, given that FACS sorting can never achieve 100% purity, there is a concern that hematopoietic cells might arise from the ones that got contaminated into the culture at the time of sorting. The sorting purity and time course analysis of ex vivo culture should be shown to exclude the possibility.

      (2) Although it was mentioned in the text that the experimental mice survived up to 12 weeks after lethal irradiation and transplantation, the time-course kinetics of donor cell repopulation (>12 weeks) would add a precise and convincing evaluation. This would be absolutely needed as the chimerism kinetics can allow us to guess what repopulation they were (HSC versus progenitors). Moreover, data on either bone marrow chimerism assessing phenotypic LT-HSC and/or secondary transplantation would dramatically strengthen the manuscript.

      (3) The conclusion by the authors, which says "Adult EHT is independent of pre-existing hematopoietic cell progenitors", is not fully supported by the experimental evidence provided (Figure 4 and Figure S3). More recipients with ZsGreen+ LSK must be tested.

      Strengths:

      The authors used multiple methods to characterize the blood-forming capacity of the genetically - and phenotypically - defined endothelial cells from several reporter mouse systems. The polylox barcoding method to trace the adult bone marrow endothelial cell contribution to hematopoiesis is a strong insight to estimate the lineage contribution.

      Weaknesses:

      It is unclear what the biological significance of the blood cells de novo generated from the adult bone marrow endothelial cells is. Moreover, since the frequency is very rare (<1% bone marrow and peripheral blood CD45+), more data regarding its identity (function, morphology, and markers) are needed to clearly exclude the possibility of contamination/mosaicism of the reporter mice system used.

    1. Perspective Institutionnelle et Historique des Droits de l’Enfant : Synthèse de l’Intervention de Marie Derain de Vaucresson

      Ce document de synthèse analyse les points clés de l'intervention de Marie Derain de Vaucresson, ancienne adjointe au Défenseur des enfants.

      Il retrace l’évolution des droits de l'enfant, du registre de la charité à celui des droits opposables, tout en examinant les cadres législatifs français et les défis persistants de la scolarisation des enfants placés.

      Résumé Exécutif

      L'approche des droits de l'enfant a connu une mutation profonde, passant d'une protection caritative au XVIIe siècle à une reconnaissance de l'enfant comme sujet de droits avec la Convention internationale des droits de l'enfant (CIDE) de 1989.

      L'intervention souligne que si tous les enfants doivent être protégés, les "enfants placés" font face à des vulnérabilités spécifiques, notamment des ruptures dans leur parcours scolaire.

      L’évolution législative française (lois de 2007, 2016 et 2022) reflète un changement de paradigme : la priorité est passée de la préservation de la famille à la satisfaction des besoins fondamentaux de l'enfant.

      La réussite de cette protection repose désormais sur une coopération pluridisciplinaire accrue entre l'Éducation nationale, les services sociaux et la santé.

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      1. Perspective Historique : De la Charité aux Droits Fondamentaux

      L'histoire de la protection de l'enfance s'articule autour de plusieurs étapes clés, souvent déclenchées a posteriori par des constats de mise en danger.

      Les racines de la protection (XVIIe - XIXe siècles)

      XVIIe siècle : L'approche caritative émerge avec Vincent de Paul, qui organise l'accueil des enfants abandonnés sur le parvis des églises.

      Milieu du XIXe siècle (1842) : Première loi organisant la protection des enfants au travail.

      Elle fixe l'âge minimum à 8 ans pour travailler dans les mines et limite le temps de travail (8h pour les 8-12 ans, 12h pour les 12-16 ans).

      Fin du XIXe siècle (1882) : L'obligation d'instruction (de 6 à 13 ans) vient concurrencer le travail des enfants dans l'industrie et les champs.

      L'émergence de la figure de l'enfant-personne (XXe siècle)

      Janusz Korczak : Médecin et pédagogue polonais, il révolutionne l'approche pédagogique en considérant l'enfant comme une personne à part entière.

      Dans son orphelinat du ghetto de Varsovie, il instaure une "mini-société" avec un tribunal des enfants et un journal, prônant l'autonomie et la participation.

      Évolutions textuelles : La première Déclaration des droits de l'enfant (1924) est impulsée par Eglantyne Jebb, suivie d'une version renforcée en 1959.

      Toutefois, ces textes restent des déclarations non opposables aux États.

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      2. La Convention Internationale des Droits de l'Enfant (CIDE)

      Adoptée à l'unanimité le 20 novembre 1989, la CIDE transforme les principes moraux en obligations juridiques pour les États.

      Principes Fondamentaux

      | Principe | Description | | --- | --- | | Opposabilité | Contrairement à une déclaration, la Convention est un traité international qui oblige les États à transposer ses dispositions en droit interne. | | Intérêt supérieur | Traduit de l'anglais the best interest, il s'agit de rechercher le "meilleur intérêt" de l'enfant face à des intérêts multiples ou conflictuels. | | Non-discrimination | Garantie d'accès aux droits sans distinction (le droit français identifiant aujourd'hui environ 24 critères de discrimination). | | Participation | L'enfant a le droit d'exprimer son opinion sur les décisions le concernant (famille, école, justice). |

      La Métaphore de la Balance

      L'intervention présente les droits de l'enfant comme une balance entre deux plateaux :

      1. Le plateau de la protection : Il pèse très lourd pour les jeunes enfants incapables de se défendre seuls.

      2. Le plateau de la participation : Il prend du poids à mesure que l'enfant grandit, lui permettant de devenir acteur de son propre destin.

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      3. L'Architecture Institutionnelle en France

      La France a ratifié la CIDE en août 1990. Depuis, plusieurs mécanismes de défense ont été mis en place.

      Le Défenseur des enfants (2000) : Institution indépendante créée pour promouvoir les droits et traiter les réclamations individuelles.

      Le Défenseur des droits (2011) : Cette instance a absorbé le Défenseur des enfants. Elle dispose de pouvoirs d'intervention accrus :

      ◦ Accès direct aux lieux fermés (centres de rétention, centres éducatifs fermés).  

      ◦ Capacité de formuler des recommandations formelles aux administrations (Rectorats, Conseils départementaux).  

      ◦ Saisine possible par les enfants eux-mêmes ou par des adultes signalant un droit bafoué.

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      4. Évolution du Cadre Législatif de la Protection de l'Enfance

      Le droit français a connu trois réformes majeures en quinze ans, marquant une rupture avec le "profamilialisme" historique.

      1. Loi de 2007 : Elle structure la décentralisation vers les départements et crée les Cellules de Recueil des Informations Préoccupantes (CRIP).

      Elle est toutefois critiquée pour avoir parfois retardé des placements nécessaires en tentant de maintenir le lien familial à tout prix.

      2. Loi de 2016 : Inversion du paradigme. On ne part plus de la famille, mais de l'enfant et de ses besoins.

      Elle insiste sur la stabilité des parcours, le maintien des liens avec les frères et sœurs, et la recherche de l'adoptabilité.

      3. Loi de 2022 (Loi Taquet) : Elle vise à remobiliser l'État aux côtés des départements.

      Elle met l'accent sur la protection des jeunes majeurs (au-delà de 18 ans) et l'implication de la société civile.

      Citation marquante : "L'approche par les droits n'a jamais été acquise en protection de l'enfance et elle est encore un combat à défendre."

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      5. Défis Spécifiques : L'Enfant Placé et l'École

      L'intervention souligne que si tous les enfants sont des élèves, ils sont avant tout des enfants dont le destin peut entraver l'apprentissage.

      Obstacles à la scolarité

      Parcours hachés : Les ruptures de placement (succession de familles d'accueil ou d'établissements) entraînent des ruptures scolaires.

      Absence de scolarisation : En 2011, environ 4 % des adolescents placés n'étaient pas scolarisés.

      Délais d'évaluation : Les périodes de transition (évaluation globale de la situation) peuvent durer plusieurs mois, privant l'enfant d'école durant des moments clés comme l'apprentissage de la lecture.

      Pistes de Solutions et Préconisations

      Pluridisciplinarité : Nécessité d'une coordination étroite entre les chargés de mission "enfants protégés" des Rectorats et les services de l'Aide Sociale à l'Enfance (ASE).

      Unités mobiles : L'idée que "l'école aille aux enfants" lors de phases critiques de placement pour éviter les déscolarisations prolongées.

      Vision immédiate : Les droits de l'enfant ne doivent pas être perçus comme la préparation d'un "citoyen de demain", mais comme des droits applicables "ici et maintenant", y compris dans le cadre du placement.

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      Conclusion

      La protection de l'enfance et le respect des droits fondamentaux sont présentés comme une responsabilité collective.

      L'enjeu actuel réside dans la capacité des acteurs institutionnels à dépasser leurs silos respectifs pour construire des réponses adaptées aux réalités territoriales, garantissant ainsi que le statut d'enfant placé ne soit plus un obstacle à la réussite scolaire et au développement personnel.

    1. Impact des Éco-émotions sur la Jeunesse : Analyse et Perspectives

      Ce document de synthèse analyse les interventions d'Arnaud Sapin concernant l'impact de la crise environnementale sur la vie affective et psychologique des jeunes.

      Il examine les mécanismes de l'éco-anxiété, la diversité des émotions climatiques et les stratégies d'accompagnement pour les éducateurs et les parents.

      Synthèse de la problématique

      L'éco-anxiété ne doit pas être perçue comme une pathologie mentale en soi, mais comme une préoccupation rationnelle face à une situation climatique objectivement alarmante. Selon les données du GIEC, la trajectoire actuelle menace la disponibilité des ressources (eau, nourriture) et la biodiversité, tout en prévoyant jusqu'à 200 millions de réfugiés climatiques d'ici 2050.

      Pour la jeunesse, cette réalité se traduit par un bouleversement profond des émotions et des projections dans l'avenir.

      Le défi majeur réside dans la transition d'une anxiété paralysante vers un engagement constructif. Alors que les émotions négatives (tristesse, colère, impuissance) prédominent chez les 16-25 ans, l'enjeu pour les adultes est de valider ces ressentis tout en stimulant le pouvoir d'agir des jeunes pour transformer ces tensions en leviers de changement.

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      1. Caractérisation de l'éco-anxiété et des éco-émotions

      Un continuum de ressentis

      L'éco-anxiété n'est pas un bloc monolithique mais un spectre d'intensités :

      Inquiétude modérée : Une préoccupation saine qui peut motiver une remise en question des habitudes.

      Anxiété profonde : Une anticipation négative et forte de l'avenir climatique, souvent liée à l'idée d'effondrement (collapsologie).

      Dimensions existentielles : Elle interroge le rapport à la finitude, à la mort et à l'identité personnelle et collective.

      La galaxie des émotions climatiques

      Au-delà de l'anxiété, une diversité de réactions affectives émerge :

      | Émotion | Caractéristiques et Origines | | --- | --- | | Tristesse et Deuil | Sentiment de perte lié à la disparition de la biodiversité ou du patrimoine. On parle de solastalgie pour la douleur liée à la perte d'un lieu naturel cher. | | Colère | Émotion politique dirigée vers des cibles identifiées comme responsables (institutions, entreprises, générations précédentes). | | Culpabilité | Responsabilité dirigée vers soi-même ("je ne fais pas assez"). | | Impuissance | Sentiment de ne disposer d'aucun levier pour remédier à la gravité de la situation. | | Espoir | Peut être irréaliste (attente d'une solution technique externe) ou constructif (engagement actif). |

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      2. État des lieux chez les jeunes (16-25 ans)

      Une étude internationale de 2021 menée dans 10 pays (dont la France) auprès de 10 000 jeunes révèle l'ampleur du phénomène.

      Données comparatives (Monde vs France)

      Les jeunes Français se distinguent par un pessimisme et un sentiment d'impuissance plus marqués que la moyenne mondiale :

      Tristesse : 66 % (Monde) / 63 % (France).

      Anxiété : Environ 60 % dans les deux cas.

      Optimisme : 30 % (Monde) / 22 % (France).

      Sentiment d'impuissance : 56 % (Monde) / 68 % (France).

      Colère : 56 % (Monde) / 60 % (France).

      « Huit personnes sur dix ressentent de l’éco-anxiété... ce n’est pas un phénomène à la marge, c’est un phénomène majoritaire. » — Arnaud Sapin.

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      3. Conséquences sur la vie et la santé mentale

      Les mécanismes de régulation (Coping)

      Pour gérer la tension émotionnelle, les individus utilisent différentes stratégies de "coping" :

      1. Centré sur l'émotion : Partage des ressentis, distraction, évitement ou déni pour se protéger.

      2. Centré sur le sens : Réévaluation positive ("voir le verre à moitié plein") ou minimisation de la gravité.

      3. Centré sur le problème : Passage à l'action pour transformer l'émotion en ressource.

      L'éco-anxiété pathologique

      Elle est identifiée selon trois critères principaux :

      Intensité et durée : Une souffrance forte qui s'installe durablement.

      Répercussions fonctionnelles : Troubles du sommeil, perte d'appétit, conflits relationnels.

      Paralysie : L'anxiété mobilise toute l'énergie de l'individu, l'empêchant paradoxalement d'agir pour l'environnement.

      Bouleversement des choix de vie

      L'avenir est perçu à travers un prisme pessimiste par 27 jeunes sur 30 interrogés (dégradation des conditions de vie, conflits). Cela influence :

      La carrière : Recherche de métiers à impact ou refus de travailler pour des entreprises polluantes.

      La parentalité : Questionnement profond sur la responsabilité de mettre au monde un enfant dans un contexte de crise (évoqué dans 23 entretiens sur 30).

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      4. Le rôle pivot de l'école et des adultes

      L'école est citée comme le premier acteur de sensibilisation, mais son action peut être contre-productive si elle se limite à une saturation d'informations sans offrir d'espace d'expression.

      Les écueils identifiés

      Saturation et fragmentation : Trop d'informations déconnectées du pouvoir d'agir.

      Fossé générationnel : Sentiment de trahison envers les aînés et désillusion face aux figures d'autorité.

      Manque d'espace émotionnel : L'école instruit mais offre rarement un cadre pour exprimer la peur ou la colère.

      Pistes d'action pour les éducateurs

      Pour accompagner les jeunes, les adultes doivent adopter une posture proactive et empathique :

      Valider l'émotion : Ne pas minimiser la peur ; confirmer que ces ressentis sont normaux et rationnels.

      Cultiver l'espoir constructif : Mettre l'accent sur les avancées (scientifiques, associatives) et les rôles modèles.

      Stimuler le sentiment de compétence : Aider les jeunes à identifier des leviers d'action locaux et concrets, sans leur faire porter seuls la responsabilité du monde.

      Inscrire l'action dans le collectif : Sortir de l'approche individuelle pour créer du lien social autour de projets communs.

      Être un modèle : Aligner ses propres comportements (mobilité, consommation) pour réduire la défiance des jeunes envers l'autorité.

      « On peut transformer des émotions négatives en émotions positives en passant à l'action... c’est plus joyeux d’agir. » — Citation d'une pâtissière de 24 ans.

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      Conclusion

      L'éco-anxiété témoigne d'une jeunesse lucide et sensible. L'enjeu de l'accompagnement n'est pas de supprimer cette anxiété par le déni ou la "pensée magique" technologique, mais de l'intégrer comme une force de transformation.

      En transformant l'information en savoir et le savoir en action, les éducateurs peuvent aider les jeunes à retrouver un sentiment de maîtrise sur leur propre futur.

    1. Briefing sur la Coéducation : Analyse des Regards Croisés entre Parents et Enseignants

      Ce document de synthèse s'appuie sur l'enquête inédite menée par Pierre Périer, sociologue et professeur en sciences de l’éducation à l’université de Rennes (laboratoire CREAD), présentée lors d'une conférence pour la plateforme Léa.fr.

      L'étude analyse les dynamiques, les représentations et les freins liés à la coéducation en France.

      Résumé Exécutif

      La coéducation, bien qu’institutionnalisée par la loi de 2013, reste un concept en quête de définition pratique sur le terrain.

      L'enquête révèle un consensus massif sur l'intérêt du principe (plus de 80 % d'adhésion), mais met en lumière une « coéducation empêchée » par des contraintes structurelles, temporelles et culturelles.

      Si les parents envisagent la coéducation comme une responsabilité partagée globale (instruction et éducation), les enseignants ont tendance à la restreindre à la sphère éducative, tout en manifestant une posture défensive face à une potentielle intrusion parentale.

      Le défi majeur réside dans la co-construction de projets locaux qui dépassent les simples routines institutionnelles (fêtes d'école, réunions de rentrée) pour devenir un véritable levier de réussite scolaire, particulièrement dans les contextes de précarité ou de transition vers le collège.

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      1. Cadre Méthodologique et Contexte de l'Enquête

      L'étude se présente comme la première étape d'un travail au long cours visant à défricher l'appropriation du terme « coéducation ».

      Échantillon : Plus de 1 000 parents (représentatifs par CSP et région) et 2 000 enseignants (membres de la plateforme Léa.fr).

      Profils : Une forte féminisation est observée (71 % de femmes chez les parents, 92 % chez les enseignants).

      La diversité des contextes est représentée, incluant l'enseignement public et privé, ainsi que l'éducation prioritaire (8 % des parents répondants).

      Méthode : Questionnaire en ligne avec questions ouvertes et fermées, permettant de recueillir plus de 5 000 termes associés spontanément à la coéducation.

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      2. Divergences de Significations et Représentations

      L'analyse des nuages de mots révèle que parents et enseignants n'attribuent pas la même portée au terme.

      | Dimension | Point de vue des Parents | Point de vue des Enseignants | | --- | --- | --- | | Concepts clés | Ensemble, valeurs, respect, responsabilité, communauté. | Collaboration, partenariat, coopération, échange, lien. | | Périmètre | Instruction scolaire + Éducation (vision globale de l'enfant). | Principalement Éducation (comportement, règles de vie). | | Objectif | Réussite globale et bien-être de l'enfant. | Préparation de l'élève à l'apprentissage (scolarisabilité). |

      Le clivage instruction/éducation : Les enseignants attendent souvent des parents qu'ils préparent l'enfant à son rôle d'élève (discipline, autonomie), tandis qu'ils conservent la primauté sur l'instruction.

      Les parents, de leur côté, ne distinguent pas aussi nettement l'enfant de l'élève.

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      3. L'Intérêt versus l'Implication : Le Paradoxe de l'Action

      Il existe un décalage significatif entre l'adhésion de principe et la pratique réelle.

      Intérêt déclaré : Massif (81 % des parents, 94 % des enseignants).

      Implication effective : Seul un tiers des parents et des enseignants se disent "très impliqués".

      La "coéducation empêchée" : Environ 50 % des deux groupes sont intéressés mais peu impliqués.

      Ce phénomène s'explique par un manque de clarté sur les "règles du jeu" et les finalités de cette coopération.

      Facteurs d'éloignement : Les parents les plus en retrait sont souvent ceux dont les enfants sont au collège, ceux qui sont peu familiers avec l'institution ou dont les enfants sont en difficulté scolaire (paradoxe de l'éloignement).

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      4. Analyse des Freins et Obstacles

      L'enquête identifie plusieurs barrières qui freinent le déploiement d'une coéducation fluide.

      Pour les Parents

      Manque de temps : Horaires de travail décalés ou indisponibilité des enseignants.

      Sentiment d'illégitimité : Certains parents ne se sentent pas autorisés à franchir le "seuil symbolique" de l'école (effet portail).

      Invisibilité : 40 % des parents pensent que les autres parents manquent d'intérêt, ce qui traduit une difficulté à créer une dynamique collective.

      Pour les Enseignants

      Crainte de l'intrusion : Peur que les parents ne s'immiscent dans les méthodes pédagogiques ou ne contrôlent le travail de l'enseignant.

      Vulnérabilité et solitude : Sentiment de solitude face à des parents parfois perçus comme agressifs ou "colonisateurs", aggravé par des événements traumatisants (assassinat de Samuel Paty, suicides dans la profession).

      Manque de formation : La relation avec les familles n'est pas une compétence innée et n'est pas suffisamment valorisée institutionnellement.

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      5. Leviers et Perspectives d'Amélioration

      Pour transformer la coéducation en un levier d'égalité des chances, plusieurs pistes sont avancées par les acteurs.

      Outils et Modalités

      Numérique : Les parents sont favorables à des applications favorisant l'interactivité et la communication immédiate (suivi des difficultés, cahiers de vie en ligne).

      Cependant, l'outil ne doit pas remplacer le lien humain, notamment pour les familles éloignées de l'écrit.

      Guides pratiques : Les enseignants privilégient l'élaboration de guides précisant les rôles et responsabilités de chacun.

      Informel et convivialité : Le développement de "cafés des parents", de lieux passerelles ou d'activités de loisirs (sport, jeux de société en maternelle) aide à briser les barrières symboliques.

      Recommandations Institutionnelles

      1. Co-construction : La coéducation ne doit pas être descendante. Les parents doivent être associés à la définition des projets dès leur genèse.

      2. Explicitation des rôles : Clarifier le périmètre d'intervention de chaque partie pour construire une relation de confiance.

      3. Gestion du temps : Aménager des temps dédiés à cette mission pour éviter qu'elle ne soit perçue comme une surcharge chronophage.

      4. Médiation : Utiliser des tiers (CPE, associations, parents traducteurs dans les contextes migratoires) pour faciliter le lien et la compréhension mutuelle.

      La Place de l'Élève

      L'enfant ne doit pas être un simple objet de communication entre adultes.

      Il doit être associé à la coéducation de manière différenciée selon son âge et le sujet traité (particulièrement sur les méthodes de travail et l'autonomie), tout en préservant la légitimité des parents sur les questions éducatives sensibles.

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      Conclusion

      La coéducation est perçue par tous comme un facteur clé de la réussite des élèves (troisième priorité des parents, deuxième pour les enseignants).

      Toutefois, son succès dépend de la capacité de l'institution à sortir des routines pour proposer des conditions concrètes de dialogue, de formation et de reconnaissance du rôle des familles, dans toute leur diversité.

    1. Reviewer #1 (Public review):

      Summary:

      Morgan et al. studied how paternal dietary alteration influenced testicular phenotype, placental and fetal growth using a mouse model of paternal low protein diet (LPD) or Western Diet (WD) feeding, with or without supplementation of methyl-donors and carriers (MD). They found diet- and sex-specific effects of paternal diet alteration. All experimental diets decreased paternal body weight and the number of spermatogonial stem cells, while fertility was unaffected. WD males (irrespective of MD) showed signs of adiposity and metabolic dysfunction, abnormal seminiferous tubules, and dysregulation of testicular genes related to chromatin homeostasis. Conversely, LPD induced abnormalities in the early placental cone, fetal growth restriction, and placental insufficiency, which were partly ameliorated by MD. The paternal diets changed the placental transcriptome in a sex-specific manner and led to a loss of sexual dimorphism in the placental transcriptome. These data provide a novel insight into how paternal health can affect the outcome of pregnancies, which is often overlooked in prenatal care.

      Strengths:

      The authors have performed a well-designed study using commonly used mouse models of paternal underfeeding (low protein) and overfeeding (Western diet). They performed comprehensive phenotyping at multiple timepoints, including the fathers, the early placenta, and the late gestation feto-placental unit. The inclusion of both testicular and placental morphological and transcriptomic analysis is a powerful, non-biased tool for such exploratory observational studies. The authors describe changes in testicular gene expression revolving around histone (methylation) pathways that are linked to altered offspring development (H3.3 and H3K4), which is in line with hypothesised paternal contributions to offspring health. The authors report sex differences in control placentas that mimic those in humans, providing potential for translatability of the findings. The exploration of sexual dimorphism (often overlooked) and its absence in response to dietary modification is novel and contributes to the evidence-base for the inclusion of both sexes in developmental studies.

      Weaknesses:

      The data are overall consistent with the conclusions of the authors. The paternal and pregnancy data are discussed separately, instead of linking the paternal phenotype to offspring outcomes. Some clarifications regarding the methods and the model would improve the interpretation of the findings.

      (1) The authors insufficiently discuss their rationale for studying methyl-donors and carriers as micronutrient supplementation in their mouse model. The impact of the findings would be better disseminated if their role were explained in more detail.

      (2) It is unclear from the methods exactly how long the male mice were kept on their respective diets at the time of mating and culling. Male mice were kept on the diet between 8 and 24 weeks before mating, which is a large window in which the males undergo a considerable change in body weight (Figure 1A). If males were mated at 8 weeks but phenotyped at 24 weeks, or if there were differences between groups, this complicates the interpretation of the findings and the extrapolation of the paternal phenotype to changes seen in the fetoplacental unit. The same applies to paternal age, which is an important known factor affecting male fertility and offspring outcomes.

      (3) The male mice exhibited lower body weights when fed experimental diets compared to the control diet, even when placed on the hypercaloric Western Diet. As paternal body weight is an important contributor to offspring health, this is an important confounder that needs to be addressed. This may also have translational implications; in humans, consumption of a Western-style diet is often associated with weight gain. The cause of the weight discrepancy is also unaddressed. It is mentioned that the isocaloric LPD was fed ad libitum, while it is unclear whether the WD was also fed ad libitum, or whether males under- or over-ate on each experimental diet.

      (4) The description and presentation of certain statistical analyses could be improved.

      (i) It is unclear what statistical analysis has been performed on the time-course data in Figure 1A (if any). If one-way ANOVA was performed at each timepoint (as the methods and legend suggest), this is an inaccurate method to analyse time-course data.

      (ii) It is unclear what methods were used to test the relative abundance of microbiome species at the family level (Figure 2L), whether correction was applied for multiple testing, and what the stars represent in the figure. 3) Mentioning whether siblings were used in any analyses would improve transparency, and if so, whether statistical correction needed to be applied to control for confounding by the father.

    2. Author response:

      Public Reviews: 

      Reviewer #1 (Public review):

      Summary:

      Morgan et al. studied how paternal dietary alteration influenced testicular phenotype, placental and fetal growth using a mouse model of paternal low protein diet (LPD) or Western Diet (WD) feeding, with or without supplementation of methyl-donors and carriers (MD). They found diet- and sex-specific effects of paternal diet alteration. All experimental diets decreased paternal body weight and the number of spermatogonial stem cells, while fertility was unaffected. WD males (irrespective of MD) showed signs of adiposity and metabolic dysfunction, abnormal seminiferous tubules, and dysregulation of testicular genes related to chromatin homeostasis. Conversely, LPD induced abnormalities in the early placental cone, fetal growth restriction, and placental insufficiency, which were partly ameliorated by MD. The paternal diets changed the placental transcriptome in a sex-specific manner and led to a loss of sexual dimorphism in the placental transcriptome. These data provide a novel insight into how paternal health can affect the outcome of pregnancies, which is often overlooked in prenatal care.

      Strengths:

      The authors have performed a well-designed study using commonly used mouse models of paternal underfeeding (low protein) and overfeeding (Western diet). They performed comprehensive phenotyping at multiple timepoints, including the fathers, the early placenta, and the late gestation feto-placental unit. The inclusion of both testicular and placental morphological and transcriptomic analysis is a powerful, non-biased tool for such exploratory observational studies. The authors describe changes in testicular gene expression revolving around histone (methylation) pathways that are linked to altered offspring development (H3.3 and H3K4), which is in line with hypothesised paternal contributions to offspring health. The authors report sex differences in control placentas that mimic those in humans, providing potential for translatability of the findings. The exploration of sexual dimorphism (often overlooked) and its absence in response to dietary modification is novel and contributes to the evidence-base for the inclusion of both sexes in developmental studies.

      Weaknesses:

      The data are overall consistent with the conclusions of the authors. The paternal and pregnancy data are discussed separately, instead of linking the paternal phenotype to offspring outcomes. Some clarifications regarding the methods and the model would improve the interpretation of the findings.

      (1) The authors insufficiently discuss their rationale for studying methyl-donors and carriers as micronutrient supplementation in their mouse model. The impact of the findings would be better disseminated if their role were explained in more detail.

      We acknowledge the Reviewer’s comments regarding the amount of detail in support of the inclusion of methyl carriers and donors within our diet. Therefore, we will revise the manuscript to include more justification, especially within the Introduction section, for their inclusion.

      (2) It is unclear from the methods exactly how long the male mice were kept on their respective diets at the time of mating and culling. Male mice were kept on the diet between 8 and 24 weeks before mating, which is a large window in which the males undergo a considerable change in body weight (Figure 1A). If males were mated at 8 weeks but phenotyped at 24 weeks, or if there were differences between groups, this complicates the interpretation of the findings and the extrapolation of the paternal phenotype to changes seen in the fetoplacental unit. The same applies to paternal age, which is an important known factor affecting male fertility and offspring outcomes.

      We thank the Reviewer for their comments regarding the ages of the males analysed. We will provide more detailed descriptions of the males in our manuscript. However, all male ages were balanced across all groups.

      (3) The male mice exhibited lower body weights when fed experimental diets compared to the control diet, even when placed on the hypercaloric Western Diet. As paternal body weight is an important contributor to offspring health, this is an important confounder that needs to be addressed. This may also have translational implications; in humans, consumption of a Western-style diet is often associated with weight gain. The cause of the weight discrepancy is also unaddressed. It is mentioned that the isocaloric LPD was fed ad libitum, while it is unclear whether the WD was also fed ad libitum, or whether males under- or over-ate on each experimental diet.

      We agree with the Reviewer that the general trend towards a lighter body weight for our experimental animals is unexpected. We can confirm that all diets were fed ad libitum. However, as males were group housed, we were unable to measure food consumption for individual males. We also observed that for males fed the high fat diets, they often shredded significant quantities of their diet, rather than eating it, so preventing accurate measurement of food intake.

      We also agree with the Reviewer that body weight can be a significant confounder for many paternal and offspring parameters. However, while the experimental males did become lighter, there were no statistical differences between groups in mean body weight. As such, body weight was not included as a variable within our statistical analysis.

      (4) The description and presentation of certain statistical analyses could be improved.

      (i) It is unclear what statistical analysis has been performed on the time-course data in Figure 1A (if any). If one-way ANOVA was performed at each timepoint (as the methods and legend suggest), this is an inaccurate method to analyse time-course data.

      (ii) It is unclear what methods were used to test the relative abundance of microbiome species at the family level (Figure 2L), whether correction was applied for multiple testing, and what the stars represent in the figure. 3) Mentioning whether siblings were used in any analyses would improve transparency, and if so, whether statistical correction needed to be applied to control for confounding by the father.

      We apologize for the lack of clarity regarding the statistical analyses. Going forward, we will revise the manuscript and include a more detailed description of the different analyses, the inclusion of siblings, and the correction for multiple testing.

      Reviewer #2 (Public review):

      Summary:

      The authors investigated the effects of a low-protein diet (LPD) and a high sugar- and fat-rich diet (Western diet, WD) on paternal metabolic and reproductive parameters and fetoplacental development and gene expression. They did not observe significant effects on fertility; however, they reported gut microbiota dysbiosis, alterations in testicular morphology, and severe detrimental effects on spermatogenesis. In addition, they examined whether the adverse effects of these diets could be prevented by supplementation with methyl donors. Although LPD and WD showed limited negative effects on paternal reproductive health (with no impairment of reproductive success), the consequences on fetal and placental development were evident and, as reported in many previous studies, were sex-dependent.

      Strengths:

      This study is of high quality and addresses a research question of great global relevance, particularly in light of the growing concern regarding the exponential increase in metabolic disorders, such as obesity and diabetes, worldwide. The work highlights the importance of a balanced paternal diet in regulating the expression of metabolic genes in the offspring at both fetal and placental levels. The identification of genes involved in metabolic pathways that may influence offspring health after birth is highly valuable, strengthening the manuscript and emphasizing the need to further investigate long-term outcomes in adult offspring.

      The histological analyses performed on paternal testes clearly demonstrate diet-induced damage. Moreover, although placental morphometric analyses and detailed histological assessments of the different placental zones did not reveal significant differences between groups, their inclusion is important. These results indicate that even in the absence of overt placental phenotypic changes, placental function may still be altered, with potential consequences for fetal programming.

      Weaknesses:

      Overall, this manuscript presents a rich and comprehensive dataset; however, this has resulted in the analysis of paternal gut dysbiosis remaining largely descriptive. While still valuable, this raises questions regarding why supplementation with methyl donors was unable to restore gut microbial balance in animals receiving the modified diets.

      We thank the Reviewer for their considered thoughts on the gut dysbiosis induced in our models the minimal impact of the methyl donors and carriers. We will include additional text within the Discussion to acknowledge this. However, at this point in time, we are unsure as to why the methyl donors had minimal impact. It could be that the macronutrients (i.e. protein, fat, carbohydrates) have more of an influence on gut bacterial profiles than micronutrients. Alternatively, due to the prolonged nature of our feeding regimens, any initial influences of the methyl donors may become diluted out over time. We will amend the text to reflect these potential factors.

    1. R0:

      Reviewer #1:

      The article is relevant and timely. Please use COREQ guidelines/other community guidelines for qualitative research listed on the EQUATOR network to improve the rigour and flow of the article. Additional suggestions for your consideration are listed below. 1. The rationale for the article as well as research question needs to be made more explicit. Additionally, the novelty of the article is relatively unclear. 2. The use of a framework devised for infectious diseases needs to be examined- why was this framework chosen? 3. Line 52-54: It may be useful to examine the cause of death in context of age; additionally, the later use of terms like young-old-middle aged are ambiguous later in the article, please provide an operational definition. 4. Line 54-57: The reason for prioritising lived experience is unclear. The logical flow establishing the rationale for this inquiry needs strengthening. 5. The linkages of NCDs with urbanisation need more citation and can be explored better. The authors may consider reassessing the introduction section which seems to appear definitive without providing a research gap. 6. Why is this research needed? What makes it relevant for global health? This needs to be elucidated in the text. 7. The use of the word "drug peddler" should be in quotes at least at first use, to ensure that it is seen in its local context. 8. The narrative findings are rich and well presented- the global health impact of these findings/future paths based on these findings need to be be made explicit.

      Reviewer #2:

      This manuscript is well-written and engaging, and I would like to commend the authors for presenting such rich and dense information.

      Here are my comments, which I believe will enhance the content of this manuscript.

      1. Page 2, line 27 (abstract section): The term "oppression" appears quite strong, and I do not find any evidence supporting it in the results section. Could the authors clarify what they mean by "oppression"? Additionally, the sentence “as gender norms and experiences of masculinities and femininities reflected the division of roles and access to resources by men and women, which in turn shaped their ability to seek early and better healthcare interventions” seems to contradict the notion of oppression if participants are able to seek early and improved healthcare interventions.

      2. Page 6, line 133: typo: a dot is in the middle of the sentence. Consider removing.

      3. Page 7 on selection of study participants: How did researchers identify participants based on diabetes and hypertension criteria, given that these medical conditions require a confirmed diagnosis from health professionals? Additionally, were any participants identified using a snowball sampling technique?

      4. It is mentioned that this study was conducted as part of the broad ARISE project. Can the authors clarify whether the study participants were enrolled into the main project and if so, to what extent this initial involvement of the main study had impacted the data collection?

      5. An important section on data collection is missing. I would like authors to include this in order to explain what specific data were collected at each round, why was it necessary to split data collection over different timepoints, where and in which language were data collected, how long lasted interviews on average, and who collected the data, any bias that may result from the way the study participants perceived the data collection team during fieldwork?

      6. The data analysis section requires further clarification. It would be beneficial if the authors could elaborate on their data coding strategy. It appears that a deductive approach was employed, yet they did not explain how the coding frame was initially developed before its application to the data, nor did they specify who conducted these procedures. Additionally, information regarding the software used to facilitate the data analysis is missing.

      7. The use of relevant qualitative data reporting guideline is missing. This is important to include as it will help ensure that ensure that the methods and findings are clearly and transparently communicated in the manuscript.

      8. In relation to adapting the framework for Infectious Diseases of Poverty Research, it would be beneficial if the authors could clarify the specific adaptations made to the framework and how it was applied during data analysis.

      9. They mentioned that participants who faced distress during the interviews were allowed to grieve and were consoled. How many participants developed such condition during interviews at which round of data collection, and to what extent did this impact on data collected?

      10. This study seeks to explore the lived experiences of individuals affected by NCDs in informal settlements. However, the findings presented in the results section, particularly the initial part titled "lived experience of NCD," fall short of adequately conveying the participants' current experiences with these health conditions. Firstly, the authors include excerpts from three informants but fail to demonstrate how these accounts are representative of all study participants. Secondly, the discussion of informants' lives prior to developing their current health conditions renders the information less pertinent to the study's objectives. It would be beneficial if the authors could focus on participants current lives to discuss the intersectionality of NCDs with social, economic, and gender factors within the participants' living environments, and how these elements contribute to shaping the management of their health conditions. This way of presenting the findings restricts our understanding, as the same observation is reiterated throughout the discussion section.

      11. In the context of chronic disease, it can be assumed that participants with diabetes, hypertension, or comorbidities might encounter similar yet distinct challenges. It would be beneficial for the authors to discuss the findings across these cases, highlighting both similarities and differences, and to establish a hierarchy within the data.

      12. The use of terms like "people," "women," or "men" in the results section can be misleading, as they might suggest that the findings apply to the general population. Instead, it is advisable to use terms such as "informants," "study participants," or "men" or "women in this study" to ensure that the information reported is limited to those individuals who participated in this data collection.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript examines the passage of an intrathecal CSF tracer into skull bone marrow, cortex, and venous compartments using serial MRI at multiple time points. The study builds on recent anatomical and imaging work suggesting direct communication between CSF spaces and bone marrow in the skull. It extends these observations to a larger, clinically heterogeneous human cohort. The imaging methodology is carefully executed, and the dataset is rich. The findings are potentially important for understanding CSF drainage pathways and their associations with inflammation, sleep quality, and cognition. However, key aspects of the interpretation - particularly regarding tracer kinetics and the definition of "clearance" - require clarification and, in my view, reconsideration.

      Strengths:

      (1) The study employs a well-established intrathecal contrast-enhanced MRI approach with multiple post-injection time points, enabling the assessment of regional tracer dynamics.

      (2) The analysis of skull bone marrow in distinct anatomical regions (near the superior sagittal sinus, lateral fissure, and cisterna magna) is novel and informative.

      (3) The cohort size is relatively large for an intrathecal tracer study in humans, and the authors make commendable efforts to relate imaging findings to clinical variables such as inflammation, sleep quality, and cognitive performance.

      (4) The manuscript is clearly written, the figures are informative, and the discussion is well grounded in recent anatomical and experimental literature on skull-meningeal connections.

      Weaknesses:

      The central interpretation that a higher percentage increase in skull bone marrow tracer signal at 4.5 hours reflects reduced clearance is not convincingly justified. Based on the existing CSF tracer literature, the 4-6 hour time window is generally considered an enrichment or inflow phase rather than a clearance phase. Later time points (15 and 39 hours) are more likely to reflect clearance or washout. An alternative interpretation - that a higher signal at 4.5 hours reflects more pronounced tracer entry - should be considered and discussed.

      Relatedly, the manuscript lacks a clear conceptual separation between tracer enrichment and clearance phases across time points. If 4.5 hours is intended to represent clearance, this assumption requires more vigorous justification and alignment with prior work.

      CSF passage via the nasal/olfactory pathway is insufficiently discussed. Previous human imaging studies have questioned the importance of peri-olfactory CSF clearance, yet the present findings suggest delayed enrichment in the nasal turbinates. This discrepancy should be explicitly addressed, including a discussion of potential methodological limitations (e.g., timing of acquisitions, ROI definition, or sensitivity to slow drainage pathways).

      More generally, given the descriptive nature of the study and the limited temporal sampling, some conclusions regarding directionality and efficiency of "drainage" may be overstated and would benefit from more cautious framing.

    1. R0:

      Reviewer #1: Title: Probabilistic Forecasting of Monthly Dengue Cases Using Epidemiological and Climate Signals: A BiLSTM–Naive Bayes Model Versus Mechanistic and Count-Model Baselines. Manuscript Number: PGPH-D-25-03170

      This manuscript presents a rigorous comparative study of probabilistic forecasting models for monthly dengue incidence in Freetown, Sierra Leone, covering the period 2015–2025. It evaluates four major model classes—NB-GLM, INGARCH-NB, Renewal-NB, and BiLSTM-NB—under a leakage-safe rolling-origin evaluation. The article demonstrates strong methodological maturity, careful control of data leakage, and thorough probabilistic evaluation using proper scoring rules, interval coverage, sharpness metrics, PIT diagnostics, and Diebold–Mariano tests. The manuscript is generally well-written, technically sound, and addresses an important operational public health problem. It positions itself as one of the few works offering aligned comparisons of mechanistic, statistical, and deep-learning models under realistic constraints for West African dengue surveillance. This article presents a methodologically rigorous comparison of four probabilistic forecasting approaches—NB-GLM, INGARCH-NB, Renewal-NB, and BiLSTM-NB—applied to monthly dengue case data from Freetown, Sierra Leone (2015–2025). The study addresses an important gap by evaluating mechanistic, statistical, and deep-learning models under aligned, leakage-safe conditions. While the work is comprehensive and technically strong, several critical issues affect its accessibility, interpretability, and broader applicability.

      Strengths The study excels in methodological rigor. Its strict leakage safeguards, careful feature-timing rules, and use of expanding-window rolling-origin evaluation significantly strengthen reliability. The inclusion of proper scoring rules, interval coverage, sharpness metrics, PIT histograms, and Diebold–Mariano tests provides a complete probabilistic evaluation rarely seen in dengue forecasting studies. The horizon-specific findings—INGARCH-NB outperforming at 1–2 months and BiLSTM-NB excelling at 3 months—are well supported by aligned comparisons and statistical significance tests. The transparency of data, code, and alignment artefacts enhances reproducibility and credibility. Additionally, the manuscript offers practical guidance for operational forecasting, including a realistic “light climate” input strategy suitable for resource-limited settings.

      Limitations Despite its strengths, the manuscript is heavily technical, with extensive mathematical exposition in the main text. This may limit accessibility for public-health practitioners who are likely part of the target audience. The mechanistic renewal model is presented as a baseline but is arguably underspecified; the use of a short, fixed 3-month kernel may not realistically capture dengue’s generation interval dynamics, likely contributing to its poor performance. This limits the interpretive value of the mechanistic comparison. This limitation should be addressed. The study’s climate treatment, while intentionally conservative, may underexploit important environmental drivers. Although justified operationally, this constraint restricts exploration of potentially meaningful lag structures or seasonal climate anomalies. The analysis is limited to a single city and monthly data frequency, raising questions about generalizability across geographies with different climate patterns and dengue transmission dynamics. Moreover, the monthly temporal resolution may obscure rapid outbreak shifts, possibly disadvantaging mechanistic and hybrid models that rely on finer-grained dynamics. This should be addressed. The manuscript makes a valuable and original contribution to dengue forecasting, offering robust methodological innovations and practical insights for real-time surveillance systems. However, improved clarity, stronger justification for mechanistic assumptions, and expanded discussion of generalizability would enhance its usefulness and scholarly impact. With revisions to improve accessibility and contextual depth, the study is well positioned for publication and for informing operational forecasting practice in similar settings.

      Reviewer #2: 1. What is PIT in the abstract stand for? The authors should avoid using abbreviations in the abstract. 2. The authors should providing some additional analysis, such as experimenting with alternative or longer serial-interval kernels, or simple sensitivity checks (e.g., different window lengths, or, if possible, finer temporal resolution). 3. Please, justifies the small climate feature set, mentioning any exploratory work with larger sets. 4. The authors should add a clearly labelled missing-data handling subsection that specifies: The imputation method, the number of imputed months, and how they were used in training/evaluation, plus any sensitivity. 5. While the architecture, optimization, and calibration steps are described, the process for choosing hyperparameters is not fully audit-ready. 6. I recommend that the authors conduct an additional experiment to demonstrate the generalizability of the proposed model.

    1. Reviewer #1 (Public review):

      Summary:

      In the work from Qiu et al., a workflow aimed at obtaining the stabilization of a simple small protein against mechanical and chemical stressors is presented.

      Strengths:

      The workflow makes use of state-of-the-art AI-driven structure generation and couples it with more classical computational and experimental characterizations in order to measure its efficacy. The work is well presented, and the results are thorough and convincing.

      Weaknesses:

      I will comment mostly on the MD results due to my expertise.

      The Methods description is quite precise, but is missing some important details:

      (1) Version of GROMACS used.

      (2) The barostat used.

      (3) pH at which the system is simulated.

      (4) The pulling is quite fast (but maybe it is not a problem)

      (5) What was the value for the harmonic restraint potential? 1000 is mentioned for the pulling potential, but it is not clear if the same value is used for the restraint, too, during pulling.

      (6) The box dimensions.

      From this last point, a possible criticism arises: Do the unfolded proteins really still stay far enough away from themselves to not influence the result? This might not be the major influence, but for correctness, I would indicate the dimensions of the box in all directions and plot the minimum distance of the protein from copies of itself across the boundary conditions over time.

      Additionally, no time series are shown for the equilibration phases (e.g., RMSD evolution over time), which would empower the reader to judge the equilibration of the system before either steered MD or annealing MD is performed.

    2. Reviewer #3 (Public review):

      Summary:

      Qiu et al. present a hierarchical framework that combines AI and molecular dynamics simulation to design an α-helical protein with enhanced thermal, chemical, and mechanical stability. Strategically, chemical modification by incorporating additional α-helix, site-specific salt bridges, and metal coordination further enhanced the stability. The experimental validation using single-molecule force spectroscopy and CD melting measurements provides fundamental physical chemical insights into the stabilization of α-helices. Together with the group's prior work on super-stable β strands (https://www.nature.com/articles/s41557-025-01998-3), this research provides a comprehensive toolkit for protein stabilization. This framework has broad implications for designing stable proteins capable of functioning under extreme conditions.

      Strengths:

      The study represents a complete framework for stabilizing the fundamental protein elements, α-helices. A key strength of this work is the integration of AI tools with chemical knowledge of protein stability.<br /> The experimental validation in this study is exceptional. The single-molecule AFM analysis provided a high-resolution look at the energy landscape of these designed scaffolds. This approach allows for the direct observation of mechanical unfolding forces (exceeding 200 pN) and the precise contribution of individual chemical modifications to global stability. These measurements offer new, fundamental insights into the physicochemical principles that govern α-helix stabilization.

      Weaknesses:

      (1) The authors report that appending an additional helix increases the overcall stability of the α-helical protein. Could the author provide a more detailed structural explanation for this? Why does the mechanical stability increase as the number of helixes increase? Is there a reported correlation between the number of helices (or the extent of the hydrophobic core) and the stability?

      (2) The author analyzed both thermal stability and mechanical stability. It would be helpful for the author to discuss the relationship between these two parameters in the context of their design. Since thermal melting probes equilibrium stability (ΔG), while mechanical stability probes the unfolding energy barriers along the pulling coordinate.

      (3) While the current study demonstrates a dramatic increase in global stability, the analysis focuses almost exclusively on the unfolding (melting) process. However, thermodynamic stability is a function of both folding (kf) and unfolding (ku) rates. It remains unclear whether the observed ultrastability is primarily driven by a drastic decrease in the unfolding rate (ku) or if the design also maintains or improves the folding rate (kf)?

      (4) The authors chose the spectrin repeat R15 as the starting scaffold for their design. R15 is a well-established model known for its "ultra-fast" folding kinetics, with folding rates (kf ~105s), near three orders of magnitude faster than its homologues like R17 (Scott et.al., Journal of molecular biology 344.1 (2004): 195-205). Does the newly designed protein, with its additional fourth helix and site-specific chemical modifications, retain the exceptionally high folding rate of the parent R15?

    1. Optimiser la Mémorisation et l'Apprentissage en Milieu Scolaire : Stratégies et Leviers Neurocognitifs

      Synthèse de Direction

      Ce document synthétise les stratégies pédagogiques visant à contrer le phénomène de l'oubli chez les élèves et à renforcer l'ancrage des connaissances sur le long terme.

      Le constat de départ souligne que l'oubli n'est pas une défaillance de l'élève, mais un processus biologique naturel illustré par la "courbe de l'oubli" d'Ebbinghaus.

      L'enjeu majeur réside dans la lutte contre l'illusion du savoir — cette fausse impression de maîtrise issue d'une relecture passive du cours.

      Les points clés pour transformer les pratiques d'apprentissage sont :

      La Métacognition : Enseigner explicitement aux élèves le fonctionnement de leur cerveau pour les rendre acteurs de leurs progrès.

      L'Enseignement Explicite : Baliser clairement les notions essentielles pour éviter la confusion entre la trace écrite et les exemples.

      L'Apprentissage Actif : Privilégier la fragmentation des informations et la réactivation régulière via des rituels (boîte à questions, mots de passe).

      La Récupération en Classe : Intégrer des temps de révision et d'analyse réflexive après les évaluations pour consolider les acquis.

      --------------------------------------------------------------------------------

      1. Comprendre les Obstacles à l'Apprentissage

      La Courbe de l'Oubli et le Fonctionnement Cérébral

      L'oubli est la réaction normale du cerveau en l'absence de réactivation régulière des connaissances.

      Selon les sources, il ne s'agit pas d'un manque de travail ou de talent, mais d'une caractéristique neurologique. L'apprentissage est décrit comme un acte physique demandant de l'investissement, des efforts et générant des émotions (joie de comprendre ou frustration de l'échec).

      L'Illusion du Savoir

      De nombreux élèves confondent « relire » et « apprendre ». Des techniques passives, telles que la relecture multiple (jusqu'à 5 ou 7 fois) ou le surlignage de mots en couleurs, créent une illusion de maîtrise sans garantir la mémorisation à long terme. Cette approche est jugée inefficace par rapport aux méthodes de récupération active.

      --------------------------------------------------------------------------------

      2. Le Levier de la Métacognition

      Pour que les élèves réussissent, il est impératif de leur apprendre comment apprendre.

      Cours de méthodologie : La mise en place d'une heure hebdomadaire (ou des séances intégrées à la vie de classe) permet d'aborder des questions fondamentales : Pourquoi j'oublie ? Comment fonctionne ma mémoire ? Quelles stratégies adopter ?

      La Pensée Positive : Développer l'idée que le progrès est accessible à tous, quel que soit le niveau initial, renforce l'état d'esprit nécessaire à l'apprentissage.

      --------------------------------------------------------------------------------

      3. Stratégies de Classe pour Favoriser la Mémorisation

      L'Enseignement Explicite

      Le contenu d'un cahier peut être confus pour un élève (mélange d'exercices, d'exemples et de leçons). Les enseignants doivent :

      • Identifier clairement ce qui doit être retenu (encadrés, soulignage en rouge).

      • Prendre 5 minutes en fin d'heure pour résumer l'essentiel de la séance.

      Gestion de l'Attention et Fragmentation

      Le cerveau traite mieux les informations courtes et structurées :

      Règle de concision : Formuler les points importants en phrases d'environ une douzaine de mots maximum.

      Alternance attentionnelle : Découper le cours en séquences (explications courtes, activités, retours au calme) pour maintenir la concentration.

      Apprentissage actif : Un élève actif retient mieux qu'un élève passif. Les mini-tâches, l'échange et la coopération sont à privilégier.

      --------------------------------------------------------------------------------

      4. Rituels de Réactivation Régulière

      La répétition est la clé de l'ancrage mémoriel. Plusieurs rituels simples peuvent être instaurés :

      | Rituel | Description | Objectif | | --- | --- | --- | | La boîte à questions | Un élève responsable crée des cartes flash sur le cours. En début d'heure, il interroge ses camarades en piochant 4 ou 5 questions. | Autonomie et révision continue des notions de l'année. | | Le mot de passe | Pour entrer en classe, l'élève doit donner la définition d'un mot complexe vu la veille. | Réactivation immédiate et interaction ludique. | | QCM Numériques | Utilisation d'outils (ex: Quiz Wizard) pour générer des tests rapides sur Pronote. | Entraînement régulier sans surcharge de correction pour le professeur. |

      --------------------------------------------------------------------------------

      5. Techniques de Révision et de Consolidation

      Les révisions ne doivent pas être cantonnées au domicile ; elles doivent s'intégrer au temps scolaire.

      Technique de la feuille blanche : À la fin d'un chapitre, les élèves notent de mémoire tout ce qu'ils ont retenu sur une feuille vierge pour identifier leurs lacunes.

      Méthode Feynman : Apprendre en enseignant aux autres. Les élèves s'expliquent mutuellement des notions avec des mots simples.

      Ateliers de révision : Rotation toutes les 15 minutes sur différents supports (cartes de géographie effaçables, création de flashcards, jeux pédagogiques comme le Timeline ou les dominos).

      --------------------------------------------------------------------------------

      6. Analyse Post-Évaluation et Réflexion

      Le processus d'apprentissage ne s'arrête pas à la note. Pour progresser, l'élève doit analyser ses erreurs :

      Écrits réflexifs : Après une évaluation importante (ex: brevet blanc), les élèves remplissent une fiche d'analyse sur leur préparation, le temps passé et les méthodes utilisées.

      Auto-conseil : L'élève définit lui-même ses axes d'amélioration pour la prochaine évaluation.

      En conclusion, la réussite des élèves repose sur la compréhension du fonctionnement cérébral, la structuration explicite des savoirs, la réactivation constante et l'entraînement actif.

    1. État des lieux de l'enseignement privé en France : Enjeux de mixité, de performance et de financement

      Résumé Exécutif

      L'enseignement privé en France traverse une période de mutations structurelles et de débats politiques intenses.

      Bien que la part des élèves scolarisés dans le privé sous contrat demeure globalement stable à l'échelle nationale (environ 17 à 18 %), l'attractivité de ce secteur s'intensifie, particulièrement dans les grandes métropoles comme Paris.

      Cette dynamique est alimentée par une recherche d'excellence académique, une offre pédagogique spécifique (bilinguisme, encadrement) et une déception croissante vis-à-vis du système public, marqué par des problématiques de non-remplacement des enseignants.

      Toutefois, cette attractivité s'accompagne d'une homogénéisation sociale accrue au sein des établissements privés, comme le révèle la publication récente de l'Indice de Position Sociale (IPS).

      Le débat public se cristallise désormais sur la question de la mixité sociale, le financement public (qui assure environ 75 % du budget du privé sous contrat) et la nécessité de renforcer les contrôles étatiques sur ces établissements, tout en préservant leur « caractère propre » garanti par la loi.

      --------------------------------------------------------------------------------

      1. La dynamique de l'enseignement privé : Chiffres et typologies

      Le paysage de l'enseignement privé se divise principalement en deux catégories, régies par des cadres légaux et des niveaux d'implication de l'État distincts.

      1.1 Distinction entre « sous contrat » et « hors contrat »

      Privé sous contrat d'association : Il représente l'immense majorité du secteur (environ 17 % des élèves).

      Ces établissements participent au service public d'éducation, suivent les programmes nationaux et voient leurs enseignants rémunérés par l'État.

      Privé hors contrat : Bien que minoritaire (un peu plus de 2 % des élèves aujourd'hui contre 1 % en 2015), ce secteur connaît une croissance notable.

      Il concerne environ 1 800 établissements sur les 57 000 que compte la France.

      1.2 Disparités territoriales

      La situation de l'enseignement privé varie considérablement selon les zones :

      Paris et grandes métropoles : Une forte pression démographique inversée.

      Alors que le public perd des classes, le privé affiche des listes d'attente importantes.

      Au collège Sévigné (Paris 5e), on compte 1 000 demandes pour seulement 150 places.

      Zones rurales et Bretagne : Dans certains territoires (Bretagne, Pays Basque), l'école privée est parfois la seule disponible dans la commune, accueillant ainsi tous les enfants du village et maintenant une mixité sociale de fait.

      --------------------------------------------------------------------------------

      2. Les facteurs d'attractivité et les motivations des parents

      Le choix du privé par les familles repose sur une combinaison de facteurs pédagogiques, institutionnels et sociaux.

      2.1 La quête de l'excellence et de l'encadrement

      Selon un sondage IPSOS de septembre 2023, les motivations principales sont :

      Recherche d'excellence : 23 %

      Enseignement religieux et tradition : 20 %

      Continuité familiale : 20 %

      Déception vis-à-vis du public : 10 %

      Les établissements de prestige, tels que le collège Sévigné, affichent des résultats exceptionnels (97 % de mentions « Très Bien » au brevet), s'appuyant sur une sélection sur dossier et une atmosphère d'exigence.

      2.2 La crise de l'offre publique

      Le non-remplacement des enseignants est un levier majeur de transfert vers le privé.

      En 2023, le Sénat rapporte une moyenne de 12 jours d'absence non remplacés dans le public contre 10 dans le privé.

      « Un élève qui fait l'ensemble de sa scolarité dans le public dans le 93 aura perdu un an de scolarité au total. »

      Cette baisse d'attractivité du métier d'enseignant dans le public, illustrée par des concours dont le nombre de candidats est inférieur au nombre de postes (notamment dans les académies de Créteil et Versailles), pousse les parents vers le privé pour garantir la continuité des cours.

      --------------------------------------------------------------------------------

      3. L'enjeu de la mixité sociale et l'Indice de Position Sociale (IPS)

      La publication de l'IPS en 2022, sous la contrainte d'une décision de justice, a mis en lumière une fracture sociale croissante.

      3.1 Comparaison des indices de position sociale

      L'IPS mesure les conditions socio-culturelles des familles (allant de 45 à 185).

      Plus il est élevé, plus le milieu est favorisé.

      | Niveau scolaire | IPS Moyen - Public | IPS Moyen - Privé sous contrat | | --- | --- | --- | | École primaire | 105,8 | 121,5 | | Collège | 100,9 | 125,0 | | Lycée | 105,0 | 125,0 |

      3.2 La « sécession scolaire »

      On observe une hausse significative de la part des élèves très favorisés dans le privé : ils représentaient 30 % des effectifs en 2003, contre 42 % aujourd'hui.

      Cette ségrégation sociale est particulièrement marquée en milieu urbain, où des collèges publics défavorisés et des collèges privés favorisés coexistent à moins de 15 minutes de marche l'un de l'autre.

      --------------------------------------------------------------------------------

      4. Cadre légal, financement et contrôle

      Le système repose sur un équilibre historique souvent qualifié de « paix scolaire », mais aujourd'hui remis en question.

      4.1 La Loi Debré (1959) et l'héritage de 1984

      La loi Debré a instauré le système de contrat entre l'État et le privé.

      En 1984, le projet de loi Savary, visant à créer un grand service public unifié, avait provoqué des manifestations massives (plus d'un million de personnes) pour la défense de « l'école libre », entraînant le retrait du texte.

      4.2 Un financement public prédominant

      L'État et les collectivités territoriales financent environ 75 % du budget des écoles privées sous contrat (soit un montant estimé entre 10 et 13 milliards d'euros).

      Pourtant, un élève du privé coûte globalement moins cher à l'État qu'un élève du public (un écart total d'environ 1,5 milliard d'euros), car certaines charges et structures diffèrent.

      4.3 Vers un renforcement des contrôles

      Historiquement faibles (seulement 5 établissements contrôlés en 2023 sur 7 500), les contrôles s'intensifient sous l'impulsion des récents rapports de la Cour des comptes et de scandales médiatisés (affaire Stanislas, Notre-Dame de Bétharram).

      Objectif : 40 % des établissements contrôlés d'ici 2027.

      Nature des contrôles : Utilisation des fonds publics, respect des programmes, vie scolaire et respect du « caractère propre ».

      --------------------------------------------------------------------------------

      5. Débats et perspectives politiques

      Le débat actuel se concentre sur la possibilité de conditionner le financement public à des objectifs de mixité sociale.

      Positions législatives : Des propositions de loi (notamment portées par les groupes socialistes et communistes au Sénat) ont visé à moduler les subventions en fonction de l'IPS des établissements.

      Ces textes ont été rejetés par la majorité sénatoriale de droite, qui privilégie la liberté de choix des parents et la spécificité du projet pédagogique privé.

      Le concept de « caractère propre » : Bien que non défini précisément par la loi, il garantit la liberté religieuse ou pédagogique (langues régionales, méthodes spécifiques) de l'établissement.

      Pour certains, c'est l'essence même du pluralisme ; pour d'autres, c'est un outil permettant l'évitement scolaire et la sélection des élèves.

      Recrutement et sélection : Contrairement au public, les directeurs du privé peuvent recruter leurs enseignants et sélectionner leurs élèves sur dossier, ce qui pose la question de l'égalité des armes entre les deux systèmes.

      En conclusion, si l'enseignement privé assure une mission de service public, son autonomie de gestion et sa capacité de sélection, couplées à un financement public massif, restent au cœur d'une tension politique majeure entre liberté d'enseignement et exigence d'égalité républicaine.

    1. Reviewer #2 (Public review):

      Summary:

      Stanojcic et al. investigate the origins of DNA replication in the unicellular parasite Trypanosoma brucei. They perform two experiments, stranded SNS-seq and DNA molecular combing. Further, they integrate various publicly available datasets, such as G4-seq and DRIP-seq, into their extensive analysis. Using this data, they elucidate the structure of the origins of replication. In particular, they find various properties located at or around origins, such as polynucleotide stretches, G-quadruplex structures, regions of low and high nucleosome occupancy, R-loops, and that origins are mostly present in intergenic regions. Combining their population-level SNS-seq and their single-molecule DNA molecular combing data, they elucidate the total number of origins as well as the number of origins active in a single cell.

      Strengths:

      (1) A very strong part of this manuscript is that the authors integrate several other datasets and investigate a large number of properties around origins of replication. Data analysis clearly shows the enrichment of various properties at the origins, and the manuscript concludes with a very well-presented model that clearly explains the authors' understanding and interpretation of the data.

      (2) The DNA combing experiment is an excellent orthogonal approach to the SNS-seq data. The authors used the different properties of the two experiments (one giving location information, one giving single-molecule information) well to extract information and contrast the experiments.

      (3) The discussion is exemplary, as the authors openly discuss the strengths and weaknesses of the approaches used. Further, the discussion serves its purpose of putting the results in both an evolutionary and a trypanosome-focused context.

      Weaknesses:

      I have major concerns about the origin of replication sites determined from the SNS-seq data. As a caveat, I want to state that, before reading this manuscript, SNS-seq was unknown to me; hence, some of my concerns might be misplaced.

      (1) I do not understand why SNS-seq would create peaks. Replication should originate in one locus, then move outward in both directions until the replication fork moving outward from another origin is encountered. Hence, in an asynchronous population average measurement, I would expect SNS data to be broad regions of + and -, which, taken together, cover the whole genome. Why are there so many regions not covered at all by reads, and why are there such narrow peaks?

      (2) I am concerned that up to 96% percent of all peaks are filtered away. If there is so much noise in the data, how can one be sure that the peaks that remain are real? Specifically, if the authors placed the same number of peaks as was measured randomly in intergenic regions, would 4% of these peaks pass the filtering process by chance?

      (3) There are 3 previous studies that map origins of replication in T. brucei. Devlin et al. 2016, Tiengwe et al. 2012, and Krasiļņikova et al. 2025 (https://doi.org/10.1038/s41467-025-56087-3), all with a different technique: MFA-seq. All three previous studies mostly agree on the locations and number of origins. The authors compared their results to the first two, but not the last study; they found that their results are vastly different from the previous studies (see Supplementary Figure 8A). In their discussion, the authors defend this discrepancy mostly by stating that the discrepancy between these methods has been observed in other organisms. I believe that, given the situation that the other studies precede this manuscript, it is the authors' duty to investigate the differences more than by merely pointing to other organisms. A conclusion should be reached on why the results are different, e.g., by orthogonally validating origins absent in the previous studies.

      (4) Some patterns that were identified to be associated with origins of replication, such as G-quadruplexes and nucleosomes phasing, are known to be biases of SNS-seq (see Foulk et al. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res. 2015;25(5):725-735. doi:10.1101/gr.183848.114).

      Are the claims well substantiated?:

      My opinion on whether the authors' results support their conclusions depends on whether my concerns about the sites determined from the SNS-seq data can be dismissed. In the case that these concerns can be dismissed, I do think that the claims are compelling.

      Impact:

      If the origins of replication prove to be distributed as claimed, this study has the potential to be important for two fields. Firstly, in research focused on T. brucei as a disease agent, where essential processes that function differently than in mammals are excellent drug targets. Secondly, this study would impact basic research analyzing DNA replication over the evolutionary tree, where T. brucei can be used as an early-divergent eukaryotic model organism.

    2. Author response:

      eLife Assessment

      The authors use sequencing of nascent DNA (DNA linked to an RNA primer, "SNS-Seq") to localise DNA replication origins in Trypanosoma brucei, so this work will be of interest to those studying either Kinetoplastids or DNA replication. The paper presents the SNS-seq results for only part of the genome, and there are significant discrepancies between the SNS-Seq results and those from other, previously-published results obtained using other origin mapping methods. The reasons for the differences are unknown and from the data available, it is not possible to assess which origin-mapping method is most suitable for origin mapping in T. brucei. Thus at present, the evidence that origins are distributed as the authors claim - and not where previously mapped - is inadequate.

      We would like to clarify a few points regarding our study. Our primary objective was to characterise the topology and genome-wide distribution of short nascent-strand (SNS) enrichments. The stranded SNS-seq approach provides the high strand-specific resolution required to analyse origins. The observation that SNS-seq peaks (potential origins) are most frequently found in intergenic regions is not an artefact of analysing only part of the genome; rather, it is a result of analysing the entire genome.

      We agree that orthogonal validation is necessary. However, neither MFA-seq nor TbORC1/CDC6 ChIP-on-chip has yet been experimentally validated as definitive markers of origin activity in T. brucei, nor do they validate each other. 

      Public Reviews:

      Reviewer #1 (Public review):

      In this paper, Stanojcic and colleagues attempt to map sites of DNA replication initiation in the genome of the African trypanosome, Trypanosoma brucei. Their approach to this mapping is to isolate 'short-nascent strands' (SNSs), a strategy adopted previously in other eukaryotes (including in the related parasite Leishmania major), which involves isolation of DNA molecules whose termini contain replication-priming RNA. By mapping the isolated and sequenced SNSs to the genome (SNS-seq), the authors suggest that they have identified origins, which they localise to intergenic (strictly, inter-CDS) regions within polycistronic transcription units and suggest display very extensive overlap with previously mapped R-loops in the same loci. Finally, having defined locations of SNS-seq mapping, they suggest they have identified G4 and nucleosome features of origins, again using previously generated data.

      Though there is merit in applying a new approach to understand DNA replication initiation in T. brucei, where previous work has used MFA-seq and ChIP of a subunit of the Origin Replication Complex (ORC), there are two significant deficiencies in the study that must be addressed to ensure rigour and accuracy.

      (1) The suggestion that the SNS-seq data is mapping DNA replication origins that are present in inter-CDS regions of the polycistronic transcription units of T. brucei is novel and does not agree with existing data on the localisation of ORC1/CDC6, and it is very unclear if it agrees with previous mapping of DNA replication by MFA-seq due to the way the authors have presented this correlation. For these reasons, the findings essentially rely on a single experimental approach, which must be further tested to ensure SNS-seq is truly detecting origins. Indeed, in this regard, the very extensive overlap of SNS-seq signal with RNA-DNA hybrids should be tested further to rule out the possibility that the approach is mapping these structures and not origins.

      (2) The authors' presentation of their SNS-seq data is too limited and therefore potentially provides a misleading view of DNA replication in the genome of T. brucei. The work is presented through a narrow focus on SNS-seq signal in the inter-CDS regions within polycistronic transcription units, which constitute only part of the genome, ignoring both the transcription start and stop sites at the ends of the units and the large subtelomeres, which are mainly transcriptionally silent. The authors must present a fuller and more balanced view of SNS-seq mapping across the whole genome to ensure full understanding and clarity.

      Regarding comparisons with previous work:

      Two other attempts to identify origins in T. brucei —ORC1/CDC6 binding sites (ChIP-on-chip, PMID: 22840408) and MFA-seq (PMID: 22840408, 27228154)—were both produced by the McCulloch group. These methods do not validate each other; in fact, MFA-seq origins overlap with only 4.4% of the 953 ORC1/CDC6 sites (PMID: 29491738). Therefore, low overlap between SNS-seq peaks and ORC1/CDC6 sites cannot disqualify our findings. Similar low overlaps are observed in other parasites (PMID: 38441981, PMID: 38038269, PMID: 36808528) and in human cells (PMID: 38567819).

      We also would like to emphasize that the ORC1/CDC6 dataset originally published (PMID: 22840408) is no longer available; only a re-analysis by TritrypDB exists, which differs significantly from the published version (personal communication from Richard McCulloch). While the McCulloch group reported a predominant localization of ORC1/CDC6 sites within SSRs at transcription start and termination regions, our re-analysis indicates that only 10.3% of TbORC1/CDC6-12Myc sites overlapped with 41.8% of SSRs.

      MFA-seq does not map individual origins, it rather detects replicated genomic regions by comparing DNA copy number between S- and G1-phases of the cell cycle (PMID: 36640769; PMID: 37469113; PMID: 36455525). The broad replicated regions (0.1–0.5 Mbp) identified by MFA-seq in T. brucei are likely to contain multiple origins, rather than just one. In that sense we disagree with the McCulloch's group who claimed that there is a single origin per broad peak. Our analysis shows that up to 50% of the origins detected by stranded SNS-seq locate within broad MFA-seq regions. The methodology used by McCulloch’s group to infer single origins from MFA-seq regions has not been published or made available, as well as the precise position of these regions, making direct comparison difficult.

      Finally, the genomic features we describe—poly(dA/dT) stretches, G4 structures and nucleosome occupancy patterns—are consistent with origin topology described in other organisms.

      On the concern that SNS-seq may map RNA-DNA hybrids rather than replication origins: Isolation and sequencing of short nascent strands (SNS) is a well-established and widely used technique for high-resolution origin mapping. This technique has been employed for decades in various laboratories, with numerous publications documenting its use. We followed the published protocol for SNS isolation (Cayrou et al., Methods, 2012, PMID: 22796403). RNA-DNA hybrids cannot persist through the multiple denaturation steps in our workflow, as they melt at 95°C (Roberts and Crothers, Science, 1992; PMID: 1279808). Even in the unlikely event that some hybrids remained, they would not be incorporated into libraries prepared using a single-stranded DNA protocol and therefore would not be sequenced (see Figure 1B and Methods).

      Furthermore, our analysis shows that only a small proportion (1.7%) of previously reported RNA-DNA hybrids overlap with SNS-seq origins. It is important to note that RNA-primed nascent strands naturally form RNA-DNA hybrids during replication initiation, meaning the enrichment of RNA-DNA hybrids near origins is both expected and biologically relevant.

      On the claim that our analysis focuses narrowly on inter-CDS regions and ignores other genomic compartments: this is incorrect. We mapped and analyzed stranded SNS-seq data across the entire genome of T. brucei 427 wild-type strain (Müller et al., Nature, 2018; PMID: 30333624), including both core and subtelomeric regions. Our findings indicate that most origins are located in intergenic regions, but all analyses were performed using the full set of detected origins, regardless of location.

      We did not ignore transcription start and stop sites (TSS/TTS). The manuscript already includes origin distribution across genomic compartments as defined by TriTrypDB (Fig. 2C) and addresses overlap with TSS, TTS and HT in the section “Spatial coordination between the activity of the origin and transcription”. While this overlap is minimal, we have included metaplots in the revised manuscript for clarity.

      Reviewer #2 (Public review):

      Summary: 

      Stanojcic et al. investigate the origins of DNA replication in the unicellular parasite Trypanosoma brucei. They perform two experiments, stranded SNS-seq and DNA molecular combing. Further, they integrate various publicly available datasets, such as G4-seq and DRIP-seq, into their extensive analysis. Using this data, they elucidate the structure of the origins of replication. In particular, they find various properties located at or around origins, such as polynucleotide stretches, G-quadruplex structures, regions of low and high nucleosome occupancy, R-loops, and that origins are mostly present in intergenic regions. Combining their population-level SNS-seq and their single-molecule DNA molecular combing data, they elucidate the total number of origins as well as the number of origins active in a single cell.

      Strengths:

      (1) A very strong part of this manuscript is that the authors integrate several other datasets and investigate a large number of properties around origins of replication. Data analysis clearly shows the enrichment of various properties at the origins, and the manuscript concludes with a very well-presented model that clearly explains the authors' understanding and interpretation of the data.

      We sincerely thank you for this positive feedback.

      (2) The DNA combing experiment is an excellent orthogonal approach to the SNS-seq data. The authors used the different properties of the two experiments (one giving location information, one giving single-molecule information) well to extract information and contrast the experiments.

      Thank you very much for this remark.

      (3) The discussion is exemplary, as the authors openly discuss the strengths and weaknesses of the approaches used. Further, the discussion serves its purpose of putting the results in both an evolutionary and a trypanosome-focused context.

      Thank you for appreciating our discussion.

      Weaknesses:

      I have major concerns about the origin of replication sites determined from the SNS-seq data. As a caveat, I want to state that, before reading this manuscript, SNS-seq was unknown to me; hence, some of my concerns might be misplaced.

      (1) I do not understand why SNS-seq would create peaks. Replication should originate in one locus, then move outward in both directions until the replication fork moving outward from another origin is encountered. Hence, in an asynchronous population average measurement, I would expect SNS data to be broad regions of + and -, which, taken together, cover the whole genome. Why are there so many regions not covered at all by reads, and why are there such narrow peaks?

      Thank you for asking these questions. As you correctly point out, replication forks progress in both directions from their origins and ultimately converge at termination sites. However, the SNS-seq method specifically isolates short nascent strands (SNSs) of 0.5–2.5 kb using a sucrose gradient. These short fragments are generated immediately after origin firing and mark the sites of replication initiation, rather than the entire replicated regions. Consequently: (i) SNS-seq does not capture long replication forks or termination regions, only the immediate vicinity of origins. (ii) The narrow peaks indicate the size of selected SNSs (0.5–2.5 kb) and the fact that many cells initiate replication at the same genomic sites, leading to localized enrichment. (iii) Regions without coverage refer to genomic areas that do not serve as efficient origins in the analyzed cell population. Thus, SNS-seq is designed to map origin positions, but not the entire replicated regions.

      (2) I am concerned that up to 96% percent of all peaks are filtered away. If there is so much noise in the data, how can one be sure that the peaks that remain are real? Specifically, if the authors placed the same number of peaks as was measured randomly in intergenic regions, would 4% of these peaks pass the filtering process by chance?

      Maintaining the strandness of the sequenced DNA fibres enabled us to filter the peaks, thereby increasing the probability that the filtered peak pairs corresponded to origins. Two SNS peaks must be oriented in a way that reflects the topology of the SNS strands within an active origin: the upstream peak must be on the minus strand and followed by the downstream peak on the plus strand.

      As suggested by the reviewer, we tested whether randomly placed plus and minus peaks could reproduce the number of filter-passing peaks using the same bioinformatics workflow. Only 1–6% of random peaks passed the filters, compared with 4–12% in our experimental data, resulting in about 50% fewer selected regions (origins). Moreover, the “origins” from random peaks showed 0% reproducibility across replicates, whereas the experimental data showed 7–64% reproducibility. These results indicate that the retainee peaks are highly unlikely to arise by chance and support the specificity of our approach. Thank you for this suggestion.

      (3) There are 3 previous studies that map origins of replication in T. brucei. Devlin et al. 2016, Tiengwe et al. 2012, and Krasiļņikova et al. 2025 (https://doi.org/10.1038/s41467-025-56087-3), all with a different technique: MFA-seq. All three previous studies mostly agree on the locations and number of origins. The authors compared their results to the first two, but not the last study; they found that their results are vastly different from the previous studies (see Supplementary Figure 8A). In their discussion, the authors defend this discrepancy mostly by stating that the discrepancy between these methods has been observed in other organisms. I believe that, given the situation that the other studies precede this manuscript, it is the authors' duty to investigate the differences more than by merely pointing to other organisms. A conclusion should be reached on why the results are different, e.g., by orthogonally validating origins absent in the previous studies.

      The MFA-seq data for T. brucei were published in two studies by McCulloch’s group: Tiengwe et al. (2012) using TREU927 PCF cells, and Devlin et al. (2016) using PCF and BSF Lister427 cells. In Krasilnikova et al. (2025), previously published MFA-seq data from Devlin et al. were remapped to a new genome assembly without generating new MFA-seq data, which explains why we did not include that comparison.

      Clarifying the differences between MFA-seq and our stranded SNS-seq data is essential. MFA-seq and SNS-seq interrogate different aspects of replication. SNS-seq is a widely used, high-resolution method for mapping individual replication origins, whereas MFA-seq detects replicated regions by comparing DNA copy number between S and G1 phases. MFA-seq identified broad replicated regions (0.1–0.5 Mb) that were interpreted by McCulloch’s group as containing a single origin. We disagree with this interpretation and consider that there are multiple origins in each broad peaks; theoretical considerations of replication timing indicate that far more origins are required for complete genome duplication during the short S-phase. Once this assumption is reconsidered, MFA-seq and SNS-seq results become complementary: MFA-seq identifies replicated regions, while SNS-seq pinpoints individual origins within those regions. Our analysis revealed that up to 50% of the origins detected by stranded SNS-seq were located within the broad MFA peaks. This pattern—broad MFA-seq regions containing multiple initiation sites—has also recently been found in Leishmania by McCulloch’s team using nanopore sequencing (PMID: 26481451). Nanopore sequencing showed numerous initiation sites within MFA-seq regions and additional numerous sites outside these regions in asynchronous cells, consistent with what we observed using stranded SNS-seq in T. brucei. We will expand our discussion and conclude that the discrepancy arises from methodological differences and interpretation. The two approaches provide complementary insights into replication dynamics, rather than ‘vastly different’ results.

      We recognize the importance of validating our results in future using an alternative mapping method and functional assays. However, it is important to emphasize that stranded SNS-seq is an origin mapping technique with a very high level of resolution. This technique can detect regions between two divergent SNS peaks, which should represent regions of DNA replication initiation. At present, no alternative technique has been developed that can match this level of resolution.

      (4) Some patterns that were identified to be associated with origins of replication, such as G-quadruplexes and nucleosomes phasing, are known to be biases of SNS-seq (see Foulk et al. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res. 2015;25(5):725-735. doi:10.1101/gr.183848.114).

      It is important to note that the conditions used in our study differ significantly from those applied in the Foulk et al. Genome Res. 2015. We used SNS isolation and enzymatic treatments as described in previous reports (Cayrou, C. et al. Genome Res, 2015 and Cayrou, C et al. Methods, 2012). Here, we enriched the SNS by size on a sucrose gradient and then treated this SNS-enriched fraction with high amounts of repeated λ-exonuclease treatments (100u for 16h at 37oC - see Methods). In contrast, Foulk et al. used sonicated total genomic DNA for origin mapping, without enrichment of SNS on a sucrose gradient as we did, and then they performed a λ-exonuclease treatment. A previous study (Cayrou, C. et al. Genome Res, 2015, Figure S2, which can be found at https://genome.cshlp.org/content/25/12/1873/suppl/DC1) has shown that complete digestion of G4-rich DNA sequences is achieved under the conditions we used.

      Furthermore, the SNS depleted control (without RNA) was included in our experimental approach. This control represents all molecules that are difficult to digest with lambda exonuclease, including G4 structures. Peak calling was performed against this background control, with the aim of removing false positive peaks resulting from undigested DNA structures. We explained better this step in the revised manuscript.

      The key benefit of our study is that the orientation of the enrichments (peaks) remains consistent throughout the sequencing process. We identified an enrichment of two divergent strands synthesised on complementary strands containing G4s. These two divergent strands themselves do not, however, contain G4s (see Fig. 8 for the model). Therefore, the enriched molecules detected in our study do not contain G4s. They are complementary to the strands enriched with G4s. This means that the observed enrichment of

      G4s cannot be an artefact of the enzymatic treatments used in this study. We added this part in the discussion of the revised manuscript.

      We also performed an additional control which is not mentioned in the manuscript. In parallel with replicating cells, we isolated the DNA from the stationary phase of growth, which primarily contains non-replicating cells. Following the three λ-exonuclease treatments, there was insufficient DNA remaining from the stationary phase cells to prepare the libraries for sequencing. This control strongly indicated that there was little to no contaminating DNA present with the SNS molecules after λ-exonuclease enrichment.

    1. Reviewer #3 (Public review):

      This study concerns how observers (human participants) detect changes in the statistics of their environment, termed regime shifts. To make this concrete, a series of 10 balls are drawn from an urn that contains mainly red or mainly blue balls. If there is a regime shift, the urn is changed over (from mainly red to mainly blue) at some point in the 10 trials. Participants report their belief that there has been a regime shift as a % probability. Their judgement should (mathematically) depend on the prior probability of a regime shift (which is set at one of three levels) and the strength of evidence (also one of three levels, operationalized as the proportion of red balls in the mostly-blue urn and vice versa). Participants are directly instructed of the prior probability of regime shift and proportion of red balls, which are presented on-screen as numerical probabilities. The task therefore differs from most previous work on this question in that probabilities are instructed rather than learned by observation, and beliefs are reported as numerical probabilities rather than being inferred from participants' choice behaviour (as in many bandit tasks, such as Behrens 2007 Nature Neurosci).

      The key behavioural finding is that participants over-estimate the prior probability of regime change when it is low, and under estimate it when it is high; and participants over-estimate the strength of evidence when it is low and under-estimate it when it is high. In other words participants make much less distinction between the different generative environments than an optimal observer would. This is termed 'system neglect'. A neuroeconomic-style mathematical model is presented and fit to data.

      Functional MRI results how that strength of evidence for a regime shift (roughly, the surprise associated with a blue ball from an apparently red urn) is associated with activity in the frontal-parietal orienting network. Meanwhile at time-points where the probability of a regime shift is high, there is activity in another network including vmPFC. Both networks show individual differences effects, such that people who were more sensitive to strength of evidence and prior probability show more activity in the frontal-parietal and vmPFC-linked networks respectively.

      Strengths

      (1) The study provides a different task for looking at change-detection and how this depends on estimates of environmental volatility and sensory evidence strength, in which participants are directly and precisely informed of the environmental volatility and sensory evidence strength rather than inferring them through observation as in most previous studies

      (2) Participants directly provide belief estimates as probabilities rather than experimenters inferring them from choice behaviour as in most previous studies

      (3) The results are consistent with well-established findings that surprising sensory events activate the frontal-parietal orienting network whilst updating of beliefs about the word ('regime shift') activates vmPFC.

      Weaknesses

      (1) The use of numerical probabilities (both to describe the environments to participants, and for participants to report their beliefs) may be problematic because people are notoriously bad at interpreting probabilities presented in this way, and show poor ability to reason with this information (see Kahneman's classic work on probabilistic reasoning, and how it can be improved by using natural frequencies). Therefore the fact that, in the present study, people do not fully use this information, or use it inaccurately, may reflect the mode of information delivery.

      In the response to this comment the authors have pointed out their own previous work showing that system neglect can occur even when numerical probabilities are not used. This is reassuring but there remains a large body of classic work showing that observers do struggle with conditional probabilities of the type presented in the task.

      (2) Although a very precise model of 'system neglect' is presented, many other models could fit the data.

      For example, you would get similar effects due to attraction of parameter estimates towards a global mean - essentially application of a hyper-prior in which the parameters applied by each participant in each block are attracted towards the experiment-wise mean values of these parameters. For example, the prior probability of regime shift ground-truth values [0.01, 0.05, 0.10] are mapped to subjective values of [0.037, 0.052, 0.069]; this would occur if observers apply a hyper-prior that the probability of regime shift is about 0.05 (the average value over all blocks). This 'attraction to the mean' is a well-established phenomenon and cannot be ruled out with the current data (I suppose you could rule it out by comparing to another dataset in which the mean ground-truth value was different).

      More generally, any model in which participants don't fully use the numerical information they were given would produce apparent 'system neglect'. Four qualitatively different example reasons are: 1. Some individual participants completely ignored the probability values given. 2. Participants did not ignore the probability values given, but combined them with a hyperprior as above. 3. Participants had a reporting bias where their reported beliefs that a regime-change had occurred tend to be shifted towards 50% (rather than reporting 'confident' values such 5% or 95%). 4. Participants underweighted probability outliers, resulting in underweighting of evidence in the 'high signal diagnosticity' environment (10.1016/j.neuron.2014.01.020 )

      In summary I agree that any model that fits the data would have to capture the idea that participants don't differentiate between the different environments as much as they should, but I think there are a number of qualitatively different reasons why they might do this - of which the above are only examples - hence I find it problematic that the authors present the behaviour as evidence for one extremely specific model.

      (3) Despite efforts to control confounds in the fMRI study, including two control experiments, I think some confounds remain.

      For example, a network of regions is presented as correlating with the cumulative probability that there has been a regime shift in this block of 10 samples (Pt). However, regardless of the exact samples shown, Pt always increases with sample number (as by the time of later samples, there have been more opportunities for a regime shift)? To control for this the authors include, in a supplementary analysis, an 'intertemporal prior.' I would have preferred to see the results of this better-controlled analysis presented in the main figure. From the tables in the SI it is very difficult to tell how the results change with the includion of the control regressors.

      On the other hand, two additional fMRI experiments are done as control experiments and the effect of Pt in the main study is compared to Pt in these control experiments. Whilst I admire the effort in carrying out control studies, I can't understand how these particular experiment are useful controls. For example, in experiment 3 participants simply type in numbers presented on the screen - how can we even have an estimate of Pt from this task?

      (4) The Discussion is very long, and whilst a lot of related literature is cited, I found it hard to pin down within the discussion, what the key contributions of this study are. In my opinion it would be better to have a short but incisive discussion highlighting the advances in understanding that arise from the current study, rather than reviewing the field so broadly.

    2. Author response:

      The following is the authors’ response to the current reviews

      eLife Assessment

      This study offers valuable insights into how humans detect and adapt to regime shifts, highlighting dissociable contributions of the frontoparietal network and ventromedial prefrontal cortex to sensitivity to signal diagnosticity and transition probabilities. The combination of an innovative instructed-probability task, Bayesian behavioural modeling, and model-based fMRI analyses provides a solid foundation for the main claims; however, major interpretational limitations remain, particularly a potential confound between posterior switch probability and time in the neuroimaging results. At the behavioural level, reliance on explicitly instructed conditional probabilities leaves open alternative explanations that complicate attribution to a single computational mechanism, such that clearer disambiguation between competing accounts and stronger control of temporal and representational confounds would further strengthen the evidence.

      Thank you. In this revision, we will focus on addressing Reviewer 3’s concern on the potential confound between posterior probability and time in neuroimaging results. First, we will present whole-brain results of subjects’ probability estimates (their subjective posterior probability of switch) after controlling for the effect of time on probability of switch (the intertemporal prior). Second, we will compare the effect of probability estimates (Pt) on vmPFC and ventral striatum activity—which we found to correlate with Pt—with and without including intertemporal prior in the GLM. Third, to address Reviewer 3’s comment that from the Tables of activation in the supplement vmPFC and ventral striatum cannot be located, we will add slice-by-slice image of the whole-brain results on Pt in the Supplemental Information in addition to the Tables of Activation.

      Public Reviews:

      Reviewer #1 (Public review):<br /> Summary:

      The study examines human biases in a regime-change task, in which participants have to report the probability of a regime change in the face of noisy data. The behavioral results indicate that humans display systematic biases, in particular, overreaction in stable but noisy environments and underreaction in volatile settings with more certain signals. fMRI results suggest that a frontoparietal brain network is selectively involved in representing subjective sensitivity to noise, while the vmPFC selectively represents sensitivity to the rate of change.

      Strengths:

      The study relies on a task that measures regime-change detection primarily based on descriptive information about the noisiness and rate of change. This distinguishes the study from prior work using reversal-learning or change-point tasks in which participants are required to learn these parameters from experiences. The authors discuss these differences comprehensively.

      The study uses a simple Bayes-optimal model combined with model fitting, which seems to describe the data well. The model is comprehensively validated.

      The authors apply model-based fMRI analyses that provide a close link to behavioral results, offering an elegant way to examine individual biases.

      Weaknesses:

      The authors have adequately addressed my prior concerns.

      Thank you for reviewing our paper and providing constructive comments that helped us improve our paper.

      Reviewer #3 (Public review):

      Thank you again for reviewing the manuscript. In this revision, we will focus on addressing your concern on the potential confound between posterior probability and time in neuroimaging results. First, we will present whole-brain results of subjects’ probability estimates (Pt, their subjective posterior probability of switch) after controlling for the effect of time on probability of switch (the intertemporal prior). Second, we will compare the effect of probability estimates (Pt) on vmPFC and ventral striatum activity—which we found to correlate with Pt—with and without including intertemporal prior in the GLM. These results will be summarized in a new figure (Figure 4).

      Finally, to address that you were not able to locate vmPFC and ventral striatum from the Tables of activation, we will add slice-by-slice image of the whole-brain results on Pt in the supplement in addition to the Tables of Activation.

      This study concerns how observers (human participants) detect changes in the statistics of their environment, termed regime shifts. To make this concrete, a series of 10 balls are drawn from an urn that contains mainly red or mainly blue balls. If there is a regime shift, the urn is changed over (from mainly red to mainly blue) at some point in the 10 trials. Participants report their belief that there has been a regime shift as a % probability. Their judgement should (mathematically) depend on the prior probability of a regime shift (which is set at one of three levels) and the strength of evidence (also one of three levels, operationalized as the proportion of red balls in the mostly-blue urn and vice versa). Participants are directly instructed of the prior probability of regime shift and proportion of red balls, which are presented on-screen as numerical probabilities. The task therefore differs from most previous work on this question in that probabilities are instructed rather than learned by observation, and beliefs are reported as numerical probabilities rather than being inferred from participants' choice behaviour (as in many bandit tasks, such as Behrens 2007 Nature Neurosci).

      The key behavioural finding is that participants over-estimate the prior probability of regime change when it is low, and under estimate it when it is high; and participants over-estimate the strength of evidence when it is low and under-estimate it when it is high. In other words participants make much less distinction between the different generative environments than an optimal observer would. This is termed 'system neglect'. A neuroeconomic-style mathematical model is presented and fit to data.

      Functional MRI results how that strength of evidence for a regime shift (roughly, the surprise associated with a blue ball from an apparently red urn) is associated with activity in the frontal-parietal orienting network. Meanwhile at time-points where the probability of a regime shift is high, there is activity in another network including vmPFC. Both networks show individual differences effects, such that people who were more sensitive to strength of evidence and prior probability show more activity in the frontal-parietal and vmPFC-linked networks respectively.

      Strengths

      (1) The study provides a different task for looking at change-detection and how this depends on estimates of environmental volatility and sensory evidence strength, in which participants are directly and precisely informed of the environmental volatility and sensory evidence strength rather than inferring them through observation as in most previous studies

      (2) Participants directly provide belief estimates as probabilities rather than experimenters inferring them from choice behaviour as in most previous studies

      (3) The results are consistent with well-established findings that surprising sensory events activate the frontal-parietal orienting network whilst updating of beliefs about the word ('regime shift') activates vmPFC.

      Weaknesses

      (1) The use of numerical probabilities (both to describe the environments to participants, and for participants to report their beliefs) may be problematic because people are notoriously bad at interpreting probabilities presented in this way, and show poor ability to reason with this information (see Kahneman's classic work on probabilistic reasoning, and how it can be improved by using natural frequencies). Therefore the fact that, in the present study, people do not fully use this information, or use it inaccurately, may reflect the mode of information delivery.

      In the response to this comment the authors have pointed out their own previous work showing that system neglect can occur even when numerical probabilities are not used. This is reassuring but there remains a large body of classic work showing that observers do struggle with conditional probabilities of the type presented in the task.

      Thank you. Yes, people do struggle with conditional probabilities in many studies. However, as our previous work suggested (Massey and Wu, 2005), system-neglect was likely not due to response mode (having to enter probability estimates or making binary predictions, and etc.).

      (2) Although a very precise model of 'system neglect' is presented, many other models could fit the data.

      For example, you would get similar effects due to attraction of parameter estimates towards a global mean - essentially application of a hyper-prior in which the parameters applied by each participant in each block are attracted towards the experiment-wise mean values of these parameters. For example, the prior probability of regime shift ground-truth values [0.01, 0.05, 0.10] are mapped to subjective values of [0.037, 0.052, 0.069]; this would occur if observers apply a hyper-prior that the probability of regime shift is about 0.05 (the average value over all blocks). This 'attraction to the mean' is a well-established phenomenon and cannot be ruled out with the current data (I suppose you could rule it out by comparing to another dataset in which the mean ground-truth value was different).

      We thank the reviewer for this comment. We do not disagree that there are alternative models that can describe over- and underreactions seen in the dataset. However, we do wish to point out that since we began with the normative Bayesian model, the natural progression in case the normative model fails to capture data is to modify the starting model. It is under this context that we developed the system-neglect model. It was a simple extension (a parameterized version) of the normative Bayesian model.

      Regarding the hyperprior idea, even if the participants have a hyperprior, there has to be some function that describes/implements attraction to the mean. Having a hyperprior itself does not imply attraction to this hyperprior. We therefore were not sure why the hyperprior itself can produce attraction to the mean.

      We do look further into the possibility of attraction to the mean. First, as suggested by the reviewer, we looked into another dataset with different mean ground-truth value. In Massey and Wu (2005), the transition probabilities were [0.02 0.05 0.1 0.2], which is different from the current study [0.01 0.05 0.1], and there they also found over- and underreactions as well. Second, we reason that for the attraction to the mean idea to work subjects need to know the mean of the system parameters. This would take time to develop because we did not tell subjects about the mean. If this is caused by attraction to the mean, subjects’ behavior would be different in the early stage of the experiment where they had little idea about the mean, compared with the late stage of the experiment where they knew about the mean. We will further analyze and compare participants’ data at the beginning of the experiment with data at the end of the experiment.

      More generally, any model in which participants don't fully use the numerical information they were given would produce apparent 'system neglect'. Four qualitatively different example reasons are: 1. Some individual participants completely ignored the probability values given. 2. Participants did not ignore the probability values given, but combined them with a hyperprior as above. 3. Participants had a reporting bias where their reported beliefs that a regime-change had occurred tend to be shifted towards 50% (rather than reporting 'confident' values such 5% or 95%). 4. Participants underweighted probability outliers, resulting in underweighting of evidence in the 'high signal diagnosticity' environment (10.1016/j.neuron.2014.01.020 )

      We thank the reviewer for pointing out these potential explanations. Again, we do not disagree that any model in which participants don’t fully use numerical information they were given would produce system neglect. It is hard to separate ‘not fully using numerical information’ from ‘lack of sensitivity to the numerical information’. We will respond in more details to the four example reasons later.

      In summary I agree that any model that fits the data would have to capture the idea that participants don't differentiate between the different environments as much as they should, but I think there are a number of qualitatively different reasons why they might do this - of which the above are only examples - hence I find it problematic that the authors present the behaviour as evidence for one extremely specific model.

      Again, we do not disagree with the reviewer on the modeling statement. However, we also wish to point out that the system-neglect model we had is a simple extension of the normative Bayesian model. Had we gone to a non-Bayesian framework, we would have faced the criticism of why we simply do not consider a simple extension of the starting model. In response, we will add a section in Discussion summarizing our exchange on this matter.

      (3) Despite efforts to control confounds in the fMRI study, including two control experiments, I think some confounds remain.

      For example, a network of regions is presented as correlating with the cumulative probability that there has been a regime shift in this block of 10 samples (Pt). However, regardless of the exact samples shown, Pt always increases with sample number (as by the time of later samples, there have been more opportunities for a regime shift)? To control for this the authors include, in a supplementary analysis, an 'intertemporal prior.' I would have preferred to see the results of this better-controlled analysis presented in the main figure. From the tables in the SI it is very difficult to tell how the results change with the includion of the control regressors.

      Thank you. In response, we will add a new figure, now Figure 4, showing the results of Pt and delta Pt from GLM-2 where we added the intertemporal prior as a regressor to control for temporal confounds. We compared Pt and delta Pt results in vmPFC and ventral striatum between GLM-1 and GLM-2. We also will show the results of intertemporal prior on vmPFC and ventral striatum under GLM-2.

      On the other hand, two additional fMRI experiments are done as control experiments and the effect of Pt in the main study is compared to Pt in these control experiments. Whilst I admire the effort in carrying out control studies, I can't understand how these particular experiment are useful controls. For example, in experiment 3 participants simply type in numbers presented on the screen - how can we even have an estimate of Pt from this task?

      We thank the reviewer for this comment. On the one hand, the effect of Pt we see in brain activity can be simply due to motor confounds and the purpose of Experiment 3 was to control for them. Our question was, if subjects saw the similar visual layout and were just instructed to press buttons to indicate two-digit numbers, would we observe the vmPFC, ventral striatum, and the frontoparietal network like what we did in the main experiment (Experiment 1)?

      On the other hand, the effect of Pt can simply reflect probability estimates of that the current regime is the blue regime, and therefore not particularly about change detection. In Experiment 2, we tested that idea, namely whether what we found about Pt was unique to change detection. In Experiment 2, subjects estimated the probability that the current regime is the blue regime (just as they did in Experiment 1) except that there were no regime shifts involved. In other words, it is possible that the regions we identified were generally associated with probability estimation and not particularly about probability estimates of change. We used Experiment 2 to examine whether this were true.

      To make the purpose of the two control experiments clearer, we updated the paragraph describing the control experiments on page 9:

      “To establish the neural representations for regime-shift estimation, we performed three fMRI experiments ( subjects for each experiment, 90 subjects in total). Experiment 1 was the main experiment, while Experiments 2 to 3 were control experiments that ruled out two important confounds (Fig. 1E). The control experiments were designed to clarify whether any effect of subjects’ probability estimates of a regime shift, , in brain activity can be uniquely attributed to change detection. Here we considered two major confounds that can contribute to the effect of . First, since subjects in Experiment 1 made judgments about the probability that the current regime is the blue regime (which corresponded to probability of regime change), the effect of  did not particularly have to do with change detection. To address this issue, in Experiment 2 subjects made exactly the same judgments as in Experiment 1 except that the environments were stationary (no transition from one regime to another was possible), as in Edwards (1968) classic “bookbag-and-poker chip” studies. Subjects in both experiments had to estimate the probability that the current regime is the blue regime, but this estimation corresponded to the estimates of regime change only in Experiment 1. Therefore, activity that correlated with probability estimates in Experiment 1 but not in Experiment 2 can be uniquely attributed to representing regime-shift judgments. Second, the effect of  can be due to motor preparation and/or execution, as subjects in Experiment 1 entered two-digit numbers with button presses to indicate their probability estimates. To address this issue, in Experiment 3 subjects performed a task where they were presented with two-digit numbers and were instructed to enter the numbers with button presses. By comparing the fMRI results of these experiments, we were therefore able to establish the neural representations that can be uniquely attributed to the probability estimates of regime-shift.”

      To further make sure that the probability-estimate signals in Experiment 1 were not due to motor confounds, we implemented an action-handedness regressor in the GLM, as we described below on page 19:

      “Finally, we note that in GLM-1, we implemented an “action-handedness” regressor to directly address the motor-confound issue, that higher probability estimates preferentially involved right-handed responses for entering higher digits. The action-handedness regressor was parametric, coding -1 if both finger presses involved the left hand (e.g., a subject pressed “23” as her probability estimate when seeing a signal), 0 if using one left finger and one right finger (e.g., “75”), and 1 if both finger presses involved the right hand (e.g., “90”). Taken together, these results ruled out motor confounds and suggested that vmPFC and ventral striatum represent subjects’ probability estimates of change (regime shifts) and belief revision.”

      (4) The Discussion is very long, and whilst a lot of related literature is cited, I found it hard to pin down within the discussion, what the key contributions of this study are. In my opinion it would be better to have a short but incisive discussion highlighting the advances in understanding that arise from the current study, rather than reviewing the field so broadly.

      Thank you. We thank the reviewer for pushing us to highlight the key contributions. In response, we added a paragraph at the beginning of Discussion to better highlight our contributions:

      “In this study, we investigated how humans detect changes in the environments and the neural mechanisms that contribute to how we might under- and overreact in our judgments. Combining a novel behavioral paradigm with computational modeling and fMRI, we discovered that sensitivity to environmental parameters that directly impact change detection is a key mechanism for under- and overreactions. This mechanism is implemented by distinct brain networks in the frontal and parietal cortices and in accordance with the computational roles they played in change detection. By introducing the framework in system neglect and providing evidence for its neural implementations, this study offered both theoretical and empirical insights into how systematic judgment biases arise in dynamic environments.”

      **Recommendations for the authors:

      Reviewer #3 (Recommendations for the authors):**

      Thank you for pointing out the inclusion of the intertemporal prior in glm2, this seems like an important control that would address my criticism. Why not present this better-controlled analysis in the main figure, rather than the results for glm1 which has no effective control of the increasing posterior probability of a reversal with time?

      Thank you for this suggestion. We added a new figure (Figure 4) that showed results from GLM-2. In this new figure, we showed whole-brain results on Pt and delta Pt, ROI results of vmPFC and ventral striatum on Pt, delta Pt, and intertemporal prior.

      The reason we kept results from GLM-1 (Figure 3) was primarily because we wanted to compare the effect of Pt between experiments under identical GLM. In other words, the regressors in GLM-1 was identical across all 3 experiments. In Experiments 1 and 2, Pt and delta Pt were respectively probability estimates and belief updates that current regime was the Blue regime. In Experiment 3, Pt and delta Pt were simply the number subjects were instructed to press (Pt) and change in number between successive periods (delta Pt).

      As a further point I could not navigate the tables of fMRI activations in SI and recommend replacing or supplementing these with images. For example I cannot actually find a vmPFC or ventral striatum cluster listed for the effect of Pt in GLM1 (version in table S1), which I thought were the main results? Beyond that, comparing how much weaker (or not) those results are when additional confound regressors are included in GLM2 seems impossible.

      The vmPFC and ventral striatum were part of the cluster labeled as Central Opercular cortex. In response, we will provide information about coordinates on the local maxima within the cluster. We will also add slice-by-slice images showing the effect of Pt.


      The following is the authors’ response to the original reviews

      eLife Assessment

      This study offers valuable insights into how humans detect and adapt to regime shifts, highlighting distinct contributions of the frontoparietal network and ventromedial prefrontal cortex to sensitivity to signal diagnosticity and transition probabilities. The combination of an innovative task design, behavioral modeling, and model-based fMRI analyses provides a solid foundation for the conclusions; however, the neuroimaging results have several limitations, particularly a potential confound between the posterior probability of a switch and the passage of time that may not be fully controlled by including trial number as a regressor. The control experiments intended to address this issue also appear conceptually inconsistent and, at the behavioral level, while informing participants of conditional probabilities rather than requiring learning is theoretically elegant, such information is difficult to apply accurately, as shown by well-documented challenges with conditional reasoning and base-rate neglect. Expressing these probabilities as natural frequencies rather than percentages may have improved comprehension. Overall, the study advances understanding of belief updating under uncertainty but would benefit from more intuitive probabilistic framing and stronger control of temporal confounds in future work.

      We thank the editors for the assessment and we appreciate your efforts in reviewing the paper. The editors added several limitations in the assessment based on the new reviewer 3 in this round, which we would like to clarify below.

      With regard to temporal confounds, we clarified in the main text and response to Reviewer 3 that we had already addressed the potential confound between posterior probability of a switch and passage of time in GLM-2 with the inclusion of intertemporal prior. After adding intertemporal prior in the GLM, we still observed the same fMRI results on probability estimates. In addition, we did two other robustness checks, which we mentioned in the manuscript.

      With regard to response mode (probability estimation rather than choice or indicating natural frequencies), we wish to point out that the in previous research by Massey and Wu (2005), which the current study was based on, the concern of participants showing system-neglect tendencies due to the mode of information delivery, namely indicating beliefs through reporting probability estimates rather than through choice or other response mode was addressed. Massy and Wu (2005, Study 3) found the same biases when participants performed a choice task that did not require them to indicate probability estimates.

      With regard to the control experiments, the control experiments in fact were not intended to address the confounds between posterior probability and passage of time. Rather, they aimed to address whether the neural findings were unique to change detection (Experiment 2) and to address visual and motor confounds (Experiment 3). These and the results of the control experiments were mentioned on page 18-19.

      We also wish to highlight that we had performed detailed model comparisons after reviewer 2’s suggestions. Although reviewer 2 was unable to re-review the manuscript, we believe this provides insight into the literature on change detection. See “Incorporating signal dependency into system-neglect model led to better models for regime-shift detection” (p.27-30). The model comparison showed that system-neglect models that incorporate signal dependency are better models than the original system-neglect model in describing participants probability estimates. This suggests that people respond to change-consistent and change-inconsistent signals differently when judging whether the regime had changed. This was not reported in previous behavioral studies and was largely inspired by the neural finding on signal dependency in the frontoparietal cortex. It indicates that neural findings can provide novel insights into computational modeling of behavior.

      To better highlight and summarize our key contributions, we added a paragraph at the beginning of Discussion:

      “In this study, we investigated how humans detect changes in the environments and the neural mechanisms that contribute to how we might under- and overreact in our judgments. Combining a novel behavioral paradigm with computational modeling and fMRI, we discovered that sensitivity to environmental parameters that directly impact change detection is a key mechanism for under- and overreactions. This mechanism is implemented by distinct brain networks in the frontal and parietal cortices and in accordance with the computational roles they played in change detection. By introducing the framework in system neglect and providing evidence for its neural implementations, this study offered both theoretical and empirical insights into how systematic judgment biases arise in dynamic environments.”    

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      The study examines human biases in a regime-change task, in which participants have to report the probability of a regime change in the face of noisy data. The behavioral results indicate that humans display systematic biases, in particular, overreaction in stable but noisy environments and underreaction in volatile settings with more certain signals. fMRI results suggest that a frontoparietal brain network is selectively involved in representing subjective sensitivity to noise, while the vmPFC selectively represents sensitivity to the rate of change.

      Strengths:

      - The study relies on a task that measures regime-change detection primarily based on descriptive information about the noisiness and rate of change. This distinguishes the study from prior work using reversal-learning or change-point tasks in which participants are required to learn these parameters from experiences. The authors discuss these differences comprehensively.

      - The study uses a simple Bayes-optimal model combined with model fitting, which seems to describe the data well. The model is comprehensively validated.

      - The authors apply model-based fMRI analyses that provide a close link to behavioral results, offering an elegant way to examine individual biases.

      We thank the reviewer for the comments.

      Weaknesses:

      The authors have adequately addressed most of my prior concerns.

      We thank the reviewer for recognizing our effort in addressing your concerns.

      My only remaining comment concerns the z-test of the correlations. I agree with the non-parametric test based on bootstrapping at the subject level, providing evidence for significant differences in correlations within the left IFG and IPS.

      However, the parametric test seems inadequate to me. The equation presented is described as the Fisher z-test, but the numerator uses the raw correlation coefficients (r) rather than the Fisher-transformed values (z). To my understanding, the subtraction should involve the Fisher z-scores, not the raw correlations.

      More importantly, the Fisher z-test in its standard form assumes that the correlations come from independent samples, as reflected in the denominator (which uses the n of each independent sample). However, in my opinion, the two correlations are not independent but computed within-subject. In such cases, parametric tests should take into account the dependency. I believe one appropriate method for the current case (correlated correlation coefficients sharing a variable [behavioral slope]) is explained here:

      Meng, X.-l., Rosenthal, R., & Rubin, D. B. (1992). Comparing correlated correlation coefficients. Psychological Bulletin, 111(1), 172-175. https://doi.org/10.1037/0033-2909.111.1.172

      It should be implemented here:

      Diedenhofen B, Musch J (2015) cocor: A Comprehensive Solution for the Statistical Comparison of Correlations. PLoS ONE 10(4): e0121945. https://doi.org/10.1371/journal.pone.0121945

      My recommendation is to verify whether my assumptions hold, and if so, perform a test that takes correlated correlations into account. Or, to focus exclusively on the non-parametric test.

      In any case, I recommend a short discussion of these findings and how the authors interpret that some of the differences in correlations are not significant.

      Thank you for the careful check. Yes. This was indeed a mistake from us. We also agree that the two correlations are not independent. Therefore, we modified the test that accounts for dependent correlations by following Meng et al. (1992) suggested by the reviewer. We updated in the Methods section on p.56-57:

      “In the parametric test, we adopted the approach of Meng et al. (1992) to statistically compare the two correlation coefficients. This approach specifically tests differences between dependent correlation coefficients according to the following equation

      Where N is the number of subjects, z<sub>ri</sub> is the Fisher z-transformed value of r<sub>i</sub>,(r<sub>1</sub> = r<sub>blue</sub> and r<sub>2</sub> = r<sub>red</sub>), and r<sub>x</sub> is the correlation between the neural sensitivity at change-consistent signals and change-inconsistent signals. The computation of h is based on the following equations

      Where is the mean of the , and f should be set to 1 if > 1.”

      We updated on the Results section on p.29:

      “Since these correlation coefficients were not independent, we compared them using the test developed in Meng et al. (1992) (see Methods). We found that among the five ROIs in the frontoparietal network, two of them, namely the left IFG and left IPS, the difference in correlation was significant (one-tailed z test; left IFG: z = 1.8908, p = 0.0293; left IPS: z = 2.2584, p = 0.0049). For the remaining three ROIs, the difference in correlation was not significant (dmPFC: z = 0.9522, p = 0.1705; right IFG: z = 0.9860, p = 0.1621; right IPS: z = 1.4833, p = 0.0690).”

      We added a Discussion on these results on p.41:

      “Interestingly, such sensitivity to signal diagnosticity was only present in the frontoparietal network when participants encountered change-consistent signals. However, while most brain areas within this network responded in this fashion, only the left IPS and left IFG showed a significant difference in coding individual participants’ sensitivity to signal diagnosticity between change-consistent and change-inconsistent signals. Unlike the left IPS and left IFG, we observed in dmPFC a marginally significant correlation with behavioral sensitivity at change-inconsistent signals as well. Together, these results indicate that while different brain areas in the frontoparietal network responded similarly to change-consistent signals, there was a greater degree of heterogeneity in responding to change-inconsistent signals.”

      Reviewer #3 (Public review):

      This study concerns how observers (human participants) detect changes in the statistics of their environment, termed regime shifts. To make this concrete, a series of 10 balls are drawn from an urn that contains mainly red or mainly blue balls. If there is a regime shift, the urn is changed over (from mainly red to mainly blue) at some point in the 10 trials. Participants report their belief that there has been a regime shift as a % probability. Their judgement should (mathematically) depend on the prior probability of a regime shift (which is set at one of three levels) and the strength of evidence (also one of three levels, operationalized as the proportion of red balls in the mostly-blue urn and vice versa). Participants are directly instructed of the prior probability of regime shift and proportion of red balls, which are presented on-screen as numerical probabilities. The task therefore differs from most previous work on this question in that probabilities are instructed rather than learned by observation, and beliefs are reported as numerical probabilities rather than being inferred from participants' choice behaviour (as in many bandit tasks, such as Behrens 2007 Nature Neurosci).

      The key behavioural finding is that participants over-estimate the prior probability of regime change when it is low, and under estimate it when it is high; and participants over-estimate the strength of evidence when it is low and under-estimate it when it is high. In other words participants make much less distinction between the different generative environments than an optimal observer would. This is termed 'system neglect'. A neuroeconomic-style mathematical model is presented and fit to data.

      Functional MRI results how that strength of evidence for a regime shift (roughly, the surprise associated with a blue ball from an apparently red urn) is associated with activity in the frontal-parietal orienting network. Meanwhile, at time-points where the probability of a regime shift is high, there is activity in another network including vmPFC. Both networks show individual differences effects, such that people who were more sensitive to strength of evidence and prior probability show more activity in the frontal-parietal and vmPFC-linked networks respectively.

      We thank the reviewer for the overall descriptions of the manuscript.

      Strengths

      (1) The study provides a different task for looking at change-detection and how this depends on estimates of environmental volatility and sensory evidence strength, in which participants are directly and precisely informed of the environmental volatility and sensory evidence strength rather than inferring them through observation as in most previous studies

      (2) Participants directly provide belief estimates as probabilities rather than experimenters inferring them from choice behaviour as in most previous studies

      (3) The results are consistent with well-established findings that surprising sensory events activate the frontal-parietal orienting network whilst updating of beliefs about the word ('regime shift') activates vmPFC.

      Thank you for these assessments.

      Weaknesses

      (1) The use of numerical probabilities (both to describe the environments to participants, and for participants to report their beliefs) may be problematic because people are notoriously bad at interpreting probabilities presented in this way, and show poor ability to reason with this information (see Kahneman's classic work on probabilistic reasoning, and how it can be improved by using natural frequencies). Therefore the fact that, in the present study, people do not fully use this information, or use it inaccurately, may reflect the mode of information delivery.

      We appreciate the reviewer’s concern on this issue. The concern was addressed in Massey and Wu (2005) as participants performed a choice task in which they were not asked to provide probability estimates (Study 3 in Massy and Wu, 2005). Instead, participants in Study 3 were asked to predict the color of the ball before seeing a signal. This was a more intuitive way of indicating his or her belief about regime shift. The results from the choice task were identical to those found in the probability estimation task (Study 1 in Massey and Wu). We take this as evidence that the system-neglect behavior the participants showed was less likely to be due to the mode of information delivery.

      (2) Although a very precise model of 'system neglect' is presented, many other models could fit the data.

      For example, you would get similar effects due to attraction of parameter estimates towards a global mean - essentially application of a hyper-prior in which the parameters applied by each participant in each block are attracted towards the experiment-wise mean values of these parameters. For example, the prior probability of regime shift ground-truth values [0.01, 0.05, 0.10] are mapped to subjective values of [0.037, 0.052, 0.069]; this would occur if observers apply a hyper-prior that the probability of regime shift is about 0.05 (the average value over all blocks). This 'attraction to the mean' is a well-established phenomenon and cannot be ruled out with the current data (I suppose you could rule it out by comparing to another dataset in which the mean ground-truth value was different).

      We thank the reviewer for this comment. It is true that the system-neglect model is not entirely inconsistent with regression to the mean, regardless of whether the implementation has a hyper prior or not. In fact, our behavioral measure of sensitivity to transition probability and signal diagnosticity, which we termed the behavioral slope, is based on linear regression analysis. In general, the modeling approach in this paper is to start from a generative model that defines ideal performance and consider modifying the generative model when systematic deviations in actual performance from the ideal is observed. In this approach, a generative Bayesian model with hyper priors would be more complex to begin with, and a regression to the mean idea by itself does not generate a priori predictions.

      More generally, any model in which participants don't fully use the numerical information they were given would produce apparent 'system neglect'. Four qualitatively different example reasons are: 1. Some individual participants completely ignored the probability values given. 2. Participants did not ignore the probability values given, but combined them with a hyperprior as above. 3. Participants had a reporting bias where their reported beliefs that a regime-change had occurred tend to be shifted towards 50% (rather than reporting 'confident' values such 5% or 95%). 4. Participants underweighted probability outliers resulting in underweighting of evidence in the 'high signal diagnosticity' environment (10.1016/j.neuron.2014.01.020)

      In summary I agree that any model that fits the data would have to capture the idea that participants don't differentiate between the different environments as much as they should, but I think there are a number of qualitatively different reasons why they might do this - of which the above are only examples - hence I find it problematic that the authors present the behaviour as evidence for one extremely specific model.

      Thank you for raising this point. The modeling principle we adopt is the following. We start from the normative model—the Bayesian model—that defined what normative behavior should look like. We compared participants’ behavior with the Bayesian model and found systematic deviations from it. To explain those systematic deviations, we considered modeling options within the confines of the same modeling framework. In other words, we considered a parameterized version of the Bayesian model, which is the system-neglect model and examined through model comparison the best modeling choice. This modeling approach is not uncommon in economics and psychology. For example, Kahneman and Tversky adopted this approach when proposing prospect theory, a modification of expected utility theory where expected utility theory can be seen as one specific model for how utility of an option should be computed.

      (3) Despite efforts to control confounds in the fMRI study, including two control experiments, I think some confounds remain.

      For example, a network of regions is presented as correlating with the cumulative probability that there has been a regime shift in this block of 10 samples (Pt). However, regardless of the exact samples shown, doesn't Pt always increase with sample number (as by the time of later samples, there have been more opportunities for a regime shift)? Unless this is completely linear, the effect won't be controlled by including trial number as a co-regressor (which was done).

      Thank you for raising this concern. Yes, Pt always increases with sample number regardless of evidence (seeing change-consistent or change-inconsistent signals). This is captured by the ‘intertemporal prior’ in the Bayesian model, which we included as a regressor in our GLM analysis (GLM-2), in addition to Pt. In short, GLM-1 had Pt and sample number. GLM-2 had Pt, intertemporal prior, and sample number, among other regressors. And we found that, in both GLM-1 and GLM-2, both vmPFC and ventral striatum correlated with Pt.

      To make this clearer, we updated the main text to further clarify this on p.18:

      “We examined the robustness of P<sub>t</sub> representations in these two regions in several follow-up analyses. First, we implemented a GLM (GLM-2 in Methods) that, in addition to P<sub>t</sub>, included various task-related variables contributing to P<sub>t</sub> as regressors (Fig. S7 in SI). Specifically, to account for the fact that the probability of regime change increased over time, we included the intertemporal prior as a regressor in GLM-2. The intertemporal prior is the natural logarithm of the odds in favor of regime shift in the t-th period, where q is transition probability and t = 1,…,10 is the period (see Eq. 1 in Methods). It describes normatively how the prior probability of change increased over time regardless of the signals (blue and red balls) the subjects saw during a trial. Including it along with P<sub>t</sub> would clarify whether any effect of P<sub>t</sub> can otherwise be attributed to the intertemporal prior. Second, we implemented a GLM that replaced P<sub>t</sub> with the log odds of P<sub>t</sub>, ln (P<sub>t</sub>/(1-P<sub>t</sub>)) (Fig. S8 in SI). Third, we implemented a GLM that examined  separately on periods when change-consistent (blue balls) and change-inconsistent (red balls) signals appeared (Fig. S9 in SI). Each of these analyses showed the same pattern of correlations between P<sub>t</sub> and activation in vmPFC and ventral striatum, further establishing the robustness of the P<sub>t</sub> findings.”

      On the other hand, two additional fMRI experiments are done as control experiments and the effect of Pt in the main study is compared to Pt in these control experiments. Whilst I admire the effort in carrying out control studies, I can't understand how these particular experiment are useful controls. For example in experiment 3 participants simply type in numbers presented on the screen - how can we even have an estimate of Pt from this task?

      We thank the reviewer for this comment. On the one hand, the effect of Pt we see in brain activity can be simply due to motor confounds and the purpose of Experiment 3 was to control for them. Our question was, if subjects saw the similar visual layout and were just instructed to press buttons to indicate two-digit numbers, would we observe the vmPFC, ventral striatum, and the frontoparietal network like what we did in the main experiment (Experiment 1)?

      On the other hand, the effect of Pt can simply reflect probability estimates of that the current regime is the blue regime, and therefore not particularly about change detection. In Experiment 2, we tested that idea, namely whether what we found about Pt was unique to change detection. In Experiment 2, subjects estimated the probability that the current regime is the blue regime (just as they did in Experiment 1) except that there were no regime shifts involved. In other words, it is possible that the regions we identified were generally associated with probability estimation and not particularly about probability estimates of change. We used Experiment 2 to examine whether this were true.

      To make the purpose of the two control experiments clearer, we updated the paragraph describing the control experiments on page 9:

      “To establish the neural representations for regime-shift estimation, we performed three fMRI experiments (n\=30 subjects for each experiment, 90 subjects in total). Experiment 1 was the main experiment, while Experiments 2 to 3 were control experiments that ruled out two important confounds (Fig. 1E). The control experiments were designed to clarify whether any effect of subjects’ probability estimates of a regime shift, P<sub>t</sub>, in brain activity can be uniquely attributed to change detection. Here we considered two major confounds that can contribute to the effect of . First, since subjects in Experiment 1 made judgments about the probability that the current regime is the blue regime (which corresponded to probability of regime change), the effect of P<sub>t</sub> did not particularly have to do with change detection. To address this issue, in Experiment 2 subjects made exactly the same judgments as in Experiment 1 except that the environments were stationary (no transition from one regime to another was possible), as in Edwards (1968) classic “bookbag-and-poker chip” studies. Subjects in both experiments had to estimate the probability that the current regime is the blue regime, but this estimation corresponded to the estimates of regime change only in Experiment 1. Therefore, activity that correlated with probability estimates in Experiment 1 but not in Experiment 2 can be uniquely attributed to representing regime-shift judgments. Second, the effect of P<sub>t</sub> can be due to motor preparation and/or execution, as subjects in Experiment 1 entered two-digit numbers with button presses to indicate their probability estimates. To address this issue, in Experiment 3 subjects performed a task where they were presented with two-digit numbers and were instructed to enter the numbers with button presses. By comparing the fMRI results of these experiments, we were therefore able to establish the neural representations that can be uniquely attributed to the probability estimates of regime-shift.”

      To further make sure that the probability-estimate signals in Experiment 1 were not due to motor confounds, we implemented an action-handedness regressor in the GLM, as we described below on page 19:

      “Finally, we note that in GLM-1, we implemented an “action-handedness” regressor to directly address the motor-confound issue, that higher probability estimates preferentially involved right-handed responses for entering higher digits. The action-handedness regressor was parametric, coding -1 if both finger presses involved the left hand (e.g., a subject pressed “23” as her probability estimate when seeing a signal), 0 if using one left finger and one right finger (e.g., “75”), and 1 if both finger presses involved the right hand (e.g., “90”). Taken together, these results ruled out motor confounds and suggested that vmPFC and ventral striatum represent subjects’ probability estimates of change (regime shifts) and belief revision.”

      (4) The Discussion is very long, and whilst a lot of related literature is cited, I found it hard to pin down within the discussion, what the key contributions of this study are. In my opinion it would be better to have a short but incisive discussion highlighting the advances in understanding that arise from the current study, rather than reviewing the field so broadly.

      Thank you. We thank the reviewer for pushing us to highlight the key contributions. In response, we added a paragraph at the beginning of Discussion to better highlight our contributions:

      “In this study, we investigated how humans detect changes in the environments and the neural mechanisms that contribute to how we might under- and overreact in our judgments. Combining a novel behavioral paradigm with computational modeling and fMRI, we discovered that sensitivity to environmental parameters that directly impact change detection is a key mechanism for under- and overreactions. This mechanism is implemented by distinct brain networks in the frontal and parietal cortices and in accordance with the computational roles they played in change detection. By introducing the framework in system neglect and providing evidence for its neural implementations, this study offered both theoretical and empirical insights into how systematic judgment biases arise in dynamic environments.”

      Recommendations for the authors:

      Reviewer #3 (Recommendations for the authors):

      Many of the figures are too tiny - the writing is very small, as are the pictures of brains. I'd suggest adjusting these so they will be readable without enlarging.

      Thank you. We apologize for the poor readability of the figures. We had enlarged the figures (Fig. 5 in particular) and their font size to make them more readable.

      1. 需要单独有一个产品价值与场景环节
      2. 详细总结团队当前 skills 和 agent rules 分发遇到的问题
      3. 推导出来我们需要有三种 skills 第一种是严肃维护团队自上而下推广的,第二种个人维护分享交流的,第三种社区优秀的便于下载的 4.skills 工程化规范章节需要单独成篇,主要讨论 skills 如何长期持续维护,需要包含示例、测试、统计机制、CR 机制等
    1. Reviewer #1 (Public review):

      Summary:

      The authors proposed a new method to infer connectivity from spike trains whose main novelty relies on their approach to mitigate the problem of model mismatch. The latter arises when the inference algorithm is trained or based on a model that does not accurately describe the data. They propose combining domain adaptation with a deep neural architecture and in an architecture called DeepDAM. They apply DeepDAM to an in vivo ground-truth dataset previously recorded in mouse CA1, show that it performs better than methods without domain adaptation, and evaluate its robustness. Finally, they show that their approach can also be applied to a different problem i.e., inferring biophysical properties of individual neurons.

      Strengths:

      (1) The problem of inferring connectivity from extracellular recording is a very timely one: as the yield of silicon probes steadily increases, the number of simultaneously recorded pairs does so quadratically, drastically increasing the possibility of detecting connected pairs.

      (2) Using domain adaptation to address model mismatch is a clever idea, and the way the authors introduced it into the larger architecture seems sensible.

      (3) The authors clearly put a great effort into trying to communicate the intuitions to the reader.

      Weaknesses:

      (1) The validation of the approach is incomplete: due to its very limited size, the single ground-truth dataset considered does not provide a sufficient basis to draw a strong conclusion. While the authors correctly note that this is the only dataset of its kind, the value of this validation is limited compared to what could be done by carefully designing in silico experiments.

      (2) Surprisingly, the authors fail to compare their method to the approach originally proposed for the data they validate on (English et al., 2017).

      (3) The authors make a commendable effort to study the method's robustness by pushing the limits of the dataset. However, the logic of the robustness analysis is often unclear, and once again, the limited size of the dataset poses major limitations to the authors.

      (4) The lack of details concerning both the approach and the validation makes it challenging for the reader to establish the technical soundness of the study.

      Although in the current form this study does not provide enough basis to judge the impact of DeepDAM in the broader neuroscience community, it nevertheless puts forward a valuable and novel idea: using domain adaptation to mitigate the problem of model mismatch. This approach might be leveraged in future studies and methods to infer connectivity.

    2. Author response:

      General Response

      We thank the reviewers for their positive assessment of our work and for acknowledging the timeliness of the problem and the novelty of using domain adaptation to address model mismatch. We appreciate the constructive feedback regarding validation and clarity. In the revised manuscript, we will address these points as follows:

      (1) Systematic Validation: We will design and perform systematic in silico experiments to evaluate the method beyond the single in vivo dataset , including robustness tests regarding recording length and network synchrony.

      (2) Recurrent Networks & Failure Analysis: We will test our method on synthetic datasets generated from highly recurrent networks and analyze exactly when the method breaks as a function of mismatch magnitude.

      (3) Method Comparisons: We will report the Matthews Correlation Coefficient (MCC) for the approach by English et al. (2017) and expand our comparison and discussion of GLM-based methods.

      (4) Clarifications: We will rigorously define the dataset details (labeling, recording methodology), mathematical notation, and machine learning terminology ('data', 'labels').

      (5) Discussion of Limitations: We will explicitly discuss the challenges and limitations inherent in generalizing to more recurrently connected regions.

      Below are our more detailed responses:

      Public Reviews:

      Reviewer #1 (Public review):

      Weaknesses:

      (1) The validation of the approach is incomplete: due to its very limited size, the single ground-truth dataset considered does not provide a sufficient basis to draw a strong conclusion. While the authors correctly note that this is the only dataset of its kind, the value of this validation is limited compared to what could be done by carefully designing in silico experiments.

      We thank the reviewer for acknowledging the scarcity of suitable in vivo ground-truth datasets and the limitations this poses. We agree that additional validation is necessary to draw strong conclusions. In the revised manuscript, we will systematically design and perform in silico experiments for evaluations beyond the single in vivo dataset.

      (2) Surprisingly, the authors fail to compare their method to the approach originally proposed for the data they validate on (English et al., 2017).

      We agree that this is an essential comparison. We will report the Matthews Correlation Coefficient (MCC) result of the approach by English et al. (2017) on the spontaneous period of the recording.

      (3) The authors make a commendable effort to study the method's robustness by pushing the limits of the dataset. However, the logic of the robustness analysis is often unclear, and once again, the limited size of the dataset poses major limitations to the authors.

      We appreciate the reviewer recognizing our initial efforts to evaluate robustness. In our original draft, we tested recording length, network model choices, and analyzed failure cases. However, we agree that the limited real data restricts the scope of these tests. To address this, we will perform more systematic robustness tests on the newly generated synthetic datasets in the revised version, allowing us to evaluate performance under a wider range of conditions.

      (4) The lack of details concerning both the approach and the validation makes it challenging for the reader to establish the technical soundness of the study.

      We will revise the manuscript thoroughly to better present the methodology of our framework and the validation pipelines. We will ensure that the figures and text clearly articulate the technical details required to assess the soundness of the study.

      Although in the current form this study does not provide enough basis to judge the impact of DeepDAM in the broader neuroscience community, it nevertheless puts forward a valuable and novel idea: using domain adaptation to mitigate the problem of model mismatch. This approach might be leveraged in future studies and methods to infer connectivity.

      We thank the reviewer again for acknowledging the novelty and importance of our work.

      Reviewer #2 (Public review):

      While the validation data set was well chosen and of high quality, it remains a single dataset and also remains a non-recurrent network. The authors acknowledge this in the discussion, but I wanted to chime in to say that for the method to be more than convincing, it would need to have been tested on more datasets. It should be acknowledged that the problem becomes more complicated in a recurrent excitatory network, and thus the method may not work as well in the cortex or in CA3.

      We will carefully revise our text to specifically discuss this limitation and the challenges inherent in generalizing to more recurrently connected regions. Furthermore, to empirically address this concern, we will test our method extensively on synthetic datasets generated from highly recurrent networks to quantify performance in these regimes.

      While the data is shown to work in this particular dataset (plus the two others at the end), I was left wondering when the method breaks. And it should break if the models are sufficiently mismatched. Such a question can be addressed using synthetic-synthetic models. This was an important intuition that I was missing, and an important check on the general nature of the method that I was missing.

      We thank the reviewer for this insight regarding the general nature of the method. While we previously analyzed failure cases regarding strong covariation and low spike counts, we agree that a systematic analysis of mismatch magnitude is missing. Building on our planned experiments with synthetic data, we will analyze and discuss exactly when the method breaks as a function of the mismatch magnitude between datasets.

      While the choice of state-of-the-art is good in my opinion, I was looking for comments on the methods prior to that. For instance, methods such based on GLMs have been used by the Pillow, Paninski, and Truccolo groups. I could not find a decent discussion of these methods in the main text and thought that both their acknowledgement and rationale for dismissing were missing.

      As the reviewer noted, we extensively compared our method with a GLM-based method (GLMCC) and CoNNECT, whose superiority over other GLM-based methods, such as extend GLM method (Ren et al., 2020, J Neurophysiol), have already been demonstrated in their papers (Endo et al., Sci Rep, 2021). However, we acknowledge that the discussion of the broader GLM literature was insufficient. To make the comparison more thorough, we will conduct comparisons with additional GLM-based methods and include a detailed discussion of these approaches.

      Endo, D., Kobayashi, R., Bartolo, R., Averbeck, B. B., Sugase-Miyamoto, Y., Hayashi, K., ... & Shinomoto, S. (2021). A convolutional neural network for estimating synaptic connectivity from spike trains. Scientific Reports, 11(1), 12087.

      Ren, N., Ito, S., Hafizi, H., Beggs, J. M., & Stevenson, I. H. (2020). Model-based detection of putative synaptic connections from spike recordings with latency and type constraints. Journal of Neurophysiology, 124(6), 1588-1604.

      While most of the text was very clear, I thought that page 11 was odd and missing much in terms of introductions. Foremost is the introduction of the dataset, which is never really done. Page 11 refers to 'this dataset', while the previous sentence was saying that having such a dataset would be important and is challenging. The dataset needs to be properly described: what's the method for labeling, what's the brain area, what were the spike recording methodologies, what is meant by two labeling methodologies, what do we know about the idiosyncrasies of the particular network the recording came from (like CA1 is non-recurrent, so which connections)? I was surprised to see 'English et al.' cited in text only on page 13 since their data has been hailed from the beginning.

      Further elements that needed definition are the Nsyn and i, which were not defined in the cortex of Equation 2-3: I was not sure if it referred to different samples or different variants of the synthetic model. I also would have preferred having the function f defined earlier, as it is defined for Equation 3, but appears in Equation 2.

      When the loss functions are described, it would be important to define 'data' and 'labels' here. This machine learning jargon has a concrete interpretation in this context, and making this concrete would be very important for the readership.

      We thank the reviewer for these constructive comments on the writing. We will clarify the introduction of the dataset (labeling method, brain area, recording methodology) and ensure all mathematical terms (such as Nsyn, i, and function f) and machine learning terminology (definitions of 'data' and 'labels' in this context) are rigorously defined upon first use in the revised manuscript.

      While I appreciated that there was a section on robustness, I did not find that the features studied were the most important. In this context, I was surprised that the other datasets were relegated to supplementary, as these appeared more relevant.

      Robustness is an important aspect of our framework to demonstrate its applicability to real experimental scenarios. We specifically analyzed how synchrony between neurons, the number of recorded spikes and the choice of the network influence the performance of our method. We also agree that these aspects are limited by the one dataset we evaluated on. Therefore, we will test the robustness of our method more systematically on synthetic datasets.

      With more extensive analysis on synthetic datasets, we believe that the results on inferring biophysical properties of single neuron and microcircuit models remain in the supplement, such that the main figures focus purely on synaptic connectivity inference.

      Some of the figures have text that is too small. In particular, Figure 2 has text that is way too small. It seemed to me that the pseudo code could stand alone, and the screenshot of the equations did not need to be repeated in a figure, especially if their size becomes so small that we can't even read them.

      We will remove the pseudo-code and equations from Figure 2 to improve readability. The pseudo-code will be presented as a distinct box in the main text.

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      Reply to the reviewers

      In our manuscript, we describe a role for the nuclear mRNA export factor UAP56 (a helicase) during metamorphic dendrite and presynapse pruning in flies. We characterize a UAP56 ATPase mutant and find that it rescues the pruning defects of a uap56 mutant. We identify the actin severing enzyme Mical as a potentially crucial UAP56 mRNA target during dendrite pruning and show alterations at both the mRNA and protein level. Finally, loss of UAP56 also causes presynapse pruning defects with actin abnormalities. Indeed, the actin disassembly factor cofilin is required for pruning specifically at the presynapse.

      We thank the reviewers for their constructive comments, which we tried to address experimentally as much as possible. To summarize briefly, while all reviewers saw the results as interesting (e. g., Reviewer 3's significance assessment: "Understanding how post-transcriptional events are linked to key functions in neurons is important and would be of interest to a broad audience") and generally methodologically strong, they thought that our conclusions regarding the potential specificity of UAP56 for Mical mRNA was not fully covered by the data. To address this criticism, we added more RNAi analyses of other mRNA export factors and rephrased our conclusions towards a more careful interpretation, i. e., we now state that the pruning process is particularly sensitive to loss of UAP56. In addition, reviewer 1 had technical comments regarding some of our protein and mRNA analyses. We added more explanations and an additional control for the MS2/MCP system. Reviewers 2 and 3 wanted to see a deeper characterization of the ATPase mutant provided. We generated an additional UAP56 mutant transgene, improved our analyses of UAP56 localization, and added a biochemical control experiment. We hope that our revisions make our manuscript suitable for publication.

      1. Point-by-point description of the revisions

      This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

      • *

      Comments by reviewer 1.

      Major comments

      1.

      For Figure 4, the MS2/MCP system is not quantitative. Using this technique, it is impossible to determine how many RNAs are located in each "dot". Each of these dots looks quite large and likely corresponds to some phase-separated RNP complex where multiple RNAs are stored and/or transported. Thus, these data do not support the conclusion that Mical mRNA levels are reduced upon UAP56 knockdown. A good quantitative microscopic assay would be something like smFISH. Additinally, the localization of Mical mRNA dots to dendrites is not convincing as it looks like regions where there are dendritic swellings, the background is generally brighter.

      Our response

      We indeed found evidence in the literature that mRNPs labeled with the MS2/MCP or similar systems form condensates (Smith et al., JCB 2015). Unfortunately, smFISH is not established for this developmental stage and would likely be difficult due to the presence of the pupal case. To address whether the Mical mRNPs in control and UAP56 KD neurons are comparable, we characterized the MCP dots in the respective neurons in more detail and found that their sizes did not differ significantly between control and UAP56 KD neurons. To facilitate interpretability, we also increased the individual panel sizes and include larger panels that only show the red (MCP::RFP) channel. We think these changes improved the figure. Thanks for the insight.

      Changes introduced: Figure 5 (former Fig. 4): Increased panel size for MCP::RFP images, left out GFP marker for better visibility. Added new analysis of MCP::RFP dot size (new Fig. 5 I).

      1.

      Alternatively, levels of Mical mRNA could be verified by qPCR in the laval brain following pan-neuronal UAP56 knockdown or in FACS-sorted fluorescently labeled da sensory neurons. Protein levels could be analyzed using a similar approach.

      Our response

      We thank the reviewer for this comment. Unfortunately, these experiments are not doable as neuron-wide UAP56 KD is lethal (see Flybase entry for UAP56). From our own experience, FACS-sorting of c4da neurons would be extremely difficult as the GFP marker fluorescence intensity of UAP56 KD neurons is weak - this would likely result in preferential sorting of subsets of neurons with weaker RNAi effects. In addition, FACS-sorting whole neurons would not discriminate between nuclear and cytoplasmic mRNA.

      The established way of measuring protein content in the Drosophila PNS system is immunofluorescence with strong internal controls. In our case, we also measured Mical fluorescence intensity of neighboring c1da neurons that do not express the RNAi and show expression levels as relative intensities compared to these internal controls. This procedure rules out the influence of staining variation between samples and is used by other labs as well.

      1.

      In Figure 5, the authors state that Mical expression could not be detected at 0 h APF. The data presented in Fig. 5C, D suggest the opposite as there clearly is some expression. Moreover, the data shown in Fig. 5D looks significantly brighter than the Orco dsRNA control and appears to localize to some type of cytoplasmic granule. So the expression of Mical does not look normal.

      Our response

      We thank the reviewer for this comment. In the original image in Fig. 5 C, the c4da neuron overlaps with the dendrite from a neighboring PNS neuron (likely c2da or c3da). The latter neuron shows strong Mical staining. We agree that this image is confusing and exchanged this image for another one from the same genotype.

      Changes introduced: Figure 5 L (former Fig. 5 C): Exchanged panel for image without overlap from other neuron.

      1.

      Sufficient data are not presented to conclude any specificity in mRNA export pathways. Data is presented for one export protein (UAP56) and one putative target (Mical). To adequately assess this, the authors would need to do RNA-seq in UAP56 mutants.

      Our response

      We thank the reviewer for this comment. To address this, we tested whether knockdown of three other mRNA export factors (NXF1, THO2, THOC5) causes dendrite pruning defects, which was not the case (new Fig. S1). While these data are consistent with specific mRNA export pathways, we agree that they are not proof. We therefore toned down our interpretation and removed the conclusion about specificity. Instead, we now use the more neutral term "increased sensibility (to loss of UAP56)".

      Changes introduced: Added new Figure S1: RNAi analyses of NXF1, THO2 and THOC5 in dendrite pruning. Introduced concluding sentence at the end of first Results paragraph: We conclude that c4da neuron dendrite pruning is particularly sensitive to loss of UAP56. (p. 6)

      1.

      In summary, better quantitative assays should be used in Figures 4 and 5 in order to conclude the expression levels of either mRNA or protein. In its current form, this study demonstrates the novel finding that UAP56 regulates dendrite and presynaptic pruning, potentially via regulation of the actin cytoskeleton. However, these data do not convincingly demonstrate that UAP56 controls these processes by regulating of Mical expression and defintately not by controlling export from the nucleus.

      Our response

      We hope that the changes we introduced above help clarify this.

      1.

      While there are clearly dendrites shown in Fig. 1C', the cell body is not readily identifiable. This makes it difficult to assess attachment and suggests that the neuron may be dying. This should be replaced with an image that shows the soma.

      Our response

      We thank the reviewer for this comment. Changes introduced: we replaced the picture in the panel with one where the cell body is more clearly visible.

      1.

      The level of knockdown in the UAS56 RNAi and P element insertion lines should be determined. It would be useful to mention the nature of the RNAi lines (long/short hairpin). Some must be long since Dcr has been co-expressed. Another issue raised by this is the potential for off-target effects. shRNAi lines would be preferable because these effects are minimized.

      Our response

      We thank the reviewer for this comment. Assessment of knockdown efficiency is a control to make sure the manipulations work the way they are intended to. As mRNA isolation from Drosophila PNS neurons is extremely difficult, RNAi or mutant phenotypes in this system are controlled by performing several independent manipulations of the same gene. In our case, we used two independent RNAi lines (both long hairpins from VDRC/Bloomington and an additional insertion of the VDRC line, see Table S1) as well as a mutant P element in a MARCM experiment, i. e., a total of three independent manipulations that all cause pruning defects, and the VDRC RNAi lines do not have any predicted OFF targets (not known for the Bloomington line). If any of these manipulations would not have matched, we would have generated sgRNA lines for CRISPR to confirm.

      Minor comments:

      1.

      The authors should explain what EB1:GFP is marking when introduced in the text.


      Our response

      We thank the reviewer for this comment. Changes introduced: we explain the EB1::GFP assay in the panel with one where the cell body is more clearly visible.

      1.

      The da neuron images throughout the figures could be a bit larger.

      Our response

      We thank the reviewer for this comment. Changes introduced: we changed the figure organization to be able to use larger panels:

      • the pruning analysis of the ATPase mutations (formerly Fig. 2) is now its own figure (Figure 3).

      • we increased the panel sizes of the MCP::RFP images (Figure 5 A - I, formerly Fig. 4).

      Reviewer #1 (Significance (Required)):

      Strengths:

      The methodology used to assess dendrite and presynaptic prunings are strong and the phenotypic analysis is conclusive.

      Our response

      We thank the reviewer for this comment.

      Weakness:

      The evidence demonstrating that UAP56 regulates the expression of Mical is unconvincing. Similarly, no data is presented to show that there is any specificity in mRNA export pathways. Thus, these major conclusions are not adequately supported by the data.

      Our response

      We hope the introduced changes address this comment.

      __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

      In this paper, the authors describe dendrite pruning defects in c4da neurons in the DEXD box ATPase UAP56 mutant or in neuronal RNAi knockdown. Overexpression UAP56::GFP or UAP56::GFPE194Q without ATPase activity can rescue dendrite pruning defects in UAP56 mutant. They further characterized the mis-localization of UAP56::GFPE194Q and its binding to nuclear export complexes. Both microtubules and the Ubiquitin-proteasome system are intact in UAP56RNAi neurons. However, they suggest a specific effect on MICAL mRNA nuclear export shown by using the MS2-MCP system., resulting in delay of MICAL protein expression in pruned neurons. Furthermore, the authors show that UAP56 is also involved in presynaptic pruning of c4da neuros in VNC and Mica and actin are also required for actin disassembly in presynapses. They propose that UAP56 is required for dendrite and synapse pruning through actin regulation in Drosophila. Following are my comments.

      Major comments

      1.

      The result that UAP56::GFPE194Q rescues the mutant phenotype while the protein is largely mis-localized suggests a novel mechanism or as the authors suggested rescue from combination of residual activities. The latter possibility requires further support, which is important to support the role mRNA export in dendrite and pre-synapse pruning. One approach would be to examine whether other export components like REF1, and NXF1 show similar mutant phenotypes. Alternatively, depleting residual activity like using null mutant alleles or combining more copies of RNAi transgenes could help.

      Our response

      We thank the reviewer for this comment. We agree that the mislocalization phenotype is interesting and could inform further studies on the mechanism of UAP56. To further investigate this and to exclude that this could represent a gain-of-function due to the introduced mutation, we made and characterized a new additional transgene, UAP56::GFP E194A. This mutant shows largely the same phenotypes as E194Q, with enhanced interactions with Ref1 and partial mislocalization to the cytoplasm. In addition, we tested whether knockdown of THO2, THOC5 or NXF1 causes pruning defects (no).

      Changes introduced:

      • added new Figure S1: RNAi analyses of NXF1, THO2 and THOC5 in dendrite pruning.

      • made and characterized a new transgene UAP56 E194A (new Fig. 2 B, E, E', 3 C, C', E, F).

      1.

      The localization of UAP56::GFP (and E194Q) should be analyzed in more details. It is not clear whether the images in Fig. 2A and 2B are from confocal single sections or merged multiple sections. The localization to the nuclear periphery of UAP56::GFP is not clear, and the existence of the E194Q derivative in both nucleus and cytosol (or whether there is still some peripheral enrichment) is not clear if the images are stacked.

      Our response

      We thank the reviewer for this comment. It is correct that the profiles in the old Figure 2 were from single confocal sections from the displayed images. As it was difficult to create good average profiles with data from multiple neurons, we now introduce an alternative quantification based on categories (nuclear versus dispersed) which includes data from several neurons for each genotype, including the new E194A transgene (new Fig 3 G). Upon further inspection, the increase at the nuclear periphery was not always visible and may have been a misinterpretation. We therefore removed this statement.

      Changes introduced:

      • added new quantitative analysis of UAP56 wt and E/A, E/Q mutant localization (new Fig 3 G).

      1.

      The Ub-VV-GFP is a new reagent, and its use to detect active proteasomal degradation is by the lack of GFP signals, which could be also due to the lack of expression. The use of Ub-QQ-GFP cannot confirm the expression of Ub-VV-GFP. The proteasomal subunit RPN7 has been shown to be a prominent component in the dendrite pruning pathway (Development 149, dev200536). Immunostaining using RPN7 antibodies to measure the RPN expression level could be a direct way to address the issue whether the proteasomal pathway is affected or not.

      Our response

      We thank the reviewer for this comment. We agree that it is wise to not only introduce a positive control for the Ub-VV-GFP sensor (the VCP dominant-negative VCP QQ), but also an independent control. As mutants with defects in proteasomal degradation accumulate ubiquitinated proteins (see, e. g., Rumpf et al., Development 2011), we stained controls and UAP56 KD neurons with antibodies against ubiquitin and found that they had similar levels (new Fig. S3).

      Changes introduced:

      • added new ubiquitin immunofluorescence analysis (new Fig. S3).

      1.

      Using the MS2/MCP system to detect the export of MICAL mRNA is a nice approach to confirm the UAP56 activity; lack of UAP56 by RNAi knockdown delays the nuclear export of MS2-MICAL mRNA. The rescue experiment by UAS transgenes could not be performed due to the UAS gene dosage, as suggested by the authors. However, this MS2-MICAL system is also a good assay for the requirement of UAP56 ATPase activity (absence in the E194Q mutant) in this process. Could authors use the MARCM (thus reduce the use of UAS-RNAi transgene) for the rescue experiment? Also, the c4da neuronal marker UAS-CD8-GFP used in Fig4 could be replaced by marker gene directly fused to ppk promoter, which can save a copy of UAS transgene. The results from the rescue experiment would test the dependence of ATPase activity in nuclear export of MICAL mRNA.

      Our response

      We thank the reviewer for this comment. This is a great idea but unfortunately, this experiment was not feasible due to the (rare) constraints of Drosophila genetics. The MARCM system with rescue already occupies all available chromosomes (X: FLPase, 2nd: FRT, GAL80 + mutant, 3rd: GAL4 + rescue construct), and we would have needed to introduce three additional ones (MCP::RFP and two copies of unmarked genomic MICAL-MS2, all on the third chromosome) that would have needed to be introduced by recombination. Any Drosophilist will see that this is an extreme, likely undoable project :-(

      1.

      The UAP56 is also involved in presynaptic pruning through regulating actin assembly, and the authors suggest that Mical and cofilin are involved in the process. However, direct observation of lifeact::GFP in Mical or cofilin RNAi knockdown is important to support this conclusion.

      Our response

      We thank the reviewer for this comment. In response, we analyzed the lifeact::GFP patterns of control and cofilin knockdown neurons and found that loss of cofilin also leads to actin accumulation (new Fig. 7 I, J).

      Changes introduced:

      • new lifeact analysis (new Fig. 7 I, J).

      Minor comments:

      1.

      RNA localization is important for dendrite development in larval stages (Brechbiel JL, Gavis ER. Curr Biol. 20;18(10):745-750). Yet, the role of UAP56 is relatively specific and shown only in later-stage pruning. It would need thorough discussion.


      Our response

      We thank reviewer 2 for this comment. We added the following paragraph to the discussion: "UAP56 has also been shown to affect cytoplasmic mRNA localization in Drosophila oocytes (Meignin and Davis, 2008), opening up the possibility that nuclear mRNA export and cytoplasmic transport are linked. It remains to be seen whether this also applies to dendritic mRNA transport (Brechbiel and Gavis, 2008)." (p.13)

      1.

      Could authors elaborate on the possible upstream regulators that might be involved, as described in "alternatively, several cofilin upstream regulators have been described (Rust, 2015) which might also be involved in presynapse pruning and subject to UAP56 regulation" in Discussion?

      Our response

      We thank reviewer 2 for this comment. In the corresponding paragraph, we cite as example now that cofilin is regulated by Slingshot phosphatases and LIM kinase (p.14).

      1.

      In Discussion, the role of cofilin in pre- and post-synaptic processes was described. The role of Tsr/Cofilin regulating actin behaviors in dendrite branching has been described in c3da and c4da neurons (Nithianandam and Chien, 2018 and other references) should be included in Discussion.

      Our response

      We thank reviewer 2 for this comment. In response we tested whether cofilin is required for dendrite pruning and found that this, in contrast to Mical, is not the case (new Fig. S6). We cite the above paper in the corresponding results section (p.12).

      Changes introduced:

      • new cofilin dendrite pruning analysis (new Fig. S6).

      • added cofilin reference in Results.

      1.

      The authors speculate distinct actin structures have to be disassembled in dendrite and presynapse pruning in Discussion. What are the possible actin structures in both sites could be elaborated.

      Our response

      We thank reviewer 2 for this comment. In response, we specify in the Discussion: "As Mical is more effective in disassembling bundled F-actin than cofilin (Rajan et al., 2023), it is interesting to speculate that such bundles are more prevalent in dendrites than at presynapses." (p14)

      Reviewer #2 (Significance (Required)):

      The study initiated a genetic screen for factors involved in a dendrite pruning system and reveals the involvement of nuclear mRNA export is an important event in this process. They further identified the mRNA of the actin disassembly factor MICAL is a candidate substrate in the exporting process. This is consistent with previous finding that MICAL has to be transcribed and translated when pruning is initiated. As the presynapses of the model c4da neuron in this study is also pruned, the dependence on nuclear export and local actin remodeling were also shown. Thus, this study has added another layer of regulation (the nuclear mRNA export) in c4da neuronal pruning, which would be important for the audience interested in neuronal pruning. The study is limited for the confusing result whether ATPase activity of the exporting factor is required.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      Summary: In the manuscript by Frommeyer, Gigengack et al. entitled "The UAP56 mRNA Export Factor is Required for Dendrite and Synapse Pruning via Actin Regulation in Drosophila" the authors surveyed a number of RNA export/processing factors to identify any required for efficient dendrite and/or synapse pruning. They describe a requirement for a general poly(A) RNA export factor, UAP56, which functions as an RNA helicase. They also study links to aspects of actin regulation.

      Overall, while the results are interesting and the impact of loss of UAP56 on the pruning is intriguing, some of the data are overinterpreted as presented. The argument that UAP56 may be specific for the MICAL RNA is not sufficiently supported by the data presented. The two stories about poly(A) RNA export/processing and the actin regulation seem to not quite be connected by the data presented. The events are rather distal within the cell, making connecting the nuclear events with RNA to events at the dendrites/synapse challenging.

      Our response

      We thank reviewer 3 for this comment. To address this, we tested whether knockdown of three other mRNA export factors (NXF1, THO2, THOC5) causes dendrite pruning defects, which was not the case (new Fig. S1). While these data are consistent with specific mRNA export pathways, we agree that they are not proof. We therefore toned down our interpretation and removed the conclusion about specificity. Instead, we now use the more neutral term "increased sensibility (to loss of UAP56)".

      We agree that it is a little hard to tie cofilin to UAP56, as we currently have no evidence that cofilin levels are affected by loss of UAP56, even though both seem to affect lifeact::GFP in a similar way (new Fig. 7 I, J). However, a dysregulation of cofilin can also occur through dysregulation of upstream cofilin regulators such as Slingshot and LIM kinase, making such a relationship possible.

      Changes introduced:

      • added new Figure S1: RNAi analyses of NXF1, THO2 and THOC5 in dendrite pruning.

      • introduced concluding sentence at the end of first Results paragraph: "We conclude that c4da neuron dendrite pruning is particularly sensitive to loss of UAP56." (p. 6)

      • add new lifeact::GFP analysis of cofilin KD (new Fig. I, J).

      • identify potential other targets from the literature in the Discussion (Slingshot phosphatases and LIM kinase, p.14).

      There are a number of specific statements that are not supported by references. See, for example, these sentences within the Introduction- "Dysregulation of pruning pathways has been linked to various neurological disorders such as autism spectrum disorders and schizophrenia. The cell biological mechanisms underlying pruning can be studied in Drosophila." The Drosophila sentence is followed by some specific examples that do include references. The authors also provide no reference to support the variant that they create in UAP56 (E194Q) and whether this is a previously characterized fly variant or based on an orthologous protein in a different system. If so, has the surprising mis-localization been reported in another system?

      Our response

      We thank reviewer 3 for this comment. We added the following references on pruning and disease:

      1) Howes, O.D., Onwordi, E.C., 2023. The synaptic hypothesis of schizophrenia version III: a master mechanism. Mol. Psychiatry 28, 1843-1856.

      2) Tang, G., et al., 2014. Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits. Neuron 83, 1131-43.

      To better introduce the E194 mutations, we explain the position of the DECD motif in the Walker B domain, give the corresponding residues in the human and yeast homologues and cite papers demonstrating the importance of this residue for ATPase activity:

      3) Saguez, C., et al., 2013. Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability. RNA 19:1363-71.

      4) Shen, J., et al., 2007. Biochemical Characterization of the ATPase and Helicase Activity of UAP56, an Essential Pre-mRNA Splicing and mRNA Export Factor. J. Biol. Chem. 282, P22544-22550.

      We are not aware of other studies looking at the relationship between the UAP56 ATPase and its localization. Thank you for pointing this out!

      Specific Comments:

      Specific Comment 1: Figure 1 shows the impact of loss of UAP56 on neuron dendrite pruning. The experiment employs both two distinct dsRNAs and a MARCM clone, providing confidence that there is a defect in pruning upon loss of UAP56. As the authors mention screening against 92 genes that caused splicing defects in S2 cells, inclusion of some examples of these genes that do not show such a defect would enhance the argument for specificity with regard to the role of UAP56. This control would be in addition to the more technical control that is shown, the mCherry dsRNA.

      Our response

      We thank reviewer 3 for this comment. To address this, we included the full list of screened genes with their phenotypic categorization regarding pruning (103 RNAi lines targeting 64 genes) as Table S1. In addition, we also tested four RNAi lines targeting the nuclear mRNA export factors Nxf1, THO2 and THOC5 which do not cause dendrite pruning defects (Fig. S1).

      Changes introduced:

      • added RNAi screen results as a list in Table S1.

      • added new Figure S1: RNAi analyses of NXF1, THO2 and THOC5 in dendrite pruning.

      Specific Comment 2: Later the authors demonstrate a delay in the accumulation of the Mical protein, so if they assayed these pruning events at later times, would the loss of UAP56 cause a delay in these events as well? Such a correlation would enhance the causality argument the authors make for Mical levels and these pruning events.

      Our response

      We thank reviewer 3 for this comment. Unfortunately, this is somewhat difficult to assess, as shortly after the 18 h APF timepoint, the epidermal cells that form the attachment substrate for c4da neuron dendrites undergo apoptosis. Where assessed (e. g., Wang et al., 2017, Development) 144: 1851–1862), this process, together with the reduced GAL4 activity of our ppk-GAL4 during the pupal stage (our own observations), eventually leads to pruning, but the causality cannot be easily attributed anymore. We therefore use the 18 h APF timepoint essentially as an endpoint assay.

      Specific Comment 3: Figure 2 provides data designed to test the requirement for the ATPase/helicase activity of UAP56 for these trimming events. The first observation, which is surprising, is the mislocalization of the variant (E194Q) that the authors generate. The data shown does not seem to indicate how many cells the results shown represent as a single image and trace is shown the UAP56::GFP wildtype control and the E194Q variant.

      Our response

      We thank reviewer 3 for this comment. It is correct that the traces shown are from single confocal sections. To better display the phenotypic penetrance, we now added a categorical analysis that shows that the UAP56 E194Q mutant is completely mislocalized in the majority of cells assessed (and the newly added E194A mutant in a subset of cells).

      Changes introduced:

      • added categorical quantification of UAP56 variant localization (new Fig. 2 G).

      __Specific Comment 4: __Given the rather surprising finding that the ATPase activity is not required for the function of UAP56 characterized here, the authors do not provide sufficient references or rationale to support the ATPase mutant that they generate. The E194Q likely lies in the Walker B motif and is equivalent to human E218Q, which can prevent proper ATP hydrolysis in the yeast Sub2 protein. There is no reference to support the nature of the variant created here.

      Our response

      We thank reviewer 3 for this comment. To better introduce the E194 mutations, we explain the position of the DECD motif in the Walker B domain, give the corresponding residues in the human and yeast homologues (Sub2) and cite papers demonstrating the importance of this residue for ATPase activity:

      1) Saguez, C., et al., 2013. Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability. RNA 19:1363-71.

      2) Shen, J., et al., 2007. Biochemical Characterization of the ATPase and Helicase Activity of UAP56, an Essential Pre-mRNA Splicing and mRNA Export Factor. J. Biol. Chem. 282, P22544-22550.

      __Specific Comment 5: __Given the surprising results, the authors could have included additional variants to ensure the change has the biochemical effect that the authors claim. Previous studies have defined missense mutations in the ATP-binding site- K129A (Lysine to Alanine): This mutation, in both yeast Sub2 and human UAP56, targets a conserved lysine residue that is critical for ATP binding. This prevents proper ATP binding and consequently impairs helicase function. There are also missense mutations in the DEAD-box motif, (Asp-Glu-Ala-Asp) involved in ATP binding and hydrolysis. Mutations in this motif, such as D287A in yeast Sub2 (corresponding to D290A in human UAP56), can severely disrupt ATP hydrolysis, impairing helicase activity. In addition, mutations in the Walker A (GXXXXGKT) and Walker B motifs are can impair ATP binding and hydrolysis in DEAD-box helicases. Missense mutations in these motifs, like G137A (in the Walker A motif), can block ATP binding, while E218Q (in the Walker B motif)- which seems to be the basis for the variant employed here- can prevent proper ATP hydrolysis.

      Our response

      We thank reviewer 3 for this comment. Our cursory survey of the literature suggested that mutations in the Walker B motif are the most specific as they still preserve ATP binding and their effects have not well been characterized overall. In addition, these mutations can create strong dominant-negatives in related helicases (e. g., Rode et al., 2018 Cell Reports, our lab). To better characterize the role of the Walker B motif in UAP56, we generated and characterized an alternative mutant, UAP56 E194A. While the E194A variant does not show the same penetrance of localization phenotypes as E194Q, it also is partially mislocalized, shows stronger binding to Ref1 and also rescues the uap56 mutant phenotypes without an obvious dominant-negative effect, thus confirming our conclusions regarding E194Q.

      Changes introduced:

      • added biochemical, localization and phenotypic analysis of newly generated UAP56 E194A variant (new Figs. 2 B, 2 E, E', 3 C, C'). categorical quantification of UAP56 variant localization (new Fig. 2 G).

      __Specific Comment 6: __The co-IP results shown in Figure 2C would also seem to have multiple potential interpretations beyond what the authors suggest, an inability to disassemble a complex. The change in protein localization with the E194Q variant could impact the interacting proteins. There is no negative control to show that the UAP56-E194Q variant is not just associated with many, many proteins. Another myc-tagged protein that does not interact would be an ideal control.

      Our response

      We thank reviewer 3 for this comment. To address this comment, we tried to co-IP UAP56 wt or UAP56 E194Q with a THO complex subunit THOC7 (new Fig. S2). The results show that neither UAP56 variant can co-IP THOC7 under our conditions (likely because the UAP56/THO complex intermediate during mRNA export is disassembled in an ATPase-independent manner (Hohmann et al., Nature 2025)).

      Changes introduced:

      • added co-IP experiment between UAP56 variants and THOC7 (new Fig. S2).

      __Specific Comment 7: __With regard to Figure 3, the authors never define EB1::GFP in the text of the Results, so a reader unfamiliar with this system has no idea what they are seeing. Reading the Materials and Methods does not mitigate this concern as there is only a brief reference to a fly line and how the EB1::GFP is visualized by microscopy. This makes interpretation of the data presented in Figure 3A-C very challenging.

      Our response

      We thank reviewer 3 for pointing this out. We added a description of the EB1::GFP analysis in the corresponding Results section (p.8).

      __Specific Comment 8: __The data shown for MICAL MS2 reporter localization in Figure 4 is nice, but is also fully expected on many former studies analyzing loss of UAP56 or UAP56 hypomorphs in different systems. While creating the reporter is admirable, to make the argument that MICAL localization is in some way preferentially impacted by loss of UAP56, the authors would need to examine several other transcripts. As presented, the authors can merely state that UAP56 seems to be required for the efficient export of an mRNA transcript, which is predicted based on dozens of previous studies dating back to the early 2000s.

      Our response

      Firstly, thank you for commenting on the validity of the experimental approach! The primary purpose of this experiment was to test whether the mechanism of UAP56 during dendrite pruning conforms with what is known about UAP56's cellular role - which it apparently does. We also noted that our statements regarding the specificity of UAP56 for Mical over other transcripts are difficult. While our experiments would be consistent with such a model, they do not prove it. We therefore toned down the corresponding statements (e. g., the concluding sentence at the end of first Results paragraphis now: "We conclude that c4da neuron dendrite pruning is particularly sensitive to loss of UAP56." (p. 6)).

      Minor (and really minor) points:

      In the second sentence of the Discussion, the word 'developing' seems to be mis-typed "While a general inhibition of mRNA export might be expected to cause broad defects in cellular processes, our data in develoing c4da neurons indicate that loss of UAP56 mainly affects pruning mechanisms related to actin remodeling."

      Sentence in the Results (lack of page numbers makes indicating where exactly a bit tricky)- "We therefore reasoned that Mical expression could be more challenging to c4da neurons." This is a complete sentence as presented, yet, if something is 'more something'- the thing must be 'more than' something else. Presumably, the authors mean that the length of the MICAL transcript could make the processing and export of this transcript more challenging than typical fly transcripts (raising the question of the average length of a mature transcript in flies?).

      Our response

      Thanks for pointing these out. The typo is fixed, page numbers are added. We changed the sentence to: "Because of the large size of its mRNA, we reasoned that MICAL gene expression could be particularly sensitive to loss of export factors such as UAP56." (p.9) We hope this is more precise language-wise.

      Reviewer #3 (Significance (Required)):

      Understanding how post-transcriptional events are linked to key functions in neurons is important and would be of interest to a broad audience.

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      Referee #3

      Evidence, reproducibility and clarity

      Summary:

      In the manuscript by Frommeyer, Gigengack et al. entitled "The UAP56 mRNA Export Factor is Required for Dendrite and Synapse Pruning via Actin Regulation in Drosophila" the authors surveyed a number of RNA export/processing factors to identify any required for efficient dendrite and/or synapse pruning. They describe a requirement for a general poly(A) RNA export factor, UAP56, which functions as an RNA helicase. They also study links to aspects of actin regulation.

      Overall, while the results are interesting and the impact of loss of UAP56 on the pruning is intriguing, some of the data are overinterpreted as presented. The argument that UAP56 may be specific for the MICAL RNA is not sufficiently supported by the data presented. The two stories about poly(A) RNA export/processing and the actin regulation seem to not quite be connected by the data presented. The events are rather distal within the cell, making connecting the nuclear events with RNA to events at the dendrites/synapse challenging.

      There are a number of specific statements that are not supported by references. See, for example, these sentences within the Introduction- "Dysregulation of pruning pathways has been linked to various neurological disorders such as autism spectrum disorders and schizophrenia. The cell biological mechanisms underlying pruning can be studied in Drosophila." The Drosophila sentence is followed by some specific examples that do include references. The authors also provide no reference to support the variant that they create in UAP56 (E194Q) and whether this is a previously characterized fly variant or based on an orthologous protein in a different system. If so, has the surprising mis-localization been reported in another system?

      Specific Comments:

      Figure 1 shows the impact of loss of UAP56 on neuron dendrite pruning. The experiment employs both two distinct dsRNAs and a MARCM clone, providing confidence that there is a defect in pruning upon loss of UAP56. As the authors mention screening against 92 genes that caused splicing defects in S2 cells, inclusion of some examples of these genes that do not show such a defect would enhance the argument for specificity with regard to the role of UAP56. This control would be in addition to the more technical control that is shown, the mCherry dsRNA. Later the authors demonstrate a delay in the accumulation of the Mical protein, so if they assayed these pruning events at later times, would the loss of UAP56 cause a delay in these events as well? Such a correlation would enhance the causality argument the authors make for Mical levels and these pruning events.

      Figure 2 provides data designed to test the requirement for the ATPase/helicase activity of UAP56 for these trimming events. The first observation, which is surprising, is the mislocalization of the variant (E194Q) that the authors generate. The data shown does not seem to indicate how many cells the results shown represent as a single image and trace is shown the UAP56::GFP wildtype control and the E194Q variant.

      Given the rather surprising finding that the ATPase activity is not required for the function of UAP56 characterized here, the authors do not provide sufficient references or rationale to support the ATPase mutant that they generate. The E194Q likely lies in the Walker B motif and is equivalent to human E218Q, which can prevent proper ATP hydrolysis in the yeast Sub2 protein. There is no reference to support the nature of the variant created here.

      Given the surprising results, the authors could have included additional variants to ensure the change has the biochemical effect that the authors claim. Previous studies have defined missense mutations in the ATP-binding site- K129A (Lysine to Alanine): This mutation, in both yeast Sub2 and human UAP56, targets a conserved lysine residue that is critical for ATP binding. This prevents proper ATP binding and consequently impairs helicase function. There are also missense mutations in the DEAD-box motif, (Asp-Glu-Ala-Asp) involved in ATP binding and hydrolysis. Mutations in this motif, such as D287A in yeast Sub2 (corresponding to D290A in human UAP56), can severely disrupt ATP hydrolysis, impairing helicase activity. In addition, mutations in the Walker A (GXXXXGKT) and Walker B motifs are can impair ATP binding and hydrolysis in DEAD-box helicases. Missense mutations in these motifs, like G137A (in the Walker A motif), can block ATP binding, while E218Q (in the Walker B motif)- which seems to be the basis for the variant employed here- can prevent proper ATP hydrolysis.

      The co-IP results shown in Figure 2C would also seem to have multiple potential interpretations beyond what the authors suggest, an inability to disassemble a complex. The change in protein localization with the E194Q variant could impact the interacting proteins. There is no negative control to show that the UAP56-E194Q variant is not just associated with many, many proteins. Another myc-tagged protein that does not interact would be an ideal control.

      With regard to Figure 3, the authors never define EB1::GFP in the text of the Results, so a reader unfamiliar with this system has no idea what they are seeing. Reading the Materials and Methods does not mitigate this concern as there is only a brief reference to a fly line and how the EB1::GFP is visualized by microscopy. This makes interpretation of the data presented in Figure 3A-C very challenging. The data shown for MICAL MS2 reporter localization in Figure 4 is nice, but is also fully expected on many former studies analyzing loss of UAP56 or UAP56 hypomorphs in different systems. While creating the reporter is admirable, to make the argument that MICAL localization is in some way preferentially impacted by loss of UAP56, the authors would need to examine several other transcripts. As presented, the authors can merely state that UAP56 seems to be required for the efficient export of an mRNA transcript, which is predicted based on dozens of previous studies dating back to the early 2000s.

      Minor (and really minor) points:

      In the second sentence of the Discussion, the word 'developing' seems to be mis-typed "While a general inhibition of mRNA export might be expected to cause broad defects in cellular processes, our data in develoing c4da neurons indicate that loss of UAP56 mainly affects pruning mechanisms related to actin remodeling."

      Sentence in the Results (lack of page numbers makes indicating where exactly a bit tricky)- "We therefore reasoned that Mical expression could be more challenging to c4da neurons." This is a complete sentence as presented, yet, if something is 'more something'- the thing must be 'more than' something else. Presumably, the authors mean that the length of the MICAL transcript could make the processing and export of this transcript more challenging than typical fly transcripts (raising the question of the average length of a mature transcript in flies?).

      Significance

      Understanding how post-transcriptional events are linked to key functions in neurons is important and would be of interest to a broad audience.

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      Referee #2

      Evidence, reproducibility and clarity

      In this paper, the authors describe dendrite pruning defects in c4da neurons in the DEXD box ATPase UAP56 mutant or in neuronal RNAi knockdown. Overexpression UAP56::GFP or UAP56::GFPE194Q without ATPase activity can rescue dendrite pruning defects in UAP56 mutant. They further characterized the mis-localization of UAP56::GFPE194Q and its binding to nuclear export complexes. Both microtubules and the Ubiquitin-proteasome system are intact in UAP56RNAi neurons. However, they suggest a specific effect on MICAL mRNA nuclear export shown by using the MS2-MCP system., resulting in delay of MICAL protein expression in pruned neurons. Furthermore, the authors show that UAP56 is also involved in presynaptic pruning of c4da neuros in VNC and Mica and actin are also required for actin disassembly in presynapses. They propose that UAP56 is required for dendrite and synapse pruning through actin regulation in Drosophila. Following are my comments.

      Major comments

      1. The result that UAP56::GFPE194Q rescues the mutant phenotype while the protein is largely mis-localized suggests a novel mechanism or as the authors suggested rescue from combination of residual activities. The latter possibility requires further support, which is important to support the role mRNA export in dendrite and pre-synapse pruning. One approach would be to examine whether other export components like REF1, and NXF1 show similar mutant phenotypes. Alternatively, depleting residual activity like using null mutant alleles or combining more copies of RNAi transgenes could help.

      2. The localization of UAP56::GFP (and E194Q) should be analyzed in more details. It is not clear whether the images in Fig. 2A and 2B are from confocal single sections or merged multiple sections. The localization to the nuclear periphery of UAP56::GFP is not clear, and the existence of the E194Q derivative in both nucleus and cytosol (or whether there is still some peripheral enrichment) is not clear if the images are stacked.

      3. The Ub-VV-GFP is a new reagent, and its use to detect active proteasomal degradation is by the lack of GFP signals, which could be also due to the lack of expression. The use of Ub-QQ-GFP cannot confirm the expression of Ub-VV-GFP. The proteasomal subunit RPN7 has been shown to be a prominent component in the dendrite pruning pathway (Development 149, dev200536). Immunostaining using RPN7 antibodies to measure the RPN expression level could be a direct way to address the issue whether the proteasomal pathway is affected or not.

      4. Using the MS2/MCP system to detect the export of MICAL mRNA is a nice approach to confirm the UAP56 activity; lack of UAP56 by RNAi knockdown delays the nuclear export of MS2-MICAL mRNA. The rescue experiment by UAS transgenes could not be performed due to the UAS gene dosage, as suggested by the authors. However, this MS2-MICAL system is also a good assay for the requirement of UAP56 ATPase activity (absence in the E194Q mutant) in this process. Could authors use the MARCM (thus reduce the use of UAS-RNAi transgene) for the rescue experiment? Also, the c4da neuronal marker UAS-CD8-GFP used in Fig4 could be replaced by marker gene directly fused to ppk promoter, which can save a copy of UAS transgene. The results from the rescue experiment would test the dependence of ATPase activity in nuclear export of MICAL mRNA.

      5. The UAP56 is also involved in presynaptic pruning through regulating actin assembly, and the authors suggest that Mical and cofilin are involved in the process. However, direct observation of lifeact::GFP in Mical or cofilin RNAi knockdown is important to support this conclusion.

      Minor comments

      1. RNA localization is important for dendrite development in larval stages (Brechbiel JL, Gavis ER. Curr Biol. 20;18(10):745-750). Yet, the role of UAP56 is relatively specific and shown only in later-stage pruning. It would need thorough discussion.

      2. Could authors elaborate on the possible upstream regulators that might be involved, as described in "alternatively, several cofilin upstream regulators have been described (Rust, 2015) which might also be involved in presynapse pruning and subject to UAP56 regulation" in Discussion?

      3. In Discussion, the role of cofilin in pre- and post-synaptic processes was described. The role of Tsr/Cofilin regulating actin behaviors in dendrite branching has been described in c3da and c4da neurons (Nithianandam and Chien, 2018 and other references) should be included in Discussion.

      4. The authors speculate distinct actin structures have to be disassembled in dendrite and presynapse pruning in Discussion. What are the possible actin structures in both sites could be elaborated.

      Significance

      The study initiated a genetic screen for factors involved in a dendrite pruning system and reveals the involvement of nuclear mRNA export is an important event in this process. They further identified the mRNA of the actin disassembly factor MICAL is a candidate substrate in the exporting process. This is consistent with previous finding that MICAL has to be transcribed and translated when pruning is initiated. As the presynapses of the model c4da neuron in this study is also pruned, the dependence on nuclear export and local actin remodeling were also shown. Thus, this study has added another layer of regulation (the nuclear mRNA export) in c4da neuronal pruning, which would be important for the audience interested in neuronal pruning. The study is limited for the confusing result whether ATPase activity of the exporting factor is required.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      This manuscript by Frommeyer et al. explores the role of the helicase and regulator of nuclear export, UAP56, in the control of dendrite and presynaptic pruning in Drosophila larval da sensory neurons. The authors present evidence showing that UAP56 regulates these processes via the actin cytoskeleton and suggest that this is occurs by controlling the expression of the actin severing enzyme, Mical.

      Major comments:

      The most signficant issue with the manuscript is that some of the major conclusions are not supported by the data. Additional experiment would need to be completed in order support these claims. These (and other) major comments are as follows:

      1. For Figure 4, the ms2/MCP system is not quantitative. Using this technique, it is impossible to determine how many RNAs are located in each "dot". Each of these dots looks quite large and likely corresponds to some phase-separated RNP complex where multiple RNAs are stored and/or transported. Thus, these data do not support the conclusion that Mical mRNA levels are reduced upon UAP56 knockdown. A good quantitative microscopic assay would be something like smFISH. Additinally, the localization of Mical mRNA dots to dendrites is not convincing as it looks like regions where there are dendritic swellings, the background is generally brighter.

      2. Alternatively, levels of Mical mRNA could be verified by qPCR in the laval brain following pan-neuronal UAP56 knockdown or in FACS-sorted fluorescently labeled da sensory neurons. Protein levels could be analyzed using a similar approach.

      3. In Figure 5, the authors state that Mical expression could not be detected at 0 h APF. The data presented in Fig. 5C, D suggest the opposite as there clearly is some expression. Moreover, the data shown in Fig. 5D looks significantly brighter than the Orco dsRNA control and appears to localize to some type of cytoplasmic granule. So the expression of Mical does not look normal.

      4. Sufficient data are not presented to conclude any specificity in mRNA export pathways. Data is presented for one export protein (UAP56) and one putative target (Mical). To adequately assess this, the authors would need to do RNA-seq in UAP56 mutants.

      5. In summary, better quantitative assays should be used in Figures 4 and 5 in order to conclude the expression levels of either mRNA or protein. In its current form, this study demonstrates the novel finding that UAP56 regulates dendrite and presynaptic pruning, potentially via regulation of the actin cytoskeleton. However, these data do not convincingly demonstrate that UAP56 controls these processes by regulating of Mical expression and defintately not by controlling export from the nucleus.

      6. While there are clearly dendrites shown in Fig. 1C', the cell body is not readily identifiable. This makes it difficult to assess attachment and suggests that the neuron may be dying. This should be replaced with an image that shows the soma.

      7. The level of knockdown in the UAS56 RNAi and P element insertion lines should be determined. It would be useful to mention the nature of the RNAi lines (long/short hairpin). Some must be long since Dcr has been co-expressed. Another issue raised by this is the potential for off-target effects. shRNAi lines would be preferable because these effects are minimized.

      Minor comments:

      1. The authors should explain what EB1:GFP is marking when introduced in the text.

      2. The da neuron images througout the figures could be a bit larger.

      Significance

      Strengths:

      The methodology used to assess dendrite and presynaptic prunings are strong and the phenotypic analysis is conclusive.

      Weakness:

      The evidence demonstrating that UAP56 regulates the expression of Mical is unconvincing. Similarly, no data is presented to show that there is any specificity in mRNA export pathways. Thus, these major conclusions are not adequately supported by the data.

      Advance:

      The findings that UAP56 regulate dendrite and synaptic pruning are novel. As is its specific regulation of the actin cytoskeleton. These findings are restricted to a phenotypic analysis and do not show that it is not simply due to the disruption of general mRNA export.

      Audience:

      In its current form the manuscript whould be of interest to an audience who specializes in the study of RNA binding proteins in the control of neurodevelopment. This would include scientists who work in invertebrate and vertebrate model systems.

      My expertise:

      My lab uses Drosophila to study the role of RNA binding proteins in neurodevelopment and neurodegeneration. Currently, we use flies as a model to better understand the molecular pathogenesis of neurodevelopmenal disorders such as FXS and ASD.

    1. Author response:

      Thank you very much for the constructive feedback on our manuscript, "Simple Methods to Acutely Measure Multiple Timing Metrics among Sexual Repertoire of Male Drosophila," and for the opportunity to address the reviewers' comments. We appreciate the time and effort the reviewers have invested in evaluating our work, and we agree that their suggestions will significantly strengthen the manuscript.

      We are currently working diligently to address all the concerns raised in the public reviews and recommendations. Below is an outline of the major revisions we plan to implement in the revised version:

      (1) Statistical Rigor and Analysis

      We acknowledge the statistical limitations pointed out by Reviewer #2. We will re-analyze the multi-group data in Figures 3 and 4 using One-way and Two-way ANOVA with appropriate post-hoc tests (e.g., Tukey's HSD), respectively, to properly account for multiple comparisons and interaction effects between genotype and training conditions.

      (2) Comparison with Existing Tools

      As suggested by both reviewers, we will provide a detailed comparison of DrosoMating with established automated tracking systems (e.g., FlyTracker, JAABA, Ctrax),and specific use cases where DrosoMating offers distinct advantages in terms of cost, accessibility, and ease of use for high-throughput screening.

      (3) Control for Locomotor Activity

      To address the potential confound of general locomotor deficits in w1118 and y1 mutants, we will calculate and present general locomotion metrics (e.g., average velocity, total distance traveled) from our tracking data to dissociate motor defects from specific courtship deficits.

      (4) Software Capabilities and Cross-Species Applicability

      We will clarify how DrosoMating handles fly identification during mating (including occlusion management). We will also discuss or test the software's applicability across different *Drosophila* species, as requested.

      (5) Minor Corrections

      We will address all textual errors, standardize terminology (e.g., "Mating Duration" vs. "Copulation Duration"), improve figure legibility, and provide complete statistical details for all figures.

      We believe these revisions will substantially improve the rigor, clarity, and utility of our manuscript. We aim to resubmit the revised version within the standard timeframe and will ensure the preprint is updated accordingly.

    1. Reviewer #1 (Public review):

      Summary:

      This fundamental study identifies a new mechanism that involves a mycobacterial nucleomodulin manipulation of the host histone methyltransferase COMPASS complex to promote infection. Although other intracellular pathogens are known to manipulate histone methylation, this is the first report demonstrating specific targeting the COMPASS complex by a pathogen. The rigorous experimental design using of state-of-the art bioinformatic analysis, protein modeling, molecular and cellular interaction and functional approaches, culminating with in vivo infection modeling provide convincing, unequivocal evidence that supports the authors claims. This work will be of particular interest to cellular microbiologist working on microbial virulence mechanisms and effectors, specifically nucleomodulins, and cell/cancer biologists that examine COMPASS dysfunction in cancer biology.

      Strengths:

      (1) The strengths of this study include the rigorous and comprehensive experimental design that involved numerous state-of-the-art approaches to identify potential nucleomodulins, define molecular nucleomodulin-host interactions, cellular nucleomodulin localization, intracellular survival, and inflammatory gene transcriptional responses, and confirmation of the inflammatory and infection phenotype in a small animal model.

      (2) The use of bioinformatic, cellular and in vivo modeling that are consistent and support the overall conclusions is a strengthen of the study. In addition, the rigorous experimental design and data analysis including the supplemental data provided, further strengthens the evidence supporting the conclusions.

      Weaknesses:

      (1) This work could be stronger if the MgdE-COMPASS subunit interactions that negatively impact COMPASS complex function were more well defined. Since the COMPASS complex consists of many enzymes, examining functional impact on each of the components would be interesting.

      (2) Examining the impact of WDR5 inhibitors on histone methylation, gene transcription and mycobacterial infection could provide additional rigor and provide useful information related to mechanisms and specific role of WDR5 inhibition on mycobacteria infection.

      (3) The interaction between MgdE and COMPASS complex subunit ASH2L is relatively undefined and studies to understand the relationship between WDR5 and ASH2L in COMPASS complex function during infection could provide interesting molecular details that are undefined in this study.

      (4) The AlphaFold prediction results for all the nuclear proteins examined could be useful. Since the interaction predictions with COMPASS subunits range from 0.77 for WDR5 and 0.47 for ASH2L, it is not clear how the focus on COMPASS complex over other nuclear proteins was determined.

      Comments on revisions:

      The authors have addressed the weaknesses that were identified by this reviewer by providing rational explanation and specific references that support the findings and conclusions.

    2. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This fundamental study identifies a new mechanism that involves a mycobacterial nucleomodulin manipulation of the host histone methyltransferase COMPASS complex to promote infection. Although other intracellular pathogens are known to manipulate histone methylation, this is the first report demonstrating the specific targeting of the COMPASS complex by a pathogen. The rigorous experimental design using state-of-the art bioinformatic analysis, protein modeling, molecular and cellular interaction, and functional approaches, culminating with in vivo infection modeling, provides convincing, unequivocal evidence that supports the authors' claims. This work will be of particular interest to cellular microbiologists working on microbial virulence mechanisms and effectors, specifically nucleomodulins, and cell/cancer biologists that examine COMPASS dysfunction in cancer biology.

      Strengths:

      (1) The strengths of this study include the rigorous and comprehensive experimental design that involved numerous state-of-the-art approaches to identify potential nucleomodulins, define molecular nucleomodulin-host interactions, cellular nucleomodulin localization, intracellular survival, and inflammatory gene transcriptional responses, and confirmation of the inflammatory and infection phenotype in a small animal model.

      (2) The use of bioinformatic, cellular, and in vivo modeling that are consistent and support the overall conclusions is a strength of the study. In addition, the rigorous experimental design and data analysis, including the supplemental data provided, further strengthen the evidence supporting the conclusions.

      Weaknesses:

      (1) This work could be stronger if the MgdE-COMPASS subunit interactions that negatively impact COMPASS complex function were better defined. Since the COMPASS complex consists of many enzymes, examining the functional impact on each of the components would be interesting.

      We thank the reviewer for this insightful comment. A biochemistry assays could be helpful to interpret the functional impact on each of the components by MgdE interaction. However, the purification of the COMPASS complex could be a hard task itself due to the complexity of the full COMPASS complex along with its dynamic structural properties and limited solubility.

      (2) Examining the impact of WDR5 inhibitors on histone methylation, gene transcription, and mycobacterial infection could provide additional rigor and provide useful information related to the mechanisms and specific role of WDR5 inhibition on mycobacterial infection.

      We thank the reviewer for the comment. A previous study showed that WIN-site inhibitors, such as compound C6, can displace WDR5 from chromatin, leading to a reduction in global H3K4me3 levels and suppression of immune-related gene expression (Hung et al., Nucleic Acids Res, 2018; Bryan et al., Nucleic Acids Res, 2020). These results closely mirror the functional effects we observed for MgdE, suggesting that MgdE may act as a functional mimic of WDR5 inhibition. This supports our proposed model in which MgdE disrupts COMPASS activity by targeting WDR5, thereby dampening host pro-inflammatory responses.

      (3) The interaction between MgdE and COMPASS complex subunit ASH2L is relatively undefined, and studies to understand the relationship between WDR5 and ASH2L in COMPASS complex function during infection could provide interesting molecular details that are undefined in this study.

      We thank the reviewer for the comment. In this study, we constructed single and multiple point mutants of MgdE at residues S<sup>80</sup>, D<sup>244</sup>, and H<sup>247</sup> to identify key amino acids involved in its interaction with ASH2L (Figure 5A and B; New Figure S4C). However, these mutations did not interrupt the interaction with MgdE, suggesting that more residues are involved in the interaction.

      ASH2L and WDR5 function cooperatively within the WRAD module to stabilize the SET domain and promote H3K4 methyltransferase activity with physiological conditions (Couture and Skiniotis, Epigenetics, 2013; Qu et al., Cell, 2018; Rahman et al., Proc Natl Acad Sci U S A, 2022). ASH2L interacts with RbBP5 via its SPRY domain, whereas WDR5 bridges MLL1 and RbBP5 through the WIN and WBM motifs (Chen et al., Cell Res, 2012; Park et al., Nat Commun, 2019). The interaction status between ASH2L and WDR5 during mycobacterial infection could not be determined in our current study.

      (4) The AlphaFold prediction results for all the nuclear proteins examined could be useful. Since the interaction predictions with COMPASS subunits range from 0.77 for WDR5 and 0.47 for ASH2L, it is not clear how the focus on COMPASS complex over other nuclear proteins was determined.

      We thank the reviewer for the comment. We employed AlphaFold to predict the interactions between MgdE and the major nuclear proteins. This screen identified several subunits of the SET1/COMPASS complex as high-confidence candidates for interaction with MgdE (Figure S4A). This result is consistent with a proteomic study by Penn et al. which reported potential interactions between MgdE and components of the human SET1/COMPASS complex based on affinity purification-mass spectrometry analysis (Penn et al., Mol Cell, 2018).

      Reviewer #2 (Public review):

      Summary:

      The manuscript by Chen et al addresses an important aspect of pathogenesis for mycobacterial pathogens, seeking to understand how bacterial effector proteins disrupt the host immune response. To address this question, the authors sought to identify bacterial effectors from M. tuberculosis (Mtb) that localize to the host nucleus and disrupt host gene expression as a means of impairing host immune function.

      Strengths:

      The researchers conducted a rigorous bioinformatic analysis to identify secreted effectors containing mammalian nuclear localization signal (NLS) sequences, which formed the basis of quantitative microscopy analysis to identify bacterial proteins that had nuclear targeting within human cells. The study used two complementary methods to detect protein-protein interaction: yeast two-hybrid assays and reciprocal immunoprecipitation (IP). The combined use of these techniques provides strong evidence of interactions between MgdE and SET1 components and suggests that the interactions are, in fact, direct. The authors also carried out a rigorous analysis of changes in gene expression in macrophages infected with the mgdE mutant BCG. They found strong and consistent effects on key cytokines such as IL6 and CSF1/2, suggesting that nuclear-localized MgdE does, in fact, alter gene expression during infection of macrophages.

      Weaknesses:

      There are some drawbacks in this study that limit the application of the findings to M. tuberculosis (Mtb) pathogenesis. The first concern is that much of the study relies on ectopic overexpression of proteins either in transfected non-immune cells (HEK293T) or in yeast, using 2-hybrid approaches. Some of their data in 293T cells is hard to interpret, and it is unclear if the protein-protein interactions they identify occur during natural infection with mycobacteria. The second major concern is that pathogenesis is studied using the BCG vaccine strain rather than virulent Mtb. However, overall, the key findings of the paper - that MgdE interacts with SET1 and alters gene expression are well-supported.

      We thank the reviewer for the comment. We agree that the ectopic overexpression could not completely reflect a natural status, although these approaches were adopted in many similar experiments (Drerup et al., Molecular plant, 2013; Chen et al., Cell host & microbe, 2018; Ge et al., Autophagy, 2021). Further, the MgdE localization experiment using Mtb infected macrophages will be performed to increase the evidence in the natural infection.

      We agree with the reviewer that BCG strain could not fully recapitulate the pathogenicity or immunological complexity of M. tuberculosis infection. We employed BCG as a biosafe surrogate model since it was acceptable in many related studies (Wang et al., Nat Immunol, 2025; Wang et al., Nat Commun, 2017; Péan et al., Nat Commun, 2017; Li et al., J Biol Chem, 2020).

      Reviewer #3 (Public review):

      In this study, Chen L et al. systematically analyzed the mycobacterial nucleomodulins and identified MgdE as a key nucleomodulin in pathogenesis. They found that MgdE enters into host cell nucleus through two nuclear localization signals, KRIR<sup>108-111</sup> and RLRRPR<sup>300-305</sup>, and then interacts with COMPASS complex subunits ASH2L and WDR5 to suppress H3K4 methylation-mediated transcription of pro-inflammatory cytokines, thereby promoting mycobacterial survival. This study is potentially interesting, but there are several critical issues that need to be addressed to support the conclusions of the manuscript.

      (1) Figure 2: The study identified MgdE as a nucleomodulin in mycobacteria and demonstrated its nuclear translocation via dual NLS motifs. The authors examined MgdE nuclear translocation through ectopic expression in HEK293T cells, which may not reflect physiological conditions. Nuclear-cytoplasmic fractionation experiments under mycobacterial infection should be performed to determine MgdE localization.

      We thank the reviewer for this insightful comment. In the revised manuscript, we addressed this concern by performing nuclear-cytoplasmic fractionation experiments using M. bovis BCG-infected macrophages to assess the subcellular localization of MgdE (New Figure 2F) (Lines 146–155). Nuclear-cytoplasmic fractionation experiments showed that WT MgdE and the NLS single mutants (MgdE<sup>ΔNLS1</sup> and MgdE<sup>ΔNLS2</sup>) could be detected both in the cytoplasm and in the nucleus, while the double mutant MgdE<sup>ΔNLS1-2</sup> was detectable only in the cytoplasm. These findings strongly indicate that MgdE is capable of translocating into the host cell nucleus during BCG infection, and that this nuclear localization relies on the dual NLS motifs.

      (2) Figure 2F: The authors detected MgdE-EGFP using an anti-GFP antibody, but EGFP as a control was not detected in its lane. The authors should address this technical issue.

      We thank the reviewer for this question. In the revised manuscript, we have included the uncropped immunoblot images, which clearly show the EGFP band in the corresponding lane. These have been provided in the New Figure 2E.

      (3) Figure 3C-3H: The data showing that the expression of all detected genes in 24 h is comparable to that in 4 h (but not 0 h) during WT BCG infection is beyond comprehension. The issue is also present in Figure 7C, Figure 7D, and Figure S7. Moreover, since Il6, Il1β (pro-inflammatory), and Il10 (anti-inflammatory) were all upregulated upon MgdE deletion, how do the authors explain the phenomenon that MgdE deletion simultaneously enhanced these gene expressions?

      We thank the reviewer for the comment. A relative quantification method was used in our qPCR experiments to normalize the WT expression levels in Figure 3C–3H, Figure 7C, 7D, and New Figure S6.

      The concurrent induction of both types of cytokines likely represents a dynamic host strategy to fine-tune immune responses during infection. This interpretation is supported by previous studies (Podleśny-Drabiniok et al., Cell Rep, 2025; Cicchese et al., Immunological Reviews, 2018).

      (4) Figure 5: The authors confirmed the interactions between MgdE and WDR5/ASH2L. How does the interaction between MgdE and WDR5 inhibit COMPASS-dependent methyltransferase activity? Additionally, the precise MgdE-ASH2L binding interface and its functional impact on COMPASS assembly or activity require clarification.

      We thank the reviewer for this insightful comment. We cautiously speculate that the MgdE interaction inhibits COMPASS-dependent methyltransferase activity by interfering with the integrity and stability of the COMPASS complex. Accordingly, we have incorporated the following discussion into the revised manuscript (Lines 303-315):

      “The COMPASS complex facilitates H3K4 methylation through a conserved assembly mechanism involving multiple core subunits. WDR5, a central scaffolding component, interacts with RbBP5 and ASH2L to promote complex assembly and enzymatic activity (Qu et al., 2018; Wysocka et al., 2005). It also recognizes the WIN motif of methyltransferases such as MLL1, thereby anchoring them to the complex and stabilizing the ASH2L-RbBP5 dimer (Hsu et al., Cell, 2018). ASH2L further contributes to COMPASS activation by interacting with both RbBP5 and DPY30 and by stabilizing the SET domain, which is essential for efficient substrate recognition and catalysis (Qu et al., Cell, 2018; Park et al., Nat Commun, 2019). Our work shows that MgdE binds both WDR5 and ASH2L and inhibits the methyltransferase activity of the COMPASS complex. Site-directed mutagenesis revealed that residues D<sup>224</sup> and H<sup>247</sup> of MgdE are critical for WDR5 binding, as the double mutant MgdE-D<sup>224</sup>A/H<sup>247</sup>A fails to interact with WDR5 and shows diminished suppression of H3K4me3 levels (Figure 5D).”

      Regarding the precise MgdE-ASH2L binding interface, we attempted to identify the key interaction site by introducing point mutations into ASH2L. However, these mutations did not disrupt the interaction (Figure 5A and B; New Figure S4C), suggesting that more residues are involved in the interaction.

      (5) Figure 6: The authors proposed that the MgdE-regulated COMPASS complex-H3K4me3 axis suppresses pro-inflammatory responses, but the presented data do not sufficiently support this claim. H3K4me3 inhibitor should be employed to verify cytokine production during infection.

      We thank the reviewer for the comment. We have now revised the description in lines 220-221 and lines 867-868 "MgdE suppresses host inflammatory responses probably by inhibition of COMPASS complex-mediated H3K4 methylation."

      (6) There appears to be a discrepancy between the results shown in Figure S7 and its accompanying legend. The data related to inflammatory responses seem to be missing, and the data on bacterial colonization are confusing (bacterial DNA expression or CFU assay?).

      We thank the reviewer for the comment. New Figure S6 specifically addresses the effect of MgdE on bacterial colonization in the spleens of infected mice, which was assessed by quantitative PCR rather than by CFU assay.

      We have now revised the legend of New Figure S6 as below (Lines 986-991):

      “MgdE facilitates bacterial colonization in the spleens of infected mice. Bacterial colonization was assessed in splenic homogenates from infected mice (as described in Figure 7A) by quantifying bacterial DNA using quantitative PCR at 2, 14, 21, 28, and 56 days post-infection.”

      (7) Line 112-116: Please provide the original experimental data demonstrating nuclear localization of the 56 proteins harboring putative NLS motifs.

      We thank the reviewer for the comment. We will provide this data in the New Table S3.

      Recommendations for the authors:

      Reviewer #2 (Recommendations for the authors):

      There are a few concerns about specific experiments:

      Major Comments:

      (1) Questions about the exact constructs used in their microscopy studies and the behavior of their controls. GFP is used as a negative control, but in the data they provide, the GFP signal is actually nuclear-localized (for example, Figure 1c, Figure 2a). Later figures do show other constructs with clear cytoplasmic localization, such as the delta-NLS-MgdE-GFP in Figure 2D. This raises significant questions about how the microscopy images were analyzed and clouds the interpretation of these findings. It is also not clear if their microscopy studies use the mature MdgE, lacking the TAT signal peptide after signal peptidase cleavage (the form that would be delivered into the host cell) or if they are transfecting the pro-protein that still has the TAT signal peptide (a form that would present in the bacterial cell but that would not be found in the host cell). This should be clarified, and if their construct still has the TAT peptide, then key findings such as nuclear localization and NLS function should be confirmed with the mature protein lacking the signal peptide.

      We thank the reviewer for this question.  EGFP protein can passively diffuse through nuclear pores due to its smaller size (Petrovic et al., Science, 2022; Yaseen et al., Nat Commun, 2015; Bhat et al., Nucleic Acids Res, 2015). However, upon transfection with EGFP-tagged wild-type MdgE and its NLS deletion mutants (MdgE<sup>ΔNLS1</sup>, MdgE<sup>ΔNLS2</sup>, and MdgE<sup>ΔNLS1-2</sup>), we observed significantly stronger nuclear fluorescence in cells expressing wild-type MdgE compared to the EGFP protein. Notably, the MdgE<sup>ΔNLS1-2</sup>-EGFP mutant showed almost no detectable nuclear fluorescence (Figure 2C, D, and E). These results indicate that (i) MdgE-EGFP fusion protein could not enter the nucleus by passive diffusion, and (ii) EGFP does not interfere with the nuclear targeting ability of MdgE.

      We did not construct a signal peptide-deleted MgdE for transfection assays. Instead, we performed an infection experiment using recombinant M. bovis BCG strains expressing Flag-tagged wild-type MgdE. The mature MgdE protein (signal peptide cleaved) can be detected in the nucleus fractionation (New Figure 2F), suggesting that the signal peptide does not play a role for the nuclear localization of MgdE.

      (2) The localization of MdgE is not shown during actual infection. The study would be greatly strengthened by an analysis of the BCG strain expressing their MdgE-FLAG construct.

      We thank the reviewer for the comment. In the revised manuscript, we constructed M. bovis BCG strains expressing FLAG-tagged wild-type MdgE as well as NLS deletion mutants (MdgE<sup>ΔNLS1</sup>, MdgE<sup>ΔNLS2</sup>, and MdgE<sup>ΔNLS1-2</sup>). These strains were used to infect THP-1 cells, and nuclear-cytoplasmic fractionation was performed 24 hours post-infection.

      Nuclear-cytoplasmic fractionation experiments showed that WT MgdE and the NLS single mutants could be detected both in the cytoplasm and in the nucleus by immunoblotting, while the double mutant MgdE<sup>ΔNLS1-2</sup> was detectable only in the cytoplasm (New Figure 2F) (Lines 146–155). These findings indicate that MdgE is capable of entering the host cell nucleus during BCG infection, and that this nuclear localization depends on the presence of both its N-terminal and C-terminal NLS motifs.

      (3) Their pathogenesis studies suggesting a role for MdgE would be greatly strengthened by studying MdgE in virulent Mtb rather than the BCG vaccine strain. If this is not possible because of technical limitations (such as lack of a BSL3 facility), then at least a thorough discussion of studies that examined Rv1075c/MdgE in Mtb is important. This would include a discussion of the phenotype observed in a previously published study examining the Mtb Rv1075c mutant that showed a minimal phenotype in mice (PMID: 31001637) and would also include a discussion of whether Rv1075c was identified in any of the several in vivo Tn-Seq studies done on Mtb.

      We thank the reviewer for this insightful comment. In the revised manuscript, we have incorporated a more thorough discussion of prior studies that examined Rv1075c/MgdE in Mtb, including the reported minimal phenotype of an Mtb MgdE mutant in mice (PMID: 31001637) (Lines 288–294).

      In the latest TnSeq studies in M. tuberculosis, Rv1075c/MgdE was not classified as essential for in vivo survival or virulence (James et al., NPJ Vaccines, 2025; Zhang et al., Cell, 2013). However, this absence should not be interpreted as evidence of dispensability since these datasets also failed to identify some well characterized virulence factors including Rv2067c (Singh et al., Nat Commun, 2023), PtpA (Qiang et al., Nat Commun, 2023), and PtpB (Chai et al., Science, 2022) which were demonstrated to be required for the virulence of Mtb.

      Minor Comments:

      (1) Multiple figures with axes with multiple discontinuities used when either using log-scale or multiple graphs is more appropriate, including 3B, 7A.

      We sincerely thank the reviewer for pointing this out. In the revised manuscript, we have updated Figure 3B and Figure 7A.

      (2) Figure 1C - Analysis of only nuclear MFI can be very misleading because it is affected by the total expression of each construct. Ratios of nuclear to cytoplasmic MFI are a more rigorous analysis.

      We thank the reviewer for this comment. We agree that analyzing the ratio of nuclear to cytoplasmic mean fluorescence intensity (MFI) provides a more rigorous quantification of nuclear localization, particularly when comparing constructs with different expression levels. However, the analysis presented in Figure 1C was intended as a preliminary qualitative screen to identify Tat/SPI-associated proteins with potential nuclear localization, rather than a detailed quantitative assessment.

      (3) Figure 5C - Controls missing and unclear interpretation of their mutant phenotype. There is no mock or empty-vector control transfection, and their immunoblot shows a massive increase in total cellular H3K4me3 signal in the bulk population, although their prior transfection data show only a small fraction of cells are expressing MdgE. They also see a massive increase in methylation in cells transfected with the inactive mutant, but the reason for this is unclear. Together, these data raise questions about the specificity of the increasing methylation they observe. An empty vector control should be included, and the phenotype of the mutant explained.

      We thank the reviewer for this comment. In the revised manuscript, we transfected HEK293T cells with an empty EGFP vector and performed a quantitative analysis of H3K4me3 levels. The results demonstrated that, at the same time point, cells expressing MdgE showed significantly lower levels of H3K4me3 compared to both the EGFP control and the catalytically inactive mutant MdgE (D<sup>244</sup>A/H<sup>247</sup>A) (New Figure 5D) (Lines 213–216). These findings support the conclusion that MdgE specifically suppresses H3K4me3 levels in cells.

      (4) Figure S1A - The secretion assay is lacking a critical control of immunoblotting a cytoplasmic bacterial protein to demonstrate that autolysis is not releasing proteins into the culture filtrate non-specifically - a common problem with secretion assays in mycobacteria.

      We thank the reviewer for this comment. To address the concerns, we examined FLAG-tagged MgdE and the secreted antigen Ag85B in the culture supernatants by monitoring the cytoplasmic protein GlpX. The absence of GlpX in the supernatant confirmed that there was no autolysis in the experiment. We could detect MgdE-Flag in the culture supernatant (New Figure S2A), indicating that MgdE is a secreted protein.

      (5) The volcano plot of their data shows that the proteins with the smallest p-values have the smallest fold-changes. This is unusual for a transcriptomic dataset and should be explained.

      We thank the reviewer for this comment. We are not sure whether the p-value is correlated with fold-change in the transcriptomic dataset. This is probably case by case.

      Reviewer #3 (Recommendations for the authors):

      There are several minor comments:

      (1) Line 104-109: The number of proteins harboring NLS motifs and candidate proteins assigned to the four distinct pathways does not match the data presented in Table S2. Please recheck the details. Figure 1A and B, as well as Figure S1A and B, should also be corrected accordingly.

      We thank the reviewer for the comment. We have carefully checked the details and the numbers were confirmed and updated.

      (2) Please add the scale bar in all image figures, including Figure 1C, Figure 2D, Figure 5C, Figure 7B, and Figure S2.

      We thank the reviewer for this suggestion. We have now added scale bars to all relevant image figures in the revised manuscript, including Figure 1C, New Figure 2C, Figure 5C, Figure 7B, and New Figure S2B.

      (3) Please add the molecular marker in all immunoblotting figures, including Figure 2C, Figure 2F, Figure 4B, Figure 4C, Figure 5B, Figure 5D, and Figure S5.

      We thank the reviewer for this suggestion. We have now added the molecular marker in all immunoblotting figures in the revised manuscript, including New Figure 2E–F, Figure 4B–C, Figure 5B and D, Figure S2A, New Figure S2E and New Figure S4C.

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    1. Reviewer #2 (Public review):

      Summary:

      AutoMorphoTrack provides an end-to-end workflow for organelle-scale analysis of multichannel live-cell fluorescence microscopy image stacks. The pipeline includes organelle detection/segmentation, extraction of morphological descriptors (e.g., area, eccentricity, "circularity," solidity, aspect ratio), tracking and motility summaries (implemented via nearest-neighbor matching using cKDTree), and pixel-level overlap/colocalization metrics between two channels. The manuscript emphasizes a specific application to live imaging in neurons, demonstrated on iPSC-derived dopaminergic neuronal cultures with mitochondria in channel 0 and lysosomes in channel 1, while asserting adaptability to other organelle pairs.

      The tool is positioned for cell biologists, including users with limited programming experience, primarily through two implemented modes of use: (i) a step-by-step Jupyter notebook and (ii) a modular Python package for scripted or batch execution, alongside an additional "AI-assisted" mode that is described as enabling analyses through natural-language prompts.

      The motivation and general workflow packaging are clear, and the notebook-plus-modules structure is a reasonable engineering choice. However, in its current form, the manuscript reads more like a convenient assembly of standard methods than a validated analytical tool. Key claims about robustness, accuracy, and scope are not supported by quantitative evidence, and the 'AI-assisted' framing is insufficiently defined and attributes to the tool capabilities that are provided by external LLM platforms rather than by AutoMorphoTrack itself. In addition, several figure, metric, and statistical issues-including physically invalid plots and inconsistent metric definitions-directly undermine trust in the quantitative outputs.

      Strengths:

      (1) Clear motivation: lowering the barrier for organelle-scale quantification for users who do not routinely write custom analysis code.

      (2) Multiple entry points: an interactive notebook together with importable modules, emphasizing editable parameters rather than a fully opaque black box.

      (3) End-to-end outputs: automated generation of standardized visualizations and tables that, if trustworthy, could help users obtain quantitative summaries without assembling multiple tools.

      Weaknesses:

      (1) "AI-assisted / natural-language" functionality is overstated.

      The manuscript implies an integrated natural-language interface, but no such interface is implemented in the software. Instead, users are encouraged to use external chatbots to help generate or modify Python code or execute notebook steps. This distinction is not made clearly and risks misleading readers.

      (2) No quantitative validation against trusted ground truth.

      There is no systematic evaluation of segmentation accuracy, tracking fidelity, or interaction/overlap metrics against expert annotations or controlled synthetic data. Without such validation, accuracy, parameter sensitivity, and failure modes cannot be assessed.

      (3) Limited benchmarking and positioning relative to existing tools.

      The manuscript does not adequately compare AutoMorphoTrack to established platforms that already support segmentation, morphometrics, tracking, and colocalization (e.g., CellProfiler) or to mitochondria-focused toolboxes (e.g., MiNA, MitoGraph, Mitochondria Analyzer). This is particularly problematic given the manuscript's implicit novelty claims.

      (4) Core algorithmic components are basic and likely sensitive to imaging conditions.

      Heavy reliance on thresholding and morphological operations raises concerns about robustness across varying SNR, background heterogeneity, bleaching, and organelle density; these issues are not explored.

      (5) Multiple figure, metric, and statistical issues undermine confidence.

      The most concerning include:<br /> (i) "Circularity (4πA/P²)" values far greater than 1 (Figures 2 and 7, and supplementary figures), which is inconsistent with the stated definition and strongly suggests a metric/label mismatch or computational error.

      (ii) A displacement distribution extending to negative values (Figure 3B). This is likely a plotting artifact (e.g., KDE boundary bias), but as shown, it is physically invalid and undermines confidence in the motility analysis.

      (iii) Colocalization/overlap metrics that are inconsistently defined and named, with axis ranges and terminology that can mislead (e.g., Pearson r reported for binary masks without clarification).

      (iv) Figure legends that do not match the displayed panels, and insufficient reporting of Ns, p-values, sampling units, and statistical assumptions.

    2. Author response:

      Reviewer #1

      We thank the reviewer for their thoughtful and constructive assessment of AutoMorphoTrack and for recognizing its potential utility as an open-source end-to-end workflow for organelle analysis.

      (1) Novelty and relationship to existing tools / FIJI workflows

      We appreciate this concern and agree that many of the underlying image-processing operations (e.g., thresholding, morphological cleanup, region properties) are well-established. Our goal with AutoMorphoTrack is not to introduce new segmentation algorithms, but rather to provide a curated, reproducible, and extensible end-to-end workflow that integrates segmentation, morphology, tracking, motility, and colocalization into a single, transparent pipeline tailored for live-cell organelle imaging.

      While an experienced user could assemble similar analyses ad hoc using FIJI or custom scripts, our contribution lies in:

      Unifying these steps into a single workflow with consistent parameterization and outputs

      Generating standardized, publication-ready visualizations and tables at each step,

      Enabling batch and longitudinal analyses across cells and conditions, and

      Lowering the barrier for users who do not routinely write custom analysis code.

      We note that the documentation-style presentation of the manuscript is intentional, as it serves both as a methods paper and a practical reference for users implementing the workflow. We agree, however, that the manuscript currently overemphasizes step-by-step execution at the expense of positioning. In revision, we will more explicitly frame AutoMorphoTrack as a workflow integration and usability contribution, rather than a fundamentally new algorithmic advance.

      We will also cite and discuss the image.sc example referenced by the reviewer, clarifying conceptual overlap and differences in scope.

      (2) Comparison to existing pipelines (Imaris, CellProfiler, CellPose, StarDist)

      We agree and thank the reviewer for highlighting this omission. In the revised manuscript, we will expand the related-work and positioning section to explicitly compare AutoMorphoTrack with established commercial (e.g., Imaris) and open-source (e.g., CellProfiler, MiNA, MitoGraph) platforms, as well as learning-based segmentation tools such as CellPose and StarDist.

      Rather than claiming superiority, we will clarify trade-offs, emphasizing that AutoMorphoTrack prioritizes:

      Transparency and parameter interpretability,

      Lightweight dependencies suitable for small live-imaging datasets

      Direct integration of morphology, tracking, and colocalization in a single workflow, and

      Ease of modification for domain-specific use cases.

      (3) AI / chatbot integration

      We appreciate this critique and agree that the current description is insufficiently precise. AutoMorphoTrack does not implement a native natural-language interface. Instead, our intent was to convey that the workflow can be executed and modified with assistance from external large language models (LLMs) in a notebook-based environment.

      In revision, we will revise this section to:

      Clearly distinguish AutoMorphoTrack’s functionality from that of external LLM tools,

      Remove any implication of a built-in AI interface, and

      Provide concrete, reproducible examples of how non-coding users may interact with the pipeline using natural-language prompts mediated by external tools.

      Reviewer #2

      We thank the reviewer for their detailed and technically rigorous evaluation. We appreciate the recognition of the workflow’s motivation and structure, and we agree that several aspects of validation, positioning, and quantitative reporting must be strengthened.

      (1) AI-assisted / natural-language functionality

      We agree with this critique. AutoMorphoTrack does not provide a native natural-language execution layer, and the manuscript currently overstates this aspect. In revision, we will explicitly scope any reference to AI assistance as external, optional support for code generation and parameter editing, with clearly documented examples and stated limitations.

      We agree that conflating external LLM capabilities with the software itself risks misleading readers, and we will correct this accordingly.

      (2) Lack of quantitative validation

      We fully agree that the current manuscript lacks formal quantitative validation. In the revised version, we will add a dedicated validation section including:

      Segmentation accuracy compared to expert annotations using overlap metrics (e.g., Dice / IoU),

      Tracking fidelity assessed using manually annotated tracks and/or synthetic ground truth,

      Sensitivity analyses for key parameters (e.g., thresholding and linking distance), and

      Explicit discussion of failure modes and quality-control indicators.

      We acknowledge that without such validation, claims of robustness are not sufficiently supported.

      (3) Benchmarking and positioning relative to existing tools

      We agree and will substantially strengthen AutoMorphoTrack’s benchmarking and positioning relative to existing platforms. Rather than framing novelty algorithmically, we will clarify that the primary contribution is a reproducible, integrated workflow designed specifically for two-organelle live imaging in neurons, with transparent parameters and standardized outputs.

      We note that our goal is not to exhaustively benchmark against all available tools, but rather to provide representative comparisons that clarify operating regimes, assumptions, and trade-offs. We will add a comparative table and/or qualitative comparison highlighting strengths, assumptions, and limitations relative to existing tools.

      (4) Core algorithms and robustness

      We agree that reliance on threshold-based segmentation introduces sensitivity to imaging conditions. In revision, we will:

      Explicitly discuss the operating regime and assumptions under which AutoMorphoTrack performs reliably,

      Clarify that the framework is modular and can accept alternative segmentation backends, and

      Include guidance on when outputs should be treated with caution.

      (5) Figure, metric, and statistical issues

      We thank the reviewer for identifying several critical issues and agree that these undermine confidence. In revision, we will correct all figure, metric-definition, and reporting inconsistencies, including:

      Resolving circularity values exceeding 1 by correcting computation and/or labeling errors,

      Revising physically invalid displacement plots and clarifying kernel-density limitations,

      Ensuring colocalization metrics are consistently defined, named, and interpreted, with explicit clarification of whether calculations are intensity- or mask-based,

      Correcting figure legends to match displayed panels, and

      Clearly reporting sample size, sampling units, and statistical assumptions, including handling of multiple comparisons where applicable.

      (6) Value-added demonstration

      We agree that the manuscript would benefit from a clearer demonstration of value-added use cases. In revision, we will include at least one realistic example showing how AutoMorphoTrack enables a complete, reproducible analysis workflow with reduced setup burden compared to manually assembling multiple tools.

      (7) Editorial suggestions

      We agree and will streamline the Results section to reduce procedural repetition and focus more on validation, limitations, and quality-control guidance.

      Reviewer #3

      We thank the reviewer for their positive assessment of clarity and organization, and for the constructive practical feedback.

      Installation issues

      We appreciate the detailed report of installation failures and acknowledge that the current packaging and distribution are inadequate. Prior to revision, we will:

      Fix the package structure to support standard installation methods,

      Ensure all required files (e.g., setup configuration, README) are correctly included,

      Test installation on clean environments across platforms, and

      Correct broken links to notebooks and documentation.

      We agree that without a functional installation pathway, the utility of the tool is severely limited.

      AI-assisted claims

      We agree with the reviewer and echo our responses above. The AI-assisted description will be clarified and appropriately scoped in the revised manuscript.

      Additional suggestions

      Color accessibility: We will revise all figures to use colorblind-safe palettes.

      Velocity–displacement diagonal: We will explicitly explain the origin of this relationship, including whether it reflects dataset properties, tracking assumptions, or minimum detectable motion.

      Integrated correlation metric: We agree that Spearman correlation is more appropriate for many of these relationships and will replace Pearson correlations accordingly.

      Supplementary movies: We agree that providing raw movies would improve interpretability and will add representative examples as supplementary material.

    1. Sutherland’s theory may explain why crime is multigenerational. A longitudinal study beginning in the 1960s found that the best predictor of antisocial and criminal behavior in children was whether their parents had been convicted of a crime (Todd and Jury 1996). Children who were younger than ten years old when their parents were convicted were more likely than other children to engage in spousal abuse and criminal behavior by their early thirties. Even when taking socioeconomic factors such as dangerous neighborhoods, poor school systems, and overcrowded housing into consideration, researchers found that parents were the main influence on the behavior of their offspring (Todd and Jury 1996).

      My husband and his siblings were raised by the same mother and father. One son, my husband, the middle child, always stayed between the lines and followed tall of he rules, he was well liked, got good grades, put himself through college while working full time and became an aeronautical engineer.and would often accept punishment for deviant behavior committed by his brother to keep the peace.He also got the least attention. He is the most conscientious loving , trustworthy and loyal person I know. To me this contradicts Edwin Sutherlands differential association theory.

      His older brother, began using drugs at a very early age, sniffed glue at 9, took downers or reds at 12, and essentially became sexually active at 12 or 13m because their adult neighbor seduced him. Much of this his parents were oblivious to. He was put on behavioral medications as a child, and commanded a lot of attention. As a young man he joined the military and after going AWOL several times was ulitimatley dishonorably discharged. In later life he became an i.v. meth user, contracted HIV from sharing needles, went through countless rehabs, and ultimately died as an overdose. To this day his parents are in denial about who he had become.

      His youngest brother by 7 years also became sexually active at a very young age. The same neighbor sexually abused him. He became a womanizer often having 3 to 4 women who all thought the were the only one. He has never held a real job, Had two children that he loves but never really helped the Mom with any sort of support , financially or in caring for the kids. but has managed to find women with lots of money support him. He lived with their parents on and off into his 50's. Just recently at 60 years old he seems to have settled down with one woman. She is also a very wealthy woman who is happy to take care of him.

    2. High rates of poverty and single-parent homes correlated with high rates of juvenile violence. Research into social disorganization theory can greatly influence public policy. For instance, studies have found that children from disadvantaged communities who attend preschool programs that teach basic social skills are significantly less likely to engage in criminal activity. (Lally 1987)

      This alligns with the fact that the first 3 to 4 years of life play the largest role in who we become.

    1. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1(Public review):

      Summary:

      In this study, the authors distinguished afferent inputs to different cell populations in the VTA using dimensionality reduction approaches and found significantly distinct patterns between normal and drug treatment conditions. They also demonstrated negative correlations of the inputs induced by drugs with gene expression of ion channels or proteins involved in synaptic transmission and demonstrated the knockdown of one of the voltage-gated calcium ion channels caused decreased inputs.

      Weaknesses:

      (1) For quantifications of brain regions in this study, boundaries were based on the Franklin-Paxinos (FP) atlas according to previous studies (Beier KT et al 2015, Beier KT et al 2019). It has been reported significant discrepancies exist between the anatomical labels on the FP atlas and the Allen Brain Atlas (ref: Chon U et al., Nat Commun 2019). Although a summary of conversion is provided as a sheet, the authors need to describe how consistent or different the brain boundaries they defined in the manuscript with Allen Brain Atlas by adding histology images. Also, I wonder how reliable the annotations were for over a hundred of animals with manual quantification. The authors should briefly explain it rather than citing previous studies in the Material and Methods Section.

      We thank the reviewer for attention to this point; indeed, neuroanatomical detail is often overlooked in modern neuroscience, occasionally leading to spurious conclusions. We acknowledge that there are significant discrepancies in brain region definitions across atlases, which can make cross-study comparisons difficult. Here, all cells were manually quantified by Dr. Kevin Beier, as in previous studies (Beier et al., Cell 2015; Nature 2017; Cell Reports 2019; Tian et al., Cell Reports 2022; Tian et al., Neuron 2024; Hubbard et al., Neuropsychopharmacology, 2025). As such, these studies are internally consistent as relates to the definition of brain regions, which is critical here since our analysis in this manuscript relates to data quantified only by a single individual. Several brain regions were quite easy to distinguish anatomically, such as the medial habenula and lateral habenula. Others, such as the extended amygdala area, are much more difficult. We have now provided example images in Figure S1 that detail the anatomical boundaries that we used, overlayed on images of Neurotrace blue (fluorescent Nissl stain).

      (2) Regarding the ellipsoids in the PC, although it's written in the manuscript that "Ellipsoids were centered at the average coordinate of a condition and stretched one standard deviation along the primary and secondary axes", it's intuitively hard to understand in some figures such as Figure 2O, P and Figure S1. The authors need to make their data analysis methods more accessible by providing source code to the public.

      The source code is now available to the public at https://github.com/ktbartas/Bartas_et_al_eLife_2024, which is noted in the Code Availability statement. The code for generating ellipsoids is in the first notebook, `0-dataexploration-master-euclidean.ipynb`, in the function `confidence_ellipse`, which is called from `make_pca_plots` and `umap_and_heatmap`. Example plots are all live in the notebooks as can be viewed directly from GitHub.

      (3) In histology images (Figure 1B and 3K), the authors need to add dashed lines or arrows to guide the reader's attention.

      Dashed lines have been added to these figure panels as requested.

      (4) In Figure 2A and G, apparently there are significant differences in other brain regions such as NAcMed or PBN. If they are also statistically significant, the authors should note them as well and draw asterisks(*).

      We appreciate the care in ensuring that statistics are being applied and shown appropriately. In panel A (now Figure 3A), the Two-way ANOVA interaction term was not significant (p = 0.9365), we did not find it justified to do further comparisons. However, for Figure 3G, the interaction term was significant (p = 0.0001), and thus further pairwise comparisons were performed with Sidak's correction for multiple comparisons. When done, the only two brain regions that were significantly different were the DStr (p = 0.0051) and GPe (p = 0.0036). While the NAcMed and PBN visually look different, according to the corrected statistics, they were not significantly different (NAcMed p = 0.5037, PBN p = 0.8123). The notations in our original figure thus accurately reflected these statistics.

      (5) In Figure 2N about the spatial distribution of starter cells, the authors need to add histology images for each experimental condition (i.e. saline, fluoxetine, cocaine, methamphetamine, amphetamine, nicotine, and morphine) as supplement figures

      We have now provided these as Figure S2.

      (6) In the manuscript, it is necessary to explain why Cacna1e was selected among other calcium ion channels.

      We have added a sentence to the "Functional validation of link between gene expression and RABV labeling" section (lines 722-724).

      Reviewer #2 (Public review):

      The application of rabies virus (RabV)-mediated transsynaptic tracing has been widely utilized for mapping celltype-specific neural connectivities and examining potential modifications in response to biological phenomena or pharmacological interventions. Despite the predominant focus of studies on quantifying and analyzing labeling patterns within individual brain regions based on labeling abundance, such an approach may inadvertently overlook systemic alterations. There exists a considerable opportunity to integrate RabV tracing data with the global connectivity patterns and the transcriptomic signatures of labeled brain regions. In the present study, the authors take an important step towards achieving these objectives. Specifically, the authors conducted an intensive reanalysis of a previously generated large dataset of RabV tracing to the ventral tegmental area (VTA) using dimension reduction methods such as PCA and UMPA. This reaffirmed the authors' earlier conclusion that different cell types in the VTA, namely dopamine neurons (DA) and GABAergic neurons, exhibit quantitatively distinct input patterns, and a single dose of addictive drugs, such as cocaine and morphine, induced altered labeling patterns. Additionally, the authors illustrate that distinct axes of PCA can discriminate experimental variations, such as minor differences in the injection site of viral tracers, from bona fide alternations in labeling patterns caused by drugs of abuse. While the specific mechanisms underlying altered labeling in most brain regions remain unclear, whether involving synaptic strength, synaptic numbers, pre-synaptic activities, or other factors, the present study underscores the efficacy of an informatics approach in extracting more comprehensive information from the RabV-based circuit mapping data. Moreover, the authors showcased the utility of their previously devised bulk gene expression patterns inferred by the Allen Gene Expression Atlas (AGEA) and "projection portrait" derived from bulk axon mapping data sourced from the Allen Mouse Brain Connectivity Atlas. The utilization of such bulk data rests upon several limitations. For instance, the collection of axon mapping data involves an arbitrary selection of both cell type-specific and non-specific data, which might overlook crucial presynaptic partners, and often includes contamination from neighboring undesired brain regions. Concerns arise regarding the quantitativeness of AGEA, which may also include the potential oversight of key presynaptic partners. Nevertheless, the authors conscientiously acknowledged these potential limitations associated with the dataset. Notably, building on the observation of a positive correlation between the basal expression levels of Ca2+ channels and the extent of drug-induced changes in RabV labeling patterns, the authors conducted a CRISPRi-based knockdown of a single Ca2+ channel gene. This intervention resulted in a reduction of RabV labeling, supporting that the observed gene expression patterns have causality in RabV labeling efficiency. While a more nuanced discussion is necessary for interpreting this result (see below), overall I commend the authors for their efforts to leverage the existing dataset in a more meaningful way. This endeavor has the potential to contribute significantly to our understanding of the mechanisms underlying alterations in RabV labeling induced by drugs of abuse. Finally, drawing upon the aforementioned reanalysis of previous data, the authors underscored that a single administration of ketamine/xylazine anesthesia could induce enduring modifications in RabV labeling patterns for VTA DA neurons, specifically those projecting to the nucleus accumbens and amygdala. Given the potential impact of such alterations on motivational behaviors at a broader level, I fully agree that prudent consideration is warranted when employing ketamine/xylazine for the investigation of motivational behaviors in mice.

      Specific Points:

      (1) Beyond advancements in bioinformatics, readers may find it insightful to explore whether the PCA/UMPAbased approach yields novel biological insights. For example, the authors are encouraged to discuss more functional implications of PBN and LH in the context of drugs of abuse, as their labeling abundance could elucidate the PC2 axis in Fig. 2M.

      Thank you for this suggestion: we added text (Lines 787-795) discussing the LH and PBN (and GPe) specifically, but also highlighted the importance of our approach in hypothesis-generating science.

      (2) While I appreciate the experimental data on Cacna1e knockdown, I am unclear about the rationale behind specifically focusing on Cacna1e. The logic behind the statement, "This means that expression of this gene is not inhibitory towards RABV transmission," is also unclear. Loss-of-function experiments only signify the necessity or permissive functions of a gene. In this context, Cacna1e expression levels are required for efficient RabV labeling, but this neither supports nor excludes the possibility that this gene expression instructively suppresses RabV labeling/transmission, which could be assessed through gain-of-function experiments.

      We thank the reviewer for their suggestions regarding this result, and agree that a gain-of-function would be required to provide clearer evidence on this point.  We therefore understand that our original phrasing may be misleading. Thus, we have edited this section to the more conservative statement: “These results indicate that reduced levels of Cacna1e likely lower the number of RABV-labeled inputs from the NAcLat, and directly link the levels of Cacna1e and RABV input labeling” (lines 742-744) - we refrain from over-interpreting the results. As mentioned above in response to R1, we added a sentence to explain the rationale behind focusing on Cacna1e (lines 722-724).

      Reviewer #3 (Public Review):

      Summary:

      Authors mapped monosynaptic inputs to dopamine, GABA, and glutamate neurons in VTA under different anesthesia methods, and under drugs (cocaine, morphine, methamphetamine, amphetamine, nicotine, fluoxetine). They found that input patterns under different conditions are separated, and identified some key brain areas to contribute to such separation. They also searched a database for gene expression patterns that are common across input brain areas with some changes by anesthesia or drug administration.

      Strengths:

      The whole-brain approach to address drug effects is appealing and their conclusion is clear. The methodology and motivation are clearly explained.

      Weaknesses:

      While gene expression analyses may not be related to their findings on the anatomical effects of drugs, this will be a nice starting point for follow-up studies. 

      We understand and agree with the suggestion that gene expression allows us to provide correlative observations between in situ hybridization datasets and rabies mapping datasets, and that these results do not show causality. As such, future studies would be needed to assess this in more detail. We have added a line in the discussion to this effect (lines 851-853).

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      Recommendations for improving the writing and presentation:

      (1) There are a couple of packages available for 3D whole-brain reconstructions based on Allen Brain Atlas (eg. https://github.com/tractatus/wholebrain, https://github.com/lahammond/BrainJ), which would be helpful to align with the gene expression or other data from Allen Institute.

      This comment is related to the noted weakness we responded to previously in this rebuttal also from R1 (see comment 1), about the discrepancies between the Franklin-Paxinos atlas and Allen Brain atlas. We agree that a systematic comparison of these two atlases using a tool like wholebrain or BrainJ would be valuable for the field. However, it would be a substantial amount of work, and likely would be an independent study in itself. We believe that the resolution of these atlases was sufficient to make our key conclusions here (e.g., identify gene expression patterns that relate to drug-induced changes rabies virus labeling patterns, and develop a testable hypothesis for CRISPR-based gene editing). They are also based on the same atlases and region definitions that have been applied in our previous studies (e.g., Beier et al., Cell 2015; Beier et al., Nature 2017; Beier et al., Cell Reports 2019; Tian et al., Cell Reports 2022; Tian et al., Neuron 2024; Hubbard et al., Neuropsychophamacology 2025, etc.)  The expression of Cacna1e is relatively consistent across the NAc, as we have now detailed in Figure S13.

      (2) There are so far two kinds of rabies virus strains available in the neuroscience field (SAD-B19 or CVS-N2c). It is recommended to describe which strain was used in the Material and Methods Section because labeling efficiency and toxicity is quite different between the strains (Reardon TR et al., Neuron 2016).

      We have now noted that we used SAD B19 for all experiments (Lines 141-142).

      Minor corrections to the text and figures:

      (1)  In Figure 1A, the color differences are not clear (i.e. light gray and dark gray). The figure can be simplified.

      In addition, generally, images/figures are recommended not to be overlapped with other figures/images (Figures 2A-F, 2G-L).

      (2)  In Figures 7C and D, the authors could add enlarged views of starter cells in VTA and NAcLat.

      We have attempted to simplify schematics and figures throughout. High-magnification images of cells have been added as insets in what is now Figure 10 (formerly Figure 7).

      Reviewer #2 (Recommendations For the authors):

      The number of animals for each graph should be explicated within the figure legend. For example, Figure 1C and Figure 7E lack this information. It is also advisable to delineate the definition of error bars within the figure legend.

      We have now added mouse numbers to all figures and/or legends, as appropriate. We also indicated in the legend at the end of Figure 1 how error bars and asterisks are defined. Furthermore, we added a sentence to the methods saying that in UMAP and PCA plots each dot is an animal (lines 244-245).

      The visual representations, particularly in Figures 1 and 3, are overcrowding. Furthermore, the arrangement of figure subpanels does not consistently adhere to the sequence of explication in the main text, significantly compromising the readability of the text. The authors are encouraged to consider the possibility of segmenting dense figures into two if there exists no upper limit for the number of figure displays. To illustrate, in Figure 3Q, crucial details about experimental conditions are denoted by numerical references, owing to spatial constraints.

      We agree that the figure layout and mis-alignment with a linear read of the text was unideal. Therefore, we broke our figures, especially the original Figures 1-4, into multiple sub-figures, including both main and supplemental figures. This facilitated the use of space to rearrange the figure panels, allowing the story to be told in a linear fashion. All figures and panels should now be read in order.

      I am seeking clarification on how to interpret the term "overlap" at the bottom of figures illustrating Gene Ontology analysis.

      We have clarified the meaning of overlap in this context (lines 324-325): The ‘overlap’ term on the x-axis of these plots means the number of genes in the correlated gene lists that were also within the list of genes for the corresponding GO term.

      The authors could provide Cacna1e gene expression patterns within the NAc from the AGEA data.

      Cacna1e expression data are now provided in Figure S13.

      Additionally, the meaning of "controls" in Figure 7F, along with the "No gRNA" condition, remains ambiguous. While the text mentions "no shRNA", the involvement of shRNA in this experiment lacks clarity.

      We now clarify that the control conditions are based on previously published data where no AAVs were injected into NAcLat. This is now clarified in the legend for Figure 10F (lines 1277-1578). We also corrected “shRNA” to “gRNA” in the text.

    1. Author response:

      The following is the authors’ response to the original reviews

      We appreciate the reviewers’ insightful comments. In response, we conducted three new experiments, summarized in Author response table 1. After the table, we provide detailed responses to each comment.

      Author response table 1.

      Summary of new experiments and results.

      Reviewer #1 (Public review):

      The authors show that corticotropin-releasing factor (CRF) neurons in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) monosynaptically target cholinergic interneurons (CINs) in the dorsal striatum of rodents. Functionally, activation of CRFR1 receptors increases CIN firing rate, and this modulation was reduced by pre-exposure to ethanol. This is an interesting finding, with potential significance for alcohol use disorders, but some conclusions could use additional support.

      Strengths:

      Well-conceived circuit mapping experiments identify a novel pathway by which the CeA and BNST can modulate dorsal striatal function by controlling cholinergic tone. Important insight into how CRF, a neuropeptide that is important in mediating aspects of stress, affective/motivational processes, and drug-seeking, modulates dorsal striatal function.

      Weaknesses:

      (1) Tracing and expression experiments were performed both in mice and rats (in a mostly nonoverlapping way). While these species are similar in many ways, some conclusions are based on assumptions of similarities that the presented data do not directly show. In most cases, this should be addressed in the text (but see point number 2).

      In the revised manuscript, we have clarified this limitation in the first paragraph of the Methods and the third paragraph of the Discussion and avoid cross-species claims, limiting our conclusions to the species in which each assay was performed. Specifically, we now state that while mice and rats share many conserved amygdalostriatal components, our tracing and expression studies were performed in a species-specific manner, and direct cross-species comparisons of CRF–CIN connectivity and CRFR1 expression were not assessed. We further note that future studies will be needed to determine the extent to which these observations are conserved across species as more tools become available.

      (2) Experiments in rats show that CRFR1 expression is largely confined to a subpopulation of striatal CINs. Is this true in mice, too? Since most electrophysiological experiments are done in various synaptic antagonists and/or TTX, it does not affect the interpretation of those data, but non-CIN expression of CRFR1 could potentially have a large impact on bath CRF-induced acetylcholine release.

      To address whether CRFR1 expression in striatal CINs is conserved across species, we performed new histological experiments using CRFR1-GFP mice. Striatal sections were immunostained with anti-ChAT, and we found that approximately 10% of CINs express CRFR1 (new Fig. 4D, 4E). This result indicates that, similar to rats, a subset of CINs in mice express CRFR1. However, the proportion of CRFR1<sup>+</sup> CINs is lower than the proportion of CRF-responsive CINs observed during electrophysiology experiments, suggesting that CRF may also modulate CIN activity indirectly through network or synaptic mechanisms. We have also noted in the revised Discussion that while CRFR1 expression is confirmed in a subset of CINs, the broader distribution of CRFR1 among other striatal cell types remains to be determined (third paragraph of Discussion).

      In our study, bath application of CRF increased striatal ACh release. Because striatal ACh is released primarily from CINs, and CRFR1 is an excitatory receptor, this effect is most likely mediated by CRF activation of CRFR1 on CINs, leading to enhanced CIN activity and ACh release. Although CRFR1 may also be expressed on other striatal neurons, these cell types—medium spiny neurons and GABAergic interneurons—are inhibitory. If CRF were to activate CRFR1 on these GABAergic neurons, the resulting increase in GABA release would suppress CIN activity and consequently reduce, rather than enhance, ACh release. Given that most CINs responded functionally while only a small subset expressed CRFR1, these findings imply that indirect mechanisms, such as CRF modulation of local circuits influencing CIN excitability, may also contribute to the observed increase in ACh release. Together, these data support a model in which CRF primarily enhances ACh release via activation of CRFR1-expressing CINs, while indirect network effects may further amplify this response.

      (3) Experiments in rats show that about 30% of CINs express CRFR1 in rats. Did only a similar percentage of CINs in mice respond to bath application of CRF? The effect sizes and error bars in Figure 5 imply that the majority of recorded CINs likely responded. Were exclusion criteria used in these experiments?

      We thank the reviewer for this insightful question. In our mouse cell-attached recordings, ~80% of CINs increased firing during CRF bath application, and all recorded cells were included in the analysis (no exclusions based on response direction/magnitude; cells were only required to meet standard recording-quality criteria such as stable baseline firing and seal).

      Using a CRFR1-GFP reporter mouse, we found that ~10% of striatal CINs are GFP+, suggesting that the high proportion of CRF-responsive CINs cannot be explained solely by somatic reporter-labeled CRFR1 expression. Importantly, the CRF-induced increase in CIN firing is blocked by the selective CRFR1 antagonist NBI 35695 (Fig. 5B–C), supporting a CRFR1-dependent mechanism at the circuit level. We now discuss several non-mutually exclusive explanations for this apparent discrepancy: (i) reporter lines (e.g., CRFR1-GFP) may underestimate functional CRFR1 expression, particularly for low-level or compartmentalized receptor pools; (ii) bath-applied CRF may act indirectly via CRFR1 on presynaptic afferents, thereby enhancing excitatory drive onto CINs; and (iii) electrical coupling among CINs could allow direct effects in a subset of CINs to propagate through the CIN network (Ren, Liu et al. 2021). We added this discussion to the revised manuscript (fourth paragraph of the Discussion).

      (4) The conclusion that prior acute alcohol exposure reduces the ability of subsequent alcohol exposure to suppress CIN activity in the presence of CRF may be a bit overstated. In Figure 6D (no ethanol preexposure), ethanol does not fully suppress CIN firing rate to baseline after CRF exposure. The attenuated effect of CRF on CIN firing rate after ethanol pre-treatment (6E) may just reduce the maximum potential effect that ethanol can have on firing rate after CRF, due to a lowered starting point. It is possible that the lack of significant effect of ethanol after CRF in pre-treated mice is an issue of experimental sensitivity. Related to this point, does pre-treatment with ethanol reduce the later CIN response to acute ethanol application (in the absence of CRF)?

      In the revised manuscript, we have tempered our interpretation in the final Results section and throughout the Discussion to emphasize that ethanol pre-exposure attenuates, rather than abolishes, the CRFinduced increase in CIN firing. We also note the reviewer’s important point that in Figure 6D, ethanol does not fully suppress firing to baseline after CRF exposure, consistent with a partial effect. Regarding the reviewer’s question, our experiments were specifically designed to test interactions between CRF and ethanol, so we did not assess whether ethanol pre-treatment alters subsequent responses to ethanol alone. We now explicitly acknowledge CRF-dependent and CRF-independent effects of ethanol on CIN activity as an important point for future studies to disentangle (sixth paragraph of the Discussion). For example, comparing ethanol responses with and without prior ethanol without any treatment with CRF could resolve this question.

      (5) More details about the area of the dorsal striatum being examined would be helpful (i.e., a-p axis).

      We now provide more detail regarding the anterior–posterior axis of the dorsal striatum examined. Most recordings and imaging were performed in the posterior dorsomedial striatum (pDMS), corresponding to coronal slices posterior to the crossing of the anterior commissure and anterior to the tail of the striatum (starting around 0.62 mm and ending at −1.3 mm relative to the Bregma). While our primary focus was on posterior slices, some anterior slices were included to increase the sample size. These details have been added to the Methods (Last sentence of the ‘Histology and cell counting’ section and of the ‘Slice electrophysiology’ section).

      Reviewer #2 (Public review):

      Essoh and colleagues present a thorough and elegant study identifying the central amygdala and BNST as key sources of CRF input to the dorsal striatum. Using monosynaptic rabies tracing and electrophysiology, they show direct connections to cholinergic interneurons. The study builds on previous findings that CRF increases CIN firing, extending them by measuring acetylcholine levels in slices and applying optogenetic stimulation of CRF+ fibers. It also uncovers a novel interaction between alcohol and CRF signaling in the striatum, likely to spark significant interest and future research.

      Strengths:

      A key strength is the integration of anatomical and functional approaches to demonstrate these projections and assess their impact on target cells, striatal cholinergic interneurons.

      Weaknesses:

      (1) The nature of the interaction between alcohol and CRF actions on cholinergic neurons remains unclear. Also, further clarification of the ACh sensor used and others is required

      We have clarified the nature of the interaction between alcohol and CRF signaling in CINs and have provided additional details regarding the acetylcholine sensor used. These issues are addressed in detail in our responses to the specific comments below.

      Reviewer #2 (Recommendations for the authors):

      (1) The interaction between the effects of alcohol and CRF is a novel and important part of this study. When considering possible mechanisms underlying the findings in the discussion, there is no mention of occlusion. Given that incubation with alcohol produced a similar increase in firing of CINs as CRF, occlusion could be a parsimonious explanation for the observed interaction. Have the author considered blocking the effects of alcohol on CIN with CRF-R1 antagonist? Another experiment that could address the occlusion would be to test if alcohol also increases ACh levels as it did CRF.

      We thank the reviewer for proposing occlusion as a potential mechanism underlying the interaction between alcohol and CRF. We agree that, in principle, alcohol-induced endogenous CRF release could occlude subsequent exogenous CRF-mediated potentiation of CIN firing, and we carefully considered this possibility.

      However, several observations from our data argue against occlusion driven by acute alcohol exposure or withdrawal in this preparation. First, as shown in Fig. 6A, bath application of alcohol transiently reduced CIN firing, and firing recovered to baseline levels after washout without any rebound increase. Second, in Fig. 6D–E, the baseline firing rates under control conditions and following alcohol pretreatment were comparable, indicating that acute alcohol exposure and short-term withdrawal did not produce a sustained increase in CIN excitability. Together, these results suggest that acute withdrawal in slices is less likely to trigger substantial endogenous CRF release capable of occluding subsequent exogenous CRF effects.

      While we and others have previously reported increased spontaneous CIN firing following prolonged in vivo alcohol exposure and extended withdrawal periods (e.g., 21 days), short-term withdrawal (e.g., 1 day) does not robustly alter baseline CIN firing (Ma, Huang et al. 2021, Huang, Chen et al. 2024). Consistent with these prior findings, the absence of a rebound or elevated baseline firing in the present slice experiments discouraged further pursuit of an endogenous CRF occlusion mechanism under acute conditions.

      We also considered experimentally testing occlusion by blocking CRFR1 signaling during alcohol pre-treatment. However, this approach is technically challenging in slice recordings, as CRFR1 antagonists require prolonged incubation (~1 hour) during alcohol exposure. Because it is unclear whether endogenous CRF release is triggered by alcohol incubation itself or by withdrawal, the antagonist would need to remain present throughout both the incubation and withdrawal periods. This leaves insufficient time for complete washout of the CRFR1 antagonist prior to subsequent bath application of exogenous CRF to assess its effects on CIN firing. Consequently, residual antagonist presence would confound the interpretation of the exogenous CRF response.

      Finally, regarding the possibility that alcohol increases acetylcholine release, we did not observe alcohol-induced increases in CIN firing in slices, arguing against elevated ACh signaling under these conditions. Consistent with prior work (Ma, Huang et al. 2021, Huang, Chen et al. 2024), alcohol-induced increases in CIN excitability and cholinergic signaling appear to depend on prolonged in vivo exposure and extended withdrawal rather than acute slice-level manipulations.

      We have now incorporated discussion of occlusion as a potential mechanism (seventh paragraph) and clarified why our data and technical considerations argue against it in the present study. We thank the reviewer for this wonderful suggestion, which we will test in future in vivo studies.

      (2) Retrograde monosynaptic tracing of inputs to CIN. Results state the finding of labeling in all previously reported area..." Can the authors report these areas? A list in the text or a bar plot, if there is quantification, will suffice. This formation will serve as important validation and replication of previous findings.

      We thank the reviewer for this constructive suggestion. We agree that summarizing the anatomical sources of CIN input provides important validation of our tracing results. In the revised Results, we now list the major input regions observed, including the striatum itself, cortex (e.g., cingulate cortex, motor cortex, somatosensory cortex), thalamus (e.g., parafascicular thalamic nucleus, centrolateral thalamic nucleus), globus pallidus, and midbrain (first paragraph of the Results). Quantitative analysis of relative input strength will be presented in a separate study that expands on these findings. Here, we limit the current manuscript to the functional characterization of CRF and alcohol modulation of CINs.

      (3) Given the difference in connectivity among striatal subregions, it would be important to describe in more detail the injection site in the results and figures. In the figure, for example, you might want to include the AP coordinates, given that it is such a zoomed-in image, it is hard to tell how anterior/posterior the site is. I imagine that the picture is a representative image of the injection site, but maybe having a side image with overlay of injection sites in all the animals used, would help.

      The anterior–posterior (AP) coordinates for representative images have been included in the panels and reiterated more clearly in the revised Results section and figure legends. In the legend for Figure 3B, a list of AP coordinates for each animal used for Figure 3A-3E has been added.

      (4) Figure 1D inset, there seem to be some double-labeled cells in the zoomed in BNST images. The authors might want to comment on this. It seemed far from the injection site. Do D1-MSN so far away show connectivity to CINs?

      Upon closer inspection of the BNST images, we noted a small number of double-labeled cells were indeed present, consistent with prior reports that a subset of D1R-expressing neurons (~10%) has been reported previously in our lab in the BNST, with the majority being D2R-expressing neurons (Lu, Cheng et al. 2021). Given the BNST’s anatomical proximity to the dorsal striatum, it is plausible that some D1Rexpressing neurons in this region provide monosynaptic input to CINs, highlighting a potential ventral-to-dorsal connection that merits further study.

      (5) Can the author provide quantification of the onset delay of the optogenetic evoked CRF+ axon responses onto CINs? The claim of monosynaptic connectivity is well supported by the TTX/4AP experiment but additional information on the timing will strengthen that conclusion.

      We thank the reviewer for this insightful suggestion. Quantifying the onset latency of optogenetically evoked CRFMsup+</sup> axon responses onto CINs provides valuable confirmation of monosynaptic connectivity. To address this, we performed new latency measurements under the same recording conditions as the TTX/4-AP experiments. The average onset latency from the start of the optical stimulation was 5.85 ± 0.37 ms (new Figure 3J), consistent with direct monosynaptic transmission.

      As an additional reference, we analyzed latency data from a separate project in which we optogenetically stimulated cholinergic interneurons and recorded synaptic responses in medium spiny neurons. This circuit, known to involve disynaptic transmission from CINs to MSNs via nAChR-expressing interneurons (Autor response image 1) (English, Ibanez-Sandoval et al. 2011), exhibited a significantly longer latency (18.34 ± 0.70 ms; t<sub>(29)</sub> = 10.3, p < 0.001) compared to CRF⁺ CeA/BNST inputs to CINs (5.85 ± 0.37 ms). Together, these results further support that CRF⁺ axons form direct functional synapses onto CINs.

      Author response image 1.

      Latency of disynaptic transmission from CINs to MSNs via interneurons A) Schematic illustrating optogenetic stimulation of Chrimson-expressing CINs, leading to excitation of nAChRexpressing interneurons that release GABA onto recorded MSNs. B) Sample trace of disynaptic transmission (left) and bar graph summarizing onset latency (right) from light stimulation to synaptic response onset (n = 23 neurons from 3 mice).

      (6) The ACh sensor reported is "AAV-GRABACh4m" but the reference is for GRAB-ACh3.0. Also, BrainVTA has GRAB-ACh4.3. Is this the vector? Could you please check the name of the construct and report the corresponding reference, as well as clarify the meaning of the additional "m". They have a mutant version of the GRAB-ACH that researchers use for control, and of course, you want to use it as a control, but not for the test experiment.

      GRAB-ACh4m is the correct acetylcholine sensor used in this study. The ACh4 series (including ACh4h, ACh4m, and ACh4l; personal communication with Dr. Yulong Li’s lab) represents an updated generation following GRAB-ACh3.0. Although the ACh4 family has not yet been formally published, these constructs are publicly available through BrainVTA (https://www.brainvta.tech/plus/view.php?aid=2680).

      The suffix “m” does not indicate a mutant control; rather, it denotes a medium-affinity variant within the ACh4 sensor family. Importantly, the mutant (non-responsive) control sensor is only available for GRAB-ACh3.0 (ACh3.0mut) and does not exist for the ACh4 series.

      Our laboratory has previously used GRAB-ACh4m in multiple peer-reviewed publications (Huang, Chen et al. 2024, Gangal, Iannucci et al. 2025, Purvines, Gangal et al. 2025), and its use has also been reported by independent groups in recent preprints (Potjer, Wu et al. 2025, Touponse, Pomrenze et al. 2025). We have now clarified the construct name, its relationship to GRAB-ACh3.0, in the Methods ‘Reagents’ section, and we have corrected the reference accordingly.

      (7) Are CRF-R1+ CINs equally abundant in the DMS and DLS? From the image in Figure 4, it seems that a larger percentage of CINs are CRFR1+ in the DLS than in DMS. Is this true? The authors probably already have this data, or it should be easy to get, and it could be additional information that was not studied before.

      We did not perform a quantitative comparison of CRFR1+ CIN abundance between the DMS and DLS in the present study. While the representative images in Figure 4 may appear to suggest regional differences, these panels were selected to illustrate labeling quality rather than relative density and should not be interpreted as evidence of unequal distribution. We have clarified this point in the revised Discussion (last sentence of the third paragraph) and note that future studies will be needed to systematically evaluate potential regional differences in CRFR1 expression, which could have important implications for dorsal striatal function.

      (8) The manuscript states several times that there are no CRF+ neurons in the dorsal striatum. At the same time, there are reports of the CRF+ neuron in the ventral striatum and its role in learning. Could the authors include mention of the studies by the Lemos group (10.1016/j.biopsych.2024.08.006)

      We have revised the Discussion section to clarify that our findings pertain specifically to the dorsal striatum and now acknowledge the presence and functional relevance of CRF+ neurons in the ventral striatum, citing the Lemos group’s study (fifth paragraph of the Discussion).

      (9) For the histology analysis, please express cell counts as "density", not just number of cells, by providing an area (e.g., "number of cell/ µm2").

      In the revised manuscript, all histological outcomes have been recalculated as cell density (cells/mm<sup>2</sup>) by normalizing raw cell counts to the measured area of each region of interest (ROI). Figures that previously displayed absolute counts now present densities (cells/mm<sup>2</sup>), with corresponding updates made to figure legends and text. We note one exception in Figure 4B, where the comparison between the total number of CINs and CRFR1+ CINs is best represented as cell counts rather than normalized values, as the counting was conducted in the same area (within the same ROI) of the dorsostriatal subregion.

      (10) Figure 2C, we can see there are some labeled fibers in the striatum cut. Would it be possible to get a better confocal image?

      Figure 2C has been replaced with a higher-quality confocal image captured at the same magnification and scale. The updated image provides improved clarity and resolution, ensuring accurate visualization of labeled CRF+ fibers, but not cell bodies, within the striatum.

      (11) The ACh measurements in the slice are very informative and an important addition. I first thought that these experiments with the GRAB-ACh sensor were performed in ChAT-eGFP mice. After reading more carefully, I realized they were done in wild-type mice. Would you include the wildtype label in the figure as well? The ChATeGFP BAC transgenic line was reported to have enhanced ACh packaging and increased ACh release, which could have magnified the signals. So, it is important to highlight the experiments were done in wildtype mice.

      We now label with ‘WT mice’ and note in the legend that all GRAB-ACh experiments were performed in wild-type mice, not ChAT-eGFP, to avoid confounds in ACh release. We thank the reviewer for this important suggestion.

      Reviewer #3 (Public review):

      The authors demonstrate that CRF neurons in the extended amygdala form GABAergic synapses onto cholinergic interneurons and that CRF can excite these neurons. The evidence is strong, however, the authors fail to make a compelling connection showing CRF released from these extended amygdala neurons is mediating any of these effects. Further, they show that acute alcohol appears to modulate this action, although the effect size is not particularly robust.

      Strengths:

      This is an exciting connection from the extended amygdala to the striatum that provides a new direction for how these regions can modulate behavior. The work is rigorous and well done.

      Weaknesses:

      (1) While the authors show that opto stim of these neurons can increase firing, this is not shown to be CRFR1 dependent. In addition, the effects of acute ethanol are not particularly robust or rigorously evaluated. Further, the opto stim experiments are conducted in an Ai32 mouse, so it is impossible to determine if that is from CEA and BNST, vs. another population of CRF-containing neurons. This is an important caveat.

      We added recordings with the CRFR1 antagonist antalarmin. Light-evoked increases in CIN firing were abolished under CRFR1 blockade, linking the effect to CRFR1 (Figure 5J, 5K). We also clarify that CRFCre;Ai32 does not isolate CeA versus BNST sources, so we temper regional claims and highlight this as a limitation. The acute ethanol effects are modest but consistent; we expanded the discussion of dose and preparation constraints in acute slice physiology and note that in vivo studies will be needed to define the network-level impact.

      Reviewer #3 (Recommendations for the authors):

      (1) The authors could bring some of this data together by examining CRFR1 dependence of optical stimulationinduced increases in firing. Further, the authors have devoted significant effort to exploring how the BNST and CEA project to the CIN, yet their ephys does not explore site-specific infusion of ChR2 into either region. How are we to be sure it is not some other population of CRF neurons mediating this effect? The alcohol data does not appear particularly robust, but I think if the authors wanted to, they could explore other concentrations. Mostly I think it is important to discuss the limitations of acute alcohol on 5a brain slice.

      We thank the reviewer for these thoughtful comments, which helped us strengthen the mechanistic interpretation of the CRF-CIN interaction. In the revised manuscript, we have addressed each point as follows:

      - CRFR1 dependence of optogenetically evoked responses: We performed new recordings in which optogenetic stimulation of CRF⁺ terminals in the dorsal striatum was conducted in the presence of the CRFR1 antagonist antalarmin. The increase in CIN firing evoked by light stimulation was abolished under CRFR1 blockade, confirming that this effect is mediated through CRFR1 activation (new Figure 5J, 5K, third paragraph of the corresponding Result section). These results directly link the functional effects of CRF⁺ terminal activation to CRFR1 signaling on CINs.

      - CeA vs. BNST projection specificity: The reviewer is correct that CeA and BNST projections were not analyzed separately. As unknown pathways, our experiment was designed to first establish the monosynaptic connections between CeA/BNST CRF neurons to striatal CINs. Future studies would further explore the specific contribution of each site. However, our data exclude the possibility of other CRF neurons as we selectively infused Cre-dependent opsins into both CeA and BNST of CRF-Cre mice (Figure 3G-3J).

      - Limitations of acute slice experiments: We have expanded the Discussion (sixth paragraph) to acknowledge that acute slice physiology cannot fully capture the dynamic and network-level effects of ethanol observed in vivo. While this preparation enables mechanistic precision, factors such as washout, diffusion constraints, and the absence of systemic feedback may underestimate ethanol’s impact on CINs. We now explicitly note this limitation and highlight the need for in vivo studies to examine behavioral and circuit-level implications of CRF–alcohol interactions.

      Collectively, these revisions clarify the CRFR1 dependence of CRF<sup>+</sup> terminal effects and reaffirm that both CeA and BNST projections contribute to CIN modulation while addressing the methodological limitations of the slice preparation.

      Reviewer #4 Public Review):

      This manuscript presents a compelling and methodologically rigorous investigation into how corticotropin-releasing factor (CRF) modulates cholinergic interneurons (CINs) in the dorsal striatum - a brain region central to cognitive flexibility and action selection-and how this circuit is disrupted by alcohol exposure. Through an integrated series of anatomical, optogenetic, electrophysiological, and imaging experiments, the authors uncover a previously uncharacterized CRF⁺ projection from the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) to dorsal striatal CINs.

      Strengths:

      Key strengths of the study include the use of state-of-the-art monosynaptic rabies tracing, CRF-Cre transgenic models, CRFR1 reporter lines, and functional validation of synaptic connectivity and neurotransmitter release. The finding that CRF enhances CIN excitability and acetylcholine (ACh) release via CRFR1, and that this effect is attenuated by acute alcohol exposure and withdrawal, provides important mechanistic insight into how stress and alcohol interact to impair striatal function. These results position CRF signaling in CINs as a novel contributor to alcohol use disorder (AUD) pathophysiology, with implications for relapse vulnerability and cognitive inflexibility associated with chronic alcohol intake. The study is well-structured, with a clear rationale, thorough methodology, and logical progression of results. The discussion effectively contextualizes the findings within broader addiction neuroscience literature and suggests meaningful future directions, including therapeutic targeting of CRFR1 signaling in the dorsal striatum.

      Weaknesses:

      (1) Minor areas for improvement include occasional redundancy in phrasing, slightly overlong descriptions in the abstract and significance sections, and a need for more concise language in some places. Nevertheless, these do not detract from the manuscript's overall quality or impact. Overall, this is a highly valuable contribution to the fields of addiction neuroscience and striatal circuit function, offering novel insights into stress-alcohol interactions at the cellular and circuit level, which requires minor editorial revisions.

      We have streamlined the abstract and significance statement, reduced redundancy, and improved conciseness throughout the text. We appreciate the reviewer’s feedback, which has helped us further strengthen the clarity and readability of the manuscript.

      Reviewer #4 (Recommendations for the authors):

      (1) Line 29-30: Slightly verbose. Consider: "Alcohol relapse is associated with corticotropin-releasing factor (CRF) signaling and altered reward pathway function, though the precise mechanisms are unclear."

      The sentence has been revised as recommended to improve clarity and conciseness in the introductory section (Lines 31-32).

      (2) Lines 39-43: Good synthesis, but could better emphasize the novelty of identifying a CRF-CIN pathway.

      The abstract has been revised to more clearly emphasize the novelty of identifying a CRF-CIN pathway and its functional significance (Line 42-43).

      (3) Lines 66-68: Consider integrating clinical relevance more directly, e.g., "AUD affects over 14 million adults in the U.S., with relapse often triggered by stress...".

      The introduction has been revised to more directly emphasize the clinical relevance of alcohol use disorder, including its high prevalence and the role of stress in relapse, thereby underscoring the translational significance of our findings (Lines 68-69).

      (4) Line 83: Repetition of "goal-directed learning, habit formation, and behavioral flexibility" appears multiple times; consider variety.

      We have varied the phrasing in the Introduction to avoid redundancy. Specifically, in place of repeating “goal-directed learning, habit formation, and behavioral flexibility,” we now use alternative terms such as “action selection,” “habitual responding,” and “cognitive flexibility,” depending on the context.

      (5) Lines 107-116: Clarify why both rats and mice were used-do they serve different experimental purposes?

      We now explain that each species was used for complementary experimental purposes. Rats were used for histological validation of CRFR1 expression using the CRFR1-Cre-tdTomato line, which has been extensively characterized in this species. Mice were used for the majority of electrophysiological, optogenetic, and GRAB-ACh sensor experiments due to the availability of well-established transgenic CRF-Cre-driver lines. This division allowed us to leverage the most appropriate tools in each species to address different aspects of the study. We have clarified this rationale in the Methods (first paragraph of the “Animals” section) and Discussion (third paragraph).

      (6) Electrophysiology section: The distinction between acute exposure vs. withdrawal could be further emphasized.

      To better highlight the distinction between acute alcohol exposure and withdrawal, we have clarified the timing and context of each condition within the Results section for Figure 6. Specifically, we now distinguish the immediate suppressive effects of alcohol observed during bath application (acute exposure) from the subsequent changes in CIN firing measured after washout (withdrawal). These revisions clarify the temporal dynamics and functional implications of CRF–alcohol interactions in our experimental design.

      (7) Lines 227-229: Reword for clarity: "Significantly more BNST neurons projected to CINs compared to the CeA...".

      The sentence has been reworded to clarify as recommended (Lines 247-248).

      (8) Lines 373-374: Consider connecting the CRF-CIN circuit to behavioral inflexibility in AUD more directly.

      We have modified the sentence (Lines 390-395) to more explicitly link alcohol-induced dysregulation of the CRF–CIN circuit to behavioral inflexibility in AUD, consistent with the established role of CINs in action selection and cognitive flexibility.

      (9) Lines 387-389: This is an excellent point about stress resilience; consider expanding with examples or potential implications.

      We thank the reviewer for this insightful suggestion. In the revised Discussion (sixth paragraph), we expanded this section to more directly connect alcohol-induced disruption of CRF–CIN signaling with impaired stress resilience and behavioral inflexibility. Specifically, we now note that such dysregulation may compromise stress resilience mechanisms mediated by CRF–cholinergic interactions in the striatum and related corticostriatal circuits. We further discuss how impaired CIN responsiveness could blunt adaptive behavioral adjustments under stress, biasing animals toward habitual or compulsive alcohol seeking. This addition highlights the broader implication that alcohol-induced alterations in CRF–CIN signaling may contribute to relapse vulnerability by undermining adaptive stress coping.

      References

      English, D. F., O. Ibanez-Sandoval, E. Stark, F. Tecuapetla, G. Buzsaki, K. Deisseroth, J. M. Tepper and T. Koos (2011). "GABAergic circuits mediate the reinforcement-related signals of striatal cholinergic interneurons." Nat Neurosci 15(1): 123–130.

      Gangal, H., J. Iannucci, Y. Huang, R. Chen, W. Purvines, W. T. Davis, A. Rivera, G. Johnson, X. Xie, S. Mukherjee, V. Vierkant, K. Mims, K. O'Neill, X. Wang, L. A. Shapiro and J. Wang (2025). "Traumatic brain injury exacerbates alcohol consumption and neuroinflammation with decline in cognition and cholinergic activity." Transl Psychiatry 15(1): 403.

      Huang, Z., R. Chen, M. Ho, X. Xie, H. Gangal, X. Wang and J. Wang (2024). "Dynamic responses of striatal cholinergic interneurons control behavioral flexibility." Sci Adv 10(51): eadn2446.

      Lu, J. Y., Y. F. Cheng, X. Y. Xie, K. Woodson, J. Bonifacio, E. Disney, B. Barbee, X. H. Wang, M. Zaidi and J. Wang (2021). "Whole-Brain Mapping of Direct Inputs to Dopamine D1 and D2 Receptor-Expressing Medium Spiny Neurons in the Posterior Dorsomedial Striatum." Eneuro 8(1).

      Ma, T., Z. Huang, X. Xie, Y. Cheng, X. Zhuang, M. J. Childs, H. Gangal, X. Wang, L. N. Smith, R. J. Smith, Y. Zhou and J. Wang (2021). "Chronic alcohol drinking persistently suppresses thalamostriatal excitation of cholinergic neurons to impair cognitive flexibility." J Clin Invest 132(4): e154969.

      Potjer, E. V., X. Wu, A. N. Kane and J. G. Parker (2025). "Parkinsonian striatal acetylcholine dynamics are refractory to L-DOPA treatment." bioRxiv.

      Purvines, W., H. Gangal, X. Xie, J. Ramos, X. Wang, R. Miranda and J. Wang (2025). "Perinatal and prenatal alcohol exposure impairs striatal cholinergic function and cognitive flexibility in adult offspring." Neuropharmacology 279: 110627.

      Ren, Y., Y. Liu and M. Luo (2021). "Gap Junctions Between Striatal D1 Neurons and Cholinergic Interneurons." Front Cell Neurosci 15: 674399.

      Touponse, G. C., M. B. Pomrenze, T. Yassine, V. Mehta, N. Denomme, Z. Zhang, R. C. Malenka and N. Eshel (2025). "Cholinergic modulation of dopamine release drives effortful behavior." bioRxiv.

    1. Reviewer #1 (Public review):

      Summary:

      This study presents an interesting behavioral paradigm and reveals interactive effects of social hierarchy and threat type on defensive behaviors. However, addressing the aforementioned points regarding methodological detail, rigor in behavioral classification, depth of result interpretation, and focus of the discussion is essential to strengthen the reliability and impact of the conclusions in a revised manuscript.

      Strengths:

      The paper is logically sound, featuring detailed classification and analysis of behaviors, with a focus on behavioral categories and transitions, thereby establishing a relatively robust research framework.

      Weaknesses:

      Several points require clarification or further revision.

      (1) Methods and Terminology Regarding Social Hierarchy:

      The study uses the tube test to determine subordinate status, but the methodological description is quite brief. Please provide a more detailed account of the experimental procedure and the criteria used for determination.

      The dominance hierarchy is established based on pairs of mice. However, the use of terms like "group cohesion" - typically applied to larger groups - to describe dyadic interactions seems overstated. Please revise the terminology to more accurately reflect the pairwise experimental setup.

      (2) Criteria and Validity of Behavioral Classification:

      The criteria for classifying mouse behaviors (e.g., passive defense, active defense) are not sufficiently clear. Please explicitly state the operational definitions and distinguishing features for each behavioral category.

      How was the meaningfulness and distinctness of these behavioral categories ensured to avoid overlap? For instance, based on Figure 3E, is "active defense" synonymous with "investigative defense," involving movement to the near region followed by return to the far region? This requires clearer delineation.

      The current analysis focuses on a few core behaviors, while other recorded behaviors appear less relevant. Please clarify the principles for selecting or categorizing all recorded behaviors.

      (3) Interpretation of Key Findings and Mechanistic Insights:

      Looming exposure increased the proportion of proactive bouts in the dominant zone but decreased it in the subordinate zone (Figure 4G), with a similar trend during rat exposure. Please provide a potential explanation for this consistent pattern. Does this consistency arise from shared neural mechanisms, or do different behavioral strategies converge to produce similar outputs under both threats?

      (4) Support for Claims and Study Limitations:

      The manuscript states that this work addresses a gap by showing defensive responses are jointly shaped by threat type and social rank, emphasizing survival-critical behaviors over fear or stress alone. However, it is possible that the behavioral differences stem from varying degrees of danger perception rather than purely strategic choices. This warrants a clear description and a deeper discussion to address this possibility.

      The Discussion section proposes numerous brain regions potentially involved in fear and social regulation. As this is a behavioral study, the extensive speculation on specific neural circuitry involvement, without supporting neuroscience data, appears insufficiently grounded and somewhat vague. It is recommended to focus the discussion more on the implications of the behavioral findings themselves or to explicitly frame these neural hypotheses as directions for future research.

    2. Reviewer #2 (Public review):

      Summary:

      The authors investigate how dominance hierarchy shapes defensive strategies in mice under two naturalistic threats: a transient visual looming stimulus and a sustained live rat. By comparing single versus paired testing, they report that social presence attenuates fear and that dominant and subordinate mice exhibit different patterns of defensive and social behaviors depending on threat type. The work provides a rich behavioral dataset and a potentially useful framework for studying hierarchical modulation of innate fear.

      Strengths:

      (1) The study uses two ecologically meaningful threat paradigms, allowing comparison across transient and sustained threat contexts.

      (2) Behavioral quantification is detailed, with manual annotation of multiple behavior types and transition-matrix level analysis.

      (3) The comparison of dominant versus subordinate pairs is novel in the context of innate fear.

      (4) The manuscript is well-organized and clearly written.

      (5) Figures are visually informative and support major claims.

      Weaknesses:

      Lack of neural mechanism insights.

    3. Author response:

      Public Reviews:

      Reviewer #1 (Public review):

      Summary: 

      This study presents an interesting behavioral paradigm and reveals interactive effects of social hierarchy and threat type on defensive behaviors. However, addressing the aforementioned points regarding methodological detail, rigor in behavioral classification, depth of result interpretation, and focus of the discussion is essential to strengthen the reliability and impact of the conclusions in a revised manuscript. 

      Strengths: 

      The paper is logically sound, featuring detailed classification and analysis of behaviors, with a focus on behavioral categories and transitions, thereby establishing a relatively robust research framework. 

      Weaknesses: 

      Several points require clarification or further revision. 

      (1) Methods and Terminology Regarding Social Hierarchy: 

      The study uses the tube test to determine subordinate status, but the methodological description is quite brief. Please provide a more detailed account of the experimental procedure and the criteria used for determination. 

      We will add more details about how the tube test was performed in the revised manuscript.

      The dominance hierarchy is established based on pairs of mice. However, the use of terms like "group cohesion" - typically applied to larger groups - to describe dyadic interactions seems overstated. Please revise the terminology to more accurately reflect the pairwise experimental setup.

      Thanks for the comment. We agree that the term “group cohesion” can be misleading and will replace it with “social engagement”.

      (2) Criteria and Validity of Behavioral Classification: 

      The criteria for classifying mouse behaviors (e.g., passive defense, active defense) are not sufficiently clear. Please explicitly state the operational definitions and distinguishing features for each behavioral category. 

      Passive defense was defined as an immobility-based defensive strategy characterized by suppression of locomotor activity. This category included freezing and tail rattling, which in our study involved minimal body displacement aside from rapid tail vibration. Active defense was defined as movement- or posture-dependent defensive strategy, encompassing behaviors that involved locomotor engagement or spatial repositioning relative to the threat, including approach, investigation, withdrawal, and stretch-attend. We will clarify this in the revised manuscript.

      How was the meaningfulness and distinctness of these behavioral categories ensured to avoid overlap? For instance, based on Figure 3E, is "active defense" synonymous with "investigative defense," involving movement to the near region followed by return to the far region? This requires clearer delineation. 

      Defensive behaviors in the rat exposure paradigm were grouped into two categories: passive and active defense, each comprising distinct behaviors. All the manually annotated behaviors were mutually exclusive; that is, each video frame was assigned a single behavioral label to avoid overlap across behaviors. Active defense includes four behaviors: approach, investigation, withdrawal, and stretch-attend. We will clarify these points in the revised manuscript.

      The current analysis focuses on a few core behaviors, while other recorded behaviors appear less relevant. Please clarify the principles for selecting or categorizing all recorded behaviors.

      Thank you for pointing this out. In the current study, we focused primarily on defensive and social behaviors. We also included several neutral solitary behaviors related to anxiety and defensive state, such as sniffing, grooming, and rearing, which were consistently expressed across animals and closely linked to our main findings. We will clarify this rationale in the revised manuscript.

      (3) Interpretation of Key Findings and Mechanistic Insights:

      Looming exposure increased the proportion of proactive bouts in the dominant zone but decreased it in the subordinate zone (Figure 4G), with a similar trend during rat exposure. Please provide a potential explanation for this consistent pattern. Does this consistency arise from shared neural mechanisms, or do different behavioral strategies converge to produce similar outputs under both threats?

      Thanks for bringing up this important question. The consistent increase in proactive bouts in dominant mice across both paradigms suggests a consistent rank-dependent reorganization of dyadic interaction under threats. We propose that this convergence reflects a shared neural mechanism that links defensive state with social-rank information, potentially mediated by overlapping hypothalamic and prefrontal circuits. We will expand the Discussion to incorporate this explanation.

      (4) Support for Claims and Study Limitations:

      The manuscript states that this work addresses a gap by showing defensive responses are jointly shaped by threat type and social rank, emphasizing survival-critical behaviors over fear or stress alone. However, it is possible that the behavioral differences stem from varying degrees of danger perception rather than purely strategic choices. This warrants a clear description and a deeper discussion to address this possibility.

      We thank the reviewer for this insightful comment. We agree that, in principle, behavioral differences could arise from variations in perceived danger rather than strategic choice. In humans, decisions can sometimes reflect value-based strategies that override perceived danger. In contrast, under naturalistic threat conditions, mice likely rely predominantly on danger perception to make behavioral decisions, and such responses are expected to be consistent with value-based strategies shaped by natural selection. In the revised manuscript, we will expand the Discussion to address the role of threat perception and its relationship to decision-making in our behavioral paradigms.

      The Discussion section proposes numerous brain regions potentially involved in fear and social regulation. As this is a behavioral study, the extensive speculation on specific neural circuitry involvement, without supporting neuroscience data, appears insufficiently grounded and somewhat vague. It is recommended to focus the discussion more on the implications of the behavioral findings themselves or to explicitly frame these neural hypotheses as directions for future research.

      We will revise the Discussion to focus more directly on behavioral findings and add explicit neural hypotheses as potential future directions.

      Reviewer #2 (Public review):

      Summary:

      The authors investigate how dominance hierarchy shapes defensive strategies in mice under two naturalistic threats: a transient visual looming stimulus and a sustained live rat. By comparing single versus paired testing, they report that social presence attenuates fear and that dominant and subordinate mice exhibit different patterns of defensive and social behaviors depending on threat type. The work provides a rich behavioral dataset and a potentially useful framework for studying hierarchical modulation of innate fear.

      Strengths:

      (1) The study uses two ecologically meaningful threat paradigms, allowing comparison across transient and sustained threat contexts.

      (2) Behavioral quantification is detailed, with manual annotation of multiple behavior types and transition-matrix level analysis.

      (3) The comparison of dominant versus subordinate pairs is novel in the context of innate fear.

      (4) The manuscript is well-organized and clearly written.

      (5) Figures are visually informative and support major claims.

      Weaknesses:

      Lack of neural mechanism insights.

      The current study focused on behavior. In the revised manuscript, we will incorporate a discussion of potential neural mechanisms and highlight this as an important direction for future work.

      Reviewer #3 (Public review):

      Summary:

      This study examines how dominance hierarchy influences innate defensive behaviors in pair-housed male mice exposed to two types of naturalistic threats: a transient looming stimulus and a sustained live rat. The authors show that social presence reduces fear-related behaviors and promotes active defense, with dominant mice benefiting more prominently. They also demonstrate that threat exposure reinforces social roles and increases group cohesion. The work highlights the bidirectional interaction between social structure and defensive behavior.

      Strengths:

      This study makes a valuable contribution to behavioral neuroscience through its well-designed examination of socially modulated fear. A key strength is the use of two ethologically relevant threat paradigms - a transient looming stimulus and a sustained live predator, enabling a nuanced comparison of defensive behaviors. The experimental design is robust, systematically comparing animals tested alone versus with their cage mate to cleanly isolate social effects. The behavioral analysis is sophisticated, employing detailed transition maps that reveal how social context reshapes behavioral sequences, going beyond simple duration measurements. The finding that social modulation is rank-dependent adds significant depth, linking social hierarchy to adaptive defense strategies. Furthermore, the demonstration that threat exposure reciprocally enhances social cohesion provides a compelling systems-level perspective. Together, these elements establish a strong behavioral framework for future investigations into the neural circuits underlying socially modulated innate fear.

      Weaknesses:

      The study exhibits several limitations. The neural mechanism proposed is speculative, as the study provides no causal evidence.

      Establishing causal evidence for neural mechanisms is beyond the scope of the current behavioral study. We highlight this as an important direction for future work.

    1. Reviewer #2 (Public review):

      Summary:

      Tan et al. examined how multivoxel patterns shift in time windows surrounding event boundaries caused by both prediction errors and prediction uncertainty. They observed that some regions of the brain show earlier pattern shifts than others, followed by periods of increased stability. The authors combine their recent computational model to estimate event boundaries that are based on prediction error vs. uncertainty and use this to examine the moment-to-moment dynamics of pattern changes. I believe this is a meaningful contribution that will be of interest to memory, attention, and complex cognition research.

      Strengths:

      The authors have shown exceptional transparency in terms of sharing their data, code, and stimuli which is beneficial to the field for future examinations and to the reproduction of findings. The manuscript is well written with clear figures. The study starts from a strong theoretical background to understand how the brain represents events and have used a well-curated set of stimuli. Overall, the authors extend the event segmentation theory beyond prediction error to include prediction uncertainty which is an important theoretical shift that has implications in episodic memory encoding, use of semantic and schematic knowledge and to attentional processing.

      Weaknesses:

      (1) I am not fully satisfied with the author's explanation of pattern shifts occurring 11.9s prior to event boundaries. The average length of time for an event was 21.4 seconds. The window around the identified event boundaries was 20 seconds on either side. The earliest identified pattern shift peaks occur at 11.9s prior to the actual event boundary. This would mean on average, a pattern shift is occurring approximately at the midway point of the event (11.9s prior to a boundary of a 21.4s event is approx. the middle of an event). The authors offer up an explanation in which top down regions signal an update that propagates to lower order regions closer to the boundary. To make this interpretation concrete, they added an example: "in a narrative where a goal is reached midway-for instance, a mystery solved before the story formally ends-higher-order regions may update the event representation at that point, and this updated model then cascades down to shape processing in lower-level regions". This might make sense in a one-off case of irregular storytelling, but it is odd to think this would generalize. If an event is occurring and a given collection of regions represent that event, it doesn't follow the accepted convention of multivariate representational analysis that that set of regions would undergo such a large shift in patterns in the middle of an event. The stabilization of these patterns taking so long is also odd to me. I suspect some of these findings may be due to the stimuli used in this experiment and I am not confident this would generalize and invite the authors to disagree and explain. In the case of the exercise routine video, I try to imagine going from the push-up event to the jumping jack event. The actor stops doing pushups, stands up, and moves minimally for 16 seconds (these lulls are not uncommon). At that point they start doing jumping jacks. It is immediately evident from that moment on that jumping jacks will be the kind of event you are perceiving which may explain the long delay in event pattern stabilisation. Then about 11.9s prior to the end of the event, when the person is still performing jumping jacks (at this point they have been performing jumping jacks for 6 seconds), I would expect the brain to still be expecting this " jumping jacks event". For some reason at this point multivariate patterns in higher order regions shift. I do not understand what kind of top down processing is happening here and the reviewers need to be more concrete in their explanation because as of right now it is ill-defined. I also recognize that being specific to jumping jacks is maybe unfair, but this would apply to the push-ups, granola bar eating, or table cleaning events in the same manner. I suspect one possibility is that the participants realize that the stereotyped action of jumping jacks is going to continue and, thus, mindwander to other thoughts while waiting for novel, informative information to be presented. This explanation would challenge the more active top down processing assumed by the authors.

      I had provided a set of concerns to the authors that were not part of the public review and were not addressed. I was unaware of the exact format of the eLife approach, but I think they are worth open discussion so I am adding them here for consideration. Apologies for any confusion.

      (2) Why did the authors not examine event boundary activity magnitude differences from the uncertainty vs error boundaries? I see that the authors have provided the data on the openneuro. However, it seems like the difference in activity maps would not only provide extra contextualization of the findings, but also be fairly trivial. Just by eye-balling the plots, it appears as though there may be activity differences in the mPFC occurring shortly after a boundary between the two. Given this regions role in prediction error and schema, it would be important to understand whether this difference is merely due to thresholding effects or is statistically meaningful.

      (3) Further, the authors omitted all subcortical regions some of which would be especially interesting such as the hippocampus, basal ganglia, ventral tegmental area. These regions have a rich and deep background in event boundary activity, and prediction error. Univariate effects in these regions may provide interesting effects that might contextualize some of the pattern shifts in the cortex.

      (3) I see that field maps were collected, but the fmriprep methods state that susceptibility distortion correction was not performed. Is there a reason to omit this?

      (4) How many events were present in the stimuli?

    2. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      This paper investigates the control signals that drive event model updating during continuous experience. The authors apply predictions from previously published computational models to fMRI data acquired while participants watched naturalistic video stimuli. They first examine the time course of BOLD pattern changes around human-annotated event boundaries, revealing pattern changes preceding the boundary in anterior temporal and then parietal regions, followed by pattern stabilization across many regions. The authors then analyze time courses around boundaries generated by a model that updates event models based on prediction error and another that uses prediction uncertainty. These analyses reveal overlapping but partially distinct dynamics for each boundary type, suggesting that both signals may contribute to event segmentation processes in the brain.

      Strengths:

      (1) The question addressed by this paper is of high interest to researchers working on event cognition, perception, and memory. There has been considerable debate about what kinds of signals drive event boundaries, and this paper directly engages with that debate by comparing prediction error and prediction uncertainty as candidate control signals.

      (2) The authors use computational models that explain significant variance in human boundary judgments, and they report the variance explained clearly in the paper.

      (3) The authors' method of using computational models to generate predictions about when event model updating should occur is a valuable mechanistic alternative to methods like HMM or GSBS, which are data-driven.

      (4) The paper utilizes an analysis framework that characterizes how multivariate BOLD pattern dissimilarity evolves before and after boundaries. This approach offers an advance over previous work focused on just the boundary or post-boundary points.

      We appreciate this reviewer’s recognition of the significance of this research problem, and of the value of the approach taken by this paper.

      Weaknesses:

      (1) While the paper raises the possibility that both prediction error and uncertainty could serve as control signals, it does not offer a strong theoretical rationale for why the brain would benefit from multiple (empirically correlated) signals. What distinct advantages do these signals provide? This may be discussed in the authors' prior modeling work, but is left too implicit in this paper.

      We added a brief discussion in the introduction highlighting the complementary advantages of prediction error and prediction uncertainty, and cited prior theoretical work that elaborates on this point. Specifically, we now note that prediction error can act as a reactive trigger, signaling when the current event model is no longer sufficient (Zacks et al., 2007). In contrast, prediction uncertainty is framed as proactive, allowing the system to prepare for upcoming changes even before they occur (Baldwin & Kosie, 2021; Kuperberg, 2021). Together, this makes clearer why these two signals could each provide complementary benefits for effective event model updating.

      "One potential signal to control event model updating is prediction error—the difference between the system’s prediction and what actually occurs. A transient increase in prediction error is a valid indicator that the current model no longer adequately captures the current activity. Event Segmentation Theory (EST; Zacks et al., 2007) proposes that event models are updated when prediction error increases beyond a threshold, indicating that the current model no longer adequately captures ongoing activity. A related but computationally distinct proposal is that prediction uncertainty (also termed "unpredictability") can serve as a control signal (Baldwin & Kosie, 2021). The advantage of relying on prediction uncertainty to detect event boundaries is that it is inherently proactive: the cognitive system can start looking for cues about what might come next before the next event starts (Baldwin & Kosie, 2021; Kuperberg, 2021). "

      (2) Boundaries derived from prediction error and uncertainty are correlated for the naturalistic stimuli. This raises some concerns about how well their distinct contributions to brain activity can be separated. The authors should consider whether they can leverage timepoints where the models make different predictions to make a stronger case for brain regions that are responsive to one vs the other.

      We addressed this concern by adding an analysis that explicitly tests the unique contributions of prediction error– and prediction uncertainty–driven boundaries to neural pattern shifts. In the revised manuscript, we describe how we fit a combined FIR model that included both boundary types as predictors and then compared this model against versions with only one predictor. This allowed us to identify the variance explained by each boundary type over and above the other. The results revealed two partially dissociable sets of brain regions sensitive to error- versus uncertainty-driven boundaries (see Figure S1), strengthening our argument that these signals make distinct contributions.

      "To account for the correlation between uncertainty-driven boundaries and error-driven boundaries, we also fitted a FIR model that predicted pattern dissimilarity from both types of boundaries (combined FIR) for each parcel. Then, we performed two likelihood ratio tests: combined FIR to error FIR, which measures the unique contribution of uncertainty boundaries to pattern dissimilarity, and combined FIR to uncertainty FIR, which measures the unique contribution of error boundaries to pattern dissimilarity. The analysis also revealed two dissociable sets of brain regions associated with each boundary type (see Figure S1)."

      (3) The authors refer to a baseline measure of pattern dissimilarity, which their dissimilarity measure of interest is relative to, but it's not clear how this baseline is computed. Since the interpretation of increases or decreases in dissimilarity depends on this reference point, more clarity is needed.

      We clarified how the FIR baseline is estimated in the methods section. Specifically, we now explain that the FIR coefficients should be interpreted relative to a reference level, which reflects the expected dissimilarity when timepoints are far from an event boundary. This makes it clear what serves as the comparison point for observed increases or decreases in dissimilarity.

      "The coefficients from the FIR model indicate changes relative to baseline, which can be conceptualized as the expected value when far from event boundaries."

      (4) The authors report an average event length of ~20 seconds, and they also look at +20 and -20 seconds around each event boundary. Thus, it's unclear how often pre- and post-boundary timepoints are part of adjacent events. This complicates the interpretations of the reported time courses.

      This is related to reviewer's 2 comment, and it will be addressed below.

      (5) The authors describe a sequence of neural pattern shifts during each type of boundary, but offer little setup of what pattern shifts we might expect or why. They also offer little discussion of what cognitive processes these shifts might reflect. The paper would benefit from a more thorough setup for the neural results and a discussion that comments on how the results inform our understanding of what these brain regions contribute to event models.

      We thank the reviewer for this advice on how better to set the context for the different potential outcomes of the study. We expanded both the introduction and discussion to better set up expectations for neural pattern shifts and to interpret what these shifts may reflect. In the introduction, we now describe prior findings showing that sensory regions tend to update more quickly than higher-order multimodal regions (Baldassano et al., 2017; Geerligs et al., 2021, 2022), and we highlight that it remains unclear whether higher-order updates precede or follow those in lower-order regions. We also note that our analytic approach is well-suited to address this open question. In the discussion, we then interpret our results in light of this framework. Specifically, we describe how we observed early shifts in higher-order areas such as anterior temporal and prefrontal cortex, followed by shifts in parietal and dorsal attention regions closer to event boundaries. This pattern runs counter to the traditional bottom-up temporal hierarchy view and instead supports a model of top-down updating, where high-level representations are updated first and subsequently influence lower-level processing (Friston, 2005; Kuperberg, 2021). To make this interpretation concrete, we added an example: in a narrative where a goal is reached midway—for instance, a mystery solved before the story formally ends—higher-order regions may update the event representation at that point, and this updated model then cascades down to shape processing in lower-level regions. Finally, we note that the widespread stabilization of neural patterns after boundaries may signal the establishment of a new event model.

      Excerpt from Introduction:

      “More recently, multivariate approaches have provided insights into neural representations during event segmentation. One prominent approach uses hidden Markov models (HMMs) to detect moments when the brain switches from one stable activity pattern to another (Baldassano et al., 2017) during movie viewing; these periods of relative stability were referred to as "neural states" to distinguish them from subjectively perceived events. Sensory regions like visual and auditory cortex showed faster transitions between neural states. Multi-modal regions like the posterior medial cortex, angular gyrus, and intraparietal sulcus showed slower neural state shifts, and these shifts aligned with subjectively reported event boundaries. Geerligs et al. (2021, 2022) employed a different analytical approach called Greedy State Boundary Search (GSBS) to identify neural state boundaries. Their findings echoed the HMM results: short-lived neural states were observed in early sensory areas (visual, auditory, and somatosensory cortex), while longer-lasting states appeared in multi-modal regions, including the angular gyrus, posterior middle/inferior temporal cortex, precuneus, anterior temporal pole, and anterior insula. Particularly prolonged states were found in higher-order regions such as lateral and medial prefrontal cortex.

      The previous evidence about evoked responses at event boundaries indicates that these are dynamic phenomena evolving over many seconds, with different brain areas showing different dynamics (Ben-Yakov & Henson, 2018; Burunat et al., 2024; Kurby & Zacks, 2018; Speer et al., 2007; Zacks, 2010). Less is known about the dynamics of pattern shifts at event boundaries (e.g. whether shifts observed in higher-order regions precedes or follow shifts observed in lower-level regions), because the HMM and GSBS analysis methods do not directly provide moment-by-moment measures of pattern shifts. Both the spatial and temporal aspects of evoked responses and pattern shifts at event boundaries have the potential to provide evidence about two potential control processes (error-driven and uncertainty-driven) for event model updating.”

      Excerpt from Discussion:

      “We first characterized the neural signatures of human event segmentation by examining both univariate activity changes and multivariate pattern changes around subjectively identified event boundaries. Using multivariate pattern dissimilarity, we observed a structured progression of neural reconfiguration surrounding human-identified event boundaries. The largest pattern shifts were observed near event boundaries (~4.5s before) in dorsal attention and parietal regions; these correspond with regions identified by Geerligs et. al as shifting their patterns on a fast to intermediate timescale (2022). We also observed smaller pattern shifts roughly 12 seconds prior to event boundaries in higher-order regions within anterior temporal cortex and prefrontal cortex, and these are slow-changing regions identified by Geerligs et. al (2022). This is puzzling. One prevalent proposal, based on the idea of a cortical hierarchy of increasing temporal receptive windows (TRWs), suggests that higher-order regions should update representations after lower-order regions do (Chang et al., 2021). In this view, areas with shorter TRWs (e.g., word-level processors) pass information upward, where it is integrated into progressively larger narrative units (phrases, sentences, events). This proposal predicts neural shifts in higher-order regions to follow those in lower-order regions. By contrast, our findings indicate the opposite sequence. Our findings suggest that the brain might engage in top-down event representation updating, with changes in coarser-grain representations propagating downward to influence finer-grain representations. (Friston, 2005; Kuperberg, 2021). For example, in a narrative where the main goal is achieved midway—such as a detective solving a mystery before the story formally ends—higher-order regions might update the overarching event representation at that point, and this updated model could then cascade down to reconfigure how lower-level regions process the remaining sensory and contextual details. In the period after a boundary (around +12 seconds), we found widespread stabilization of neural patterns across the brain, suggesting the establishment of a new event model. Future work could focus on understanding the mechanisms behind the temporal progression of neural pattern changes around event boundaries.”

      Reviewer #2 (Public review):

      Summary:

      Tan et al. examined how multivoxel patterns shift in time windows surrounding event boundaries caused by both prediction errors and prediction uncertainty. They observed that some regions of the brain show earlier pattern shifts than others, followed by periods of increased stability. The authors combine their recent computational model to estimate event boundaries that are based on prediction error vs. uncertainty and use this to examine the moment-to-moment dynamics of pattern changes. I believe this is a meaningful contribution that will be of interest to memory, attention, and complex cognition research.

      Strengths:

      The authors have shown exceptional transparency in terms of sharing their data, code, and stimuli, which is beneficial to the field for future examinations and to the reproduction of findings. The manuscript is well written with clear figures. The study starts from a strong theoretical background to understand how the brain represents events and has used a well-curated set of stimuli. Overall, the authors extend the event segmentation theory beyond prediction error to include prediction uncertainty, which is an important theoretical shift that has implications in episodic memory encoding, the use of semantic and schematic knowledge, and attentional processing.

      We thank the reader for their support for our use of open science practices, and for their appreciation of the importance of incorporating prediction uncertainty into models of event comprehension.

      Weaknesses:

      The data presented is limited to the cortex, and subcortical contributions would be interesting to explore. Further, the temporal window around event boundaries of 20 seconds is approximately the length of the average event (21.4 seconds), and many of the observed pattern effects occur relatively distal from event boundaries themselves, which makes the link to the theoretical background challenging. Finally, while multivariate pattern shifts were examined at event boundaries related to either prediction error or prediction uncertainty, there was no exploration of univariate activity differences between these two different types of boundaries, which would be valuable.

      The fact that we observed neural pattern shifts well before boundaries was indeed unexpected, and we now offer a more extensive interpretation in the discussion section. Specifically, we added text noting that shifts emerged in higher-order anterior temporal and prefrontal regions roughly 12 seconds before boundaries, whereas shifts occurred in lower-level dorsal attention and parietal regions closer to boundaries. This sequence contrasts with the traditional bottom-up temporal hierarchy view and instead suggests a possible top-down updating mechanism, in which higher-order representations reorganize first and propagate changes to lower-level areas (Friston, 2005; Kuperberg, 2021). (See excerpt for Reviewer 1’s comment #5.)

      With respect to univariate activity, we did not find strong differences between error-driven and uncertainty-driven boundaries. This makes the multivariate analyses particularly informative for detecting differences in neural pattern dynamics. To support further exploration, we have also shared the temporal progression of univariate BOLD responses on OpenNeuro (BOLD_coefficients_brain_animation_pe_SEM_bold.html and BOLD_coefficients_brain_animation_uncertainty_SEM_bold.html in the derivatives/figures/brain_maps_and_timecourses/ directory; https://doi.org/10.18112/openneuro.ds005551.v1.0.4) for interested researchers.

      Reviewer #3 (Public review):

      Summary:

      The aim of this study was to investigate the temporal progression of the neural response to event boundaries in relation to uncertainty and error. Specifically, the authors asked (1) how neural activity changes before and after event boundaries, (2) if uncertainty and error both contribute to explaining the occurrence of event boundaries, and (3) if uncertainty and error have unique contributions to explaining the temporal progression of neural activity.

      Strengths:

      One strength of this paper is that it builds on an already validated computational model. It relies on straightforward and interpretable analysis techniques to answer the main question, with a smart combination of pattern similarity metrics and FIR. This combination of methods may also be an inspiration to other researchers in the field working on similar questions. The paper is well written and easy to follow. The paper convincingly shows that (1) there is a temporal progression of neural activity change before and after an event boundary, and (2) event boundaries are predicted best by the combination of uncertainty and error signals.

      We thank the reviewer for their thoughtful and supportive comments, particularly regarding the use of the computational model and the analysis approaches.

      Weaknesses:

      (1) The current analysis of the neural data does not convincingly show that uncertainty and prediction error both contribute to the neural responses. As both terms are modelled in separate FIR models, it may be that the responses we see for both are mostly driven by shared variance. Given that the correlation between the two is very high (r=0.49), this seems likely. The strong overlap in the neural responses elicited by both, as shown in Figure 6, also suggests that what we see may mainly be shared variance. To improve the interpretability of these effects, I think it is essential to know whether uncertainty and error explain similar or unique parts of the variance. The observation that they have distinct temporal profiles is suggestive of some dissociation,but not as convincing as adding them both to a single model.

      We appreciate this point. It is closely related to Reviewer 1's comment 2; please refer to our response above.

      (2) The results for uncertainty and error show that uncertainty has strong effects before or at boundary onset, while error is related to more stabilization after boundary onset. This makes me wonder about the temporal contribution of each of these. Could it be the case that increases in uncertainty are early indicators of a boundary, and errors tend to occur later?

      We also share the intuition that increases in uncertainty are early indicators of a boundary, and errors tend to occur later. If that is the case, we would expect some lags between prediction uncertainty and prediction error. We examined lagged correlation between prediction uncertainty and prediction error, and the optimal lag is 0 for both uncertainty-driven and error-driven models. This indicates that when prediction uncertainty rises, prediction error also simultaneously rises.

      Author response image 1.

      (3) Given that there is a 24-second period during which the neural responses are shaped by event boundaries, it would be important to know more about the average distance between boundaries and the variability of this distance. This will help establish whether the FIR model can properly capture a return to baseline.

      We have added details about the distribution of event lengths. Specifically, we now report that the mean length of subjectively identified events was 21.4 seconds (median 22.2 s, SD 16.1 s). For model-derived boundaries, the average event lengths were 28.96 seconds for the uncertainty-driven model and 24.7 seconds for the error-driven model.

      " For each activity, a separate group of 30 participants had previously segmented each movie to identify fine-grained event boundaries (Bezdek et al., 2022). The mean event length was 21.4 s (median 22.2 s, SD 16.1 s). Mean event lengths for uncertainty-driven model and error-driven model were 28.96s, and 24.7s, respectively (Nguyen et al., 2024)."

      (4) Given that there is an early onset and long-lasting response of the brain to these event boundaries, I wonder what causes this. Is it the case that uncertainty or errors already increase at 12 seconds before the boundaries occur? Or if there are other makers in the movie that the brain can use to foreshadow an event boundary? And if uncertainty or errors do increase already 12 seconds before an event boundary, do you see a similar neural response at moments with similar levels of error or uncertainty, which are not followed by a boundary? This would reveal whether the neural activity patterns are specific to event boundaries or whether these are general markers of error and uncertainty.

      We appreciate this point; it is similar to reviewer 2’s comment 2. Please see our response to that comment above.

      (5) It is known that different brain regions have different delays of their BOLD response. Could these delays contribute to the propagation of the neural activity across different brain areas in this study?

      Our analyses use ±20 s FIR windows, and the key effects we report include shifts ~12s before boundaries in higher-order cortex and ~4.5s pre-boundary in dorsal attention/parietal areas. Given the literature above, region-dependent BOLD delays are much smaller (~1–2s) than the temporal structure we observe (Taylor et al., 2018), making it unlikely that HRF lag alone explains our multi-second, region-specific progression.

      (6) In the FIR plots, timepoints -12, 0, and 12 are shown. These long intervals preclude an understanding of the full temporal progression of these effects.

      For page length purposes, we did not include all timepoints. We uploaded a brain animation of all timepoints and coefficients for each parcel in Openneuro (PATTERN_coefficients_brain_animation_human_fine_pattern.html and PATTERN_coefficients_lines_human_fine.html in the derivatives/figures/brain_maps_and_timecourses/ directory; https://doi.org/10.18112/openneuro.ds005551.v1.0.4) for interested researchers.

      References

      Taylor, A. J., Kim, J. H., & Ress, D. (2018). Characterization of the hemodynamic response function across the majority of human cerebral cortex. NeuroImage, 173, 322–331. https://doi.org/10.1016/j.neuroimage.2018.02.061

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript offers a careful and technically impressive dissection of how subpopulations within the subthalamic nucleus support reward‑biased decision‑making. The authors recorded from STN neurons in monkeys performing an asymmetric‑reward version of a visual motion discrimination task and combined single‑unit analyses, regression modeling, and drift‑diffusion framework fitting to reveal functionally distinct clusters of neurons. Each subpopulation demonstrated unique relationships to decision variables - such as the evidence‑accumulation rate, decision bound, and non‑decision processes - as well as to post‑decision evaluative signals like choice accuracy and reward expectation. Together, these findings expand our understanding of the computational diversity of STN activity during complex, multi‑attribute choices.

      Strengths:

      (1) The use of an asymmetric‑reward paradigm enables a clean separation between perceptual and reward influences, making it possible to identify how STN neurons blend these different sources of information.

      (2) The dataset is extensive and well‑controlled, with careful alignment between behavioral and neural analyses.

      (3) Relating neuronal cluster activity to drift‑diffusion model parameters provides an interpretable computational link between neural population signals and observed behavior.

      (4) The clustering analyses, validated across multiple parameters and distance metrics, reveal robust functional subgroups within STN. The differentiation of clusters with respect to both evidence and reward coding is an important advance over treating the STN as a unitary structure.

      (5) By linking neural activity to predicted choice accuracy and reward expectation, the study extends the discussion of the STN beyond decision formation to include outcome monitoring and post‑decision evaluation.

      Weaknesses:

      (1) The inferred relationships between neural clusters and specific drift‑diffusion parameters (e.g., bound height, scaling factor, non‑decision time) are intriguing but inherently correlational. The authors should clarify that these associations do not necessarily establish distinct computational mechanisms.

      (2) While the k‑means approach is well described, it remains somewhat heuristic. Including additional cross‑validation (e.g., cluster reproducibility across monkeys or sessions) would strengthen confidence in the four‑cluster interpretation.

      (3) The functional dissociations across clusters are clearly described, but how these subgroups interact within the STN or through downstream basal‑ganglia circuits remains speculative.

      (4) A natural next step would be to construct a generative multi‑cluster model of STN activity, in which each cluster is treated as a computational node (e.g., evidence integrator, bound controller, urgency or evaluative signal).

      (5) Such a low‑dimensional, coupled model could reproduce the observed diversity of firing patterns and predict how interactions among clusters shape decision variables and behavior.

      (6) Population‑level modeling of this kind would move the interpretation beyond correlational mapping and serve as an intermediate framework between single‑unit analysis and in‑vivo perturbation.

      (7) Causal inference gap - Without perturbation data, it is difficult to determine whether the identified neural modulations are necessary or sufficient for the observed behavioral effects. A brief discussion of this limitation - and how future causal manipulations could test these cluster functions - would be valuable.

    2. Reviewer #3 (Public review):

      Summary:

      In this study, the authors investigate single neuron activity in the subthalamic nucleus (STN) of two monkeys performing a perceptual decision-making task in which both perceptual evidence and reward were manipulated. They find rich representations of decision variables (such as choice, perceptual evidence and reward) in neural activity, and following prior work, cluster a subset of these neurons into subpopulations with varying activity profiles. Further, they relate the activity of neurons within these clusters to parameters of drift diffusion models (DDMs) fit to animal behaviour on trial subsets by neural firing rates, finding heterogeneous and temporally varying relationships between different clusters and DDM parameters, suggesting that STN neurons may play multiple roles in decision formation and evaluation.

      Strengths:

      The behavioural task used by the authors is rich and affords disambiguation between decision variables such as perceptual evidence, value and choice, by independently manipulating stimulus strength and reward size. Both their monkeys show good performance on the task, and their population of ~150 neurons across monkeys reveals a rich repertoire of decision-related activity in single neurons, with individual neurons showing strong tuning to choice, stimulus strength and reward bias. There is little doubt that neurons in the STN are tuned to several decision variables and show heterogeneous tuning profiles.

      Weaknesses:

      The primary weakness of the paper lies in the claim that STN contains multiple sub-populations with distinct involvements in decision making, which is inadequately supported by the paper's methods and analyses.

      First, while it is clear that the ~150 recorded neurons across 2 monkeys (91, 59 respectively) display substantial heterogeneity in their activity profiles across time and across stimulus/reward conditions, the claim of sub-populations largely rests on clustering a *subset of less than half the population - 66 neurons (48, 15 respectively) - chosen manually by visual inspection*. The full population seems to contain far more decision-modulated neurons, whose response profiles seem to interpolate between clusters. Moreover, it is unclear if the 4 clusters hold for each of the 2 monkeys, and the choice of 4-5 clusters does not seem well supported by metrics such as silhouette score, etc, that peak at 3 (1 or 2 were not attempted). From the data, it is easier to draw the conclusion that the STN population contains neurons with heterogeneous response profiles that smoothly vary in their tuning to different decision variables, rather than distinct sub-populations.

      Second, assuming the existence of sub-populations, it is unclear how their time- and condition-varying relationship with DDM parameters is to be interpreted. These relationships are inferred by splitting trials based on individual neurons' firing rates in different task epochs and reward contexts, and regressing onto the parameters of separate DDMs fit to those subsets of trials. The result is that different sub-populations show heterogeneous relationships to different DDM parameters over time - a result that, while interesting, leaves the computational involvement of these sub-populations/implementation of the decision process unclear.

      Outlook:

      This is a paper with a rich dataset of neural activity in the STN in a rich perceptual decision-making task, and convincing evidence of heterogeneity in choice, value and evidence tuning across the STN, suggesting the STN may be involved in several aspects of decision-making. However, the authors' specific claims about sub-populations in the STN, each having distinct relationships to decision processes, are not adequately supported by their analyses.

    1. Reviewer #2 (Public review):

      Summary:

      This study, conducted by Esmaeili and colleagues, investigates the functional connectivity signatures of different auditory, visual, and motor states in 42 ECoG patients. Patients performed three tasks: picture naming, visual word reading, and auditory word repetition. They use an SVM classifier on correlation patterns across electrodes during these tasks, separating speech production from sensory perception, and incorporating baseline silence as another state. They find that it is possible to classify five states (auditory perception, picture viewing, word reading, speech production, and baseline) based on their connectivity patterns alone. Furthermore, they find a sparser set of "discriminative connections" for each state that can be used to predict each of these states. They then relate these connectivity matrices to high-gamma evoked data, and show largely overlapping relationships between the discriminative connections and the active high-gamma electrodes. However, there are still some connectivity nodes that are important in discriminating states, but that do not show high evoked activity, and vice versa. Overall, the study has a large number of patients, and the ability to decode cognitive state is compelling. The main weaknesses of the work are in placing the findings into a wider context for what additional information the connectivity analysis provides about brain processing of speech, since, as it stands, the analysis mostly reidentifies areas already known to be important for speaking, listening, naming, and visual processing.

      Strengths:

      (1) The authors were able to assess their connectivity analysis on a large cohort of patients with wide coverage across speech and language areas.

      (2) The use of controlled tasks for picture naming, visual word reading, and auditory word repetition allows for parcellating specific components of stimulus perception and speech production.

      (3) The authors chose not to restrict their connectivity analysis to previously identified high amplitude responses, which allowed them to find regions that are discriminative between different states in their speech tasks, but not necessarily highly active.

      Weaknesses:

      (1) Although the work identifies some clear connectivity between brain areas during speech perception and production, it is not clear whether this approach allows us to learn anything new about brain systems for speech. The areas that are identified have been shown in other studies and are largely unsurprising - the auditory cortex is involved in hearing words, picture naming involves frontal and visual cortical interactions, and overt movements include the speech motor cortex. The temporal pole is a new area that shows up, but (see below) it is important to show that this region is not affected by artifacts. Overall, it would help if the authors could expand upon the novelty of their approach.

      (2) Because the connectivity is derived from single trials, it is possible that some of the sparse connectivity seen in noncanonical areas is due to a common artifact across channels. The authors do employ a common average reference, which should help to reduce common-mode noise across all channels, but not smaller subsets. Could the authors include more information to show that this is not the case in their dataset? For example, the temporal pole electrodes show strong functional connectivity, but these areas can tend to include more EMG artifact or ocular artifact. Showing single-trial traces for some of these example pairs of electrodes and their FC measures could help in interpreting how robust the findings are.

      (3) The connectivity matrices are defined by taking the correlation between all pairs of electrodes across 500-ms epochs for each cognitive state, presumably for electrodes that are time-aligned. However, it is likely that different areas will interact with different time delays - for example, activity in one area may lead to activity in another. It might be helpful to include some time lags between different brain areas if the authors are interested in dynamics between areas that are not simultaneous.

      (4) In Figure 3, the baseline is most commonly confused with other categories (most notably, speech production, 22% of the time). Is there any intuition for why this might be? Could some of this confusion be due to task-irrelevant speech occurring during the baseline / have the authors verified that all pre-stimulus time periods were indeed silent?

      (5) How similar are discriminative connections across participants? Do they tend to reflect the same sparse anatomical connections? It is not clear how similar the results are across participants.

      (6) The results in Figure 5F are interesting and show that frontal electrodes are often highly functionally connected, but have low evoked activity. What do the authors believe this might reflect? What are these low-evoked activity electrodes potentially doing? Some (even speculative) mention might be helpful.

      (7) One comparison that seems to be missing, if the authors would like to claim the utility of functional connectivity over evoked measures, is to directly compare a classifier based on the high gamma activity patterns alone, rather than the pairwise connectivity. Does the FC metric outperform simply using evoked activity?

    1. Analyse Approfondie du Conflit : Stratégies, Bénéfices et Psychologie

      Résumé Exécutif

      Ce document synthétise une analyse approfondie de la nature des disputes, s'éloignant de la perception traditionnelle du conflit comme étant purement négatif.

      La thèse centrale est que le conflit, loin d'être un obstacle au bien-être, est un phénomène naturel et un moteur essentiel de développement personnel, relationnel et sociétal.

      Son caractère constructif ou destructeur dépend entièrement de la manière dont il est géré.

      Les points clés révèlent que la maîtrise du conflit repose sur la régulation émotionnelle, l'application de techniques de communication spécifiques et une volonté de remettre en question ses propres certitudes.

      Les bénéfices d'une dispute bien menée sont multiples : elle permet * d'affirmer ses valeurs, de poser des limites, * de renforcer les liens en créant un sentiment d'appartenance et de confiance, * et même de stimuler l'excellence en milieu professionnel.

      La gestion efficace des émotions, notamment le stress et la colère, est fondamentale.

      Des stratégies comme la reconnaissance de ses émotions, le recadrage de la montée d'adrénaline en énergie positive et des techniques d'ancrage physique sont des outils puissants.

      L'analyse met également en lumière l'influence déterminante des expériences de l'enfance sur notre rapport adulte au conflit, soulignant que des schémas de communication peuvent être consciemment modifiés.

      Enfin, des méthodes concrètes pour désamorcer les tensions et dialoguer, même avec des interlocuteurs aux opinions radicalement opposées, sont présentées, insistant sur l'importance de présumer la bonne foi et de rechercher des solutions communes.

      --------------------------------------------------------------------------------

      1. La Perception Évolutive du Conflit

      La compréhension psychologique du conflit a radicalement changé.

      Si elle a longtemps prôné l'évitement des disputes, considérées comme nuisibles au bien-être, la perspective moderne est tout autre.

      Vision Traditionnelle vs. Moderne :

      Anciennement : La psychologie défendait l'idée qu'il fallait "éviter les conflits autant que possible".

      Le bonheur était assimilé à l'absence de conflit.   

      Aujourd'hui : Le conflit est considéré comme "tout à fait naturel".

      Ce qui est déterminant n'est pas le conflit en soi, mais "la façon dont on se dispute".

      Le Conflit comme Moteur Social :

      ◦ Le sociologue Georg Simmel est cité pour affirmer que "le conflit est ce qui fait bouger une société".  

      ◦ L'harmonie et le consensus total mènent à la stagnation : "quand on vient en harmonie et qu'on est tous du même avis, il ne se passe rien, tout se met à l'arrêt".  

      ◦ Éviter systématiquement la confrontation ne résout pas les problèmes sous-jacents, qui continuent de "s'aggraver jusqu'à ce que plus rien ne puisse sauver le couple".

      2. Les Multiples Bénéfices d'un Conflit Constructif

      Lorsqu'il est abordé de manière saine, le conflit devient une source de force et de croissance à plusieurs niveaux.

      Développement Personnel :

      Affirmation de soi : Une dispute est une occasion "d'affirmer ses propres valeurs, poser ses limites et savoir qui on est".

      Même sans solution, elle permet de "s'exprimer" et de "formuler son opinion à haute voix".  

      Connaissance de soi : La confrontation peut mener à une meilleure connaissance de soi-même et des autres.

      Andj, musicien de punk hardcore, témoigne qu'une "confrontation violente avec ses parents" lui a permis de mieux se connaître et de les voir sous un autre jour, améliorant considérablement leurs relations.

      Renforcement des Relations :

      Création de liens : Le conflit peut paradoxalement créer un "sentiment d'appartenance" lorsqu'on réalise qu'on est finalement d'accord avec l'autre sur certains points.   

      Signe de confiance : Une première dispute avec une nouvelle connaissance peut "briser la glace".

      Andj déclare : "je ne me dispute qu'avec les personnes qui comptent pour moi [...] si j'ai une confrontation avec quelqu'un, ça veut dire que je tiens à cette relation".  

      Évolution commune : Le plus grand bonheur réside dans le fait que l'autre "n'a pas pris la fuite" et a surmonté l'épreuve ensemble, ce qui "nous a fait évoluer ensemble".

      Efficacité Professionnelle :

      ◦ En management, des équipes "un peu trop harmonieuses" où la critique constructive est absente n'atteignent qu'un "résultat moyen".  

      ◦ Pour "atteindre l'excellence, on a besoin du conflit".

      3. La Psychologie du Conflit : Maîtriser le "Tsunami Émotionnel"

      La clé d'une dispute constructive réside dans la capacité à gérer le flux d'émotions intenses qu'elle génère.

      La Nature des Émotions en Conflit :

      ◦ La dispute est décrite comme un "grand tsunami émotionnel".  

      ◦ Les émotions proviennent de trois sources :      

      1. L'objet direct de la dispute.     

      2. Les "émotions coptées" : stress ou frustration accumulés durant la journée.    

      3. Les émotions de la petite enfance.

      Stratégies de Régulation Émotionnelle :

      Reconnaissance et acceptation : La première étape est de reconnaître ses émotions ("oui je suis en colère et c'est légitime").

      Le simple fait d'accepter et d'accueillir mentalement le stress "le réduit déjà de 30 %".  

      Recadrage cognitif : Il est possible de "requalifier ce sentiment" et de voir la montée d'adrénaline comme un "surcroix d'énergie" positif.

      Cette technique, enseignée en négociation, peut réduire la tension de 40 % supplémentaires.  

      Ancrage physique : En cas de confusion mentale ou émotionnelle, il est important de "sentir le sol sous mes pieds" pour retrouver un ancrage.  

      La pause stratégique : Proposer de "quitter la pièce" lorsque la discussion tourne en rond est "très efficace".

      En 3 minutes, "le stress disparaît, la colère s'estompe".  

      L'expression physique : Le cri, pratiqué par Andj, est présenté comme "une forme puissante d'expression de soi et un outil pour réguler ses émotions".

      Le Rôle Positif de la Colère :

      ◦ La colère n'est pas systématiquement négative. Hasnain Kazim, écrivain, déclare : "j'aime la colère, je trouve qu'elle a quelque chose d'extrêmement direct.

      Elle dit clairement : ça me va, ça ça ne me convient pas".  

      ◦ Il la préfère à l'agressivité passive : "ce que je trouve horrible, c'est quand tu vois bien que tu as vexé une personne [...] et qu'elle ne dit rien".

      4. Techniques Pratiques pour une Communication Efficace en Situation de Conflit

      Des outils rhétoriques et des approches spécifiques peuvent transformer une querelle en un dialogue productif.

      | Technique | Description | Exemple / Citation | | --- | --- | --- | | Éviter les Généralisations | Les mots comme "jamais" ou "toujours" enferment l'autre dans une position inconfortable et ferment le dialogue. Il faut "débarrasser le dialogue de ces mots". | "Il est toujours de mauvaise humeur." | | Utiliser le "Je" | Remplacer le "tu accusateur" par des formulations commençant par "je" pour désamorcer l'agressivité et prendre la responsabilité de sa propre perception. | Au lieu de "Tu ne m'as pas compris", dire "Je me suis mal exprimé". | | Formuler son Point de Vue | Argumenter en commençant par "de mon point de vue" pour éviter de présenter son opinion comme une vérité absolue et agressive. | "De mon point de vue, si je dis à l'autre c'est toi qui est stressé, ça ne va faire qu'attiser le débat." | | Poser des Questions Ouvertes | Utiliser des questions qui commencent par "pourquoi", "comment" ou "qu'est-ce que" pour encourager une réponse développée et maintenir la discussion ouverte. | "Qu'est-ce que tu veux dire exactement ?" | | Utiliser les Questions Fermées | Poser des questions auxquelles on ne peut répondre que par "oui", "non" ou "peut-être" pour "fixer quelque chose qui n'était pas clair". | "Ça, ça te dérange ? - Oui." | | Désamorcer les Attaques Déloyales | Identifier les techniques rhétoriques visant à déstabiliser (ex: ad hominem), puis les neutraliser en recentrant calmement le débat sur le sujet principal. | Attaque : "Ça ne vous dérange pas de passer à la télé avec la même veste qu'avanthière ?" Réponse : "Bon, à part la couleur de la veste \[...\] comment peut-on faire pour avancer sur notre sujet ?" |

      5. Le Conflit Idéologique : Dialoguer avec l'Opposition

      Le journaliste Hasnain Kazim partage son expérience sur la nécessité et la méthode pour engager le dialogue avec des personnes aux opinions radicalement différentes, notamment politiques.

      Principes Fondamentaux :

      Quitter sa zone de confort : Il faut être prêt à "remettre en question ses propres certitudes".  

      Éviter l'étiquetage : Ne pas immédiatement ranger l'autre dans un camp ("droitard", "réac", "gauchiste") pour ne pas le considérer comme un "ennemi" et couper l'échange.  

      Présomption de bonne foi : Partir du principe que "l'autre ne me tend pas de piège" et qu'il est "honnête et sincère".

      Si ses propos semblent contradictoires, au lieu de le juger "stupide", il faut chercher à comprendre "ce qu'il y a derrière".  

      Courtoisie et absence de condescendance : Ne pas arriver en "donneur de leçon" ou en "maître d'école".

      Stratégies d'Engagement :

      L'importance d'initier le dialogue : Le plus important est "déjà de le faire, tout simplement".  

      Se fixer des limites : Il est crucial de savoir se protéger et de "riposter" si nécessaire.

      On n'est pas obligé de "témoigner de l'empathie à tout le monde".  

      Les effets à retardement : Même si un débat semble échouer sur le moment, il peut avoir des "effets rétroactifs".

      L'interlocuteur peut plus tard réfléchir au courage de la démarche, ce qui peut faire évoluer sa pensée.

      6. L'Héritage de l'Enfance et son Impact sur le Conflit

      Notre manière de gérer les disputes à l'âge adulte est profondément façonnée par les modèles et les expériences de notre enfance.

      L'Apprentissage par l'Exemple :

      ◦ La relation à la dispute est "héritière de ce que nous ont montré nos modèles d'identification" (parents, figures d'autorité).  

      ◦ Si un enfant apprend qu'il est aimé lorsqu'il est "passif", "discret" et "d'accord avec tout le monde", il évitera la confrontation à l'âge adulte.

      Exemple d'Andj :

      ◦ Il a grandi dans une famille où les disputes étaient gérées par des cris et parfois des "agressions physiques".   

      ◦ Ses parents "réprimaient leurs propres besoins", générant une "énorme frustration".   

      ◦ Enfant, il essayait de se "rendre invisible".   

      ◦ La musique punk lui a permis de réaliser qu'il n'était pas seul, en entendant d'autres "hurler cette injustice".   

      ◦ Le processus de guérison a nécessité plusieurs années de confrontations directes avec ses parents, où il a pu "verbaliser tout ce qui [l]'avait blessé", ce qui a finalement permis de rétablir une relation saine.

      7. La Résolution : Clore la Dispute de Manière Constructive

      Une bonne dispute doit avoir une bonne conclusion pour que ses effets soient bénéfiques.

      Rituels de Réconciliation : Des rituels comme "la bise de réconciliation" ou une poignée de main sont particulièrement efficaces, notamment avec les enfants, pour clore un conflit.

      Règles Familiales : Établir des règles claires, comme celle de ne pas "emporter la dispute au lit", aide à contenir le conflit et à préserver la relation.

      Savoir Ne Pas Disputer : Parfois, la dispute "n'en vaut pas la peine".

      Lors de retrouvailles familiales, la préservation des liens peut être "plus importante que le sujet qui vient de surgir".

      Le Piège de l'Empathie pour les Spectateurs : Lors d'une dispute de groupe, les spectateurs doivent se méfier de l'empathie, qui peut les transformer en "partie prenante", leur faisant voir le débat d'un seul côté et transformant les autres en "adversaires".

    1. Document d'Information : Les Fonctions Exécutives chez l'Enfant

      Résumé Exécutif

      Ce document synthétise les perspectives de Julie Beaulieu, docteure en psychopédagogie, sur les fonctions exécutives chez l'enfant.

      Considérées comme le « chef d'orchestre » ou la « tour de contrôle » du cerveau, ces habiletés cognitives de haut niveau, situées dans le lobe frontal, sont fondamentales pour la régulation des pensées, des émotions et des comportements.

      Elles permettent à l'individu de s'adapter à son environnement et d'atteindre des objectifs.

      Leur développement s'étend sur une longue période, débutant dès les premiers mois de la vie pour atteindre leur plein potentiel vers l'âge de 25 ans, avec des phases de progression accélérée durant la période préscolaire (3-7 ans) et au début de l'adolescence.

      Si la maturation cérébrale est un facteur essentiel, l'environnement et la diversité des expériences vécues par l'enfant jouent un rôle crucial pour « aiguiser » ces fonctions.

      Quatre composantes principales sont identifiées : la mémoire de travail, l'inhibition, la flexibilité cognitive et la planification.

      Ces fonctions, bien que distinctes, sont interdépendantes et fonctionnent en synergie, à l'image d'une « toile d'araignée ».

      Elles sont la fondation de nombreuses aptitudes et ont un impact direct sur la réussite éducative et le développement global de l'enfant (social, langagier, moteur).

      Le soutien parental est déterminant et s'intègre dans les activités quotidiennes.

      Les stratégies efficaces incluent le jeu, les interactions sociales, le questionnement (notamment le « pourquoi »), l'offre de choix, la stimulation de l'autonomie et de la créativité, ainsi que la mise en place d'un environnement sécurisant et positif.

      L'objectif n'est pas d'entraîner ces fonctions comme un muscle, mais de les mobiliser à travers des expériences ludiques et variées.

      --------------------------------------------------------------------------------

      1. Définition et Nature des Fonctions Exécutives

      Les fonctions exécutives sont définies comme un ensemble de processus cognitifs de haut niveau localisés dans le lobe frontal du cerveau.

      Elles sont essentielles pour la réalisation de tâches complexes et pour l'adaptation de l'individu à son environnement.

      Leur rôle principal est de réguler trois aspects fondamentaux du comportement humain :

      Les pensées : Organiser ses idées, raisonner, réfléchir.

      Les comportements : Contrôler ses actions, initier des tâches, s'ajuster.

      Les émotions : Gérer ses réactions émotionnelles.

      Ces fonctions sont mobilisées en continu dans la vie quotidienne, dès qu'une tâche n'est pas automatique ou routinière et demande de réfléchir, raisonner, anticiper, planifier ou diriger son attention.

      La Métaphore du Chef d'Orchestre

      Pour illustrer leur rôle, Julie Beaulieu utilise deux analogies principales :

      La tour de contrôle : Elles permettent de se contrôler et de diriger ses actions.

      Le chef d'orchestre : C'est la métaphore la plus développée.

      Les fonctions exécutives agissent comme le chef d'orchestre du cerveau, coordonnant l'ensemble des autres habiletés cognitives (les "instruments") pour produire un comportement harmonieux et adapté à un objectif précis.

      Elles dirigent les pensées, les émotions et les comportements pour permettre à l'individu de s'adapter et de répondre efficacement aux exigences de son environnement.

      2. Le Développement des Fonctions Exécutives

      Le développement des fonctions exécutives est un processus long et progressif, influencé par la maturation biologique et les expériences environnementales.

      Chronologie du Développement

      Début : Les composantes des fonctions exécutives commencent à se développer dès les premiers mois de la vie.

      Maturité : Elles atteignent leur plein potentiel au début de l'âge adulte, vers 25 ans.

      Périodes Clés : Deux périodes sont identifiées comme particulièrement propices à une progression importante :

      1. L'âge préscolaire (3 à 6-7 ans).    2. Le début de l'adolescence.

      Il est crucial de noter que le rythme de développement varie d'un enfant à l'autre, en fonction de facteurs individuels comme la maturation de leur propre cerveau.

      Le Rôle Crucial de l'Environnement

      La maturation du cerveau est une composante essentielle, mais l'environnement dans lequel l'enfant évolue est un moteur fondamental du développement.

      L'expérience : C'est principalement à travers les expériences vécues que les enfants mobilisent et développent leurs fonctions exécutives.

      L'aiguisage : Plus les fonctions sont mobilisées, plus elles s'« aiguisent », devenant « sophistiquées et spécialistes ».

      La variété : Proposer des contextes et des expériences variés permet de mobiliser un plus large éventail de fonctions exécutives et de favoriser un développement plus complet.

      Le soutien offert par l'entourage (parents, éducateurs) est donc primordial.

      3. Contextes Favorables au Développement

      Certains contextes et situations sont particulièrement efficaces pour mobiliser et renforcer les fonctions exécutives, car ils empêchent l'enfant de recourir à des automatismes.

      Situations nouvelles : Toute situation où l'enfant n'a pas de routine établie l'oblige à s'adapter et donc à mobiliser ses fonctions exécutives.

      Tâches complexes : Les activités qui demandent plusieurs étapes, actions ou une réflexion soutenue.

      Situations inattendues : Les imprévus qui forcent une réévaluation et une adaptation.

      Résolution de problèmes : Chercher des solutions à un conflit ou à une difficulté pratique.

      Le jeu : Un contexte extrêmement favorable, en particulier :

      Le jeu symbolique (« faire semblant ») : Se mettre dans la peau d'un personnage.  

      Les jeux de société : Qui demandent de la stratégie, de la mémoire et de l'anticipation.

      Interactions sociales : Les discussions, les échanges et les conversations demandent une réflexion constante, une anticipation des réponses et une adaptation à l'interlocuteur.

      Découvertes et nature : Explorer de nouveaux environnements.

      Projets à long terme : Les projets qui s'étalent sur plusieurs jours ou semaines et nécessitent de la planification.

      Environnement stimulant et sécurisant : Un climat familial positif, où l'enfant se sent en sécurité, est plus propice à la mobilisation des fonctions exécutives.

      Un stress élevé ou des émotions négatives intenses peuvent entraver leur fonctionnement.

      4. Les Quatre Composantes Principales des Fonctions Exécutives

      L'exposé se concentre sur quatre composantes fondamentales, qui sont interdépendantes et fonctionnent comme une « toile d'araignée ».

      | Composante | Description | Exemple Clé | | --- | --- | --- | | Mémoire de travail | Capacité à retenir temporairement et à manipuler l'information pour l'utiliser dans une tâche. Elle fait le pont entre la mémoire sensorielle et la mémoire à long terme, et filtre les informations pertinentes. C'est l'aspect "actif" ou "dynamique" de la mémoire. | Le calcul mental (ex: 52 + 48). Il faut retenir les chiffres et activement les additionner pour trouver le résultat. | | Inhibition | Capacité à résister aux impulsions, aux distractions et aux habitudes pour adopter un comportement plus approprié. C'est le « frein » du cerveau qui permet de prendre un temps de recul avant d'agir et de diriger son attention. | Un jeune enfant à l'épicerie qui s'empêche de faire un commentaire à voix haute sur l'apparence physique d'une personne. | | Flexibilité cognitive | Capacité à se désengager d'une tâche ou d'une perspective pour s'adapter à une nouvelle situation, un nouveau point de vue ou de nouvelles règles. Elle permet de changer de stratégie quand la première ne fonctionne pas. | Un enfant qui accepte d'arrêter son jeu vidéo pour venir souper en famille, se désengageant cognitivement de la première activité pour se réengager dans la seconde. | | Planification | Capacité à anticiper des événements futurs, à se fixer un but et à organiser une séquence d'actions pour l'atteindre. Son développement est plus tardif, car il dépend de l'acquisition de la notion du temps. | Prévoir le trajet pour aller au parc, ou suivre les étapes d'une recette de cuisine. |

      5. L'Importance Fondamentale des Fonctions Exécutives

      Les fonctions exécutives sont décrites comme des compétences de base, constituant la fondation de multiples aptitudes et habiletés. Leur importance est capitale car :

      • Elles soutiennent la réussite éducative à tous les niveaux (primaire, secondaire, et même post-secondaire).

      • Elles ont un impact sur l'ensemble des sphères de développement de l'enfant : langagier, social, cognitif et même moteur.

      • Elles sont essentielles pour fonctionner efficacement dans la vie de tous les jours et pour le bien-être futur de l'individu.

      6. Stratégies de Soutien Parental pour le Développement

      Les parents peuvent jouer un rôle actif dans le développement des fonctions exécutives de leur enfant à travers des actions intégrées au quotidien, sans nécessiter de matériel sophistiqué.

      Approches Générales

      Développer le langage intérieur : Verbaliser à voix haute ses propres pensées pour modéliser, puis encourager l'enfant à se parler dans sa tête pour retenir des consignes ou se réguler.

      Soutenir les habiletés sociales : Favoriser les interactions sociales positives et variées.

      Offrir un soutien émotionnel : Accueillir les émotions de l'enfant avec empathie pour créer un climat de sécurité.

      Pratiquer des activités sportives et artistiques : Ces activités mobilisent l'ensemble des fonctions exécutives.

      Utiliser l'étayage et le questionnement : Accompagner l'enfant et le questionner sur ce qu'il pense, et surtout pourquoi il le pense, pour l'amener à verbaliser son raisonnement.

      Offrir des choix : Faire un choix implique de prioriser et de renoncer, ce qui mobilise la réflexion.

      Favoriser l'autonomie : Encourager l'enfant à faire les choses par lui-même.

      Tenir compte de ses intérêts : Un contexte agréable et motivant est plus propice à la mobilisation cognitive.

      Proposer des défis : Offrir des défis légèrement supérieurs à ses capacités actuelles, tout en l'accompagnant.

      Stratégies Ciblées par Composante

      Pour la Mémoire de Travail

      Stratégies de mémorisation : Utiliser des images mentales, la répétition (avec le langage intérieur), le regroupement d'informations (ex: retenir trois légumes), des associations ou des moyens mnémotechniques (chansons, rythmes).

      Repères visuels : Utiliser des listes, des pictogrammes pour les routines afin de se souvenir des étapes.

      Résumer et reformuler : Demander à l'enfant de redire avec ses propres mots ce qu'il a entendu ou lu.

      Réduire la charge cognitive : Éviter de surcharger l'enfant d'informations, surtout dans les moments de transition (ex: le matin avant de partir à l'école).

      Pour l'Inhibition

      Prendre un temps de recul : Apprendre à l'enfant à compter jusqu'à trois avant de réagir.

      Communiquer les attentes : Avoir des attentes réalistes et les expliquer clairement à l'enfant.

      Gestion des émotions : Mettre en place des stratégies pour gérer la colère ou la tristesse (respiration, visualisation, relaxation).

      Jeux d'adresse et d'équilibre : Ces jeux demandent de contrôler ses gestes et d'éviter les mouvements impulsifs.

      Jeux de vitesse et de réflexes : Jouer à des jeux où il faut réagir rapidement mais au bon moment (ex: le jeu Dobble).

      Pour la Flexibilité Cognitive

      Laisser prendre des décisions : Permettre à l'enfant de prendre des décisions par lui-même.

      Déroger aux routines : Modifier occasionnellement une routine pour l'amener à s'adapter.

      Préparer les transitions : Aviser l'enfant à l'avance d'un changement d'activité pour lui permettre de s'y préparer cognitivement.

      Stimuler la créativité : Encourager les activités créatives, les jeux de rôle et l'improvisation.

      Changer la fin des histoires : Avant de lire la fin d'un livre, demander à l'enfant d'inventer sa propre conclusion.

      Pour la Planification

      Utiliser des repères de temps visuels : Calendriers, horaires de la journée ou de la fin de semaine.

      Amener à anticiper : Poser des questions comme : «

      • Qu'est-ce que tu penses qu'il va se passer après ? »,
      • « De quoi auras-tu besoin pour cette activité ? ».

      Créer des plans : Avant une construction en blocs LEGO, suggérer de dessiner un petit plan.

      Remettre en séquence : Après avoir lu une histoire ou vu un film, demander à l'enfant de raconter ce qui s'est passé au début, au milieu et à la fin.

    1. Synthèse sur la Gestion de la Colère et de la Frustration chez l'Enfant

      Résumé Exécutif

      Ce document de synthèse présente les stratégies et les concepts clés pour aider les enfants à gérer leur colère et leur frustration, basés sur l'expertise de Madame Ly Massy, docteure en psychologie de l'éducation.

      L'approche fondamentale est de considérer la colère non pas comme une émotion négative à supprimer, mais comme une émotion normale, un signal d'alarme indiquant une injustice ou un préjudice.

      Elle devient problématique uniquement lorsqu'elle est mal exprimée, trop intense ou injustifiée.

      La gestion de ces émotions repose sur le développement de la régulation émotionnelle, une compétence qui s'articule autour de trois habiletés fondamentales :

      1. L'identification des émotions et de leurs sources.

      2. La modulation de l'intensité émotionnelle pour prévenir l'escalade et retrouver le calme.

      3. L'expression adéquate des émotions pour résoudre les situations conflictuelles.

      Le rôle des parents est central et s'articule autour de la prévention et de l'enseignement de stratégies concrètes.

      La prévention consiste à instaurer un environnement sécurisant par le biais de routines prévisibles et de saines habitudes de vie, notamment une activité physique suffisante et un sommeil adéquat.

      Pour l'enseignement, il est crucial d'aider l'enfant à développer un vocabulaire émotionnel, à reconnaître ses propres signaux (physiques, cognitifs, comportementaux) et à maîtriser un éventail de stratégies d'apaisement (techniques physiques, diversion de l'attention).

      Le modelage parental — la manière dont les parents gèrent et expriment leurs propres émotions — est l'outil le plus puissant pour guider l'enfant.

      --------------------------------------------------------------------------------

      1. Comprendre la Colère : Une Émotion Normale

      Selon Madame Ly Massy, la perception de la colère comme une émotion purement négative est une erreur. Elle doit être comprise comme un mécanisme de signalisation fondamental.

      Une Fonction d'Alarme : La colère est décrite comme "un petit bouton d'alarme" qui signale qu'une situation est perçue comme injuste, moqueuse, ou qu'elle nuit au développement personnel.

      Quand la Colère Devient Problématique : L'enjeu n'est pas de supprimer la colère, mais de gérer son expression. Elle devient problématique dans trois cas de figure :

      1. Expression Inadéquate : L'enfant "explose au lieu de dire tranquillement qu'est-ce qui nous fait vivre l'émotion négative".   

      2. Intensité Excessive : L'émotion est trop intense et l'enfant ne parvient pas à la contrôler.  

      3. Justification Inappropriée : La colère est déclenchée par une mauvaise interprétation de la situation (par exemple, percevoir un accident comme un acte intentionnel).

      2. La Régulation Émotionnelle : Un Apprentissage en Trois Temps

      La régulation émotionnelle est une habileté qui se développe tout au long de la vie, mais dont les bases devraient être acquises au début du primaire. Elle se compose de trois compétences interdépendantes.

      | Habileté | Description | | --- | --- | | 1\. Identification des Émotions | Comprendre l'émotion ressentie et ses sources. C'est le prérequis à toute gestion. | | 2\. Modulation des Émotions | Être capable de gérer l'intensité de l'émotion pour éviter une escalade ou pour s'apaiser après une crise et retrouver son calme. | | 3\. Expression des Émotions | Communiquer de manière adéquate ce que l'on ressent et ce qui a causé l'émotion, dans le but de résoudre la situation. |

      3. Facteurs de Difficulté dans la Régulation Émotionnelle

      Plusieurs caractéristiques individuelles peuvent expliquer pourquoi certains enfants ont plus de difficultés à gérer leurs émotions.

      Hypersensibilité ou Hyperréactivité : Une réaction plus intense aux émotions, aux situations et à l'environnement. Le "moindre petit élément va susciter une réaction négative".

      Seuil de Frustration Bas : L'image d'une "mèche courte" est utilisée. Il en faut très peu pour que l'enfant se sente frustré ou en colère.

      Faible Tolérance au Délai de Gratification : Une difficulté à attendre. Le besoin doit être satisfait immédiatement, sinon une réaction négative s'ensuit.

      Rigidité Mentale : Un besoin que les choses soient faites d'une manière précise ou selon leurs goûts.

      Un changement dans la routine peut susciter des émotions négatives.

      C'est un trait commun chez les enfants avec un trouble du spectre de l'autisme.

      Perceptions Faussées : Une tendance à interpréter les situations neutres comme hostiles.

      L'enfant est en état de vigilance constant, "comme s'il était toujours en vigilance d'avoir peur d'être attaqué".

      Cela peut mener à généraliser ou dramatiser les événements.

      Trauma : Un traumatisme peut accentuer tous les facteurs précédents et mener à un "surétiquetage" des émotions négatives comme de la colère, l'enfant cherchant inconsciemment à se sentir moins victime et plus combatif.

      4. Stratégies Parentales : Prévention et Anticipation

      La première étape pour un parent est de mettre en place des conditions pour "prévenir les pertes de contrôle" plutôt que de simplement réagir aux crises.

      4.1. Un Environnement Sécurisant

      Instauration de Routines : Mettre en place des routines prévisibles, surtout pour les moments de la journée identifiés comme difficiles (le matin avant l'école, la période des devoirs, le coucher, la fin de l'effet de la médication).

      La prévisibilité rend la gestion des frustrations plus facile pour l'enfant.

      4.2. Saines Habitudes de Vie

      Activité Physique : Essentielle pour évacuer les tensions accumulées.

      Un enfant qui ne bouge pas assez (par exemple, en s'installant directement pour les devoirs après l'école) aura plus de mal à gérer un événement frustrant.

      Sommeil : Le manque de sommeil est un des "premiers facteurs qui va nuire à la régulation émotionnelle". Les besoins varient selon l'âge :

      Début du primaire : Entre 11 et 13 heures.    ◦ Début du secondaire : Environ 9 heures.

      4.3. Identification des Déclencheurs de Crises

      Il est crucial de distinguer deux types de facteurs :

      Facteurs Précipitants (Déclencheurs) : Ce qui initie la crise. Exemples : l'envahissement de l'espace personnel, une blessure d'estime de soi, l'accumulation de stress, la faim.

      En identifiant la source, on peut agir en amont (par exemple, prévoir une collation dans la voiture au retour de l'école).

      Facteurs Aggravants : Ce qui intensifie la crise. Exemples : la fatigue de fin de journée, la fin de l'effet d'une médication pour un TDAH.

      5. Développer les Compétences de Régulation Émotionnelle

      Le parent doit activement enseigner les trois habiletés fondamentales de la régulation, en choisissant un moment où l'enfant est calme.

      5.1. Habileté 1 : Identifier les Émotions

      Enrichir le Vocabulaire : Aider l'enfant à nommer ses émotions avec précision (irrité, frustré, furieux) et à décrire ses sensations physiques ("vidé", "submergé").

      Reconnaître les Signaux : L'aider à identifier les signaux précurseurs de la colère :

      Signaux Corporels : Externes (tension, rougeur, froncement des sourcils) et internes (cœur qui bat vite, souffle court).   

      Comportements : Tendance à frapper, à se replier sur soi.  

      Pensées : Identifier les pensées qui nourrissent la colère ("il l'a fait exprès").

      Outils et Approches :

      Refléter : "Tu as l'air déçu, est-ce qu'il s'est passé quelque chose ?".  

      Utiliser des supports ludiques : Livres, histoires, jeux de mimes, affiches, collages, création d'un "dictionnaire des émotions".

      5.2. Habileté 2 : Moduler les Émotions

      Évaluer l'Intensité : Utiliser une échelle simple comme un thermomètre des émotions (vert, jaune, orange, rouge) pour aider l'enfant à prendre conscience de son état et à choisir la bonne stratégie.

      Enseigner Deux Grandes Catégories de Stratégies :

      1. Stratégies Physiques : Très efficaces pour réduire la tension physique lors d'émotions intenses.    

      Respiration profonde : "La plus simple et la plus efficace".     

      Relaxation : Relaxation musculaire, méditation de pleine conscience.    

      Stimulation Sensorielle : Objets lourds (couverture, toutou), se bercer, se retirer dans un endroit calme et peu stimulant (tente, garde-robe), manipuler des objets (balles de tension, "putty").  

      2. Stratégies de Diversion de l'Attention : Idéales pour interrompre les pensées négatives en boucle qui nourrissent la colère.     

      Activités exigeant de la concentration : Compter à l'envers (de 20 à 1, ou de 200 à 0 par bonds de 4), dessiner des mandalas, jouer aux LEGO, faire une activité artistique.    

      Objets Visuels : Regarder des objets calmants (spirales liquides, lumières douces).

      Stratégie Cognitive : Les "Pensées Froides"

      ◦ Aider l'enfant à remplacer les "pensées chaudes" qui attisent la colère par des "pensées froides" qui apaisent.

      Exemples : "Il ne l'a pas fait exprès", "Ça va me couler sur le dos comme sur un canard".

      5.3. Habileté 3 : Exprimer les Émotions

      Le Bon Moment et le Bon Endroit : Cette étape ne peut se faire que lorsque l'enfant s'est calmé, dans un lieu propice à la discussion.

      Le "Message au Je" : Enseigner à l'enfant à structurer son expression :

      1. Je me sens... (nommer l'émotion).   

      2. Parce que... (expliquer la cause).  

      3. J'aimerais que... (proposer une solution pour que cela ne se répète pas).

      Le Modelage Parental : "Ici encore plus, le modelage est important".

      Les parents doivent eux-mêmes utiliser le "message au je" et parler de leurs propres émotions. C'est en voyant ses parents le faire que l'enfant apprendra le mieux.

      6. Messages Clés et Conclusion

      Madame Ly Massy conclut avec plusieurs messages fondamentaux pour les parents et les éducateurs :

      La Normalité de la Colère : Il faut cesser de diaboliser cette émotion.

      La Primauté de l'Identification : "Si on n'a pas identifié comment on se sent, on ne sera pas capable de prendre les bons moyens pour se contrôler".

      La Diversité des Stratégies : Il faut enseigner un large éventail de stratégies, car aucune n'est universellement efficace.

      Les Exutoires Sains : Proposer des moyens sains de se défouler quand l'expression verbale directe n'est pas possible (écriture, art, sport intense).

      Le Rôle du Parent : Être un modèle de calme, éviter de "mettre de l'huile sur le feu" et savoir prendre soi-même une grande respiration pour garder son calme face à l'enfant.

    1. Soutenir la Participation Parentale à l'École : Synthèse et Stratégies Clés

      Résumé Exécutif

      Ce document de synthèse analyse les stratégies et les concepts fondamentaux pour favoriser une participation parentale efficace et constructive dans le milieu scolaire, en se basant sur l'expertise d'Elodie Marion, docteure en administration publique et professeure à l'Université de Montréal.

      L'implication parentale est définie comme un ensemble de pratiques à la maison, à l'école et dans la communauté, qui soutiennent le développement de l'enfant.

      Son efficacité dépend moins des attentes de l'école envers les parents que des pratiques mises en place par l'école elle-même pour la soutenir.

      Trois facteurs principaux influencent l'implication des parents : leur contexte de vie, leur perception d'être invités à participer et leur sentiment de compétence.

      Pour surmonter les obstacles et catalyser l'engagement, une approche proactive fondée sur les "Cinq R" est proposée :

      • Reconnaissance,
      • Respect,
      • Rôle,
      • Résultats et
      • Relation.

      Cette approche transforme la dynamique de simple diffusion d'information en une véritable collaboration.

      Enfin, la mise en place d'un plan de communication stratégique par les enseignants est essentielle.

      Ce plan doit être structuré autour de quatre axes : la fréquence, la bidirectionnalité, les moyens adaptés et la diversité des thèmes abordés.

      L'objectif ultime est d'instaurer une coéducation, une responsabilité partagée qui non seulement bénéficie à l'élève, mais brise également l'isolement de l'enseignant et augmente l'efficacité collective.

      --------------------------------------------------------------------------------

      1. Définition et Modalités de l'Implication Parentale

      L'implication parentale englobe toutes les pratiques que les parents mettent en œuvre en lien avec l'éducation de leur enfant.

      La recherche la catégorise généralement en trois sphères distinctes, chacune avec des actions concrètes.

      1.1. Implication à la Maison

      Rôle Parental : Création d'un environnement familial propice à l'apprentissage et au développement.

      Cela inclut la gestion des habitudes de vie (sommeil, alimentation), du stress et le soutien général au bien-être de l'enfant.

      Soutien Scolaire : Actions directement liées à la scolarité de l'enfant, telles que les communications sur l'école, les encouragements, et la transmission d'attentes claires concernant la réussite scolaire.

      1.2. Implication à l'École

      Communications : Échanges formels et informels avec le personnel de l'école.

      Communications en personne : Rencontres parents-enseignants, réunions avec la direction.   

      Communications écrites : Messages quotidiens, signatures sur les évaluations (noté comme étant souvent une diffusion d'information plutôt qu'une communication bilatérale).

      Participation à la Prise de Décision : Engagement actif dans les instances de gouvernance de l'école, comme les comités d'école, les comités au centre de services scolaires ou la participation aux rencontres pour l'élaboration de plans d'intervention.

      Volontariat : Implication bénévole dans les activités de l'école.

      1.3. Implication dans la Communauté

      Bien que mentionnée comme une troisième catégorie par les chercheurs, cette dimension n'est pas détaillée dans la source.

      2. Facteurs d'Influence sur l'Implication Parentale

      L'engagement des parents n'est pas inné ; il est influencé par un ensemble de facteurs personnels et contextuels. L'école doit les comprendre pour adapter ses propres pratiques.

      Le Contexte de Vie des Familles : Les conditions de vie des parents (statut d'emploi, stress, monoparentalité, statut socio-économique) déterminent leur capacité à s'impliquer.

      Par exemple, un parent d'un enfant autiste peut trouver le soutien scolaire à la maison extrêmement difficile, non par manque de volonté, mais en raison du contexte familial.

      La Perception d'Être Invité : L'implication dépend de la manière dont le parent perçoit et reçoit l'invitation de l'école à participer.

      Une invitation envoyée n'est pas nécessairement une invitation ressentie comme telle.

      Le Sentiment de Compétence Parentale : Les parents, particulièrement ceux d'enfants en difficulté, peuvent se sentir démunis ou incompétents pour aider leur enfant.

      Ce manque de confiance peut être interprété à tort comme un désengagement.

      De plus, un sentiment de ne pas être à la hauteur des attentes de l'école peut paralyser leur implication.

      La Reconnaissance de l'Expertise Parentale : Les parents possèdent une connaissance fine et continue de leur enfant. Ils sont "le filon du parcours de son enfant".

      Ils souhaitent que cette expertise, acquise au quotidien et souvent documentée, soit reconnue et valorisée par l'école, plutôt que de se sentir dans une relation hiérarchique face à "l'expert" scolaire.

      3. Une Approche Stratégique : Le Cadre des "Cinq R" pour Favoriser l'Engagement

      Pour passer d'une posture d'attente à une posture de soutien actif, Elodie Marion propose un cadre mnémonique basé sur "cinq R".

      Cette approche vise à regarder ses propres pratiques ("se regarder dans le miroir") plutôt que de juger celles des parents ("regarder par la fenêtre").

      | Le "R" | Définition | Actions Concrètes | | --- | --- | --- | | Reconnaissance | Valider et reconnaître les efforts et contributions des parents, passés et présents. | \- Mentionner les progrès accomplis les années précédentes.<br>\- Souligner les efforts actuels, même s'ils sont modestes.<br>\- Éviter de se concentrer uniquement sur les nouveaux défis sans reconnaître le travail déjà fait. | | Respect | Tenir compte de l'opinion, de l'expertise, de la culture et du contexte de vie des parents. | \- Pratiquer l'écoute active lors des rencontres.<br>\- Arriver en mode discussion plutôt qu'avec un ordre du jour rigide.<br>\- Adapter les attentes de l'école à la réalité des familles (ex: trouver des alternatives au soutien scolaire à la maison si nécessaire). | | Rôle | Clarifier et définir explicitement le rôle attendu du parent pour éviter les malentendus. | \- Expliciter ce que l'école attend (ex: encourager, questionner) et ce qu'elle n'attend pas (ex: ré-enseigner la matière).<br>\- Co-construire un rôle clair et accepté par le parent.<br>\- Donner des pistes concrètes sur comment le parent peut questionner son enfant sur les apprentissages. | | Résultats | Mettre en évidence les progrès et les objectifs pour que l'engagement soit perçu comme utile. | \- Définir des objectifs clairs pour chaque rencontre.<br>\- Communiquer fréquemment les améliorations observées chez l'enfant suite à l'implication du parent.<br>\- Célébrer les succès pour motiver la poursuite des efforts, au lieu d'attendre les bilans officiels. | | Relation | Bâtir une véritable relation de confiance et un sentiment d'appartenance. | \- Organiser des moments de rencontre informels (ex: fête de la rentrée).<br>\- Concevoir les rencontres pour permettre un réel échange et pas seulement une transmission d'information.<br>\- Intégrer un temps pour la "présentation des parents" lors des réunions de début d'année. |

      4. Mettre en Place un Plan de Communication Efficace à l'École

      Un plan de communication réfléchi est un outil puissant pour structurer et améliorer les interactions avec les parents tout au long de l'année.

      Il doit s'articuler autour de quatre axes.

      4.1. Fréquence

      Planification : Établir dès le début de l'année un rythme de communication réaliste pour l'enseignant et pertinent pour les parents.

      Routine : Créer des habitudes de communication (ex: un courriel hebdomadaire) pour éviter les contacts sporadiques ou uniquement déclenchés par des problèmes.

      4.2. Bidirectionnalité

      Passer de la diffusion à l'échange : Concevoir les communications pour solliciter l'avis et les informations des parents.

      Poser des questions ouvertes : Utiliser des questions commençant par "comment" ou "pourquoi" pour inviter à des réponses développées, plutôt que des questions fermées (oui/non).

      Expliciter les besoins : Indiquer clairement aux parents les types d'informations que l'enseignant souhaite recevoir (ex: un changement à la maison, une inquiétude de l'enfant).

      4.3. Moyens de Communication

      Adaptation : Choisir des moyens de communication adaptés au contexte des parents (ex: langue, niveau de littératie).

      Simplification : Alléger les communications écrites pour en faciliter la compréhension. Éviter les lettres de plusieurs pages.

      Le Piège Technologique : La multiplication des plateformes de communication (portails, applications) augmente souvent la quantité d'informations diffusées sans pour autant améliorer la relation.

      Il est recommandé de réduire le nombre de canaux pour se concentrer sur la qualité des échanges.

      4.4. Diversité des Thèmes

      Prévoir à l'avance : Planifier la diversité des sujets à aborder durant l'année pour éviter de ne communiquer que sur les difficultés.

      Inciter à l'action : Pour chaque information transmise, se poser la question : "Qu'est-ce que je veux que le parent fasse de cette information ?".

      Cela permet de formuler des suggestions concrètes (ex: "Vous pourriez l'encourager en...", "N'hésitez pas à lui demander comment...").

      5. Vers la Coéducation : Bénéfices d'une Responsabilité Partagée

      L'instauration d'une collaboration solide et d'une relation de confiance entre l'école et la famille mène à une véritable coéducation.

      Cette responsabilité partagée génère des bénéfices significatifs pour toutes les parties prenantes.

      Pour l'enseignant :

      Briser l'isolement : Le parent devient un allié dans la résolution de problèmes.  

      Stimuler la créativité : L'échange avec le parent apporte de nouvelles perspectives et idées d'intervention.  

      Gagner en efficacité : Bien que la collaboration demande du temps initialement, elle permet de comprendre plus vite les situations et de trouver des solutions plus durables, rendant le travail plus efficace à long terme.

      Pour le parent :

      ◦ Se sent valorisé, compétent et partie prenante de la réussite de son enfant.

      Pour l'élève :

      ◦ Bénéficie d'un soutien cohérent et aligné entre la maison et l'école, favorisant sa réussite scolaire et éducative.

    1. Document de Synthèse : Contrer l'Absentéisme au Secondaire

      Résumé Exécutif

      Ce document synthétise une approche innovante pour la gestion de l'absentéisme dans une école secondaire, développée et présentée par Véronique Sir, directrice d'établissement et candidate au doctorat.

      Le projet marque une transition fondamentale d'un modèle punitif, jugé lourd et inefficace, vers un modèle relationnel qui responsabilise et outille les enseignants.

      Cette nouvelle stratégie a permis de réduire de 50 % le nombre d'élèves présentant plus de 15 absences non motivées en une seule année scolaire.

      Au-delà des chiffres, la retombée la plus significative est l'amélioration notable de la relation entre les enseignants et les élèves, les premiers n'étant plus perçus comme des "polices de la retenue" mais comme des adultes bienveillants et soucieux de la présence de chaque jeune.

      La mise en œuvre s'est articulée en cinq étapes clés, incluant une analyse rigoureuse, la création d'un sous-comité stratégique, une approche pilote par "petits pas", une intégration systémique et un partage des connaissances.

      Le projet met en lumière l'importance du temps, de l'adhésion des équipes et de la focalisation sur le pouvoir d'agir collectif de l'école plutôt que sur des facteurs externes.

      Contexte et Problématique Initiale

      À l'arrivée de la nouvelle direction il y a trois ans, deux irritants majeurs étaient palpables et verbalisés par le personnel de l'école :

      1. Un manque de cohérence dans l'application du code de vie.

      2. Une gestion des absences perçue comme excessivement lourde et inefficace.

      Cette dernière tâche était si pesante que la majorité des enseignants souhaitaient s'en dégager.

      L'analyse initiale des données a permis de "neutraliser l'effet négatif" des perceptions en démontrant que le problème, bien que réel, ne concernait que deux ou trois élèves par groupe, et non une majorité comme il était parfois ressenti.

      Le Projet de Gestion des Absences : Une Approche Relationnelle

      Philosophie et Changement de Paradigme

      Le cœur du projet est un changement radical de philosophie, passant d'un système répressif à une approche humaine et proactive.

      D'un modèle punitif à un modèle relationnel : L'ancienne méthode, qui consistait à sanctionner l'absence (par exemple, par une retenue), est abandonnée au profit d'une démarche qui cherche à comprendre les causes de l'absence et à outiller l'élève.

      Comme le résume Mme Sir : "On est passé d'un modèle punitif à un modèle relationnel et outillé soutenu par des facilitateurs à l'école."

      Le rôle central de l'enseignant : Le projet repose sur l'implication directe des enseignants, qui deviennent les premiers intervenants.

      Ils sont responsables des sept premières interventions auprès de leurs élèves tuteurs, incluant deux appels aux parents pour les sensibiliser.

      Cette approche s'oppose au réflexe de déléguer cette responsabilité à l'équipe de soutien, reconnaissant qu'une poignée d'intervenants ne peut gérer efficacement les absences de plus de 900 élèves.

      La présence des enseignants est donc jugée "essentielle".

      Résultats Quantitatifs

      Le projet, axé sur une gestion par les résultats, a démontré un impact mesurable et significatif sur la réduction de l'absentéisme chronique non motivé.

      | Période | Contexte | Nombre d'élèves avec >15 absences non motivées | | --- | --- | --- | | Juin 2024 | Fin de la phase pilote (3 mois, 3 groupes sur 35) | Environ 120 élèves | | Juin 2025 | Fin de la première année complète (tous les groupes) | Environ 60 élèves | | 31 octobre 2025 | Début de l'année scolaire en cours | 6 élèves |

      Ces chiffres représentent une diminution d'environ 50 % des cas d'absentéisme chronique en un an.

      Il est noté que le mois de juin tend à augmenter le nombre d'absences, ce qui rend la comparaison encore plus probante.

      Le principal fait saillant est que tous les élèves de l'école (clientèle d'environ 950 jeunes) sont désormais connus et suivis, ne permettant à personne de "passer sous la craque".

      Les Cinq Étapes de la Mise en Œuvre

      Le cheminement réflexif du projet a été structuré en cinq phases distinctes, menées en collaboration avec des chercheurs universitaires.

      1. Analyse de la situation : La première étape a consisté à faire émerger des données factuelles pour objectiver les deux irritants majeurs (code de vie et gestion des absences).

      2. Création du sous-comité : Considérée comme le "cœur de la démarche", cette étape a impliqué la sélection stratégique de ses membres.

      Le comité inclut non seulement des personnes ouvertes au changement, mais aussi des enseignants plus critiques et des membres du personnel encore attachés au modèle punitif.

      L'objectif était de créer un espace de réflexion pour confirmer la fin du statu quo et construire une vision commune.

      3. Culture des "petits pas" : Pour gérer le changement, le projet a débuté par un pilote limité : trois groupes, trois enseignants volontaires, pendant trois mois.

      Ce n'est que la deuxième année que l'approche a été étendue à toute l'école.

      Cette phase a été marquée par des "allers-retours constants" et un "droit à l'erreur", permettant d'ajuster les moyens tout en gardant le cap sur la finalité (le modèle relationnel).

      4. Veilles et intégration systémique : Cette étape, imbriquée dans les autres, a consisté à ancrer le projet dans toutes les instances de l'école :

      Comité projet éducatif : Intégration d'indicateurs sur l'assiduité.   

      Plan de lutte contre la violence et l'intimidation : Favoriser un climat scolaire sécuritaire.  

      Assemblées générales : Véhiculer l'importance du projet, en faisant témoigner les "agents facilitateurs".  

      Rencontres de niveaux : Instaurer un point statutaire toutes les deux semaines pour suivre les élèves absentéistes.

      5. Partage à la communauté : La dernière étape consiste à diffuser le projet pour "faire gagner du temps" à d'autres équipes-écoles, évitant ainsi de réinventer des solutions existantes.

      Défis, Facteurs de Succès et Recommandations

      Défis Rencontrés

      La gestion du temps et des attentes : Les résultats ne sont pas immédiats.

      Comprendre les causes profondes de l'absentéisme prend du temps, ce qui peut être un défi dans une culture axée sur les résultats rapides.

      L'adhésion de l'équipe : La deuxième année, lorsque tout le personnel est impliqué, est cruciale et peut voir émerger plus de résistance.

      Le sous-comité joue un rôle fondamental pour accueillir ces résistances sans reculer.

      La gestion des cas chroniques : Certains élèves, aux prises avec des enjeux de santé mentale ou de démotivation scolaire importants, résistent aux interventions.

      L'implication des professionnels (psychoéducateurs, conseillers d'orientation) est ici fondamentale.

      Le roulement du personnel : L'arrivée de personnel non formé en pédagogie peut rendre la création de liens plus difficile, nécessitant un soutien accru de la part des "agents facilitateurs" internes.

      Principale Réussite : L'Amélioration de la Relation Enseignant-Élève

      Le gain le plus "magnifique" et le plus positif du projet est l'amélioration de la qualité des relations.

      Les enseignants ne sont plus vus comme des agents de sanction. Un enseignant a partagé une anecdote révélatrice :

      "Les élèves m'ont dit à plusieurs reprises cette année : 'Cou'donc, avez-vous une vie à part nous regarder à l'école ?'".

      Pour l'équipe, cette remarque est une "victoire", car elle signifie que chaque élève sait qu'au moins un adulte se soucie de sa présence.

      Erreurs à Éviter

      1. Aller trop vite : Le changement culturel et la compréhension des causes profondes de l'absence exigent du temps.

      2. Remettre le sort aux parents : Plutôt que de se concentrer sur les motifs d'absence (sur lesquels l'école a peu de contrôle), la discussion doit être réorientée vers le "pouvoir d'agir collectif" à l'interne.

      3. Utiliser les données à mauvais escient : Un outil de suivi (Power BI) a été développé pour fournir des données quotidiennes.

      La vigilance est de mise pour que ces données servent à comprendre et agir, et non à "masquer artificiellement" les problèmes ou à créer une compétition entre les écoles.

      Retombées Stratégiques et Pérennité du Projet

      Outre la baisse de l'absentéisme et l'amélioration des relations, le projet a généré plusieurs impacts positifs durables :

      Approche personnalisée : L'école est passée d'une généralisation ("tous les élèves de 4e secondaire s'absentent") à une analyse fine et personnalisée des besoins de chaque élève.

      Standardisation des interventions : Un protocole écrit garantit la qualité et la pérennité des interventions, indépendamment du personnel en place.

      Autonomisation et résilience des équipes : Les enseignants ont développé une autonomie ("empowerment") et une résilience face à la problématique, conscients de leur pouvoir d'agir collectif.

      Préparation à la croissance : La structure mise en place est comparée aux "fondations d'une maison", rendant l'école prête à accueillir une hausse de sa clientèle.

      Pérennité du modèle : Le projet est conçu pour être durable. L'objectif final est de développer une autonomie telle que le projet puisse survivre au départ de la direction actuelle.

      Comme le conclut Mme Sir : "demain matin si je pars comme direction d'établissement, le projet va survivre grâce à nos agents facilitateurs qui vont assurer la pérennité du projet."

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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      Reply to the reviewers

      *Reviewer #1 (Evidence, reproducibility and clarity (Required): *

      *Using genetics and microscopy approaches, Cabral et al. investigate how fission yeast regulates its length and width in response to osmotic, oxidative, or low glucose stress. Miller et al. have recently found that the cell cycle regulators Cdc25, Cdc13 and Cdr2 integrate information about cell volume, time and cell surface area into the cellular decision when to divide. Cabral now build on this work and test how disruption of these regulators affects cell size adaptation. They find that each stress condition shows a distinct dependence on the individual regulators, suggesting that the complex size control network enables optimized size adaptation for each condition. Overall, the manuscript is clear and the detailed methods ensure that the experiments can be replicated.

      Major comments:

      1.) It would be much easier to follow the authors' conclusions, if in addition to surface area to volume ratio, length and width, they would also plot cell volume at division in Figs. 1-4.*

      AUTHOR RESPONSE: Due to space constraints in the main (and supplemental) figures, we focused on SA:Vol ratio together with cell length and width, which directly define cell geometry in rod-shaped fission yeast. Surface area and volume are derived from these measurements and can be misleading when considered alone, as similar surface area or volume values can arise from distinct combinations of length and width. The SA:Vol ratio therefore serves as a robust integrative metric for capturing coordinated changes in length and width that reshape cell geometry. We would be happy to include individual surface area and volume plots if requested.

      2.) To me, it seems that maybe even more than upon osmotic stress, the cdc13-2x strain differs qualitatively from WT in low glucose conditions, where the increased SA-V ratio is almost completely abolished.

      AUTHOR RESPONSE: We agree with the reviewer and have revised the manuscript text to point out this difference. The newly added text states: “Under low glucose, cdc13-2x cells also showed a WT-like response, decreasing length and increasing in SA:Vol ratio (Figures 3B-D). However, this SA:Vol increase was reduced compared to WT (1% vs 8.5%; Figures 1D and 3B), suggesting impaired geometric remodeling under glucose limitation.”

      3.) It is not entirely clear to me why two copies of Cdc13 would qualitatively affect the responses. Shouldn't the extra copy behave similarly to the endogenous one and therefore only lead to quantitative changes? Maybe the authors can discuss this more clearly or even test a strain in which Cdc13 function is qualitatively disrupted.

      AUTHOR RESPONSE: Increased Cdc13 protein concentration in cdc13-2x cells disrupts the typical time-scaling of Cdc13 protein. Consistent with this, cdc13-2x cells enter mitosis at a smaller cell size. We have modified the text to clarify this point. The new text states: “To access the role of the Cdc13 time-sensing pathway, we disrupted Cdc13 protein abundance by creating a cdc13-2x strain carrying an additional copy of cdc13 integrated at an exogenous locus. cdc13-2x cells divided at a smaller size than WT, reflecting accelerated mitotic entry upon disruption of typical time-scaling of Cdc13 protein (Figure S1A).”

      4.) I don't see why the authors come to the conclusion that under osmotic stress cells would maximize cell volume. It leads to a decreased cell length, doesn't it?

      AUTHOR RESPONSE: WT cells under osmotic stress do decrease in length, but this is accompanied by an increase in cell width. Because width contributes disproportionately to cell volume in rod-shaped cells, this change results in a modest but reproducible reduction in the SA:Vol ratio relative to WT cells in control medium (Figure 1D). We note that the degree of this change under osmotic stress is small (-0.4%), although statistically significant (p * Likewise, in Figure 2B, they interpret tiny changes in the SA/V. By my estimation, the difference between control and osmotic stress is only 2% (1.195/1.17), less that the wild-type case, which appears to be twice that (which is still pretty modest). The small amplitude of these changes is obscured by the fact that the graphs do not have a baseline at zero, which, as a matter of good data-presentation practice, they should.

      *

      AUTHOR RESPONSE: We appreciate the reviewer’s distinction between statistical and biological significance and agree that this is an important point to clarify. We now note in the revised text that changes in SA:Vol ratio under osmotic stress are numerically small and should not be overinterpreted. Our revised text now states: “Under oxidative and osmotic stress, the SA:Vol ratio decreased, indicating greater cell volume expansion relative to surface area (Figure 1D). However, we note that the reduction in SA:Vol under osmotic stress, while statistically significant, was modest in magnitude (−0.4%).”

      Although small in absolute terms, even subtle geometric changes can be biologically meaningful in fission yeast due to the small size of these cells, where minor shifts in length or width translate into measurable differences in membrane area relative to cytoplasmic volume. Importantly, in Figure 2B, the key observation is not the magnitude of the change but its direction: cdc25-degron-DaMP cells exhibit a ~2% increase in SA:Vol ratio under osmotic stress, in contrast to the decrease observed in WT cells under the same condition. This opposite response reflects altered cell geometry and is supported by corresponding changes in cell length and width. We have revised the Results text to emphasize both the modest magnitude and the directional nature of these effects: “Under osmotic stress, cdc25-degron-DaMP cells exhibited a ~2% increase in SA:Vol ratio, opposite to the modest decrease observed in WT cells. This increase arose from increased cell length and reduced width (Figures 2B-D).”

      Regarding data presentation, because SA:Vol ratios vary over a narrow numerical range, setting the y-axis minimum to zero would compress the data and obscure all detectable differences. Instead, we have modifed our SA:Vol ratio graphs in Fig. 1-4 to have consistent axis scaling across panels to accurately convey relative changes while maintaining visual clarity. We are happy to provide full data tables and statistical outputs upon request.

      * I am also concerned about the use of manual measurement of width at a single point along the cell. This approach is very sensitive to the choice of width point and to non-cylindrical geometries, several of which are evident in the images presented. MATLAB will return the ??? as well as the length from a mask, but even better, one can more accurately calculate the surface area and volume by assuming rotational symmetry of the mask. Given that surface area and volume calculation need to be redone anyway, as discussed below, I encourage the authors to calculate them directly from the mask, instead of using the cylindrical assumption.*

      AUTHOR RESPONSE: In initial experiments to calculate surface area and volume of fission yeast cells for prior work (Miller et al., 2023, Current Biology) we found that automated width measurements by MATLAB or ImageJ were inaccurate for a subset of cells leading to noisy cell surface area and volume values. Measuring cell width by hand and assuming that each cell in a given strain had the same cell radius (average of population) for calculation of cell surface area and volume gave more consistent results and recapitulated established conclusions regarding size control mechanisms.

      In this previous work and the current study, abnormally skinny or wide regions of a cell were avoided when drawing a line to measure the cell width by hand. For each strain and condition, an average cell width was determined per independent experiment and used for surface area and volume calculations. Additionally, previous analysis demonstrated that this approach yields results consistent with a rotation method derived directly from cell masks, which does not assume a cylindrical cell shape (Facchetti et al., 2019, Current Biology; Miller et al., 2023, Current Biology).

      To test the validity of our size measurements and confirm the robustness of our results in this study we compared the surface area and volume of cells by this rotation method. We have added this additional information to our revised methods section and also added SA:Vol ratio graphs generated from the rotation size measurement to our revised Figure S1 E-J. Importantly, both approaches used to measure cell size gave consistent results and supported the same conclusions.*

      The authors also need to be more careful about their claims about size-dependent scaling. The concentration of both Cdc13 and Cdc25 scale with size (perhaps indirectly, in the case of Cdc13), but Cdr2 does not. Cdr2 activity has been proposed to scale with size, and its density at cortical nodes has been reported to scale with size, although that claim has been challenged .*

      AUTHOR RESPONSE: We have modified text in the Introduction and Results to address this point. Our revised text in the introduction states: “Recent work has shown that Cdk1 activation integrates size- and time-dependent inputs: the Wee1-inhibitory kinase Cdr2 cortical node density scales with cell surface area (Pan et al., 2014; Facchetti et al., 2019); Cdc25 nuclear accumulation scales with cell volume; and cyclin Cdc13 accumulates over time in the nucleus (Miller et al., 2023) (Figure 1B).” Our revised text in the results section states: “Cdr2 functions as a cortical scaffold that regulates Wee1 activity in relation to cell size, with Cdr2 nodal density reported to scale with cell surface area, enforcing a surface area threshold for mitotic entry (Pan et al., 2014; Allard et al., 2018; Facchetti et al., 2019; Sayyad and Pollard, 2022).”*

      Even taking the authors approach at face value, there are observations that do not seem to make sense, which led me to realize that the wrong formulae were used to calculate surface area and volume.

      In Figure 1E,F, the KCl-treated cells get shorter and wider; surely, that should result in a lower SA/V ratio. However, as noted above, in Figure 1D, they are shown to have a similar ratio. As a sanity check, I eye-balled the numbers off of the figure (control: 14 µm x 3.6 µm and KCl: 11 µm x 3.8 µm) and calculated their surface area and volume using the formula for a capsule (i.e., a cylinder with hemispheric ends).

      SA = the surface area of the two hemispheres + the surface are of the cylinder in between = 4*pi*(width/2)^2 + pi*width*(length-width), the length-width term calculates the side length of the capsule (length without the hemispheres) from the full length of the capsule (length including the hemispheres)

      V = the volume of the two hemispheres + the volume of the cylinder in between = 4/3*pi*(width/2)^3 + pi*(width/2)^2*(length-width).

      I got SA/V ratios of around 2, which are way off from what is presented in Figure 1D, but my calculated ratio goes down in KCl, as expected, but not as reported.

      To make sure I was not doing something wrong, I was going to repeat my calculations with the formulae in Table 1, which made me realize both are incorrect. The stated formula for the cell surface area-2*pi*RL-only represents to surface area of the cylindrical side of the cells, not its hemispherical ends. And it is not even the correct formula for the surface area of the side, because that calls for L to be the length of the side (without the hemispherical ends) not the length of the cell (which includes the hemispherical ends). L here is stated to be cell length (which is what is normally measured in the field, and which is consistent with the reported length of control cells in Figure 1E being 14 µm). The formula for the volume of a capsule in the form use in Table 1 (volume of a cylinder of length L - the volume excluded from the hemispherical ends) is pi*R^2*L - (8-(4/3*pi))*R^3.

      Given these problems, I think I spent too much time thinking about the rest of the paper, because all of the calculations, and perhaps their interpretations, need to be redone.*

      AUTHOR RESPONSE: The surface area and volume equations for a cylinder with hemispherical ends used in our study and listed in our table are correct and widely used in other work with fission yeast cells (Navarro and Nurse, 2012; Pan et al., 2014; Facchetti et al., 2019; BayBay et al., 2020; and Miller et al., 2023). We write our equations with variables for cell length and radius because these are biologically relevant and measured parameters for fission yeast cells. Cell length (L) refers to the total tip-to-tip length of the cell, including the hemispherical ends, and radius (R) refers to half the measured cell width. We have revised the Methods section to clarify this definition and avoid ambiguity (Please see methods section “Cell geometry measurements”)

      Additionally, SA or Vol calculations were performed using the length of each individual cell and the average cell radius of the population. We did not use mean cell length of the population for our calculations like the reviewer assumed in their “sanity check” above. Please see methods section “Cell geometry measurements”. We hope that these clarifications and text revisions improve transparency and reproducibility.

      * Minor Points:

      Strains should be identified by strain number is the text and figure legends.*

      AUTHOR RESPONSE: For clarity and readability, we refer to strains by genotype in the main text and figure legends, which we believe is more informative for readers than strain numbers. All strain numbers corresponding to each genotype are provided in Table S1, ensuring traceability and reproducibility without compromising clarity in data presentation.*

      In the Introduction, "Most cell control their size" should be "Most eukaryotic cell control their size".*

      • *

      AUTHOR RESPONSE: The text has been corrected as suggested.*

      Reviewer #2 (Significance (Required)):

      Nothing to add.*

      *Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      Summary This manuscript reports that fission yeast cells exhibit distinct cell size and geometry when exposed to osmotic, oxidative, or low-glucose stress. Based on quantitative measurements of cell length and width, the authors propose that different stress conditions trigger specific 'geometric adaptation' patterns, suggesting that cell size homeostasis is flexibly modulated depending on environmental cues. The study provides phenotypic evidence that multiple environmental stresses lead to distinct outcomes in the balance between cell surface area and volume, which the authors interpret as stress-specific modes of size control.

      Major comments 1) The authors define the 48-hour time point as the 'long-term response', but no justification is provided for why 48 hours represents a physiologically relevant adaptation phase. It is unclear whether the size-control mode has stabilized by that time, or whether it may continue to change afterward. At minimum, the authors should provide a rationale (e.g., growth recovery dynamics, transcriptional adaptation plateau, or pilot time-course observations) to demonstrate that 48 hours corresponds to the steady-state adaptive phase rather than an arbitrarily selected time point.*

      AUTHOR RESPONSE: We thank the reviewer for this important point and agree that the definition of the long-term response should be clarified. We have addressed this with new experiments and revised text. We now incorporate growth curve data and doubling time analyses for all yeast strains grown under control and stress conditions (See new Figure S3). These analyses show that following an initial transient stress-induced cell cycle delay, growth rates stabilize well before 48 hours. Notably, the slowest growth rate observed was in 1M KCl, with a doubling time of ~4 hours across all yeast strains tested. Thus, by 48 hours, cells in this condition have undergone more than 12 generations of growth, while cells in all other conditions with shorter doubling times have undergone even more divisions. So by allowing cells to grow for 48 hours prior to imaging, we are capturing cells that have resumed sustained cell cycle progression following transient stress-induced cell cycle delays. Because cell size control is tightly linked to the cell cycle, we define 48 hours as a physiologically relevant time point where cells have adapted to stress conditions.

      Our revised methods now states: “Cultures were incubated at 25°C while shaking at 180 rpm for 48 h prior to imaging. This time point was chosen to ensure that cells had progressed beyond the initial transient stress response and reached a stable, condition-specific growth state, as confirmed by growth curve and doubling time analyses showing stabilization well before 48 h (Figure S3), including in the slowest growing condition (1 M KCl; doubling time ~4 h).”

      * 2*)Related to the above comment, the authors propose that different stresses lead to distinct cell size adaptations, yet the rationale for the chosen stress intensities and exposure times is insufficiently described. It remains unclear whether the osmotic, oxidative, and low-glucose conditions used here induce comparable levels of cellular stress. Dose-response and time-course analyses would greatly strengthen the conclusions. Without such analyses, it is difficult to support the interpretation that geometry modulation represents a direct adaptive response.

      AUTHOR RESPONSE: * *We selected the specific stress conditions based on previously published work showing that these doses elicit robust responses while preserving overall cell viability and the capacity for recovery. We note that osmotic, oxidative, and low glucose conditions perturb fundamentally different cellular systems (turgor pressure and cell wall mechanics, redox balance, and metabolism etc.) and therefore do not generate directly comparable levels of cellular stress in a quantitative sense. Our goal was not to equalize stress intensity across conditions, but to examine how cells change their geometry in response to distinct classes of stressors.

      We have clarified the rationale for specific stress conditions in the revised methods: “These stress intensities were selected based on prior studies demonstrating robust cellular responses while preserving cell viability and the capacity for recovery (Fantes and Nurse, 1977, Shiozaki and Russell, 1995, Degols, et al., 1996; López-Avilés et al., 2008; Sansó et al., 2008; Satioh et al., 2015, Salat-Canela et al., 2021, Bertaux et al., 2023).”

      * 3) The authors describe stress-induced size changes as an 'adaptive' response. While this is an appealing hypothesis, the presented data do not demonstrate that the change in cell size itself confers a fitness advantage. Evidence showing that blocking the size change reduces stress survival-or that the altered size improves growth recovery- would be required to support this claim. Without such data, the use of the term 'geometric adaptation' seems overstated.*

      AUTHOR RESPONSE: We have revised the text to remove the term “adaptive” and now describe stress-induced size changes in descriptive terms. As discussed further in response to Comment 4, new growth curve and doubling time analyses show that defects in surface area or volume expansion do not uniformly impair growth or survival over the stress exposure examined here, reinforcing the decision to avoid fitness-based language.*

      4) The authors conclude that mutants exhibit no major defects in growth or viability during 48-hour stress exposure based on comparable septation index values (Fig. S2). However, septation index alone does not fully capture growth performance or cell-cycle progression and is not sufficient to support claims regarding fitness or robustness of proliferation. If the authors intend to make statements about 'growth', 'viability', or 'cell-cycle progression', additional quantitative measures (e.g., growth curves, doubling time, colony-forming units, or microcolony growth measurements) would be necessary. Alternatively, the claims should be toned down to align with the measurements currently provided.*

      AUTHOR RESPONSE: We have addressed this concern with new experiments and revised text. In addition to septation index measurements (now analyzed using chi-square tests of proportions; Figure S2), we performed growth curve experiments and doubling time analyses for all genotypes under control and stress conditions (new Figure S3). These additional data show that growth rates are largely comparable across genotypes in control, oxidative, and low-glucose conditions, with more pronounced genotype-dependent differences emerging under osmotic stress. Defects in surface area or volume expansion did not uniformly correspond to impaired population growth, indicating that geometric remodeling is not strictly required for proliferation over the 48-hour stress exposure examined here. We have refined our conclusion to emphasize that defects in surface area or volume expansion do not uniformly impair growth or survival. See revised Results text under the heading “Defects in surface area or volume expansion do not uniformly compromise growth or survival”.*

      5) Related to the above comment, the manuscript does not adequately rule out the possibility that the decreased division size simply results from slower growth or delayed cell-cycle progression rather than a shift in the size-control mechanism. Measurements and normalizations of growth rate are required; without them, the interpretation remains speculative.*

      AUTHOR RESPONSE: We agree that changes in growth rate or altered cell cycle timing are important to consider. We have revised our text: “Changes in growth rate or cell cycle progression under stress may influence division size by altering mitotic regulator accumulation. Future studies measuring mitotic regulator dynamics alongside growth rates will be needed to distinguish direct changes in size control mechanisms from growth- or timing-dependent effects.”

      * 6) Regarding the phenotypes of wee1-2x cells, it is interesting that they increase the SA:Vol ratio under all stress conditions and show phenotypes distinct from cdr2Δ cells. From these observations, the authors claims that Cdr2 and Wee1 function as a surface-area-sensing module that complements the volume-sensing and time-sensing pathways to maintain geometric homeostasis. To support this interpretation, the authors could consider additional experiments, such as analyzing cdr2Δ + wee1-2x cells under the same stress conditions. Such data would test whether increased Wee1 can rescue or modify the cdr2Δ phenotype, providing functional evidence for the proposed Cdr2-Wee1-Cdk1 regulatory relationship. Measurements of cell length, width, SA:Vol ratio, and, if feasible, Cdk1 activity markers in the strain would greatly strengthen the mechanistic claims.*

      AUTHOR RESPONSE: We thank the reviewer for this insightful suggestion. While analysis of a cdr2Δ wee1-2x strain could provide additional mechanistic detail, such experiments address a distinct question beyond the scope of our current study, which focuses on how cell geometry changes under different stress conditions in cells with perturbed surface area-, volume-, or time-sensing pathways. Our conclusions regarding a surface area-sensing role for Cdr2-Wee1 signaling are based on previous studies (Pan et al., 2014; Facchetti et al., 2019; Miller et al., 2023) and the cell geometry phenotypes we observe of cdr2Δ and wee1-2x cells under stress conditions. *

      Minor comments 1) The manuscript focuses on adaptation through changes in the surface-to-volume ratio; however, only the ratio is shown. Presenting the underlying values of surface area and volume would clarify which geometric parameter primary contributes to the observed changes.*

      AUTHOR RESPONSE: Please see our response to Reviewer 1 major comment 1.*

      *2) Statistical analysis for Fig.S2 should be provided.

      AUTHOR RESPONSE: We have completed this. See revised Figure S2 and methods.*

      3) The paper by Kellog and Levin 2022 is missing from the reference list.*

      AUTHOR RESPONSE: Thank you for catching this. This reference has now been added. *

      **Referees cross-commenting**

      After reading the other reviewer's reports, I recognize that focal points differ, but they appear sequential rather than contradictory.

      Reviewer 2 raises concerns regarding the surface area/volume calculations, which-if incorrect-would influence many of the quantitative conclusions. I agree that confirming the validity of these calculations (and recalculating if necessary) should be the top priority before evaluating the biological interpretations.

      Reviewer 1 raises more mechanistic biological questions. These are certainly important, but in my view they depend on the robustness of the quantitative analysis highlighted by Reviewer 2.

      Therefore, I regard the reports as complementary rather than conflicting. Once the analytical issue pointed out by Reviewer 2 is resolved, the field will be in a better position to assess the significance of the mechanistic points raised by Reviewer 1 (as well as those in my own report).

      Reviewer #3 (Significance (Required)):

      General assessment One of the major strengths of this manuscript is its quantitative, side-by-side comparison of multiple environmental stresses under a unified experimental and analytical framework. The authors provide well-controlled morphometric measurements, allowing direct comparison of geometry changes that would otherwise be difficult to evaluate across studies. The observation that different stress types generate distinct geometric outcomes is particularly intriguing and has the potential to stimulate new conceptual thinking in the field of size control. However, the strength of the conceptual conclusion is currently limited by several aspects of the experimental design and interpretation. In particular, it remains unclear whether the observed geometry changes represent active adaptive responses rather than non-specific consequences of prolonged or string stress exposure. Demonstrating whether geometry remodeling provides a fitness advantage, clarifying whether the changes reach a steady-state rather than reflecting slow drift over time, or identifying upstream stress pathways that govern the response would substantially strengthen the conceptual advance. Even if additional mechanistic or fitness-related data cannot be added, refining the interpretation so that it remains aligned with the present evidence will enhance the clarity, and impact of the study.

      Advance Previous study - including the 2023 publication by the James B. Moseley group - established that fission yeast integrates distinct size-control pathways related to surface area, volume, and time under normal growth conditions. The present manuscript extends this line of work to stressed environments and argues that each stress condition elicits a distinct size-control pattern. To our knowledge, a systematic comparison of cell geometry across multiple stress types in the context of size-control pathways has not been reported, and this represents a potentially valuable conceptual advance. The advance is primarily phenomenological and conceptual rather than mechanistic: the work presents new correlation between stress types and geometry but does not yet elucidate the pathways governing these responses or demonstrate a functional advantage. With additional evidence - or with qualifiers ensuring that claims match the current data - the study could make an important contribution to understanding how cells integrate environmental cues into size-control strategies.

      Audience Although the primary audience consists of researchers in the fields of cell growth, cell-cycle control, and stress responses in yeast, the conceptual contribution may interest broader fields such as growth homeostasis, metabolic adaptation, and pathological cell size changes in higher eukaryotes. Beyond yeast biology, the modular view of size regulation proposed here may inspire new investigations in stem cell biology, cancer research, and biotechnology where environmental adaptation and cell size are closely linked.

      Expertise: nuclear morphology; cell morphology; cell growth; cell cycle; cytoskeleton*

    2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #3

      Evidence, reproducibility and clarity

      Summary

      This manuscript reports that fission yeast cells exhibit distinct cell size and geometry when exposed to osmotic, oxidative, or low-glucose stress. Based on quantitative measurements of cell length and width, the authors propose that different stress conditions trigger specific 'geometric adaptation' patterns, suggesting that cell size homeostasis is flexibly modulated depending on environmental cues. The study provides phenotypic evidence that multiple environmental stresses lead to distinct outcomes in the balance between cell surface area and volume, which the authors interpret as stress-specific modes of size control.

      Major comments

      1) The authors define the 48-hour time point as the 'long-term response', but no justification is provided for why 48 hours represents a physiologically relevant adaptation phase. It is unclear whether the size-control mode has stabilized by that time, or whether it may continue to change afterward. At minimum, the authors should provide a rationale (e.g., growth recovery dynamics, transcriptional adaptation plateau, or pilot time-course observations) to demonstrate that 48 hours corresponds to the steady-state adaptive phase rather than an arbitrarily selected time point.

      2)Related to the above comment, the authors propose that different stresses lead to distinct cell size adaptations, yet the rationale for the chosen stress intensities and exposure times is insufficiently described. It remains unclear whether the osmotic, oxidative, and low-glucose conditions used here induce comparable levels of cellular stress. Dose-response and time-course analyses would greatly strengthen the conclusions. Without such analyses, it is difficult to support the interpretation that geometry modulation represents a direct adaptive response.

      3) The authors describe stress-induced size changes as an 'adaptive' response. While this is an appealing hypothesis, the presented data do not demonstrate that the change in cell size itself confers a fitness advantage. Evidence showing that blocking the size change reduces stress survival-or that the altered size improves growth recovery- would be required to support this claim. Without such data, the use of the term 'geometric adaptation' seems overstated.

      4) The authors conclude that mutants exhibit no major defects in growth or viability during 48-hour stress exposure based on comparable septation index values (Fig. S2). However, septation index alone does not fully capture growth performance or cell-cycle progression and is not sufficient to support claims regarding fitness or robustness of proliferation. If the authors intend to make statements about 'growth', 'viability', or 'cell-cycle progression', additional quantitative measures (e.g., growth curves, doubling time, colony-forming units, or microcolony growth measurements) would be necessary. Alternatively, the claims should be toned down to align with the measurements currently provided.

      5) Related to the above comment, the manuscript does not adequately rule out the possibility that the decreased division size simply results from slower growth or delayed cell-cycle progression rather than a shift in the size-control mechanism. Measurements and normalizations of growth rate are required; without them, the interpretation remains speculative.

      6) Regarding the phenotypes of wee1-2x cells, it is interesting that they increase the SA:Vol ratio under all stress conditions and show phenotypes distinct from cdr2Δ cells. From these observations, the authors claims that Cdr2 and Wee1 function as a surface-area-sensing module that complements the volume-sensing and time-sensing pathways to maintain geometric homeostasis. To support this interpretation, the authors could consider additional experiments, such as analyzing cdr2Δ + wee1-2x cells under the same stress conditions. Such data would test whether increased Wee1 can rescue or modify the cdr2Δ phenotype, providing functional evidence for the proposed Cdr2-Wee1-Cdk1 regulatory relationship. Measurements of cell length, width, SA:Vol ratio, and, if feasible, Cdk1 activity markers in the strain would greatly strengthen the mechanistic claims.

      Minor comments

      1) The manuscript focuses on adaptation through changes in the surface-to-volume ratio; however, only the ratio is shown. Presenting the underlying values of surface area and volume would clarify which geometric parameter primary contributes to the observed changes.

      2) Statistical analysis for Fig.S2 should be provided.

      3) The paper by Kellog and Levin 2022 is missing from the reference list.

      Referees cross-commenting

      After reading the other reviewer's reports, I recognize that focal points differ, but they appear sequential rather than contradictory.

      Reviewer 2 raises concerns regarding the surface area/volume calculations, which-if incorrect-would influence many of the quantitative conclusions. I agree that confirming the validity of these calculations (and recalculating if necessary) should be the top priority before evaluating the biological interpretations.

      Reviewer 1 raises more mechanistic biological questions. These are certainly important, but in my view they depend on the robustness of the quantitative analysis highlighted by Reviewer 2.

      Therefore, I regard the reports as complementary rather than conflicting. Once the analytical issue pointed out by Reviewer 2 is resolved, the field will be in a better position to assess the significance of the mechanistic points raised by Reviewer 1 (as well as those in my own report).

      Significance

      General assessment

      One of the major strengths of this manuscript is its quantitative, side-by-side comparison of multiple environmental stresses under a unified experimental and analytical framework. The authors provide well-controlled morphometric measurements, allowing direct comparison of geometry changes that would otherwise be difficult to evaluate across studies. The observation that different stress types generate distinct geometric outcomes is particularly intriguing and has the potential to stimulate new conceptual thinking in the field of size control. However, the strength of the conceptual conclusion is currently limited by several aspects of the experimental design and interpretation. In particular, it remains unclear whether the observed geometry changes represent active adaptive responses rather than non-specific consequences of prolonged or string stress exposure. Demonstrating whether geometry remodeling provides a fitness advantage, clarifying whether the changes reach a steady-state rather than reflecting slow drift over time, or identifying upstream stress pathways that govern the response would substantially strengthen the conceptual advance. Even if additional mechanistic or fitness-related data cannot be added, refining the interpretation so that it remains aligned with the present evidence will enhance the clarity, and impact of the study.

      Advance

      Previous study - including the 2023 publication by the James B. Moseley group - established that fission yeast integrates distinct size-control pathways related to surface area, volume, and time under normal growth conditions. The present manuscript extends this line of work to stressed environments and argues that each stress condition elicits a distinct size-control pattern. To our knowledge, a systematic comparison of cell geometry across multiple stress types in the context of size-control pathways has not been reported, and this represents a potentially valuable conceptual advance. The advance is primarily phenomenological and conceptual rather than mechanistic: the work presents new correlation between stress types and geometry but does not yet elucidate the pathways governing these responses or demonstrate a functional advantage. With additional evidence - or with qualifiers ensuring that claims match the current data - the study could make an important contribution to understanding how cells integrate environmental cues into size-control strategies.

      Audience

      Although the primary audience consists of researchers in the fields of cell growth, cell-cycle control, and stress responses in yeast, the conceptual contribution may interest broader fields such as growth homeostasis, metabolic adaptation, and pathological cell size changes in higher eukaryotes. Beyond yeast biology, the modular view of size regulation proposed here may inspire new investigations in stem cell biology, cancer research, and biotechnology where environmental adaptation and cell size are closely linked.

      Expertise: nuclear morphology; cell morphology; cell growth; cell cycle; cytoskeleton.

    3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      Cabral et al. present a analysis of the effects of environmental stress of cellular geometry in the fission yeast S. pombe. The stresses they study-oxidative, osmotic and nutritional-have previously been shown to affect cell size in fission yeast. Here, the authors do a more sophisticated analysis, measuring surface area as well as volume (for which length had previously been used as a proxy, assuming fission yeast cells are cylinders of constant width). In addition, they investigate the effect of mutations in three cell-cycle control proteins that have been proposed to regulate cell geometry: Cdc13, Cdc25 and Cdr2. It is an interesting study that could provide insight into cell-size control and environmental-stress response in fission yeast. However, I have serious concerns about the analysis of the data. In fact, as I was writing up my concerns, I noticed that the formulae in Table 1 for surface area and volume are incorrect, so the whole paper appears to require reanalysis.

      One general problem is that the authors seem to confuse statistical significance with biological significance. They claim that both oxidative and osmotic stress cause a reduction in SA/V ratio. For oxidative stress, the difference is evident, but the control and KCl-treated cells look to have indistinguishable distributions. Perhaps there is a significant statistical difference between the, but I am skeptical. (I would ask for the data table to try out the stats myself, but given the revelation below that the number will all need to be recalculated, that point is moot). In any case, the difference is certainly not biologically significant.

      Likewise, in Figure 2B, they interpret tiny changes in the SA/V. By my estimation, the difference between control and osmotic stress is only 2% (1.195/1.17), less that the wild-type case, which appears to be twice that (which is still pretty modest). The small amplitude of these changes is obscured by the fact that the graphs do not have a baseline at zero, which, as a matter of good data-presentation practice, they should.

      I am also concerned about the use of manual measurement of width at a single point along the cell. This approach is very sensitive to the choice of width point and to non-cylindrical geometries, several of which are evident in the images presented. MATLAB will return the ??? as well as the length from a mask, but even better, one can more accurately calculate the surface area and volume by assuming rotational symmetry of the mask. Given that surface area and volume calculation need to be redone anyway, as discussed below, I encourage the authors to calculate them directly from the mask, instead of using the cylindrical assumption.

      The authors also need to be more careful about their claims about size-dependent scaling. The concentration of both Cdc13 and Cdc25 scale with size (perhaps indirectly, in the case of Cdc13), but Cdr2 does not. Cdr2 activity has been proposed to scale with size, and its density at cortical nodes has been reported to scale with size, although that claim has been challenged <https://pubmed.ncbi.nlm.nih.gov/36093997>.

      Even taking the authors approach at face value, there are observations that do not seem to make sense, which led me to realize that the wrong formulae were used to calculate surface area and volume.

      In Figure 1E,F, the KCl-treated cells get shorter and wider; surely, that should result in a lower SA/V ratio. However, as noted above, in Figure 1D, they are shown to have a similar ratio. As a sanity check, I eye-balled the numbers off of the figure (control: 14 µm x 3.6 µm and KCl: 11 µm x 3.8 µm) and calculated their surface area and volume using the formula for a capsule (i.e., a cylinder with hemispheric ends).

      SA = the surface area of the two hemispheres + the surface are of the cylinder in between = 4pi(width/2)^2 + piwidth(length-width), the length-width term calculates the side length of the capsule (length without the hemispheres) from the full length of the capsule (length including the hemispheres)

      V = the volume of the two hemispheres + the volume of the cylinder in between = 4/3pi(width/2)^3 + pi(width/2)^2(length-width).

      I got SA/V ratios of around 2, which are way off from what is presented in Figure 1D, but my calculated ratio goes down in KCl, as expected, but not as reported.

      To make sure I was not doing something wrong, I was going to repeat my calculations with the formulae in Table 1, which made me realize both are incorrect. The stated formula for the cell surface area-2piRL-only represents to surface area of the cylindrical side of the cells, not its hemispherical ends. And it is not even the correct formula for the surface area of the side, because that calls for L to be the length of the side (without the hemispherical ends) not the length of the cell (which includes the hemispherical ends). L here is stated to be cell length (which is what is normally measured in the field, and which is consistent with the reported length of control cells in Figure 1E being 14 µm). The formula for the volume of a capsule in the form use in Table 1 (volume of a cylinder of length L - the volume excluded from the hemispherical ends) is piR^2L - (8-(4/3pi))R^3.

      Given these problems, I think I spent too much time thinking about the rest of the paper, because all of the calculations, and perhaps their interpretations, need to be redone.

      Minor Points:

      Strains should be identified by strain number is the text and figure legends.

      In the Introduction, "Most cell control their size" should be "Most eukaryotic cell control their size".

      Significance

      Nothing to add.

    4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

      Learn more at Review Commons


      Referee #1

      Evidence, reproducibility and clarity

      Using genetics and microscopy approaches, Cabral et al. investigate how fission yeast regulates its length and width in response to osmotic, oxidative, or low glucose stress. Miller et al. have recently found that the cell cycle regulators Cdc25, Cdc13 and Cdr2 integrate information about cell volume, time and cell surface area into the cellular decision when to divide. Cabral now build on this work and test how disruption of these regulators affects cell size adaptation. They find that each stress condition shows a distinct dependence on the individual regulators, suggesting that the complex size control network enables optimized size adaptation for each condition. Overall, the manuscript is clear and the detailed methods ensure that the experiments can be replicated.

      Major comments:

      1. It would be much easier to follow the authors' conclusions, if in addition to surface area to volume ratio, length and width, they would also plot cell volume at division in Figs. 1-4.
      2. To me, it seems that maybe even more than upon osmotic stress, the cdc13-2x strain differs qualitatively from WT in low glucose conditions, where the increased SA-V ratio is almost completely abolished.
      3. It is not entirely clear to me why two copies of Cdc13 would qualitatively affect the responses. Shouldn't the extra copy behave similarly to the endogenous one and therefore only lead to quantitative changes? Maybe the authors can discuss this more clearly or even test a strain in which Cdc13 function is qualitatively disrupted.
      4. I don't see why the authors come to the conclusion that under osmotic stress cells would maximize cell volume. It leads to a decreased cell length, doesn't it?

      Significance

      Fission yeast has long been used as a model for eukaryotic cell size regulation. So far, this research has been mostly focused on steady state size regulation. While it has long been clear that cells across organisms adapt their size in response to environmental changes, little is known about how these external inputs are processed through the size control network. Dissecting how disruption of the various branches of the size control network affects size adaptation is an important step towards a mechanistic understanding of this process. Future studies will have to build on these observations and investigate how each stress mechanistically affects the respective regulator(s). While the details of the molecular players and their contribution to size adaptation are likely specific to fission yeast, the concept of stress type-specific size adaptation that is mediated through different regulators is likely conserved and thus of broader relevance.

    1. The difference between the negative and positive ends of political trustgenerates a 9% increase in the specific support for democracy. Although thisis a large and expected effect, it may be an artifact of measurement. 11 Thesubstantive effects of tolerance and interpersonal trust are modest, and thelatter makes a difference only for specific support. More interestingly, religi-osity has no discernable effect, whereas stronger preferences for religiousindividuals in public offices decrease diffuse support by only 2% and specificsupport by 4%. This result shows that at the attitudinal level, being a piousMuslim does not necessarily have a negative impact on attitudes towarddemocracy (Esposito & Mogahed, 2007; Hassan, 2008; Jamal, 2006; Rose,2002; Tessler, 2002)

      Вывод

    1. Pratiques Punitives en Milieu Scolaire : Analyse, Effets et Recommandations

      Résumé Exécutif

      Ce document de breffage synthétise les conclusions de l'expert Vincent Bernier, docteur en psychopédagogie, concernant les pratiques punitives en milieu scolaire, et plus particulièrement la suspension.

      La recherche, unanime depuis près de 50 ans, démontre que ces pratiques sont non seulement inefficaces, mais aussi profondément néfastes pour les élèves.

      Loin de corriger les comportements problématiques, elles les exacerbent et entraînent une cascade d'effets négatifs à court, moyen et long terme, incluant la détérioration du rendement scolaire, le décrochage, l'augmentation des inégalités sociales et des problèmes de santé mentale et physique graves.

      Face à ce constat, le document expose des alternatives fondées sur des approches éducatives qui visent à développer les compétences des élèves plutôt qu'à les sanctionner.

      Ces alternatives se déclinent en deux catégories : des pratiques concrètes de gestion de classe, comme les conséquences éducatives, et des programmes structurants d'alternative à la suspension. Six recommandations stratégiques sont formulées pour le système éducatif québécois, appelant à un changement de paradigme.

      Celles-ci incluent la sensibilisation du personnel, l'interdiction de la suspension externe sans service, et le financement de programmes alternatifs éprouvés, soulignant que l'investissement dans la prévention est socialement et économiquement plus judicieux que la gestion des conséquences coûteuses de l'inaction.

      --------------------------------------------------------------------------------

      1. Définition et Typologie des Pratiques Punitives

      Selon Vincent Bernier, professeur et chercheur à l'Université de Sherbrooke, il est essentiel de distinguer clairement les pratiques punitives des pratiques éducatives.

      Les pratiques punitives s'inscrivent dans une logique de répression plutôt que d'éducation.

      Leur objectif principal est d'infliger une sanction en réponse à une faute afin de dissuader l'élève de répéter un comportement.

      Ces pratiques se situent sur un continuum d'intensité et peuvent être regroupées en trois grandes catégories :

      Les punitions classiques : Ces mesures visent à sanctionner directement un élève.

      ◦ La copie de lignes ou de textes.   

      ◦ L'assignation de travaux supplémentaires.  

      ◦ Le retrait de privilèges ou de droits.

      Les pratiques d'exclusion : Ces mesures consistent à retirer l'élève d'une situation ou d'un milieu.

      ◦ La retraite de classe (qui peut être punitive ou éducative selon son application).   

      ◦ Les retenues. 

      ◦ La suspension scolaire (interne ou externe).

      Les punitions physiques : Bien que moins courantes au Québec, elles existent dans certains contextes.

      ◦ Les châtiments corporels.  

      ◦ La contention physique.

      En opposition, les pratiques éducatives visent à aider l'élève à développer ses compétences socio-émotionnelles, son autonomie et sa responsabilité par des mesures d'aide et de soutien.

      2. Les Effets Négatifs Documentés des Pratiques Punitives

      La littérature scientifique, québécoise et internationale, documente les effets des pratiques punitives depuis les années 1970 et est décrite comme "unanime" sur leurs conséquences négatives.

      Impacts Comportementaux et Académiques

      Contrairement à l'objectif visé, les pratiques punitives ne règlent pas les problèmes de comportement ; elles les aggravent.

      Escalade des comportements : On observe une détérioration et une aggravation des comportements problématiques chez les élèves exposés à ces pratiques.

      Désengagement scolaire : Les élèves développent des trajectoires d'évitement, une réduction de la motivation à apprendre et un désengagement progressif de l'école, menant souvent à l'absentéisme.

      Baisse du rendement : Une diminution significative du rendement scolaire est fréquemment constatée.

      Décrochage scolaire : La suspension scolaire est fortement corrélée au risque de décrochage.

      Conséquences Sociales et Relationnelles

      Ces pratiques endommagent le lien entre l'élève et l'école.

      Relations négatives : Elles contribuent au développement de relations conflictuelles et négatives avec le personnel scolaire.

      Sentiment d'exclusion : Les élèves se sentent isolés, mis de côté et exclus, ce qui renforce leur marginalisation.

      Affiliation à des pairs déviants : L'exclusion du milieu scolaire augmente le risque d'affiliation à des groupes de pairs déviants et l'enrôlement potentiel dans des gangs de rue.

      Exacerbation des Inégalités

      Les pratiques punitives ne sont pas appliquées uniformément et ont pour effet d'aggraver les inégalités sociales, socio-économiques et culturelles existantes.

      Marginalisation des groupes vulnérables : Elles touchent de manière disproportionnée certains groupes, notamment :

      ◦ Les garçons. 

      ◦ Les élèves ayant des difficultés d'apprentissage.  

      ◦ Les minorités ethniques.  

      ◦ Les élèves issus de milieux défavorisés.

      Effets à Long Terme et sur la Santé

      Les études longitudinales démontrent que les conséquences de l'exposition à ces pratiques se prolongent bien au-delà de la période scolaire et affectent la santé globale des individus.

      Santé mentale et physique : Une diminution du bien-être général est observée, avec des risques accrus de :

      ◦ Dépression.  

      ◦ Consommation de drogues.  

      ◦ Automutilation.  

      ◦ Grossesses à risque.

      Judiciarisation : Le risque d'arrestation à l'âge adulte est plus élevé pour les élèves ayant été fréquemment suspendus.

      3. Alternatives aux Pratiques Punitives : Une Approche Éducative

      Pour remplacer les pratiques punitives, deux grandes catégories d'alternatives sont proposées, toutes deux centrées sur l'éducation et le développement de compétences.

      Catégorie 1 : Pratiques Éducatives Concrètes

      Ces alternatives peuvent être mises en œuvre quotidiennement par le personnel scolaire pour prévenir et gérer les écarts de conduite.

      Enseignement explicite des comportements attendus.

      Stratégies de gestion de classe proactives (ex: précorrection, proximité).

      Développement de l'autorégulation (ex: autoévaluation, autotraitement).

      Mise en place de conséquences éducatives : Cette approche est particulièrement efficace. Elle se distingue de la punition par sa finalité.

      La maxime qui la guide est que "la sanction elle est pas là pour faire mal mais elle est là pour faire sens".

      Logique et naturelle : La conséquence découle directement du comportement problématique.  

      Réparatrice : Elle vise à réparer le tort causé.  

      Responsabilisante : Elle implique l'élève dans la solution et l'aide à assumer ses responsabilités.  

      Éducative : Elle est présentée comme une mesure d'aide et une occasion d'apprentissage pour développer des compétences.

      Catégorie 2 : Alternatives Structurantes

      Ces approches sont plus globales et s'appliquent à l'échelle de l'école ou du centre de services scolaire.

      Programmes d'alternative à la suspension scolaire :

      Ces programmes, souvent menés par des organismes externes comme le YMCA, offrent un cadre structuré aux élèves qui auraient autrement été suspendus à la maison sans services.

      Fonctionnement :     

      Matin : Réalisation des travaux scolaires envoyés par l'école.     

      Après-midi : Participation à des groupes animés pour développer des compétences spécifiques (socio-émotionnelles, résolution de conflits, etc.).  

      Efficacité prouvée : Les études sur ces programmes démontrent des effets positifs à court et moyen terme, incluant une amélioration des comportements et une diminution du recours futur à la suspension.

      4. Recommandations Stratégiques pour le Système Éducatif Québécois

      Vincent Bernier formule six recommandations claires pour systématiser le passage d'une approche punitive à une approche éducative au Québec.

      | N° | Recommandation | Description | | --- | --- | --- | | 1 | Sensibiliser le personnel scolaire | Il est impératif d'informer l'ensemble du personnel scolaire sur les effets négatifs documentés des pratiques punitives afin de créer une prise de conscience collective. | | 2 | Soutenir le développement professionnel | Offrir de la formation continue pour outiller le personnel avec des pratiques alternatives efficaces et pour déconstruire les croyances erronées sur l'efficacité des punitions. | | 3 | Interdire la suspension externe sans services | Modifier les règlements d'école, les codes de vie et la Loi sur l'instruction publique pour interdire formellement la suspension scolaire à l'externe (à la maison) sans aucune mesure d'aide ou de soutien. | | 4 | Recadrer la suspension comme mesure de dernier recours | Si une suspension est inévitable, elle doit être utilisée de manière constructive : soit à l'interne avec des services et des mesures d'aide, soit à l'externe mais en référant l'élève à un programme d'alternative structuré. L'objectif doit être de "servir plutôt que de sévir". | | 5 | Financer et déployer les programmes alternatifs | Investir dans le déploiement de programmes d'alternative à la suspension, via des organismes communautaires locaux, pour que chaque école au Québec puisse avoir accès à ce type de service. | | 6 | Consigner systématiquement les suspensions | Mettre en place un système provincial pour consigner toutes les suspensions (internes et externes) afin d'obtenir un portrait juste et clair du phénomène (fréquence, durée, profils d'élèves) et ainsi prendre des décisions éclairées. |

      5. Conclusion : L'Urgence d'un Virage vers la Prévention

      La discussion met en lumière une réalité documentée depuis un demi-siècle : les pratiques punitives sont contre-productives.

      L'expert souligne que le Québec doit réfléchir aux services qu'il souhaite offrir à ses jeunes.

      Il est démontré que le coût sociétal de l'inaction (gestion de la criminalité, des problèmes de santé mentale, du décrochage) est largement supérieur au coût d'investissement dans des mesures de prévention et des programmes alternatifs.

      Le passage d'une culture de la punition à une culture du soutien n'est pas seulement une question de bienveillance, mais une décision stratégique et économique pour l'avenir.

    1. Analyse de la Psychologie des Séparations

      Résumé Exécutif

      Ce document de synthèse analyse les multiples facettes de la séparation, qu'elle soit amoureuse ou amicale, en se basant sur une combinaison de témoignages personnels et d'analyses psychologiques.

      Il en ressort que la séparation est un processus psychobiologique profond, dont la tolérance se construit dès l'enfance à travers les mécanismes d'attachement.

      La douleur de la rupture n'est pas purement émotionnelle ; elle se manifeste par des symptômes physiques concrets et des changements hormonaux mesurables, activant dans le cerveau des zones similaires à celles de la douleur physique.

      Le processus de deuil est une étape cruciale et individuelle, où le refoulement des émotions peut mener à une "explosion" ou une "implosion".

      La guérison passe souvent par la capacité à donner un sens à l'événement, en construisant un récit cohérent de ce qui s'est passé. Des approches thérapeutiques comme l'EMDR peuvent aider à retraiter les souvenirs traumatiques.

      Enfin, la séparation, bien que douloureuse, peut se révéler être une opportunité de croissance, permettant une meilleure connaissance de soi, la redéfinition de ses propres limites et une forme de "renaissance" personnelle.

      Le soutien social et l'expression des émotions, notamment par l'écriture, sont des aides précieuses tout au long de ce processus.

      --------------------------------------------------------------------------------

      1. La Nature Fondamentale de la Séparation : Attachement et Anxiété

      La séparation est un événement complexe qui puise ses racines dans les fondements psychologiques de l'individu et génère une anxiété profonde.

      L'Importance de l'Attachement Primal : La capacité à gérer une séparation et à tolérer la solitude est directement liée aux expériences d'attachement vécues dès les premiers instants de la vie.

      Les relations avec les parents et les amis durant l'enfance déterminent la manière dont un individu entrera en relation et vivra les ruptures futures.

      La Douleur Partagée de la Rupture : Contrairement à une idée reçue, l'acte de quitter une personne peut être aussi difficile et douloureux que d'être quitté.

      La décision de rompre est souvent lourde et entraîne des conséquences importantes pour celui qui la prend. Un témoin exprime ce dilemme : "je savais que j'allais lui faire très mal mais je pouvais pas faire autrement".

      Anxiété et Peur de la Solitude : La séparation engendre une forte anxiété, souvent liée à la peur de se retrouver seul et d'affronter les étapes du deuil.

      Perte d'Identité et Confiance en Soi : Pour certains, la relation est si fusionnelle que la séparation entraîne un sentiment de néant.

      Une personne témoigne : "à partir du moment où il est parti vraiment et c'est là que j'ai commencé à ressentir que j'étais plus rien quoi comme si ma vie finalement était suspendue à la sienne".

      La capacité à se détacher est donc intrinsèquement liée à l'estime de soi et à la conviction de pouvoir exister indépendamment de l'autre.

      Le Poids des Modèles Familiaux : Les schémas parentaux peuvent influencer la perception de la séparation.

      Une personne ayant vu sa mère "tout supporter" pour "préserver la famille" a ressenti sa propre rupture comme un échec à maintenir ce modèle, faisant le "deuil de la famille".

      2. Les Manifestations Physiques et Neurologiques de la Douleur

      La souffrance d'une rupture n'est pas une simple métaphore ; elle s'inscrit dans le corps et le cerveau de manière quantifiable et observable.

      Réactions Organiques Généralisées : L'ensemble de l'organisme réagit à une séparation. Les symptômes physiques courants incluent :

      ◦ Changements d'appétit (perte de 6 kg en trois mois de grossesse pour une témoin).   

      ◦ Maux de tête et maux d'estomac.  

      ◦ Troubles du sommeil et du rythme cardiaque.  

      ◦ Sensations localisées, comme une "boule au ventre" persistante.   

      ◦ Réactions de stress physique comme la transpiration des mains.

      La Douleur Émotionnelle et la Douleur Physique : Des études montrent que la souffrance émotionnelle d'une rupture active des zones cérébrales très proches de celles activées par une douleur physique intense.

      La source cite : "la souffrance qu'on ressent quand quelqu'un qu'on aime n'est plus à nos côtés cette douleur est vraiment très proche de la douleur physique qu'on ressent quand on se fait très mal".

      Changements Hormonaux et État de Manque : La période qui suit immédiatement la rupture est marquée par des bouleversements hormonaux significatifs, créant un état de manque comparable à une addiction.

      Le "système de récompense" du cerveau "réclame la personne qui est partie".

      | Hormone | Niveau après la rupture | Rôle et Conséquence | | --- | --- | --- | | Dopamine | Très élevé | Associée au système de récompense, son taux élevé explique les pensées obsessionnelles envers l'autre. | | Ocytocine | Baisse | "L'hormone de l'attachement", sa diminution accentue le sentiment de détachement et de perte. | | Sérotonine | Très faible | "L'hormone du contentement", son faible niveau contribue à l'état de tristesse et de nostalgie profonde. |

      3. Le Processus de Deuil et les Stratégies de Guérison

      La traversée de la séparation est un processus long et individuel, qui nécessite d'affronter la douleur pour pouvoir la surmonter.

      La Nécessité de Vivre ses Émotions : Le refoulement est une stratégie à risque. Il est essentiel de "s'autoriser à vivre son émotion, son chagrin, sa tristesse".

      Le refoulement mène à deux issues possibles : "soit au bout d'un moment on explose ou soit on implose en soi".

      La Création d'un Récit Cohérent : Une étape clé de la guérison est la capacité à élaborer un récit, à trouver un sens à ce qui s'est passé.

      Se poser les questions "pourquoi ça m'est arrivé ?", "quelles leçons j'en tire ?" et "qu'est-ce que je ferais différemment ?" permet de créer une histoire cohérente qui aide à "surmonter les coups durs".

      Thérapies et Neuroplasticité :

      Traumatisme et Système Limbique : Un événement traumatisant comme une rupture peut rester "bloqué dans le cerveau émotionnel" (le système limbique).  

      La Thérapie EMDR : Cette thérapie utilise des stimulations bilatérales alternées (mouvements oculaires) pour permettre au cerveau de "retraiter" le souvenir traumatique.

      Grâce à la neuroplasticité, elle aide à créer de nouvelles connexions synaptiques, permettant de "porter un nouveau regard sur ce qui nous est arrivé et de soulager son vécu émotionnel".

      L'Importance du Soutien Social : L'isolement aggrave la douleur. Rompre l'isolement en se reconnectant à sa "communauté" et à ses amis est crucial.

      Le soutien consiste principalement à "être à ses côtés et de l'écouter".

      Autres Mécanismes d'Adaptation :

      L'Écriture : Écrire, comme rédiger des lettres (même non envoyées), est un moyen de se "plonger en soi" et de clarifier ses pensées.  

      Le Temps : Le processus est individuel et ne suit pas un schéma unique. Il est essentiel de "laisser du temps au processus de séparation".

      4. La Rupture Amicale : Une Douleur Similaire

      La source souligne que la fin d'une amitié profonde peut être aussi dévastatrice qu'une rupture amoureuse, car les mêmes mécanismes d'attachement sont en jeu.

      Une Souffrance Équivalente : La douleur ressentie est décrite comme étant la même que pour une perte amoureuse.

      Un témoin décrit son état : "c'était douloureux c'était comme une rupture active", "j'avais une boule dans la gorge parfois j'avais envie de pleurer".

      Le Manque de Communication : Contrairement aux ruptures amoureuses, qui sont souvent officialisées, les amitiés ont tendance à "se défaire lentement".

      Le manque de communication directe est souvent au cœur du problème.

      "Ce qui nous a manqué avec mon ami cette communiquer", constate un témoin.

      L'idéal serait de "rompre avec un ami comme on rond avec un amoureux ou une amoureuse".

      L'Incompréhension et les Questions Sans Réponse : L'absence d'explication claire laisse la personne dans le questionnement et la douleur.

      "Je sais pas exactement ce qui s'est passé pour ça il aurait fallu qu'on s'explique".

      5. La Séparation comme Catalyseur de Croissance Personnelle

      Bien qu'étant une épreuve difficile, la séparation peut paradoxalement devenir une opportunité de transformation et de renouveau.

      Une "Renaissance" : Plusieurs témoignages évoquent un sentiment de renaissance après avoir traversé le deuil.

      Une personne décrit cette étape comme "une renaissance de maréchaux", où elle a retrouvé la force et le courage d'agir selon ses propres envies.

      Meilleure Connaissance de Soi : La rupture force à l'introspection.

      Elle "peut aussi nous permettre de nous connaître et de savoir qu'est-ce qu'on veut qu'est-ce qu'on ne veut plus pour sa vie future".

      Redéfinition des Limites : C'est l'occasion de définir des limites personnelles qui n'étaient pas respectées.

      Une conclusion forte émerge d'un témoignage : "on ne doit pas douter de quelque chose qui concerne son intégrité. [...] je veux plus que mon intégrité soit atteint".

      Vers des Relations Futures Plus Stables :

      L'expérience montre que les personnes qui se donnent le temps de vivre pleinement le processus de séparation "parviennent généralement mieux à nouer une relation stable par la suite".

      La Possibilité d'une Nouvelle Relation : Une séparation amoureuse peut évoluer.

      En se débarrassant des "restes de relations amoureuses" et en trouvant un "nouveau cadre", il est possible de rester amis.

    1. Author response:

      The following is the authors’ response to the previous reviews

      Reviewer 1

      Minor

      The main substance of my previous comment I suppose targeted a deeper issue - namely whether such a result is reflecting a resolution to a 'neural prediction' puzzle or a 'perceptual prediction' puzzle. Of course, these results tell us a great deal about a potential resolution for how dampening and sharpening might co-exist in the brain - but in the absence of corresponding perceptual effects (or a lack of correlation between neural and perceptual variables - as outlined in this revision) I do wonder if any claims about implications for perception might need moderation or caveating. To be honest, I don't think the authors *need* to make any more changes along these lines for this paper to be acceptable - it is more an issue they might wish to consider themselves when contextualizing their findings.

      Thank you for the thoughtful comment. We have now added a caveat to the relevant section of the discussion to make it clearer that we are discussing neural results, not perceptual results (p.20, lines 378-379).

      I am also happy with the changes that the authors have made justifying which claims can and cannot made based on a statistical decoding test against 'chance' in a single condition using t-tests. I was perhaps a little unclear when I spoke about 'comparisons against 0' in my original review, when the key issue (as the authors have intuited!) is about comparisons against 'chance' (where e.g., 0% decoding above chance is the same thing as 'chance'!). The authors are of course correct in the amendment they have made on p.29 to make clear this is a 'fixed effects analysis' - though I still worry this could be a little cryptic for the average reader. I am not suggesting that the authors run more analyses, or revise any conclusions, but I think it would be more transparent if a note was added along the lines of "while the fixed effects approach (one-sample t-test) enables us to establish whether some consistent informative patterns are detectable in these particular subjects, the results from our paired t-tests support inference to the wider population".

      This sentence has been added for increased transparency (p. 27, lines 544-547).

      Reviewer 3

      Major

      (1) In the previous round of comments, I noted that: "I am not fully convinced that Figures 3A/B and the associated results support the idea that early learning stages result in dampening and later stages in sharpening. The inference made requires, in my opinion, not only a significant effect in one-time bin and the absence of an effect in other bins. Instead to reliably make this inference one would need a contrast showing a difference in decoding accuracy between bins, or ideally an analysis not contingent on seemingly arbitrary binning of data, but a decrease (or increase) in the slope of the decoding accuracy across trials. Moreover, the decoding analyses seem to be at the edge of SNR, hence making any interpretation that depends on the absence of an effect in some bins yet more problematic and implausible". The authors responded: "we fitted a logarithmic model to quantify the change of the decoding benefit over trials, then found the trial index for which the change of the logarithmic fit was < 0.1%. Given the results of this analysis and to ensure a sufficient number of trials, we focused our further analyses on bins 1-2". However, I do not see how this new analysis addresses the concern that the conclusion highlights differences in decoding performance between bins 1 and 2, yet no contrast between these bins are performed. While I appreciate the addition of the new model, in my current understanding it does not solve the problem I raised. I still believe that if the authors wish to conclude that an effect differs between two bins they must contrast these directly and/or use a different appropriate analysis approach.

      Relatedly, the logarithmic model fitting and how it justifies the focus on analysis bin 1-2 needs to be explained better, especially the rationale of the analysis, the choice of parameters (e.g., why logarithmic, why change of logarithmic fit < 0.1% as criterion, etc), and why certain inferences follow from this analysis. Also, the reporting of the associated results seems rather sparse in the current iteration of the manuscript.

      We thank the reviewer for this important point. Following your suggestion, we conducted additional post-hoc tests directly comparing the first and second bins. We found significant differences between bins in the invalid trials, but not the valid trials, suggesting that sharpening/dampening effects are condition specific. This is discussed in the manuscript on p.14, lines 268-271; p.15, 280-284; p.20, lines 382-386.

      A logarithmic analysis was chosen as learning is usually found to be a nonlinear process; learning effects occur rapidly before stabilising relatively early, as seen in Fig. 2D. This is consistent with other research which found that logarithmic fits efficiently describe learning curves in statistical learning (Kang et al., 2023; Siegelman et al., 2018; Choi et al., 2020). By utilising a change of logarithmic fit at <0.1% as a criterion, it is ensured that virtually zero learning took place after that point, allowing us to focus our analysis on learning effects as they developed and providing a more accurate model of representational change. This is explained in the manuscript on p.13, lines 250-251; p.27-28, lines 557-563.

      (2) A critical point the authors raise is that they investigate the buildup of expectations during training. They go on to show that the dampening effect disappears quickly, concluding: "the decoding benefit of invalid predictions [...] disappeared after approximately 15 minutes (or 50 trials per condition)". Maybe the authors can correct me, but my best understanding is as follows: Each bin has 50 trials per condition. The 2:1 condition has 4 leading images, this would mean ~12 trials per leading stimulus, 25% of which are unexpected, so ~9 expected trials per pair. Bin 1 represents the first time the participants see the associations. Therefore, the conclusion is that participants learn the associations so rapidly that ~9 expected trials per pair suffice to not only learn the expectations (in a probabilistic context) but learn them sufficiently well such that they result in a significant decoding difference in that same bin. If so, this would seem surprisingly fast, given that participants learn by means of incidental statistical learning (i.e. they were not informed about the statistical regularities). I acknowledge that we do not know how quickly the dampening/sharpening effects develop, however surprising results should be accompanied with a critical evaluation and exceptionally strong evidence (see point 1). Consider for example the following alternative account to explain these results. Category pairs were fixed across and within participants,i.e. the same leading image categories always predicted the same trailing image categories for all participants. Some category pairings will necessarily result in a larger representational overlap (i.e., visual similarity, etc.) and hence differences in decoding accuracy due to adaptation and related effects. For example, house  barn will result in a different decoding performance compared to coffee cup  barn, simply due to the larger visual and semantic similarity between house and barn compared to coffee cup and barn. These effects should occur upon first stimulus presentation, independent of statistical learning, and may attenuate over time e.g., due to increasing familiarity with the categories (i.e., an overall attenuation leading to smaller between condition differences) or pairs.

      We apologise for the confusion, there are 50 expected trials per bin per condition. The trial breakdown is as follows. Each participant completed 1728 trials, split equally across 3 mappings (two 2:1 maps and one 1:2 map), giving 1152 trials in the 2:1 mapping. Stimuli were expected in 75% of trials (864), leaving 216 per bin, and 54 per leading image in each bin. We have clarified this in the script (p.14, line 267; p.15, line 280). This is in line with similar studies in the field (e.g. Han et al., 2019).

      (3) In response to my previous comment, why the authors think their study may have found different results compared to multiple previous studies (e.g. Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011), particularly the sharpening to dampening switch, the authors emphasize the use of non-repeated stimuli (no repetition suppression and no familiarity confound) in their design. However, I fail to see how familiarity or RS could account for the absence of

      sharpening/dampening inversion in previous studies.

      First, if the authors argument is about stimulus novelty and familiarity as described by Feuerriegel et al., 2021, I believe this point does not apply to the cited studies. Feuerriegel et al., 2021 note: "Relative stimulus novelty can be an important confound in situations where expected stimulus identities are presented often within an experiment, but neutral or surprising stimuli are presented only rarely", which indeed is a critical confound. However, none of the studies (Han et al., 2019; Richter et al., 2018; Kumar et al., 2017; Meyer and Olson, 2011) contained this confound, because all stimuli served as expected and unexpected stimuli, with the expectation status solely determined by the preceding cue. Thus, participants were equally familiar with the images across expectation conditions.

      Second, for a similar reason the authors argument for RS accounting for the different results does not hold either in my opinion. Again, as Feuerriegel et al. 2021 correctly point out: "Adaptation-related effects can mimic ES when the expected stimuli are a repetition of the last-seen stimulus or have been encountered more recently than stimuli in neutral expectation conditions." However, it is critical to consider the precise design of previous studies. Taking again the example of Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011. To my knowledge none of these studies contained manipulations that would result in a more frequent or recent repetition of any specific stimulus in the expected compared to unexpected condition. The crucial manipulation in all these previous studies is not that a single stimulus or stimulus feature (which could be subject to familiarity or RS) determines the expectation status, but rather the transitional probability (i.e. cue-stimulus pairing) of a particular stimulus given the cue. Therefore, unless I am missing something critical, simple RS seems unlikely to differ between expectation condition in the previous studies and hence seems implausible to account for differences in results compared to the current study.

      Moreover, studies cited by the authors (e.g. Todorovic & de Lange, 2012) showed that RS and ES are separable in time, again making me wonder how avoiding stimulus repetition should account for the difference in the present study compared to previous ones. I am happy to be corrected in my understanding, but with the currently provided arguments by the authors I do not see how RS and familiarity can account for the discrepancy in results.

      The reviewer is correct in that the studies cited (Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011) ensure that participants are equally familiar with the images across expectation conditions. Where the present study differs is that participants are not familiar with individual exemplars at all. Han et al., 2019 used a pool of 30 individual images, and subjects underwent exposure sessions lasting two hours each daily for 34 days prior to testing. Kumar et al., 2017 used a pool of 12 images with subjects being exposed to each sequential pair 816 times over the course of the training period. Meyer & Olsen, 2011 used pure tones at five different pitch levels. While familiarity of stimuli across conditions was controlled for in these studies in the sense that familiarity was constant across conditions, novelty was not controlled for. The present study uses a pool of ~3500 images, which are unrepeated across trials.

      Feuerriegel et al., 2021 also points out: “There are also effects of adaptation that are dependent on the recent stimulation history extending beyond the last encountered stimulus and long-lag repetition effects that occur when the first and second presentation of a stimulus is separated by tens or even hundreds of intervening images”. Bearing this in mind, and given the very small pool of stimuli being used by Han et al., 2019; Kumar et al., 2017; Meyer and Olson, 2011, it stands to reason that these studies may still have built-in but unaccounted for effects relating to the repetition of exemplars. Thus, our avoidance of those possible confounds, in addition to foregoing any prior training, may elicit differing results. Furthermore, as pointed out by Walsh et al. 2020, methodological heterogeneity (such as subject training) can produce contrasting results as PP makes divergent predictions regarding the properties of prediction error given different permutations of variables such as training, transitional probabilities, and conditional probabilities. In our case, the use of differing methodology was intentional. These issues have been discussed in more detail on p.5, lines 112-115; p.19, lines 368-377; p.20, lines 378-379).

      Minor

      (1) The authors note in their reply to my previous questions that: "As mentioned above, we opted to target our ERP analyses on Oz due to controversies in the literature regarding univariate effects of ES (Feuerriegel et al., 2021)". This might be a lack of understanding on my side, but how are concerns about the reliability of ES, as outlined by Feuerriegel et al. (2021), an argument for restricting analyses to 1 EEG channel (Oz)? Could one not argue equally well that precisely because of these concerns we should be less selective and instead average across multiple (occipital) channels to improve the reliability of results?

      The reviewer is correct in suggesting that a cluster of occipital electrodes may be more reliable than reporting one single electrode. We have amended the analysis to examine electrodes Oz, O1, and O2 (p.9, lines 187-188; p.11, lines 197-201).

      (2) The authors provide a github link for the dataset and code. However, I doubt that github is a suitable location to share EEG data (which at present I also cannot find linked in the github repo). Do the authors plan to share the EEG data and if so where?

      Thank you for bringing this to my attention. EEG data has now been uploaded at osf.io/x7ydf and linked to the github repository (p.28, lines 569-570).

      (3) The figure text could benefit from additional information; e.g. Fig.1C and Fig.3 do not clarify what the asterisk indicates; p < ? with or without multiple comparison correction?

      Thank you for pointing out this oversight, the figure texts have been amended (p. 9, line 168; p.16, line 289).

    1. Reviewer #3 (Public review):

      Summary:

      Brands and colleagues investigate how temporal adaptation can aid object recognition, and what neural computations may underlie these effects. They employed a previously published experimental paradigm to study how adaptation to temporally constant distractor input facilitates the recognition of a newly appearing target object. Specifically, they studied how this effect is modulated by the contrast of the target object.

      They found that adaptation enhances the recognition of high-contrast objects more than that of low-contrast objects. This behavioral effect was mirrored by a larger effect of adaptation on the response to the high-contrast objects in relatively higher visual areas.

      To investigate what neural computations can support this interaction, they implement several candidate neural mechanisms in a deep convolutional neural network: additive suppression, divisive suppression, and lateral recurrence. The authors conclude that divisive and additive suppression, which are intrinsic to the neuron, best explain the interaction between contrast and adaptation in the human data. They further show that these mechanisms, and divisive suppression in particular, show increased robustness to spatial shifts of the adaptor stimulus, hinting and potential perceptual benefits.

      Strengths:

      (1) Overall, this is a well-written paper, supported by thorough analyses and illustrated with clear, well-designed figures that effectively show overall trends as well as data variance. The authors tell a compelling story while responsibly steering away from overreaching conclusions.

      (2) What makes this paper stand out is its comprehensive approach to understanding the behavioral benefit of neural adaptation and its mechanistic underpinnings. The authors effectively achieve this through integrating new behavioral and neural data with simulations using neural network models.

      (3) The findings convincingly demonstrate that neuronally intrinsic adaptation mechanisms are sufficient to explain the observed interaction between temporal adaptation, contrast, and object recognition. Furthermore, the paper highlights that these intrinsic mechanisms offer superior robustness compared to learned lateral recurrence mechanisms, which, while being more expressive, can also be more brittle.

      Weaknesses:

      While the results and conclusion are well supported, there were a few major points that need clarification for me.

      (1) Divisive normalization

      I was confused by the author's classification of divisive normalization as a neuronally intrinsic mechanism, that is, one that operates within a single neuron, independent of interactions with other neurons.

      My understanding is that divisive normalization, as originally proposed by Heeger in the early nineties, describes a mechanism where neurons integrate pooled activity from neighboring cells to mutually inhibit one another. In this form, divisive normalization is fundamentally an interneuronal mechanism involving recurrence. Adding to the confusion, the authors highlight in the introduction their interest in divisive normalization for its relation to stimulus contrast, a relation likely linked to neuronal pooling.

      However, my reading of the methods section (Equations 6 and 7) suggests the authors implemented only a temporal feedback component, leaving out the pooling across neurons (Equation 5). This distinction should be disambiguated early in the paper. I recommend choosing a less ambiguous term than "divisive normalization". Even "temporal divisive normalization" is still ambiguous, as lateral neuronal interactions are also inherently temporal.

      (2) Parietal electrodes

      The paper's adapter-specific effects are centered around the P9/P10 electrodes, which the authors identify as "parietal." However, it is unclear to me which part of the cortex drives these electrodes, particularly whether it is actually the parietal cortex. I am no expert in EEG, but based on the topomaps in Figures 4 and 5, it appears that these electrodes cover more posterior occipito-temporal regions rather than truly parietal regions. Given the central role of P9/P10 to the main findings, the paper would be significantly improved for non-EEG readers by clarifying which cortical regions are covered by these electrodes.

      (3) Interpretation of non-significant statistical results

      In some places, the authors attach relatively strong claims to non-significant statistical results. For example, in Figure 5D, they claim that there is no effect of contrast on occipital electrodes, based on a non-significant p-value. P-values do not quantify evidence for the null hypothesis, so the authors should be careful with such claims. In fact, Figure 5D shows such a clear negative slope, with variance comparable to Figure 5A, that I am surprised that the p-value for the slope of Figure 5D was in fact so large. A similar issue arises in the discussion for Figure 6, where the authors claim that the effect of contrast is adapter-specific. However, this claim is based on the observation that is significant for same-noise trials, but not for different-noise or blank trials. To statistically substantiate the claims that there is an adapter-specific effect, the authors should directly compare the slope for same-noise trials with the slope for different-noise/blank trials.

      (4) The match between behavior and models

      The authors' claim that models with intrinsic adaptation better match the interaction between contrast and temporal adaptation observed in human behavior is not fully substantiated. This conclusion appears to be based on a qualitative assessment of Figure 8, which, in my view, does not unambiguously rule out an interaction for lateral recurrence. Furthermore, a potential confounding factor is the ceiling effect that limits higher accuracy values. Indeed, conditions where the interaction was not/less (i.e., shorter time sequences and lateral inhibition) are also the conditions where accuracy values are closer to this ceiling, which may mask a potential interaction.

    1. Reviewer #2 (Public review):

      Summary:

      This work presents a reproducible, scalable workflow for spike sorting that leverages parallelization to handle large neural recording datasets. The authors introduce both a processing pipeline and a benchmarking framework that can run across different computing environments (workstations, HPC clusters, cloud). Key findings include demonstrating that Kilosort4 outperforms Kilosort2.5 and that 7× lossy compression has minimal impact on spike sorting performance while substantially reducing storage costs.

      Strengths:

      (1) Extremely high-quality figures with clear captions that effectively communicate complex workflow information.

      (2) Very detailed, well-written methods section providing thorough documentation.

      (3) Strong focus on reproducibility, scalability, modularity, and portability using established technologies (Nextflow, SpikeInterface, Code Ocean).

      (4) Pipeline publicly available on GitHub with documentation.

      (5) Clear cost analysis showing ~$5/hour for AWS processing with transparent breakdown.

      (6) Good overview of previous spike sorting benchmarking attempts in the introduction.

      (7) Practical value for the community by lowering barriers to processing large datasets.

      Weaknesses:

      No significant weaknesses were identified, although it is noted that the limitations section of the discussion could be expanded.

    2. Reviewer #3 (Public review):

      Summary:

      The authors provide a highly valuable and thoroughly documented pipeline to accelerate the processing and spike sorting of high-density electrophysiology data, particularly from Neuropixels probes. The scale of data collection is increasing across the field, and processing times and data storage are growing concerns. This pipeline provides parallelization and benchmarking of performance after data compression that helps address these concerns. The authors also use their pipeline to benchmark different spike sorting algorithms, providing useful evidence that Kilosort4 performs the best out of the tested options. This work, and the ability to implement this pipeline with minimal effort to standardize and speed up data processing across the field, will be of great interest to many researchers in systems neuroscience.

      Strengths:

      The paper is very well written and clear in most places. The accompanying GitHub and ReadTheDocs are well organized and thorough. The authors provide many benchmarking metrics to support their claims, and it is clear that the pipeline has been very thoroughly tested and optimized by users at the Allen Institute for Neural Dynamics. The pipeline incorporates existing software and platforms that have also been thoroughly tested (such as SpikeInterface), so the authors are not reinventing the wheel, but rather putting together the best of many worlds. This is a great contribution to the field, and it is clear that the authors have put a lot of thought into making the pipeline as accessible as possible.

      Weaknesses:

      There are no major weaknesses. I have only a handful of very minor questions and suggestions that could clarify/generalize aspects of the pipeline or make the text more understandable to non-specialists.

      (1) Could the authors please expand on the statement on line 274, that processing their test dataset serially "on a single GPU-capable cloud workstation... would take approximately 75 hours and cost over 90 USD." How were these values calculated? I was a bit surprised that this is a >4-fold slow-down from their pipeline, but only increases the cost by ~1.35x, if I understood correctly. More context on why this is, and maybe some context on what a g4dn.4xlarge is compared to the other instances, might help readers who are less familiar with AWS and cloud computing.

      (2) One of the most commonly used preprocessing pipelines for Neuropixels data is the CatGT/ecephys pipeline from the developers of SpikeGLX at Janelia. It may be worth commenting very briefly, either in the preprocessing section or in the discussion, on how the preprocessing steps available in this pipeline compare to the steps available in CatGT. For example, is "destriping" similar to the "-gfix" option in catGT to remove high-amplitude artifacts?

      (3) Why are there duplicate units (line 194), and how often is this an issue? I understand that this is likely more of a spike sorter issue than an issue with this pipeline, but 1-2 sentences elaborating why might be helpful for readers.

      (4) It seems from the parameter files on GitHub that the cluster curation parameters are customizable - correct? If so, it may be worth explicitly saying so in the curation section of the text, as the presented recipe will not always be appropriate. A presence ratio of >0.8 could be particularly problematic for some recordings, for example, if a cell is only active during a specific part of the behavior, that may be a feature of the experiment, or the animal could be transitioning between sleep and wake states, in which different units may become active at different times.

      (5) The axis labels in Figures 3d-e are too small to see, and Figure 3d would benefit from a brief description of what is shown.

      (6) What is the difference between "neural" and "passing QC" in Figure 4?

      (7) I understand the current paper is focused on spike data, so there may not be an answer to this, but I am curious about the NP2.0 probes that save data in wideband. Does the lossy compression negatively affect the LFP data? Is software filtering applied for the spike band before or after compression?

    1. Reviewer #2 (Public review):

      This manuscript presents an impressive and novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms. The proposed multi-scale nested structure offers valuable new insights into functional brain states and their dynamics. Importantly, investigation of global brain states in the context of a naturalistic viewing context represents an important and timely contribution that addresses unresolved issues about global signals and anticorrelations in resting-state fMRI. This manuscript presents a novel investigation of organizational principles governing brain activity at both global and local scales during naturalistic viewing paradigms. The authors demonstrate that brain activity during naturalistic viewing is dominated by two anti-correlated states that toggle between each other with a third transitional state mediating between them. The successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) is a particular strength. The successful replication across three independent datasets (StudyForrest, NarrattenTion, and CamCAN) is a particular strength, and I appreciate the authors' careful documentation of both convergent and divergent findings across these samples.

      Overall, this manuscript makes important contributions to our understanding of large-scale brain organization during naturalistic cognition. The multi-scale framework and robust replication across datasets are notable strengths. Addressing the concerns raised below will substantially strengthen the impact and interpretability of this work.

      (1) Network Definition and Specificity

      (a) The authors adopt an overly broad characterization of the Default Mode Network (DMN). The statement that "areas most active in the default mode state... consist of the precuneus, angular gyrus, large parts of the superior and middle temporal cortex, large parts of the somatomotor areas, frontal operculi, insula, parts of the prefrontal cortex and limbic areas" includes regions typically assigned to other networks. The insula is canonically considered a core node of the Salience Network/Ventral Attention Network (VAN), not the DMN. Also, not clear which limbic areas? The DMN findings reported need to be critically reassessed in this context.

      (b) Given the proposed role of state switching in your framework, a detailed analysis of salience network nodes (particularly insula and dorsal ACC) would be highly informative.

      (c) While you report transition-related signals in the visual and auditory cortex, the involvement of insular and frontal control systems in state transitions remains unaddressed.

      (d) My recommendation is to provide a more anatomically precise characterization of network involvement, particularly distinguishing DMN from salience/VAN regions, and analyze the specific role of salience network nodes in mediating state transitions.

      (2) Distinguishing Top-Down from Stimulus-Driven Effects

      (a) The finding that "the superior parietal lobe (SPL) and the frontal eye fields (FEF) show the greatest overlap between their local ROI state switches and the global state switches" raises an important question: To what extent are these effects driven by overt changes in visual gaze or attention shifts triggered by stimulus features versus internally-generated state changes?

      (b) Similarly, the observation that DAN areas show the highest overlap with global state changes in StudyForrest and NarrattenTion, while VAN shows the highest overlap in CamCAN, lacks sufficient anatomical detail regarding which specific nodes are involved. This information would help clarify whether insular regions and other VAN components play distinct roles in state switching.

      (c) It will be important to (i) discuss potential confounds from eye movements and stimulus-driven attention shifts; (ii) provide detailed anatomical breakdowns of network nodes involved in state transitions, particularly for VAN; (iii) if eye-tracking data or any other relevant stimulus-related data are available, include analyses examining relationships between these measures and state transitions.

      (3) Physiological Interpretation of the "Down" State

      The linkage between the "Down" state and the Default Mode State (DMS) is intriguing but requires deeper physiological grounding. Recent work by Epp et al. (Nature Neuroscience, 2025) demonstrates that decreased BOLD signal in DMN regions does not necessarily indicate reduced metabolic activity and can reflect neurovascular coupling modes with specific metabolic profiles. It would be useful to discuss whether your "Down" state might represent a particular neurovascular coupling mode with distinct metabolic demands rather than simply reduced neural activity. Alternatively, your analytical approach might be insensitive to or unconfounded by such neurovascular uncoupling. This discussion would substantially enrich the biological interpretation of the DMS versus TPS dual mechanism framework.

      (4) Statistical Validation of Bimodality Detection

      The method of selecting bimodal timepoints using the Dip test followed by sign-alignment is novel and creative. However, this filter-then-align procedure could potentially introduce circularity by imposing the anticorrelated structure the authors aim to detect. It would be important to implement validation analyses to confirm that anticorrelation is an intrinsic property rather than a methodological artifact. Approaches include leave-one-subject-out cross-validation, unsupervised dimensionality reduction (e.g., PCA) applied independently to verify the anticorrelated structure, and split-half reliability analysis. Such validation would significantly strengthen the statistical foundation of findings.

      (5) Quantifying Hyperalignment Contribution

      The appendix notes that non-hyperaligned data show a coarser structure, but the specific contribution of hyperalignment to your findings requires more thorough quantification. Please provide a systematic comparison of results with and without hyperalignment, demonstrating that similar (even if weaker) anatomical correspondence exists in native subject space. This would establish that the mesoscale organizational principles you identify are not artifacts of the alignment procedure but reflect genuine neurobiological organization. Consider presenting correlation coefficients or overlap metrics quantifying the similarity of state structures before and after hyperalignment.

      (6) Functional Characterization of the Unimodal State

      The observation that the brain spends approximately 34% of its time in a "Unimodal State" is presented primarily as a transition period. This is an interesting observation. However, it would be useful to characterize the functional connectivity profile of the unimodal state. Specifically, investigate whether it represents a distinct functional state with its own characteristic connectivity pattern. More detailed analysis would provide a more complete picture of temporal brain dynamics during naturalistic viewing and could yield new perspectives on how the brain reorganizes between stable states.

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      We sincerely appreciate the feedback, attention to detail and timeliness of the referees for our manuscript. Below, we provide a point-by-point response to all comments from the referees, detailing the changes we have already made, and those that are in progress. Referee's comments will appear in bolded text, while our responses will be unbolded. Any text quoted directly from the manuscript will be italicised and contained within "quotation marks". Additionally, we have grouped all comments into four categories (structural changes, minor text changes, experimental changes, figure changes), comments are numbered 1-n in each of these categories. Please note: this response to reviewer's comments included some images that cannot be embedded in this text-only section.

      1. General Statements

      We appreciate the overall highly positive and enthusiastic comments from all reviewers, who clearly appreciated the technical difficulty of this study, and noted amongst other things that this study represents" a major contribution to the future advancement of oocyst-sporozoite biology" and the development of the segmentation score for oocysts as a "major advance[ment]". We apologise for the omission of line numbers on the document sent to reviewers, we removed these for the bioRxiv submission without considering that this PDF would be transferred across to Review Commons.

      We have responded to all reviewers comments through a variety of text changes, experimental inclusions, or direct query response. Significant changes to the manuscript since initial submission are as follows:

      1. Refinement of rhoptry biogenesis model: Reviewers requested more detail around the content of the AORs, which we had previously suggested were a vehicle for rhoptry biogenesis as we saw they carried the rhoptry neck protein RON4. To address this, we first attempted to address this using antibodies against rhoptry bulb proteins but were unsuccessful. We then developed a * berghei* line where there rhoptry bulb protein RhopH3 was GFP-tagged. Using this parasite line, we observed that the earliest rhoptry-like structure, which we had previously interpreted as an AOR contained RhopH3. By contrast, RhopH3 was absent from AORs. Reflecting these observations we have renamed this initial structure the 'pre-rhoptry' and suggested a model for rhoptry biogenesis where rhoptry neck cargo are trafficked via the AOR but rhoptry bulb cargo are trafficked by small vesicles that move along the rootlet fibre (previously observed by EM).
      2. Measurement of rhoptry neck vs bulb: While not directly suggested by the reviewers, we have also included an analysis that estimates the proportion of the sporozoite rhoptry that represents the rhoptry neck. By contrast to merozoites, which we show are overwhelmingly represented by the rhoptry bulb, the vast majority of the sporozoite rhoptry represents the rhoptry neck.
      3. Measurement of subpellicular microtubules: One reviewer asked if we could measure the length of subpellicular microtubules where we had previously observed that they were longer on one side of the sporozoite than the other. We have now provided absolute and relative (% sporozoite length) length measurements for these subpellicular microtubules and also calculated the proportion of the microtubule that is polyglutamylated.
      4. More detailed analysis of RON11cKD rhoptries: Multiple comments suggested a more detailed analysis of the rhoptries that were formed/not formed in RON11cKD We have included an updated analysis that shows the relative position of these rhoptries in sporozoites.

      2. Point-by-point description of the revisions

      Reviewer #1

      Minor text changes (Reviewer #1)

      1. __Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. __

      We agree with the reviewer that it is a reasonable suggestion. After obtaining additional data on the contents of the AOR (as described in General Statements #1), this section has been significantly rewritten to highlight these findings. 2.

      __Add reference on page 3 after 'disrupted parasites' __

      This sentence has been rewritten slightly with some references included and now reads: "Most data on these processes comes from electron microscopy studies 6-8, with relatively few functional reports on gene deleted or disrupted parasites9-11. 3.

      __Change 'the basal complex at the leading edge' - this seems counterintuitive __

      This change has been made. 4.

      __Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? __

      This was supposed to read "SG invasion" and has now been fixed. 5.

      __On page 4: 'handful of proteins' __

      This error has been corrected. 6.

      __What are the 'three microtubule spindle structures'? __

      The three microtubule spindle structures: hemispindle, mitotic spindle, and interpolar spindle are now listed explicitly in the text. 7.

      __On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? __

      The following sentence already detailed that we had recently used U-ExM to visualise rhoptry biogenesis in blood-stage parasites, but the following two sentences have been added to provide extra detail on these findings: "In that study, we defined the timing of rhoptry biogenesis showing that it begun prior to cytokinesis and completed approximate coincident with the final round of mitosis. Additionally, we observed that rhoptry duplication and inheritance was coupled with centriolar plaque duplication and nuclear fission." 8.

      __change 'rhoptries golgi-derived, made de novo' __

      This has been fixed. 9.

      __change 'new understand to' __

      This change has been made 10.

      __'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? __

      We assume this is in reference to mention of "rhoptry malformations" in the abstract. In the RON11 merozoite study (PMID:39292724) the authors noted no gross rhoptry malformations, only that one was not formed/missing. The abstract sentence has been changed to the following to better reflect this nuance: "*We show that stage-specific disruption of RON11 leads to a formation of sporozoites that only contain half the number of rhoptries of controls like in merozoites, however unlike in merozoites the majority of rhoptries appear grossly malformed."

      * 11.

      __What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? __

      We are unaware of any studies that specifically look at sporozoites crossing the SG basal lamina. A review, although now ~15 years old stated that "No information is available as to how the sporozoites traverse the basal lamina" (PMID:19608457) and we don't know any more information since then. To try and better define our understanding of rhoptry secretion during SG invasion, we have added the following sentence:

      "It is currently unclear precisely when during these steps of SG invasion rhoptry proteins are required, but rhoptry secretion is thought to begin before in the haemolymph before SG invasion16." 12.

      __On page change/specify: 'wide range of parasite structures' __

      The structures observed have been listed: centriolar plaque, rhoptry, apical polar rings, rootlet fibre, basal complex, apicoplast. 13.

      __On page 7: is Airyscan2 a particular method or a specific microscope? __

      Airyscan2 is a detector setup on Zeiss LSM microscopes, this was already detailed in the materials and methods sections, but figure legends have been clarified to read: "...imaged by an LSM900 microscopy with an Airyscan2 detector". 14.

      __how large is RON11? __

      RON11 is 112 kDa in * berghei*, as noted in the text. 15.

      __There is no causal link between ookinete invasion and oocyst developmental asynchrony __

      We have deleted the sentence that implied that ookinete invasion was responsible for oocyst asynchrony. This section now simply states that "Development of each oocyst within a midgut is asynchronous..." 16.

      __First sentence of page 24 appears to contradict what is written in results____ I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp __

      This sentence was worded confusingly, making it appear contradictory when that was not the intention. The sentence has been changed to more clearly support what is written in the discussion and now reads: "Our extensive analysis only found one additional ultrastructural difference between Plasmodium spp."

      __On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. __

      It is not entirely clear which new data should be specifically discussed based on this comment. However, we have added a new paragraph that broadly compares MoTissU-ExM and our findings with other imaging methods previously used on mosquito-stage malaria parasites:

      "*Comparison of MoTissU-ExM and other imaging modalities

      Prior to the development of MoTissU-ExM, imaging of mosquito-stage malaria parasites in situ had been performed using electron microscopy7,8,11,28, conventional immunofluorescence assays (IFA)10, and live-cell microscopy25. MoTissU-ExM offers significant advantages over electron microscopy techniques, especially volume electron microscopy, in terms of accessibility, throughput, and detection of multiple targets. While we have benchmarked many of our observations against previous electron microscopy studies, the intracellular detail that can be observed by MoTissU-ExM is not as clear as electron microscopy. For example, previous electron microscopy studies have observed Golgi-derived vesicles trafficking along the rootlet fibre8 and distinguished the apical polar rings44; both of which we could not observe using MoTissU-ExM. Compared to conventional IFA, MoTissU-ExM dramatically improves the number and detail of parasite structures/organelles that can be visualised while maintaining the flexibility of target detection. By contrast, it can be difficult or impossible to reliably quantify fluorescence intensity in samples prepared by expansion microscopy, something that is routine for conventional IFA. For studying temporally complex processes, live-cell microscopy is the 'gold-standard' and there are some processes that fundamentally cannot be studied or observed in fixed cells. We attempt to increase the utility of MoTissU-ExM in discerning temporal relationships through the development of the segmentation score but note that this cannot be applied to the majority of oocyst development. Collectively, MoTissU-ExM offers some benefits over these previously applied techniques but does not replace them and instead serves as a novel and complementary tool in studying the cell biology of mosquito-stage malaria parasites.**"

      *

      __First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. __

      The sentence in question is "To the best of our knowledge, the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported."

      This sentence has now been changed to read as follows: "While numerous studies have characterized proteins whose disruption inhibited SG invasion9,10,15,59-63, to the best of our knowledge the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported ."

      __On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. __

      In our opinion, this statement is already qualified in the following sentence which reads: "Recent studies have shown that while oocysts increase in size initially, their size eventually plateaus (11 days pot infection (dpi) in P. falciparum4)."

      __On page 16 the authors state that different rhoptries might have different function. This is an interesting hypothesis/result that could be mentioned in the abstract. __

      The abstract already contains the following statement: "...and provide the first evidence that rhoptry pairs are specialised for different invasion events." We see this as an equivalent statement.


      Experimental changes (Reviewer #1)

      1. On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries?

      The answer to this is not completely clear. We have added the following data to Figures 6 and 8 where we quantify the proportion of rhoptries that are either apical or cytoplasmic: In both wildtype parasites and RON11ctrl parasites, oocyst spz rhoptries are roughly 50:50 apical:cytoplasmic (with a small but consistent majority apical), while almost all rhoptries are found at the apical end (>90%) in SG spz. Presumably, after the initial apical rhoptries are 'used up' during SG invasion, the rhoptries that were previously cytoplasmic take their place. In RON11cKD the ratio of apical:cytoplasmic rhoptries is fairly similar to control oocyst spz. In RON11cKD SG spz, the proportion of cytoplasmic rhoptries decreases but not to the same extent as in wildtype or RON11Ctrl. From this, we infer that the two rhoptries that are lost/not made in RON11cKD sporozoites are likely a combination of both the apical and cytoplasmic rhoptries we find in control sporozoites.

      __in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? __

      We have labelled all of Figure 1 more clearly to point out that the 'dark lines' are indeed rhoptries. Additionally, we have renamed the 'protein-dense granules' to 'protein-rich granules', as it seems we are suggesting that these structures are dense granules the secretory organelle. At this stage we simply do not know what all of these granules are. The observation that some but not all of these granules contain CSP (Supplementary Figure 2) suggests that they may represent heterogenous structures. It is indeed possible that some are micronemes, however, we think it is unlikely that they are all micronemes for a number of reasons: (1) micronemes are not nearly this protein dense in other Plasmodium lifecycle stages, (2) some of them carry CSP which has not been demonstrated to be micronemal, (3) very few of these granules are present in SG sporozoites, which would be unexpected because microneme secretion is required for hepatocyte invasion.

      __Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. __

      We agree that Figure 2b adds little and so have moved that to Supplementary Figure 2, but think that the relative ease at which it can be distinguished if sporozoites are in the secretory cavity or SG epithelial cell is a key observation because of the difficulty in doing this by conventional IFA.

      __On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. __

      When writing the manuscript, we were unsure whether to include this data at all as it is a purely incidental finding. We had no intention of investigating CSP specifically, but anti-CSP antibodies were included in most of the salivary gland imaging experiments so we could more easily find sporozoites. Given the tremendous importance of CSP to the field, we figured that these observations were potentially important enough that they should be reported in the literature even though they are not something we have the intention or resources to investigate subsequently. Additionally, after consultation with other microscopists we think there is a reasonable chance that this double-layer effect could be a product of chemical fixation. To account for this, we have qualified the paragraph on CSP with this sentence:

      "We cannot determine if there is any functional significance of this second CSP layer and considering that it has not been observed previously it may well represent an artefact of chemical (paraformaldehyde) fixation."

      __Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. __

      We have moved the images of developing rhoptries in * falciparum *(previously Figure S6a and b) into figure 4, which now looks as follows:

      Figure S8 (previously S6c) now consists only of the MG spz rhoptry quantification

      Manuscript structural changes (Reviewer #1)

      1. Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

      2. 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral.

      3. 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings.

      4. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study.____ Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries.

      5. __Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily' __

      Response to Reviewer#1 manuscript structural changes 1-5: This reviewer considers the findings related to rhoptry biology as the most significant aspect of the study and suggests rewriting the manuscript to emphasize these findings specifically. Doing so might make the key findings easier to interpret. However, in our view, this approach could misrepresent how the study originated and what we see as the most important outcomes. We did not develop MoTissU-ExM specifically to investigate rhoptry biology. Instead, this technique was created independently of any particular biological question, and once established, we asked what questions it could answer, using rhoptry biology as a proof of concept. Given the authors' previous work and available resources, we chose to focus on rhoptry biology. Since this was driven by basic research rather than a specific hypothesis, it's important to acknowledge this in the manuscript. While we agree that the findings related to rhoptry biology are valuable, we believe that highlighting the technique's ability to observe organelles, structures, and phenotypes with unprecedented ease and detail is more important than emphasizing the rhoptry findings alone. For these reasons, we have decided not to restructure the manuscript as suggested.


      Reviewer #2

      Minor text changes (Reviewer #2)

      1. __The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial. __

      In the legend of Figure 1, the image Z-depth has been clarified as "sum distance of Z-slices in max intensity projection". 2.

      __Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins __

      This has been corrected. 3.

      __Paragraph 5 of the introduction - line 7, "also able to observed" should be observe __

      This has been changed. 4.

      __In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding __

      This has been fixed. 5.

      __The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs." __

      This has been removed. 6.

      __The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins __

      This mistake has been corrected. 7.

      __One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place. __

      We agree that this was not highlighted enough in the text. The final sentence of the results section about this now reads:

      "While diameter is a useful readout for oocyst development in the early stages of its growth, this suggests that diameter is a poor readout for oocyst development once sporozoite formation has begun and highlights the usefulness of the segmentation score as an alternative.", and the final sentence of the discussion section about this now reads "Considering that oocyst size does not plateau until cytokinesis begins4, measuring oocyst diameter may represent a useful biological clock specifically when investigating the early stages of oocyst development." 8.

      __How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite. __

      Our hypothesis is that apical polarity is established by the positioning and attachment of the centriolar plaque to the parasite plasma membrane in both forming merozoites and sporozoites. While the apical polar ring proteins are obviously present at the apical end, and have important functions, we think that they themselves are unlikely to regulate polarity establishment directly. Additionally, it seems that the apical polar rings are visible in forming sporozoites far before the comparable stages of merozoite formation. An important note here is that at this point, this is largely inferences based on observational differences and there is relatively little functional data on proteins that regulate polarity establishment at any stage of the Plasmodium 9.

      __Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures __

      We have added a paragraph in the discussion that provides a more clear comparison between MoTissU-ExM and other imaging modalities previously applied on mosquito-stage parasites (see response to Reviewer#1 (Minor text changes) comment #17). 10.

      __The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. __

      In our opinion this can be inferred through the more general statement that "development of each oocyst within a midgut is asynchronous..."


      Figure changes (Reviewer #2)

      1. __Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it. __

      We completely agree with the reviewer that the stage 2 image would benefit from the addition of a DNA stain. Many of the images in Figure 3b were done on samples that did not have a DNA stain and so in these * yoelii samples we did not find examples of all segmentation scores with the DNA stain. Examples of segmentation score 2 and 6 for P. berghei, and 6 for P. falciparum* can be found with DNA stains in Figure S8. 2.

      __For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious. __

      The CPs in Figure 1a and 1b have been circled on the NHS ester only panel for clarity. +

      __Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone. __

      The image in Figure 1c has been replaced with one that more clearly shows the rootlet fibre.

      Based on electron microscopy studies, the rootlet fibre has been defined as a protein dense structure that connects the centriolar plaque to the apical polar rings (PMID: 17908361). Through NHS ester and tubulin staining, we could identify the apical polar rings and centriolar plaque as sites on the apical end of the parasite and nucleus that microtubules are nucleated from. There is a protein dense fibre that connects these two structures. Based on the fact that the protein density of this structure was previously considered sufficient for its identification by electron microscopy, we consider its visualisation by NHS ester staining sufficient for its identification by U-ExM.

      __Fig 1B - could the tubulin image in the hemispindle panel be made brighter? __

      The tubulin staining in this panel was not saturated, and so this change has been made.

      __Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case __

      We have made all microscopy labels legible at least when printed in A4/Letter size.

      __Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed. __

      The way Figure 6a was previously designed and described was an oversight, that wrongly suggested we had quantified a >99% reduction in *ron11 * The 99% reduction has been removed from Figure 6a and the corresponding part of the figure legend has been rewritten to emphasise that this was previously established:

      "(a) Schematic showing previously established Ron11Ctrl and Ron11cKD parasite lines where ron11 expression was reduced by >99%9."

      As to the second part of the question, we did not independently test either protein or RNA level expression of RON11, but we were gifted the clonal parasite lines established by Prof. Ishino's lab in PMID: 31247198 not just the genetic constructs.

      __Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text. __

      Thank you for pointing out this mistake, the colours have now been corrected.

      __Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35 __

      The text saying 100% (35/35) only considered salivary glands that were infected (ie. Uninfected SGs were removed from the count. The two sentences that report this data have been clarified to reflect this better:

      "Of *PbRON11ctrl SGs that were infected (35/39), 100% (35/35) contained sporozoites in the secretory cavity (Figure S8c). Conversely of infected PbRON11cKD SGs (59/82), only 24% (14/59) contained sporozoites within the secretory cavity (Figure S9d)."

      *

      __Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%. __

      Please see the response to Reviewer#2 Figure Changes Comment #8 where this was addressed.


      Experimental changes (Reviewer #2)

      1. __Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries? __

      We hypothesise that this morphological difference arises because the congruent rhoptries are 'used' during SG invasion, while the dimorphic rhoptries are utilized during hepatocyte invasion. It is not straightforward to test this functionally at this point, as no protein is known to have differential localization between the two. Additionally, RON11 is likely directly involved in both SG and hepatocyte invasion through a secreted portion of the protein (as seen in RBC invasion). Therefore, RON11cKD sporozoites may have combined defects, meaning we cannot assume any defect is solely due to the absence of two rhoptries. Determining this functionally is of high interest to our research groups and remains an area of ongoing study, but it is beyond the scope of this study. 2.

      Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?

      __ __We do not have access to a parasite line that expresses a tagged copy of RON11, or anti-PbRON11 antibodies. Based on previously published localisation data, however, it seems likely that RON11 localises to both sets of rhoptries. Below are excerpts from Figure 1c of PMID: 31247198, where RON11 (in green) seems to have a more basally-extended localisation in midgut (MG) sporozoites than in salivary gland (SG) sporozoites. From this we infer that in the MG sporozoite you're seeing RON11 in both pairs of rhoptries, but only the one remaining pair in the SG sporozoite.


      __The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function. __

      Based on our analysis, it does indeed seem that RON11 is important for both types of rhoptries as when RON11 isn't expressed sporozoites still have both apical and cytoplasmic rhoptries (ie. Not just one pair is lost; see Reviewer #1 Experimental changes comment #1).

      __The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair. __

      We do not agree that this constitutes a contradiction; instead, more nuance is needed to fully explain the phenotype. As shown in the new graph added in response to Reviewer#1 Figure changes comment #1 in RON11cKD oocyst sporozoites, 64% of all rhoptries are located at the apical end. Our hypothesis is that these rhoptries are used for SG invasion and, therefore, would not be present in RON11cKD SG sporozoites. Consequently, the fact that 64% of RON11cKD sporozoites lack rhoptries is exactly what we would expect. Essentially, we predict three slightly different 'pathways' for RON11cKD sporozoites: If they had 2 apical rhoptries in the oocyst, we predict they would have zero rhoptries in the SG. If they had 2 cytoplasmic rhoptries in the oocyst, we predict they would have two rhoptries in the SG. If they had one apical and one cytoplasmic rhoptry in the oocyst, we predict they would have one rhoptry in the SG. In any case, we expect the apical rhoptries to be 'used up,' which appears to be supported by the data.

      __Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy? __

      We have performed an analysis of subpellicular microtubules which is now included as Supplementary Figure 2. We could not always distinguish every SPMT from each other and so have not quantified SPMT number. We have, however, quantified their absolute length on both the 'long side' and 'short side', their relative length (as % sporozoite length) and the degree to which they are polyglutamylated.

      A description of this analysis is now found in the results section as follows: "*We quantified the length and degree of polyglutamylation of SPMTs on the 'long side' and 'short side' of the sporozoite (Figure S2). 'Short side' SPMTs were on average 33% shorter (mean = 3.6 µm {plus minus}SD 1.0 µm) than 'long side' SPMTs (mean = 5.3 µm {plus minus}SD 1.5 µm) and extended 17.4% less of the total sporozoite length. While 'short side' SPMTs were significantly shorter, a greater proportion of their length (87.9% {plus minus}SD 11.2%) was polyglutamylated compared to 'long side' SPMTs (69.4% {plus minus}SD 13.8%)." *

      Supplementary Figure 2: Analysis of sporozoite subpellicular microtubules. Isolated P. yoelii salivary gland sporozoites were prepared by U-ExM and stained with anti-tubulin (microtubules) and anti-PolyE (polyglutamylated SPMTs) antibodies. SPMTs were defined as being on either the 'long side' (nucleus distant from plasma membrane) or 'short side' (nucleus close to plasma membrane) of the sporozoite as depicted in Figure 1f. (a) SPMT length along with (b) SPMT length as a proportion of sporozoite length were both measured. (c) Additionally, the proportion of the SPMT that was polyglutamylated was measured. Analysis comprises 25 SPMTs (11 long side, 14 short side) from 6 SG sporozoites. ** = p The following section has also been added to the methods to describe this analysis: * "Subpellicular microtubule measurement

      • To measure subpellicular microtubule length and polyglutamylation maximum intensity projections were made of sporozoites stained with NHS Ester, anti-tubulin and anti-PolyE antibodies, and SYTOX Deep Red. The side where the nucleus was closest to the parasite plasma membrane was defined as the 'short side', while the side where the nucleus was furthest from the parasite plasma membrane was defined as the 'long side'. Subpellicular microtubules were then measured using a spline contour from the apical end of the sporozoite to the basal-most end of the microtubule with fluorescence intensity across the contour plotted (Zeiss ZEN 3.8). Sporozoite length was defined as the distance from the sporozoite apical polar rings to the basal complex, measuring through the centre of the cytoplasm. The percentage of the subpellicular microtubule that was polyglutamylated was determined by assessing when along the subpellicular microtubule contour the anti-PolyE fluorescence intensity last dropped below a pre-defined threshold."

      *

      __In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already? __

      Please see the response to Reviewer #2 Experimental Changes Comment #4.

      __Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion?____ Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries. __

      In theory, it is possible that the dimorphic rhoptries are precursors to the uniform rhoptries, specifically how the larger one of the two in the dimorphic pair might be a precursor. Maybe the smaller one is, but we have no evidence to suggest that this rhoptry lengthens after SG invasion. We are interested in isolating sporozoites from SGs to add a temporal perspective, but currently, this isn't feasible. When sporozoites are isolated from SGs, they are collected at all stages of invasion. Additionally, we don't know how long each step of SG invasion takes, so a time-based method might not be effective either. We are developing an assay to better determine the timing of events during SG invasion with MoTissU-ExM, but this is beyond the scope of this study.

      __In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection). __

      It has already been shown that RON11cKD sporozoites fail hepatocyte invasion (PMID: 31247198), even when isolated from the haemolymph and so it seems very unlikely that they would be invasive following SG isolation. As mentioned in the discussion, RON11 in merozoites has a 'dual-function' where it is partially secreted during merozoite invasion in addition to its rhoptry biogenesis functions. Assuming this is also the case in sporozoites, using the RON11cKD parasite line we cannot differentiate these two functions and therefore cannot ascribe invasion defects purely to issues with rhoptry biogenesis. In order to answer this question functionally, we would need to identify a protein that only has roles in rhoptry biogenesis and not invasion directly.

      Reviewer #3

      Minor text changes (Reviewer #3)

      1. __Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood. __

      This has been corrected 2.

      __The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context. __

      The text does not state that the APR is a tubulin structure. Given that it is a proteinaceous structure, we visualise the APRs through protein density (NHS Ester). It has been standard for decades to define APRs by protein density using electron microscopy, and it has previously been sufficient in Plasmodium using expansion microscopy (PMIDs: 41542479, 33705377) so it is unclear why it should not be done so in this study. 3.

      __I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended. __

      This study includes at least 50 references from papers broadly related to sporozoite biology, covering publications from every decade since the 1970s. The most recent review that discusses salivary gland invasion cites 11 proteins involved in SG invasion. We have replaced "handful" with a more precise term, as it is not the best adjective, but it is hardly an exaggeration.


      Figure changes (Reviewer #3)

      1. __The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature. __

      We are unaware of any direct protein localisation evidence suggesting that the rhoptry pairs may carry different cargo. However, only a few proteins have been localised in a way that would allow us to determine if they are associated with distinct rhoptry pairs, so this possibility cannot be ruled out either. It seems unlikely that the parasite 'selects' a specific pair, as rhoptries are typically always found at the apical end. What appears more plausible is that the "docked pair" forms first and immediately occupies the apical docking site, preventing the cytoplasmic pair from docking there. Regarding any evidence that the second pair is activated during liver cell invasion, it has been well documented over decades that rhoptries are involved in hepatocyte invasion. If the dimorphic rhoptries are the only ones present in the parasite during hepatocyte invasion, then they must be used for this process. 2.

      __The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining. __

      This is a logical misinterpretation based on the image provided in Figure 1c. Our intention was not to imply that PolyE staining enables us to see the rootlet fibre but that PolyE and tubulin allow us to see the APR to which the rootlet fibre is connected. There is some PolyE staining that likely corresponds to the early SPMTs that in 1c appears to run along the rootlet fibre but this is a product of the max-intensity projection. Please see Reviwer#2 Figure Changes Comment #3 for the updated Figure 1c. 3.

      __More arrows should be added to Figures 6b and 6c to guide readers and improve clarity. __

      We have added arrows to Figure 6b and 6c which point out what we have defined as normal and aberrant rhoptries more clearly. These panels now look like this: 4.

      __Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square. __

      We agree that the highlighted square and the zoomed area appear different, but this is due to the differing amounts of light captured by the objectives used in these two panels. The entire SG panel was captured with a 5x objective, while the zoomed panel was captured with a 63x objective. Because of this difference, the plane of focus of the zoomed area is hard to distinguish in the whole SG image. The zoomed image is on the 'top' of the SG (closest to the coverslip), while most of the signal you see in the whole SG image comes from the 'middle' of the SG. To demonstrate this more clearly, we have provided the exact region of interest shown in the 63x image alongside a 5x image and an additional 20x image, all of which are clearly superimposable.__

      __ 5.

      __Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days. __

      We are unsure what the suggested change here is. We do not know what is wrong with the statement about day 15 post infection, that is when these midguts were dissected. __ Experimental Changes (Reviewer #3)__

      1. __The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis. __

      It is important to note that the hypothesis that AORs, or rhoptry anlagen, carry rhoptry cargo and serve as vehicles of rhoptry biogenesis was proposed long before this study (PMID: 17908361). In that study, it was assumed that structures now called AORs or rhoptry anlagen were developing rhoptries. Although often visualised by EM and presumed to carry rhoptry cargo (PMID: 33600048, 26565797, 25438048), it was only more recently that AORs became the subject of dedicated investigation (PMID: 31805442), where the authors stated that "...AORs could be immature rhoptr[ies]...". Our observation that AORs contain the rhoptry protein RON4, which is not known to localize to any other organelle, we therefore consider sufficient to conclude that AORs carry rhoptry cargo and are thus vehicles for rhoptry biogenesis. 2.

      __The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11. __

      We are unsure what suggests that RON11 lacks a clear orthologue. RON11 is conserved across all apicomplexans and is also present in Vitrella brassicaformis (OrthoMCL orthogroup: OG7_0028843). A phylogenetic comparison of RON11 across apicomplexans has previously been performed (PMID: 31247198), and this study provides a structural prediction of PbRON11 with the dual EF-hand domains annotated (Supplementary Figure 9). 3.

      __The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs. __

      This is already stated verbatim in the results "Our data cannot directly confirm that membrane fusion occurs between rhoptries and AORs..." 4.

      __It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. __

      We are unclear about the comment or suggestion here, as the claims that RON11cKD does not help interpret rhoptry pair specialization, and that these parasites have combined defects, are both directly stated in the manuscript. 5.

      __Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)? __

      Not all RON11cKD SG sporozoites 'use up' their rhoptries during SG invasion. This is quantified in both Figure 7a and the text, which states: "64% of *PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries."

      * 6.

      Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?

      __ __We agree that a direct comparison between for example * yoelii and P. berghei *oocyst size would be inappropriate, however Figure 3c and Supplementary Figure 4 are not direct comparisons between two species, but a summation of all oocysts measured in this study to indicate that the trends we observe transcend parasite/mosquito species differences. Our study was not set up with the experimental power to determine if mosquito host species alter oocyst size. 7.

      __While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution. __

      The purpose of this study is not to discredit, undermine, or supersede other imaging techniques. It is simply to use U-ExM to answer biological questions that cannot or have not been answered using other techniques. Please refer to Reviewer # 1 Minor text changes comment#17 to see the new paragraph "Comparison of MoTissU-ExM and other imaging modalities" that addresses this

      Both conventional IFA and immunoEM have already been performed on RON11 in sporozoites before (PMID: 31247198). When assessing defects caused by RON11 knockdown, conventional IFA isn't especially helpful because it doesn't allow visualization of individual rhoptries. Thin-section TEM also doesn't provide the whole-cell view needed to draw these kinds of conclusions. Volume EM could likely support these observations, but we don't have access to or expertise in this technique, and we believe it is beyond the scope of this study. It's also important to note that for the defect we observe-missing or abnormal rhoptries-the visualization with NHS ester isn't significantly different from what would be seen with EM-based techniques, where rhoptries are easily identified based on their protein density.

      The statement that "UExM can sometimes produce unexpected localisation patterns due to the denaturation process..." is partially correct but lacks important nuance in this context. Based on our extensive experience with U-ExM, there are two main reasons why the localisation of a single protein may look different when comparing U-ExM and traditional IFA images. First, denaturation: in conventional IFAs, antibodies need to recognize conformational epitopes to bind to their target, whereas in U-ExM, antibodies must recognize linear epitopes. This doesn't mean the target protein's localisation changes, only that the antibody's ability to recognize it does. Second, antibody complexes seem unable to freely diffuse out of the gel, which can result in highly fluorescent signals not related to the target protein appearing in the image, as we have previously reported (PMID: 36993603). Importantly, neither of these factors applies to our phenotypic analysis of RON11 knockdown. All phenotypes described are based solely on NHS Ester (total protein) staining, so the considerations about changes in the localisation of individual proteins are not relevant.

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      Referee #3

      Evidence, reproducibility and clarity

      Overall, the manuscript is well-written and -structured. However, I would like to raise several major points for consideration:

      1. While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution.
      2. The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis.
      3. The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature.
      4. The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11.
      5. The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs.
      6. It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. I like the introduction of a segmentation score to Plasmodium oocyst maturation.

      Minor comments:

      1. The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context.
      2. The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining.
      3. Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square.
      4. Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days.
      5. More arrows should be added to Figures 6b and 6c to guide readers and improve clarity.
      6. Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)?
      7. Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?
      8. I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended.
      9. Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood.

      Significance

      In this study, the authors explore Ultrastructure Expansion Microscopy (U-ExM) in Plasmodium-infected mosquito tissue with the aim to enhance the visualization of parasite ultrastructure. For this purpose, they revisit sporogony, the maturation of sporozoites inside oocysts, and sporozoite invasion of salivary glands, which has been studied both by cell biological methods and experimental genetics over four decades. They focus their analysis on the biogenesis and function of key secretory organelles, termed rhoptries, which are central to parasite invasion and, again, have been studied extensively.

      This study is a follow-up of a previous study by the same authors (Ref. 19). In the former study the authors showed that U-ExM allows to visualize subcellular structures in sporozoites, including the nucleus, rhoptries, Golgi, apical polar rings (APR), and basal complex, as well as midgut-associated oocysts with developing sporozoites. Here, the authors claim a new finding by stating that sporozoites possess two distinct rhoptry pairs. Supposedly, only one pair is utilized during salivary gland invasion. The authors suggest specialization of rhoptries for different cell invasion events. The authors also revisit a RON11 knock-down parasite line, which was previously shown to be deficient in salivary gland invasion, host cell attachment, gliding locomotion, and liver invasion (Ref. 14).

      I find it difficult to estimate the significance. Obviously, attention will be limited to Plasmodium researchers only, as this study is descriptive and revisits a well-studied aspect of the Plasmodium life cycle in the Anopheles vector.

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      Referee #2

      Evidence, reproducibility and clarity

      The manuscript by Liffner et al have used the modified expansion microscopy as they term Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM) to study a cell biology of temporal development of malaria parasite sporozoite biogenesis within mosquito host. They employed three different malaria parasite models Plasmodium yoelii, P.beghei and P falciparum and infected them in mosquito host.

      The application of MoTissU-ExM to infected mosquito tissues is a significant technical advance, enabling visualizations previously only achievable with electron microscopy.

      The major conclusion and advances are as following

      • The establishment of a "segmentation score" as a great tool for staging asynchronous oocyst development.
      • The location of Centriolar plaques, rootlet and other structures which are difficult to analyse
      • The first detailed timeline for sporozoite rhoptry biogenesis.
      • Clear quantification showing that sporozoites possess four rhoptries and utilise two during salivary gland (SG) invasion.
      • A characterization of the RON11 knockout phenotype, linking it to defects in rhoptry biogenesis and a specific block in SG epithelial cell invasion. The following points are intended to further strengthen the paper for publication.

      Points for Revision

      1. For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious.
      2. The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial.
      3. Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone.
      4. Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries?
      5. Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?
      6. The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function.
      7. The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair.
      8. Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy?
      9. Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion? Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries.
      10. In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already?
      11. In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection).

      There are a few typing errors in the document:

      1. Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins
      2. Paragraph 5 of the introduction - line 7, "also able to observed" should be observe
      3. In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding
      4. The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs."
      5. The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins

      Some suggestions for figures

      Fig 1B - could the tubulin image in the hemispindle panel be made brighter?

      Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it.

      Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case

      Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed.

      Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text.

      Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35

      Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%.

      Some point to discuss

      1.One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place.<br /> 2. The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. 3. How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite.

      Significance

      The following aspects are important:

      This is novel and more cell biology approach to study the challenging stage of malaria parasite within mosquito. By using MoTissU-ExM, the authors have enabled the three-dimensional observation of ultrastructures of oocyst-sporozoite development that were previously difficult to observe with conventional electron microscopy alone. This includes the developmental process and entire ultrastructure of oocysts and sporozoites, and even the tissue architecture of the mosquito salivary gland and its epithelia cells.

      Advances:

      By observing sporozoites formation within the oocyst and the overall ultrastructure of the sporozoite with MoTissU-ExM, the authors have provided detailed descriptions of the complete structure and three-dimensional spatial relationships of the rhoptries, rootlet fibre, nucleus, and other organelles. Furthermore, their detailed localisation analysis of sporozoites within the salivary gland is also a great achievement. Considering that such observations were technically and laboriously very difficult with conventional electron microscopy, enabling these analyses with higher efficiency and relatively lower difficulty represents a major contribution to the future advancement of oocyst-sporozoite biology. The development of the 'segmentation score' for sporozoite formation within the oocyst is another major advance. I think this will enable detailed descriptions of structural changes at each developmental stage and of the molecular mechanisms involved in the development of oocysts-sporozoites This has its advantages if antibodies can be used and somewhat reduces the need for immuno-EM. Secondly, in terms of sporozoite rhoptry biology, the Schrevel et al Parasitology 2007 seems to only focus on oocyst sporozoite rhoptries as they say that the sporozoites have 4 rhoptries. This study on the other hand also looks at salivary gland sporozoites and shows that there are potentially important differences between the two - namely the reduction from 4 rhoptries to two. This also leads to further questions about the different types of rhoptries in oocyst sporozoites and whether they're adapted to invasion of different cell types (sal gland epithelial cells or liver hepatocytes)

      Limitation

      It would be that expansion microscopy alone still has its limits when it comes to observing ultra-fine structures. For example, visualising the small vesicular structures that Schrevel et al. observed in detail with electron microscopy, or seeing ultra-high resolution details such as the fusion of membrane structures and their interactions with structures like the rootlet fibre and microtubules. Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures The real impact of this work is mostly cell biologist working with malaria parasite and more in mosquito stages. But the approaches can be applied to any material from any species where temporal dynamics need to be studied with tissue related structures and where UExM can be applied. I am parasite cell biologist working with parasites stages within mosquito vector host.

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      Referee #1

      Evidence, reproducibility and clarity

      In this paper the authors use ultrastructure expansion microscopy to investigate the mosquito stages of the malaria parasite, specifically the stage called oocyst and the process of sporozoite development. They report a number of observations of which the ones concerning rhoptries are the most interesting. There are four of these organelles in the first form of sporozoites in the oocyst and only two in the mature form in the salivary gland. Using a gene knockout of a protein that was reported to be important for rhoprty formation in merozoites, the parasites invading into human red blood cells, they found that fewer rhoptries are formed also in sporozoites and that these cannot enter into the salivary gland cells any more. The presented data are in my view conclusive and no additional experiments are needed for this work to be published. The described experiments should be readily reproducible and have a high statistical power. The text is mostly clearly written but could be improved to make it more concise and more precise and to avoid overstatements. Some references could be added. It would have helped to have line numbers in the manuscript. My suggestions are as following:

      Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

      Add reference on page 3 after 'disrupted parasites' Change 'the basal compelx at the leading edge' - this seems counterintuitive Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? On page 4: 'handful of proteins' 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral. what are the 'three microtubule spindle structures'? 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings. On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? change 'rhoptries golgi-derived, made de novo' change 'new understand to' 'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? On page change/specify: 'wide range of parasite structures' On page 7: is Airyscan2 a particular method or a specific microscope? what are the dark lines in panel E? in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study. Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. On page 16 the authors state that different rhoptries might have different function. This is an interesting hyopthesis/result that could be mentioned in the abstract. how large is RON11? On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries? Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries. There is no causal link between ookinete invasion and oocyst developmental asynchrony First sentence of page 24 appears to contradict what is written in results I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily'

      Significance

      This interesting study investigates the development of malaria parasites in the mosquito using ultrastructure expansion microscopy adapted to mosquito tissue. It provides new and beautiful views of the process of sporozoite formation. The authors discovered that four secretory vesicles called rhoptires are formed in the sporozoites with two pairs being important for distinct functions, one pair functions during invasion of the salivary glands of the mosquito and the other in liver infection, although the latter is not shown but inferred from prior data.

      This study will thus be of interest to scientists investigating malaria parasites in the mosquito as well as to scientist working on vesicle secretion and invasion in these parasites.

      The authors use a previously generated parasite line that lack a protein to investigate its function in rhoptry biogenesis and find that its absence leads to fewer rhoptries which impacts the capacity of the parasite to enter into salivary gland cells. This is a nice functional addition to an otherwise largely descriptive study, but mimics largely the previously reported results from the blood stages. It is not clear to this reviewer how much the study advances the field over the many previous electron microscopy studies. This could be better elaborated in the text.

      Strength of the study: beautiful microscopy, new insights into rhoptry formation and function, new technique to study malaria parasites in the mosquito

      Weakness of the study: Some loose ends in the description of spindles and CSP layers, text could be more focussed on the key advancements reported

    1. Clawdbot Realistic Costs:Software: Free (MIT licensed, forever)Hardware: VPS $4-5/month, or Raspberry Pi ~$50-100 upfront, or old laptop free, or Mac Mini ~$600AI Model: Claude Pro $20/month (casual) to Claude Max $200/month (heavy use like Viticci)Realistic minimum: ~$25/monthBut remember: that $300+ in 2 days user is real. Heavy agentic use burns through tokens fast.

      Assuming cloud based models. Why? You could drop up to 200 month on a VPS and be really self sufficient, but probably wouldn't need a VPS that heavy?

    1. Violences Institutionnelles : Analyse et Perspectives Juridiques et Pratiques

      Synthèse Exécutive

      Ce document de synthèse analyse les dimensions multiples des violences institutionnelles, en s'appuyant sur une expertise croisée du droit, des politiques publiques et de la recherche en sciences sociales.

      Il ressort que la notion de "violence institutionnelle" est complexe, marquée par une ambiguïté juridique persistante malgré des avancées législatives récentes.

      Le terme de "maltraitance institutionnelle" est souvent privilégié pour souligner la relation de pouvoir asymétrique inhérente entre l'institution et l'usager.

      Les points critiques à retenir sont les suivants :

      1. Une Définition Juridique Incomplète : La loi du 7 février 2022 a introduit dans le Code de l'Action Sociale et des Familles (art. L119-1) une définition de la maltraitance qui englobe l'origine institutionnelle.

      Cependant, elle ne définit pas spécifiquement ce que constitue la "maltraitance institutionnelle", laissant une marge d'interprétation et posant des défis en matière de qualification et de traitement.

      2. Un Phénomène Peu Quantifié : Il existe une carence significative de données statistiques publiques permettant de mesurer l'ampleur des violences institutionnelles en France.

      Les données disponibles indiquent toutefois une forte exposition des professionnels du secteur social et de la santé à la violence, à des niveaux comparables à ceux des forces de l'ordre, ce qui témoigne d'un climat de travail particulièrement difficile.

      3. Des Responsabilités Partagées : La lutte contre la maltraitance institutionnelle ne peut se limiter à la sanction des fautes individuelles.

      Elle engage des chaînes de responsabilité plurielles et complexes, impliquant les professionnels, les institutions, et plus largement la société dans sa capacité à définir des seuils de tolérance et à protéger les plus vulnérables.

      4. L'Importance Cruciale du Soutien Organisationnel : Une étude menée à la Ville de Paris révèle que le bien-être des professionnels du travail social n'est pas corrélé au nombre d'actes de violence subis, mais plutôt à la qualité du soutien organisationnel perçu.

      La "détresse morale", liée au manque de marges de manœuvre pour répondre adéquatement aux besoins des usagers, est également un facteur déterminant.

      Ces constats identifient le soutien aux équipes et le renforcement de l'autonomie professionnelle comme des leviers d'action stratégiques pour la prévention.

      1. Le Cadre Conceptuel et Juridique des Violences Institutionnelles

      1.1. Ambiguïtés Sémantiques : Violence vs. Maltraitance

      Une distinction fondamentale est établie entre les notions de "violence" et de "maltraitance".

      Alors que la violence peut survenir dans n'importe quel contexte, la maltraitance se caractérise par une relation asymétrique de pouvoir ou de dépendance entre l'auteur et la victime.

      Dans le contexte institutionnel, la victime se trouve dans une position d'infériorité dont il lui est difficile de s'extraire.

      Perspective de la recherche : La littérature scientifique suggère de privilégier le terme de "maltraitance institutionnelle", car elle implique une relation de pouvoir où la victime est en position d'infériorité, ce qui est particulièrement vrai pour les enfants relevant de l'aide sociale à l'enfance.

      Perspective des personnes concernées :

      Le plaidoyer d'ATD Quart Monde ("Stop à la maltraitance institutionnelle", septembre 2024) met en lumière le caractère systémique du phénomène et la forte exposition des personnes en situation de pauvreté.

      Une citation issue de ce travail illustre la dépendance de la personne vis-à-vis de l'institution :

      La maltraitance institutionnelle peut prendre deux formes :

      1. Une réalité factuelle et objectivable : Des actes pouvant constituer des infractions pénales (violences, négligences graves).

      2. Une réalité subjective : Le vécu ou le ressenti d'une personne qui s'estime victime, même en l'absence d'infraction pénale caractérisée.

      1.2. L'Évolution du Droit et des Politiques Publiques

      La reconnaissance des violences institutionnelles dans le droit et les politiques publiques a progressé par à-coups successifs.

      | Année | Événement Clé | Contribution | | --- | --- | --- | | 1970 | Opération "pouponnière" lancée par Simone Veil. | Première action ciblée sur les violences institutionnelles envers les enfants, en parallèle des travaux sociologiques d'Erving Goffman sur l'"institution totale". | | Années 2000 | Loi du 2 janvier 2002. | Promotion des droits des usagers pour pallier l'asymétrie de la relation avec l'institution et favoriser l'expression des victimes. | | 2008 | Réforme constitutionnelle. | Création du Défenseur des droits, permettant notamment au Défenseur des enfants de recevoir des réclamations individuelles. | | 2022 | Loi du 7 février 2022. | Première définition légale de la maltraitance dans le secteur social et médico-social. | | 2022 | Loi du 21 mars 2022. | Amélioration de la protection des lanceurs d'alerte, un enjeu connexe à la révélation des dysfonctionnements institutionnels. |

      En matière de protection de l'enfance spécifiquement, la terminologie a évolué, passant des "maltraitances" et "mauvais traitements" (loi de 1989) à la notion de "danger" (loi de 2007), pour finalement réintégrer les termes d'"enfant victime de violence" et d'"enfant maltraité" dans les lois de 2016 et 2022.

      1.3. La Définition de la Maltraitance par la Loi du 7 février 2022

      L'article L119-1 du Code de l'Action Sociale et des Familles (CASF) constitue une avancée majeure. Il définit la maltraitance comme suit :

      "La maltraitance [...] vise toute personne en situation de vulnérabilité lorsqu'un geste, une parole, une action ou un défaut d'action compromet ou porte atteinte à son développement, à ses droits, à ses besoins fondamentaux ou à sa santé [...] et que cette atteinte intervient dans une relation de confiance, de dépendance, de soin ou d'accompagnement.

      Les situations de maltraitance peuvent être ponctuelles ou durables, intentionnelles ou non.

      Leur origine peut être individuelle, collective ou institutionnelle."

      Analyse de cette définition :

      Points positifs : Elle est large, reconnaît la vulnérabilité de la personne et la relation de dépendance.

      Elle dissocie la maltraitance de l'infraction pénale, permettant de qualifier des situations sans qu'un délit soit nécessairement constitué.

      Elle nomme explicitement l'origine "institutionnelle".

      Limites : Le texte ne définit pas ce qu'est la maltraitance institutionnelle en soi.

      Par ailleurs, cette approche se heurte à la logique du droit pénal, qui repose sur le principe de la responsabilité personnelle et ne prévoit pas d'infraction spécifique liée au contexte institutionnel ou à la vulnérabilité des publics accompagnés.

      2. Quantification et Mesure du Phénomène

      2.1. Un Manque de Données Statistiques

      Un obstacle majeur à la compréhension et à la lutte contre les violences institutionnelles est l'absence de quantification claire dans la statistique publique.

      Les enquêtes nationales (ONPE, INED) fournissent peu d'éléments spécifiques sur ce phénomène, ce qui rend son ampleur difficile à évaluer.

      2.2. L'Exposition des Professionnels à la Violence

      Malgré le manque de données globales, les chiffres sur la violence subie par les professionnels sont révélateurs du climat dans le secteur social.

      • Les données de la fonction publique montrent que les professions intermédiaires de la santé et du travail social sont particulièrement victimes de violence dans l'exercice de leurs fonctions.

      • Leur niveau d'exposition à la violence est presque aussi élevé que celui des forces de l'ordre, ce qui souligne l'intensité des tensions et la possible banalisation de la violence dans ce champ.

      • Un très faible pourcentage de ces violences fait l'objet d'une plainte et aboutit à une condamnation pénale, ce qui constitue un enjeu majeur pour la reconnaissance des préjudices subis.

      3. La Question Centrale de la Responsabilité

      3.1. Dépassement de la Responsabilité Individuelle

      La Commission nationale de lutte contre les maltraitances souligne que la maltraitance institutionnelle et la responsabilité individuelle ne sont pas exclusives l'une de l'autre.

      Il est essentiel de distinguer les comportements individuels déviants des dysfonctionnements collectifs ou systémiques qui engagent la société tout entière.

      L'enjeu est de ne pas réduire la maltraitance institutionnelle à une simple somme de fautes professionnelles.

      3.2. Des Chaînes de Responsabilité Plurielles

      La protection de l'enfant, en particulier, met en jeu des chaînes de responsabilité complexes et entremêlées :

      Responsabilité familiale : Souvent déjà mise à mal dans les situations de protection.

      Responsabilité des professionnels : Directement en contact avec les usagers.

      Responsabilité des institutions : Liée à l'organisation, aux moyens, à la culture interne.

      Responsabilité sociétale : Reflétant les seuils de tolérance collectifs et les dispositifs mis en place pour protéger les plus vulnérables.

      De plus, la jurisprudence européenne se montre de plus en plus ferme, ayant déjà condamné la France pour des dysfonctionnements dans son dispositif de protection de l'enfance sur le motif de traitement inhumain et dégradant.

      4. Perspectives et Leviers d'Action : L'Étude de la Ville de Paris

      En partenariat avec l'Université de Lille, l'Observatoire social de la Ville de Paris a lancé en 2023 une étude sur les violences institutionnelles, axée sur le vécu des professionnels des politiques sociales (protection de l'enfance, autonomie, etc.).

      4.1. Principaux Enseignements Préliminaires

      1. Forte Exposition, Fort Engagement : L'étude confirme une forte exposition des professionnels à la violence, mais révèle également un niveau d'engagement au travail particulièrement élevé.

      2. Le Rôle Clé du Soutien Organisationnel : De manière contre-intuitive, le bien-être au travail des professionnels n'est pas directement corrélé au nombre d'actes de violence subis.

      Le facteur le plus déterminant est le soutien organisationnel perçu par les agents.

      Un professionnel qui se sent soutenu par son institution vivra mieux son quotidien, même dans un contexte de violence.

      3. L'Impact de la "Détresse Morale" : Le second facteur déterminant est la "détresse morale".

      Ce concept, issu de travaux canadiens, décrit le sentiment d'impuissance des professionnels qui estiment ne pas avoir les marges de manœuvre ou les moyens nécessaires pour répondre de manière satisfaisante aux besoins des usagers.

      4.2. Pistes de Travail Identifiées

      Ces résultats, bien que préliminaires, ouvrent des pistes d'action concrètes pour prévenir la maltraitance institutionnelle en agissant sur le climat de travail et le bien-être des professionnels.

      Les leviers identifiés sont :

      Renforcer le soutien organisationnel : Mettre en place des dispositifs d'écoute, de reconnaissance et d'appui concrets pour les équipes.

      Améliorer le soutien en équipe : Favoriser la cohésion et l'entraide entre collègues.

      Accroître les marges de manœuvre : Redonner aux professionnels la capacité d'agir de manière adaptée aux situations, réduisant ainsi la détresse morale.

      Travailler sur l'éthique et les valeurs partagées : Consolider une culture professionnelle commune pour guider l'action dans des contextes complexes.

    1. Learning for me is easy. I don’t even have to think about it. I have a preferred learning style. If I can't learn something right away, I have difficulty staying with it. I think my teachers are the most significant aspect of my learning.

      All of them depend on what I am learning. #1 -3 I've learned about being motivated and interested in what I'm learning which is one of the major aspects of my learning. A preferred learning style is just what it says, how one PREFERS to learn something. I agree that a preference on learning something shouldn't be categorized in a box, however it should just be looked at a a preference by that individual. 4- Somewhat, I believe that teachers are a big part, but the most significant aspect is me.

    1. suspiciously precise floats, or,how I got Claude's real limits

      Summary: Claude Usage Limits & Cost Analysis

      Subscription vs. API Efficiency * Massive Cost Savings: Claude subscriptions can be up to 36x cheaper than using the API for equivalent token throughput. * The "Max 5x" Sweet Spot: The $100/mo Max 5x plan is identified as the most optimized tier, offering roughly 8.3x more weekly usage than the Pro plan (exceeding its "5x" marketing). * The "Max 20x" Diminishing Returns: While the $200/mo tier provides 4x higher short-term (5-hour) burst limits than the Max 5x, its weekly ceiling is only ~2x higher, making it less efficient for consistent long-term work.

      Dual-Layer Usage Framework * 5-Hour Rolling Window: Controls "burst" activity. The counter starts at your first prompt; once reached, you must wait for the window to reset. * 7-Day Weekly Ceiling: A hard cap on total "active compute hours" (time spent processing tokens/reasoning). This acts as a global safety valve for the system. * Unified Quota: All usage across the browser (claude.ai), Claude Desktop, and Claude Code (terminal) counts toward the same unified limit.

      Token Consumption Dynamics * Exponential Context Cost: Claude re-reads the entire chat history for every new message. A 50-message thread uses significantly more tokens (and quota) than five separate 10-message chats. * Input-Heavy Bias: Large file attachments, long project instructions, and extensive "Extended Thinking" sessions consume the quota much faster than short, text-only queries.

      Optimization Strategies * The /compact Command: Users are encouraged to use /compact (in Claude Code) or manually summarize/restart chats every 15–20 messages to reset the "token tax" of long histories. * Lean Context: Keeping CLAUDE.md and project documentation concise prevents "context bloat" from draining limits prematurely. * Strategic Timing: Since the 5-hour window starts with the first prompt, power users should time their first interaction to align with their most intensive coding blocks.

      Recommendation: Which Claude Tier is Right for You?

      1. The "Value King": Max 5x ($100/mo) * Why it’s recommended: Data analysis shows this tier is "over-provisioned." While marketed as 5x, it often provides ~6x higher session limits and ~8.3x higher weekly limits than the Pro plan. * The Sweet Spot: It offers the best balance between a massive increase in capacity and price. Most daily professional users and developers find it nearly impossible to hit these limits even with "all-day" use. * Best For: Professional developers, heavy researchers, and those who want consistent access to the Claude 3.5 Opus model without hitting daily caps.

      2. The "Sprint Specialist": Max 20x ($200/mo) * The Caveat: Despite the name, the weekly ceiling is only ~2x higher than the Max 5x plan, not 4x higher. You are essentially paying for a much higher "burst" capacity. * Why choose it: It allows for extremely high-intensity sessions (up to 900+ messages in 5 hours). This is useful if you do massive "sprints" where you need Claude to process huge amounts of data or code in a very short window. * Best For: Solopreneurs building products in rapid bursts, or users who never want to think about rate limits during a 4–6 hour deep-work session.

      3. The "Standard Choice": Pro ($20/mo) * Why it’s recommended: It is 5x cheaper than the next tier and sufficient for 90% of users who use Claude for general writing, light coding, or occasional complex tasks. * Best For: Students, casual coders, and users with small projects (under 1,000 lines of code).

      Summary Table * Quick Comparison Summary * Pro: $20/mo | Baseline Capacity | Best for personal/standard use. * Max 5x: $100/mo | ~8.3x Weekly Capacity | Best overall value for power users. * Max 20x: $200/mo | ~16x Weekly Capacity | Best for high-intensity "burst" work.

    1. Scientific evidence indicates thatregular physical activity1. helps to prevent disease;2. promotes a sense of wellbeing, self-esteem, and overallwellness;3. aids in fighting obesity;

      I find it interesting that this information is so well known, yet so poorly utilized. In particular, I was considering schools with lesser funding and/or in areas of increased poverty. These locations often appear to have greater health issues and issues regarding behaviour and attendance. To be able to have a fun, active physical education would likely help students in a broader sense. Unfortunately (to my understanding), many parents and teachers do not see physical education as a priority for students. As such, these benefits are not seen as much as they could be.

    1. This is very telling in today's society. The lower you are on the totem poll (working class), the less access you have to resources to live (the rich have access to almost all the resources). That is why we are very divided and always in conflict because while we cant have very basic resources, the rich have them all and control who can access them.

    1. 6 pillars of Well Architect Framework

      1. Operation Excellence
        • perform operation as code (iaas)
        • learn from operational failiure
        • anticipate failure
      2. Reliability
        • automatically recover from failure
        • scale horzontally
        • stop guessing capacity
        • manage change with automation
      3. Security.
        • principle of least privilage, IAM
        • protect data in transit and at rest
        • security at all layers
      4. Performance Efficiency
        • go global in minutes
        • experiment frequently,
        • user serverless architecture
      5. Sustainability.
        • adopt efficient tech
        • used managed services
      6. Cost Optimization
        • pay for what you suse
        • use cloudwatch to mesure efficiecy
        • use tags to meaure roi
        • use managed service to reduce cost
    1. La Maison Perchée : Analyse d'une Communauté de Rétablissement

      Résumé Exécutif

      Ce document de synthèse analyse les thèmes, les témoignages et la structure de l'association "La Maison Perchée", tels que présentés dans le contexte source.

      Fondée en juin 2020 par un groupe de jeunes adultes, dont trois sur quatre sont directement concernés par des troubles psychiques,

      La Maison Perchée se positionne comme "la brique manquante du rétablissement".

      Son objectif principal est de créer une communauté d'entraide non médicalisée pour les 18-40 ans vivant avec un trouble psychique, en se basant sur le principe fondamental de la pair-aidance.

      L'analyse révèle plusieurs axes majeurs :

      1. Le Vécu Intime des Troubles : Des récits personnels poignants sur la bipolarité et la schizophrénie illustrent la réalité des symptômes (manie, dépression, psychose, paranoïa), la violence des crises, l'épreuve de l'hospitalisation et la souffrance psychique intense, souvent qualifiée de "handicap invisible".

      2. La Philosophie du Rétablissement : Le concept de rétablissement est central. Il ne s'agit pas d'une guérison, mais d'un processus d'apprentissage pour "vivre et non plus survivre" avec une maladie chronique. Ce chemin est jalonné de difficultés et de petites victoires qui doivent être célébrées.

      3. La Puissance de la Pair-Aidance : L'entraide entre pairs est le cœur du projet.

      Le partage d'un vécu similaire offre des modèles, une compréhension authentique et un soutien que le système de soin traditionnel ne fournit pas toujours.

      L'association forme activement ses membres à cette pratique.

      4. La Création d'un Espace Physique : Le projet s'est concrétisé par l'ouverture d'un café-lieu de rencontre à Paris.

      Cet espace vise à déstigmatiser la santé mentale et à offrir un refuge où les conversations profondes et l'acceptation sont la norme, créant ainsi des liens sociaux essentiels.

      En somme, La Maison Perchée émerge comme une initiative vitale, une "pulsion de vie" qui répond à un besoin criant de soutien communautaire, de reconnaissance et d'espoir pour une population souvent marginalisée par la maladie et la stigmatisation.

      --------------------------------------------------------------------------------

      1. Origine et Mission de La Maison Perchée

      1.1. Fondation et Équipe

      L'association "La Maison Perchée" a été officiellement fondée en juin 2020. L'équipe fondatrice est composée de quatre jeunes adultes, tous âgés de moins de 30 ans au moment de la création :

      Maxime : Diagnostiqué bipolaire.

      Victoria : Diagnostiquée d'un trouble bipolaire psychotique.

      Lucile : Atteinte de schizophrénie.

      Caroline Mats : Non concernée directement par un trouble psychique.

      Cette composition, où trois des quatre fondateurs sont "concernés", incarne la philosophie de l'association.

      L'idée est née d'expériences personnelles directes et indirectes avec la maladie psychique, l'hospitalisation et le sentiment d'injustice face au manque de soutien.

      Lucile évoque le déclenchement de sa schizophrénie en 2016 et son expérience en tant qu'enfant d'un parent malade. Maxime mentionne l'injustice ressentie à l'hôpital en voyant d'autres patients sans visites, ce qui a été un moteur initial : "je me suis dit il leur faut un endroit".

      1.2. Philosophie et Objectifs

      La mission de La Maison Perchée repose sur des concepts forts visant à combler un vide dans le parcours de soin.

      "La brique manquante du rétablissement" : L'association se définit comme le "lieu manquant de la psychiatrie", un espace non médicalisé essentiel qui complète le suivi clinique.

      "Apprendre à vivre et non plus survivre" : L'objectif est de dépasser la simple gestion de la maladie pour permettre aux individus de s'épanouir et de mener une vie riche de sens.

      C'est une démarche décrite comme une "p***** de pulsion de vie".

      La Pair-Aidance : Le principe de l'aide par les pairs (P.A.I.R.) est central.

      Il s'agit de s'appuyer sur "une personne avec un vécu similaire au sien" pour obtenir des conseils, des modèles de rétablissement et une compréhension authentique de ce qu'ils traversent.

      Une Communauté Jeune : Le ciblage d'une tranche d'âge spécifique (18-40 ans) répond à un besoin d'être avec des personnes de son âge, une problématique rencontrée par certains dans des structures de soin plus traditionnelles (C.T.T.P.).

      2. Témoignages : Le Vécu des Troubles Psychiques

      Le documentaire est jalonné de témoignages directs et crus sur l'expérience de la maladie mentale.

      2.1. Le Trouble Bipolaire

      L'expérience de Maxime : Il décrit une phase de manie survenue à 22 ans en Chine, où il avait créé quatre entreprises.

      Cette période était caractérisée par un sentiment de toute-puissance ("devant moi un vieux sage"), des délires mystiques ("prières cheloues avec des couleurs") et des comportements obsessionnels ("je compte tout"). Cette phase a été suivie d'une hospitalisation sous contention, puis de 17 mois de profonde dépression et une tentative de suicide.

      L'expérience de Victoria : Diagnostiquée à 20 ans, elle décrit le trouble comme une "alternation assez extrême de l'humeur". La maladie et les effets secondaires du traitement ont été un "parcours du combattant" qui l'a contrainte à interrompre ses études de design pendant un an.

      2.2. La Schizophrénie

      Le vécu de Lucile : Elle insiste sur le fait qu'il n'y a pas "une schizophrénie, mais des schizophrénies". Son quotidien est marqué par des épisodes psychotiques avec une "impression de mort imminente" et la peur que les lieux publics "explosent".

      Elle décrit une anxiété constante et une "impression paranoïque que les gens parlent sur nous" dans les environnements bruyants. Elle qualifie sa condition de "handicap invisible", car la souffrance n'est pas apparente.

      2.3. Crises, Hospitalisation et Souffrance

      Les récits des crises psychotiques sont particulièrement intenses.

      Dissociation et mise en danger : Victoria raconte des "bouffées délirantes" où, se sentant "complètement enfermée", elle a couru nue dans Paris et s'est "tabassée contre le sol", animée par un désir de "désexister" et de "s'atomiser". Une "toute petite partie" d'elle restait consciente, rendant l'expérience "terrible".

      L'expérience de l'hôpital : Les séjours en psychiatrie sont décrits comme des périodes d'"incertitude et de solitude".

      Les témoignages évoquent l'isolement (se faire livrer des choses "à travers la grille"), le choc de la contention et la froideur du système.

      La réalité de la souffrance : Un point est martelé : "les maladies psy c'est une p***** de souffrance". La douleur psychique est comparée à la douleur physique, avec une échelle de souffrance de 0 à 10.

      Le suicide est présenté comme une pensée qui émerge de cette souffrance extrême, non par désir de mort mais par besoin que "ça s'arrête".

      3. Le Rétablissement et la Pair-Aidance

      3.1. Définir le Rétablissement

      Le concept de rétablissement est présenté de manière nuancée, loin de "vendre du rêve".

      Un Processus, pas une Guérison : Il s'agit d'apprendre à mener une vie épanouissante malgré la maladie. C'est un compromis, car "vivre avec une maladie chronique, c'est un compromis".

      Un Chemin Laborieux : Le processus peut être long et difficile ("Est-ce que c'est facile ? Non. Est-ce qu'on en chie par un moment ? Oui.").

      Célébrer les Petites Victoires : Le rétablissement passe par la reconnaissance des progrès, même les plus simples, comme "reprendre les transports" ou "sortir de chez soi".

      Les Marqueurs du Rétablissement : Pour Maxime, être rétabli signifie avoir surmonté les prédictions négatives de ses psychiatres ("vous pourrez plus jamais entreprendre, vous pourrez pas avoir de vie amoureuse") et simplement "être content de se lever le matin".

      3.2. Le Pouvoir de la Pair-Aidance

      La pair-aidance est le pilier de La Maison Perchée. Son efficacité réside dans le partage d'expériences vécues.

      Compréhension Mutuelle : Elle permet des "échanges de gens qui peuvent réellement comprendre ce que vous vivez".

      Source d'Espoir : Recevoir une carte postale d'un pair en étant hospitalisé est décrit comme un contact qui rend l'hôpital "un peu moins froid, moins impressionnant".

      Formation Active : L'association identifie des membres "plus ou moins rétablis" et les forme à la pair-aidance pour qu'ils puissent à leur tour "donner en retour".

      4. La Concrétisation du Projet : Du Virtuel au Physique

      4.1. Les Débuts et la Stratégie

      La création de l'association a suivi une approche pragmatique et résiliente. Face à l'hésitation initiale, la stratégie de Maxime était directe : "Bon les gars, vous allez chercher trois potes d'HP et on va faire un zoom". Ils ont persisté en créant des événements en ligne, même lorsque personne ne s'inscrivait au début.

      4.2. Le Lieu : Un Café Communautaire

      Le projet a franchi une étape décisive avec l'ouverture d'un lieu physique.

      Adresse : Avenue de la République, à Paris.

      Concept : Un café ouvert à tous, conçu pour parler de santé mentale et déstigmatiser les troubles psychiques.

      Ambiance : Le lieu favorise des interactions immédiates et profondes. Un témoignage décrit la magie du lieu : "tout le monde se parle de trucs méga deep et méga perso, genre au bout de 1 seconde 30". Il attire un public mixte : membres, proches, et simples passants.

      Financement : L'aménagement du lieu a été en partie financé par l'obtention d'un "appel à projet" de 20 000 €, destinés à l'achat de matériel, dont une machine à café professionnelle estimée à 10 000 €.

      4.3. Activités et Communauté

      Le lieu est un hub pour diverses activités qui renforcent les liens et le soutien.

      Écriture de cartes : Des ateliers sont organisés pour écrire des mots de soutien à des personnes actuellement hospitalisées.

      Discussions thématiques : Des événements comme les "Tête Perchée" permettent de discuter publiquement de sujets comme la vie professionnelle avec un trouble psychique.

      Création de liens : Le but premier pour beaucoup de membres est simple : "j'ai surtout envie d'avoir des amis". Le lieu offre cet espace de socialisation sécurisant.

      5. Thèmes Transversaux et Enjeux

      5.1. La Stigmatisation

      La lutte contre la stigmatisation est un combat permanent.

      Dans le monde professionnel : La réponse à la question "Comment avez-vous géré le regard d'autrui ?" est "En disant rien pendant longtemps".

      Préjugés du public : Une anecdote relate une rencontre avec une personne exprimant sa peur des "fous" et des "schizophrènes", à qui les fondatrices ont dû répondre : "on vous fait peur ?".

      Action de déstigmatisation : La Maison Perchée combat ces stéréotypes par la prise de parole publique et l'ouverture de son lieu à tous.

      5.2. Le Rôle des Proches

      La famille et les amis jouent un rôle ambivalent mais souvent crucial.

      Souffrance collatérale : Lucile et Caroline témoignent de la souffrance vécue "en tant que proche", une expérience qui a motivé leur engagement.

      Soutien fondamental : Maxime souligne l'importance du soutien de sa mère, sa tante et sa sœur à sa sortie d'hôpital, le qualifiant de "chance dans sa malchance".

      5.3. Identité et Créativité

      La maladie est également présentée comme une facette de l'identité, parfois source de force et de créativité.

      Une vision positive : Un membre compare les personnes atteintes de troubles à des "X-Men", possédant "un truc en plus que les autres n'ont pas".

      L'art comme exutoire : Plusieurs membres canalisent leurs expériences à travers l'art : une membre travaille sur un roman graphique de sa vie de 15 ans à aujourd'hui, Lucile lit des poèmes, un autre écrit, chante et filme.

      La citation inspirante : Une phrase partagée lors d'un atelier d'écriture résume cette dualité : "Si tu es tombé si bas, dis-toi que tu peux monter aussi haut".

    1. Synthèse sur la Santé Mentale : Témoignages et Enjeux

      Résumé Exécutif

      Ce document de synthèse analyse un ensemble de témoignages et d'expertises sur la santé mentale, en se concentrant sur les expériences vécues par des athlètes de haut niveau et des jeunes.

      Il met en lumière le caractère universel des troubles psychiques, qui touchent toutes les strates de la société, indépendamment du succès ou du statut social. Les principaux points à retenir sont les suivants :

      1. Universalité du Mal-être : La santé mentale est une composante de la vie de chacun, susceptible de se dégrader. Les témoignages révèlent que même les champions du monde, au sommet de leur carrière, peuvent souffrir de dépression sévère, illustrant la dichotomie fréquente entre la réussite professionnelle et la détresse personnelle.

      2. Stigmatisation et Silence : La honte, la peur d'être perçu comme "faible" et la pression sociale, particulièrement dans des environnements compétitifs comme le sport de haut niveau, constituent des obstacles majeurs à l'expression de la souffrance. Beaucoup souffrent en silence, cachant leur mal-être derrière une façade de normalité.

      3. Facteurs de Risque Spécifiques :

      Pour les Athlètes : La pression de la performance, la gestion difficile de la "redescente" après un grand succès, l'isolement lié aux blessures mal gérées, et la culture du vestiaire où "il n'y a pas de place pour les faibles".    ◦ Pour les Jeunes : La pression scolaire, les traumatismes infantiles, l'environnement familial, le harcèlement (scolaire et en ligne), et l'influence des algorithmes des réseaux sociaux qui peuvent créer des "bulles numériques" toxiques.

      4. Symptômes et Conséquences : La détresse psychologique se manifeste par un large éventail de symptômes : isolement social, anxiété, crises d'angoisse, troubles du comportement alimentaire (TCA), scarifications, idées suicidaires, et symptômes physiques chroniques. La solitude est décrite comme une force destructrice capable de "tuer une personne".

      5. Voies de Guérison : Le déclencheur de la guérison est souvent une rencontre ou la décision de parler. Les solutions passent par la recherche d'une aide professionnelle adaptée (psychologues, coachs mentaux), le soutien de l'entourage, l'expression des émotions (par l'écriture, le sport, l'art) et l'acceptation que la vulnérabilité n'est pas une faiblesse.

      6. Initiatives et Prise de Conscience : Des initiatives comme le fonds de dotation "Génération 2018", créé par des footballeurs champions du monde, témoignent d'une volonté croissante de "mettre le sujet sur la table", de déstigmatiser la santé mentale et d'avoir un impact positif sur la jeunesse.

      1. Définitions et Perceptions de la Santé Mentale

      La santé mentale est décrite comme une composante fondamentale et universelle de l'être humain, souvent comparée à la santé physique. Elle n'est pas un état statique mais un équilibre fluctuant.

      Définitions Personnelles :

      ◦ "La santé mentale au sens large, on en a tous une, plus ou moins bonne, plus ou moins efficiente."    ◦ C'est "comment tu te sens à l'intérieur de soi", le "bien-être du cerveau", et se ressent "dans la tête, dans le cœur, dans le ventre".    ◦ Elle est perçue comme un équilibre essentiel : "si on n'a pas ça, ben on a plus envie de rien faire."    ◦ Elle inclut la connaissance de sa "propre écologie", l'harmonie entre le mental et le physique.

      Déterminants Multiples : La santé mentale est influencée par une multitude de facteurs : le logement, le travail, l'ambiance sociale, les amis, le sport, et même la météo ou le contexte international. L'état psychologique (bonheur, conflits, questionnements) en est un déterminant central.

      Dégradation Générale : Des études médicales sérieuses indiquent une "aggravation constante sur les 10 dernières années" des troubles anxieux et dépressifs, une tendance accentuée par la crise du Covid-19. La santé mentale des jeunes semble s'être "considérablement dégradée depuis 2019".

      2. Le Sport de Haut Niveau : Une Arène de Pression Psychologique

      Les athlètes, bien que perçus comme des modèles de force, font face à des défis psychologiques uniques et intenses, souvent cachés derrière l'image du succès.

      La Dichotomie entre la Réussite et la Souffrance

      Le succès professionnel peut coexister avec une profonde détresse personnelle, créant un décalage difficile à gérer.

      Témoignage d'un Champion du Monde : "Quand tu es champion du monde, ben on voit la coupe et elle est en or quoi. Et on voit que ça et mais derrière il y a beaucoup de choses qui se passent dans la tête des joueurs."

      Expérience Personnelle : Un joueur décrit son arrivée en Espagne à 18 ans : "à la fois, je vivais le rêve au niveau professionnel et à la fois au niveau personnel, j'étais pas bien, j'étais en dépression."

      La "Redescente" Post-Victoire et les Blessures

      L'après-succès et la gestion des blessures sont des périodes de grande vulnérabilité.

      L'Après-Graal : "Quand tu es arrivé tout en haut, tu as atteint le graal, une redescente après qui n'est pas facile à gérer." Un joueur mentionne qu'Adil Rami a vécu un "espèce de burnout après le mondial".

      L'Impact des Blessures : Une blessure peut signifier la perte de son identité. Un joueur raconte : "j'avais du mal à même le matin me réveiller, c'était dur. Je me demandais ce que j'allais faire parce que pour moi, il y avait que le foot dans la vie."

      Mauvaise Gestion par les Clubs : Un joueur témoigne avoir été mal accompagné, poussé à jouer malgré une blessure pour les besoins de l'équipe, ce qui a aggravé son état physique. Les fuites d'informations confidentielles dans la presse l'ont fait passer pour "un poids pour ce club-là", entraînant un harcèlement de la part des supporters et un profond isolement.

      La Culture du Silence et la Peur de la Faiblesse

      Le milieu sportif impose une culture où la vulnérabilité est souvent assimilée à une faiblesse inacceptable.

      La Loi du Vestiaire : "Quand tu arrives dans le vestiaire, il y a pas de place pour les faibles. [...] Il faut être meilleur que l'autre pour avoir sa place le weekend."

      Stigmatisation par l'Encadrement : Une joueuse de handball en national raconte avoir été renvoyée par sa coach avec cette justification : "je préfère quelqu'un de moins fort que toi mais de stable mentalement plutôt que toi qui est instable mentalement." Elle ajoute : "je me suis vraiment rendu compte que la santé mentale dans le sport c'était tabou."

      Auto-Censure : Les athlètes eux-mêmes intériorisent cette norme. L'un d'eux déclare : "Le fond du problème, c'est que eux-mêmes se l'interdisent."

      3. La Vulnérabilité de la Jeunesse

      Les jeunes sont confrontés à une série de pressions et de traumatismes qui peuvent déclencher des crises de santé mentale sévères.

      Pression Scolaire et Épuisement

      L'environnement scolaire et la pression à la réussite sont des facteurs de stress majeurs.

      Origine des Troubles : Une jeune femme lie sa dépression aux cours : "très vite on m'a mis la pression et je me suis mise la pression. Et donc ça, ça s'est enchaîné à la phobie scolaire et puis après [...] mes TCA."

      Statistiques : Il est noté que "7-8 jeunes sur 10 ont une pression à la réussite plus ou moins forte et plus ils se la mettent forte et plus effectivement ils ont un risque anxio-dépressif qui est élevé."

      Surmenage (Burnout) : Le surmenage n'est pas exclusif au monde du travail. Une jeune femme engagée dans une association décrit un épuisement physique et émotionnel : "Le rythme il baissait pas, il baissait jamais. [...] je pense qu'à un moment mon cerveau il s'est dit stop."

      Traumatismes et Environnement Familial

      Les expériences vécues dans l'enfance et la dynamique familiale jouent un rôle crucial.

      Traumatisme Précoce : Un jeune homme raconte la mort de son petit frère de deux mois alors qu'il n'avait que 14 ans, et le sentiment de culpabilité qui l'a envahi : "je me suis senti coupable. Je me suis dit c'est de ma faute." Il a gardé ce fardeau pour lui pendant des mois.

      Environnement Familial :

      ◦ Certaines familles sont "assez réservées" et ont du mal à exprimer leurs problèmes.    ◦ Une jeune femme décrit une famille "dans le paraître", où il faut "paraître forte, faut pas montrer ses faiblesses", ce qui l'a empêchée de parler de sa scarification. Sa mère a qualifié ses blessures de "foutaises", provoquant un sentiment de non-compréhension et de repli sur soi.    ◦ Un autre jeune, ayant grandi dans un quartier populaire, a endossé très tôt le rôle de "sauveur" familial, portant une pression immense.

      Harcèlement et L'Influence des Réseaux Sociaux

      Le harcèlement, qu'il soit physique, psychologique ou en ligne, a des conséquences dévastatrices.

      Définition du Harcèlement : "Si quelqu'un dit qu'il est harcelé, il est harcelé. C'est ça la définition du harcèlement."

      Violence Psychologique : Une jeune femme décrit un harcèlement moral au collège : "de m'humilier, de me dire des mots méchants. Il y a aussi du chantage", ce qui a "ruiné" son estime de soi.

      Algorithmes Toxiques : Les réseaux sociaux peuvent enfermer les utilisateurs dans une "bulle numérique".

      ◦ Un jeune homme raconte comment, en état de dépression, les algorithmes lui proposaient des contenus "qui glamorisaient, qui incitaient au comportement autodommageable".    ◦ Une autre témoigne : "j'avais peur d'avoir une maladie grave, bah moi mon algorithme vraiment il y avait que des personnes qui allaient mourir."    ◦ Ces contenus, bien que procurant un sentiment paradoxal de "réconfort" et de "compréhension", aggravent le mal-être.

      4. Les Chemins vers le Rétablissement

      Malgré la profondeur de la souffrance, les témoignages montrent que la guérison est possible, bien que le chemin soit souvent long et difficile.

      L'Importance Cruciale de la Parole

      Briser le silence est la première étape, et la plus fondamentale, vers la guérison.

      Le Déclic : Souvent, "le déclic, il vient souvent d'une rencontre".

      Parler pour Aller Mieux : "On va mieux grâce à l'échange, à pouvoir parler de sa situation." L'important est de trouver une "oreille attentive", que ce soit un ami, un professionnel, ou un guide spirituel.

      Lignes d'Écoute : L'appel à une ligne d'écoute, même tenue par des étudiants, a été une porte d'entrée pour "déballer son histoire".

      La Recherche d'un Soutien Professionnel

      Trouver le bon professionnel est un processus parfois ardu mais essentiel.

      La Difficulté de Trouver le Bon Psy : Un jeune homme exprime son blocage : "je suis tombé sur des psy qui qui était pas bien après avoir entendu mon histoire. C'est à dire voir un psy pleurer."

      Persévérance : La recommandation est de ne pas abandonner : "c'est pas parce que tu en as tu es tombé sur trois pas bien que le 4e ou la 4e ou la 5e ou le 5e va pas être bien."

      Terminologie : Dans le sport, il est plus courant de parler de "prépa mental" ou "coach mental" que de "psychologue", car le terme "psy" "fait un peu peur". L'important reste la démarche.

      Médicaments : Les traitements comme les antidépresseurs sont mentionnés comme une aide potentielle, souvent mal vue mais qui, "bien utilisé, ça peut vraiment aider."

      Stratégies Personnelles de Guérison

      Chaque individu développe ses propres outils pour surmonter les épreuves.

      Faire ce qu'on a pas envie de faire : "Quand tu fais une grosse dépression, tout ce que tu tout ce que tu as pas envie de faire, c'est ce qu'il faut que tu fasses." Cela inclut bien manger, faire du sport, et parler à un psychologue.

      L'Écriture : "J'ai acheté un cahier. J'écrivais ma colère, ma tristesse. Je cherchais à comprendre." Noter les déclencheurs des crises d'angoisse permet de "déconstruire" le processus.

      Activités Passion : L'équitation est citée comme un refuge permettant de "stopper mon cerveau". Retrouver goût à la vie passe par "faire les choses que j'aime", comme le dessin, sortir, manger.

      L'Humour et le Soutien des Pairs : Un joueur raconte comment ses coéquipiers ont désamorcé un titre de presse ("le mal-aimé") en chantant la chanson, transformant une situation douloureuse en un moment de camaraderie.

      Le Rôle des Initiatives Collectives

      La prise de conscience collective mène à la création de structures de soutien.

      Génération 2018 : Ce fonds de dotation, créé par les joueurs de l'équipe de France de football 2018, a choisi la santé mentale comme thématique pour "laisser une trace, un héritage" et "mettre le sujet sur la table". Leur but est d'aider les autres, mais aussi de "s'aider nous-mêmes".

      5. Ressources et Lignes d'Aide

      Plusieurs organisations sont mentionnées comme des ressources pour obtenir de l'aide et de l'information :

      E-Enfance 3018 : Lutte contre le harcèlement et les violences numériques. Numéro d'appel : 3018.

      Nightline : Association visant à améliorer la santé mentale des jeunes et étudiants. Site web : nightline.fr.

      Psycom : Propose de l'information fiable et accessible sur la santé mentale. Site web : psycom.org.

    1. La Crise de la Santé Mentale Étudiante : Pressions, Impacts et Initiatives

      Synthèse

      Ce document de synthèse analyse la crise profonde et multifactorielle de la santé mentale qui affecte la population étudiante. La détresse psychologique, déjà présente avant la pandémie de COVID-19 (touchant 20-25% des étudiants), a été exacerbée par la crise sanitaire, atteignant un pic de 43% en 2021. Cette situation critique est alimentée par une convergence de facteurs de stress : une pression académique intense, la précarité financière, l'isolement social, et une anxiété profonde face à un avenir jugé incertain, marqué par le changement climatique et l'instabilité économique.

      Le système de soins en santé mentale s'avère largement insuffisant pour répondre à cette demande croissante, caractérisé par un manque criant de professionnels (un psychologue pour 15 000 étudiants en milieu universitaire) et des listes d'attente prohibitively longues. Dans ce contexte, des initiatives d'aide par les pairs, telles que l'association Nightline, émergent comme une ressource essentielle, offrant un soutien accessible, anonyme et gratuit par des étudiants formés à l'écoute.

      Malgré l'ampleur de leur souffrance, les étudiants ne se positionnent pas comme une génération décliniste. Au contraire, ils manifestent une forte volonté d'agir et de s'engager, cherchant à construire un avenir où le bien-être prime sur la réussite matérielle, et affirmant leur capacité à être des acteurs du changement dès maintenant.

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      1. Un Constat Alarmant : L'ampleur de la détresse psychologique

      Les enquêtes épidémiologiques successives révèlent une dégradation continue de la santé mentale des étudiants.

      Avant la crise sanitaire : Les études montraient déjà des taux de détresse psychologique élevés, oscillant entre 20% et 25%. Ce mal-être étudiant, documenté depuis des années, n'a pas été suffisamment pris en compte par les pouvoirs publics, malgré des rapports comme celui de Danièle Viard en 2006.

      L'effet "loupe" du COVID-19 : La pandémie a agi comme un catalyseur et un amplificateur d'une souffrance préexistante. Le taux de détresse psychologique a grimpé à 43% en septembre 2021. Le nombre d'étudiants présentant des symptômes dépressifs a doublé entre l'avant et l'après-COVID, un constat partagé dans plusieurs pays européens.

      Nature de la détresse : Il ne s'agit pas d'une maladie psychiatrique en soi, mais d'un syndrome qui affecte plusieurs sphères de la vie :

      Affective et émotionnelle : idées tristes, morosité, irritabilité.    ◦ Somatique : troubles du sommeil, fatigue intense, incapacité à accomplir des tâches simples.    ◦ Cognitive : baisse de la concentration, de la mémorisation et de la motivation, ce qui impacte directement la capacité à étudier.

      2. Les Multiples Facteurs de Stress des Étudiants

      La détresse étudiante est le résultat d'une accumulation de facteurs de stress spécifiques à leur condition et à l'époque actuelle.

      Pression Académique et Élitiste

      Un environnement universitaire extrêmement compétitif est une source majeure de stress.

      Discours élitiste : Certains professeurs instaurent une pression intense dès le début du cursus avec des phrases telles que : "Vous avez choisi un cursus très difficile, il va falloir s'accrocher. Votre voisin de table ne sera pas là dans un an."

      Surcharge de travail : Des étudiants témoignent d'un épuisement total, sacrifiant leur vie sociale et leurs loisirs. Une étudiante déclare : "J'ai arrêté le sport, j'ai arrêté de voir des gens. Je me concentre vraiment sur mes études [...] Les études, c'est toute ma vie."

      Conséquences directes : Cette pression mène à des burnouts, des dépressions et une angoisse constante de l'échec. Un professeur de japonais admet : "On le sait tous les ans, il y a plusieurs suicides. Ils s'en vantent pas mais on le sait quand même."

      Précarité et Conditions de Vie

      Les difficultés matérielles sont un fardeau quotidien.

      Précarité financière : La gestion du loyer, des courses, des transports et le manque de soutien familial pour certains créent une anxiété permanente.

      Pression familiale : Pour certains, la réussite est une obligation due à l'investissement des parents. Une étudiante aînée de trois sœurs confie : "Je me dis que je n'ai pas le droit à l'échec."

      L'Impact Dévastateur de la Crise Sanitaire

      La pandémie a eu des effets particulièrement destructeurs sur les jeunes.

      Isolation et perte de socialité : La privation de contacts sociaux a été qualifiée de "cataclysmique" pour une tranche d'âge où la vie relationnelle est fondamentale.

      Traumatisme psychique : Les confinements ont été vécus comme un traumatisme, générant des états de stress post-traumatique, un phénomène inédit à cette échelle. Les peurs (mourir, que les proches meurent, un prochain confinement) ont été des facteurs traumatisants majeurs.

      Stigmatisation : Les étudiants ont été désignés comme un des vecteurs de la circulation virale, ce qui a eu un "effet terrible sur leur santé mentale".

      Conséquences à long terme : Les experts estiment que les conséquences de cette période se feront sentir pendant des décennies, tant sur la santé mentale que sur les parcours de formation et professionnels.

      Anxiété face à un Avenir Incertain

      Le climat général de la société est perçu comme particulièrement anxiogène.

      Crises multiples : Le changement climatique, l'inflation, la guerre, le chômage et les violences sexistes et sexuelles créent un contexte d'incertitude généralisée.

      Écoanxiété : Il ne s'agit pas d'un trouble, mais d'une peur légitime et saine face à un danger concret et actuel. Un expert précise : "Quand on n'a pas peur face à un danger, c'est pas qu'on est courageux, c'est qu'on est inconscient."

      Perte de prévisibilité : Les structures sociales et les projections (formation menant à un métier, planète viable pour fonder une famille) sont disloquées, ce qui est particulièrement déstabilisant pour des psychismes en construction.

      Vulnérabilités Spécifiques

      | Catégorie | Description de la Vulnérabilité | | --- | --- | | L'Âge (15-25 ans) | Le cerveau continue sa maturation jusqu'à 25 ans, le rendant particulièrement sensible aux influences environnementales ("plasticité pour le meilleur et pour le pire"). 75% des pathologies psychiatriques (schizophrénie, trouble bipolaire) se déclarent durant cette période, soulignant l'importance de la prévention et de la détection précoce. | | Étudiants Étrangers | Ils subissent une double pression : la pression académique commune, et une pression supplémentaire pour être "excellents" afin d'assurer le renouvellement de leur titre de séjour. À cela s'ajoutent les défis de l'intégration (logement, solitude, choc culturel). |

      3. Nightline : Une Réponse par les Pairs

      Face à la saturation des services professionnels, l'association étudiante Nightline offre un modèle de soutien alternatif et vital.

      Mission et Dispositifs

      Objectif : Améliorer la santé mentale des jeunes, en particulier des étudiants.

      Principe fondamental : L'aide par les pairs. Le service est composé et destiné aux étudiants.

      Actions : L'association informe, déstigmatise, soutient et oriente à travers :

      ◦ Une ligne d'écoute nocturne (tous les soirs de 21h à 2h30).  

      ◦ Des formations dans les établissements d'enseignement supérieur.  

      ◦ Des actions de terrain et des campagnes sur les réseaux sociaux.  

      ◦ Des actions de plaidoyer pour améliorer les politiques de santé mentale.

      Le Rôle des Bénévoles Étudiants

      Profil : Ce ne sont pas des professionnels, mais des étudiants formés à l'écoute active et bienveillante.

      Anonymat : C'est une règle cardinale, valable pour l'appelant comme pour le bénévole. Les bénévoles ne doivent pas révéler leur engagement à leur entourage pour protéger le service et ne pas décourager des proches d'appeler. La seule rupture de l'anonymat est envisagée en cas de risque suicidaire immédiat pour appeler les secours, et l'appelant en est prévenu.

      Soutien des bénévoles : Un cadre strict est mis en place pour les protéger :

      Débriefings à chaud entre bénévoles juste après les appels difficiles.    ◦ Réunions de partage mensuelles et obligatoires, encadrées par une psychologue, pour extérioriser et analyser les appels marquants.

      Un Mouvement International

      Le concept de Nightline est né dans les années 1970 au Royaume-Uni. Il s'est depuis étendu en Irlande, en Allemagne, en Suisse, en Autriche et en France. Des projets sont en cours pour une implantation en Belgique, montrant que le problème de la santé mentale étudiante est une préoccupation européenne.

      4. Les Défaillances du Système de Soins en Santé Mentale

      L'accès aux soins professionnels est un parcours du combattant pour les étudiants en souffrance.

      Pénurie de professionnels : Le ratio dans les services de santé universitaires est de un psychologue pour 15 000 étudiants, soit dix fois moins que la recommandation internationale.

      Listes d'attente "lunaires" : Les délais pour obtenir un rendez-vous dans le secteur public (Centres Médico-Psychologiques) sont de 6 à 18 mois, ce qui est incompatible avec l'urgence d'une détresse psychique. Le système est décrit comme "bouché de chez boucher".

      Coût des soins : Le secteur privé est souvent inaccessible financièrement pour une population précaire, ce qui conduit de nombreux étudiants à renoncer aux soins.

      5. Au-delà du Désarroi : L'Engagement et la Résilience Étudiante

      Contrairement à une vision parfois décliniste, les témoignages révèlent une jeunesse engagée et tournée vers l'action.

      Rejet du catastrophisme : Les experts notent que les étudiants, malgré leurs difficultés, ne sont pas dans une vision catastrophique du monde. Ils conservent le sentiment d'avoir les moyens d'intervenir sur leur avenir grâce à leur formation et leur vitalité.

      Volonté d'agir maintenant : Le discours dominant est celui de l'action immédiate. Une étudiante déclare : "On est acteur et actrice déjà maintenant [...] on leur dise pas vous ferez plus tard, mais ils peuvent faire maintenant et ils ont envie de faire maintenant."

      Redéfinition de la réussite : Il y a une prise de conscience que le bien-être et un bon cadre de vie sont plus importants qu'un "cadre de vie luxueux" ou un gros salaire. La quête de sens prime sur la seule réussite professionnelle.

    1. L'Anxiété chez les Jeunes : Synthèse et Analyse

      Résumé Exécutif

      Ce document de synthèse analyse le phénomène croissant de l'anxiété chez les jeunes, un enjeu de plus en plus qualifié d'« épidémie » ou de « maladie de l'heure ».

      Les témoignages de jeunes et d'experts révèlent un problème multifactoriel profondément ancré dans les structures sociales, éducatives et familiales contemporaines.

      Les principaux moteurs de cette anxiété incluent une pression de performance omniprésente, des horaires surchargés qui éliminent le temps de repos et de jeu, et l'influence des réseaux sociaux qui promeuvent une culture de la perfection irréaliste.

      L'analyse scientifique distingue le stress (une réponse à une menace immédiate) de l'anxiété (une menace anticipée), tout en soulignant que le cerveau y réagit de manière identique.

      Des facteurs déclencheurs universels — un faible sens de contrôle, l'imprévisibilité, la nouveauté et une menace à l'ego (acronyme SPIN) — sont identifiés comme étant au cœur des expériences anxieuses des jeunes. Parallèlement, l'omniprésence du numérique contribue à une régression de l'intelligence émotionnelle en réduisant les interactions en face à face.

      Les experts appellent à un « grand réveil », arguant que l'anxiété juvénile n'est pas un simple problème individuel à traiter, mais un symptôme d'un système qui « use le cerveau » des enfants.

      Les solutions résident non seulement dans des stratégies d'adaptation individuelles comme la respiration ou la méditation, mais aussi dans des changements systémiques, notamment une remise en question du modèle éducatif axé sur la performance et la création de conditions plus propices à une croissance saine.

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      1. Le Phénomène de l'Anxiété : Une "Épidémie" Moderne

      L'anxiété chez les jeunes est décrite comme un phénomène majeur et en pleine expansion.

      Elle est qualifiée de « maladie de l'heure », succédant à d'autres diagnostics prévalents comme le TDAH il y a quelques années.

      Prévalence et Perception

      Consultations en hausse : Un professionnel de la santé mentale note une augmentation significative du phénomène depuis environ six à sept ans, déclarant que l'anxiété représente aujourd'hui la majorité de ses consultations.

      Impression générale : La narratrice, travaillant dans les médias, a constaté à travers diverses statistiques que le problème est bien plus répandu qu'elle ne l'imaginait initialement.

      Les jeunes eux-mêmes décrivent l'anxiété comme une expérience quasi universelle dans leur entourage.

      Manifestations et Symptômes

      Les témoignages des jeunes décrivent un large éventail de manifestations physiques, cognitives et émotionnelles.

      | Type de Symptôme | Description | Exemples de Témoignages | | --- | --- | --- | | Physique | Réactions corporelles involontaires déclenchées par la panique ou le stress. | Maux de ventre (Julian, Sopiane), pleurs incontrôlables, hyperventilation, respiration rapide (Magalie). | | Cognitif | Pensées envahissantes et scénarios catastrophiques. | Le "cerveau qui arrête jamais" (Alexandra), se concentrer uniquement sur la panique au point d'en oublier la cause initiale (Sopiane). | | Émotionnel | Sentiment de perte de contrôle, de solitude et de peur. | Peur du jugement et du rejet (Alexandra), impression que l'anxiété "finit plus", difficulté à gérer ses émotions et à montrer sa vulnérabilité (Magalie). | | Comportemental | Réactions externes observables, souvent interprétées différemment par l'entourage. | Colère et agressivité (Julian), comportement très oppositionnel lors des crises (Sopiane), perfectionnisme extrême (arracher une feuille de dessin pour une erreur). |

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      2. Les Causes Multifactorielles de l'Anxiété Juvénile

      L'anxiété des jeunes ne peut être attribuée à une cause unique. Elle émerge de l'intersection de pressions sociales, de l'environnement numérique et de dynamiques scolaires et familiales.

      La Pression de la Performance

      Un thème central est la quête incessante de la performance dans toutes les sphères de la vie.

      Horaires surchargés : Les jeunes comme Magalie et Marie décrivent des emplois du temps extrêmement chargés, remplis d'études, de tutorat, d'activités parascolaires et de travail, ne leur laissant jamais le temps de se reposer. Marie répond "jamais" à la question "Tu te reposes quand ?".

      Compétition interne et externe : Magalie décrit son besoin de tout faire comme "une compétition contre [elle]-même".

      Alexandra exprime un désir de plaire à tout le monde et d'être perçue comme "parfaite".

      Cette pression est si normalisée que pleurer à cause des devoirs devient un sujet de conversation banal entre amis.

      Pression sociétale et parentale : Les jeunes ressentent que la performance à l'école, dans les sports et sur les réseaux sociaux est ce qui est valorisé par la société.

      Les parents, même avec de bonnes intentions, peuvent amplifier cette pression, comme le reconnaît la narratrice à propos des cours de violon de sa fille Julian.

      L'Influence du Monde Numérique et des Réseaux Sociaux

      L'environnement numérique joue un rôle significatif dans l'amplification de l'anxiété.

      Culture de la perfection : Les plateformes comme Instagram présentent une vision idéalisée de la vie ("la photo parfaite avec le l'auto parfaite avec la maison parfaite").

      Cette exposition constante à la perfection crée un décalage avec la réalité et renforce le sentiment de ne jamais être à la hauteur.

      Régression de l'intelligence émotionnelle : Un spécialiste du non-verbal explique que la diminution des interactions en face à face au profit des communications numériques appauvrit la capacité à décoder les émotions.

      Une étude comparant deux camps de vacances (avec et sans téléphones) a montré qu'après une semaine, les jeunes privés de leur appareil étaient devenus plus aptes à juger les émotions des autres.

      Le Rôle du Système Scolaire

      Le système éducatif est identifié comme un facteur de stress majeur.

      Surcharge d'examens : La chercheuse Sonia Lupien note une augmentation constante du nombre d'examens, qu'elle qualifie scientifiquement de "périodes de stress". L'accumulation de ces déclencheurs de stress épuise les élèves.

      Contagion du stress : Des études montrent qu'un professeur souffrant d'épuisement professionnel peut voir les enfants de sa classe produire davantage d'hormones de stress, car ils "raisonnent avec le professeur".

      Le modèle finlandais comme contre-exemple : La professeure Tina Montreuil décrit le système finlandais, qui minimise les facteurs de stress :

      ◦ Journées d'école plus courtes (fin vers 14h30-15h).  

      ◦ Pauses déjeuner très longues.  

      ◦ Absence quasi totale de devoirs et d'examens formels.  

      ◦ L'évaluation se fait par des projets, des discussions et des mises en contexte.  

      ◦ Bien que non utopique, ce système génère des niveaux d'anxiété de performance bien plus faibles qu'en Amérique du Nord.

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      3. Perspectives d'Experts et Compréhension Scientifique

      Les experts consultés appellent à un changement de perspective, passant d'une vision individualisée de l'anxiété à une compréhension systémique.

      Un Problème Systémique, Pas Individuel

      Le professeur Carl la Charité soutient que l'anxiété des enfants est un message adressé à la société.

      Du corps au cerveau : Il établit un parallèle historique : "Il y a [...] un siècle, la façon dont on traitait les enfants, ça nous amenait à user leur corps. [...] Aujourd'hui, en fait, ce qu'on use, c'est leur cerveau."

      Un appel au "grand réveil" : Il considère l'anxiété comme un signal que les méthodes éducatives actuelles ne fonctionnent plus. La solution n'est pas de "réparer" l'enfant, mais de changer les conditions dans lesquelles il grandit.

      Démystifier le Stress et l'Anxiété (Approche de Sonia Lupien)

      La chercheuse Sonia Lupien propose un cadre scientifique pour comprendre et gérer le stress.

      Stress vs. Anxiété : L'analogie du "mammouth" illustre la différence :

      Stress : La réponse biologique à une menace réelle et présente ("le mammouth est devant toi").  

      Anxiété : La réponse à une menace anticipée ou imaginaire ("le mammouth est dans ta tête").  

      ◦ Pour le cerveau, la production d'hormones de stress est identique dans les deux cas.

      Les quatre déclencheurs du stress (SPIN) : Le cerveau détecte une menace et déclenche une réponse de stress lorsqu'il est confronté à une ou plusieurs de ces situations.

      | Acronyme | Caractéristique | Description | | --- | --- | --- | | S | Sens de contrôle faible | Impression de ne pas avoir le contrôle sur la situation. | | P | Personnalité menacée | L'ego ou les compétences sont remis en question. | | I | Imprévisibilité | La situation est nouvelle ou inattendue. | | N | Nouveauté | La situation n'a jamais été rencontrée auparavant. |

      Le danger de la narration "stress toxique" : Sonia Lupien soutient que le discours médiatique et scientifique présentant tout stress comme négatif est contre-productif.

      Il peut engendrer une "sensibilité à l'anxiété" ou la "peur d'avoir peur", où une personne panique face aux sensations physiques normales du stress, croyant qu'elles sont dangereuses.

      Il est crucial de normaliser la réponse de stress comme étant nécessaire à la survie.

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      4. Stratégies d'Adaptation et Solutions

      Face à ce constat, des solutions émergent à la fois au niveau individuel, familial et sociétal.

      Techniques Personnelles et Familiales

      Suivre son intuition : Les jeunes gèrent souvent mieux le stress en suivant leur intuition : jouer, chanter, rire pour dépenser l'énergie accumulée.

      Techniques de régulation simples : Des méthodes scientifiquement prouvées sont souvent les plus simples.

      Respiration abdominale : Une "belle grosse respiration avec une belle grosse bédaine" active le système parasympathique et arrête la réponse de stress.  

      Chanter : A un effet similaire à la respiration profonde. 

      Méditation : Pratiquée par Magalie pour faire le vide et analyser sa journée.

      Communication et soutien : L'écoute est fondamentale.

      Le père de Sopiane a appris qu'au lieu de proposer des solutions, il est plus efficace d'écouter, de valider l'émotion et d'offrir un soutien inconditionnel ("je suis là pour toi").

      Changement de vocabulaire et mantras : Adopter un langage qui normalise l'inconfort sans le dramatiser.

      Le mantra utilisé par Judith et Sopiane est un exemple puissant : "C'est vraiment inconfortable en ce moment mais ça va pas durer."

      Prise de conscience parentale : Les parents doivent réfléchir à l'impact de leur propre anxiété de performance sur leurs enfants et ajuster leurs attentes, en se concentrant sur le processus d'apprentissage plutôt que sur le résultat.

      Pistes de Réflexion Sociétales

      Créer des conditions de croissance : Selon Carl la Charité, les enfants n'ont pas besoin qu'on "tire sur la fleur pour qu'elle pousse". Ils ont besoin de conditions propices :

      ◦ Jouer dehors.  

      ◦ Interagir avec d'autres enfants, y compris dans le conflit.   

      ◦ Avoir des adultes qui fixent des limites mais prennent aussi le temps d'écouter.

      Réformer le système éducatif : S'inspirer de modèles moins axés sur la performance et les examens, comme le système finlandais, pour recentrer l'éducation sur le goût d'apprendre.

      Parler ouvertement : La démarche de la narratrice montre que le fait de parler ouvertement de l'anxiété, de poser des questions et de partager des expériences permet de briser l'isolement, de valider les ressentis et d'ouvrir la voie à des solutions collectives.

    1. Document d'information : Jeunes Sans-Abri à 20 ans

      Synthèse Exécutive

      Ce document de synthèse analyse les témoignages de plusieurs jeunes sans-abri, mettant en lumière les causes multifactorielles de leur situation, les dures réalités de leur quotidien et leurs sources de résilience.

      Les points clés sont les suivants :

      Des parcours de rupture profonde : L'itinérance des jeunes est rarement un choix, mais plutôt la conséquence de traumatismes et de ruptures systémiques.

      Les causes principales identifiées sont le rejet familial violent, l'abandon à la majorité par les services de l'Aide Sociale à l'Enfance (ASE), et les parcours migratoires périlleux en tant que demandeurs d'asile.

      Une survie quotidienne brutale : La vie dans la rue est une lutte incessante contre l'épuisement physique et psychologique. Elle est marquée par la mendicité ("la manche"), l'exposition aux éléments, le manque d'hygiène, et un sentiment d'invisibilité et de danger permanent. La précarité expose à la violence, au vol et à une déconnexion progressive avec la réalité.

      La résilience face à l'adversité : Malgré des conditions extrêmes, ces jeunes font preuve d'une forte résilience. Ils s'accrochent à des passions personnelles (musique, sport, jeux), à des relations humaines (amour, amitié) et à des aspirations fortes pour l'avenir, comme la poursuite des études, la fondation d'une famille ou l'obtention d'un logement stable, qui deviennent des moteurs pour s'en sortir.

      Les obstacles systémiques et les lueurs d'espoir : La dépendance aux drogues apparaît comme un facteur aggravant majeur, créant un cercle vicieux difficile à briser seul.

      L'accès aux services d'urgence (115) est souvent saturé.

      Cependant, l'engagement collectif, la solidarité entre pairs et l'aide des associations spécialisées offrent des perspectives concrètes de réinsertion, menant dans certains cas à des réussites tangibles (obtention de statut, logement, reprise des études).

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      1. Origines de l'itinérance : Des parcours de rupture

      L'entrée dans la rue à un jeune âge est systématiquement liée à des événements traumatisants et à des failles dans les filets de sécurité sociaux et familiaux.

      1.1. La rupture familiale

      Le rejet et les violences intra-familiales sont une cause directe de l'itinérance pour plusieurs jeunes. L'environnement familial est décrit non pas comme un refuge mais comme la source du problème.

      Le rejet parental dès la naissance : Une jeune femme raconte avoir été un enfant non désiré, un sentiment verbalisé par sa mère et renforcé par l'abandon de son père.

      La fuite comme seule issue : Face à un harcèlement familial constant (rabaissement, dénigrement), la fugue est perçue comme un acte de survie pour éviter une implosion.

      1.2. La sortie de l'Aide Sociale à l'Enfance (ASE)

      La fin de la prise en charge à 18 ans constitue un point de bascule critique, projetant des jeunes sans soutien familial directement dans la précarité.

      L'abandon institutionnel : Un jeune homme, placé de 2 à 18 ans, décrit sa mise à la rue comme une conséquence directe du refus de son contrat jeune majeur.

      1.3. Les parcours migratoires

      Pour les jeunes étrangers, l'itinérance est une conséquence directe de leur parcours d'exil et des difficultés rencontrées à leur arrivée en France.

      La fuite de la violence : Un jeune homme brésilien explique avoir fui la favela en raison des menaces des narcotrafiquants et des dettes de drogue de sa famille.

      La désillusion à l'arrivée : Un jeune demandeur d'asile pensait être pris en charge immédiatement, mais a été confronté à la réalité de la rue dès sa première nuit.

      2. La réalité quotidienne de la rue : Survie et vulnérabilité

      La vie sans-abri est un combat permanent marqué par la précarité matérielle, la détresse psychologique et une exposition constante aux dangers.

      2.1. Les difficultés matérielles et physiques

      Le quotidien est une lutte pour subvenir aux besoins les plus élémentaires.

      L'absence de ressources : Une jeune femme enceinte décrit son dénuement total.

      La mendicité ("la manche") : C'est une activité épuisante et nécessaire pour se nourrir.

      L'épuisement et l'inconfort : L'exposition aux intempéries et la marche continue mènent à un épuisement extrême.

      2.2. L'impact psychologique

      La rue a des effets dévastateurs sur la santé mentale.

      Le sentiment d'invisibilité et de solitude : L'isolement est profond, menant à la peur de mourir seul et sans que personne ne s'en aperçoive.

      La confusion entre rêve et réalité : L'épuisement et le stress permanent créent un état de déphasage mental.

      La survie plutôt que la vie : L'existence se réduit à une lutte pour les besoins primaires, effaçant toute notion de vie épanouie.

      2.3. Les dangers et l'insécurité

      La rue est un environnement hostile où la méfiance est une règle de survie.

      La violence et le vol : Les agressions sont une réalité tangible et le vol est fréquent, même pendant le sommeil.

      La vulnérabilité des femmes : Les femmes seules sont particulièrement exposées aux agressions, notamment la nuit.

      Le squat dans des lieux dangereux : Pour se mettre à l'abri, certains occupent des bâtiments délabrés et dangereux.

      3. Mécanismes de résilience et sources d'espoir

      Face à cette brutalité, les jeunes développent des stratégies de survie psychologique et s'accrochent à des projets et des relations qui leur donnent la force de continuer.

      3.1. Les passions personnelles comme échappatoires

      Les activités personnelles permettent de s'évader mentalement de la dureté du quotidien.

      Le Rubik's Cube : Pour une jeune réfugiée, ce jeu fait disparaître les problèmes.

      Le piano : Pour un jeune brésilien, jouer du piano est un lien avec son passé et sa grand-mère, apportant joie et motivation.

      Le roller : Cette activité est synonyme de liberté et permet de combattre la solitude.

      3.2. L'importance des relations et de la parentalité

      Les liens affectifs sont un puissant soutien. L'arrivée d'un enfant devient un objectif majeur pour s'en sortir.

      L'amour dans la rue : Être en couple est une épreuve mais aussi une force.

      La grossesse comme moteur : Un enfant à naître transforme la lutte pour la survie en un projet de construction d'un avenir stable, motivé par la peur d'un placement.

      3.3. La quête de dignité et de normalité

      Conserver une part de "vie normale" est essentiel pour le moral.

      L'apparence : Rester bien habillé et propre grâce aux associations est une façon de maintenir son estime de soi.

      La douche : Ce moment est décrit comme une renaissance, un moyen de laver la saleté et de retrouver une part de dignité aux yeux des autres.

      4. La dépendance comme facteur aggravant

      Pour une des jeunes femmes, la consommation de crack est à la fois une conséquence et une cause de son maintien dans la rue, créant un cycle d'autodestruction.

      La conscience de l'addiction : Elle reconnaît lucidement sa dépendance et le fait qu'elle l'empêche d'avancer.

      L'isolement social : La drogue et la mendicité qui en découle l'ont coupée de toute relation sociale normale.

      La prise de conscience : Elle réalise qu'elle ne peut pas s'en sortir seule et qu'elle doit accepter de l'aide extérieure.

      5. L'engagement collectif et la recherche de solutions

      Face à l'inertie des institutions, certains jeunes choisissent l'action collective pour faire entendre leur voix.

      La solidarité entre pairs : L'union est vue comme une nécessité pour trouver des solutions.

      L'action politique : Des actions comme des occupations sont menées pour interpeller la mairie et exiger des solutions de logement, considéré comme un droit fondamental.

      L'épreuve des démantèlements : Les expulsions de campements sont vécues comme des traumatismes répétés, aggravant l'épuisement physique et moral.

      6. Les perspectives d'avenir : Entre aspiration et précarité

      Malgré les obstacles, tous les jeunes expriment des rêves et des projets concrets. La fin du documentaire montre que des évolutions positives sont possibles.

      | Individu | Situation Initiale | Aspiration / Projet | Évolution à la fin | | --- | --- | --- | --- | | La jeune étudiante | Vit en campement avec sa famille. | Poursuivre ses études, devenir avocate ou médecin, avoir sa propre chambre. | A obtenu son brevet avec mention, est affectée en lycée général. Vit en hôtel avec sa famille. | | Le jeune demandeur d'asile (africain) | Dort dans le parc de Belleville. | Être considéré comme mineur, aller à l'école, avoir un toit. | Est reconnu mineur, suit un CAP et a obtenu un hébergement. | | Le jeune demandeur d'asile (brésilien) | Squatte un bâtiment incendié. | Travailler déclaré, quitter la précarité. | Travaille au noir, a pu louer une petite chambre, fait des économies. | | Le couple attendant un enfant | Vit dans la rue. | Trouver un logement stable pour accueillir leur enfant et éviter le placement. | Sont toujours à la recherche d'une solution rapide. | | La jeune femme dépendante | Lutte contre son addiction et la vie dans la rue. | Partir faire les saisons avec un camion aménagé. | A pris contact avec une association, initiant une première étape vers la sortie de rue. |

      Ces parcours montrent que si la sortie de l'itinérance est un processus long et difficile, l'accès à un statut, un logement, une formation ou un accompagnement social constitue le point de départ indispensable à la reconstruction.

    1. Document d'information : La mouvance du "mâle alpha"

      Synthèse

      Ce document d'information synthétise les thèmes, les figures clés et les impacts de la mouvance du "mâle alpha", un phénomène social émanant principalement des réseaux sociaux.

      Porté par des influenceurs comme Andrew Tate à l'international et des figures québécoises telles que Julien Bournival, ce mouvement prône un retour aux valeurs traditionnelles et à des rôles de genre strictement définis, où l'homme est le pourvoyeur et le leader, et la femme, plus soumise, se consacre au foyer.

      L'idéologie fondamentale repose sur une forme de déterminisme biologique, affirmant que les hommes et les femmes possèdent des caractéristiques innées et distinctes qui les destinent à des rôles différents.

      Ce discours trouve un écho particulier auprès de jeunes hommes en quête de repères, attirés par un message mêlant développement personnel (discipline, forme physique, succès entrepreneurial) et une rhétorique de rébellion contre un establishment perçu comme hostile.

      Les experts analysent cette mouvance comme une manifestation contemporaine d'un discours antiféministe récurrent, intrinsèquement misogyne, qui exprime une crainte de la perte des privilèges masculins face à l'avancée de l'égalité des genres.

      Ce phénomène est étroitement lié à une méfiance généralisée envers les institutions (gouvernement, médias, science), à l'adhésion à des théories du complot concernant une "élite" manipulatrice, et à une convergence avec les idéologies de la droite conservatrice, incluant un retour à la religion chrétienne.

      Socialement, cette mouvance contribue à une polarisation idéologique croissante entre les jeunes hommes, qui tendent à devenir plus conservateurs, et les jeunes femmes, de plus en plus progressistes.

      Son influence est désormais palpable jusque dans les salles de classe, où des discours rétrogrades et masculinistes refont surface, témoignant de la nécessité d'une vigilance continue face à la remise en question des acquis en matière d'égalité.

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      1. Définition et idéologie du mouvement "mâle alpha"

      Le mouvement "mâle alpha" est défini comme un phénomène émanant d'influenceurs du web et des réseaux sociaux qui prônent un retour à certaines valeurs traditionnelles. Son idéologie repose sur plusieurs piliers fondamentaux.

      Principes fondamentaux :

      Rôles de genre traditionnels : L'homme assume le rôle de leader et de pourvoyeur ("provider", "chef à la maison"), tandis que la femme est plus soumise et se consacre au foyer et à la famille ("nurture").

      Un influenceur affirme : "La règle c'est que l'homme est un homme puis est masculin puis il doit être le chef à la maison puis c'est lui le provider."

      Force et responsabilité masculine : L'homme "alpha" doit être fort physiquement et mentalement, prendre ses responsabilités, protéger et subvenir aux besoins de sa famille.

      Contrôle dans la relation : Certains discours promeuvent un contrôle sur la partenaire féminine. Un extrait viral stipule : "Quand tu es en couple, tu laisses pas ta blonde sortir d'un club.

      Tu laisses pas ta blonde aller dans un festival. Tu laisses pas ta blonde de mettre des photos de ses fesses en Gstring sur Instagram."

      Justification par le déterminisme biologique :

      Différences innées : Les partisans soutiennent que les hommes et les femmes sont biologiquement différents, ce qui détermine leurs traits de caractère. L'homme serait naturellement "assertif", "direct" et "fonceur", tandis que la femme serait dotée d'une "sensibilité" et d'une "intuition" supérieures.

      Rejet de l'égalité des compétences : L'idée que les hommes et les femmes sont égaux en aptitudes et en compétences est jugée "complètement ridicule" par Julien Bournival.

      Hypergamie : Un coach en dating du mouvement affirme que les femmes sont biologiquement attirées par des hommes qui leur sont supérieurs en termes de confiance, charisme, salaire, grandeur et force, un concept qu'il nomme "hypergamie".

      La "crise de la masculinité" :

      • Les influenceurs du mouvement estiment qu'il existe une "crise de la masculinité" causée par une société qui perçoit la masculinité comme "toxique" et tente d' "émasculiner" les hommes.

      • Cette perception est partagée par de jeunes hommes qui se sentent attaqués ou dévalorisés. Une jeune femme observe : "À force de se faire dire qu'on est méchant, qu'on est pas bon, qu'on est un problème, mais je pense que leur réaction c'est la colère."

      2. Figures clés et leurs discours

      Plusieurs influenceurs sont identifiés comme des figures centrales de ce mouvement, chacun avec un style et une portée distincts.

      Andrew Tate : La figure de proue internationale

      Profil : Influenceur britanno-américain, ancien champion de kickboxing, décrit comme une "méga star" et l'une des personnes les plus recherchées sur Google. Il a été arrêté en Roumanie pour trafic d'êtres humains, viol et formation d'un gang criminel.

      Message double : Son discours est un mélange de développement personnel (discipline, détermination, prise de responsabilité) et de propos jugés "irrespectueux, misagènes [sic] envers la femme".

      Défense de ses partisans : Ses adeptes, comme Julien Bournival, défendent "l'essence de son message" tout en minimisant ses controverses, les qualifiant de "jokes déplacés" ou d'actes d'un "personnage" destiné à provoquer. Un jeune homme affirme : "si m'aide à faire de l'argent. Je vois pas pourquoi je veux dire c'est une mauvaise personne".

      Julien Bournival : Le modèle québécois en Floride

      Profil : Entrepreneur québécois installé en Floride, il se décrit comme faisant partie du "1 % en terme de revenu" et du "1 % en terme de fitness". Il a quitté le Québec, qu'il qualifiait de "République socialiste" durant la pandémie.

      Discours : Il prône un retour aux valeurs traditionnelles, se définit comme un "pourvoyeur" et vit une relation où sa femme s'occupe de la maison et de la famille. Il lie de plus en plus ses valeurs à sa foi chrétienne.

      Activité entrepreneuriale : Il dirige une entreprise (Global) dans le domaine de l'amélioration énergétique, mais utilise ses réunions d'employés comme des séances de "croissance personnelle" où il promeut sa vision du monde, affirmant que les entrepreneurs ont une "responsabilité morale" de bâtir un peuple fort contre les "dirigeants" qui veulent un peuple faible et contrôlable.

      Louis Rassico : L'influenceur repenti

      Parcours : Jeune entraîneur québécois, il a été l'une des premières figures "mâle alpha" au Québec. Il admet avoir été influencé par Andrew Tate et avoir copié son style "intense" ("Ferme ta gueule") pour gagner en popularité, ce qui a fonctionné.

      Prise de distance : Il a depuis changé de discours, qualifiant Tate de "manipulateur" et décrivant son propre parcours comme une "déprogrammation" ou une "déradicalisation". Il a réalisé qu'il "perdai[t] contact avec la vraie réalité des choses".

      Chloé Roma : La défenseure des droits des hommes

      Position : Canadienne connaissant un grand succès en défendant les droits des hommes. Elle soutient que les hommes sont en crise, manquent de modèles positifs et sont toujours soumis à l'attente d'être "protecteur et pourvoyeur", contrairement aux femmes qui sont maintenant perçues comme capables de multiples rôles.

      Analyse sur Tate : Elle pense que le succès de Tate s'explique par le fait qu'il a touché une audience d'hommes sans figure paternelle ou modèle masculin positif, mais critique le fait que son message renforce les attentes négatives déjà pesantes sur les hommes.

      3. Analyse critique et impacts sociétaux

      Des experts et des acteurs de la société civile offrent une analyse critique de ce mouvement et de ses conséquences.

      Perspective sociologique (Francis Dupuis-Déri) :

      Discours récurrent : La "crise de la masculinité" n'est pas un phénomène nouveau. Des discours similaires existent depuis l'Antiquité romaine et à chaque siècle depuis, quel que soit le contexte politique ou culturel.

      Nature misogyne : Le discours de la crise est "nécessairement misogyne" car il postule que (1) les hommes vont mal, (2) c'est à cause des femmes, et (3) la solution est un retour à une masculinité traditionnelle.

      Réponse à l'égalité : Ce mouvement est une forme d'antiféminisme porté par des hommes qui "ne veulent pas de l'égalité" et voient le progrès des droits des femmes comme une "menace" à leurs privilèges.

      Réfutation du déterminisme : L'idée de rôles biologiquement définis est contredite par l'histoire de l'humanité, qui montre une grande diversité de rôles assumés par les hommes et les femmes. La différence des rôles est avant tout liée à la "socialisation et des éducations différentes".

      Impact en milieu scolaire (Véronique Guitras, enseignante) :

      Retour de discours rétrogrades : L'enseignante a constaté un "clash de discours" dans sa classe après un congé de maternité. Des élèves masculins tiennent désormais des propos "conservateurs, traditionnels, masculinistes".

      Exemples concrets : Un élève lui a affirmé que l'aspiration de toutes les femmes est d'être "invité sur un yat à Dubaï", et qu'elles ne sont pas des "bâtisseuses" comme les hommes. Elle décrit ce phénomène comme un retour "60 ans en arrière".

      Polarisation idéologique croissante :

      Fossé de genre : Un fossé idéologique se creuse chez les jeunes en Occident : les jeunes femmes deviennent de plus en plus progressistes et féministes, tandis que les jeunes hommes deviennent de plus en plus conservateurs.

      Débat "l'homme ou l'ours" : Ce débat viral illustre la méfiance des femmes envers les hommes.

      Une jeune femme explique préférer rencontrer un ours dans la forêt, car "l'ours quand il va m'attaquer, on va pas me demander comment j'étais habillée avant".

      Une autre affirme qu'il est "nécessaire pour nous de se méfier de tous les hommes" pour leur propre sécurité.

      4. Liens avec le conservatisme et les théories du complot

      Le discours "mâle alpha" est intrinsèquement lié à une méfiance envers les institutions et à une adhésion à des idéologies conservatrices et conspirationnistes.

      Méfiance envers les institutions :

      Rejet de l'autorité : Il existe une perte de confiance généralisée envers la science, la médecine, le gouvernement et surtout les médias, qualifiés d' "agence de publicité du gouvernement".

      Ce phénomène a été "considérablement accéléré" par la pandémie.

      Sentiment d'abandon : Selon l'anthropologue Samuel Viger, ce rejet peut provenir d'un sentiment d'abandon par le système (crises du logement, de la santé, inégalités croissantes), poussant certains individus vers des discours marginaux.

      Rhétorique conspirationniste :

      L'élite manipulatrice : Les influenceurs de la mouvance véhiculent l'idée qu'une "élite" satanique contrôle le monde et cherche à affaiblir la population en s'attaquant à la famille traditionnelle, en "brainwashant" les enfants et en promouvant une société de "weak person".

      La posture de rébellion : Adopter les valeurs "mâle alpha" est présenté comme "l'ultime rébellion" contre ce système de contrôle.

      Convergence avec la droite et la religion :

      Idéologie de droite : Le mouvement s'aligne sur des valeurs conservatrices. Julien Bournival admire Donald Trump et s'est installé en Floride pour le mode de vie républicain promu par Ron DeSantis.

      Retour à la foi chrétienne : Plusieurs figures du mouvement, dont Bournival, se tournent vers la Bible pour justifier les valeurs traditionnelles.

      Le passage biblique sur la soumission de la femme à l'homme (Éphésiens 5:22-33) est cité comme un "code d'éthique". La foi est présentée comme une garantie morale pour la soumission de la femme.

      Hostilité envers les minorités de genre :

      Vision rigide des genres : L'existence de personnes transgenres et de drag queens est perçue comme une attaque directe à leur conception "biologisante" et naturelle de l'homme et de la femme.

      Accusations de "grooming" : Les drag queens qui lisent des contes aux enfants sont accusées de "grooming" et de faire partie d'un "agenda satanique".

      Cette rhétorique escalade jusqu'à des comparaisons avec la pédophilie : "c'est quoi la prochaine affaire [...] c'est on va accepter les pédophiles".

      5. Citations marquantes

      | Thème | Citation | Locuteur | | --- | --- | --- | | Idéologie Mâle Alpha | "Chris, allez au gym, arrêtez de faire vos couches de guilleir." | Extrait audio d'influenceur | | Rôles Traditionnels | "La règle c'est que l'homme est un homme puis est masculin puis il doit être le chef à la maison puis c'est lui le provider." | Julien Bournival | | Soumission féminine | "Moi j'aime mieux être dans le shadow, m'occuper de notre maison \[...\] Va à la guerre, va au front, moi je reste derrière." | Partenaire de Julien Bournival | | Critique d'Andrew Tate | "On s'entend que Andw Tate a des propos irrespectueux, misagène envers la femme en général." | Journaliste | | Défense d'Andrew Tate | "L'essence de son message \[...\] c'est respecte-toi, respecte les autres, prends soin de toi. Assure-toi que quand tu dis de quoi, ta parole vaut de quoi." | Julien Bournival | | Impact scolaire | "Je me retrouve devant des jeunes qui ont des discours conservateurs, traditionnels, masculinistes \[...\] A on est revenu 60 ans en arrière." | Véronique Guitras, enseignante | | Analyse sociologique | "Le discours de crise \[...\] il dit les hommes vont mal. Ils vont mal à cause de qui ? Ils vont mal à cause des femmes." | Francis Dupuis-Déri, sociologue | | Polarisation | "L'ours quand il va m'attaquer, on va pas me demander comment j'étais habillée avant." | Jeune femme | | Théorie du complot | "Ceux qui contrôlent le monde sont satanique. Ils contrôlent les gouvernements." | Julien Bournival | | Repentir | "\[Andrew Tate\] est manipulateur clairement. \[...\] Moi-même je me suis fait influencer par lui \[...\] Je me suis déprogrammé." | Louis Rassico, entraîneur | | Vigilance | "J'ai deux filles. J'ai pas envie qu'elle vivent dans un monde inégalitaire. J'ai pas envie qu'elle soit soumise à quiconque. \[...\] il y a rien de gagner pour toujours." | Journaliste |

    1. Synthèse sur le Harcèlement Scolaire : Causes, Conséquences et Stratégies de Prévention

      Résumé Exécutif

      Ce document de synthèse analyse en profondeur la problématique du harcèlement scolaire en France, en s'appuyant sur des témoignages et des études de cas poignants.

      Il met en lumière les conséquences tragiques du harcèlement à travers les suicides de Dina et Lucas, deux adolescents de 13 ans, tout en explorant les mécanismes sous-jacents, les défaillances institutionnelles et les stratégies de prévention émergentes.

      Les principaux points à retenir sont les suivants :

      1. Les Visages de la Tragédie : Les cas de Dina et Lucas illustrent la violence extrême du harcèlement. Les motifs sont multiples et ciblent la différence :

      • l'intelligence perçue,
      • l'apparence physique,
      • l'exploration de l'identité sexuelle (Dina),
      • ou encore l'homosexualité affirmée et la réussite scolaire (Lucas).

      Ces agressions répétées, verbales et physiques, créent un sentiment insupportable de honte et d'isolement menant à des actes désespérés.

      2. Défaillances Systémiques : Les témoignages révèlent de graves lacunes dans la réponse des institutions scolaires et judiciaires.

      Les collèges ont souvent minimisé les faits, tardé à agir ou adopté des approches inefficaces qui ont aggravé la souffrance des victimes.

      Le système judiciaire a initialement classé sans suite l'affaire de Dina et annulé en appel la condamnation des harceleurs de Lucas, niant le lien entre le harcèlement et le suicide, et laissant les familles dans un profond désarroi.

      3. Un Changement de Paradigme : Face à ces échecs, de nouvelles stratégies proactives sont mises en œuvre.

      Le programme national Phare et la méthode de la préoccupation partagée, adaptée par Jean-Pierre Bélon, marquent une rupture avec l'approche punitive traditionnelle.

      Cette méthode non-accusatoire vise à responsabiliser les intimidateurs en les impliquant dans la recherche de solutions pour le bien-être de la victime, transformant ainsi leur comportement par l'empathie.

      4. Prévention et Reconstruction : La prévention commence dès l'école primaire, avec des programmes axés sur l'intelligence émotionnelle, la communication non-violente ("messages clairs") et le développement de la confiance en soi.

      Pour les victimes, la reconstruction est un long processus impliquant des thérapies (comme celles de l'association Marion la main tendue) pour surmonter des traumatismes profonds tels que la phobie scolaire et la perte d'estime de soi, qui peuvent persister des décennies plus tard.

      En conclusion, si le harcèlement scolaire a longtemps été banalisé, une prise de conscience sociétale, catalysée par des drames comme celui de Dina, a conduit à une évolution législative et à l'adoption de méthodes préventives prometteuses.

      Celles-ci reposent sur une approche systémique, éducative et empathique, visant à transformer le climat scolaire pour garantir la sécurité et le bien-être de chaque élève.

      --------------------------------------------------------------------------------

      I. Études de Cas : Les Histoires de Dina et Lucas

      A. Le Cas de Dina : Une Spirale de Jalousie et d'Homophobie

      Dina, 13 ans, est décrite par son frère, Ryan Gontier, comme une jeune fille "en avance sur son temps", intelligente, curieuse de la politique et de l'actualité, ce qui a suscité la jalousie de ses amies.

      Facteurs Déclencheurs : Le harcèlement a commencé en 4ème (2019) lorsque ses amies ont commencé à lui reprocher son intelligence et sa manière de s'exprimer : "tu es toujours en train de nous corriger parce qu'on parle pas bien français par rapport à toi." Le harcèlement s'est intensifié lorsque Dina a commencé à s'intéresser au courant LGBT et à évoquer la possibilité d'aimer aussi bien les filles que les garçons, menant à son exclusion du groupe.

      La Trahison : Un tournant tragique a eu lieu lorsqu'une de ses amies a prétendu être lesbienne pour sortir avec elle.

      Après une vingtaine de jours, elle a rompu brutalement en révélant la supercherie : "Mais moi je t'aime pas, c'est pas vrai. Je tiens à inventer cette histoire."

      L'Escalade du Harcèlement : La nouvelle a été propagée dans la cour du collège, déclenchant des insultes homophobes ("guine", "lesbienne") et des violences physiques.

      Le harcèlement s'est ensuite étendu à son physique ("trop grosse") et à ses vêtements.

      Sa mère témoigne : "Elle pouvait porter n'importe quoi. C'était prétexte à se faire insulter."

      Première Tentative de Suicide : En mars 2021, profondément blessée, Dina fait une première tentative de suicide par ingestion de médicaments.

      Hospitalisée, elle explique ne plus vouloir vivre pour ne pas affronter les moqueries à son retour au collège.

      Le Retour Impossible : Son retour au collège est un calvaire. Sa mère raconte recevoir des appels de détresse : "Maman, je suis aux toilettes. Maman, je vais mourir. Maman, il faut venir me chercher."

      Le Suicide : Malgré un changement de lycée en septembre 2021, une rencontre fortuite avec ses anciennes harceleuses à la cantine un mois plus tard a ravivé son traumatisme. "Je pense qu'elle s'est dit ça va recommencer", analyse son frère.

      Une semaine après, Dina met fin à ses jours en se pendant dans son armoire.

      B. Le Cas de Lucas : La Différence Ciblée

      Lucas, 13 ans, s'est suicidé en janvier 2023 après avoir été harcelé en raison de son homosexualité et de sa réussite scolaire au collège Louis Armand de Golbey.

      Affirmation de Soi : Lucas avait fait son coming-out à sa mère, qui l'avait pleinement soutenu : "Sois heureux mon fils parce que je le savais déjà en fait."

      Il est décrit comme un très bon élève, assidu et participatif, ce qui aurait provoqué la jalousie d'autres élèves.

      Harcèlement Continu : Dès son arrivée au collège, Lucas subit des insultes sur son physique, sa manière de s'habiller et de parler, et son orientation sexuelle. Sa mère explique : "Ça a été crescendo avec les quatre élèves dont il m'avait donné les noms déjà."

      Un Refuge dans la Danse : Lucas trouvait un exutoire et un espace d'acceptation dans un club de danse hip-hop, basé sur des valeurs de diversité et d'unité.

      L'Acte Final : Le harcèlement s'est poursuivi jusqu'à la veille de sa mort.

      Sa mère pense que les insultes du vendredi 6 janvier ont été "la goutte de trop."

      Il a été retrouvé pendu par sa petite sœur de 5 ans et son ex-conjoint.

      Sa mère interprète son geste : "Je pense que c'était pour couper sa voix en fait. C'était pour dire stop."

      II. Défaillances Institutionnelles et Judiciaires

      A. La Réponse des Établissements Scolaires

      Dans les deux cas, la réponse des institutions scolaires est présentée comme largement insuffisante.

      Pour Dina : Les parents ont rencontré la Conseillère Principale d'Éducation (CPE) à plusieurs reprises.

      Au lieu de confronter les harceleuses, la CPE a demandé à Dina d'écrire leurs noms sur un papier, une tâche que l'adolescente, tremblante, n'a pas pu accomplir.

      Cette approche a mis le fardeau de la preuve et de la dénonciation sur la victime déjà fragilisée.

      Pour Lucas : Sa mère a signalé les faits et les noms des harceleurs.

      Seul un professeur a réagi en envoyant un communiqué à ses collègues, mais sans suite. "Il y a pas eu de convocation des parents, il y a rien eu." Les sanctions n'ont été prises qu'après le drame : les quatre élèves responsables ont été exclus 14 jours après le décès de Lucas. "C'est avant qu'il aurait fallu le protéger."

      Banalisation du Harcèlement : Un expert souligne une tendance historique en France à banaliser les brimades, considérées à tort comme "formatrices".

      Or, "on sait aujourd'hui que ces brimades répétées, ces insultes à répétition, elles sont dévastatrices."

      B. Les Obstacles Judiciaires

      Les familles ont également dû faire face à un système judiciaire qui a, dans un premier temps, nié la gravité des faits.

      Affaire Dina : L'enquête a été initialement classée sans suite par la procureure. La famille a appris cette décision par la presse.

      Le procureur a suggéré que Dina s'était suicidée en raison de sa "fragilité psychologique" et que "rien ne s'était passé dans le collège".

      La famille a dû se constituer partie civile pour que l'enquête reprenne finalement en novembre 2023, sur la base de chefs d'accusation incluant harcèlement, provocation au suicide et homicide involontaire.

      Affaire Lucas : En juin 2023, les quatre harceleurs ont été reconnus coupables de harcèlement, mais le tribunal a refusé d'établir un lien avec le suicide.

      En novembre 2023, la condamnation a été annulée par la cour d'appel de Nancy, qui a estimé que le harcèlement n'était pas prouvé, ni son effet sur la santé mentale de Lucas.

      Sa mère a annoncé un pourvoi en cassation. Les harceleurs, se sentant acquittés, continueraient de rire en parlant de Lucas.

      L'affaire Dina a cependant eu un impact législatif majeur, conduisant au vote en mars 2022 d'une nouvelle loi créant le délit de harcèlement scolaire, passible de peines allant jusqu'à 10 ans de prison et 150 000 € d'amende en cas de tentative de suicide de la victime.

      III. L'Émergence de Nouvelles Stratégies de Prévention

      Face à l'échec des approches traditionnelles, des méthodes alternatives axées sur la prévention et l'empathie sont mises en place dans certains établissements, notamment dans le cadre du programme national Phare.

      A. La Méthode de la Préoccupation Partagée

      Adaptée en France par Jean-Pierre Bélon, cette méthode vise à résoudre les situations de harcèlement sans passer par l'accusation et la sanction, qui se révèlent souvent contre-productives.

      Principe Clé : Au lieu de confronter l'intimidateur, l'équipe éducative le convoque en lui exprimant son inquiétude pour la victime.

      L'objectif est de le transformer en acteur de la solution. On lui demande : "qu'est-ce qu'il pourrait mettre en place pour cet élève ?"

      Posture de l'Adulte : L'entretien doit être court, direct et ferme, mais courtois.

      L'adulte ne doit pas entrer dans un débat argumentatif ("c'est elle qui a commencé") mais maintenir le cap sur le mal-être de la victime.

      L'autorité est incarnée par la certitude que les brimades doivent cesser.

      Efficacité : Cette méthode montre des résultats très positifs.

      Elle permet de "casser ce cercle vicieux" et de créer une nouvelle dynamique. Un principal de collège, Philippe Weis, témoigne que grâce à cette approche, de nombreuses situations (46 en avril) sont réglées avant de devenir graves.

      B. L'Implication des Élèves : Les Ambassadeurs Anti-Harcèlement

      Le programme Phare prévoit la formation d'élèves ambassadeurs pour jouer un rôle de "sentinelle".

      Rôle : Ces élèves sont formés pour détecter les situations de harcèlement, écouter leurs camarades et alerter les adultes.

      Ils ne sont pas des "balances" mais des relais de confiance. Comme l'explique un élève : "Nous, on est des enfants et eux aussi et ils se sentent plus peut-être plus en sécurité."

      Motivation : Beaucoup d'ambassadeurs, comme Charlotte, ont eux-mêmes été victimes de harcèlement, ce qui renforce leur engagement. Dans un collège, 30 élèves se sont portés volontaires pour 12 places initialement prévues.

      C. La Prévention dès l'École Primaire

      La lutte contre le harcèlement commence dès le plus jeune âge par le développement de compétences psychosociales.

      Intelligence Émotionnelle : Une école primaire de la banlieue parisienne a mis en place des outils pour aider les enfants à identifier et nommer leurs émotions (par exemple, un système de "souris" de différentes couleurs à l'entrée de la classe).

      Méthode des "Messages Clairs" : Les élèves de CM2 apprennent à gérer les conflits en exprimant leur ressenti de manière structurée et non-violente, à l'écart des autres.

      Un élève dit à un autre : "Quand tu m'as déplacé mon manteau, je me suis senti mal parce que tu fais ça tout le temps. J'aimerais que tu arrêtes s'il te plaît."

      Confiance en Soi : La directrice de l'école insiste sur l'importance de la confiance en soi, qui permet à un enfant de "réagir face à des brimades" et de "solliciter l'adulte" si besoin.

      IV. La Reconstruction et les Séquelles à Long Terme

      A. Le Traumatisme des Victimes Survivantes

      Le harcèlement laisse des cicatrices psychologiques profondes et durables.

      Savana : Harcelée pour ses bonnes notes, elle a développé une phobie scolaire et sociale qui l'a contrainte à arrêter ses activités extrascolaires. "Ils ont complètement gâché ma vie en fait."

      Grâce à l'association Marion la main tendue, elle participe à des groupes de parole et des séances de thérapie où elle apprend à "mettre des mots sur ses maux (m-a-u-x)".

      Julien Masquino : Harcelé 20 ans avant Lucas dans le même collège pour son "côté un peu efféminé", son "embonpoint" et son "langage châtié", il a souffert de l'effet de meute.

      Le harcèlement a détruit son estime de soi : "Vous vous dites qu'un moment peut-être que si je suis victime c'est que je le mérite."

      Il a repris confiance en lui grâce au sport et enseigne aujourd'hui le self-défense à des adolescents.

      B. Le Deuil Impossible des Familles

      Pour les familles des victimes, le deuil est inextricablement lié à la reconnaissance du harcèlement comme cause du décès.

      • Le frère de Dina déclare : "Je pense pas que mes parents et moi on puisse faire notre deuil sans que son harcèlement scolaire soit reconnu comme étant la cause de son suicide."

      • La douleur est constante, comme en témoigne le texte lu à la fin du document :

      Ton visage, ton sourire me hante, tes câlins, tes mots doux me manquent. [...] J'aimerais tellement savoir si de là-haut tu pouvais me voir, si de là où tu pouvais comprendre que sans toi plus rien n'a de sens.

    1. Synthèse du débat sur la santé mentale et la schizophrénie

      Résumé Exécutif

      Ce document synthétise les échanges d'un débat entre experts, soignants, représentants d'associations et une personne concernée, centré sur la schizophrénie et les troubles psychiques en France.

      Les discussions mettent en lumière plusieurs constats critiques : la stigmatisation profondément ancrée autour des maladies psychiques sévères, qui engendre honte et isolement, reste un obstacle majeur.

      Le parcours de soin est un véritable "labyrinthe", caractérisé par un délai de prise en charge moyen d'un an et demi après les premiers symptômes et des diagnostics initiaux souvent complexes et incertains.

      Une révolution s'opère cependant dans l'approche des soins, passant d'un modèle paternaliste à une vision centrée sur le "rétablissement" de la personne, où le patient devient l'acteur principal de son projet de vie, soutenu par sa famille qui est désormais considérée comme un partenaire essentiel.

      Enfin, le débat souligne les faiblesses structurelles du système psychiatrique français, marqué par un manque de moyens endémique, de fortes inégalités territoriales d'accès aux soins et un besoin crucial de décloisonnement entre les différents acteurs sanitaires, sociaux et professionnels.

      L'espoir, porté par les pairs-aidants et une approche plus humaine, émerge comme le moteur fondamental de la reconstruction des parcours de vie.

      --------------------------------------------------------------------------------

      Introduction

      Suite à la diffusion du documentaire "Nous, schizophrènes et combattants", un débat a réuni plusieurs acteurs clés du domaine de la santé mentale pour discuter des défis liés à la schizophrénie et aux troubles psychiques.

      Les intervenants ont partagé leurs expertises et expériences sur la détection, la prise en charge et les perspectives de rétablissement, tout en abordant l'état du système psychiatrique en France.

      Participants au débat :

      Léopole de Pomier : Patient et pair-aidant à l'association Père et danse Montpellier.

      Alexandre de Conor : Docteur en psychologie, responsable du centre de rétablissement et de réhabilitation Jean Mainviel à Montpellier.

      Étienne Verry : Praticien hospitalier au CHU de Toulouse et au centre expert schizophrénie.

      Michel Comte : Délégué régional de l'Unafam Occitanie (Union nationale de familles et amis de personnes malades et/ou handicapées psychiques).

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      Thème 1 : La Stigmatisation et le Tabou Persistants

      La perception sociale des troubles psychiques, et en particulier de la schizophrénie, reste un obstacle majeur au diagnostic, à la prise en charge et au rétablissement.

      Persistance des clichés : La schizophrénie est encore largement et à tort associée à la "double personnalité" dans les médias et l'imaginaire collectif.

      Alexandre de Conor souligne que "c'est encore un mot qui est utilisé pour désigner un changement de comportement brusque".

      Honte et auto-stigmatisation : Les personnes concernées intériorisent cette image négative. Julien, dans le documentaire, se dit "rouge de honte", ce à quoi sa mère répond : "Un diabétique n'a pas honte d'être malade".

      Cette stigmatisation a des conséquences concrètes : isolement, difficultés d'accès aux relations amicales, amoureuses, au logement.

      Vulnérabilité accrue : Contrairement à l'image du "fou dangereux", les chiffres montrent que les personnes souffrant de schizophrénie sont plus souvent victimes de personnes malveillantes que auteurs de violences. Alexandre de Conor insiste : "les chiffres nous montrent que c'est des gens qui sont plus victimes".

      Barrières culturelles et religieuses : Léopole de Pomier témoigne de l'influence de certaines croyances qui associent la maladie à une forme de possession : "on pense que c'est des esprits qui nous habitent et tout. Ça, je trouve que c'est une vraie bêtise".

      Thème 2 : Le Parcours du Dépistage et du Diagnostic

      L'accès aux soins est un processus long et complexe, souvent qualifié de "parcours du combattant" pour les personnes et leurs familles.

      Délai de prise en charge : En France, comme en Europe, la durée moyenne entre l'apparition des premiers symptômes psychotiques et la première prise en charge spécialisée est d'environ un an et demi.

      Ce délai est un enjeu majeur sur lequel il est possible d'agir.

      Difficultés du repérage précoce : Les premiers signes apparaissent souvent à l'adolescence et peuvent être confondus avec une "crise d'adolescence un peu violente", retardant ainsi le début d'un accompagnement adapté.

      Un système de soins labyrinthique : Alexandre de Conor décrit le système comme "un labyrinthe en fait entre la psychiatrie, le médico-social".

      Cette complexité et le manque de communication entre les structures peuvent décourager les familles et faire perdre un temps précieux.

      La prudence du diagnostic :

      ◦ Étienne Verry explique qu'un diagnostic posé trop rapidement lors d'un premier épisode psychotique se révèle erroné dans un cas sur deux à cinq ans.

      La prudence est donc de mise, en parlant initialement de "premier épisode psychotique" plutôt que de schizophrénie.  

      ◦ Alexandre de Conor ajoute que des diagnostics hâtifs peuvent être dangereux, notamment pour les minorités ethniques ou les populations défavorisées, et mener à des propositions de soins inadaptées (ex: prescription de neuroleptiques non justifiée).

      Thème 3 : La Prise en Charge et le Concept de Rétablissement

      Une transformation profonde de l'approche des soins est en cours, axée sur la personne, son projet de vie et son rétablissement.

      L'hospitalisation sous contrainte : Souvent redoutée, elle est décrite par la mère de Julien comme "la pire décision de ma vie".

      Michel Comte la qualifie d'expérience "violente" et "terrible" pour une famille. Les intervenants s'accordent sur la nécessité de l'éviter au maximum grâce à une intervention précoce et des équipes mobiles, tout en reconnaissant sa nécessité dans certaines situations de perte de contact avec la réalité.

      Du soin à la guérison, au rétablissement : La schizophrénie étant une maladie chronique, on ne parle pas de guérison mais de "rétablissement".

      Il s'agit de "vivre une vie satisfaisante et qui a du sens pour la personne malgré cette présence de la maladie" (Alexandre de Conor).

      L'approche centrée sur la personne :

      ◦ Cette approche constitue une "petite révolution" face à une médecine historiquement "patriarcale et très autoritaire" (Étienne Verry).   

      ◦ Elle implique de ne plus parler des personnes sans leur présence et de partir de leurs projets (emploi, logement) pour proposer un accompagnement.  

      Exemple concret : Si un jeune veut reprendre un emploi mais est gêné par des voix, on lui propose une aide pour gérer ce symptôme au service de son objectif professionnel.  

      ◦ Cela implique d'accepter une prise de risque accompagnée, par exemple en soutenant un jeune qui souhaite essayer d'arrêter son traitement, car "s'il a décidé de le faire, si on lui dit non, il le fera sans nous" (Alexandre de Conor).

      Thème 4 : Le Rôle Central des Familles et des Proches

      La famille, longtemps tenue à l'écart ou culpabilisée par la psychiatrie, est aujourd'hui reconnue comme un pilier du rétablissement.

      De la culpabilité à la solution : Michel Comte décrit le parcours des familles, qui passent souvent par le déni, la sidération et un sentiment de culpabilité ("Qu'est-ce que j'ai loupé ?") avant de comprendre qu'elles ne sont "pas le problème" mais "la solution".

      L'impact du soutien familial : Les chiffres confirment que lorsque les familles comprennent la maladie et sont soutenantes, "clairement ça a un impact très fort sur le rétablissement".

      L'intégration des familles aux soins : Des programmes de psychoéducation, comme le programme BREF, sont mis en place pour intégrer les familles, les personnes concernées et les soignants dès le début de la maladie.

      Soutien par les pairs-aidants familiaux : Des associations comme l'Unafam, composées de proches et d'aidants, offrent un espace d'écoute, de partage et de "reconstruction" pour les familles qui se sentent "en miettes".

      Thème 5 : Les Défis du Système Psychiatrique en France

      Le débat a mis en exergue les failles structurelles du système de santé mentale français, qui freinent l'accès à des soins de qualité pour tous.

      Manque de moyens et de personnel : Le secteur souffre d'un "manque de moyen endémique". Une grande proportion des postes de psychiatres dans les établissements publics ne sont pas pourvus.

      Inégalités territoriales : L'accès aux soins est très inégal. Des départements comme le Gers ou l'Ariège comptent moins de 6 psychiatres pour 100 000 habitants.

      Les Centres Médico-Psychologiques (CMP), essentiels dans la psychiatrie de secteur, connaissent des dysfonctionnements majeurs dans certains territoires, avec des délais de rendez-vous de plusieurs mois.

      Nécessité de décloisonnement : Il y a un manque critique de coordination entre la psychiatrie, les autres spécialités médicales (pour les maladies somatiques comme l'obésité liée aux traitements) et les secteurs social et professionnel. La fonction de "case manager" est présentée comme un outil pour orchestrer le parcours du jeune.

      Nouveaux modèles à développer : L'importance de l'approche "aller vers", qui consiste à se déplacer pour rencontrer les jeunes là où ils sont, est soulignée.

      Ce modèle est efficace mais coûteux car il requiert d'importantes ressources humaines.

      Thème 6 : L'Espoir, la Pair-Aidance et la Force du Collectif

      Malgré les difficultés, un message d'espoir traverse l'ensemble du débat, incarné par de nouvelles approches et la valorisation de l'expérience vécue.

      Le pouvoir de l'espoir : Il est crucial de lutter contre la représentation fataliste de la schizophrénie. Étienne Verry rappelle que 20% des patients connaissent un rétablissement complet, parfois même sans traitement, et que le pronostic est donc extrêmement variable. La maladie ne doit pas "tuer l'ambition, le rêve".

      La pair-aidance : Le rôle de Léopole de Pomier est emblématique.

      En tant que pair-aidant, il utilise son propre parcours pour aider les autres : "Si j'ai le contrôle sur ma maladie, je peux aider les autres". Cette transmission d'expérience donne de l'espoir et des outils concrets.

      La force du collectif : Les groupes de parole, pour les personnes concernées comme pour les proches, sont des lieux d'enrichissement mutuel où les expériences et les stratégies sont partagées.

      Le combat pour l'inclusion : Le mot "combattant" du titre du documentaire est validé par tous les participants.

      C'est un combat pour le rétablissement personnel, mais aussi un combat sociétal pour "abattre les stéréotypes" et pour l'inclusion.

      Comme le conclut Étienne Verry, améliorer le pronostic des jeunes est "une affaire de tous. C'est pas juste une affaire de la santé".

    1. Begin by reading the assignment instructions. Your instructor will likely have clear goals for the peer review process, and following the instructions will help you provide significant and meaningful revision ideas for your peer. Read your peer’s essay from the beginning to the end without adding any comments. This first read allows you to grasp your peer’s intentions and focus. Complete a second reading of your peer’s draft and start looking for strengths and weaknesses. Make comments on the margins of your peer’s essay. Later, you can further expand on these comments when you complete the peer review form. Stop when you feel stuck and ask yourself, “If this were my paper, how would I revise it?” Set aside time to review the organization of your peer’s essay. Read their thesis statement and make sure their body paragraphs have topic sentences that connect to their thesis statement. If there isn’t a clear connection, consider helping your peer revise their topic sentence so the connection between the thesis and body paragraph is easy to understand. Be honest. Your peers want to earn the best grade they can, and your advice during peer review will help them achieve this goal. Think of every piece of advice as constructive criticism. Your advice will help them to create a stronger, more focused writing sample.

      1.Read the assignment 2.read peer essay begging to end with no comments 3.finish second read to look for strength and weakness 4.stop when stuck 5.set time to review 6.be honest.

  2. Jan 2026
    1. ¿Qué cinco acciones

      1 Create a blank Logic app

      2 Add a logic app trigger that fires when one or more messages arrive in the queue

      3 Add an action that reads IoT temperature data from the Service Bus queue

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    2. Seleccionar y colocar:

      Orden Acción 1 Create a blank Logic app

      2 Add a logic app trigger that fires when one or more messages arrive in the queue

      3 Add an action that reads IoT temperature data from the Service Bus queue

      4 Add a condition that compares the temperature against the upper and lower thresholds

      5 Add an action that sends an email if temperature is outside thresholds

    1. Rapport de Synthèse : Crise et Dérives du Secteur Périscolaire et de l’Enseignement Privé

      Résumé Exécutif

      Une enquête approfondie de l'émission « Cash Investigation » révèle des défaillances systémiques graves au sein du secteur périscolaire et de certains établissements d'enseignement privé sous contrat en France.

      Le constat est alarmant : violences physiques et verbales, suspicions d'agressions sexuelles, et manque de surveillance généralisé.

      Les points clés à retenir :

      Ampleur nationale : À Paris, 128 animateurs ont été suspendus en 10 ans pour suspicion de violences sexuelles.

      Le phénomène touche l'ensemble du territoire, des grandes métropoles aux zones rurales.

      Failles de recrutement : Une infiltration de 27 heures a suffi pour constater des comportements inappropriés (cris, usage de téléphones, gestes déplacés) et un non-respect flagrant des quotas d'encadrement.

      Inertie institutionnelle : Des signalements d'abus restent parfois sans réponse pendant plusieurs années avant qu'une action concrète ne soit entreprise.

      Échec judiciaire : Seules 3 % des plaintes pour violences sexuelles sur mineurs aboutissent à une condamnation, tandis que 73 % sont classées sans suite.

      Manque de contrôle du privé : Les établissements privés sous contrat, financés à 75 % par l'État, échappent à une surveillance rigoureuse malgré des cas de maltraitance institutionnalisée.

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      I. Analyse du Secteur Périscolaire : Un Système en Souffrance

      L'enquête met en lumière une réalité quotidienne brutale dans les accueils périscolaires (cantines, garderies), souvent méconnue des parents qui supposent leurs enfants protégés par l'institution scolaire.

      A. Des conditions d'exercice dégradées

      L'infiltration d'une journaliste, embauchée sans expérience préalable malgré la possession du BAFA, révèle des dérives immédiates après seulement 27 heures d'observation :

      Comportements inappropriés : Animateurs focalisés sur leurs téléphones portables, hurlements constants contre les enfants, et gestes graves comme une animatrice embrassant des enfants de 4 ans sur la bouche.

      Sous-effectif chronique : Dans certains cas, le nombre d'enfants par animateur est plus du double du quota légal (par exemple, une seule personne pour 23 enfants en cour de récréation).

      Manque de considération : Le métier souffre de bas salaires, d'un recrutement précaire et d'une absence de reconnaissance professionnelle, favorisant un climat d'agressivité.

      B. Problématiques de recrutement et de formation

      Les municipalités, responsables du périscolaire, font face à des difficultés majeures pour trouver du personnel qualifié.

      Règles floues : Le recrutement se base parfois uniquement sur la "bienveillance et le bon sens", sans formation approfondie.

      Hétérogénéité de gestion : La qualité de l'encadrement dépend directement des choix budgétaires et politiques de chaque mairie.

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      II. Violences Sexuelles et Impunité

      Le document souligne une défaillance critique dans la protection des mineurs face aux prédateurs potentiels au sein des structures d'accueil.

      A. Données chiffrées et réalité du terrain

      Il n'existe pas de statistiques officielles nationales, forçant les enquêteurs à compiler leurs propres données à partir de la presse quotidienne régionale.

      Suspensions : 128 animateurs suspendus à Paris en une décennie.

      Répartition : Des affaires sont recensées partout en France (Nancy, Marseille, Haute-Savoie, Moselle).

      B. Le traitement des signalements

      Un problème majeur réside dans la lenteur de réaction des autorités face aux alertes :

      Cas du 15e arrondissement : Un animateur a été interpellé en 2023 pour des jeux et danses à caractère sexuel, alors que des signalements le concernant existaient depuis 2019.

      Culture du silence : Trop souvent, les signalements ne sont pas correctement pris en compte, permettant à des individus suspects de rester au contact des enfants pendant des mois, voire des années.

      C. Statistiques Judiciaires (Source : Ciivise)

      | Type de procédure | Pourcentage | | --- | --- | | Plaintes classées sans suite | 73 % | | Plaintes aboutissant à une condamnation | 3 % |

      Cette difficulté de condamnation s'explique souvent par la situation de "parole contre parole", où la parole de l'enfant n'est pas toujours recueillie dans des conditions optimales.

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      III. L'Enseignement Privé sous Contrat : Une Zone d'Ombre

      L'enquête s'étend aux établissements privés catholiques sous contrat, qui scolarisent près de 2 millions d'élèves en France.

      Financement Public : Ces établissements sont financés à environ 75 % par l'argent public.

      Maltraitance Institutionnalisée : Des témoignages font état de violences physiques (ex: "roustes" en public au self) sur des enfants dès l'âge de 12 ans.

      Absence de contrôle : Malgré l'importance des fonds publics engagés, l'État n'exerce pas de contrôle suffisant sur ces établissements.

      Les syndicats dénoncent un silence persistant (omerta) au sein de l'Enseignement catholique.

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      IV. Recommandations et Solutions

      A. Modèles de réussite

      L'enquête cite l'exemple d'un village ayant réussi à transformer son périscolaire en augmentant son budget de seulement 8 %. Cette hausse a permis :

      • La prise en compte du temps de préparation des ateliers.

      • Une formation accrue des animateurs.

      • Un environnement où les enfants se sentent en sécurité et épanouis.

      B. Vigilance des parents

      Face à ces risques, les pédopsychiatres conseillent d'être attentifs aux changements brusques de comportement chez l'enfant :

      • Troubles du sommeil (énurésie nocturne/pipi au lit).

      • Troubles alimentaires (perte d'appétit).

      • Changements d'humeur ou repli sur soi.

      Il est préconisé de favoriser des moments de "parole libre" lors de situations calmes (bain, trajet en voiture, lecture du soir) pour permettre à l'enfant d'exprimer un éventuel malaise.

    1. État des Lieux du Secteur Périscolaire à Paris : Dysfonctionnements et Dérives

      Résumé Exécutif

      Ce document de synthèse expose les défaillances systémiques au sein des structures périscolaires de la Ville de Paris, telles que révélées par une enquête en immersion.

      Le constat met en lumière une gestion de crise permanente caractérisée par un recrutement fondé sur la simple disponibilité plutôt que sur les compétences, une absence de formation réelle et des violations récurrentes des taux d'encadrement légaux.

      L'analyse révèle un environnement où la sécurité affective et physique des enfants est compromise par des violences verbales, un désinvestissement de certains agents et une impunité institutionnalisée pour les titulaires problématiques via un système de "chaises musicales".

      Le manque de contrôle et d'exigence de la hiérarchie favorise l'émergence de dérives graves, allant de la négligence à des signalements d'attouchements sexuels.

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      1. Un Processus de Recrutement Fondé sur l'Urgence

      Le recrutement des animateurs vacataires semble dicté par une nécessité de combler les effectifs plutôt que par une évaluation des aptitudes pédagogiques.

      Critères de sélection minimaux : La mairie exige uniquement d'avoir 18 ans, d'être motivé, d'avoir un casier judiciaire vierge (vérification du FIJAIS) et des vaccins à jour.

      Absence d'évaluation des compétences : Les entretiens ne comportent aucune question sur la gestion des enfants ou l'expérience éducative. L'atout majeur retenu est la disponibilité du candidat.

      Le dogme du "bon sens" : À défaut de directives claires, le recrutement repose sur deux notions vagues : la "bienveillance" et le "bon sens", y compris pour prodiguer des soins en l'absence d'infirmière.

      Profils à risque : Le manque d'exigence conduit au recrutement de profils inadaptés.

      Un témoignage interne (Karim, responsable éducatif ville) rapporte l'envoi de remplaçants arrivant en état d'ébriété ou issus de parcours de réinsertion sans évaluation préalable suffisante, malgré des avis de recrutement signalant une instabilité.

      2. Déficience de la Formation et de l'Intégration

      L'immersion démontre un décalage profond entre les ambitions affichées par la Ville et la réalité du terrain.

      L'inexistence du parcours d'intégration : La formation se résume à un "briefing express" (chronométré à 6 minutes et 42 secondes dans un cas précis) avant une mise en poste immédiate.

      La Charte de l'animateur ignorée : Bien que ce document définisse les missions éducatives et proscrive les comportements ambigus, il n'est souvent ni présenté ni signé par les nouveaux arrivants.

      Improvisation pédagogique : Alors que le temps périscolaire est censé être éducatif, aucune consigne ne soutient les projets pédagogiques.

      Les animateurs sont invités à "faire des dessins" ou à improviser des jeux sur leur temps personnel.

      3. Conditions de Travail et Non-Respect de la Légalité

      La gestion des effectifs se heurte à une pénurie chronique de personnel qualifié, entraînant des entorses régulières à la réglementation.

      Taux d'encadrement

      | Norme Légale (moins de 6 ans) | Réalité constatée sur le terrain | | --- | --- | | 1 animateur pour 14 enfants | Jusqu'à 1 animateur pour 23 enfants | | Surveillance active requise | Sous-effectifs fréquents (ex: 2 animateurs pour 70 enfants) |

      Précarité et rotation : Les postes de vacataires (payés 15 € brut de l'heure) servent à "boucher les trous" sans continuité éducative, les remplaçants changeant de groupe quotidiennement sans présentation préalable.

      Difficulté de recrutement : La mairie peine à trouver des profils compétents en raison des plannings morcelés et des temps partiels, ne parvenant à compléter ses équipes qu'en période de fin d'études universitaires.

      4. Dérives Comportementales et Climat de Violence

      Le manque de cadre et de formation génère des comportements abusifs au sein des écoles.

      Violences verbales systématiques : L'usage des cris, des menaces ("vous allez rien manger") et des humiliations est une pratique quotidienne pour obtenir le silence ou le respect des règles à la cantine.

      Négligence et désinvestissement : De nombreux animateurs privilégient l'usage personnel de leur smartphone au détriment de la surveillance active des enfants, en violation directe de la charte professionnelle.

      Absence de limites physiques : L'immersion a révélé des gestes inappropriés, tels que des baisers forcés sur la bouche imposés aux enfants par certains membres du personnel sous couvert d'affection.

      5. Défaillances de la Hiérarchie et Impunité

      Le système de contrôle interne semble incapable de réguler ou d'écarter les profils dangereux.

      Pilotage à distance : La hiérarchie supérieure se rend rarement sur le terrain (environ trois fois par an), adoptant une posture de "no news good news".

      Le système des "chaises musicales" : Pour les agents titulaires (fonctionnaires) faisant l'objet de signalements pour maltraitance (fessées, secousses par les oreilles), la Ville privilégie le déplacement géographique plutôt que la sanction disciplinaire ou l'exclusion.

      Cela permet à des individus problématiques de poursuivre leur carrière en changeant simplement d'établissement.

      Gravité des faits signalés : L'enquête mentionne une plainte pour attouchements sexuels sur une enfant pendant la sieste, ayant entraîné la suspension d'un agent au nom du principe de précaution.

      Citations Clés

      « Si un enfant se blesse, elle fait comment ? [...] Voilà, c'est votre bon sens. » — Recruteuse de la Ville de Paris.

      « On forme sur le tas. [...] Allez, bienvenue à bord du briefing express. » — Responsable Éducatif Ville (REV).

      « Si ça te fait de la peine [que les enfants pleurent], c'est pas fait pour toi ce travail. Parce que sinon tu vas te faire bouffer. » — Animatrice à une nouvelle recrue.

      « On peut faire une carrière entière en fait en étant déplacé tout le temps, ça il n'y a aucun problème. » — Karim, responsable périscolaire anonyme.

    1. Synthèse Analytique : Les Mécanismes du Contrôle Coercitif et de la Violence Intrafamiliale

      Résumé Exécutif

      Ce document analyse les dynamiques du contrôle coercitif à travers le prisme des audiences judiciaires et des témoignages d'experts présentés dans l'enquête d'ARTE.

      Le contrôle coercitif ne se limite pas à des actes isolés de violence physique, mais constitue un système délibéré de domination visant à aliéner la liberté de la victime. Les points clés identifiés incluent :

      La nature systémique du contrôle : Il s'agit d'une stratégie globale incluant la micro-surveillance, l'isolement social et la dévalorisation psychologique.

      L'arsenal tactique : L'utilisation de technologies (GPS, caméras, accès aux réseaux sociaux) et de pressions économiques pour maintenir une emprise totale.

      La rhétorique de l'agresseur : Une tendance systématique à l'inversion de la culpabilité, à la minimisation des faits et à l'utilisation de prétextes émotionnels pour justifier la violence.

      L'évolution juridique : La nécessité d'intégrer la notion de contrôle coercitif dans le droit pour déconstruire les rapports de domination ancrés historiquement dans le Code civil.

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      1. Définition et Stratégies du Contrôle Coercitif

      Le contrôle coercitif est décrit comme une « arme par excellence » pour soumettre l'autre.

      Contrairement à la violence ponctuelle, il s'inscrit dans la durée et l'omniprésence.

      Mécanismes de surveillance et de micro-contrôle

      L'agresseur cherche à envahir l'espace psychique, intime et professionnel de la victime par divers moyens :

      Surveillance technologique : Installation de traceurs GPS sous les véhicules, utilisation de caméras de surveillance au domicile, et exigence des codes d'accès aux téléphones et réseaux sociaux.

      Intrusion nocturne : Privation de sommeil par la musique forte ou réveils forcés durant la nuit pour obtenir des « aveux » d'infidélité imaginaire.

      Contrôle du corps : Surveillance de la tenue vestimentaire et, dans des cas extrêmes, inspection des sous-vêtements pour déceler des preuves de rapports extra-conjugaux ou de prostitution.

      Isolement et dévalorisation

      Le contrôle passe par la création d'un désert social autour de la victime :

      Rupture des liens : Interdiction ou limitation des visites à la famille (notamment la mère) et aux amis, sauf en présence de l'agresseur.

      Atteinte à la dignité : Utilisation d'un langage dégradant (« pute », « salope », « moins que rien ») et dénigrement constant des capacités professionnelles ou maternelles.

      Pathologisation de la victime : Traiter la victime d'« hystérique » ou de « folle » pour discréditer sa parole et justifier le contrôle.

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      2. Le Cycle de la Domination et de la Contrainte

      Le passage de la violence verbale à la violence physique et à la séquestration suit une progression souvent prévisible.

      La contrainte physique et matérielle

      Séquestration : Fermer la maison à clé pour empêcher la victime de sortir, ou s'enfermer avec elle pour l'empêcher de fuir une dispute.

      Contrôle des besoins vitaux : Interdiction d'accès à la cuisine pour les enfants ou la conjointe, contrôle strict des courses alimentaires (ne rapporter que de l'eau, forcer la victime à consommer des produits périmés).

      Emprise économique : Captation des prestations sociales et reproches systématiques sur la gestion financière, visant à créer une dépendance totale.

      Menaces et terrorisme domestique

      Le climat de peur est maintenu par des menaces de mort explicites et récurrentes :

      Menaces d'homicide : SMS répétés (« je vais te tuer », « je vais te crever »), mise en joue avec une arme à feu chargée, ou menaces de précipiter la victime d'un pont ou contre un mur sur l'autoroute.

      Chantage au suicide : Utilisation de la menace de se donner la mort pour manipuler la victime et l'empêcher de rompre.

      Violence physique directe : Crachats au visage, strangulation jusqu'à la perte de connaissance, coups de tête et pressions physiques pour « faire taire » la victime.

      --------------------------------------------------------------------------------

      3. Analyse de la Défense des Agresseurs

      L'analyse des audiences révèle des schémas de défense récurrents chez les auteurs de violences, visant à éluder leur responsabilité.

      | Tactique de défense | Manifestation constatée dans les sources | | --- | --- | | Inversion de la culpabilité | Affirmer que la victime est « capricieuse », « exigeante » ou qu'elle a « provoqué » l'acte par son comportement ou son infidélité supposée. | | Minimisation | Qualifier un crachat de « simple réaction », ou des menaces de mort de « mots dits sous le coup de la colère ». | | Justification par le trauma personnel | Invoquer le décès d'un proche, une éducation violente ou une surcharge de travail pour excuser le passage à l'acte. | | Déni de la réalité | Contester les faits malgré les preuves matérielles (SMS, rapports de police, expertises médicales). | | Présentation de soi comme victime | Se décrire comme le « véritable lésé » de l'histoire, celui qui a donné tout son amour sans retour. |

      --------------------------------------------------------------------------------

      4. Impact Traumatique et Conséquences Sociales

      La violence ne s'arrête pas à la victime directe ; elle irradie sur l'ensemble du cercle familial.

      Impact sur les enfants : Les enfants sont témoins et parfois cibles des violences.

      Ils vivent dans une atmosphère de terreur (« se cacher dans la chambre », « avoir peur de son père »).

      L'agresseur peut même les rendre responsables de son propre état émotionnel.

      Le cycle intergénérationnel : Les experts et magistrats soulignent le risque que les enfants reproduisent ces schémas de violence une fois adultes s'ils ne sont pas interrompus.

      La période de séparation : Identifiée comme la phase la plus dangereuse.

      C'est souvent au moment où la victime tente de reprendre sa liberté que la violence culmine (harcèlement par SMS, rodéos autour du domicile, usage de traceurs).

      --------------------------------------------------------------------------------

      5. Perspectives Institutionnelles et Juridiques

      Le document souligne le décalage entre la perception de l'agresseur et la norme légale.

      L'héritage historique : Le rappel de l'ancien article 213 du Code civil (1803), qui imposait l'obéissance de la femme à son mari, explique la persistance de structures de domination archaïques dans l'esprit de certains agresseurs.

      Le rôle de la justice : La magistrature, aujourd'hui majoritairement féminine, a pour mission de rappeler la loi et de déconstruire ces rapports de force.

      Le droit doit s'immiscer « au cœur des rapports intimes » pour protéger la liberté individuelle.

      Sanctions et obligations : Les condamnations citées incluent des peines de prison (dont certaines sous surveillance électronique), des amendes pour préjudice moral, des interdictions de paraître au domicile et l'obligation de suivre des stages de responsabilisation contre les violences sexistes.

      --------------------------------------------------------------------------------

      Note finale : Le contrôle coercitif se définit par la volonté de « conserver sous un contrôle permanent, total et absolu » une personne, la réduisant à un objet de propriété plutôt qu'à un sujet de droit.

      Sa reconnaissance judiciaire est l'outil essentiel pour briser ce système d'oppression.

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      Reviewer #1 Reviewer 1 Point 1- The authors describe cortical neuronal counts across several mammalian species, which is quite impressive, but the information on the methods of counting is lacking: how representative are the data used / shown; how many individuals / brains / sections were used for each species considered? Much more detailed description of the quantifications should be provided to judge the validity of this first conclusion.

      Response: We sincerely thank the reviewer for this insightful and constructive suggestion. We agree that the methodological description of our comparative histological analysis, which is the fundamental basis of this study, was insufficient in the original manuscript. Following the reviewer’s advice, we have extensively revised the Materials and Methods section entitled “Nissl staining and neuronal cell number count” (Page 32, Line 15).

      Reviewer 1 Point 2- The authors use several markers of cortical neuron identity to confirm their neuron number measurements, but from the data shown in Figure 1D,E it seems that only some markers (Satb2) show species-differences while others do not (CTIP2 / Tbr1). How do the authors explain this discrepancy - does this mean that it is mainly Satb2 neurons that are increased in number? But if so how to explain the relative increase in subcortical projections shown in Figure S7?

      Response: We appreciate the reviewer’s insightful comments regarding the marker expression patterns. Upon re-evaluating our data in light of your feedback, we agree that the species differences in deep-layer (DL) markers such as Ctip2 and Tbr1 in the adult stage appear relatively modest compared to the robust differences observed in Satb2 and the projection data shown in Figure S8.

      To address this point, we have incorporated a comparison between the adult data (Figure 1) and our findings from P7 (Figure S2). As shown in the revised manuscript, the species differences for all markers are significantly more pronounced at P7 than in the adult. Notably, in the lower layers, rats exhibit a significantly higher number of marker-positive cells across all markers, including those newly added in this revision, compared to mice.

      We offer the following interpretation regarding these temporal differences:

      1. Developmental Relevance: The marker molecules analyzed are well-established regulators of neuronal subtype fate and projection identity during development. Their critical fate-determining functions are primarily exercised during the migration and maturation phases of nascent neurons.
      2. Postnatal Expression Shifts: Whether these molecules maintain functional roles in the fully matured adult brain remains less certain. It is plausible that marker expression may diminish in certain neuronal populations during late postnatal development, leading to the attenuated species differences observed in adults. Consequently, we believe the strong correlation between P7 quantitative data and projection fate provides a biologically sound validation of our hypothesis.

      While we have kept the discussion in the main text concise to maintain focus for the general reader, we have provided comprehensive data in Figure 1 and Figure S2. This ensures that the necessary evidence is readily available for specialists interested in these developmental dynamics.

      Reviewer 1 Point 3- The authors focus their study almost exclusively on somatosensory cortex, but can they comment on other areas (motor, visual for instance)? It would be nice to provide additional comparative data on other areas, at least for some of the parameters examined across mouse and rat. Alternatively the authors should be more explicit in the abstract and description of the study that it is limited to a single area.

      Response: We sincerely appreciate the reviewer’s insightful comment. As suggested, we have revised the Abstract to explicitly state that our current analysis is focused on the somatosensory cortex. Furthermore, as demonstrated in Figure 1B, we have added a discussion regarding the possibility that the species differences observed in the primary somatosensory cortex may be a general feature shared across the entire cerebral cortex, as follows: “This DL-biased thickening in rats was evident in the primary somatosensory area, but is consistently observed throughout the rostral-caudal cortical regions. (Page 19, Lines 29-31)“

      Reviewer 1 Point 4- The authors provide convincing evidence of increased Wnt signaling pathway in the rat. They should show more explicitly how other classical pathways of neurogenic balance / temporal patterning are expressed in their mouse and rat transcriptome data sets. These would include Notch, FGF, BMP, for which all the data should be available to provide meaningful species comparison.

      Response: We sincerely thank the reviewer for this insightful suggestion. Following your advice, we have newly included comparative data on key signaling pathways essential for cortical development—namely Wnt, FGF, NOTCH, mTOR, SHH, and BMP—across different species. These results are now presented in Figure S17. Rat progenitors show comparable patterns to other species for FGF, mTOR, and Notch signaling, but elevated Wnt and BMP expression, especially at early stages. A detailed heatmap of raw Wnt pathway gene expression across species is also included in the same supplementary figure. We believe these additions provide a more comprehensive evolutionary perspective and significantly strengthen our findings.

      Reviewer 1 Point 5- The alignment of mouse and rat trajectories is very nicely showing a delay at early-mid-corticogenesis. But there is also heterochronic transcriptome at latest stages (end of 5). How can this be interpreted? Does this mean potentially prolonged astrogliogenesis in the rat cortex?

      Response: We sincerely appreciate the reviewer’s insightful comment and the meticulous attention given to our data. Regarding the heterochronic shift observed at Day 5, we agree that this point was not sufficiently addressed in the original manuscript.

      We would like to clarify the two primary reasons for this omission, which are inherent to the current study’s design:

      1. Resolution of Stage Alignment at Temporal Extremes: In our developmental stage alignment analysis, corresponding stages are defined by pairs showing the highest transcriptomic similarity within the sampled range. By definition, the precision of this alignment tends to decrease at the earliest and latest time points of a dataset. Since the "true" biological equivalent might lie outside our sampling window, we must be cautious in interpreting shifts at these temporal boundaries.
      2. Difference in Validation Rigor: Our study prioritized the early stages of deep-layer (DL) neuron production. Consequently, we rigorously defined the onset of neurogenesis in rats (Day 1) using multiple independent methods, including clonal analysis, immunohistochemistry, and gene expression. In contrast, Day 5 was defined simply as five days post-initiation of neurogenesis, without equivalent multi-modal validation. Given that our primary focus is the early phase of neurogenesis, the precision of the transition from late neurogenesis to gliogenesis is relatively lower. For these reasons, we believe that an in-depth discussion of the heterochronic shift at Day 5 might lead to over-interpretation. To reflect this more accurately and avoid misleading the reader, we have revised Figure 6F to de-emphasize the Day 5 shift. In addition, we revised the manuscript as “Importantly, while this analysis identified stage pairs with the highest similarity, the correspondence at the edges of the temporal sampling window is inherently less certain than at the center. Consequently, we focus on the notable reflection point at the center of our dataset. (Page 13, Lines 37-39)”.

      We believe these changes more faithfully represent the biological scope of our data while maintaining the scientific integrity of our primary conclusions.

      Reviewer 1 Point 6- Figure 7: description implies that module 3 is a subset of module 4, but this is not obvious at all from the panels shown. Please clarify.

      Response: We sincerely appreciate the reviewer’s careful reading of our manuscript. As suggested, we have revised Figure 7 to clarify the hierarchical relationship between Module 3 and Module 4, ensuring that their inclusion is now explicitly presented.


      Reviewer #2 Reviewer 2 Point 1. The introduction lacks sufficient background and fails to convey the significance of the study. Specifically, why the research was undertaken, what knowledge gap it addresses, and how the findings could be applied. Addressing these questions already in the introduction would enhance the impact of the work and broaden its readership.

      Response: We sincerely appreciate the reviewer’s insightful comment on this point. Our study reports evolutionary insights gained through an unconventional approach: a single-cell level comparison between mice and rats. We agree that clarifying the necessity of this specific approach is crucial for the manuscript. Accordingly, we have added the following two points to the Introduction:

      1. At the end of the first paragraph, we emphasized the current lack of research on the evolutionary adaptation of cortical circuits, despite the established functional importance of evolutionarily conserved circuits. (Page 3, Lines 7-10); “Paradoxically, despite the importance of these variations, research has predominantly focused on the conserved aspects of cortical architecture. Consequently, the degree of evolutionary plasticity inherent in these circuits and the cell-intrinsic mechanisms driving their modification remain profoundly enigmatic.”)
      2. At the end of the third paragraph, we revised and added text (Page3, Lines 26-27; “This lack of comparative insight represents a significant gap in our understanding of how conserved developmental programs give rise to species-specific brain architectures.”).

      Reviewer 2 Point 2. In figure 5 the authors conclude that "differences in cell cycle kinetics and indirect neurogenesis are unlikely to be the primary factors driving the species-specific variation in DL neuron production. Instead, the temporal regulation of progenitor neurogenic competence, which determines the duration of the DL production phase, provides a more plausible explanation for the greater number of DL subtypes observed in rats". It is not clear to this reviewer how the authors come to this conclusion. Authors observe a significant proportion of mitotic cells in rat VZ from day 1, and a higher constant proportion of mitotic progenitors in SVZ rats compared to mouse (Figure 5C). This points to an early difference in mitotic progenitors that may also lead to increased IP numbers, and potentially an increased number in DL cells, even before day 1. In addition, the higher abundance of IPs in the G2/S phase (statistically significant in 4 of the 7 time points) (Figure 5F), would suggest that this difference might play a role in the species-specific variation of DL neuron production. The authors should estimate cell cycle length instead of just measuring proportions to conclude something about cell cycle kinetics. They can then model growth curves to predict the effect caused if there were differences in cell cycle length between equivalent cell types across species.

      Response: We sincerely thank the reviewer for their careful reading of our manuscript and for pointing out the overstatements in our original descriptions. We agree that a more nuanced interpretation of the data was necessary. In response to these constructive suggestions, we have made the following revisions:

      1. Refinement of Descriptions: We have revised the text to more accurately reflect our findings, specifically noting that the increase in RG division on Day 1 and IP proliferation throughout the neurogenic period showed a significant trend. These features are now described more fairly and cautiously in the revised manuscript. (Page 11, Lines 42-46; “Remarkably, while the temporal dynamics of mitotic density were strikingly conserved between the two species, subtle yet discernible species-specific signatures emerged. Specifically, rats exhibited a higher ratio of mitotic cells in the VZ at the onset of neurogenesis, the precise period when DL subtypes are generated in both species. Further assessment of G2/S-phase cells via pulse-EdU labeling (Figure 5D, E) “)
      2. Inclusion of Time-lapse Imaging Data: The reviewer is correct that measuring the proportions of M and G2/S phases provides only a limited snapshot of cell cycle dynamics. To gain a more precise insight, we performed primary cultures of neural progenitor cells (NPCs) from Day 1 and conducted live-cell time-lapse imaging. This allowed us to directly quantify the cell cycle duration of mouse and rat NPCs (Figure S9A-C).
      3. Comparative Analysis and Mathematical Modeling: Our new data revealed that the cell cycle lengths of the two species are remarkably similar, with no significant differences observed under these culture conditions. Furthermore, to validate the impact of these findings on overall brain development, we developed a mathematical model based on our experimental data. This model predicts the total number of cells produced over the five-day neurogenic period, providing a more robust theoretical framework for our conclusions (Figure S9D). We believe these additions significantly strengthen the manuscript and address the reviewer's concerns regarding the physiological relevance of our observations.

      Reviewer 2 Point 3. In Figure 6 the authors focus only on the mouse and rat datasets. Given the availability of datasets from primates that the author used already for Figure 7, it would give the reader a broader prospective if also these datasets would be integrated in the analysis done for Figure 6, particularly it would be interesting to integrate them in the pseudotime alignment of cortical progenitor. How do human and/or macaque early and late neurogenic phase would compare to mouse and rat in this model?

      Response: We sincerely appreciate the reviewer’s insightful suggestion. In accordance with this comment, we have now incorporated pseudotime alignments of cortical progenitors between primates (human, macaque) and rodents (mouse, rat), presented as pairwise gene expression distance matrices with dynamic time warping in Figure S13. These heatmaps illustrate temporal compression or stretching in progenitor gene expression progression across species. Notably, macaque progenitors show no definitive deviations from rodents, whereas human progenitors exhibit distinct protraction relative to rats and even more so to mice. These additions provide a more comprehensive cross-species perspective without altering the study's core conclusions.

      Reviewer 2 Point 4. In Figures 6C and 6D, the authors distinguish between cycling and non-cycling NECs and RGCs. Could the authors clarify the rationale behind making this distinction? Could the authors comment on how they interpret the impact of cycling versus non-cycling states on species-specific non-uniform scaling? Do they consider the observed non-linear correspondences to be driven by differences in cell cycle activity?

      Response: We are grateful to the reviewer for their insightful observation. We agree that our initial classification of neural progenitor cell (NPC) populations based on proliferation marker expression levels followed a convention used in other studies but was, in the context of this work, unnecessary and potentially misleading. To avoid further confusion and focus on the core biological question, we have re-organized the data by pooling these populations into a single group. Regarding the concern about species differences in cell cycle kinetics, we believe there is no significant divergence between mice and rats that could explain the observed developmental patterns in temporal progression of neurogenesis. This is supported by two lines of evidence:

      1. Quantitative analysis of pH3-positive cells (Figure 5).
      2. New time-lapse imaging data of primary cultured NPCs, which shows no substantial difference in cell cycle length between the two species (Figure S9). These results indicate that the species-specific differences in deep-layer (DL) neuron production are not driven by cell division kinetics. Consequently, we conclude that the non-linear developmental progression of NPCs occurs independently of cell cycle regulation.

      Reviewer 2 Point 5. For the non-uniform scaling in Figure 6F, the authors identify critical inflection points and mention that "the largest delay in rat progenitors occurring where Day 1 and Day 3 progenitors overlapped". It would be good if the authors could discuss what they think all the inflection points represents. How much can it be explained by the heterogeneity within progenitors per time point? There is a clear higher spread of histograms at days 3 and 5, and the histogram at day 5 almost overlaps with day 1. I wonder if the same conclusion about non-uniform scaling would be detected if the distance matrix was built separately for specific cell types, for example only looking at NECs or RGCs.

      Response: We sincerely appreciate the reviewer’s insightful perspective on this point. In alignment with the suggestions from both this reviewer and Reviewer 1 (Point 5), we have updated the manuscript to discuss all identified inflection points. Specifically, we have clarified why our discussion focuses on the correspondence between Mouse D1 and Rat Day 3.

      A recognized limitation of our current analytical approach is that it identifies the closest matching expression profiles within the specific timeframes sampled for each species. For stages at the beginning or end of our sampling window, the "true" corresponding stage in the other species may lie outside our sampled range, which naturally limits the strength of any conclusions regarding those boundary points. Consequently, while we can confidently confirm the correspondence between Mouse Day 1 and Rat Day 3—both of which sit centrally within our sampled window—we have intentionally avoided over-interpreting data near the temporal boundaries.

      Regarding the cell types analyzed, this specific analysis was conducted exclusively on NECs and RGs (now shown in Figure 6F). Extensive prior research (Susan McConnell lab, Sally Temple lab, Fumio Matsuzaki lab, Dennis Jabaudon lab, and more) has established that the time-dependent mechanisms governing the fate determination of cortical excitatory neuron subtypes are encoded within RGs. Therefore, we focused our investigation on these lineages and did not include other cell types in this study. We believe this focused approach maintains the highest degree of biological relevance for our conclusions.

      Reviewer 2 Point 6. The authors conclude that the elevated and prolonged expression of Wnt-ligand genes in rat RGs extend the DL neurogenic window and contribute to rat-specific expansion of deep cortical layer. In order to validate this finding it would be good for the authors to perform a perturbation experiment and reduce Wnt signalling/ Axin 2 levels in rats or depleted the Lmx1a and Lhx2 double-positive population. Response: __We thank the reviewer for this insightful suggestion. We agree that providing direct experimental evidence is crucial to demonstrating that elevated Wnt signaling in RG progenitors drives the production of DL subtype neurons in rats. To address this, we performed a functional intervention on Day 3, a stage when Wnt signaling (indicated by Axin2 expression) is significantly higher in rats than in mice (__Figure 7C, D). By introducing a dominant-negative form of TCF7L2 (dnTCF7L2) to inhibit Wnt signaling specifically in RG progenitors, we tracked the fate of the resulting neurons (Figure 7I, J). Our results showed a clear reduction in the proportion of DL neurons, accompanied by a reciprocal increase in upper-layer (UL) neurons. These findings demonstrate that maintained high levels of Wnt signaling are essential for the prolonged neurogenic capacity for DL neurons in rats. This new data has been incorporated into Figure 7.

      Reviewer 2 Point 7. The authors conclude that Wnt signaling is a rat specific effect since they did not observe any clear temporal change in wnt receptors in gyrencephalic species, and only a subset of RG in rats co-express Lmx1a and Lhx2. However, specific Wntligands and receptors (Wnt5a, Fzd and Lrp6) seem to be upregulated in human as well (Fig 7G), non RG cells could act as wnt ligand inducers in other species, and it has not been demonstrated that Lmx1a and Lhx2 are the source for Wntligand production. I wonder if the authors can completely rule out a role for Wnt in the protracted neurogenesis of other species.

      Response: We sincerely appreciate the reviewer’s insightful and broad perspective regarding Wnt signaling dynamics across diverse species. In this study, our primary focus was to elucidate the specific mechanisms underlying the differences between mice and rats. Consequently, we did not initially explore Wnt dynamics in other species or their roles in developmental timing in great depth in the original manuscript. We fully acknowledge that lineage-specific adaptations occur at the individual gene level; for instance, Silver and colleagues have reported that human-specific upregulation of Wnt receptor gene FZD8 modulates neural progenitor behavior (Boyd et al., Current Biology 2008, Liu et al., Nature 2025). However, our comparative analysis of five mammalian species—carefully aligned by developmental stage—reveals a distinct global trend. While individual gene variations exist like human FZD8, the expression levels of multiple Wnt-related genes, particularly ligands, are markedly higher in rats than in the other four species.

      Following the reviewer’s insightful suggestion, we examined the potential role of Lmx1a in activating Wnt ligand transcription in rat cortical progenitors by analyzing their expression correlation at the single-cell level. Our analysis revealed that several Wnt ligand genes are co-expressed with Lmx1a with a remarkably strong positive correlation. While we have not yet experimentally demonstrated the direct transcriptional activation of Wnt ligands by Lmx1a in these cells, this robust correlation at single-cell resolution strongly suggests that Lmx1a regulates Wnt ligand expression. These new findings are now included in Figure 7 and Figure S16, and the corresponding results section (Page 15, Lines 42-44) has been revised accordingly.

      __Reviewer 2 Point 8 __Minor comments: The RNAscope experiment is currently qualitative. Is it the mRNA copy number per cell equal in both species but more cells are positive in rat, or are there differences in number of mRNA molecules as well? It is not indicated if the RNAscopeprobes are the same for mouse and rat.

      Response: We sincerely thank the reviewer for this insightful suggestion. Following the comment, we performed RNAscope analysis for Axin2 in both mice and rats and quantified the results (now included in Figure 7D). The new data successfully validate the species differences initially observed in our scRNAseq analysis: specifically, the period of high-level Axin2 expression is significantly extended in rats compared to mice. These findings provide histological evidence that reinforces our conclusions regarding the distinct temporal dynamics between the two species.

      Regarding probe design, the Axin2 RNAscope probes target conserved and corresponding sequences between mouse and rat, with species-specific probes optimized for each organism to ensure maximal specificity and sensitivity. We have updated the Methods section ("Fluorescent in situ hybridization with RNAscope") to include these details.

      Reviewer #3

      Reviewer 3 Point 1. Satb2 is also widely recognized as a deep layer marker. The authors need to perform analysis and quantification in Figs 1 and 4 with other II/III and IV markers such as Cux1 and Rorb.

      Response: We thank the reviewer for their insightful comments regarding the marker specificity. We fully agree that while Satb2 is a robust marker for callosal projection identity, its broad distribution across both deep and upper layers limits its utility as a layer-specific marker. As the reviewer suggested, Cux1 (Layers 2/3) and Rorb (Layer 4) are indeed superior markers for defining laminar identity.

      To address this, we have incorporated new immunohistochemical data for these markers in both the quantification of somatosensory cortical neurons (Figure S2) and the birth-dating analysis (Figure 4).

      Our new findings are as follows:

      1. Layer Quantification (Figure S2): By utilizing Cux1 and Rorb as more specific upper-layer (UL) markers, we confirmed that there are no significant differences in the number of these neurons between mice and rats.
      2. Birth-dating Analysis (Figure 4): These markers allowed us to more precisely define the timing of Cux1/Rorb-positive cell generation, revealing subtle but important differences between the two species. While these additions do not alter the fundamental narrative of the original manuscript, they have significantly enhanced the precision and rigor of our analysis. We are grateful to the reviewer for guiding us toward this more robust validation.

      Reviewer 3 Point 2. Rats have larger cortices. Therefore, quantification of neurons should also be normalized to cortical thickness in Fig 1E and also represented with individual data points.

      Response: We sincerely appreciate the reviewer’s constructive suggestion. We agree that normalizing the number of cortical neurons by thickness provides a more rigorous comparison. Accordingly, we have calculated the neuronal density (cell count per unit thickness) for Tbr1- and Ctip2-positive cells and included these data in Figure S2C. Our analysis confirms that these populations are distributed at a significantly higher density in mice compared to rats.

      Furthermore, we have updated the visualization in Figure 1E to display individual data points, ensuring full transparency of the underlying distribution. We believe these revisions, prompted by the reviewer’s insight, have substantially strengthened the clarity and persuasiveness of our manuscript.

      Reviewer 3 Point 3. The clonal analysis in Figs 2 and 3 quantifies GFP and RFP and reports these as neurons. However, without using cell-specific markers, it seems the authors cannot exclude that some progeny are also glia derived from a radial glial progeny. I don't expect all experiments to have this but they must have some measures of both populations to address this possibility. This needs to be addressed to build confidence in the conclusion that there is clonal production of neurons.

      Related to this, the relationship between position and fate is not always 1 to 1. The data summarized in Fig 2G are based on position and not using subtype markers. They should include assessment of markers as they do in Fig 4.

      Response: We sincerely thank the reviewer for this insightful comment. We agree that a clear definition of cell types is essential for the accuracy of clonal analysis.

      In this study, we primarily identified neurons based on their distinct morphological characteristics and performed measurements specifically on these cells. To validate this approach, we confirmed that the vast majority of cells identified as neurons were positive for NeuN and cortical excitatory neuron markers, while remaining negative for glial markers such as Olig2 and SOX9. (Notably, at postnatal day 7, most cells in the glial lineage exist as undifferentiated Olig2-positive progenitors). These observations support our conclusion that the cells analyzed based on morphology are indeed cortical excitatory neurons.

      As the reviewer rightly pointed out, evaluating cell composition using fate-specific marker expression is the ideal approach. However, our current experimental setup required multiple fluorescence channels for DAPI staining (to assess tissue architecture) and immunostaining for GFP and RFP (to identify labeled clones). Due to these technical constraints regarding available detection channels and host species compatibility, we relied on morphological criteria for the primary analysis.

      To address this concern and ensure the reliability of our findings, we performed additional analyses using a subset of samples. By co-staining retrovirally labeled neurons with cell-fate markers, we obtained results consistent with our other data (Figures 1 and 4) regarding laminar position and marker expression. Based on this consistency, we are confident that our classification based on morphology and laminar position does not alter the fundamental conclusions of this study.

      Reviewer 3 Point 4. In Fig 5, the authors use PH3 as well as EdU to measure differences in indirect neurogenesis. Using EdU and Tbr2 they report more dividing IPs. However they need to measure this over the total number of Tbr2 cells as it is not normalized to differences in Tbr2 cells between species. Are there total differences in Tbr2+ cells when normalized to DAPI as well? Moreover, little analyses is performed to measure any impact on radial glia. As no striking differences were observed in IPs this leaves the cellular mechanism a bit unclear and begs the impact on radial glia. Measuring PH3+ cells in VZ and SVZ is not cell specific nor does it yield information to support the prolonged neurogenesis.

      Response: We sincerely thank the reviewer for this insightful suggestion. We agree that quantifying Tbr2+/EdU+ double-positive cells alone was insufficient to fully capture the IP dynamics. Following the reviewer’s advice, we have now quantified the total population of Tbr2+ cells, normalized to the number of DAPI-stained nuclei. This new analysis reveals that mice and rats exhibit nearly indistinguishable temporal dynamics (Figure S10). When integrated with the original Tbr2+/EdU+ data in Figure 5, these findings suggest that rats maintain a slightly higher IP pool throughout the neurogenic period. This implies that the increased neuronal production in rats is not restricted to a specific phase, but rather occurs consistently across all developmental stages. We believe these additional data significantly strengthen our conclusions.


      Reviewer 3 Point 5. The sc-seq is done in rat and compared to published mouse data from corresponding stages. They conclude species specific differences in progenitor gene expression. I am unsure how appropriate this is. Are similar sequencing platforms used? Can they find similar results if using multiple dataset? There are other datasets that may be used to validate these findings beyond DiBella et al.

      Response: We sincerely thank the reviewer for this insightful comment. We agree that establishing the validity of our analytical approach is crucial for the reader’s confidence in our findings. To address this, we have explicitly stated in the revised manuscript that both our rat scRNAseq data and the publicly available datasets were generated using consistent experimental platforms. This ensures that the integration process is technically sound.

      Revised text (Page 13, Lines 16-18): “After quality control, we integrated these profiles with previously published mouse cortical cell data from corresponding neurogenic stages, which is prepared using the consistent platform with ours (35) (Figure S11).”

      Furthermore, to ensure the robustness of our comparative analysis, we have incorporated an additional independent dataset (Ruan et al., PNAS 2021) in addition to the Di Bella et al. Nature 2021 data used in the original manuscript. We confirmed that the results obtained using this second dataset are highly consistent with our initial findings, further validating our conclusions across different studies (Figure S13A).

      Reviewer 3 Point 6. Wnt ligand analysis requires validation in situ across developmental stages, to support their conclusions. Ideally they might consider doing some manipulations to provide context to this observation.

      Response: We sincerely thank the reviewer for these insightful suggestions. We agree that validating the spatial expression patterns of Wnt ligands and confirming their expression in rat-specific RG, as suggested by our scRNAseq data, is crucial for strengthening our conclusions.

      Regarding the expression of Wnt3a, a key ligand in cortical development: although immunohistochemical analysis clearly identified Wnt3a expression in the cortical hem, the expression levels in RG within the cortical area were substantially lower than those in the hem, making definitive visualization challenging. To complement these findings and provide more robust evidence, we performed the following additional experiments:

      1. Validation of Wnt signaling levels: Using RNAscope-based in situ hybridization for Axin2, we successfully confirmed the elevated Wnt signaling levels in rat-specific RG (Figure 7C, D), consistent with our scRNAseq findings.
      2. Elucidating strikingly high correlated expressions of Lmx1a and Wnt ligand genes in the rat cortical progenitors in our scRNAseq dataset (Figure S16B).
      3. Functional analysis: To test the functional significance of this signaling, we inhibited Wnt signaling by electroporating dominant-negative TCF7L2 into rat RG at E15.5. This manipulation resulted in a subtype shift of the generated neurons toward an upper-layer identity (Figure 7I, J). These new results demonstrate that the rat-specific extension of high Wnt signaling levels serves as a fundamental mechanism for the prolonged production of deep-layer (DL) neurons. We are grateful to the reviewer for these suggestions; these additional data have significantly strengthened our core argument that the heterochronic regulation of Wnt signaling states drives the evolution of cortical neuronal composition.

      __Reviewer 3 Point 7 __Minor concerns-1

      Please separate images in Fig 1D it is very strange to have them all on top of each other.

      Response: We sincerely thank the reviewer for this suggestion. As requested, we have provided individual channel images alongside the merged multicolor panels. We agree that this modification significantly enhances the clarity of our data and makes the results much easier to interpret.

      __Reviewer 3 Point 8 __Minor concerns-2

      Are data in Fig 4E Edu+Tbr1+EdU+? This should be clarified and would be most accurate.

      Response: We appreciate the reviewer’s suggestion. We added the label of Y axes of the plots in Figure 4E-K. The procedure of cell count in these analyses are documented in the caption of Figure 4E-K, “Normalized counts of neurons colabeled for EdU and projection-specific markers, relative to the peak of EdU+ and marker+ cells.”.

      __Reviewer 3 Point 9 __Minor concerns-3

      Fig 4 graphs only have titles without Y axis. Please adjust location of title or repeat for clarity.

      Response: We thank the reviewer for this helpful suggestion. To clarify the definition of the Y-axis, we have now added a descriptive label to the axis in the revised figure.

      __Reviewer 3 Point 10 __Minor concerns-4

      Fig 4A implies cumulative incorporation which I don't think is being performed here. They should clarify this in the figure.

      Response: We appreciate the reviewer’s insightful comment. To avoid any potential misunderstanding regarding the additivity of the effect, we have revised the illustration in Figure 4A for greater clarity.

      __Reviewer 3 Point 11 __Minor concerns-5

      Fig 5 needs labels for the actual stages assayed, as illustrated in Fig 4A.

      Response: We thank the reviewer for this helpful suggestion. Following your comment, we have added the developmental stage information (expressed as embryonic days) for both mice and rats in the revised manuscript.

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      Referee #3

      Evidence, reproducibility and clarity

      In this study the authors investigate differences between two closely related species, rats and mice, in terms of cortical development and neuronal composition. They first perform comparative analysis of cortical layers which revealed the density and markers of deep layer neurons of rats is disproportionately larger compared to adult mice. They then use retroviruses for lineage analysis from embryonic stages to P7. They find in general that there are temporal differences in when mice and rats produce upper versus deep layer neurons, with the process being protracted in rats. EdU injections were used to report differences in the timing of cortical neuron generation between species and they note no striking differences in IPs. Sc-sequencing of rat cortices at different stages was then used to measure temporal changes in gene expression and compared to published mouse data. They note that rats have sustained Wnt ligand expression in radial glia highlighting that as a potential mechanism of action.

      Major concerns 1. Satb2 is also widely recognized as a deep layer marker. The authors need to perform analysis and quantification in Figs 1 and 4 with other II/III and IV markers such as Cux1 and Rorb. 2. Rats have larger cortices. Therefore, quantification of neurons should also be normalized to cortical thickness in Fig 1E and also represented with individual data points. 3. The clonal analysis in Figs 2 and 3 quantifies GFP and RFP and reports these as neurons. However, without using cell-specific markers, it seems the authors cannot exclude that some progeny are also glia derived from a radial glial progney. I don't expect all experiments to have this but they must have some measures of both populations to address this possibility. This needs to be addressed to build confidence in the conclusion that there is clonal production of neurons. Related to this, the relationship between position and fate is not always 1 to 1. The data summarized in Fig 2G are based on position and not using subtype markers. They should include assessment of markers as they do in Fig 4. 4. In Fig 5, the authors use PH3 as well as EdU to measure differences in indirect neurogenesis. Using EdU and Tbr2 they report more dividing IPs. However they need to measure this over the total number of Tbr2 cells as it is not normalized to differences in Tbr2 cells between species. Are there total differences in Tbr2+ cells when normalized to DAPI as well? Moreover, little analyses is performed to measure any impact on radial glia. As no striking differences were observed in IPs this leaves the cellular mechanism a bit unclear and begs the impact on radial glia. Measuring PH3+ cells in VZ and SVZ is not cell specific nor does it yield information to support the prolonged neurogenesis. 5. The sc-seq is done in rat and compared to published mouse data from corresponding stages. They conclude species specific differences in progenitor gene expression. I am unsure how appropriate this is. Are similar sequencing platforms used? Can they find similar results if using multiple dataset? There are other datasets that may be used to validate these findings beyond DiBella et al. 6. Wnt ligand analysis requires validation in situ across developmental stages, to support their conclusions. Ideally they might consider doing some manipulations to provide context to this observation.

      Minor concerns 1. Please separate images in Fig 1D it is very strange to have them all on top of each other. 2. Are data in Fig 4E Edu+Tbr1+EdU+? This should be clarified and would be most accurate. 3. Fig 4 graphs only have titles without Y axis. Please adjust location of title or repeat for clarity. 4. Fig 4A implies cumulative incorporation which I don't think is being performed here. They should clarify this in the figure. 5. Fig 5 needs labels for the actual stages assayed, as illustrated in Fig 4A.

      Significance

      Strengths:

      The finding that there are differences in cortical composition between rats and mice and that this is linked to prolonged neurogenesis in rats Use of careful and detailed lineage analysis to define differences in temporal production of neurons Inclusion of single cell sequencing

      Limitations:

      Largely descriptive Requires additional investigation to support some conclusions about neurons Concerns about inferring too much from single cell sequencing done by the authors but compared to publication

      Advance: Finding that there are differences in neurogenesis between closely related species is interesting and provides insight into mechanisms of cortical evolution.

      Audience: Evolution, cortical development

      Expertise: Cortical development, evolution

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      Referee #2

      Evidence, reproducibility and clarity

      Summary:

      Yamauchi et al. performed a comparative anatomical analysis of the layer architecture in the primary somatosensory cortex across 8 mammalian species. Unlike primates, which show an expansion of upper layers (UL), rodents, especially rats, display a pronounced thickening of deep layers (DL). In this study they focus on comparing rats and mice, given the higher abundance of DL neuron subtypes in rats. Using histological analysis, they showed that rats possess significantly more DL neurons per cortical column than mice, while UL neuron counts remain similar. Clonal lineage tracing showed that rat radial glial (RG) progenitors generate more DL neurons, indicating species-specific differences in progenitor neurogenic activity. Birth dating assays confirmed an extended DL neurogenesis phase in rats, followed by a conserved UL generation phase. Single-cell RNA sequencing further revealed that rats maintain an early progenitor state longer than mice, marked by sustained expression of DL-associated genes. Specifically, rat RG progenitors exhibit prolonged and elevated expression of Wnt signaling genes, particularly Wnt ligands. Comparative analysis of published single-cell RNA-Seq across species highlighted that this extended Wnt-high period in rats is exceptional, suggesting a species-specific extension of a conserved neurogenic program.

      Major comments:

      This reviewer thinks the topic is exciting, and the experiments elegant, insightful and well described. The paper is well written and follows a very logical flow, the conclusion for each experiment is supported by the data and they are carefully stated. This reviewer really appreciated the summary illustration included as a panel in each figure, they think that this greatly enhanced the clarity and accessibility of the data presented, especially because species comparison can be difficult to follow.

      In this reviewer's opinion, there are some aspects of the findings that the authors would need to clarify/address to explain in clarify the phenotype observed and to enhance the overall significance of this very well-made paper: 1. The introduction lacks sufficient background and fails to convey the significance of the study. Specifically, why the research was undertaken, what knowledge gap it addresses, and how the findings could be applied. Addressing these questions already in the introduction would enhance the impact of the work and broaden its readership. 2. In figure 5 the authors conclude that "differences in cell cycle kinetics and indirect neurogenesis are unlikely to be the primary factors driving the species-specific variation in DL neuron production. Instead, the temporal regulation of progenitor neurogenic competence, which determines the duration of the DL production phase, provides a more plausible explanation for the greater number of DL subtypes observed in rats". It is not clear to this reviewer how the authors come to this conclusion. Authors observe a significant proportion of mitotic cells in rat VZ from day 1, and a higher constant proportion of mitotic progenitors in SVZ rats compared to mouse (Figure 5C). This points to an early difference in mitotic progenitors that may also lead to increased IP numbers, and potentially an increased number in DL cells, even before day 1. In addition, the higher abundance of IPs in the G2/S phase (statistically significant in 4 of the 7 time points) (Figure 5F), would suggest that this difference might play a role in the species-specific variation of DL neuron production. The authors should estimate cell cycle length instead of just measuring proportions to conclude something about cell cycle kinetics. They can then model growth curves to predict the effect caused if there were differences in cell cycle length between equivalent cell types across species. 3. In Figure 6 the authors focus only on the mouse and rat datasets. Given the availability of datasets from primates that the author used already for Figure 7, it would give the reader a broader prospective if also these datasets would be integrated in the analysis done for Figure 6, particularly it would be interesting to integrate them in the pseudotime alignment of cortical progenitor. How do human and/or macaque early and late neurogenic phase would compare to mouse and rat in this model? 4. In Figures 6C and 6D, the authors distinguish between cycling and non-cycling NECs and RGCs. Could the authors clarify the rationale behind making this distinction? Could the authors comment on how they interpret the impact of cycling versus non-cycling states on species-specific non-uniform scaling? Do they consider the observed non-linear correspondences to be driven by differences in cell cycle activity? 5. For the non-uniform scaling in Figure 6F, the authors identify critical inflection points and mention that "the largest delay in rat progenitors occurring where Day 1 and Day 3 progenitors overlapped". It would be good if the authors could discuss what they think all the inflection points represents. How much can it be explained by the heterogeneity within progenitors per time point? There is a clear higher spread of histograms at days 3 and 5, and the histogram at day 5 almost overlaps with day 1. I wonder if the same conclusion about non-uniform scaling would be detected if the distance matrix was built separately for specific cell types, for example only looking at NECs or RGCs. 6. The authors conclude that the elevated and prolonged expression of Wnt-ligand genes in rat RGs extend the DL neurogenic window and contribute to rat-specific expansion of deep cortical layer. In order to validate this finding it would be good for the authors to perform a perturbation experiment and reduce Wnt signalling/ Axin 2 levels in rats or depleted the Lmx1a and Lhx2 double-positive population. 7. The authors conclude that Wnt signaling is a rat specific effect since they did not observe any clear temporal change in wnt receptors in gyrencephalic species, and only a subset of RG in rats co-express Lmx1a and Lhx2. However, specific Wnt ligands and receptors (Wnt5a, Fzd and Lrp6) seem to be upregulated in human as well (Fig 7G), non RG cells could act as wnt ligand inducers in other species, and it has not been demonstrated that Lmx1a and Lhx2 are the source for Wnt ligand production. I wonder if the authors can completely rule out a role for Wnt in the protracted neurogenesis of other species.

      Minor comments:

      The RNAscope experiment is currently qualitative. Is it the mRNA copy number per cell equal in both species but more cells are positive in rat, or are there differences in number of mRNA molecules as well? It is not indicated if the RNAscope probes are the same for mouse and rat.

      Significance

      How different species achieve such remarkable differences in brain shape and size remains poorly understood. A critical aspect of this process is the duration of the neurogenic phase: the period during which neural progenitors generate neurons. This phase tends to be extended in species with larger brains and contains multiple neuronal stem cell types in varying proportions. It is thought that this accounts for their increased neuronal numbers. In their search for mechanisms that prolong neurogenesis across species, the authors propose a rat-specific role for Wnt ligands in expanding the neurogenic period in the rat brain. Importantly, they rule out that this mechanism operates in other species, such as primates or ferrets, to achieve similar extensions.

      The study is of high quality, incorporating rigorous lineage-tracing experiments in two species and single-cell RNA sequencing. Previous work established a role for Wnt signaling in regulating early neurogenesis in mice. Here, the authors characterize a novel population of radial glial cells (Lmx1a and Lhx2 double-positive) that may explain increased Wnt ligand secretion in rats. However, functional validation of this mechanism is still lacking. To strengthen its evolutionary relevance, it would be important to determine whether similar effects occur during earlier neural stages in other species (such as neuroepithelium thickening), or whether other cell types have co-opted the proposed Lmx1a-Lhx2 regulatory module in other species.

      From the perspective of a researcher with a stem cell and developmental background focused on neural evo-devo, this manuscript represents a solid and novel contribution. The proposed model of a rat-specific mechanism for extending the neurogenic phase contrasts with the prevailing concept of convergence in mechanisms underlying species-specific cortical development. This raises intriguing questions about how multiple molecular pathways have been co-opted to achieve similar developmental outcomes. Furthermore, we know very little about what determines the duration of specific developmental processes. This work suggests that extended Wnt signaling may account for prolonged neurogenesis in rats compared to mice. Future studies should aim to validate the proposed rat-specific co-option of an Lmx1a-Wnt ligand cascade in cortical radial glia, potentially through relief of Lhx2-mediated repression of Lmx1a.

    1. 1)How did he learn to read if it was forbidden? So brave! I feel bad for his suffering, it’s so unfair. Wow! His mistress changed because of slavery, that’s sad but interesting. I like how he used kids to learn, so smart. “Columbian Orator”? I need to look this up, sounds important. It’s so intense that learning to read made him suffer more… cruel that freedom was only a dream.

      2) Chattel -someone’s property (like an object) Stratagems -strategies, tricks Emancipation - freedom from slavery Vindication - defense or justification Writhed -twisted, squirmed Unabated - without decreasing

      3) Frederick Douglass was born a slave in Maryland around 1817. - Origin He went to Baltimore and learned to read with his master’s wife’s help. - Learning He escaped in 1838, got married, and changed his name. -Freedom

      Became a speaker, wrote his autobiography, fought for Black rights. - Activism In Master Hugh’s house, he had to use strategies to keep learning, because his mistress stopped teaching him. -Obstacles His mistress was kind at first, but slavery made her harsh and cruel. - Change of people Douglass befriended white kids to teach him how to read more. -Cleverness The book Columbian Orator taught him about freedom and arguing against slavery. - Education Learning to read caused pain because he saw his situation but couldn’t change it. -Pain

      4) “Slavery proved as injurious to her as it did to me.” “Education and slavery were incompatible with each other.” “The more I read, the more I was led to abhor and detest my enslavers.” “The silver trump of freedom had roused my soul to eternal wakefulness.” Freedom was always on his mind, motivating and torturing him at the same time. - Desire for freedom

      5) My thesis: Learning to read was a double-edged sword for Frederick Douglass: it freed him intellectually and gave him power to fight for freedom, but it also caused him pain by revealing the cruelty of slavery and his own oppression.

    1. Database

        1. Relational Database
      • helpful for storing massive amounts of data -able to store diiffernt types of data
      • managed services, provided backup,high availibility, high storage,
      • across muiltiple az
      • able to recover fast --AWS reposibility - making sure its operational, security of storgae, infasturcture mangaement and safaty -Coustomer Resposibil;ity= mangament of data, encryption and access contraol and monitor performance Types" 1) RDS
      • non flexible scheme
      • SQL, POSTGRESS SQL,
      • relational dataset 2) NoSQL
      • flexible scheme
      • 3)Data Warehousing 4)In memory caching Aurora:
      • faster thatn RDS -SQL

      RedShift- Data Warehouse ElasticCache : inmemory cacheing service

    1. It's probably worth mentioning that much of this work creating maize from teosinte, learning to freeze-dry potatoes and detoxify cassava, and identifying wheat and rice as valuable plants, was probably done by women.

      Just think it's fascinating how many of these techniques are still used in today's society, like refrigerating / learning to freeze dry a variety of things.

    1. Taken as a list or a single sentence, this can be a bit overwhelming to digest. There are many things being said at the same time, and they may not all be immediately clear. To better understand what each of the “areas of concern” are and how they can impact learning, each has been separated and explained in the table below. Area of ConcernWhat it means for youHow it applies to learningReal-world example 1. Social and psychological origins of …Your motivations, not only as a student but as a person, and both the social and psychological factors that influence youThis can be everything from the original motivation behind enrolling in school in the first place, down to more specific goals like why you want to learn to write and communicate well.A drive to be self-supporting and to take on a productive role in society. 2. needs, which generate …Better job, increased income, satisfying career, prestigeThis can include the area of study you select and the school you choose to attend.Pursuing a degree to seek a career in a field you enjoy. 3. expectations of …Expectation and perception (preconceived and continuing) of educational materialWhat you expect to learn to fulfill goals and meet needs.Understanding what you need to accomplish the smaller goals. An example would be “study for an exam.” 4. the mass media or other sources, which lead to…The content and learning activities of the programSelection of content aimed at fulfilling needs. Results are student satisfaction, perceived value, and continued enrollment.Choosing which learning activities to use (e.g., texts, watch videos, research alternative content, etc.). 5. differential patterns of media exposure, resulting in …Frequency and level of participationHow you engage with learning activities and how often. Results are student satisfaction and perceived value, and continued enrollment.When, how often, and how much time you spend in learning activities. 6. needs gratification and …Better job, increased income, satisfying career, prestige, more immediate goals like pass an exam, earn a good grade, etc.Needs fulfillment and completion of goals.Learning activities that meet your learning needs, including fulfillment of your original goals. 7. other unintended consequences.Increased skills and knowledge, entertainment, social involvement and networkingCauses positive loop-back into 4, 5, and 6, reinforcing those positive outcomes.Things you learn beyond your initial goals.

      Helpful.

    2. Some forms of learning are extremely difficult in activities delivered in one style or another. Subjects like computer programming would be almost impossible to learn using an aural learning style. And, while it is possible to read about a subject such as how to swing a bat or how to do a medical procedure, actually applying that knowledge in a learning environment is difficult if the subject is something that requires a physical skill.

      Taking this into account makes sense, as there are so many subjects that one can learn, those subjects may only allow one maybe two learning styles to be active.

    3. Kinesthetic: The student prefers using their body, hands, and sense of touch. An example would be doing something physical, such as examining an object rather than reading about it or looking at an illustration.

      I took a test and found that I am a kinesthetic learner. This is very true as I look back on what I've learned and how, it all came down to me actually performing the task.

    1. Analyse de la Tendance « Mamans Ghettossori » : Entre Lutte des Classes et Réalités Éducatives

      Résumé Exécutif

      L'émergence de la tendance « mamans ghettossori » sur TikTok à la fin de l'année 2024 marque un tournant dans la représentation de la parentalité sur les réseaux sociaux.

      Née en réaction au modèle « maman Montessori » — perçu comme bourgeois, permissif et déconnecté — cette tendance met en scène une éducation ancrée dans la réalité des milieux populaires.

      Si ce mouvement permet de déculpabiliser de nombreux parents face aux injonctions de la parentalité positive, il soulève également des inquiétudes quant à la validation potentielle des Violences Éducatives Ordinaires (VEO).

      Au-delà du simple contenu viral, ce phénomène cristallise une « lutte des classes 2.0 » et une réponse aux accusations de démission parentale, soulignant la nécessité de retrouver des espaces de dialogue neutres et bienveillants, tels que les associations de parents.

      --------------------------------------------------------------------------------

      1. Genèse et Mécanismes du Phénomène TikTok

      La tendance a été initiée par l'influenceuse niçoise Jessica French Riviera le 13 décembre 2024, avant de se propager massivement sous forme de « reels ».

      Le format « Ghettossori »

      Le modèle type des vidéos repose sur une litanie de phrases commençant par : « Je suis une maman ghettossori, alors bien entendu que... ». Parmi les exemples cités dans les sources :

      Gestion des loisirs : Accepter des soirées pyjamas avec de nombreux copains, pop-corn et chips sans restriction.

      Défense personnelle : Inscrire l'enfant au kickboxing pour qu'il puisse se défendre en cas de conflit (« heja »).

      Rapport à l'autorité : L'enfant craint davantage la réaction de sa mère s'il ne s'est pas défendu que celle de l'école.

      Réalité quotidienne : Commander une pizza par « flemme » de cuisiner tout en le présentant comme un cadeau à l'enfant.

      Compétition : Ne pas laisser gagner l'enfant aux jeux de société (Mario Kart, galette des rois).

      La figure de proue Montessori : Armelle

      En opposition, la maman Montessori type est représentée par Armelle (@moharmelle).

      Ses vidéos, où elle prône une bienveillance absolue (comme attendre des heures dans une voiture qu'un enfant se réveille), cumulent des millions de vues mais suscitent l'incrédulité, voire la moquerie.

      Elle est devenue la cible principale des pastiches et le contre-modèle sur lequel s'est construit l'archétype ghettossori.

      --------------------------------------------------------------------------------

      2. Une Lutte des Classes 2.0 par Réseaux Interposés

      Le conflit entre « Montessori » et « Ghettossori » dépasse le cadre pédagogique pour devenir un enjeu sociologique majeur.

      Une fracture socio-économique : En France, le réseau Montessori est majoritairement privé et coûteux (sur 300 établissements, seuls trois sont sous contrat avec l'État en 2022).

      Cela associe cette pédagogie aux classes supérieures, créant une opposition naturelle avec les milieux populaires.

      Perception culturelle :

      Modèle Montessori : Perçu comme celui des « parents bobos permissifs », prescripteurs d'une morale normative.   

      Modèle Ghettossori : Revendiqué comme celui de la « vraie vie », valorisant l'imperfection et l'authenticité face aux contraintes du quotidien.

      Effet de halo en Belgique : Bien que les vidéos soient principalement d'origine française, le phénomène touche pleinement la Belgique francophone, les archétypes étant facilement identifiables par le public belge.

      --------------------------------------------------------------------------------

      3. Enjeux Éducatifs et Risques de Dérapages

      L'analyse de la FAPEO souligne une tension entre la déculpabilisation nécessaire et le risque de dérive comportementale.

      Le risque de banalisation des VEO

      Certaines affirmations dans les vidéos ghettossori inquiètent les spécialistes. Sous couvert d'humour et de sarcasme, certains propos pourraient valider des Violences Éducatives Ordinaires (VEO).

      Les journaux (Le Figaro, Le Point, RTL) notent que la frontière entre l'éloge de la « parentalité imparfaite » et la promotion d'une éducation « à la dure » est parfois poreuse.

      Une réponse à la « démission parentale »

      La tendance est analysée comme une réponse indirecte aux discours politiques et médiatiques stigmatisants sur la prétendue démission des parents des classes populaires.

      • C'est un refus de la morale normative qui juge les mères.

      • C'est une revendication du droit à l'erreur et une manifestation d'un ras-le-bol face aux injonctions académiques.

      --------------------------------------------------------------------------------

      4. Dynamiques Sociales et Limites de l'Humour

      Un phénomène genré

      Il est frappant de noter l'absence quasi totale de « papas Montessori » ou « papas ghettossori ».

      L'éducation des jeunes enfants reste un domaine fortement imprégné par le genre, où les mères portent l'essentiel de la charge et du jugement social.

      La dérive du « Roasting »

      L'humour utilisé sur TikTok s'inscrit dans la tendance actuelle du roasting (mise en boîte). Cependant, l'analyse rappelle que :

      • Du rire à l'humiliation, il n'y a qu'un pas.

      • La moquerie répétée peut s'apparenter à du harcèlement ou de la maltraitance.

      • L'utilisation de « l'écriture en creux » permet de railler un modèle (et la personne qui l'incarne) sans la nommer explicitement, renforçant l'agressivité du sous-texte.

      --------------------------------------------------------------------------------

      5. Conclusions et Perspectives : Vers un Espace de Dialogue

      Pour sortir de la logique clivante des réseaux sociaux et des algorithmes qui favorisent le « drama », la FAPEO propose plusieurs pistes de réflexion :

      Unir plutôt que diviser : Au-delà des méthodes, toutes ces mères partagent le désir d'épanouissement de leurs enfants.

      Valoriser les associations de parents : Ces structures sont présentées comme des lieux idéaux pour un échange de pair à pair, sans jugement, loin de l'agressivité numérique.

      Promouvoir la « finesse » de l'humour : S'inspirer de figures comme Coluche, qui ridiculisait les faits et les travers collectifs sans jamais s'attaquer à l'individu ou tomber dans la vulgarité.

      Adopter un « regard qui écoute » : Selon Max Dorra, il est crucial de libérer l'autre de toute évaluation permanente pour restaurer un lien social authentique.

      En définitive, l'opposition entre Montessori et Ghettossori ne doit pas masquer l'essentiel : la nécessité de soutenir tous les parents dans leur réalité quotidienne, en favorisant l'entraide plutôt que le clash médiatique.

    1. Author response:

      We thank all reviewers for their comments. We appreciate the acknowledgement that the paper is important and that results support the major conclusions. We are planning to address the specific concerns as noted by the reviewers in the following way:

      Public Reviews:

      Reviewer #2 (Public review):

      (1) The authors generate a new tool, a Gal4 knock-in of the jam2b locus, to track EGFP-expressing cells over time and follow the developmental trajectory of jam2b-expressing cells. Figure 1 characterizes the line. However, it lacks quantification, e.g., how many etv2-expressing cells also show EGFP expression or the contribution of EGFP-expressing cells to different types of blood vessels. This type of quantification would be useful, as it would also allow for comparison of their findings to their previous data examining the contribution of SVF cells to different types of blood vessels. All the authors state that at 30 hpf, EGFP-expressing cells can be seen in the vasculature (apparently the PCV).

      It is not clear why the authors do not use a nuclear marker for both ECs (as they did in their previous publication) and for jam2b-expressing cells. UAS:nEGFP and UAS:NLS-mcherry (e.g. pt424tg) transgenic lines are available. This would circumvent the problem the authors encounter with the strong fluorescence visible in the yolk extension. It would also facilitate quantifying the contribution of jam2b cells to different types of blood vessels.

      We agree with the importance of quantification. We had performed quantification of jam2b<sup>Gt(2A-Gal4)</sup>;UAS:GFP contribution to different vascular beds, which was shown in Suppl. Fig. S3. We will clarify this in the revision. We also agree that nuclear GFP or mCherry would help to visualize and quantify cells. Unfortunately, we do not have nuclear UAS:GFP or UAS:mCherry line in our possession, and it will take too long to import it for the standard revision timeline. We are working on the construct, and will attempt to establish the line; therefore we are hoping to clarify these results with the nuclear line in the revised manuscript.

      (2) The time-lapse movie in Figure 2 is not very informative, as it just provides a single example of a dividing cell contributing to the PCV. Also, quantifications are needed. As SVF cells appear to expand significantly after their initial specification, it would be informative to know how many cell divisions and which types of blood vessels jam2b-expressing cells contribute to. Can the authors observe cells that give rise to different types of blood vessels? Jam2b expression in LPM cells apparently precedes expression of etv2. Is etv2 needed for maintenance, or do Jam2b-expressing cells contribute to different types of tissues in etv2 mutant embryos? Comparing time-lapse analysis in wildtype and etv2 mutant embryos would address this question.

      The time-lapse was meant to serve as an illustration and confirmation of jam2b cell contribution to vasculature. As noted above, Suppl. Fig. S3 provides quantification of jam2b cell contribution to different vascular beds. We had previously performed detailed time-lapse analysis and quantification of SVF cell migration to PCV, SIA and SIV using etv2-2A-Venus line (Metikala et al 2022, Dev Cell), which has some of the same (or similar) information. It is very challenging to obtain this data using jam2b reporter line due to extensive and bright GFP expression in the mesothelial layer over the yolk and yolk extension; for that reason we can only trace some GFP cells but not all of them. Regarding etv2 requirement for jam2b maintenance, we intend to address this question by analyzing jam2b cell contribution in etv2 MO injected embryos, which recapitulates the phenotype in jam2b mutants.

      (3) In Figure 3, the authors generate UAS:Cre and UAS:Cre-ERT2 transgenic lines to lineage trace the jam2b-expressing cells. It is again not clear why the authors do not use a responder line containing nuclear-localized fluorescent proteins to circumvent the strong expression of fluorescent proteins in the yolk extension. It is also unclear why the two transgenic lines give very different results regarding the number of cells being labelled. The ERT2 fusions label around 3 cells in the SIA, while the Cre line labels only about 1.5 cells per embryo, with very little contribution of labelled cells to other blood vessels. One would expect the Cre line requiring tamoxifen induction to label fewer cells when compared to the constitutive Cre line. What is the reason for this discrepancy? Are the lines single integration? Is there silencing? This needs to be better characterized, also regarding the reproducibility of the experiments. If the Cre lines were to be multiple copy integrations, outcrossing the line might lead to lower expression levels in future generations. 

      It is also not clear how the authors conclude from these findings that "SVF cells show major contribution to the SIA and SIV" when only 1.5 or 3 cells of the SIA are labelled, with even fewer cells labelled in other blood vessels. They speculate that this might be due to low recombination efficiency, a question they then set out to answer using photoconversion of etv2:KAEDE expressing cells, an experiment that they also performed in their 2014 and 2022 publications. To check for low recombination efficiency, the authors could examine the expression of Cre mRNA in their transgenic embryos. Do many more jam2b expressing cells express Cre mRNA than they observe in their switch lines? They could also compare their experiments using Cre recombinase with those using EGFP expression in jam2b cells. EGFP is relatively stable, and the time frames the authors analyze are short. As no quantification of EGFP-expressing cells is provided in Figure 1, this comparison is currently not possible. Do these two different approaches answer different questions here? 

      The reviewer brings up important points, we appreciate that. Unfortunately, we do not have a nuclear switch line in our possession, and it is not possible to obtain it in the normal manuscript revision time line. Regarding UAS:Cre and UAS:CreERT2 lines, they both show rather similar labeling, with most labeled cells present in the SIA. The difference in cell number (1.5 versus 3) is likely due to different levels of Cre expression, which may vary dependent on the integration site. The lines most likely are multi-copy integrations, which can be helpful, as this would result in higher Cre expression. We will address the silencing question by performing in situ hybridization or HCR analysis for Cre or CreERT2 and comparing it with endogenous jam2b expression, as the reviewer suggested. We have noticed that the switch line used, actb2:loxP-BFP-loxP-dsRed, exhibits lower recombination frequency compared to other switch lines (we used it because it was compatible with endothelial fli1:GFP line). We will attempt to answer this question by crossing to other switch lines, which may exhibit higher recombination frequency. In principle, UAS:GFP and switch lines should produce a similar result, except that GFP decays over time and therefore our initial expectation was that switch lines may produce a more accurate result. However, this may not be the case due to low recombination efficiency, which we will attempt to address in the revision.

      (4) Concerning the etv2:KAEDE photoconversion experiments: The percentages the authors report for SVF cells' contribution to the SIV and SIA differ from their previous study (Dev Cell, 2022). In that publication, SVF cells contributed 28% to the SIA and 48% to the SIV. In the present study, the numbers are close to 80% for both vessels. The difference is that the previous study analyzed 2dpf old embryos and the new one 4dpf old embryos. Do SVF-derived cells proliferate more than PCV-derived cells, or is there another explanation for this change in percentage contribution? 

      These numbers refer to different experiments; we apologize for the confusion. As reported earlier in Metikala et 2022, 28% of SVF cells contributed to the SIA and 48% to the SIV by 3 dpf (not 2 dpf; only PCV analysis was done at 2 dpf); SIA and SIV analysis was done based on time-lapse image analysis of etv2-2A-Venus line at 3 dpf, shown in Fig. 3C in Metikala et al. However, this only refers to SVF cell contribution. It does not mean that 28% or 48% cells in SIA or SIV are derived from SVF. The total fraction of SIA and SIV cells that are derived from SVF has not been quantified in the previous study, because that would require accurate tracking of all SVF cells, which is experimentally challenging. Etv2:Kaede experiment is slighly different, because it reports newly formed cells after 24 hpf. It cannot tell if new cells are all derived from SVF cells, although we are not aware of any other source of new endothelial cells at these stages. In the previous study by Metikala et al 2022, we reported ~22 newly formed SIA and ~50 newly formed cells in SIV by 3 dpf (Fig. 1 in Metikala et al 2022), although the entire number of cells was not quantified, therefore the percentage was not known. In the current study, we attempted to estimate the entire percentage of green only Kaede cells, which was close to 80% in both SIA or SIV at 4 dpf. Please note that this estimate was performed in the posterior portion of SIA and SIV that overlies the yolk extension and where SVF cells are observed. We did not quantify cells in the anterior SIV portion, which forms the basket over the yolk.

      (5) Single-cell sequencing data: Why do the authors not show jam2b expression in their single-cell sequencing data? They sorted for (presumably) jam2b-expressing cells and hypothesize that jam2b expression in ECs at this time point is important for the generation of intestinal vasculature. Do ECs in cluster 15 express jam2b? Why are no other top marker genes (tal1, etv2, egfl7, npas4l) included in the dot blot in Figure 5b?

      We appreciate the suggestion and will include additional marker genes as well as jam2b in the revised version of the manuscript.

      (6) Concerns about cell autonomy of mutant phenotypes: The authors need to perform in situ hybridization to characterize jam2a expression. Can it be seen in SVF cells? The double mutants show a clear phenotype in intestinal vessel development; however, it is unclear whether this is due to a cell-autonomous function of jam2a/b within SVF cells. The authors need to address this issue, as jam2b and potentially also jam2a are expressed within the tissue surrounding the forming SVF. For instance, do transplanted mutant cells contribute to the intestinal vasculature to the same extent as wild-type cells do?

      jam2a expression has been characterized in the previous studies and it is shown in the Suppl. Fig. S4E. It is primarily enriched in the skeletal muscle. However, our single-cell RNA-seq analysis shows that SVF cells also express jam2a. We will include additional data on jam2a expression in the revised manuscript. We agree that transplation to address cell autonomy is an important experiment, yet there are some practical challenges to it. Jam2a,jam2b mutant phenotype is only partially penetrant, and about 50% reduction in SVF cell number, as well as partial SIA and SIV phenotypes are observed. Only a small number of transplanted cells may contribute to intestinal vasculature, therefore it may be challenging to see the differences, given the partial penetrance. In an attempt to address cell -autonomy question, we will try a different approach. We will overexpress jam2b labeled with 2A-mCherry, and test if it can rescue the mutant phenotype in cell autonomous manner. Overexpression will be done in a mosaic manner, with higher number of cells labeled than in a typical transplantation experiment.

      (7) Finally, the authors analyze the phenotypes of hand2 mutants and their impact on the expression of jam2b and etv2. They observe a reduction in jam2b and etv2 expression in SVF cells. However, they do not show the vascular phenotypes of hand2 mutants. Is the formation of the SIA and SIV disturbed? Is hand2 cell autonomously needed in ECs? The authors suggest that hand2 controls SVF development through the regulation of jam2b. However, they also show that jam2b mutants do not have a phenotype on their own. Clearly, hand2, if it were to be required in ECs, regulates other genes important for SVF development. These might then regulate jam2b expression. The clear linear relationship, as the title suggests, is not convincingly shown by the data.

      As suggested, we will add the analysis of SIA and SIV in hand2 mutants during the revision process. We could not assess that easily because the line was not maintained in vascular fli1:GFP background. We do not know if hand2 is required cell-autonomously. This is an important question, but it may be answered better in a separate study. Regarding hand2-jam2b axis, it is very clear that jam2b expression in the posterior lateral plate mesoderm is completely lost in hand2 mutants, except for its more anterior domain over the yolk. This does support the idea that hand2 functions upstream of jam2b. However, the relationship may not be necessarily direct. We agree that hand2 may regulate additional genes involved in SVF cell development. We will attempt to clarify this relationship and test if jam2b overexpression may rescue hand2 mutant phenotype.

      Reviewer #3 (Public review):

      (1) Overall molecular mechanisms of Jam2 function are not fully uncovered in the study. How do the adhesion molecules Jam2a and Jam2b regulate SVF cell formation? Are they responsible for migration, adhesion or fate determination of these structures? The authors should provide a more in-depth study of the jam2a, jam2b mutations and assess the processes affected in these mutants. Combining these mutants with etv2:Kaede can also provide a stronger causative link between their functions and defects in SVF formation.

      Our data argue that the initial SVF cell specification (based on etv2 expression) is reduced in jam2a;jam2b mutants. We do not know if the migration or fate determination of the remaining SVF cells is also affected, although this may be more challenging to answer, as there are only few SVF cells remaining. We agree that further mechanistic studies of jam2a,jam2b function are needed. However, we think that this would be better addressed in a separate study. We are currently raising mutants crossed into fli1:Kaede line, which should confirm that there are fewer new cells that emerge after Kaede photoconversion in jam2a,jam2b mutants.

      (2) Have the authors tested the specificity of the jam2b knock-in reporter line? This is an important experiment, as many of the conclusions derive from lineage tracing and fluorescence reporting from this knock-in line. One suggestion is to cross the jam2b:GFP or jam2b:Gal4, UAS:GFP line to the generated jam2b mutants, and examine the expression pattern of these lines. Considering that the ISH experiment showed lack of jam2b expression, the reporter line should not be expressed in the jam2b mutants.

      We show in Suppl. Fig. 2 that jam2b<sup>Gt(2A-Gal4)</sup>;UAS:GFP knock-in line has similar expression pattern as jam2b mRNA by in situ hybridization, which argues for its specificity. In the revision, we plan to use HCR analysis to confirm than jam2b mRNA is expressed in the same cells as jam2b<sup>Gt(2A-Gal4)</sup>;UAS:GFP, as an additional evidence for its specificity. Unfortunately, it is not feasible to cross jam2b knock-in line into jam2b mutants, as suggested by the reviewer. Because jam2b knock-in line targets the endogenous jam2b genomic locus, which is very close in the genome to jam2b promoter deletion in jam2b mutants, the recombination frequency would be very low, and we would not get double jam2b knock-in and knock-out events in the same chromosome.

      (3) The rationale behind the regeneration study is not clear, and the mechanisms underlying the phenotype are not well described. How do the authors explain the phenotype with the impaired regeneration, and what is the significance of this finding as it relates to SVF formation and function? 

      We apologize for this omission. This experiment was more thouroughly described in our previous study by Metikala et al 2022. In that study we showed that when endothelial cells are ablated by treating with MTZ from 6 to 45 hpf, this results in ablation of all vascular endothelial cells except for SVF cells, because they originate later than other cells. We subsequently showed that these SVF cells can partially form PCV and intestinal vasculature, helping them regenerate, which was confirmed by time-lapse imaging. In the current study, we tested if jam2a; jam2b double mutants show defects in such vascular regeneration. Indeed, regeneration after cell ablation was reduced, which correlated with reduction in SVF cell number. This argues that jam2a/b function is required for SVF cell emergence and vascular recovery after endothelial cell ablation. We will provide better description of this experiment and discuss interpretations in the revised manuscript.

      (4) The authors need to include representative images of jam2b>CreERT2 with 4-OH activation at different timepoints in Figure 3.

      Yes, thanks for noting this; these images will be included in the revised manuscript.

      (5) The etv2:Kaede photoconversion experiment to show that the majority of intestinal vasculature derives after 24 hours needs to be supplemented with additional data on photoconverted post-24-hour-old endothelial cells, with the expectation that the majority of intestinal endothelial cells at 4 days will then be labeled with red Kaede. In addition, there have been data that show the red Kaede protein is not stable past several days in vivo, and 3 days might be sufficient for the removal or degradation of this photoconverted protein. Thus, the statement that intestinal vasculature forms largely by new vasculogenesis might be too strong based on existing data.

      It is apparent from Fig. 4B that many other vessels, such as the dorsal aorta and many intersegmental vessels show robust red Kaede expression at 4 dpf, arguing that there is sufficient photoconverted Kaede present at this stage, and its degradation is unlikely to be the reason. However, we are planning to include additional control experiments, as suggested by the reviewer, to make this argument stronger.

      (6) To strengthen the claim that hand2 acts upstream of jam2b, the authors can perform combinatorial genetic epistatic analysis and examine whether jam2b mutations worsen hand2 homozygous or heterozygous effects on the SVF. Similarly, overexpressing jam2b might rescue the loss of SVF/etv2 expression in hand2 mutants. 

      We appreciate this suggestion. Double epistatic analysis, while informative, can be tricky. In this case, we are dealing with jam2a; jam2b redundancy and also the maternal effect. It may take a while considerable effort to generate different combinations of tripple mutant lines (jam2a,jam2b,hand2), and it is unclear whether double or tripple heterozygous embryos will show any defects to clarify their epistatic relationship. Instead, as suggested, we are planning to overexpress jam2b in wild-type and hand2 mutants to address this point.

    2. Reviewer #2 (Public review):

      Summary:

      Griciunaite et al. report on the function of jam2b and hand2 in the formation of the intestinal vasculature derived from late-forming endothelial cells (ECs) within the secondary vascular field (SVF). They generate transgenic lines that allow for the tracking of jam2b-expressing cells, both with fluorescent proteins and through Cre-mediated recombination in reporter lines. They also show that double maternal zygotic mutants in jam2a and jam2b, as well as hand2 mutants, display defects in the formation of the intestinal vasculature.

      Strengths:

      The results are interesting, as they address the important question of how blood vessels form during later developmental time points and potentially identify specific genes regulating this process.

      Weaknesses:

      (1) The authors generate a new tool, a Gal4 knock-in of the jam2b locus, to track EGFP-expressing cells over time and follow the developmental trajectory of jam2b-expressing cells. Figure 1 characterizes the line. However, it lacks quantification, e.g., how many etv2-expressing cells also show EGFP expression or the contribution of EGFP-expressing cells to different types of blood vessels. This type of quantification would be useful, as it would also allow for comparison of their findings to their previous data examining the contribution of SVF cells to different types of blood vessels. All the authors state that at 30 hpf, EGFP-expressing cells can be seen in the vasculature (apparently the PCV).

      It is not clear why the authors do not use a nuclear marker for both ECs (as they did in their previous publication) and for jam2b-expressing cells. UAS:nEGFP and UAS:NLS-mcherry (e.g. pt424tg) transgenic lines are available. This would circumvent the problem the authors encounter with the strong fluorescence visible in the yolk extension. It would also facilitate quantifying the contribution of jam2b cells to different types of blood vessels.

      (2) The time-lapse movie in Figure 2 is not very informative, as it just provides a single example of a dividing cell contributing to the PCV. Also, quantifications are needed. As SVF cells appear to expand significantly after their initial specification, it would be informative to know how many cell divisions and which types of blood vessels jam2b-expressing cells contribute to. Can the authors observe cells that give rise to different types of blood vessels? Jam2b expression in LPM cells apparently precedes expression of etv2. Is etv2 needed for maintenance, or do Jam2b-expressing cells contribute to different types of tissues in etv2 mutant embryos? Comparing time-lapse analysis in wildtype and etv2 mutant embryos would address this question.

      (3) In Figure 3, the authors generate UAS:Cre and UAS:Cre-ERT2 transgenic lines to lineage trace the jam2b-expressing cells. It is again not clear why the authors do not use a responder line containing nuclear-localized fluorescent proteins to circumvent the strong expression of fluorescent proteins in the yolk extension. It is also unclear why the two transgenic lines give very different results regarding the number of cells being labelled. The ERT2 fusions label around 3 cells in the SIA, while the Cre line labels only about 1.5 cells per embryo, with very little contribution of labelled cells to other blood vessels. One would expect the Cre line requiring tamoxifen induction to label fewer cells when compared to the constitutive Cre line. What is the reason for this discrepancy? Are the lines single integration? Is there silencing? This needs to be better characterized, also regarding the reproducibility of the experiments. If the Cre lines were to be multiple copy integrations, outcrossing the line might lead to lower expression levels in future generations.

      It is also not clear how the authors conclude from these findings that "SVF cells show major contribution to the SIA and SIV" when only 1.5 or 3 cells of the SIA are labelled, with even fewer cells labelled in other blood vessels. They speculate that this might be due to low recombination efficiency, a question they then set out to answer using photoconversion of etv2:KAEDE expressing cells, an experiment that they also performed in their 2014 and 2022 publications. To check for low recombination efficiency, the authors could examine the expression of Cre mRNA in their transgenic embryos. Do many more jam2b expressing cells express Cre mRNA than they observe in their switch lines? They could also compare their experiments using Cre recombinase with those using EGFP expression in jam2b cells. EGFP is relatively stable, and the time frames the authors analyze are short. As no quantification of EGFP-expressing cells is provided in Figure 1, this comparison is currently not possible. Do these two different approaches answer different questions here?

      (4) Concerning the etv2:KAEDE photoconversion experiments: The percentages the authors report for SVF cells' contribution to the SIV and SIA differ from their previous study (Dev Cell, 2022). In that publication, SVF cells contributed 28% to the SIA and 48% to the SIV. In the present study, the numbers are close to 80% for both vessels. The difference is that the previous study analyzed 2dpf old embryos and the new one 4dpf old embryos. Do SVF-derived cells proliferate more than PCV-derived cells, or is there another explanation for this change in percentage contribution?

      (5) Single-cell sequencing data: Why do the authors not show jam2b expression in their single-cell sequencing data? They sorted for (presumably) jam2b-expressing cells and hypothesize that jam2b expression in ECs at this time point is important for the generation of intestinal vasculature. Do ECs in cluster 15 express jam2b? Why are no other top marker genes (tal1, etv2, egfl7, npas4l) included in the dot blot in Figure 5b?

      (6) Concerns about cell autonomy of mutant phenotypes: The authors need to perform in situ hybridization to characterize jam2a expression. Can it be seen in SVF cells? The double mutants show a clear phenotype in intestinal vessel development; however, it is unclear whether this is due to a cell-autonomous function of jam2a/b within SVF cells. The authors need to address this issue, as jam2b and potentially also jam2a are expressed within the tissue surrounding the forming SVF. For instance, do transplanted mutant cells contribute to the intestinal vasculature to the same extent as wild-type cells do?

      (7) Finally, the authors analyze the phenotypes of hand2 mutants and their impact on the expression of jam2b and etv2. They observe a reduction in jam2b and etv2 expression in SVF cells. However, they do not show the vascular phenotypes of hand2 mutants. Is the formation of the SIA and SIV disturbed? Is hand2 cell autonomously needed in ECs? The authors suggest that hand2 controls SVF development through the regulation of jam2b. However, they also show that jam2b mutants do not have a phenotype on their own. Clearly, hand2, if it were to be required in ECs, regulates other genes important for SVF development. These might then regulate jam2b expression. The clear linear relationship, as the title suggests, is not convincingly shown by the data.

    3. Reviewer #3 (Public review):

      Summary:

      This study by Griciunaite et al. investigates the function of the adhesion molecule Jam2 in initiating the formation of organ (intestinal)-specific vasculature in zebrafish. Their previous studies identified a group of late-forming vascular progenitors from the lateral plate mesoderm along the yolk extension termed the secondary vascular field (SVF), which can contribute to intestinal vasculature. Transcriptomic analysis of the zebrafish trunk region identified SVF-enriched marker genes, which include jam2b. They then performed expression analysis of jam2b using whole-mount in situ hybridization and Gal4 knock-in transgenic line analysis. These analyses show that jam2b is expressed in the SVF cells that correspond to etv2 and kdrl expression past 24 hours. Lineage tracing combining jam2b:Gal4 and UAS:Cre or UAS:CreERT2 show the contribution of jam2b in SVF and intestinal vasculature formation. jam2b mutations did not cause observable defects in the vasculature, but combined jam2a; jam2b mutations led to impaired ISV, PCV, SIA, SIV and thoracic duct lymphatic vasculature formation. Finally, the authors show that mutations in the transcription factor hand2 led to reduced jam2b expression and impaired SVF formation.

      Strengths:

      The authors accomplished many feats in generating new reporter lines and mutations that are valuable to the community. The study provided an interesting perspective on organ-specific vascular development and origin heterogeneity. The genetic aspects of the study are clean, and the mutational phenotypes are convincing.

      Several suggestions and major comments that can improve the manuscript include:

      (1) Overall molecular mechanisms of Jam2 function are not fully uncovered in the study. How do the adhesion molecules Jam2a and Jam2b regulate SVF cell formation? Are they responsible for migration, adhesion or fate determination of these structures? The authors should provide a more in-depth study of the jam2a, jam2b mutations and assess the processes affected in these mutants. Combining these mutants with etv2:Kaede can also provide a stronger causative link between their functions and defects in SVF formation.

      (2) Have the authors tested the specificity of the jam2b knock-in reporter line? This is an important experiment, as many of the conclusions derive from lineage tracing and fluorescence reporting from this knock-in line. One suggestion is to cross the jam2b:GFP or jam2b:Gal4, UAS:GFP line to the generated jam2b mutants, and examine the expression pattern of these lines. Considering that the ISH experiment showed lack of jam2b expression, the reporter line should not be expressed in the jam2b mutants.

      (3) The rationale behind the regeneration study is not clear, and the mechanisms underlying the phenotype are not well described. How do the authors explain the phenotype with the impaired regeneration, and what is the significance of this finding as it relates to SVF formation and function?

      (4) The authors need to include representative images of jam2b>CreERT2 with 4-OH activation at different timepoints in Figure 3.

      (5) The etv2:Kaede photoconversion experiment to show that the majority of intestinal vasculature derives after 24 hours needs to be supplemented with additional data on photoconverted post-24-hour-old endothelial cells, with the expectation that the majority of intestinal endothelial cells at 4 days will then be labeled with red Kaede. In addition, there have been data that show the red Kaede protein is not stable past several days in vivo, and 3 days might be sufficient for the removal or degradation of this photoconverted protein. Thus, the statement that intestinal vasculature forms largely by new vasculogenesis might be too strong based on existing data.

      (6) To strengthen the claim that hand2 acts upstream of jam2b, the authors can perform combinatorial genetic epistatic analysis and examine whether jam2b mutations worsen hand2 homozygous or heterozygous effects on the SVF. Similarly, overexpressing jam2b might rescue the loss of SVF/etv2 expression in hand2 mutants.

    1. Author response:

      The following is the authors’ response to the previous reviews

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      Wu and colleagues aimed to explain previous findings that adolescents, compared to adults, show reduced cooperation following cooperative behaviour from a partner in several social scenarios. The authors analysed behavioural data from adolescents and adults performing a zero-sum Prisoner's Dilemma task and compared a range of social and non-social reinforcement learning models to identify potential algorithmic differences. Their findings suggest that adolescents' lower cooperation is best explained by a reduced learning rate for cooperative outcomes, rather than differences in prior expectations about the cooperativeness of a partner. The authors situate their results within the broader literature, proposing that adolescents' behaviour reflects a stronger preference for self-interest rather than a deficit in mentalising.

      Strengths:

      The work as a whole suggests that, in line with past work, adolescents prioritise value accumulation, and this can be, in part, explained by algorithmic differences in weighted value learning. The authors situate their work very clearly in past literature, and make it obvious the gap they are testing and trying to explain. The work also includes social contexts that move the field beyond non-social value accumulation in adolescents. The authors compare a series of formal approaches that might explain the results and establish generative and modelcomparison procedures to demonstrate the validity of their winning model and individual parameters. The writing was clear, and the presentation of the results was logical and wellstructured.

      We thank the reviewer for recognizing the strengths of our work.

      Weaknesses:

      (Q1) I also have some concerns about the methods used to fit and approximate parameters of interest. Namely, the use of maximum likelihood versus hierarchical methods to fit models on an individual level, which may reduce some of the outliers noted in the supplement, and also may improve model identifiability.

      We thank the reviewer for this suggestion. Following the comment, we added a hierarchical Bayesian estimation. We built a hierarchical model with both group-level (adolescent group and adult group) and individual-level structures for the best-fitting model. Four Markov chains with 4,000 samples each were run, and the model converged well (see Figure supplement 7)

      We then analyzed the posterior parameters for adolescents and adults separately. The results were consistent with those from the MLE analysis (see Figure 2—figure supplement 5). These additional results have been included in the Appendix Analysis section (also see Figure supplement 5 and 7). In addition, we have updated the code and provided the link for reference. We appreciate the reviewer’s suggestion, which improved our analysis.

      (Q2) There was also little discussion given the structure of the Prisoner's Dilemma, and the strategy of the game (that defection is always dominant), meaning that the preferences of the adolescents cannot necessarily be distinguished from the incentives of the game, i.e. they may seem less cooperative simply because they want to play the dominant strategy, rather than a lower preferences for cooperation if all else was the same.

      We thank the reviewer for this comment and agree that adolescents’ lower cooperation may partly reflect a rational response to the incentive structure of the Prisoner’s Dilemma.

      However, our computational modeling explicitly addressed this possibility. Model 4 (inequality aversion) captures decisions that are driven purely by self-interest or aversion to unequal outcomes, including a parameter reflecting disutility from advantageous inequality, which represents self-oriented motives. If participants’ behavior were solely guided by the payoff-dominant strategy, this model should have provided the best fit. However, our model comparison showed that Model 5 (social reward) performed better in both adolescents and adults, suggesting that cooperative behavior is better explained by valuing social outcomes beyond payoff structures.

      Besides, if adolescents’ lower cooperation is that they strategically respond to the payoff structure by adopting defection as the more rewarding option. Then, adolescents should show reduced cooperation across all rounds. Instead, adolescents and adults behaved similarly when partners defected, but adolescents cooperated less when partners cooperated and showed little increase in cooperation even after consecutive cooperative responses. This pattern suggests that adolescents’ lower cooperation cannot be explained solely by strategic responses to payoff structures but rather reflects a reduced sensitivity to others’ cooperative behavior or weaker social reciprocity motives. We have expanded our Discussion to acknowledge this important point and to clarify how the behavioral and modeling results address the reviewer’s concern.

      “Overall, these findings indicate that adolescents’ lower cooperation is unlikely to be driven solely by strategic considerations, but may instead reflect differences in the valuation of others’ cooperation or reduced motivation to reciprocate. Although defection is the payoffdominant strategy in the Prisoner’s Dilemma, the selective pattern of adolescents’ cooperation and the model comparison results indicate that their reduced cooperation cannot be fully explained by strategic incentives, but rather reflects weaker valuation of social reciprocity.”

      Appraisal & Discussion:

      (Q3) The authors have partially achieved their aims, but I believe the manuscript would benefit from additional methodological clarification, specifically regarding the use of hierarchical model fitting and the inclusion of Bayes Factors, to more robustly support their conclusions. It would also be important to investigate the source of the model confusion observed in two of their models.

      We thank the reviewer for this comment. In the revised manuscript, we have clarified the hierarchical Bayesian modeling procedure for the best-fitting model, including the group- and individual-level structure and convergence diagnostics. The hierarchical approach produced results that fully replicated those obtained from the original maximumlikelihood estimation, confirming the robustness of our findings. Please also see the response to Q1.

      Regarding the model confusion between the inequality aversion (Model 4) and social reward (Model 5) models in the model recovery analysis, both models’ simulated behaviors were best captured by the baseline model. This pattern arises because neither model includes learning or updating processes. Given that our task involves dynamic, multi-round interactions, models lacking a learning mechanism cannot adequately capture participants’ trial-by-trial adjustments, resulting in similar behavioral patterns that are better explained by the baseline model during model recovery. We have added a clarification of this point to the Results:

      “The overlap between Models 4 and 5 likely arises because neither model incorporates a learning mechanism, making them less able to account for trial-by-trial adjustments in this dynamic task.”

      (Q4) I am unconvinced by the claim that failures in mentalising have been empirically ruled out, even though I am theoretically inclined to believe that adolescents can mentalise using the same procedures as adults. While reinforcement learning models are useful for identifying biases in learning weights, they do not directly capture formal representations of others' mental states. Greater clarity on this point is needed in the discussion, or a toning down of this language.

      We sincerely thank the reviewer for this professional comment. We agree that our prior wording regarding adolescents’ capacity to mentalise was somewhat overgeneralized. Accordingly, we have toned down the language in both the Abstract and the Discussion to better align our statements with what the present study directly tests. Specifically, our revisions focus on adolescents’ and adults’ ability to predict others’ cooperation in social learning. This is consistent with the evidence from our analyses examining adolescents’ and adults’ model-based expectations and self-reported scores on partner cooperativeness (see Figure 4). In the revised Discussion, we state:

      “Our results suggest that the lower levels of cooperation observed in adolescents stem from a stronger motive to prioritize self-interest rather than a deficiency in predicting others’ cooperation in social learning”.

      (Q5) Additionally, a more detailed discussion of the incentives embedded in the Prisoner's Dilemma task would be valuable. In particular, the authors' interpretation of reduced adolescent cooperativeness might be reconsidered in light of the zero-sum nature of the game, which differs from broader conceptualisations of cooperation in contexts where defection is not structurally incentivised.

      We thank the reviewer for this comment and agree that adolescents’ lower cooperation may partly reflect a rational response to the incentive structure of the Prisoner’s Dilemma. However, our behavioral and computational evidence suggests that this pattern cannot be explained solely by strategic responses to payoff structures, but rather reflects a reduced sensitivity to others’ cooperative behavior or weaker social reciprocity motives. We have expanded the Discussion to acknowledge this point and to clarify how both behavioral and modeling results address the reviewer’s concern (see also our response to Q2).

      (Q6) Overall, I believe this work has the potential to make a meaningful contribution to the field. Its impact would be strengthened by more rigorous modelling checks and fitting procedures, as well as by framing the findings in terms of the specific game-theoretic context, rather than general cooperation.

      We thank the reviewer for the professional comments, which have helped us improve our work.

      Reviewer #2 (Public review):

      Summary:

      This manuscript investigates age-related differences in cooperative behavior by comparing adolescents and adults in a repeated Prisoner's Dilemma Game (rPDG). The authors find that adolescents exhibit lower levels of cooperation than adults. Specifically, adolescents reciprocate partners' cooperation to a lesser degree than adults do. Through computational modeling, they show that this relatively low cooperation rate is not due to impaired expectations or mentalizing deficits, but rather a diminished intrinsic reward for reciprocity. A social reinforcement learning model with asymmetric learning rate best captured these dynamics, revealing age-related differences in how positive and negative outcomes drive behavioral updates. These findings contribute to understanding the developmental trajectory of cooperation and highlight adolescence as a period marked by heightened sensitivity to immediate rewards at the expense of long-term prosocial gains.

      Strengths:

      (1) Rigid model comparison and parameter recovery procedure.

      (2) Conceptually comprehensive model space.

      (3) Well-powered samples.

      We thank the reviewer for highlighting the strengths of our work.

      Weaknesses:

      (Q1) A key conceptual distinction between learning from non-human agents (e.g., bandit machines) and human partners is that the latter are typically assumed to possess stable behavioral dispositions or moral traits. When a non-human source abruptly shifts behavior (e.g., from 80% to 20% reward), learners may simply update their expectations. In contrast, a sudden behavioral shift by a previously cooperative human partner can prompt higher-order inferences about the partner's trustworthiness or the integrity of the experimental setup (e.g., whether the partner is truly interactive or human). The authors may consider whether their modeling framework captures such higher-order social inferences. Specifically, trait-based models-such as those explored in Hackel et al. (2015, Nature Neuroscience)-suggest that learners form enduring beliefs about others' moral dispositions, which then modulate trial-bytrial learning. A learner who believes their partner is inherently cooperative may update less in response to a surprising defection, effectively showing a trait-based dampening of learning rate.

      We thank the reviewer for this thoughtful comment. We agree that social learning from human partners may involve higher-order inferences beyond simple reinforcement learning from non-human sources. To address this, we had previously included such mechanisms in our behavioral modeling. In Model 7 (Social Reward Model with Influence), we tested a higher-order belief-updating process in which participants’ expectations about their partner’s cooperation were shaped not only by the partner’s previous choices but also by the inferred influence of their own past actions on the partner’s subsequent behavior. In other words, participants could adjust their belief about the partner’s cooperation by considering how their partner’s belief about them might change. Model comparison showed that Model 7 did not outperform the best-fitting model, suggesting that incorporating higher-order influence updates added limited explanatory value in this context. As suggested by the reviewer, we have further clarified this point in the revised manuscript.

      Regarding trait-based frameworks, we appreciate the reviewer’s reference to Hackel et al. (2015). That study elegantly demonstrated that learners form relatively stable beliefs about others’ social dispositions, such as generosity, especially when the task structure provides explicit cues for trait inference (e.g., resource allocations and giving proportions). By contrast, our study was not designed to isolate trait learning, but rather to capture how participants update their expectations about a partner’s cooperation over repeated interactions. In this sense, cooperativeness in our framework can be viewed as a trait-like latent belief that evolves as evidence accumulates. Thus, while our model does not include a dedicated trait module that directly modulates learning rates, the belief-updating component of our best-fitting model effectively tracks a dynamic, partner-specific cooperativeness, potentially reflecting a prosocial tendency.

      (Q2) This asymmetry in belief updating has been observed in prior work (e.g., Siegel et al., 2018, Nature Human Behaviour) and could be captured using a dynamic or belief-weighted learning rate. Models incorporating such mechanisms (e.g., dynamic learning rate models as in Jian Li et al., 2011, Nature Neuroscience) could better account for flexible adjustments in response to surprising behavior, particularly in the social domain.

      We thank the reviewer for the suggestion. Following the comment, we implemented an additional model incorporating a dynamic learning rate based on the magnitude of prediction errors. Specifically, we developed Model 9:  Social reward model with Pearce–Hall learning algorithm (dynamic learning rate), in which participants’ beliefs about their partner’s cooperation probability are updated using a Rescorla–Wagner rule with a learning rate dynamically modulated by the Pearce–Hall (PH) Error Learning mechanism. In this framework, the learning rate increases following surprising outcomes (larger prediction errors) and decreases as expectations become more stable (see Appendix Analysis section for details).

      The results showed that this dynamic learning rate model did not outperform our bestfitting model in either adolescents or adults (see Figure supplement 6). We greatly appreciate the reviewer’s suggestion, which has strengthened the scope of our analysis. We now have added these analyses to the Appendix Analysis section (also Figure Supplement 6) and expanded the Discussion to acknowledge this modeling extension and further discuss its implications.

      (Q3) Second, the developmental interpretation of the observed effects would be strengthened by considering possible non-linear relationships between age and model parameters. For instance, certain cognitive or affective traits relevant to social learning-such as sensitivity to reciprocity or reward updating-may follow non-monotonic trajectories, peaking in late adolescence or early adulthood. Fitting age as a continuous variable, possibly with quadratic or spline terms, may yield more nuanced developmental insights.

      We thank the reviewer for this professional comment. In addition to the linear analyses, we further conducted exploratory analyses to examine potential non-linear relationships between age and the model parameters. Specifically, we fit LMMs for each of the four parameters as outcomes (α+, α-, β, and ω). The fixed effects included age, a quadratic age term, and gender, and the random effects included subject-specific random intercepts and random slopes for age and gender. Model comparison using BIC did not indicate improvement for the quadratic models over the linear models for α<sup>+</sup> (ΔBIC<sub>quadratic-linear</sub> = 5.09), α<sup>-</sup>(ΔBIC<sub>quadratic-linear</sub> = 3.04), β (ΔBIC<sub>quadratic-linear</sub> = 3.9), or ω (ΔBIC<sub>quadratic-linear</sub>= 0). Moreover, the quadratic age term was not significant for α<sup>+</sup>, α<sup>−</sup>, or β (all ps > 0.10). For ω, we observed a significant linear age effect (b = 1.41, t = 2.65, p = 0.009) and a significant quadratic age effect (b = −0.03, t = −2.39, p = 0.018; see Author response image 1). This pattern is broadly consistent with the group effect reported in the main text. The shaded area in the figure represents the 95% confidence interval. As shown, the interval widens at older ages (≥ 26 years) due to fewer participants in that range, which limits the robustness of the inferred quadratic effect. In consideration of the limited precision at older ages and the lack of BIC improvement, we did not emphasize the quadratic effect in the revised manuscript and present these results here as exploratory.

      Author response image 1.

      Linear and quadratic model fits showing the relationship between age and the ω parameter, with 95% confidence intervals.

      (Q4) Finally, the two age groups compared - adolescents (high school students) and adults (university students) - differ not only in age but also in sociocultural and economic backgrounds. High school students are likely more homogenous in regional background (e.g., Beijing locals), while university students may be drawn from a broader geographic and socioeconomic pool. Additionally, differences in financial independence, family structure (e.g., single-child status), and social network complexity may systematically affect cooperative behavior and valuation of rewards. Although these factors are difficult to control fully, the authors should more explicitly address the extent to which their findings reflect biological development versus social and contextual influences.

      We appreciate this comment. Indeed, adolescents (high school students) and adults (university students) differ not only in age but also in sociocultural and socioeconomic backgrounds. In our study, all participants were recruited from Beijing and surrounding regions, which helps minimize large regional and cultural variability. Moreover, we accounted for individual-level random effects and included participants’ social value orientation (SVO) as an individual difference measure.

      Nonetheless, we acknowledge that other contextual factors, such as differences in financial independence, socioeconomic status, and social experience—may also contribute to group differences in cooperative behavior and reward valuation. Although our results are broadly consistent with developmental theories of reward sensitivity and social decisionmaking, sociocultural influences cannot be entirely ruled out. Future work with more demographically matched samples or with socioeconomic and regional variables explicitly controlled will help clarify the relative contributions of biological and contextual factors. Accordingly, we have revised the Discussion to include the following statement:

      “Third, although both age groups were recruited from Beijing and nearby regions, minimizing major regional and cultural variation, adolescents and adults may still differ in socioeconomic status, financial independence, and social experience. Such contextual differences could interact with developmental processes in shaping cooperative behavior and reward valuation. Future research with demographically matched samples or explicit measures of socioeconomic background will help disentangle biological from sociocultural influences.”

      Reviewer #3 (Public review):

      Summary:

      Wu and colleagues find that in a repeated Prisoner's Dilemma, adolescents, compared to adults, are less likely to increase their cooperation behavior in response to repeated cooperation from a simulated partner. In contrast, after repeated defection by the partner, both age groups show comparable behavior.

      To uncover the mechanisms underlying these patterns, the authors compare eight different models. They report that a social reward learning model, which includes separate learning rates for positive and negative prediction errors, best fits the behavior of both groups. Key parameters in this winning model vary with age: notably, the intrinsic value of cooperating is lower in adolescents. Adults and adolescents also differ in learning rates for positive and negative prediction errors, as well as in the inverse temperature parameter.

      Strengths:

      The modeling results are compelling in their ability to distinguish between learned expectations and the intrinsic value of cooperation. The authors skillfully compare relevant models to demonstrate which mechanisms drive cooperation behavior in the two age groups.

      We thank the reviewer’s recognition of our work’s strengths.

      Weaknesses:

      (Q1) Some of the claims made are not fully supported by the data:

      The central parameter reflecting preference for cooperation is positive in both groups. Thus, framing the results as self-interest versus other-interest may be misleading.

      We thank the reviewer for this insightful comment. In the social reward model, the cooperation preference parameter is positive by definition, as defection in the repeated rPDG always yields a +2 monetary advantage regardless of the partner’s action. This positive value represents the additional subjective reward assigned to mutual cooperation (e.g., reciprocity value) that counterbalances the monetary gain from defection. Although the estimated social reward parameter ω was positive, the effective advantage of cooperation is Δ=p×ω−2. Given participants’ inferred beliefs p, Δ was negative for most trials (p×ω<2), indicating that the social reward was insufficient to offset the +2 advantage of defection. Thus, both adolescents and adults valued cooperation positively, but adolescents’ smaller ω and weaker responsiveness to sustained partner cooperation suggest a stronger weighting on immediate monetary payoffs.

      In this light, our framing of adolescents as more self-interested derives from their behavioral pattern: even when they recognized sustained partner cooperation and held high expectations of partner cooperation, adolescents showed lower cooperative behavior and reciprocity rewards compared with adults. Whereas adults increased cooperation after two or three consecutive partner cooperations, this pattern was absent among adolescents. We therefore interpret their behavior as relatively more self-interested, reflecting reduced sensitivity to the social reward from mutual cooperation rather than a categorical shift from self-interest to other-interest, as elaborated in the Discussion.

      (Q2) It is unclear why the authors assume adolescents and adults have the same expectations about the partner's cooperation, yet simultaneously demonstrate age-related differences in learning about the partner. To support their claim mechanistically, simulations showing that differences in cooperation preference (i.e., the w parameter), rather than differences in learning, drive behavioral differences would be helpful.

      We thank the reviewer for raising this important point. In our model, both adolescents and adults updated their beliefs about partner cooperation using an asymmetric reinforcement learning (RL) rule. Although adolescents exhibited a higher positive and a lower negative learning rate than adults, the two groups did not differ significantly in their overall updating of partner cooperation probability (Fig. 4a-b). We then examined the social reward parameter ω, which was significantly smaller in adolescents and determined the intrinsic value of mutual cooperation (i.e., p×ω). This variable differed significantly between groups and closely matched the behavioral pattern.

      Following the reviewer’s suggestion, we conducted additional simulations varying one model parameter at a time while holding the others constant. The difference in mean cooperation probability between adults and adolescents served as the index (positive = higher cooperation in adults). As shown in the Author response image 2, decreases in ω most effectively reproduced the observed group difference (shaded area), indicating that age-related differences in cooperation are primarily driven by variation in the social reward parameter ω rather than by others.

      Author response image 2.

      Simulation results showing how variations in each model parameter affect the group difference in mean cooperation probability (Adults – Adolescents). Based on the bestfitting Model 8 and parameters estimated from all participants, each line represents one parameter (i.e., α+, α-, ω, β) systematically varied within the tested range (α±:0.1–0.9; ω, β:1–9) while other parameters were held constant. Positive values indicate higher cooperation in adults. Smaller ω values most strongly reproduced the observed group difference, suggesting that reduced social reward weighting primarily drives adolescents’ lower cooperation.

      (Q3) Two different schedules of 120 trials were used: one with stable partner behavior and one with behavior changing after 20 trials. While results for order effects are reported, the results for the stable vs. changing phases within each schedule are not. Since learning is influenced by reward structure, it is important to test whether key findings hold across both phases.

      We thank the reviewer for this thoughtful and professional comment. In our GLMM and LMM analyses, we focused on trial order rather than explicitly including the stable vs. changing phase factor, due to concerns about multicollinearity. In our design, phases occur in specific temporal segments, which introduces strong collinearity with trial order. In multi-round interactions, order effects also capture variance related to phase transitions.

      Nonetheless, to directly address this concern, we conducted additional robustness analyses by adding a phase variable (stable vs. changing) to GLMM1, LMM1, and LMM3 alongside the original covariates. Across these specifications, the key findings were replicated (see GLMM<sub>sup</sub>2 and LMM<sub>sup</sub>4–5; Tables 9-11), and the direction and significance of main effects remained unchanged, indicating that our conclusions are robust to phase differences.

      (Q4) The division of participants at the legal threshold of 18 years should be more explicitly justified. The age distribution appears continuous rather than clearly split. Providing rationale and including continuous analyses would clarify how groupings were determined.

      We thank the reviewer for this thoughtful comment. We divided participants at the legal threshold of 18 years for both conceptual and practical reasons grounded in prior literature and policy. In many countries and regions, 18 marks the age of legal majority and is widely used as the boundary between adolescence and adulthood in behavioral and clinical research. Empirically, prior studies indicate that psychosocial maturity and executive functions approach adult levels around this age, with key cognitive capacities stabilizing in late adolescence (Icenogle et al., 2019; Tervo-Clemmens et al., 2023). We have clarified this rationale in the Introduction section of the revised manuscript.

      “Based on legal criteria for majority and prior empirical work, we adopt 18 years as the boundary between adolescence and adulthood (Icenogle et al., 2019; Tervo-Clemmens et al., 2023).”

      We fully agree that the underlying age distribution is continuous rather than sharply divided. To address this, we conducted additional analyses treating age as a continuous predictor (see GLMM<sub>sup</sub>1 and LMM<sub>sup</sub>1–3; Tables S1-S4), which generally replicated the patterns observed with the categorical grouping. Nevertheless, given the limited age range of our sample, the generalizability of these findings to fine-grained developmental differences remains constrained. Therefore, our primary analyses continue to focus on the contrast between adolescents and adults, rather than attempting to model a full developmental trajectory.

      (Q5) Claims of null effects (e.g., in the abstract: "adults increased their intrinsic reward for reciprocating... a pattern absent in adolescents") should be supported with appropriate statistics, such as Bayesian regression.

      We thank the reviewer for highlighting the importance of rigor when interpreting potential null effects. To address this concern, we conducted Bayes factor analyses of the intrinsic reward for reciprocity and reported the corresponding BF10 for all relevant post hoc comparisons. This approach quantifies the relative evidence for the alternative versus the null hypothesis, thereby providing a more direct assessment of null effects. The analysis procedure is now described in the Methods and Materials section:

      “Post hoc comparisons were conducted using Bayes factor analyses with MATLAB’s bayesFactor Toolbox (version v3.0, Krekelberg, 2024), with a Cauchy prior scale σ = 0.707.”

      (Q6) Once claims are more closely aligned with the data, the study will offer a valuable contribution to the field, given its use of relevant models and a well-established paradigm.

      We are grateful for the reviewer’s generous appraisal and insightful comments.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) I commend the authors on a well-structured, clear, and interesting piece of work. I have several questions and recommendations that, if addressed, I believe will strengthen the manuscript.

      We thank the reviewer for commending the organization of our paper.

      (2) Introduction: - Why use a zero-sum (Prisoner's Dilemma; PD) versus a mixed-motive game (e.g. Trust Task) to study cooperation? In a finite set of rounds, the dominant strategy can be to defect in a PD.

      We thank the reviewer for this helpful comment. We agree that both the rationale for using the repeated Prisoner’s Dilemma (rPDG) and the limitations of this framework should be clarified. We chose the rPDG to isolate the core motivational conflict between selfinterest and joint welfare, as its symmetric and simultaneous structure avoids the sequential trust and reputation dependencies/accumulation inherent to asymmetric tasks such as the Trust Game (King-Casas et al., 2005; Rilling et al., 2002).

      Although a finitely repeated rPDG theoretically favors defection, extensive prior research shows that cooperation can still emerge in long repeated interactions when players rely on learning and reciprocity rather than backward induction (Rilling et al., 2002; Fareri et al., 2015). Our design employed 120 consecutive rounds, allowing participants to update expectations about partner behavior and to establish stable reciprocity patterns over time. We have added the following clarification to the Introduction:

      “The rPDG provides a symmetric and simultaneous framework that isolates the motivational conflict between self-interest and joint welfare, avoiding the sequential trust and reputation dynamics characteristic of asymmetric tasks such as the Trust Game (Rilling et al., 2002; King-Casas et al., 2005)”

      (3) Methods:

      Did the participants know how long the PD would go on for?

      Were the participants informed that the partner was real/simulated?

      Were the participants informed that the partner was going to be the same for all rounds?

      We thank the reviewer for the meticulous review work, which helped us present the experimental design and reporting details more clearly. the following clarifications: I. Participants were not informed of the total number of rounds in the rPDG. This prevented endgame expectations and avoided distraction from counting rounds, which could introduce additional effects. II. Participants were told that their partner was another human participant in the laboratory. However, the partner’s behavior was predetermined by a computer program. This design enabled tighter experimental control and ensured consistent conditions across age groups, supporting valid comparisons. III. Participants were informed that they would interact with the same partner across all rounds, aligning with the essence of a multiround interaction paradigm and stabilizing partner-related expectations. For transparency, we have clarified these points in the Methods and Materials section:

      “Participants were told that their partner was another human participant in the laboratory and that they would interact with the same partner across all rounds. However, in reality, the actions of the partner were predetermined by a computer program. This setup allowed for a clear comparison of the behavioral responses between adolescents and adults. Participants were not informed of the total number of rounds in the rPDG.”

      (4) The authors mention that an SVO was also recorded to indicate participant prosociality. Where are the results of this? Did this track game play at all? Could cooperativeness be explained broadly as an SVO preference that penetrated into game-play behaviour?

      We thank the reviewer for pointing this out. We agree that individual differences in prosociality may shape cooperative behavior, so we conducted additional analyses incorporating SVO. Specifically, we extended GLMM1 and LMM3 by adding the measured SVO as a fixed effect with random slopes, yielding GLMM<sub>sup</sub>3 and LMM<sub>sup</sub>6 (Tables 12–13). The results showed that higher SVO was associated with greater cooperation, whereas its effect on the reward for reciprocity was not significant. Importantly, the primary findings remained unchanged after controlling for SVO. These results indicate that cooperativeness in our task cannot be explained solely by a broad SVO preference, although a more prosocial orientation was associated with greater cooperation. We have reported these analyses and results in the Appendix Analysis section.

      (5) Why was AIC chosen rather an BIC to compare model dominance?

      Sorry for the lack of clarification. Both the Akaike Information Criterion (AIC, Akaike, 1974) and Bayesian Information Criterion (BIC, Schwarz, 1978) are informationtheoretic criterions for model comparison, neither of which depends on whether the models to be compared are nested to each other or not (Burnham et al., 2002). We have added the following clarification into the Methods.

      “We chose to use the AICc as the metric of goodness-of-fit for model comparison for the following statistical reasons. First, BIC is derived based on the assumption that the “true model” must be one of the models in the limited model set one compares (Burnham et al., 2002; Gelman & Shalizi, 2013), which is unrealistic in our case. In contrast, AIC does not rely on this unrealistic “true model” assumption and instead selects out the model that has the highest predictive power in the model set (Gelman et al., 2014). Second, AIC is also more robust than BIC for finite sample size (Vrieze, 2012).”

      (6) I believe the model fitting procedure might benefit from hierarchical estimation, rather than maximum likelihood methods. Adolescents in particular seem to show multiple outliers in a^+ and w^+ at the lower end of the distributions in Figure S2. There are several packages to allow hierarchical estimation and model comparison in MATLAB (which I believe is the language used for this analysis;

      see https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1007043).

      We thank the reviewer for this helpful comment and for referring us to relevant methodological work (Piray et al., 2019). We have addressed this point by incorporating hierarchical Bayesian estimation, which effectively mitigates outlier effects and improves model identifiability. The results replicated those obtained with MLE fitting and further revealed group-level differences in key parameters. Please see our detailed response to Reviewer#1 Q1 for the full description of this analysis and results.

      (7) Results: Model confusion seems to show that the inequality aversion and social reward models were consistently confused with the baseline model. Is this explained or investigated? I could not find an explanation for this.

      The apparent overlap between the inequality aversion (Model 4) and social reward (Model 5) models in the recovery analysis likely arises because neither model includes a learning mechanism, making them unable to capture trial-by-trial adjustments in this dynamic task. Consequently, both were best fit by the baseline model. Please see Response to Reviewer #1 Q3 for related discussion.

      (8) Figures 3e and 3f show the correlation between asymmetric learning rates and age. It seems that both a^+ and a^- are around 0.35-0.40 for young adolescents, and this becomes more polarised with age. Could it be that with age comes an increasing discernment of positive and negative outcomes on beliefs, and younger ages compress both positive and negative values together? Given the higher stochasticity in younger ages (\beta), it may also be that these values simply represent higher uncertainty over how to act in any given situation within a social context (assuming the differences in groups are true).

      We appreciate this insightful interpretation. Indeed, both α+ and α- cluster around 0.35–0.40 in younger adolescents and become increasingly polarized with age, suggesting that sensitivity to positive versus negative feedback is less differentiated early in development and becomes more distinct over time. This interpretation remains tentative and warrants further validation. Based on this comment, we have revised the Discussion to include this developmental interpretation.

      We also clarify that in our model β denotes the inverse temperature parameter; higher β reflects greater choice precision and value sensitivity, not higher stochasticity. Accordingly, adolescents showed higher β values, indicating more value-based and less exploratory choices, whereas adults displayed relatively greater exploratory cooperation. These group differences were also replicated using hierarchical Bayesian estimation (see Response to Reviewer #1 Q1). In response to this comment, we have added a statement in the Discussion highlighting this developmental interpretation.

      “Together, these findings suggest that the differentiation between positive and negative learning rates changes with age, reflecting more selective feedback sensitivity in development, while higher β values in adolescents indicate greater value sensitivity. This interpretation remains tentative and requires further validation in future research.”

      (9) A parameter partial correlation matrix (off-diagonal) would be helpful to understand the relationship between parameters in both adolescents and adults separately. This may provide a good overview of how the model properties may change with age (e.g. a^+'s relation to \beta).

      We thank the reviewer for this helpful comment. We fully agree that a parameter partial correlation matrix can further elucidate the relationships among parameters. Accordingly, we conducted a partial correlation analysis and added the visually presented results to the revised manuscript as Figure 2-figure supplement 4.

      (10) It would be helpful to have Bayes Factors reported with each statistical tests given that several p-values fall within the 0.01 and 0.10.

      We thank the reviewer for this important recommendation. We have conducted Bayes factor analyses and reported BF10 for all relevant post hoc comparisons. We also clarified our analysis in the Methods and Materials section:

      “Post hoc comparisons were conducted using Bayes factor analyses with MATLAB’s bayesFactor Toolbox (version v3.0, Krekelberg, 2024), with a Cauchy prior scale σ = 0.707.”

      (11) Discussion: I believe the language around ruling out failures in mentalising needs to be toned down. RL models do not enable formal representational differences required to assess mentalising, but they can distinguish biases in value learning, which in itself is interesting. If the authors were to show that more complex 'ToM-like' Bayesian models were beaten by RL models across the board, and this did not differ across adults and adolescents, there would be a stronger case to make this claim. I think the authors either need to include Bayesian models in their comparison, or tone down their language on this point, and/or suggest ways in which this point might be more thoroughly investigated (e.g., using structured models on the same task and running comparisons: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087619).

      We thank the reviewer for the comments. Please see our response to Reviewer 1 (Appraisal & Discussion section) for details.

      Reviewer #2 (Recommendations for the authors):

      (1) The authors may want to show the winning model earlier (perhaps near the beginning of the Results section, when model parameters are first mentioned).

      We thank the reviewer for this suggestion. We agree that highlighting the winning model early improves clarity. Currently, we have mentioned the winning model before the beginning of the Results section. Specifically, in the penultimate paragraph of the Introduction we state:

      “We identified the asymmetric RL learning model as the winning model that best explained the cooperative decisions of both adolescents and adults.”

      Reviewer #3 (Recommendations for the authors):

      (1) In addition to the points mentioned above, I suggest the following:

      Clarify plots by clearly explaining each variable. In particular, the indices 1 vs. 1,2 vs 1,2,3 were not immediately understandable.

      We thank the reviewer for this suggestion. We agree that the indices were not immediately clear. We have revised the figure captions (Figure 1 and 4) to explicitly define these terms more clearly:

      “The x-axis represents the consistency of the partner’s actions in previous trials (t<sub>−1</sub>: last trial; t<sub>−1,2</sub>: last two trials;<sub>t−1,2,3</sub>: last three trials).”

      (2) It's unclear why the index stops at 3. If this isn't the maximum possible number of consecutive cooperation trials, please consider including all relevant data, as adolescents might show a trend similar to adults over more trials.

      We thank the reviewer for raising this point. In our exploratory analyses, we also examined longer streaks of consecutive partner cooperation or defection (up to four or five trials). Two empirical considerations led us to set the cutoff at three in the final analyses. First, the influence of partner behavior diminished sharply with temporal distance. In both GLMMs and LMMs, coefficients for earlier partner choices were small and unstable, and their inclusion substantially increased model complexity and multicollinearity. This recency pattern is consistent with learning and decision models emphasizing stronger weighting of recent evidence (Fudenberg & Levine, 2014; Fudenberg & Peysakhovich, 2016). Second, streaks longer than three were rare, especially among some participants, leading to data sparsity and inflated uncertainty. Including these sparse conditions risked biasing group estimates rather than clarifying them. Balancing informativeness and stability, we therefore restricted the index to three consecutive partner choices in the main analyses, which we believe sufficiently capture individuals’ general tendencies in reciprocal cooperation.

      (3) The term "reciprocity" may not be necessary. Since it appears to reflect a general preference for cooperation, it may be clearer to refer to the specific behavior or parameter being measured. This would also avoid confusion, especially since adolescents do show negative reciprocity in response to repeated defection.

      We thank you for this comment. In our work, we compute the intrinsic reward for reciprocity as p × ω, where p is the partner cooperation expectation and ω is the cooperation preference. In the rPDG, this value framework manifests as a reciprocity-derived reward: sustained mutual cooperation maximizes joint benefits, and the resulting choice pattern reflects a value for reciprocity, contingent on the expected cooperation of the partner. This quantity enters the trade-off between U<sub>cooperation</sub> and U<sub>defection</sub> and captures the participant’s intrinsic reward for reciprocity versus the additional monetary reward payoff of defection. Therefore, we consider the term “reciprocity” an acceptable statement for this construct.

      (4) Interpretation of parameters should closely reflect what they specifically measure.

      We thank the reviewer for pointing this out. We have refined the relevant interpretations of parameters in the current Results and Discussion sections.

      (5) Prior research has shown links between Theory of Mind (ToM) and cooperation (e.g., Martínez-Velázquez et al., 2024). It would be valuable to test whether this also holds in your dataset.

      We thank the reviewer for this thoughtful comment. Although we did not directly measure participants’ ToM, our design allowed us to estimate participants’ trial-by-trial inferences (i.e., expectations) about their partner’s cooperation probability. We therefore treat these cooperation expectations as an indirect representation for belief inference, which is related to ToM processes. To test whether this belief-inference component relates to cooperation in our dataset, we further conducted an exploratory analysis (GLMM<sub>sup</sub>4) in which participants’ choices were regressed on their cooperation expectations, group, and the group × cooperation-expectation interaction, controlling for trial number and gender, with random effects. Consistent with the ToM–cooperation link in prior research (MartínezVelázquez et al., 2024), participants’ expectations about their partner’s cooperation significantly predicted their cooperative behavior (Table 14), suggesting that decisions were shaped by social learning about others’ inferred actions. Moreover, the interaction between group and cooperation expectation was not significant, indicating that this inference-driven social learning process likely operates similarly in adolescents and adults. This aligns with our primary modeling results showing that both age groups update beliefs via an asymmetric learning process. We have reported these analyses in the Appendix Analysis section.

      (6) More informative table captions would help the reader. Please clarify how variables are coded (e.g., is female = 0 or 1? Is adolescent = 0 or 1?), to avoid the need to search across the manuscript for this information.

      We thank the reviewer for raising this point. We have added clear and standardized variable coding in the table notes of all tables to make them more informative and avoid the need to search the paper. We have ensured consistent wording and formatting across all tables.

      (7) I hope these comments are helpful and support the authors in further strengthening their manuscript.

      We thank the three reviewers for their comments, which have been helpful in strengthening this work.

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      (9) Rilling, J. K., Gutman, D. A., Zeh, T. R., Pagnoni, G., Berns, G. S., & Kilts, C. D. (2002).A neural basis for social cooperation. Neuron, 35(2), 395-405. https://doi.org/10.1016/s0896-6273(02)00755-9

      (10) Fareri, D. S., Chang, L. J., & Delgado, M. R. (2015). Computational substrates of social value in interpersonal collaboration. Journal of Neuroscience, 35(21), 8170-8180. https://doi.org/10.1523/JNEUROSCI.4775-14.2015

      (11) Akaike, H. (2003). A new look at the statistical model identification. IEEE transactions on automatic control, 19(6), 716-723. https://doi.org/10.1109/TAC.1974.1100705

      (12) Schwarz, G. (1978). Estimating the dimension of a model. The annals of statistics, 461464. https://doi.org/10.1214/aos/1176344136

      (13) Burnham, K. P., & Anderson, D. R. (2002). Model selection and multimodel inference: A practical information-theoretic approach (2nd ed.). Springer.https://doi.org/10.1007/b97636

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      (16) Vrieze, S. I. (2012). Model selection and psychological theory: A discussion of the differences between the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC). Psychological Methods, 17(2), 228–243. https://doi.org/10.1037/a0027127

    1. Author response:

      The following is the authors’ response to the original reviews.

      Public Reviews:

      Reviewer #1 (Public review):

      This work by Reitz, Z. L. et al. developed an automated tool for high-throughput identification of microbial metallophore biosynthetic gene clusters (BGCs) by integrating knowledge of chelating moiety diversity and transporter gene families. The study aimed to create a comprehensive detection system combining chelator-based and transporter-based identification strategies, validate the tool through large-scale genomic mining, and investigate the evolutionary history of metallophore biosynthesis across bacteria.

      Major strengths include providing the first automated, high-throughput tool for metallophore BGC identification, representing a significant advancement over manual curation approaches. The ensemble strategy effectively combines complementary detection methods, and experimental validation using HPLC-HRMS strengthens confidence in computational predictions. The work pioneers a global analysis of metallophore diversity across the bacterial kingdom and provides a valuable dataset for future computational modeling.

      Some limitations merit consideration. First, ground truth datasets derived from manual curation may introduce selection bias toward well-characterized systems, potentially affecting performance assessment accuracy. Second, the model's dependence on known chelating moieties and transporter families constrains its ability to detect novel metallophore architectures, limiting discovery potential in metagenomic datasets. Third, while the proposed evolutionary hypothesis is internally consistent, it lacks direct validation and remains speculative without additional phylogenetic studies.

      The authors successfully achieved their stated objectives. The tool demonstrates robust performance metrics and practical utility through large-scale application to representative genomes. Results strongly support their conclusions through rigorous validation, including experimental confirmation of predicted metallophores via HPLC-HRMS analysis.

      The work provides a significant and immediate impact by enabling the transition from labor-intensive manual approaches to automated screening. The comprehensive phylogenetic framework advances understanding of bacterial metal acquisition evolution, informing future studies on microbial metal homeostasis. Community utility is substantial, since the tool and accompanying dataset create essential resources for comparative genomics, algorithm development, and targeted experimental validation of novel metallophores.

      We thank the reviewer for their valuable feedback. We appreciate the positive words, and agree with their listed limitations. Regarding the following comment:

      “Third, while the proposed evolutionary hypothesis is internally consistent, it lacks direct validation and remains speculative without additional phylogenetic studies.”

      We agree that additional phylogenetic analyses are needed in future studies. For the revised manuscript, we have validated our evolutionary hypotheses by additionally analyzing two gene families using the likelihood-based tool AleRax, which implements a probabilistic DTL model. The results were consistent with the eMPRess parsimony-based reconstructions, showing comparable patterns of rare duplication, moderate gene loss, and extensive horizontal transfer. Both methods identified similar lineages as the most probable origin and major recipients of transfer events. This agreement between independent reconciliation frameworks supports the reliability of our evolutionary conclusions. We have added a statement referencing this cross-method validation in the revised manuscript.

      Reviewer #2 (Public review):

      Summary:

      This study presents a systematic and well-executed effort to identify and classify bacterial NRP metallophores. The authors curate key chelator biosynthetic genes from previously characterized NRP-metallophore biosynthetic gene clusters (BGCs) and translate these features into an HMM-based detection module integrated within the antiSMASH platform.

      The new algorithm is compared with a transporter-based siderophore prediction approach, demonstrating improved precision and recall. The authors further apply the algorithm to large-scale bacterial genome mining and, through reconciliation of chelator biosynthetic gene trees with the GTDB species tree using eMPRess, infer that several chelating groups may have originated prior to the Great Oxidation Event.

      Overall, this work provides a valuable computational framework that will greatly assist future in silico screening and preliminary identification of metallophore-related BGCs across bacterial taxa.

      Strengths:

      (1) The study provides a comprehensive curation of chelator biosynthetic genes involved in NRP-metallophore biosynthesis and translates this knowledge into an HMM-based detection algorithm, which will be highly useful for the initial screening and annotation of metallophore-related BGCs within antiSMASH.

      (2) The genome-wide survey across a large bacterial dataset offers an informative and quantitative overview of the taxonomic distribution of NRP-metallophore biosynthetic chelator groups, thereby expanding our understanding of their phylogenetic prevalence.

      (3) The comparative evolutionary analysis, linking chelator biosynthetic genes to bacterial phylogeny, provides an interesting and valuable perspective on the potential origin and diversification of NRP-metallophore chelating groups.

      We greatly appreciate these comments.

      Weaknesses:

      (1) Although the rule-based HMM detection performs well in identifying major categories of NRP-metallophore biosynthetic modules, it currently lacks the resolution to discriminate between fine-scale structural or biochemical variations among different metallophore types.

      We agree that this is a current limitation to the methodology. More specific metallophore structural prediction is among our future goals for antiSMASH. We have added a statement to this effect in the conclusion.

      (2) While the comparison with the transporter-based siderophore prediction approach is convincing overall, more information about the dataset balance and composition would be appreciated. In particular, specifying the BGC identities, source organisms, and Gram-positive versus Gram-negative classification would improve transparency. In the supplementary tables, the "Just TonB" section seems to include only BGCs from Gram-negative bacteria - if so, this should be clearly stated, as Gram type strongly influences siderophore transport systems.

      The reviewer raises good points here. An additional ZIP file containing all BGCs used for the manual curation was inadvertently left out of the supplemental dataset for the first version of the manuscript. We have added columns with source organisms and Gram stain (retrieved from Bacdive) to Table S2. F1 scores were similar for Gram positive and negative subsets, as seen in the new Table S2.

      We thank the reviewer for suggesting this additional analysis, and have added a brief statement in the revised manuscript.

      The “Just TonB” section (in which we tested the performance of requiring TonB without another transporter) was not used for the manuscript. We will preserve it in the revised Table S2 for transparency.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) In line 43:

      "excreted" should be replace by "secreted".

      Done.

      (2) In lines 158-159:

      "we manually predicted metallophore production among a large set of BGCs."

      If they are first "annotated with default antiSMASH v6.1", then it is not entirely manual, right? I would suggest making this sentence clearer.

      We have revised the language.

      (3) In lines 165-169:

      It would be good to show the confusion matrix of these results.

      The confusion matrices are found in Table S2, columns AL-AR.

      (4) In Table 1:

      Method names (AntiSMASH rules/Transporter genes) could be misleading, since they are all AntiSMASH-based, right?

      We have adjusted the methods to clarify that while the transporter genes were detected using a modified version of antiSMASH, they are not related to our chelator-based detection rule (which is now correctly singular throughout the text).

      (5) Line 198:

      There are accidental spaces and characters inserted here.

      We could not find any accidental spaces and characters here.

      (6) Line 209:

      "In total, 3,264 NRP metallophore BGC regions were detected"

      Is this number correct? I don't see a correspondence in Table 1.

      We have added the following sentence to the Table 1 legend: “An additional 54 BGC regions were detected as NRP metallophores without meeting the requirements for the antiSMASH NRPS rule.”

      (7) Line 294:

      "From B. brennerae, we identified four catecholic compounds"

      From the bacterial cells or the culture supernatant? I think it is important to state this in a more precise way. If it is from the supernatant, it could be from EVs.

      We state in line 292 that “organic compounds were extracted from the culture supernatants”. As our goal was only to confirm the ability of the strains to produce the predicted metallophores, the precise localization (including cell pellet or EVs) was not explored.

      (8) Lines 349-357:

      These results would benefit greatly from a visualization strategy.

      Thank you, we have added a reference to the existing visualization in Fig. 5, Ring C.

      (9) Lines 452-454:

      How could clusters be de-replicated? Is there an identity equivalence scheme or similarity metric?

      The BGC regions were de-replicated with BiG-SCAPE, which uses multiple similarity metrics as described in Navarro-Muñoz et al, 2020. Clusters could be dereplicated further using a more strict cutoff.

      (10) Line 457:

      "relatively low number of published genomes."

      Could metagenome-assembled genomes help in that matter?

      This is a good question, but we find that MAGs are usually too fragmented to yield complete NRPS BGC regions. We’ve added additional sentences earlier in the discussion: “Detection rates were also lower for fragmented genomes; unfortunately, this limitation (inherent to antiSMASH itself) may hinder the identification of metallophore biosynthesis in metagenomes. As long-read sequencing of metagenomes becomes more common, we expect that detection will improve.”

      (11) Lines 514-515:

      "Adequately-performing pHMMs for Asp and His β-hydroxylase subtypes could not be constructed using the above method."

      What is the overall impact of this discrepancy in the methodology for these specific groups?

      The phylogeny-based methodology was used to reduce false positives. We expect this method will have improved precision at the possible expense of recall.

      (12) Lines 543-545:

      "RefSeq representative bacterial genomes were dereplicated at the genus level using R, randomly selecting one genome for each of the 330 genera determined by GTDB"

      Isn't it more of a random sampling than a dereplication? Dereplication would involve methods such as ANI computation.

      You are correct; we have adjusted the language to clarify.

      (13) Lines 559-560: "were filtered to remove clusters on contig edges."

      This sentence is confusing because networks will be mentioned soon, and they also have edges (not the edges mentioned here), and they could also be clustered (not the clusters mentioned here). Is there a way to make the terminology clearer?

      Thank you, we have adjusted the text to read “BGC regions on contig boundaries”

      (14) Line 560:

      "The resulting 2,523 BGC regions, as well as 78 previously reported BGCs "

      How many were there before filtering?

      We have added the number: 3,264

      (15) Lines 579-580:

      Confusing terminology, as mentioned in Lines 559-560.

      Adjusted as above.

      General comments and questions:

      An objective suggestion to enrich the discussion is to address the role of bacterial extracellular vesicles (EVs) as metallophore carriers. Studies show that EVs, such as outer membrane vesicles, can transport siderophores or other metallophores for iron acquisition in various bacteria, functioning as "public goods" for community-wide nutrient sharing. Highlighting this mechanism would add ecological and functional context to the manuscript. In the future, EV-associated metallophore transport could also be considered for integration into computational detection tools.

      We thank the reviewer for the suggestion; however, we do not think that such a discussion is needed. We briefly discuss the ecological function of metallophores as public goods (and public bads) in the first paragraph of the introduction. We did not find any reports that EV-associated genes co-localize with metallophore BGCs, which would be required for their presence to be a useful marker of metallophore production.

      Is there a feasible path to more generalizable detection of chelating motifs using chemistry-aware features? For example, a machine learning classifier trained on submolecular descriptors (e.g., functional groups, coordination motifs, SMARTS patterns, graph fingerprints, metal-binding propensity scores) could complement the current genome-based approach and broaden coverage beyond known metallophore families. While the discussion mentions future extensions centered on genomic features, integrating chemical information from predicted or known products (or biosynthetic logic inferred from BGC composition) could be explored. A hybrid framework-linking BGC-derived features with chemistry-derived features-may improve both recall for novel metallophore classes and precision in distinguishing true chelators from confounders, thereby increasing overall accuracy.

      We can envision a classifier that uses submolecular descriptors to predict the ability of a molecule to bind metal ions. However, starting with a BGC and accurately predicting the structure of a hitherto unknown chelating moiety will likely prove difficult.  We have added a sentence to the discussion stating that a future tool could use accessory genes to more completely predict chemical structure.

      Although the initial analysis was conducted using RefSeq genomes, what are the anticipated challenges and limitations when scaling this method for BGC prospecting in metagenome-assembled genomes (MAGs), particularly considering the inherent quality differences, assembly fragmentation, and taxonomic uncertainties that characterize MAG datasets compared to curated reference genomes?

      Please see our response to comment 10, line 457. Our pHMM-based approach is designed to be robust to organism taxonomy; however, fragmentation is a significant barrier to accurate antiSMASH-based BGC detection (including in contig-level single-isolate genomes, see Table 1).

      Reviewer #2 (Recommendations for the authors):

      (1) In the "Chemical identification of genome-predicted siderophores across taxa" section, it would be helpful to annotate the cross-species similarities between predicted metallophore BGCs and their reference clusters (Ref BGCs). As currently described, the main text seems to highlight the cross-species resolving power of BiG-SCAPE itself rather than demonstrating the taxonomic generalizability of the chelator HMM-based detection module.

      Thank you for this comment. We intended to display that the new rule is useful for detecting BGCs in unexplored taxa, but we acknowledge that there is not a great diversity in the strains we selected. We have removed “across taxa” to avoid misleading the reader and clarify our intent.

      (2) In addition to using eMPRess for gene-species reconciliation, it may be beneficial to explore or at least reference alternative reconciliation tools to validate the inferred duplication, transfer, and loss (DTL) scenarios. Incorporating such cross-method comparisons would enhance the robustness and credibility of the evolutionary conclusions.

      We appreciate this valuable suggestion. To validate the robustness of our reconciliation-based inferences, we additionally analyzed two gene families using the likelihood-based tool AleRax, which implements a probabilistic DTL model. The results were consistent with the eMPRess parsimony-based reconstructions, showing comparable patterns of rare duplication, moderate gene loss, and extensive horizontal transfer. Both methods identified similar lineages as the most probable origin and major recipients of transfer events. This agreement between independent reconciliation frameworks supports the reliability of our evolutionary conclusions. We have added a brief statement referencing this cross-method validation in the revised manuscript.

    1. Manifeste de l'hostilité misogyne : Analyse de la haine systémique contre les femmes dans l'espace public

      Résumé Exécutif

      Le présent document synthétise les mécanismes, les acteurs et les conséquences de la recrudescence de la violence misogyne, telle qu'analysée dans le contexte européen et international.

      Il apparaît que les femmes occupant des postes à responsabilité ou s'exprimant avec conviction dans la sphère publique — politiciennes, journalistes, activistes — sont les cibles privilégiées d'une haine organisée.

      Cette hostilité se manifeste par un cyber-harcèlement d'une violence extrême, allant de l'insulte sexiste aux menaces de viol et de mort.

      L'analyse révèle que cette haine n'est pas fortuite : elle est instrumentalisée par des mouvements populistes et d'extrême droite, et théorisée par des groupes masculinistes qui perçoivent l'égalité des genres comme une menace identitaire et sociale.

      Le monde numérique, régi par des algorithmes privilégiant l'engagement (souvent généré par la haine) et protégé par un certain vide juridique, sert d'incubateur à une radicalisation pouvant mener au terrorisme (mouvement « Incel »).

      Face à l'insuffisance des réponses institutionnelles, des initiatives de soutien émergent, tandis que des voix s'élèvent pour appeler à une déconstruction systémique des stéréotypes de masculinité.

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      1. Une Violence Ciblée et Systémique

      La haine envers les femmes n'est pas le fait d'individus isolés, mais un phénomène structurel visant à exclure les femmes de la sphère publique.

      Les cibles privilégiées

      Le mépris et l'agressivité visent particulièrement les femmes qui manifestent de l'assurance et occupent des domaines traditionnellement masculins :

      Femmes politiques : En Allemagne (34 % de députées au Bundestag) comme en France (40 % à l'Assemblée nationale), elles subissent des interruptions systématiques et une remise en question de leur légitimité.

      Journalistes sportives : L'exemple de Claudia Neumann illustre l'hostilité rencontrée lorsqu'une femme investit le "pré carré" masculin du football.

      Activistes et Écrivaines : Des figures comme Alice Barbe ou Leila Slimani subissent des campagnes de dénigrement massives en raison de leurs engagements pour les réfugiés ou les droits des femmes.

      La nature des agressions

      Contrairement aux hommes, les femmes reçoivent des attaques "taillées sur mesure" liées à leur genre :

      Sexualisation et humiliation : Envoi de "dickpics", insultes à caractère sexuel, commentaires sur le physique ou l'âge.

      Menaces extrêmes : Descriptions détaillées de viols, menaces de mutilations génitales ou de mort.

      Invisibilisation : Au sein même des institutions, la parole des femmes est souvent accueillie par un brouhaha ou un désintérêt feint de la part des collègues masculins.

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      2. Les Racines Idéologiques et l'Instrumentalisation Politique

      La misogynie moderne s'appuie sur des discours structurés et une exploitation politique délibérée.

      Le mouvement masculiniste

      Les masculinistes considèrent que les femmes ont pris le dessus et opprimeraient désormais les hommes.

      Rhétorique de la crise : Ils présentent l'égalité comme une "maladie" ou une "injustice" envers les jeunes hommes.

      Défense du patriarcat : Des auteurs comme Julien Rochedy prônent un retour aux codes virils traditionnels, affirmant que le manque de patriarcat est la cause du féminisme actuel.

      L'extrême droite et le populisme

      Des partis comme l'AfD en Allemagne ou des figures comme Éric Zemmour en France utilisent le sexisme comme stratégie électorale :

      Provocation délibérée : Utilisation de termes sexistes pour susciter l'émotion et gagner en visibilité médiatique.

      Lien avec d'autres haines : La misogynie s'accompagne souvent d'idéologies racistes et antisémites. Le féminisme est présenté comme un facteur de "décadence civilisationnelle".

      Déstabilisation : Au Parlement, l'entrée de partis d'extrême droite a entraîné une augmentation nette des exclamations et comportements sexistes.

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      3. L'Espace Numérique : Un "Lieu de Terreur"

      Internet et les réseaux sociaux agissent comme des catalyseurs de haine en raison de leur fonctionnement intrinsèque.

      La mécanique du cyber-harcèlement

      L'anonymat : Il favorise la levée des inhibitions. Les agresseurs peuvent être des "monsieur tout le monde" (professeurs, ingénieurs, avocats).

      Les algorithmes : La haine et l'incitation à la violence génèrent plus de vues et de clics, ce qui est financièrement profitable pour les plateformes.

      La radicalisation en ligne : Des forums privés permettent à des hommes de s'enfermer dans une vision étriquée du monde, se confortant mutuellement dans leur détestation des femmes.

      La menace "Incel" (Célibataires involontaires)

      Cette sous-culture numérique représente un danger sécuritaire réel :

      Idéologie : Conviction que les femmes sont des êtres inférieurs et que l'accès à leur corps est un droit fondamental.

      Apologie de la violence : Célébration de terroristes comme Elliot Rodger ou Alec Minassian.

      Passage à l'acte : Plusieurs attentats (Santa Barbara, Toronto, Halle) ont été motivés, totalement ou partiellement, par cette haine des femmes.

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      4. Conséquences Sociales et Réponses Institutionnelles

      L'impact de cette violence dépasse les victimes individuelles pour menacer la démocratie elle-même.

      L'impact sur les victimes et la démocratie

      Charge mentale et traumatisme : Les victimes doivent mettre en place des protocoles de sécurité lourds et subissent un stress psychologique intense.

      Le recul de la parole : Une femme sur deux craint d'exprimer son opinion en ligne. Ce retrait forcé crée un déséquilibre dans le débat public, laissant le champ libre aux harceleurs.

      Le sentiment d'isolement : Les militantes se sentent souvent seules face à des réseaux d'agresseurs très bien organisés.

      Un cadre juridique et policier défaillant

      Sous-estimation des faits : Les plaintes sont souvent classées sans suite ou banalisées par les forces de l'ordre ("supprimez votre compte").

      Complexité législative : Les plateformes, souvent basées à l'étranger, collaborent peu avec la justice. En France, des tentatives de régulation ont été déclarées inconstitutionnelles au nom de la liberté d'expression.

      Initiatives de soutien : Des structures comme HateAid en Allemagne offrent désormais un accompagnement juridique et psychologique pour pallier les carences de l'État.

      --------------------------------------------------------------------------------

      5. Perspectives de Lutte et Déconstruction

      Pour enrayer ce phénomène, l'analyse suggère des actions à la racine du problème.

      | Axe d'intervention | Actions préconisées | | --- | --- | | Éducation | Déconstruire les stéréotypes de genre dès le plus jeune âge et remettre en question la "masculinité toxique". | | Engagement masculin | Impliquer les hommes dans le discours féministe. Comme le souligne Leila Slimani, les hommes ont tout à gagner à un monde moins violent et moins régi par l'obligation de virilité brute. | | Justice | Former la police et le parquet à reconnaître la misogynie comme un délit grave. Créer une jurisprudence forte, à l'instar du procès remporté par Alice Barbe. | | Régulation | Imposer des règles communes au niveau européen pour contraindre les plateformes à transmettre les données des auteurs de messages haineux. |

      Conclusion : La lutte contre la misogynie organisée est un enjeu de sécurité publique et de survie démocratique.

      Refuser de céder à la peur et briser le silence sont les premiers remparts contre cette tentative de "silencier" les femmes dans l'espace public.

    1. Masculinité en crise : la fin d’Homo Virilus ?

      Ce document de synthèse analyse les fondements, l'évolution historique et la remise en question contemporaine de l'archétype de la virilité, désigné sous le terme d'« Homo Virilus ».

      À travers un prisme historique, sociologique et personnel, il explore comment ce modèle de domination, longtemps hégémonique, fait face à une crise profonde et à une nécessité de redéfinition.

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      Résumé exécutif

      L’idéal de l'« Homo Virilus » repose sur un triptyque de puissance, de courage et de domination.

      Historiquement ancré dans l'héroïsme guerrier, ce modèle a subi des chocs successifs : la déshumanisation par la guerre industrielle en 1914, l'autonomisation des femmes lors des conflits mondiaux, et les révolutions féministes des années 1970.

      Aujourd'hui, la masculinité est à la croisée des chemins. D'un côté, une réaction masculiniste et « viriliste » s'intensifie, portée par la manosphère et des discours réactionnaires.

      De l'autre, des mouvements de déconstruction émergent, portés par de nouvelles formes de paternité et une distinction claire entre masculinité et domination.

      Le coût social de la virilité obligatoire est désormais quantifié (environ 100 milliards d'euros par an en France), soulignant l'urgence d'une transition vers des modèles plus équitables.

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      1. L'Archétype d'Homo Virilus : Un Idéal de Domination

      Le concept d'Homo Virilus n'est pas une donnée biologique, mais une construction culturelle héritée de l'Antiquité gréco-romaine.

      Les piliers de la virilité : Ce modèle exige puissance, courage et domination. Il impose une maîtrise de soi constante, tout en justifiant une violence explosive si nécessaire.

      Une hiérarchie sociale : La vertu virile justifie la domination des femmes et des enfants, mais aussi celle des hommes jugés « non virils ».

      Une fragilité intrinsèque : Contrairement à l'apparence de force, la virilité est décrite comme une « insoutenable fragilité » car elle doit être prouvée et maintenue en permanence. On ne naît pas Homo Virilus, on le devient par des rites de passage, souvent liés au combat.

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      2. Les Ruptures Historiques : De la Tranchée au Foyer

      Le XXe siècle a marqué le début du déclin de l'héroïsme viril traditionnel.

      L'épreuve de la Grande Guerre (1914-1918)

      La fin du romantisme militaire : La guerre industrielle, avec son « orage d'acier », a rendu le courage individuel obsolète face aux obus.

      Le traumatisme et la « dévirilisation » : Les survivants sont revenus démembrés ou atteints de blessures invisibles (« l'obusite » ou syndrome post-traumatique). À l'époque, la psychiatrie associait ces troubles nerveux à une forme de féminisation ou de lâcheté.

      Inversion des rôles : Pendant que les hommes étaient au front, les femmes (« munitionnettes », conductrices, postières) ont prouvé leur capacité à faire fonctionner la société, brisant le mythe de leur infériorité physique et morale.

      La réaction totalitaire des années 1930

      Le fascisme comme rempart : Face à la fluidité de la République de Weimar, les régimes totalitaires (Hitler, Mussolini) ont prôné un retour à une virilité antique et implacable.

      Idéologie et exclusion : La virilité nazie était indissociable de l'antisémitisme et de l'homophobie. Les homosexuels, perçus comme des « hommes-femmes » incapables de combattre, ont été persécutés et envoyés en camps (marqués du triangle rose).

      Relégation des femmes : Le modèle « Kinder, Kuche, Kirche » (Enfant, Cuisine, Église) visait à supprimer les droits politiques des femmes pour les cantonner au rôle d'épouse-mère du guerrier.

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      3. Les Révolutions du Corps et du Pouvoir

      À partir des années 1960, l'hégémonie virile est attaquée de front par les mouvements sociaux.

      L'ère des luttes LGBT et féministes : La dépénalisation de l'homosexualité (1982 en France) et les victoires législatives (contraception, IVG avec la loi Veil) ont dissocié la sexualité de la reproduction.

      La fin du Pater Familias : Le passage de l'autorité paternelle à l'autorité parentale conjointe a mis fin à la domination juridique absolue du père dans le foyer.

      Le sentiment d'humiliation : Pour les nostalgiques de la domination, ces changements sont vécus comme une dépossession de pouvoir et une « féminisation » de la société.

      --------------------------------------------------------------------------------

      4. La Virilité comme Performance Sexuelle et Sociale

      La puissance masculine reste souvent indexée sur des attributs physiques et des performances quantifiables.

      Le Phallus comme sceptre : La puissance virile est fréquemment réduite à la puissance phallique, créant une obsession pour la taille, la durée et le nombre de conquêtes.

      La culture du vestiaire : Dès l'adolescence, la pression des pairs impose un masque de dureté. La sensibilité est assimilée à une défaillance.

      La domination économique : Au travail, Homo Virilus adopte l'uniforme du cadre ou du manager performant. Les « broligarches » (contraction de brothers et oligarques) utilisent des codes de guerre pour écraser la concurrence.

      La détresse silencieuse : L'échec professionnel est vécu comme une perte de valeur sociale totale. Le tabou de la santé mentale masculine mène à une « épidémie silencieuse » de suicides et de consommations excessives de drogues.

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      5. Violences et Consentement : L'impact de MeToo

      La dénonciation des violences sexistes et sexuelles remet en cause le « droit de cuissage » implicite de l'Homo Virilus.

      De la culture du viol au consentement : Des procès historiques (menés par Gisèle Halimi) à l'affaire Mazan, la société prend conscience que le viol n'est pas le fait de marginaux, mais d'hommes « ordinaires » issus de tous les milieux.

      Prise de conscience masculine : Le mouvement MeToo a forcé certains hommes à réévaluer leurs comportements passés, notamment sur la notion de rapport forcé ou de pression exercée sur les femmes.

      Le coût de la virilité : Les statistiques révèlent que les hommes sont responsables de :

      ◦ 91 % des tentatives d'homicides.    ◦ 99 % des incendies volontaires.    ◦ 84 % des accidents de la route mortels.    ◦ Coût total : Près de 100 milliards d'euros par an en France.

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      6. La Montée du Masculinisme et la « Manosphère »

      En réaction à ces évolutions, un courant réactionnaire puissant s'organise, notamment sur les réseaux sociaux.

      Le discours du déclin : Des personnalités politiques et médiatiques prédisent l'effondrement de la civilisation face à la « castration » de l'homme blanc.

      L'influence de TikTok : La « manosphère » attire les jeunes hommes avec des conseils de drague et de fitness qui cachent un agenda antiféministe radical. Les femmes y sont souvent désignées comme des « féminazis ».

      Le mouvement Incel : Les « célibataires involontaires » développent une haine profonde des femmes, allant parfois jusqu'à l'apologie du terrorisme ou au passage à l'acte meurtrier (Montréal, Californie).

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      7. Vers une Déconstruction et de Nouveaux Modèles

      Des alternatives à l'Homo Virilus émergent, cherchant à dissocier la masculinité de la domination.

      La paternité active : Des ateliers de préparation à la paternité aident les hommes à s'approprier les gestes du soin et de l'éducation, sortant du rôle binaire « autorité et finance ».

      La masculinité au pluriel : Les sociologues et les personnes trans (comme Léon) soulignent que la testostérone ne crée pas le comportement dominateur ; c'est l'éducation qui façonne la virilité toxique.

      Distinction clé :

      Les hommes se trouvent aujourd'hui à la croisée des chemins : perpétuer un stéréotype obsolète et coûteux, ou explorer des formes de masculinité fondées sur l'équité, la sensibilité et le partage des responsabilités.

    1. Briefing : Sensibilisation au Trouble du Développement Intellectuel (TDI)

      Ce document de synthèse analyse les enjeux, les caractéristiques et les modalités d'accompagnement liés au trouble du développement intellectuel (TDI), sur la base des expertises de Fabienne Rudler et Marie-Thé (Job coaches et spécialistes en neurodéveloppement).

      Résumé Exécutif

      Le trouble du développement intellectuel (TDI), autrefois nommé handicap mental ou déficience intellectuelle, est un trouble neurodéveloppemental (TND) apparaissant durant l'enfance (avant 18 ans).

      Il se définit par l'association de déficits des fonctions intellectuelles et de limitations significatives du comportement adaptatif. Contrairement aux troubles "Dys", le TDI impacte l'intelligence de manière globale, bien que les profils soient hétérogènes.

      La prise en charge efficace repose sur une distinction cruciale entre le langage expressif (ce que la personne dit) et le langage réceptif (ce qu'elle comprend réellement), ainsi que sur une adaptation de l'environnement face aux difficultés d'abstraction, de repérage temporel et de généralisation des apprentissages.

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      I. Cartographie et Typologie du Handicap

      Il est essentiel de situer le TDI parmi les différentes catégories de handicap pour éviter les confusions diagnostiques.

      | Type de Handicap | Caractéristiques Principales | | --- | --- | | Moteur | Limitation de la mobilité (membres) ou troubles d'origine motrice (ex: visuels). | | Sensoriel | Touche la vision et l'auditif (le goût/l'odorat ne sont pas encore reconnus suite au COVID). | | Psychique | Pathologies mentales (psychoses, bipolarité, dépression, TOC). Apparition souvent à l'adolescence ou à l'âge adulte. | | Dégénératif | État qui s'aggrave par paliers (pas de retour en arrière possible). | | Neurodéveloppemental (TND) | Apparaît durant le développement neurologique (0-18 ans). Inclut les troubles "Dys", le TDAH, l'autisme (TSA) et le TDI. |

      Distinction entre "Dys" et TDI

      Troubles Spécifiques des Apprentissages (Dys) : L'intelligence est préservée. Le trouble est spécifique à un domaine (lecture, calcul, geste).

      TDI : Les capacités cognitives globales sont impactées. La pédagogie doit être radicalement différente car elle ne peut pas s'appuyer sur le même bagage cognitif.

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      II. Étiologie et Critères Diagnostiques du TDI

      Le TDI est un trouble dynamique. L'origine du trouble est multiple, bien qu'une part importante reste inexpliquée.

      Les causes (Étiologie)

      80 % d'origine prénatale (anténatale) : Aberrations chromosomiques (Trisomie 21), malformations cérébrales, prématurité, infections (rubéole, toxoplasmose) ou intoxications durant la grossesse.

      10 % d'origine périnatale : Accidents lors de l'accouchement (anoxie cérébrale, hémorragies).

      10 % d'origine postnatale : Séquelles de méningites, traumatismes crâniens (bébé secoué), noyades ou intoxications (plomb).

      Donnée clé : 40 % à 50 % des TDI restent d'origine indéterminée, ce chiffre montant à 80 % pour les déficiences légères.

      Les trois piliers du diagnostic

      1. Déficit des fonctions intellectuelles : Évalué par des tests de QI (inférieur à 70).

      2. Limitation du comportement adaptatif : Difficultés dans la vie quotidienne (autonomie, relations sociales).

      3. Apparition durant la période développementale : Avant l'âge de 18 ans.

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      III. Analyse des Fonctions Cognitives Impactées

      Le TDI se manifeste par des altérations dans plusieurs domaines clés du traitement de l'information.

      1. Le repérage Espace-Temps

      C'est l'un des domaines les plus lourdement impactés.

      Le Temps : Étant abstrait, il est plus difficile à saisir que l'espace.

      Les personnes peuvent savoir lire l'heure (apprentissage mécanique) sans comprendre la notion de durée ou d'anticipation (ex: estimer le temps de trajet ou comprendre qu'un interrupteur s'éteint en secondes et non en minutes).

      L'Espace : Le repérage sur plan est complexe. L'apprentissage est souvent pragmatique (un trajet précis appris par cœur) plutôt que conceptuel (comprendre le réseau de transport).

      2. Les fonctions exécutives

      Inhibition : Difficulté à s'empêcher de dire ou de faire quelque chose (impulsivité sociale).

      Flexibilité : Difficulté à passer d'une tâche à une autre ou à gérer l'imprévu.

      Mémoire de travail : Capacité limitée à maintenir une information à court terme pour réaliser une action immédiate.

      3. La catégorisation

      L'incapacité à regrouper des objets par concepts (ex: fruits et légumes) rend le quotidien coûteux en énergie. Au supermarché, une personne ne sachant pas catégoriser cherchera chaque produit individuellement plutôt que par rayon.

      --------------------------------------------------------------------------------

      IV. Le Comportement Adaptatif

      Aujourd'hui, le degré de sévérité du TDI (léger, moyen, grave, profond) est déterminé par le fonctionnement adaptatif et non plus seulement par le score de QI.

      Les habiletés évaluées (échelle Vineland-II)

      Conceptuelles : Lecture, écriture, calcul, gestion de l'argent.

      Pratiques : Tâches ménagères, déplacements, utilisation d'appareils (automates, machine à laver).

      Sociales : Demander de l'aide, éviter les abus, respecter les distances sociales.

      La généralisation : un défi majeur

      Une personne avec TDI peut apprendre à utiliser une machine à laver spécifique, mais ne saura pas forcément utiliser un autre modèle. Elle ne généralise pas le principe de fonctionnement. L'apprentissage doit être multiplié dans divers contextes.

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      V. Focus sur la Communication et le Langage

      La communication orale est un piège fréquent dans l'accompagnement, car elle repose sur de nombreux codes implicites.

      Le paradoxe "Expressif vs Réceptif"

      Le "beau vernis" : Certaines personnes s'expriment très bien (langage expressif) mais ne comprennent pas les mots qu'elles emploient ou ceux des autres (langage réceptif).

      Vigilance : Il est impératif de vérifier systématiquement la compréhension, même si la personne semble éloquente.

      Les obstacles linguistiques

      L'abstraction : Les mots ne renvoyant pas à un objet physique (ex: "attendre", "organiser", "ailleurs") sont difficiles à saisir.

      Les doubles sens : Mots phonétiquement identiques mais de sens différents (ex: "glace", "moule", "vase").

      Les petits mots "outils" : Les pronoms (je/tu/moi), les articles et les prépositions peuvent brouiller le message.

      La conditionnalité : Les phrases commençant par "Si..." (ex: "Si tu es malade...") sont souvent incomprises car elles demandent de se projeter dans une situation inexistante. La réponse sera souvent : "Mais je ne suis pas malade".

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      VI. Recommandations pour l'Accompagnement

      Pour favoriser l'inclusion et l'apprentissage des personnes avec TDI, plusieurs leviers doivent être activés :

      1. Respecter le temps de latence : Le traitement de l'information est plus lent. Il faut ralentir le débit, poser une seule question à la fois et attendre la réponse sans reformuler trop vite.

      2. Passer par le concret : Utiliser des supports visuels, des objets réels et éviter les métaphores ou le second degré.

      3. Décomposer les tâches : Ce qui semble être une étape pour une personne ordinaire (ex: traverser la rue) est en réalité une succession de traitements cognitifs complexes (vitesse, distance, durée).

      4. Partir de la réponse de la personne : Si une personne répond "à côté", il faut analyser sa logique pour adapter notre message (ex: si elle ne comprend pas "fromage", utiliser le mot "camembert" si c'est sa référence).

      5. Anticiper les ruptures : Les changements de trajets, les grèves ou les pannes d'appareils sont des situations de mise en échec si elles n'ont pas été travaillées de manière pragmatique.

      Conclusion sur l'autonomie

      L'autonomie n'est pas de savoir tout faire seul, mais de savoir quand et comment demander de l'aide.

      C'est sur ce levier social et adaptatif que les éducateurs et employeurs peuvent avoir le plus d'impact, car si le QI est fixe, le comportement adaptatif peut toujours progresser par l'expérience.

    1. Guide de Référence : Le Programme Google Ad Grants pour les Associations

      Résumé Exécutif

      Le programme Google Ad Grants offre aux associations de loi 1901 une enveloppe de publicité gratuite sur le moteur de recherche Google s'élevant à 10 000 dollars par mois.

      Malgré son potentiel massif pour accroître la notoriété, recruter des bénévoles ou collecter des fonds, ce programme reste largement sous-exploité en France, avec seulement 2 000 à 3 000 associations actives sur les millions existantes.

      Ce document détaille les mécanismes du référencement payant, les critères d'éligibilité technique pour les structures, le processus d'activation en quatre étapes, ainsi que les stratégies optimales pour structurer des campagnes performantes.

      Il souligne également les limites du programme, notamment la priorité donnée aux annonceurs payants et la nécessité d'une gestion rigoureuse pour maximiser l'impact du crédit quotidien de 329 dollars.

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      1. Fondamentaux du Référencement Payant (SEA)

      Le programme Google Ad Grants s'inscrit dans le cadre du référencement payant (SEA), qu'il convient de distinguer du référencement naturel (SEO).

      Différences Clés : SEA vs SEO

      | Caractéristique | Référencement Payant (SEA) | Référencement Naturel (SEO) | | --- | --- | --- | | Position | Haut de la page (résultats sponsorisés) | Sous les annonces sponsorisées | | Délai de résultat | Court terme (immédiat après lancement) | Long terme et incertain | | Coût | Paiement au clic (offert par Ad Grants) | "Gratuit" (nécessite du temps/contenu) | | Contrôle | Choix précis des mots-clés et zones | Dépend de l'algorithme de Google |

      Spécificités du Compte Ad Grants

      Contrairement à un compte Google Ads classique, le compte Ad Grants présente des particularités :

      Enveloppe virtuelle : Aucun budget réel n'est déboursé par l'association ; Google déduit les frais de l'enveloppe de 10 000 $.

      Hiérarchie de diffusion : Les annonces Ad Grants apparaissent en dessous des annonces payantes des entreprises privées ou des institutions disposant d'un budget marketing.

      En cas de forte concurrence (ex: "collecte de dons"), il est parfois impossible de diffuser si les espaces publicitaires sont déjà saturés par des annonceurs payants.

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      2. Éligibilité et Critères Techniques

      Pour bénéficier du programme, une organisation doit remplir des critères statutaires et techniques précis.

      Structures Éligibles

      • Associations loi 1901.

      • Fonds de dotation et fondations reconnues d'utilité publique.

      Exclusions : Les entités gouvernementales, les hôpitaux, les centres de soins et les écoles ne sont pas éligibles directement (sauf via une fondation ou une structure associative dédiée).

      Exigences pour le Site Web

      Google effectue une vérification manuelle du site Internet lors de la demande. Celui-ci doit présenter :

      1. Un nom de domaine propre : Les sites hébergés sur des sous-domaines gratuits (ex: .wix.com, .google.site) sont refusés.

      2. Un contenu substantiel : Un minimum de 5 pages est requis.

      3. Une clarté institutionnelle : La mission et le statut associatif doivent être mentionnés en page d'accueil, dans une page "À propos" et dans le pied de page (footer).

      4. Performance technique : Le site doit être "responsive" (adapté aux mobiles) et avoir une vitesse de chargement satisfaisante (idéalement un score > 50/100 sur PageSpeed Insights).

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      3. Processus d'Activation en 4 Étapes

      Le lancement d'un compte Ad Grants suit un parcours structuré :

      1. Création du compte Google pour les associations : Utiliser de préférence une adresse email professionnelle liée au domaine de l'association pour simplifier la validation.

      2. Validation de l'identité : Google vérifie le statut juridique de l'association (via le numéro RNA). Cette étape prend généralement 24 heures.

      3. Activation de Google Ad Grants : Soumission du site web pour examen des critères de contenu et de performance. Le délai varie de 2 à 14 jours.

      4. Configuration finale : Validation du profil de paiement (sans carte bancaire) et accès définitif au compte.

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      4. Stratégie et Structuration des Campagnes

      Une gestion efficace repose sur une structure logique et l'alignement entre l'intention de l'utilisateur et le contenu proposé.

      Les 4 Piliers du Succès

      Le Ciblage : Sélection de mots-clés pertinents (volume de recherche > 50/mois) et spécifiques à la cause, en évitant les termes trop génériques ou ultra-concurrentiels.

      Les Annonces : Rédaction de titres percutants (jusqu'à 15 variantes) qui reprennent les mots-clés tapés par l'utilisateur.

      Les Enchères : Utilisation impérative de la stratégie "Maximiser les conversions" pour permettre à l'algorithme de Google d'optimiser la diffusion.

      Le Tracking : Connexion indispensable avec Google Analytics pour mesurer les actions concrètes (dons, inscriptions bénévoles, téléchargements).

      Exemple de Structure de Compte (Cas d'un refuge animalier)

      Campagne Adoptions : Groupes d'annonces séparés pour "Adopter un chien" et "Adopter un chat" renvoyant vers les pages respectives du site.

      Campagne Bénévolat : Mots-clés sur le don de temps, le soin aux animaux ou le travail associatif.

      Campagne Marque : Protection du nom de l'association pour apparaître systématiquement en haut lors d'une recherche directe.

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      5. Outils et Maintenance

      Le maintien de la performance nécessite l'usage d'outils complémentaires et une surveillance régulière.

      | Outil | Utilité | Niveau de difficulté | | --- | --- | --- | | Google Keyword Planner | Trouver des mots-clés et analyser leur volume/concurrence. | Débutant | | IA (ChatGPT, Gemini) | Aide à la rédaction des titres et descriptions d'annonces. | Débutant | | Google Analytics | Analyser le comportement des visiteurs après le clic. | Intermédiaire | | Google Tag Manager | Installer des marqueurs de conversion précis sans code. | Avancé |

      Conseils de Gestion

      Ne jamais supprimer de campagne : Il est préférable de mettre en pause les campagnes inactives pour conserver l'historique et gagner du temps lors de la réactivation.

      Utilisation du budget : Le plafond de 10 000 $ est réparti à hauteur de 329 $ par jour. Les crédits non utilisés un jour donné sont définitivement perdus et ne sont pas reportables.

      Sécurité des accès : Il est crucial de nommer plusieurs administrateurs pour éviter la perte du compte en cas de départ d'un collaborateur ou d'un bénévole.

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      6. Éthique et Transparence

      Bien que les annonces soient financées par Google, elles portent la mention "Sponsorisé".

      Cette transparence est renforcée par le Google Ads Transparency Center, qui permet au public de consulter les publicités diffusées par n'importe quelle entité.

      Le programme s'inscrit dans la politique de Responsabilité Sociétale des Entreprises (RSE) de Google, agissant comme un don en nature sous forme d'espace publicitaire.

    1. Reviewer #3 (Public review):

      Summary:

      The manuscript by Poh et al. investigated whether dopamine release in the ventral medial striatum integrates information about action selection, controllability of reward pursuit, effort, and reward approach. Rats were implanted with FSCV probes and trained in four Go/No Go task variants:

      (1) trials were self-initiated and had two trial types (Go vs. No Go) that were auditorily cued,

      (2) trials were cue-initiated and had two trial types (Go vs. No Go) that were auditorily cued,

      (3) trials were self-initiated and had three trial types (Go vs. No Go vs. free reward) that were auditorily cued, and effort was increased,

      (4) trials were cue-initiated and had three trial types (Go vs. No Go vs. free reward) that were auditorily cued.

      The authors report that dopamine levels rose during Go trials and slowly rose in No Go trials, but this pattern did not differ across task variants that modified effort and whether trials were cued or initiated. They also report that dopamine levels rose as rats approached the reward location and were greater in rats that bit the noseport while holding during the No Go response.

      Strengths:

      (1) Interesting task and variants within the task paradigm that would allow the authors to isolate specific behavioral metrics.

      (2) The goal of determining precisely what VMS dopamine signals do is highly significant and would be of interest to many researchers.

      Weaknesses:

      (1) This Go/No-Go procedure is different from the traditional tasks, and this leads to several problems with interpreting the results:

      (a) Go/No Go tasks typically require subjects to refrain from doing any action. In this task, a response is still required for the No Go trials (e.g., continue holding the nosepoke). The problem with this modified design is that failure to withhold a response on No Go trials could be because i) rats could not continue holding the response, as holding responses are difficult for rodents, or ii) rats could not suppress the prepotent go response. This makes interpreting the behavior and the dopamine signal in No Go trials very difficult.

      (b) Most Go/No Go tasks bias or overrepresent Go trials so that the Go response is prepotent, and consequently, successful suppression of the Go response is challenging. I didn't see any information in the manuscript about how often each trial type was presented or how the authors ensured that No Go responses (or lack thereof) were reflecting a suppression of the Go response.

      (2) The authors observe relatively consistent differences in the DA signal between Go and No Go trials after the action-cue onset. However, the response type was not randomized between trial type, so there is a confound between trial type (Go/No Go) and response (lever/nosepoke). The difference in DA signal may have nothing to do with the cue type, but reflects differences in DA signal elicited by levers vs. nosepokes.

      (3) Both Go and No Go trials start with the rat having their nose in the noseport. One cue (Go cue) signals the rat to remove their nose from the noseport and make two lever responses in 5 seconds, whereas the other cue (No Go cue) signals the rat to keep their nose in the noseport for an additional 1.7-1.9 s. The authors state that the time between cue onset and reward delivery was kept the same for all trial types, and Figure 1 suggests this is 2 s, so was reward delivered before rats completed the two lever presses? I would imagine reward was only delivered if rats completed the FR requirement, but again, the descriptions in the text and figures are incongruent.

      (4) The manuscript is difficult to understand because key details are not in the main text or are not mentioned at all. I've outlined several points below:

      (a) The author's description in the manuscript makes it appear as a discrimination task versus a Go/No Go task. I suggest including more details in the main text that clarify what is required at each step in the task. Additionally, providing clarity regarding what task events the voltammetry traces are aligned to would be very useful.

      (b) How many subjects were included in each task variant? The text makes it seem like all rats complete each task variant, but the behavioral data suggest otherwise. Moreover, it appears that some rats did more than one version. Was the order counterbalanced? If not, might this influence the DA signal?

      (5) There is a major challenge in their design and interpretation of the dopamine signal. Both trial types (Go and No Go) start with the rat having their nose in the noseport. An auditory cue is presented for 2-3 s signaling to the rat to either leave the noseport and make a lever response (Go trial) or to stay in the noseport (No Go trial). The timing of these actions and/or decisions is entirely independent, so it is not clear to me how the authors would ever align these traces to the exact decision point for each trial type. They attempt to do this with the nose-port exit analysis, but exiting the noseport for a Go trial (a rat needs to make 2 lever presses and then get a reward) versus a No Go trial (a rat needs to go retrieve the reward) is very different and not comparable.

      (6) The voltammetry analysis did not appear to test the hypotheses the authors outlined in the intro. All comparisons were done within task variants (DA dynamics in Go vs. No Go trials, aligned to different task events), but there were no comparisons across task variants to determine if the DA signal differed in cued vs self-initiated trials.

      (7) Classification of No Go behaviors was interesting, but was not well integrated with the rest of the paper and was underdeveloped. It also raised more questions for me than answers. For example:

      (a) Was the behavior classification consistent across rats for all No Go trials? If not, did the DA signal change within subjects between biting vs digging vs calm?

      (b) If "biting rats" were not always biting rats on every No Go trial, then is it fair to collapse animals into a single measure (Figure 3C).

      (c) Some of the classification groups only had 2 or fewer rats in them, making any statistical comparison and inference difficult.

    1. positive association of screen media with academic performance when adolescents spent from 2 to 4 hours on screen-based activities, but a negative association was found when they spent 7 or more hours per day.

      Negative effects happen at higher amounts of screen time. Does the amount of time correlate to the content?

    1. AbstractThe growth of metagenomics-derived amino acid sequence data has transformed our understanding of protein function, microbial diversity and evolutionary relationships. However, the vast majority of these proteins remain functionally uncharacterized. Grouping the millions of such uncharacterised sequences with the few experimentally characterised ones allows the transfer of annotations, while the inspection of conserved residues with multiple sequence alignments can provide clues to function, even in the absence of existing functional information. To address the challenges associated with this data surge and the need to group sequences, we present a scalable, open-source, parametrizable Nextflow pipeline (nf-core/proteinfamilies) that generates protein nascent families or assigns new proteins to existing families. The computational benchmarks demonstrated that resource usage can scale approximately linearly with input size, while the biological benchmarks showed that the generated protein families closely resemble manually curated families found in widely used databases.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag009), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 2: Castrense Savojardo

      This manuscript presents a Nextflow pipeline (nf-core/proteinfamilies) for large-scale protein-family generation. Overall, I think the paper is well written and clear. The pipeline appears very useful, and the reported results show good performance in both family reproducibility and computational efficiency.

      I have a few minor comments requesting additional details:

      1) Does the quality-check step only compute statistics, or is it also used to filter/clean the input set? If so, please specify the criteria and whether filtered sequences are excluded downstream.

      2) Which MMseqs2 clustering mode is used (set cover, connected components, or greedy)? Can this be changed within the pipeline? If configurable, please indicate the relevant parameters.

      3) In the reproducibility benchmark, you use DIAMOND BLASTp to assess similarity between the initial sequence set for the selected families and additional Swiss-Prot sequences. Which sequence identity and alignment coverage (if any) thresholds were applied?

      4) Counts and coverage (p. 6): You state that "These 709 families captured 96.66% of the original unique sequence identifiers (103,385 out of 106,959).". However, a few lines above the final input set is reported as 169,605 unique protein sequences. Could you please clarify the initial number of sequences and the actual coverage after family generation and redundancy reduction?

      5) Figures S1 and S2 are difficult to read due to low resolution.

    2. AbstractThe growth of metagenomics-derived amino acid sequence data has transformed our understanding of protein function, microbial diversity and evolutionary relationships. However, the vast majority of these proteins remain functionally uncharacterized. Grouping the millions of such uncharacterised sequences with the few experimentally characterised ones allows the transfer of annotations, while the inspection of conserved residues with multiple sequence alignments can provide clues to function, even in the absence of existing functional information. To address the challenges associated with this data surge and the need to group sequences, we present a scalable, open-source, parametrizable Nextflow pipeline (nf-core/proteinfamilies) that generates protein nascent families or assigns new proteins to existing families. The computational benchmarks demonstrated that resource usage can scale approximately linearly with input size, while the biological benchmarks showed that the generated protein families closely resemble manually curated families found in widely used databases.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag009), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 1: Vikram Alva

      The authors present nf-core/proteinfamilies, a standardized Nextflow workflow that constructs protein families de novo or classifies sequences against existing families. Using a curated 200-family benchmark and a UniRef90-scale run, the authors show that the pipeline attains high recall with efficient runtimes. Given the ever-increasing size of sequence databases, this work is timely and fills a practical gap in reproducible, at-scale family curation; I expect it to be adopted widely by many research groups.

      I have several comments and suggestions below:

      1. In my view, this workflow will, by construction, yield a mixture of families: some anchored on a single conserved domain/segment, others centered on recurrent multi-domain cores, and some that capture the full-length sequence. This differs from widely used family databases: Pfam is largely domain-level, whereas HAMAP and NCBIFAM are mostly full-length/isofunctional (with PANTHER sitting in between). The resulting granularity is largely determined by MMseqs2 settings (sequence identity, query/target coverage, coverage mode) and by any alignment trimming, which biases toward conserved cores. Please add a brief discussion making this explicit, with practical guidance for tuning toward full-length versus domain-centric generation of families.

      I also recommend a parameter-sensitivity analysis on the 200-family set: sequence identity (30-70%), coverage thresholds (50-95%), and coverage mode (query/target/both), with and without trimming. For each setting, report (i) total families and split/merge rates per curated family, and (ii) a simple granularity readout, the proportion classified as domain-anchored, multi-domain, or full-length. This would clarify how parameter choices drive family counts and domain/full-length centricity, and help readers select defaults aligned with their use case.

      1. In the results, the splits/misses are concentrated in Pfam/PANTHER, while HAMAP/NCBIFAM are much closer to one-to-one (HAMAP 50/50). This suggests the inflated family count is driven, in part, by the domain-centric portion of the benchmark rather than the method itself. Please add a brief note in the Discussion to make this explicit.

      2. Since AFDB has models for most UniProt entries, could these models be used as an orthogonal purity check of the generated families; e.g., map members to AFDB and ask whether they cluster to the same fold by TM-score/Foldseek (allowing full-length differences when the family is domain-anchored)?

      3. HHsearch-based merging of divergent splits. In my view, and the authors note this, several curated families split simply because sequences are very divergent. An optional HHsearch (HMM-HMM) pass could merge these back: merge only at high probability (≈≥95%) with reciprocal coverage of the shorter model (≥0.6). It would be useful to include this as an optional stage in the pipeline.

      4. Optional annotation of de novo families. I think it would be useful to add an annotation step that compares each de novo family (family HMM or MSA) against curated resources (Pfam, NCBIFAM, PANTHER/HAMAP).

      5. Could you briefly outline your expectations for how the pipeline handles transmembrane segments, coiled-coils, repeats, and IDRs, classes prone to over-splitting under MMseqs2 seeding and trimming due to short-motif signal, low complexity, and variable lengths?

    1. AbstractCancer cells are heterogeneous, each harboring distinct molecular aberrations and are dependent on different genes for their survival and proliferation. While successful targeted therapies have been developed based on driver DNA mutations, many patient tumors lack druggable mutations and have limited treatment options. Here, we hypothesize that new precision oncology targets may be identified through “expression-driven dependency”, whereby cancer cells with high expression of a targeted gene are more vulnerable to the knockout of that gene. We introduce a Bayesian approach, BEACON, to identify such targets by jointly analyzing global transcriptomic and proteomic profiles with genetic dependency data of cancer cell lines across 17 tissue lineages. BEACON identifies known druggable genes, e.g., BCL2, ERBB2, EGFR, ESR1, MYC, while revealing new targets confirmed by both mRNA- and protein-expression driven dependency. Notably, the identified genes show an overall 3.8-fold enrichment for approved drug targets and enrich for druggable oncology targets by 7 to 10-fold. We experimentally validate that the depletion of GRHL2, TP63, and PAX5 effectively reduce tumor cell growth and survival in their dependent cells. Overall, we present the catalog of express-driven dependency targets as a resource for identifying novel therapeutic targets in precision oncology.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag011), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 2

      The authors introduce BEACON, a Bayesian correlation approach designed to identify expression-driven dependency in cancer. Their hypothesis suggests that cancer cells with elevated expression of specific genes demonstrate increased vulnerability to the knockout of those same genes, thereby unveiling a promising new category of targets in precision oncology—particularly valuable for targeting cancer cells lacking druggable mutations. BEACON models expression levels and dependency scores as bivariate Gaussians and employs Markov Chain Monte Carlo (MCMC) sampling to estimate the correlation coefficient between them. They then compute p-values followed by rigorous multiple testing correction (BH based FDR correction). A notable strength of their approach lies in the integration of mass spectrometry proteomics data alongside transcriptomic and perturbation screening data, enhancing the robustness of their findings. Their work highlights some key insights: - Gene expression-driven dependency (GED) candidates identified across lineages demonstrate enrichment for "DNA-binding transcription activator activity" and "DNA-binding transcription activator activity, RNA polymerase II-specific" pathways. - The analysis successfully identifies compelling candidates with robust signals in both GED and PED (FERMT2, GRHL2, KLF5, CDK6, and CCND1), which are well-supported by existing drug evidence or established literature - Clustering analyses reveal that cancer cells from pancreas and biliary tract tissues, as well as kidney and urinary tract tissue lineages, exhibit remarkably similar expression-driven dependency profiles. Additionally, lineage-specific genes such as transcription factors, cluster together in a manner consistent with existing literature - Through Fisher's exact test, the authors demonstrate significant enrichments of druggable gene lists from DrugBank with expression-driven dependency patterns at both proteomic and transcriptomic levels - Experimental validation shows that PAX5 is essential for PAX5-high B cell lymphoma cell growth, while TP63 and GRHL2 are essential for LSCC cell growth.

      However, I have several principal concerns about the study that should be addressed to demonstrate the robust and superior performance of this proposed approach.

      Major Comments:

      1.Quantitative benchmarking: While the authors present a valuable contribution, the concept of correlating gene dependency scores to expression has been explored previously through approaches like Project DRIVE (E. Robert McDonald, III et al.) and APSIC (Montazeri et al.). BEACON demonstrates strong correlations across multiple lineages, representing broader scope compared to existing methods that appear more lineage-restricted. However, establishing BEACON's comparative advantages requires more rigorous evaluation. Notably, Project DRIVE—a foundational paper in this field—already identified several BEACON candidates in their "Expression Correlation Analysis Identifies Oncogenes and Lineage-Specific Transcription Factors" section, while APSIC characterized many lineage-specific discoveries as tumor effector genes. BEACON's strength lies in integrating proteomic data with transcriptomic and perturbation screens, enabling identification of additional candidates like PAX5 for hematopoietic and lymphoid tissue. To demonstrate the method's impact, I recommend systematic quantitative benchmarking against existing approaches.

      Importantly, BEACON utilizes richer/complementary datasets than previous studies. Disentangling contributions of data richness versus methodological innovation would provide valuable insights into whether enhanced performance stems from improved data availability or genuine method improvements.

      Overall for benchmarking, the authors are strongly encouraged to utilize any comprehensive datasets that best demonstrate their method's competitive advantage and are not limited to the specific comparisons recommended above.

      2.Correlation method comparisons: Figure S2 shows that BEACON exhibits higher MSE at extremes, and the claimed advantage over Pearson for small sample sizes is difficult to quantify from the current visualization. While the theoretical expectation that BEACON should outperform Pearson in small samples is reasonable, the practical significance remains unclear from these simulations. I recommend demonstrating BEACON's advantage using real data by creating a curated list of established GEDs/PEDs and comparing performance between the two methods. This is particularly important since several of BEACON's hits were previously reported by Project DRIVE using simple Pearson correlations. Alternatively, if BEACON's advantage is indeed significant, please elaborate on the simulation results to better justify this claim with clearer quantitative metrics.

      3.Validation experiments: I'm seeking clarification on the validation experiments for TP63 and GRHL2. These candidates were not sensitive to predicted dependency and the authors say that "pan-lineage targets may represent universal vulnerability and their inhibition may lead to undesired off-target effects on other cells". Are the authors positioning them as weaker candidates to illustrate the superiority of lineage-specific predictions like PAX5? Additionally, why were different experimental approaches used—CRISPR for PAX5 versus shRNA for TP63 and GRHL2? For a method aimed at identifying druggable targets, would drug based experiments be more relevant than knockdown approaches to better demonstrate clinical applicability?

      Minor comments

      1. In Figure 4A, the caption refers to the plot as a heatmap, but the visualization appears to be a scatterplot. Please clarify whether the heatmap is missing or modify the caption appropriately. Additionally, I recommend using a different shade of green, as the current color choice makes some gene names difficult to read.
      2. In Fig S5A, please add a legend for tumor and normal
      3. For the TP63 and GRHL2 validation experiments, please include results for all four cell lines. The current manuscript is missing HCC15-shTP63, HCC15-shGRHL2, and HARA-shGRHL2 plots.
      4. How many replicates were the experiments performed on? Is it N= 3 for all experiments?
      5. Missing some text here - "BEACON offers the unique advantage of utilizing prior distributions that are less susceptible to outliers, especially in multiple lineages where the number of cell lines."
    2. AbstractCancer cells are heterogeneous, each harboring distinct molecular aberrations and are dependent on different genes for their survival and proliferation. While successful targeted therapies have been developed based on driver DNA mutations, many patient tumors lack druggable mutations and have limited treatment options. Here, we hypothesize that new precision oncology targets may be identified through “expression-driven dependency”, whereby cancer cells with high expression of a targeted gene are more vulnerable to the knockout of that gene. We introduce a Bayesian approach, BEACON, to identify such targets by jointly analyzing global transcriptomic and proteomic profiles with genetic dependency data of cancer cell lines across 17 tissue lineages. BEACON identifies known druggable genes, e.g., BCL2, ERBB2, EGFR, ESR1, MYC, while revealing new targets confirmed by both mRNA- and protein-expression driven dependency. Notably, the identified genes show an overall 3.8-fold enrichment for approved drug targets and enrich for druggable oncology targets by 7 to 10-fold. We experimentally validate that the depletion of GRHL2, TP63, and PAX5 effectively reduce tumor cell growth and survival in their dependent cells. Overall, we present the catalog of express-driven dependency targets as a resource for identifying novel therapeutic targets in precision oncology.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag011), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 4

      Reproducibility report for: Expression-Driven Genetic Dependency Reveals Targets for Precision Oncology Journal: Gigascience ID number/DOI: GIGA-D-25-00147 Reviewer(s): Laura Caquelin, Department of Clinical Neuroscience, Karolinska Institutet, Sweden


      1. Summary of the Study The authors developed a Bayesian method called BEACON to integrate multi-omics data. The method was tested on cancer cell lines across 17 tissue types to identify expression- driven dependencies. The method recovered known drug targets and identified novel candidates. The study concludes this method provides a systematic approach to identify precision oncology targets.

      1. Scope of reproducibility According to our assessment the primary objective is: to identify expression-driven dependencies across cancer cell lines from multiple lineages enabling the discovery of genes whose expression levels correlate with cancer cell dependency scores.
      2. Outcome: Identification of genes with significant expression-driven dependencies across pan-lineage cancer cell lines.
      3. Analysis method outcome: "BEACON calculated the Bayesian correlation between the gene's expressions and CERES cancer dependency scores 25 across the pan-lineage cell lines. BEACON modeled expression levels and dependency scores as the bivariate Gaussians and used Markov Chain Monte Carlo (MCMC) sampling to estimate the correlation coefficient rho between them. Given the null hypothesis that the uncorrelated expression and dependency of a gene has the 0 rho coefficient, we statistically tested each gene's rho estimate obtained from the MCMC simulation as follows. Assume that the MCMC sampling is carried out for a null gene's expression and dependency, then we expect that the distribution of the rho estimate accumulated over the MCMC iterations will be centered at zero. Based on this rationale, we computed the z-score of i-th gene as the deviation of the MCMC estimate of rho from the expected (null) value (i.e., zero) in terms of the standard deviation observed in the simulated distribution, i.e., z(i) = rhoMCMC(i) / SDMCMC(i). Since the z-values, by nature, follow a normal distribution with zero-mean and unit-variance, then we computed the p- value for each gene's rho estimate as the probability of observing a value as extreme as the computed z-value for that gene. We multi-testing corrected the resulting p-values using the BH procedure for FDR." (page 19 -Methods section / mRNA expression-driven dependency (GED))
      4. Main result: "We first analyzed the pan-lineage GED by using mRNA levels and the corresponding dependency scores from 854 cell lines with available data across 17 lineages and identified 244 genes showing significant association (correlation coefficient, rho < -0.25, FDR < 0.05)" (page 7 - Results section / Cancer vulnerability targets showing gene expression-driven dependency (GED))

      1. Availability of Materials a. Data
      2. Data availability: Open
      3. Data completeness: Complete, all data necessary to reproduce main results are available.
      4. Access Method: Repository
      5. Repository: https://doi.org/10.6084/m9.figshare.19700056.v2 -Data quality: Structured

      b. Code - Code availability: Open - Programming Language(s): R - Repository link: https://github.com/Huang-lab/BEACON - License: MIT license - Repository status: Public - Documentation: Readme file


      1. Computational environment of reproduction analysis
      2. Operating system for reproduction: MacOS 15.5
      3. Programming Language(s): R
      4. Code implementation approach: Using shared code
      5. Version environment for reproduction: R version 4.5.0/RStudio 2025.05.1

      1. Results 5.1 Original study results
      2. Results 1: Supplementary table S2 5.2 Steps for reproduction -> Run the code PanLineageMCMC.R
      3. Issue 1: File import paths and incorrect file name -- Resolved: In the original code, there were fixed file paths that only worked on one specific computer. This caused problems when running the code on other computers. To fix this, I recommended to use relative paths, which are based on where the script is located. This way, the code can be run on any computer without needing to change the paths each time.

      ------------------ Start of script ------------------ sam.dep = read.csv(file.path(getwd(), "DepMap_data", "sample_info.csv")) ------------------- End of script -------------------

      • Issue 2: Missing function "intsect" at line 162 -- Resolved: The script called a function intsect that was not defined, leading to an error. Upon request, the authors provided the missing function and added it to the main script (PanLineageMCMC.R).

      • Issue 3: Output directory not created. -- Resolved: The script attempted to write output files to a directory that was not created beforehand. This caused errors during the loop execution when trying to save results. A directory check and automatic creation script was added. If the output folder does not exist, it is now created automatically before the loop runs.

      ------------------ Start of script ------------------ dir_path <- paste0('../out/jags.nadapt',n.adapt,'.update',n.update,'.mcmc ',n.iter,'.simulation_SD_22Q2') if (!dir.exists(dir_path)) { dir.create(dir_path, recursive = TRUE) } ------------------- End of script -------------------

      5.3 Statistical comparison Original vs Reproduced results - Results: Table.mRNA.dependency.Bayesian.pancancer file attached - Comments: The Bayesian PanCancer analysis was re-run, but only on the 244 significant genes listed in Supplementary Table S2, not on the full set of 17 285 genes. This choice was made due to limited computational resources, as running the full model would have required an estimated 100 hours. - Errors detected: - - Statistical Consistency: Among the 244 significant genes originally reported, the reproduced analysis confirmed the statistical significance of these same genes. However, the exact numerical values (Mean, standard deviation, Z value, P-value and adjusted P-value) differed slightly. These discrepancies are expected due to the nature of Bayesian inference, the absence of a random seed, and the relatively low number of MCMC iterations used (n.iter = 500). These settings may not be sufficient to ensure full convergence or reproducibility of posterior estimates and should be interpreted with caution. We were unable to compare the rho values because they were not available in the provided Supplementary table S2, nor extracted in the R code to be include in the resulting output files.


      1. Conclusion
      2. Summary of the computational reproducibility review The results of the Supplementary table S2 in the original study was partially reproduced. We were able to confirm the statistical significance of the 244 genes reported in Supplementary Table S2 using the Bayesian PanCancer model in the provided code. However, the numerical results were not always identical. This is expected because Bayesian methods involve random sampling, the original code did not set a fixed random seed, and the number of iterations used was relatively low. Furthermore, the rho values were not available for comparison, limiting a full reproducibility assessment. Several technical issues were also fixed during the reproduction process, such as hardcoded file paths, a missing function, and the absence of output directories, which were resolved to allow the code to run correctly on a different system. Due to computational limitations, running the full model on all 17,285 genes was not performed.

      3. Recommendations for authors While the original analysis code was successfully used to confirm the statistical significance of the 244 genes, we recommend several improvements to enhance reproducibility: -- Code annotation: Adding more detailed comments within the scripts would help users understand the logic behind each step and the purpose of specific commands or operations. -- Set a random seed: Include set.seed() in all scripts to improve reproducibility across different runs. -- Specify R and package versions: Provide the R version and exact package versions needed to run the code, via a requirements file for example. -- Use relative file paths: Ensure that all necessary folders and functions are created or included by default to avoid path issues. -- Increase MCMC robustness: Use a higher number of iterations and appropriate parameter settings to ensure better convergence and stability of posterior estimates. -- Inform users about computation time: Clearly indicate in the README or publication the expected runtime of the code, especially if it requires several hours or days to complete.

    3. AbstractCancer cells are heterogeneous, each harboring distinct molecular aberrations and are dependent on different genes for their survival and proliferation. While successful targeted therapies have been developed based on driver DNA mutations, many patient tumors lack druggable mutations and have limited treatment options. Here, we hypothesize that new precision oncology targets may be identified through “expression-driven dependency”, whereby cancer cells with high expression of a targeted gene are more vulnerable to the knockout of that gene. We introduce a Bayesian approach, BEACON, to identify such targets by jointly analyzing global transcriptomic and proteomic profiles with genetic dependency data of cancer cell lines across 17 tissue lineages. BEACON identifies known druggable genes, e.g., BCL2, ERBB2, EGFR, ESR1, MYC, while revealing new targets confirmed by both mRNA- and protein-expression driven dependency. Notably, the identified genes show an overall 3.8-fold enrichment for approved drug targets and enrich for druggable oncology targets by 7 to 10-fold. We experimentally validate that the depletion of GRHL2, TP63, and PAX5 effectively reduce tumor cell growth and survival in their dependent cells. Overall, we present the catalog of express-driven dependency targets as a resource for identifying novel therapeutic targets in precision oncology.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag011), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 3

      The authors develop a method for correlating gene and protein expression with cellular dependencies using the resources of DepMap. The innovation appears to be a Bayesian approach to the correlation analysis. They use this approach to identify potential therapeutic targets and evaluate some top candidates using in vitro experiments. The paper is fairly straightforward to follow.

      Major comments:

      1. Benchmarking - given the non-linear relationships shown in Fig 2, is a comparison with the Pearson method the most appropriate? Would a Spearman's be better?

      2. The analysis identifies dependencies that are proposed as therapeutic targets, however while the proteins can be druggable, what about normal tissue effects? Some of these are likely lineage-defining proteins that could be highly expressed in normal tissues. Is is notable that in Fig 5B, C that the existing drug targets have a lower association strength than other GEDs identified. Does this suggest that the strongest correlations might be lineage-crucial genes that are too important for normal tissue function to make good drug targets? This needs further consideration in the discussion. Are there any pathways differences between these groups (known drug targets vs others)? For example you might expect more tissue lineage Tfs in the "other" category, while the approved drug targets perhaps more cell surface receptors.

      3. The cell assays performed should effectively be replicating the results of the dependencies on which BEACON is based (DepMap), so why do you get different results? Is it because of the different methods used ie shRNA (not seeing the correlation between expression and dependency) vs CRISPR (replicating the correlation)? If you look at older DepMap scores when they used knockdown rather than CRISPR can you replicate your results?

      4. Although mycoplasma testing was done, were the cell lines re-authenticated by STR profiling at any point?

      5. QPCR is mentioned n the methods but not provided in the results that I can find. Did this validate gene knockdown by shRNA? Any correlation between % KD and proliferation/colony forming effect?

      6. In the discussion it should be acknowledged that cancer subtypes exist within lineages that are molecularly and clinically distinct and so the method might be missing targets specific for these eg ER+ and ER- breast cancer.

      Minor comments: 1. Results para 1 "especially in multiple lineages where the number of cell lines." Missing something in this sentence?

      1. Needs some grammar review

      3, Please italicise all gene names (when referring to gene, not protein) eg CCNE1 amplification etc

      1. Fig S5A - legend or axis labels for N and T needed.
      2. Fig S5C, D - these are proliferation not colony forming assays as stated in the text.

      3. Please include number of replicates and type of error bars in figure legends for cell assays

    1. NOTA: Cada respuesta correcta vale un punto.

      1.- El código registrará la hora en la que el pedido fue procesado desde la cola.

      2.- Cuando la función ProcessOrders falla, la función se reintentará hasta cinco veces para un pedido determinado, incluyendo el primer intento.

      3.- Cuando hay varios pedidos en la cola, se recuperará un lote de pedidos desde la cola y la función ProcessOrders ejecutará múltiples instancias de forma concurrente para procesar los pedidos.

      4.- La función ProcessOrders enviará el pedido como salida a una tabla Orders en Azure Table Storage.

    1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

      Learn more at Review Commons


      Reply to the reviewers

      We thank the reviewers and editors for their careful evaluation of our manuscript and their positive comments on the importance and rigor of the work. Below you will find our point-by-point response to each reviewer's suggestions. We believe that we have addressed (in the response and the revised manuscript) all of the concerns. Please note that in some cases, we have numbered a reviewer's comments for clarity, however beyond this, we have not altered any of the reviewers' text.

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      Lo et al., report a high-throughput functional profiling study on the gene encoding for argininosuccinate synthase (ASS1), done in a yeast experimental system. The study design is robust (see lines 141-143, main text, Methods), whereby "approximately three to four independent transformants of each variant would be isolated and assayed." (lines 140 - 141, main text, Methods). Such a manner of analysis will allow for uncertainty of the functional readout for the tested variants to be accounted for.

      This is an outstanding study providing insights on the functional landscape of ASS1. Functionally impaired ASS1 may cause citrullinemia type I, and disease severity varies according to the degree of enzyme impairment (line 30, main text; Abstract). Data from this study forms a valuable resource in allowing for functional interpretation of protein-altering ASS1 variants that could be newly identified from large-scale whole-genome sequencing efforts done in biobanks or national precision medicine programs. I have some suggestions for the Authors to consider:

      1. The specific function of ASS1 is to condense L-citrulline and L-aspartate to form argininosuccinate. Instead of measuring either depletion of substrate or formation of product, the Authors elected to study 'growth' of the yeast cells. This is a broader phenotype which could be determined by other factors outside of ASS1. Whereas i agree that the experiments were beautifully done, the selection of an indirect phenotype such as ability of the yeast cells to grow could be more vigorously discussed.

      We appreciate the reviewer's point regarding the indirect nature of growth as a functional readout. In our system, yeast growth is tightly and specifically coupled to ASS enzymatic activity. The strains used are isogenic and lack the native yeast argininosuccinate synthetase, such that arginine biosynthesis, and therefore yeast replication on minimal medium lacking arginine, depends exclusively on the activity of human ASS1. Under these defined and limiting conditions, growth provides a quantitative proxy for ASS1 function. However, we acknowledge that this assay does not resolve specific molecular mechanisms underlying reduced function, such as altered catalytic activity versus effects on protein stability. We have updated the text to clarify these points.

      "While growth is an indirect phenotype relative to direct measurement of substrate turnover or product formation, it is tightly coupled to ASS enzymatic activity in this system and is expected to be impaired by amino acid substitutions that reduce catalytic activity or protein stability. Therefore, growth on minimal medium lacking arginine is a quantitative measure of ASS enzyme function, allowing the impact of ASS1 missense variants to be assessed at scale through a high-throughput growth assay, in a single isogenic strain background, under controlled, defined conditions that limit confounding factors unrelated to ASS1 activity. We expect that the assay will detect reductions in both catalytic activity and protein stability but will not distinguish between these mechanisms."

      1. One of the key reasons why studies such as this one are valuable is due to the limitations of current variant classification methods that rely on 'conservation' status of amino acid residues to predict which variants might be 'pathogenic' and which variants might be 'likely benign'. However, there are serious limitations, and Figures 2 and 6 in the main text shows this clearly. Specifically, there is an appreciable number of variants that, despite being classified as "ClinVar Pathogenic", were shown by the assay to unlikely be functionally impaired. This should be discussed vigorously. Could these inconsistencies be potentially due to the read out (growth instead of a more direct evaluation of ASS1 function)?

      We interpret this discrepancy as reflecting a sensitivity limitation of the growth-based readout rather than a fundamental disagreement between functional effect and clinical annotation. Specifically, we believe that our assay is unable to resolve the very mildest hypomorphic variants from true wild type, i.e., the residual activity of these variants is sufficient to fully support yeast growth under the conditions used. On this basis, we have chosen not to treat wild-type-like growth in our assay as informative for benignity; conversely, reduced growth provides evidence supporting pathogenicity (all clinically validated variants examined in this range are pathogenic).

      We have revised the manuscript to clarify this point explicitly and to frame these variants as lying outside the effective resolution limit of the assay rather than representing true false positives. Additional discussion of this limitation and its implications is provided in our responses to Reviewer 2 (points 1 and 4) along with specific changes made to the text.

      1. Figure 3 is very interesting, showing a continuum of functional readout ranging from 'wild-type' to 'null'. It is very interesting that the Authors used a threshold of less than 0.85 as functionally hypomorphic. What does this mean? It would be very nice if they have data from patients carrying two hypomorphic ASS1 alleles, and correlate their functional readout with severity of clinical presentation. The reader might be curious as to the clinical presentation of individuals carrying, for example, two ASS1 alleles with normalized growth of 0.7 to 0.8.

      I hope you will find these suggestions helpful.

      We thank the reviewer for this thoughtful comment. Figure 3 indeed illustrates a continuum of functional effects, and we agree that careful interpretation of the thresholds used is important. To clarify the rationale for the hypomorphic threshold, the interpretation of intermediate growth values, and to emphasize that these labels reflect only behavior in the functional assay, we have rewritten the relevant section of the Results:

      "The normalized growth scores of the 2,193 variants tested in our functional assay form a clear bimodal distribution (Figure 3), with two distinct peaks corresponding to functional extremes, as is commonly reported in large-scale functional assays of protein function [9, 10]. The smaller peak, centered around the null control (normalized growth = 0), represents variants that fail to support growth in the assay (growth 0.85). Variants with growth values falling between these two peak-based thresholds display partial functional impairment and are classified as functionally hypomorphic (n = 323). Crucially, these classifications are entirely derived from the observed peaks in the distribution of growth values and reflect differences in functional activity under the assay conditions. They do not provide direct evidence for clinical pathogenicity or benignity and should not be used for clinical variant interpretation without proper benchmarking against clinical reference datasets, as implemented below within an OddsPath framework."

      We agree with the reviewer that correlating functional measurements with clinical severity in individuals carrying two hypomorphic ASS1 alleles would be highly informative, particularly given that ASS1 deficiency is an autosomal recessive disorder. While mild hypomorphic variants (for example, variants with normalized growth values of 0.7-0.8 in our assay) could plausibly contribute to disease when paired with a complete loss-of-function allele, systematic analysis of combinatorial genotype effects and genotype-phenotype correlations is beyond the scope of the present study, which focuses on the functional effects of individual variants. We view this as an important direction for future work.

      Reviewer #1 (Significance (Required)):

      This is an outstanding study providing insights on the functional landscape of ASS1. Functionally impaired ASS1 may cause citrullinemia type I, and disease severity varies according to the degree of enzyme impairment (line 30, main text; Abstract). Data from this study forms a valuable resource in allowing for functional interpretation of protein-altering ASS1 variants that could be newly identified from large-scale whole-genome sequencing efforts done in biobanks or national precision medicine programs.

      Reviewer #2 (Evidence, reproducibility and clarity (Required)):

      In this manuscript, Lo et al characterize the phenotypic effect of ~90% of all possible ASS1 missense mutations using an elegant yeast-based system, and use this dataset to aid the interpretation of clinical ASS1 variants. Overall, the manuscript is well-written and the experimental data are interpretated rigorously. Of particular interest is the identification of pairs of deleterious alleles that rescue ASS1 activity in trans. My comments mainly pertain to the relevance of using a yeast screening methodology to infer functional effects of human ASS1 mutations.

      1. Since human ASS1 is heterologously expressed in yeast for this mutational screen, direct comparison of native expression levels between human cells and yeast is not possible. Could the expression level of human ASS1 (driven by the pARG1 promoter) in yeast alter the measured fitness defect of each variant? For instance, if ASS1 expression in yeast is sufficiently high to mask modest reductions in catalytic activity, such variants may be misclassified as hypomorphic rather than amorphic. Conversely, if expression is intrinsically low, even mild catalytic impairments could appear deleterious. While it is helpful that the authors used non-human primate SNV data to calibrate their assay, experiments could be performed to directly address this possibility.

      The nature of the relationship between yeast growth and availability of functional ASS1 could also influence the interpretation of results from the yeast-based screen. Does yeast growth scale proportionately with ASS1 enzymatic activity?

      We completely agree that the expression level of human ASS1 in yeast could influence the measured fitness effects of individual variants. We expect the rank ordering of variants in our growth assay to reflect their relative enzymatic activity (i.e. a monotonic relationship) but acknowledge that the precise mapping between activity and growth is unknown and may include ceiling and floor effects that limit the assay's dynamic range. As the reviewer notes, under high expression conditions moderate loss-of-function variants could appear indistinguishable from wild type (ceiling effect), whereas under lower expression the same variants could behave closer to the null control (floor effect).

      In our system, ASS1 is expressed from the pARG1 promoter, chosen under the assumption that the native expression level of ARG1 (the yeast ASS1 ortholog) is appropriately tuned for yeast growth. Crucially, rather than assuming a fixed mapping from assay growth to clinical pathogenicity (given potential nonlinearities in the relationship between ASS function and growth) we benchmark the assay against external data, including known pathogenic and benign variants and non-human primate SNVs, to calibrate thresholds and guide interpretation within an OddsPath framework. This benchmarking indicates that ceiling effects are likely present, with some mild loss-of-function pathogenic variants appearing indistinguishable from wild type in the growth assay. We explicitly account for this by not using high-growth scores as evidence toward benignity. We have made the following changes the manuscript:

      "A subset of clinically pathogenic ASS1 variants exhibit near-wild-type growth in our yeast assay. In general, we expect a monotonic relationship between ASS function and yeast growth, but with the potential for floor and ceiling effects that constrain the assay's dynamic range. In this context, we interpret high-growth pathogenic variants as likely causing mild loss of function that cannot be distinguished from wild type in our assay"

      "Based on these findings and given that 22/56 pathogenic variants show >85% growth, we conclude that growth above this threshold should not be used as evidence toward benignity."

      1. It would be helpful to add an additional diagram to Figure 1A explaining how the screen was performed, in particular: when genotype and phenotype were measured, relative to plating on selective vs non-selective media? This is described in "Variant library sequence confirmation" and "Measuring the growth of individual isolates" of the Methods section but could also be distilled into a diagram.

      We thank the reviewer for this helpful suggestion. We have updated Figure 1 by adding a new schematic panel (Figure 1C) that distills the experimental workflow into a visual overview. This diagram is intended to complement the detailed descriptions in the Methods and improve clarity for the reader.

      1. The authors rationalize the biochemical consequences of ASS1 mutations in the context of ASS1 per se - for example, mutations in the active site pocket impair substrate binding and therefore catalytic activity, which is expected. Does ASS1 physically interact with other proteins in human cells, and could these interactions be altered in the presence of specific ASS1 mutations? Such effects may not be captured by performing mutational scanning in yeast.

      We are not aware of any specific protein-protein interactions involving ASS that are required for its enzymatic function. However, we agree that ASS could engage in non-essential interactions with other human proteins that might be altered by specific missense variants and that such interactions would not necessarily be captured in a yeast-based assay.

      Importantly, our complementation system depends on human ASS providing the essential enzymatic activity required for arginine biosynthesis in yeast. If ASS1 required obligate human-specific protein interactions to function, even the wild-type enzyme would fail to support yeast growth, which is clearly not the case. We therefore conclude that the assay robustly reports on the intrinsic enzymatic activity of ASS, while acknowledging that non-essential human-specific interactions may not be assessed. We have updated the manuscript to reflect this point.

      "Importantly, successful functional complementation indicates that ASS enzymatic activity does not depend on any obligate human-specific protein interactions."

      1. The authors note that only a small number (2/11) of mutations at the ASS1 monomer-monomer interface lead to growth defects in yeast. It would be helpful for the authors to discuss this further.

      As discussed in response to the reviewer's comments on the relationship between ASS activity and yeast growth (point 1 above), we expect growth to be a monotonic but nonlinear function of enzymatic activity, with potential ceiling effects at high activity. Under this model, variants causing weak or moderate loss of function may remain indistinguishable from wild type when residual activity is sufficient to support normal growth. We favor this explanation for the observation that only 2/11 interface variants show reduced growth, as many pathogenic interface substitutions are associated with milder disease presentations, consistent with higher residual enzyme function. Consistent with this interpretation, variants affecting the active site, where substitutions are expected to cause large reductions in catalytic activity, are readily detected by the assay.

      Although we cannot exclude partial buffering of dimerization defects in yeast, we interpret the reduced sensitivity to interface variants primarily as a general limitation of growth-based assays. Accordingly, our decision not to use growth >85% as evidence toward benignity is conservative relative to approaches that would classify high-growth variants as benign except at the monomer-monomer interface, avoiding reliance on structural subclassification and minimizing the risk of false benign interpretation. Reduced growth, by contrast, provides strong evidence of loss of ASS1 function and pathogenicity, validated under the OddsPath framework.

      We have updated the Results and Discussion sections to clarify these points (also see response to the reviewer's point 1).

      "A subset of clinically pathogenic ASS1 variants exhibit near-wild-type growth in our yeast assay. In general, we expect a monotonic relationship between ASS function and yeast growth, but with the potential for floor and ceiling effects that constrain the assay's dynamic range. In this context, we interpret high-growth pathogenic variants as likely causing mild loss of function that cannot be distinguished from wild type in our assay. Consistent with this view, many pathogenic variants with high assay growth are located at the monomer-monomer interface rather than the active site, and are associated with milder or later-onset clinical presentations, suggesting partial enzymatic impairment that is clinically relevant in humans but not resolved by the yeast assay."

      "Based on these findings and given that 22/56 pathogenic variants show >85% growth, we conclude that growth above this threshold should not be used as evidence toward benignity. Notably, this approach is conservative relative to treating high-growth variants as benign except at the monomer-monomer interface, avoiding reliance on structural subclassification and minimizing the risk of false benign interpretation arising from assay ceiling effects. Conversely, the variants with

      Reviewer #2 (Significance (Required)):

      This study presents the first comprehensive mutational profiling of human ASS1 and would be of broad interest to clinical geneticists as well as those seeking biochemical insights into the enzymology of ASS1. The authors' use of a yeast system to profile human mutations would be particularly useful for researchers performing deep mutational scans, given that it provides functional insights in a rapid and inexpensive manner.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      Section 1 - Evidence, reproducibility, and clarity Summary This manuscript presents a comprehensive functional profiling of 2,193 ASS1 missense variants using a yeast complementation assay, providing valuable data for variant interpretation in the rare disease citrullinemia type I. The dataset is extensive, technically sound, and clinically relevant. The demonstration of intragenic complementation in ASS1 is novel and conceptually important. Overall, the study represents a substantial contribution to functional genomics and rare disease variant interpretation.

      Major comments 1. This is an exciting paper as it can provide support to clinicians to make actionable decisions when diagnosing infants. I have a few major comments, but I want to emphasize the label of "functionally unimpaired" variants to be misleading. The authors explain that there are several pathogenic ClinVar variants that fall into this category (above the >.85 growth threshold) but I think this category needs a more specific name and I would ask the authors to reiterate the shortcomings of the assay again in the Discussion section.

      We thank the reviewer for raising this important point. We agree that the label "functionally unimpaired" could be misleading if interpreted as implying clinical benignity rather than assay behavior. We have therefore clarified that this designation refers strictly to variant behavior in the yeast growth assay and does not imply absence of pathogenicity.

      In addition, we have expanded the Discussion to explicitly address the existence of clinically pathogenic variants with high growth scores (>0.85), emphasizing that these likely reflect a ceiling effect of the assay and represent a key limitation for interpretation. This clarification reiterates that high-growth scores should not be used as evidence toward benignity, while reduced growth provides strong functional evidence of pathogenicity. Relevant revisions are described in our responses to Reviewers 1 and 2.

      1. I think there's an important discussion to be had here, is the assay detecting variants that alter the function of ASS or is it detecting a complete ablation of enzymatic activity? The results might be strengthened with a follow-up experiment that identifies stably expressed ASS1 variants.

      We agree with the review that distinguishing between stability and enzyme activity would be valuable information. Unfortunately, we do not currently have the resources to perform this type of large-scale study. We have acknowledged in the text that our assay does not distinguish between enzyme activity and protein stability:

      "We expect that the assay will detect reductions in both catalytic activity and protein stability, but will not distinguish between these mechanisms."

      At the very least, it would be great to see the authors replicate some of their interesting results from the high-throughput screen by down-selecting to ~12 variants of uncertain significance that could be newly considered pathogenic.

      We have included new analysis of all 25 VUS variants falling in the pathogenic range of our assay (Supplemental Table S7). Reclassification under current guidelines (in the absence of our data) shifts six variants to Pathogenic/Likely Pathogenic and 11 more are reclassified to Likely Pathogenic with the application of our functional data as PS3_Supporting. The remaining eight VUS are all reclassified to Likely Pathogenic when inclusion of homozygous PrimateAI-benign variants allows the assay to satisfy full PS3 criteria.

      1. I would ask the authors to provide more citations of the literature in the introduction of the manuscript. I would be especially interested in knowing more about human ASS being identified as a homolog of yeast ARG1, as they share little sequence similarity (27.5%) at the protein level. That said, I find the yeast complementation assay exciting.

      We thank the reviewer for this suggestion. Human ASS and yeast Arg1 catalyze the same biochemical reaction and share approximately 49% amino acid sequence identity. We have revised the Introduction to clarify this relationship and to note explicitly that the Saccharomyces Genome Database (SGD) identifies the human gene encoding argininosuccinate synthase (ASS1) as the ortholog of yeast ARG1. An appropriate citation has been added to support this statement. The protein alignments have been provided as File S2.

      "This assay is based on the ability of human ASS to functionally replace (complement) its yeast ortholog (Arg1) in S. cerevisiae (Saccharomyces Genome Database, 2026). Importantly, successful functional complementation indicates that ASS enzymatic activity does not depend on any obligate human-specific protein interactions. At the protein level, human ASS and yeast Arg1 display 49% sequence identity (File S2) and share identical enzymatic roles in converting citrulline and aspartate into argininisuccinate."

      1. I appreciate the efforts made by the authors to share their work and make this study more reproducible, such as sharing the hASS1 and yASS1 plasmids being shared on NCBI Genbank (Line 121) and publishing the ONT reads on SRA (Line 154). I made a requests for additional data to be shared, such as the custom method/code for codon optimization and a table of Twist variant cassettes that were ordered. I would also love to see these results shared on MaveDB.org.

      We thank the reviewer for these suggestions regarding data sharing and reproducibility. As requested, we have provided the custom codon optimization script as File S1 and the amino acid alignment used to perform codon harmonization as File S2. The sequence of the underlying variant cassette is included in the corresponding GenBank entry, and we have clarified this point in the legend of Figure 1. For each amino acid substitution, Twist Bioscience used a yeast-specific codon scheme with a single consistent codon per amino acid; accordingly, the sequence of each variant cassette can be inferred from the base construct and the specified amino acid change. A complete list of variant amino acid substitutions used in this study is provided in Table S3.

      1. I find this manuscript very exciting as the authors have a compelling assay that identifies pathogenic variants, but I was generally disappointed by the quality and organization of the figures. For example, Figure 4 provides very little insight, but could be dramatically improved with an overlay of the normalized growth score data or highlighting variants surrounding the substrate or ATP interfaces. There are some very interesting aspects of this manuscript that could be shine through with some polished figures.

      We thank the reviewer for this feedback and agree that clear and well-organized figures are essential for conveying the key results of the study. In response, we have substantially revised Figure 4 by adding colored overlays showing residue conservation and median normalized growth scores (new panels Figure 4C and 4D), which more directly link structural context to functional outcomes and highlight patterns surrounding the active site and substrate interfaces.

      I would also encourage the authors to generate a heatmap of the data represented in Figure 2 (see Fowler and Fields 2014 PMID 25075907, Figure 2), this would be more helpful reference to the readers.

      The reviewer also suggested that a heatmap representation, similar to that used in Fowler and Fields (2014), might aid interpretation of the data shown in Figure 2. Because our dataset consists of sparse single-amino acid substitutions rather than a complete mutational scan, such heatmaps are inherently less dense and less effective at conveying patterns than in saturation mutagenesis studies. Nevertheless, to aid readers who may find this visualization useful, we have generated and included a single-nucleotide variant heatmap as Supplemental Figure S1.

      My major comments are as follows: 6. Citations needed - especially in the introduction and for establishing that hASS is a homolog of yARG1

      We have added the requested citations and clarified the ASS1-ARG1 orthology in the Introduction, as described in our response to point 3 above.

      1. Generally, the authors do a nice job distinguishing the ASS1 gene from the ASS enzyme, though I found some ambiguities (Line 685). Please double-check the use of each throughout the manuscript.

      We have edited the manuscript to ensure consistent and unambiguous use of gene and enzyme nomenclature throughout.

      1. Generally, I'm confused about what strain was used for integrating all these variants, was is the arg1 knock-out strain from the yeast knockout collection or was it FY4? I think FY4 was used for the preliminary experiments, then the KO collection strain was used for making the variant library but I think this could be made more clear in the text and figures. Lines 226-229 describes introducing the hASS1 and yASS1 sequences into the native ARG1 locus in strain FY4, but the Fig1A image depicts the ASS1 variants going into arg1 KO locus. Fig1A should be moved to Fig2.

      We agree that the strain construction steps were not described as clearly as they could have been. We have therefore clarified the strain construction workflow in the Materials & Methods and Results sections, as well as in the Figure 1 legend, to explicitly distinguish preliminary experiments performed in strain FY4 from construction of the variant library in the arg1 knockout background.

      As we have also added an additional panel to Figure 1 that schematically explains how the screen was performed (per Reviewer #2's request), we believe that Figure 1A is appropriately placed and should remain in Figure 1.

      1. Line 303 - "We classify these variants as 'functionally unimpaired'", this is not an accurate description of these variants as Figure 2 highlights 24 pathogenic ClinVar variants that would fall into this category of "functionally unimpaired". The yeast growth assay appears to capture pathogenic variants, but there is likely some nuance of human ASS functionality that is not being assessed here. I would make the language more specific, e.g. "complementary to Arg1" or "growth-compatible".

      We agree that the label "functionally unimpaired" could be misinterpreted if read as implying clinical benignity. We have therefore clarified within the manuscript that this designation refers strictly to variant behavior in the yeast growth assay (i.e., wild-type-like growth under assay conditions) and does not imply absence of pathogenicity. We also expanded the Discussion to explicitly address the subset of clinically pathogenic variants with high growth scores (>0.85), consistent with a ceiling effect of the assay and a key limitation for interpretation. See response to reviewer #3 point 1. Relevant revisions are also discussed in our responses to Reviewers #1 and #2.

      1. Lines 345-355 - It is interesting that there are variants that appear functional at the substrate interfacing sites. Is there anything common across these variants? Are they maintaining the polarity or hydrophobicity of the WT residue? Are any of these variants included in ClinVar or gnomAD? Are pathogenic variants found at any of these sites

      Yes. For highly sensitive active-site residues that have few permissible variants, the vast majority of amino acid substitutions that do retain activity preserve key physicochemical properties of the wild-type residue, such as hydrophobicity or charge. We have added this important observation to the manuscript:

      "Any variants at these sensitive residues that are permissive for activity in our assay retain hydrophobicity or charged states relative to the original amino acid side chain (Figure 5A & Table S5)."

      None of these variants are present in ClinVar. Only L15V and E191D are present in gnomAD (Table S4).

      1. Lines 423-430 - The OddsPath calculation would seem to rely heavily on the thresholds of .85 for normalized growth. The OddsPath calculation could be bolstered with some additional analysis that emphasizes the robustness to alternative thresholds.

      We agree that the sensitivity of the OddsPath calculation to the choice of growth thresholds is an important consideration. In our assay, benign ClinVar variants and non-human primate variants are observed exclusively within the peak centered on wild-type growth, whereas clinically annotated variants falling below this peak are exclusively pathogenic. On this basis, we defined the upper boundary of the assay range interpreted as supporting pathogenicity as the lower boundary of the wild-type-centered peak in the growth distribution (as defined in Figure 3), rather than selecting a cutoff by direct optimization of the OddsPath. This choice reflects the observed concordance, in our dataset, between the onset of measurable functional impairment in the assay and clinical pathogenic annotation. Importantly, in practice the OddsPath value is locally robust to the precise placement of this boundary, remaining invariant across the range 0.82-0.88. Supporting our chosen threshold of 0.85, the lowest-growth benign or primate variant observed has a normalized growth value of 0.88, while the lowest growth observed among variants present as homozygotes in gnomAD was 0.86. We have clarified this rationale and analysis in the revised manuscript.

      "Notably, the "Among all nine of the human ASS1 missense variants observed as homozygotes in gnomAD which were tested as amino acid substitutions in our assay, the lowest observed growth value was 0.86 (Ala258Val) consistent with the lower boundary of the PrimateAI variants which was a growth value of 0.87 (Ala81Thr) (Figure 6) and with our use of a 0.85 classification threshold."

      "If we treat PrimateAI variants as benign (solely for OddsPath calculation purposes), the OddsPath for growth

      1. Lines 432-441 - This is an interesting idea to use variants observed in primates, has ACMG weighed in on this? I understand that CTLN1 is an autosomal recessive disorder but I'd still be interested in seeing how the observed ASS1 missense variants in gnomAD perform in your growth assay, possibly a supplemental figure?

      To our knowledge, the ACMG/AMP guidelines do not currently address the use of homozygous missense variants observed in non-human primates. We are currently in discussion with two ClinGen working groups to discuss the possibility of formalizing the use of this data source.

      We agree that comparison with human population data is also important. Accordingly, total gnomAD allele counts and homozygous counts for all applicable ASS1 missense variants are provided in Table S4, and the growth behavior of ASS1 missense variants observed in the homozygous state in gnomAD is shown in Figure 6. These homozygous variants uniformly exhibit high growth in our assay, consistent with the absence of strong loss-of-function effects. We have updated the manuscript text to clarify these points.

      Minor comments 1. Lines 53-59 - This paragraph needs to cite the literature, especially lines 56, 57, and 59 2. Line 61 - no need to repeat "citrullinemia type I", just use the abbreviation as it was introduced in the paragraph above 3. Lines 61-71 - again, this paragraph needs more literature citations 4. Line 62 - change to "results"

      The changes suggested in points 1-4 have all been implemented in the revised manuscript.

      1. Line 74-75 - "RUSP" acronym not needed as it's never used in the manuscript, the same goes for "HHS"

      We agree that the acronyms "RUSP" and "HHS" are not reused elsewhere in the manuscript. We have nevertheless retained them at first mention, alongside the expanded names, because these acronyms are commonly used in newborn screening and public health policy contexts and may be more familiar to some readers than the expanded terms. We would be happy to remove the acronyms if preferred.

      1. Line 86 - "ASS1" I think is referring to the enzyme and should just be "ASS"? If referring to the gene then italicize to "ASS1"
      2. Lines 91-93 - It would be helpful to mention this is a functional screen in yeast
      3. Line 101 - It would be helpful to the readers to define SD before using the acronym, consider changing to "minimal synthetic defined (SD) medium" and afterwards can refer to as "SD medium"
      4. 109-114 - It would be great if you could share your method for designing the codon-harmonized yASS1 gene, consider sharing as a supplemental script or creating a GitHub repository linked to a Zenodo DOI for publication.

      The changes suggested in points 6-9 have all been implemented in the revised manuscript. The codon harmonization script has been provided as File S1.

      1. Lines 135-137 - I think it's helpful to provide a full table of the cassettes ordered from Twist as well as the primers used to amplify them, consider a supplemental table.

      Details of Twist cassette and the primer sequences used for amplification have been added to the Materials & Methods.

      1. Line 138 - "standard methods" is a bit vague, I'm guessing this is a Geitz and Schiestl 2007 LiAc/ssDNA protocol (PMID 17401334)? Also, was ClonNAT used to select for natMX colonies?

      The reviewer is correct about which protocol was used, and we have added the citation. We have also clarified that selection was carried out based on resistance to nourseothricin.

      1. Line 150 - change to "sequence the entire open reading frame, as previously described [4]."
      2. Line 222-223 - remove "replace" and just use "complement" (and remove the parenthesis)
      3. Line 249 - It would be great to see a supplemental alignment of the hASS1 and yASS1 sequences.
      4. Line 261 - spelling "citrullemia" should be corrected to "citrullinemia"
      5. Line 280 - "using Oxford Nanopore sequencing" is a bit vague, I suggest specifying the equipment used (e.g. Oxford Nanopore Technologies MinION platform) or simplify to "via long-read sequencing (see Materials & Methods)"

      The changes suggested in points 12-16 have all been implemented in the revised manuscript. An alignment of the ASS and Arg1 protein sequences has been provided as File S2.

      1. Line 287-289 - It would be great to see the average number of isolates per variant, as well as a plot of the variant growth estimate vs individual isolate growth.

      We agree with the reviewer that conveying measurement precision is important. The number of isolates assayed per variant is provided in Table S4, and we have added explicit mention of this in the text. Because variants were assayed with a mixture of 1, 2, or {greater than or equal to}3 independent isolates, a scatterplot of variant-level growth estimates versus individual isolate measurements would be difficult to interpret and potentially misleading. Instead, we report standard error estimates for each variant in Table S4, derived from the linear model used to estimate growth effects, which more appropriately summarizes measurement uncertainty given the experimental design.

      1. Lines 324-25 - consider removing the last sentence of this paragraph, it is redundant as the following paragraph starts with the same statement.

      We have removed this sentence.

      1. Lines 327-335 - This is interesting and would benefit from its own subpanel or plot in which the normalized growth score is plotted against variants that are at conserved or diverse residues in human ASS, and see if there's a statistical difference in score between the two groupings.

      As suggested by the reviewer, we have added Supplemental Figure 2 (Figure S2) in which the normalized growth score of each variant is plotted against the conservation of the corresponding residue, as measured by ConSurf. The manuscript already includes a statistical analysis of the relationship between residue conservation and functional impact, showing that amorphic variants occur significantly more frequently at highly conserved residues than unimpaired variants do (one-sided Fisher's exact test). We now refer to this new supplemental figure in the relevant Results section.

      1. Lines 339-341 - As written, it is unclear if aspartate interacts with all of the same residues as citrulline or just Asn123 and Thr119.
      2. Lines 345-355 - As with my above comment, I find this interesting and would
      3. Line 353 - add a period to "al" in "Diez-Fernandex et al."

      The issues raised in points 20 and 22 have all addressed. Point 21 appears to be truncated.

      1. Figure 1 a. Remove "Figure" from the subpanels and show just "A" and "B" (as you do for Figure 4) and combine the two images into a single image. Also make this correction to Figure 5 and Figure 8. b. Panel A - I thought the hASS1 and yASS1 were dropped into FY4, not the arg1 KO strain. This needs clarification. c. Panel A - I'm assuming the natMX cassette contains its own promoter, you could use a right-angled arrow to indicate where the promotors are in your construct. d. Panel B - I'm not sure the bar graph is necessary, it would be more helpful to see calculations of the colony size (or growth curves for each strain) and plot the raw values (maybe pixel counts?) for each replicate rather than normalizing to yeast ARG1. I would be great to have a supplemental figure showing all the replicates side-by-side. e. Panel B - Would be helpful to denote the pathogenic and benign ClinVar variants with an icon or colored text.

      f. Figure 1 Caption - make "A)" and "B)" bold.

      We have implemented the requested changes in Figure 1 with the following exceptions. We have retained panels A and B as separate subfigures because they illustrate distinct experimental concepts. In addition, we respectfully disagree with point (d). The bar graph is intended to provide a clear, high-level comparison of functional complementation by hASS1 versus yASS1 and to illustrate the gross differences in growth between benign and pathogenic proof-of-principle variants. As the bar graph includes error bars for standard deviations, presenting raw colony size measurements or growth curves for individual replicates would substantially complicate the figure without materially improving interpretability for this purpose.

      1. Figure 2 a. "Shown in magenta are amino acid substitutions corresponding to ClinVar pathogenic, pathogenic/likely pathogenic, and likely pathogenic variants" is repeated in the figure caption. b. "Shown in green are amino acid substitutions corresponding to ClinVar benign and likely benign variants." I don't see any green points. c. Identify the colors used for ASS1 substrate binding residues. d. This plot would benefit from a depiction of the human ASS secondary structure and any protein domains (nucleotide-binding domain, synthase domain, and C-terminal helix from Fig4B)

      e. Line 685 675 - "ASS1" is being used in reference to the enzyme, is this correct or should it be "ASS"?

      We have made the requested changes to Figure 2. The repeated caption text has been removed, and references to green points have been corrected to orange points to match the figure. The colors used to indicate ASS substrate-binding residues are explicitly described in the figure key. Secondary structure annotations have been added. References to the enzyme have been corrected to "ASS" rather than "ASS1" where appropriate.

      1. Figure 3 a. Rename the "unimpaired" category as there are several pathogenic ClinVar variants that fall into this category.

      To address this point, we have clarified the labeling by adding "in our yeast assay" to the figure legend, making explicit that the "unimpaired" category refers only to wild-type-like behavior under assay conditions and does not imply clinical benignity. See also response to Reviewer #3, Major Comment 1.

      1. Figure 4 a. List the PDB or AlphaFold accession used for this structure b. Panel A - state which colors are used for to depict each monomer. It is confusing to see several shades of pink/purple used to depict a single monomer in Panel A. c. It is very difficult to make out the aspartate and citrulline substrates in the catalytic binding activity, consider making an inset zooming-in on this domain and displaying a ribbon diagram of the structure rather than the surface. d. Generally, it would be more helpful here to label any particular residues that were identified as pathogenic from your screen, or to overlay average grow scores per residue data onto the structure

      We have implemented the requested changes to Figure 4. The relevant PDB/AlphaFold accession is now listed, and the colors used to depict each monomer in Panel A are clarified in the figure legend. An inset focusing on the active site has been added to improve visualization of the citrulline and aspartate substrates. In addition, we have added new panels (Figure 4C and 4D) overlaying pathogenic residues and average growth scores onto the structure to more directly link structural context with functional data.

      1. Figure 5 a. Line 716 - Insert a page break to place Figure 5 on its own page b. I suggest using a heatmap for this type of plot, as it is very difficult to track which color corresponds to which residue.

      c. Fig5A - This plot could be improved by identifying which residue positions interface with which substrate.

      We have placed Figure 5 on its own page and added information to the legend identifying which residue positions interface with each substrate. We have retained the active-site variant strip charts raised in point (b), as we believe they effectively illustrate how the distribution of variant effects differs between residues. In addition, we have provided a supplemental heatmap showing variant growth across the entire protein (Figure S1), and individual variant scores for all residues are provided in Table S4.

      1. Figure 7 a. Line 735 - Insert page break to place figure on a new page

      List the PDB accession used for these images. c. For clarity I would mention "human ASS" in the figure title d. State the colors of the substrates e. Panels A and B could be combined into a single panel, making it easier to distinguish the active site and dimerization variants.

      f. Could be interesting to get SASA scores for the ClinVar structural variants to determine if they are surface-accessible

      We have implemented the requested changes in Figure 7 with the following exceptions. For point (e), there is no single orientation of the structure that allows a clear simultaneous view of both active-site and dimerization variants; accordingly, we have retained panels A and B as separate subfigures to preserve clarity. With respect to point (f), we agree that solvent accessibility analysis could be informative in other contexts. However, such an analysis does not integrate naturally with the functional and assay-based framework of the present study and was therefore not included.

      1. Figure 8 a. Panel B - overlay a square frame in the larger protein structure that depicts where the below inset is focused, and frame inset image as well.

      We have framed the inset image as requested. We did not add a corresponding frame to the full protein structure, as doing so obscured structural details in the region of interest.

      Reviewer #3 (Significance (Required)):

      Section 2 - Significance This study represents a substantial technical, functional, and translational advance in the interpretation of missense variation in ASS1, a gene of high clinical relevance for the rare disease citrullinemia type I. Its principal strength lies in the generation of an experimentally validated functional atlas of ASS1 missense variants that covers ~90% of all SNV-accessible substitutions. The scale, internal reproducibility, and careful benchmarking of the yeast complementation assay against known pathogenic and benign variants provide a robust foundation for identifying pathogenic ASS1 variants. Particularly strong aspects include the rigorous quality control of variant identities, the quantitative nature of the functional readout, and the thoughtful integration of results into the ACMG/AMP OddsPath framework. The discovery of intragenic complementation between variants affecting distinct structural regions of the enzyme is a notable conceptual and mechanistic contribution. Limitations include the assay's reduced sensitivity to variants impacting oligomerization or subtle folding defects, and the use of yeast as a heterologous system, which may mask disease-relevant mechanisms as several pathogenic ClinVar variants were found to be "functionally unimpaired". Future work extending functional testing to additional cellular contexts or expanding genotype-level combinatorial analyses would further enhance clinical applicability. Relative to prior studies, which have relied on small numbers of patient-derived variants or low-throughput biochemical assays, this work extends the field decisively by delivering a comprehensive, variant-resolved functional map for ASS1. To the best of my current knowledge, this is the first systematic functional screen of ASS1 at this scale and the first direct experimental demonstration that ASS active sites span multiple subunits, enabling intragenic complementation consistent with Crick and Orgel's classic variant sequestration model. As such, the advance is simultaneously technical (high-throughput functional genomics), mechanistic (defining structural contributors to catalysis and epistasis), and clinical (enabling evidence-based reclassification of VUS). I find the use of homozygous non-human primate variants as an orthogonal benign calibration set both creative and controversial, my hope would be that this manuscript will prompt a productive discussion.

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      Referee #3

      Evidence, reproducibility and clarity

      Summary

      This manuscript presents a comprehensive functional profiling of 2,193 ASS1 missense variants using a yeast complementation assay, providing valuable data for variant interpretation in the rare disease citrullinemia type I. The dataset is extensive, technically sound, and clinically relevant. The demonstration of intragenic complementation in ASS1 is novel and conceptually important. Overall, the study represents a substantial contribution to functional genomics and rare disease variant interpretation.

      Major comments

      This is an exciting paper as it can provide support to clinicians to make actionable decisions when diagnosing infants. I have a few major comments, but I want to emphasize the label of "functionally unimpaired" variants to be misleading. The authors explain that there are several pathogenic ClinVar variants that fall into this category (above the >.85 growth threshold) but I think this category needs a more specific name and I would ask the authors to reiterate the shortcomings of the assay again in the Discussion section. I think there's an important discussion to be had here, is the assay detecting variants that alter the function of ASS or is it detecting a complete ablation of enzymatic activity? The results might be strengthened with a follow-up experiment that identifies stably expressed ASS1 variants. At the very least, it would be great to see the authors replicate some of their interesting results from the high-throughput screen by down-selecting to ~12 variants of uncertain significance that could be newly considered pathogenic. I would ask the authors to provide more citations of the literature in the introduction of the manuscript. I would be especially interested in knowing more about human ASS being identified as a homolog of yeast ARG1, as they share little sequence similarity (27.5%) at the protein level. That said, I find the yeast complementation assay exciting. I appreciate the efforts made by the authors to share their work and make this study more reproducible, such as sharing the hASS1 and yASS1 plasmids being shared on NCBI Genbank (Line 121) and publishing the ONT reads on SRA (Line 154). I made a requests for additional data to be shared, such as the custom method/code for codon optimization and a table of Twist variant cassettes that were ordered. I would also love to see these results shared on MaveDB.org. I find this manuscript very exciting as the authors have a compelling assay that identifies pathogenic variants, but I was generally disappointed by the quality and organization of the figures. For example, Figure 4 provides very little insight, but could be dramatically improved with an overlay of the normalized growth score data or highlighting variants surrounding the substrate or ATP interfaces. There are some very interesting aspects of this manuscript that could be shine through with some polished figures. I would also encourage the authors to generate a heatmap of the data represented in Figure 2 (see Fowler and Fields 2014 PMID 25075907, Figure 2), this would be more helpful reference to the readers.

      My major comments are as follows:

      1. Citations needed - especially in the introduction and for establishing that hASS is a homolog of yARG1
      2. Generally, the authors do a nice job distinguishing the ASS1 gene from the ASS enzyme, though I found some ambiguities (Line 685). Please double-check the use of each throughout the manuscript
      3. Generally, I'm confused about what strain was used for integrating all these variants, was is the arg1 knock-out strain from the yeast knockout collection or was it FY4? I think FY4 was used for the preliminary experiments, then the KO collection strain was used for making the variant library but I think this could be made more clear in the text and figures. Lines 226-229 describes introducing the hASS1 and yASS1 sequences into the native ARG1 locus in strain FY4, but the Fig1A image depicts the ASS1 variants going into arg1 KO locus. Fig1A should be moved to Fig2.
      4. Line 303 - "We classify these variants as 'functionally unimpaired'", this is not an accurate description of these variants as Figure 2 highlights 24 pathogenic ClinVar variants that would fall into this category of "functionally unimpaired". The yeast growth assay appears to capture pathogenic variants, but there is likely some nuance of human ASS functionality that is not being assessed here. I would make the language more specific, e.g. "complementary to Arg1" or "growth-compatible".
      5. Lines 345-355 - It is interesting that there are variants that appear functional at the substrate interfacing sites. Is there anything common across these variants? Are they maintaining the polarity or hydrophobicity of the WT residue? Are any of these variants included in ClinVar or gnomAD? Are pathogenic variants found at any of these sites
      6. Lines 423-430 - The OddsPath calculation would seem to rely heavily on the thresholds of <.05 and >.85 for normalized growth. The OddsPath calculation could be bolstered with some additional analysis that emphasizes the robustness to alternative thresholds.
      7. Lines 432-441 - This is an interesting idea to use variants observed in primates, has ACMG weighed in on this? I understand that CTLN1 is an autosomal recessive disorder but I'd still be interested in seeing how the observed ASS1 missense variants in gnomAD perform in your growth assay, possibly a supplemental figure?

      Minor comments

      1. Lines 53-59 - This paragraph needs to cite the literature, especially lines 56, 57, and 59
      2. Line 61 - no need to repeat "citrullinemia type I", just use the abbreviation as it was introduced in the paragraph above
      3. Lines 61-71 - again, this paragraph needs more literature citations
      4. Line 62 - change to "results"
      5. Line 74-75 - "RUSP" acronym not needed as it's never used in the manuscript, the same goes for "HHS"
      6. Line 86 - "ASS1" I think is referring to the enzyme and should just be "ASS"? If referring to the gene then italicize to "ASS1"
      7. Lines 91-93 - It would be helpful to mention this is a functional screen in yeast
      8. Line 101 - It would be helpful to the readers to define SD before using the acronym, consider changing to "minimal synthetic defined (SD) medium" and afterwards can refer to as "SD medium"
      9. 109-114 - It would be great if you could share your method for designing the codon-harmonized yASS1 gene, consider sharing as a supplemental script or creating a GitHub repository linked to a Zenodo DOI for publication.
      10. Lines 135-137 - I think it's helpful to provide a full table of the cassettes ordered from Twist as well as the primers used to amplify them, consider a supplemental table
      11. Line 138 - "standard methods" is a bit vague, I'm guessing this is a Geitz and Schiestl 2007 LiAc/ssDNA protocol (PMID 17401334)? Also, was ClonNAT used to select for natMX colonies?
      12. Line 150 - change to "sequence the entire open reading frame, as previously described [4]."
      13. Line 222-223 - remove "replace" and just use "complement" (and remove the parenthesis)
      14. Line 249 - It would be great to see a supplemental alignment of the hASS1 and yASS1 sequences
      15. Line 261 - spelling "citrullemia" should be corrected to "citrullinemia"
      16. Line 280 - "using Oxford Nanopore sequencing" is a bit vague, I suggest specifying the equipment used (e.g. Oxford Nanopore Technologies MinION platform) or simplify to "via long-read sequencing (see Materials & Methods)"
      17. Line 287-289 - It would be great to see the average number of isolates per variant, as well as a plot of the variant growth estimate vs individual isolate growth
      18. Lines 324-25 - consider removing the last sentence of this paragraph, it is redundant as the following paragraph starts with the same statement
      19. Lines 327-335 - This is interesting and would benefit from its own subpanel or plot in which the normalized growth score is plotted against variants that are at conserved or diverse residues in human ASS, and see if there's a statistical difference in score between the two groupings
      20. Lines 339-341 - As written, it is unclear if aspartate interacts with all of the same residues as citrulline or just Asn123 and Thr119.
      21. Lines 345-355 - As with my above comment, I find this interesting and would
      22. Line 353 - add a period to "al" in "Diez-Fernandex et al."
      23. Figure 1

      a. Remove "Figure" from the subpanels and show just "A" and "B" (as you do for Figure 4) and combine the two images into a single image. Also make this correction to Figure 5 and Figure 8

      b. Panel A - I thought the hASS1 and yASS1 were dropped into FY4, not the arg1 KO strain. This needs clarification

      c. Panel A - I'm assuming the natMX cassette contains its own promoter, you could use a right-angled arrow to indicate where the promotors are in your construct

      d. Panel B - I'm not sure the bar graph is necessary, it would be more helpful to see calculations of the colony size (or growth curves for each strain) and plot the raw values (maybe pixel counts?) for each replicate rather than normalizing to yeast ARG1. I would be great to have a supplemental figure showing all the replicates side-by-side

      e. Panel B - Would be helpful to denote the pathogenic and benign ClinVar variants with an icon or colored text

      f. Figure 1 Caption - make "A)" and "B)" bold 24. Figure 2

      a. "Shown in magenta are amino acid substitutions corresponding to ClinVar pathogenic, pathogenic/likely pathogenic, and likely pathogenic variants" is repeated in the figure caption

      b. "Shown in green are amino acid substitutions corresponding to ClinVar benign and likely benign variants." I don't see any green points

      c. Identify the colors used for ASS1 substrate binding residues

      d. This plot would benefit from a depiction of the human ASS secondary structure and any protein domains (nucleotide-binding domain, synthase domain, and C-terminal helix from Fig4B)

      e. Line 685 - "ASS1" is being used in reference to the enzyme, is this correct or should it be "ASS"? 25. Figure 3

      a. Rename the "unimpaired" category as there are several pathogenic ClinVar variants that fall into this category 26. Figure 4

      a. List the PDB or AlphaFold accession used for this structure

      b. Panel A - state which colors are used for to depict each monomer. It is confusing to see several shades of pink/purple used to depict a single monomer in Panel A

      c. It is very difficult to make out the aspartate and citrulline substrates in the catalytic binding activity, consider making an inset zooming-in on this domain and displaying a ribbon diagram of the structure rather than the surface.

      d. Generally, it would be more helpful here to label any particular residues that were identified as pathogenic from your screen, or to overlay average grow scores per residue data onto the structure 27. Figure 5

      a. Line 716 - Insert a page break to place Figure 5 on its own page

      b. I suggest using a heatmap for this type of plot, as it is very difficult to track which color corresponds to which residue

      c. Fig5A - This plot could be improved by identifying which residue positions interface with which substrate 28. Figure 7

      a. Line 735 - Insert page break to place figure on a new page

      b. List the PDB accession used for these images

      c. For clarity I would mention "human ASS" in the figure title

      d. State the colors of the substrates

      e. Panels A and B could be combined into a single panel, making it easier to distinguish the active site and dimerization variants

      f. Could be interesting to get SASA scores for the ClinVar structural variants to determine if they are surface-accessible 29. Figure 8

      a. Panel B - overlay a square frame in the larger protein structure that depicts where the below inset is focused, and frame inset image as well.

      Significance

      This study represents a substantial technical, functional, and translational advance in the interpretation of missense variation in ASS1, a gene of high clinical relevance for the rare disease citrullinemia type I. Its principal strength lies in the generation of an experimentally validated functional atlas of ASS1 missense variants that covers ~90% of all SNV-accessible substitutions. The scale, internal reproducibility, and careful benchmarking of the yeast complementation assay against known pathogenic and benign variants provide a robust foundation for identifying pathogenic ASS1 variants. Particularly strong aspects include the rigorous quality control of variant identities, the quantitative nature of the functional readout, and the thoughtful integration of results into the ACMG/AMP OddsPath framework. The discovery of intragenic complementation between variants affecting distinct structural regions of the enzyme is a notable conceptual and mechanistic contribution. Limitations include the assay's reduced sensitivity to variants impacting oligomerization or subtle folding defects, and the use of yeast as a heterologous system, which may mask disease-relevant mechanisms as several pathogenic ClinVar variants were found to be "functionally unimpaired". Future work extending functional testing to additional cellular contexts or expanding genotype-level combinatorial analyses would further enhance clinical applicability.

      Relative to prior studies, which have relied on small numbers of patient-derived variants or low-throughput biochemical assays, this work extends the field decisively by delivering a comprehensive, variant-resolved functional map for ASS1. To the best of my current knowledge, this is the first systematic functional screen of ASS1 at this scale and the first direct experimental demonstration that ASS active sites span multiple subunits, enabling intragenic complementation consistent with Crick and Orgel's classic variant sequestration model. As such, the advance is simultaneously technical (high-throughput functional genomics), mechanistic (defining structural contributors to catalysis and epistasis), and clinical (enabling evidence-based reclassification of VUS). I find the use of homozygous non-human primate variants as an orthogonal benign calibration set both creative and controversial, my hope would be that this manuscript will prompt a productive discussion.

    1. IAM Guidelines & Best Practices 1) MFA 2) IMA Roles instead of USER 3) Follow the principle of least provilage 4) rotate user credentials 5) use managed policies

      Shared Responsibility | AWS | Security of infastructure , Avalibity of IAM services, Offer Policies for common scenarios.

      | Customer | manage and secure accss keys and user accounts, implement strong passwods, utlized mFA, IAM permision must be implemented correctly and follow the principle of least privilage.

    1. Briefing : L'émancipation de l'Éducation nationale face au monopole de Microsoft

      Ce document synthétise les enjeux de la dépendance technologique de l'Éducation nationale française envers Microsoft et l'émergence d'une alternative structurée autour du logiciel libre et de la collaboration enseignante.

      Résumé Exécutif

      L'Éducation nationale française fait face à une dépendance coûteuse et structurelle vis-à-vis des solutions propriétaires, principalement Microsoft.

      Le passage imposé de Windows 10 à Windows 11 illustre cette vulnérabilité : l'obsolescence logicielle pourrait coûter jusqu'à un milliard d'euros à l'échelle nationale pour le renouvellement du parc informatique.

      Face à ce constat, une "guérilla" de l'open source s'organise. Portée par la Direction du numérique pour l'éducation (DNE) et des initiatives comme « La Forge », cette dynamique mobilise désormais 10 000 enseignants-développeurs.

      L'objectif est de substituer aux licences onéreuses des « communs numériques » (Linux, BigBlueButton, NextCloud), garantissant la souveraineté des données, la pérennité des investissements publics et une pédagogie adaptée aux besoins réels du terrain.

      --------------------------------------------------------------------------------

      1. Le constat d'une dépendance critique : Le "cas d'école" Microsoft

      La relation entre l'institution scolaire et Microsoft est décrite comme une forme d'addiction budgétaire et technique.

      Le coût de l'obsolescence imposée

      L'exemple des Hauts-de-France : Suite à une cyberattaque par ransomware, la région a dû envisager la migration vers Windows 11.

      Un membre de la DSI a estimé à 100 millions d'euros le coût pour renouveler 30 000 PC incapables de supporter cette mise à jour.

      Extrapolation nationale : Les Hauts-de-France représentant environ 10 % de l'éducation nationale, le coût total pour la mise à jour forcée du parc (300 000 machines) est estimé à 1 milliard d'euros.

      La vente liée : Le monopole s'appuie sur le mécanisme de la vente liée, où le système d'exploitation est pré-installé sans distinction de prix entre le matériel et le logiciel, imposant une solution "clé en main" qui freine l'adoption d'alternatives.

      Limites des services propriétaires

      Coûts récurrents : Des dizaines de millions d'euros sont versés chaque année en licences.

      Failles systémiques : La crise du Covid-19 a révélé les carences du système numérique éducatif, notamment sa dépendance à des solutions propriétaires onéreuses et son manque de cohérence globale.

      --------------------------------------------------------------------------------

      2. La stratégie de rupture par le Logiciel Libre

      Face au monopole, des solutions basées sur Linux et l'open source prouvent leur viabilité sur le terrain.

      Distributions Linux dédiées à l'éducation

      Il existe des alternatives robustes permettant d'adapter l'ordinateur aux besoins pédagogiques :

      PrimTux : Système d'exploitation spécifique pour les écoles primaires.

      ND (Numérique Inclusif, Responsable et Durable) : Distribution destinée au secondaire.

      Obstacles et leviers d'adoption

      | Obstacle | État des lieux | Perspectives | | --- | --- | --- | | Logiciels métiers | Certains éditeurs (SVT, physique, techno) ne développent que pour Windows. | Pression par la masse : l'augmentation du parc Linux doit forcer les éditeurs à s'adapter. | | Logiciels de vie scolaire | Pronote dispose d'un client Windows complet mais d'une version web dégradée sous Linux. | Nécessité d'une évolution des clients vers des standards interopérables. | | Résilience | En cas d'attaque (ransomware), les systèmes sous Windows ont été paralysés. | Des lycées sous Linux (ex: Lycée Carnot à Bruay-la-Buissière) ont pu proposer leur aide et leurs outils. |

      --------------------------------------------------------------------------------

      3. « La Forge » : L'industrialisation de l'innovation enseignante

      « La Forge » représente un changement de paradigme : passer de l'enseignant "bricoleur" isolé à une communauté structurée de développeurs au sein de l'État.

      Un modèle collaboratif massif

      Effectifs : 10 000 enseignants inscrits.

      Volume : 6 500 projets (dépôts de code) enregistrés.

      Fonctionnement : Outil de travail collaboratif (basé sur le modèle GitHub) permettant de fédérer, tester et partager des codes sources et des ressources pédagogiques.

      Exemples de projets emblématiques

      MindStory : Alternative open source à Minecraft, permettant aux élèves de collaborer sur des constructions sans dépendre d'un compte Microsoft payant.

      Philo GPT : Interface permettant de dialoguer avec des simulations de grands philosophes.

      Execubot : Outil d'apprentissage de la programmation via un robot virtuel.

      Créa-appli : Outil utilisant l'IA pour aider les profs à générer des prototypes d'applications (HTML/JS) via le "vibe coding" (codage par prompt).

      --------------------------------------------------------------------------------

      4. Souveraineté, Communs Numériques et Commande Publique

      L'enjeu n'est pas seulement technique, il est politique et financier : assurer que l'argent public finance des biens publics.

      La notion de "Communs Numériques"

      Un commun numérique repose sur trois piliers : une ressource, une communauté et une gouvernance. L'idée est que l'amélioration d'un logiciel par le ministère bénéficie à tous.

      Les services souverains déjà déployés

      Le ministère opère et héberge ses propres instances de logiciels libres pour s'affranchir des GAFAM :

      BigBlueButton : Alternative à Zoom/Meet pour la visioconférence (participation financière du ministère au développement du projet global).

      Apps.education.fr : Portail regroupant des outils comme Tube (alternative à YouTube basée sur PeerTube) ou NextCloud (alternative à Google Drive).

      Critique du modèle traditionnel de commande publique

      Par le passé, l'État stimulait les start-ups ("EdTech") via des marchés publics sans exiger la propriété intellectuelle :

      1. Les entreprises conservaient le code source et les données.

      2. L'État devait payer des abonnements pour continuer à utiliser ce qu'il avait financé.

      3. Résultat : Aucune capitalisation sur le long terme.

      La nouvelle approche privilégie la pérennité : Investir dans l'open source permet à l'institution de conserver la maîtrise de ses outils, même après la fin d'un contrat avec un prestataire.

      --------------------------------------------------------------------------------

      Citations Clés

      « L'éducation nationale est accro à Microsoft. Chaque année, des dizaines de millions d'euros s'envolent en licences. »

      « Le slogan de la forge c'est : "L'union fait la forge". »

      « On a oublié que nos profs étaient aussi capables de fabriquer leurs propres ressources... On a passé des marchés avec ces EdTech où on n'exigeait rien en termes de propriété intellectuelle. Les boîtes repartaient avec l'ensemble du code. »

      « Un milliard pour faire une mise à jour de système d'exploitation qui était imposée par Microsoft parce que Microsoft a déclaré qu'ils arrêtent le support de Windows 10. »

    1. Author response:

      The following is the authors’ response to the original reviews

      Reviewer #1:

      (1) Legionella effectors are often activated by binding to eukaryote-specific host factors, including actin. The authors should test the following: a) whether Lfat1 can fatty acylate small G-proteins in vitro; b) whether this activity is dependent on actin binding; and c) whether expression of the Y240A mutant in mammalian cells affects the fatty acylation of Rac3 (Figure 6B), or other small G-proteins.

      We were not able to express and purify the full-length recombinant Lfat1 to perform fatty acylation of small GTPases in vitro. However, In cellulo overexpression of the Y240A mutant still retained ability to fatty acylate Rac3 and another small GTPase RheB (see Figure 6-figure supplement 2). We postulate that under infection conditions, actin-binding might be required to fatty acylate certain GTPases due to the small amount of effector proteins that secreted into the host cell.

      (2) It should be demonstrated that lysine residues on small G-proteins are indeed targeted by Lfat1. Ideally, the functional consequences of these modifications should also be investigated. For example, does fatty acylation of G-proteins affect GTPase activity or binding to downstream effectors?

      We have mutated K178 on RheB and showed that this mutation abolished its fatty acylation by Lfat1 (see Author response image 1 below). We were not able to test if fatty acylation by Lfat1 affect downstream effector binding.

      Author response image 1.

      (3) Line 138: Can the authors clarify whether the Lfat1 ABD induces bundling of F-actin filaments or promotes actin oligomerization? Does the Lfat1 ABD form multimers that bring multiple filaments together? If Lfat1 induces actin oligomerization, this effect should be experimentally tested and reported. Additionally, the impact of Lfat1 binding on actin filament stability should be assessed. This is particularly important given the proposed use of the ABD as an actin probe.

      The ABD domain does not form oligomer as evidenced by gel filtration profile of the ABD domain. However, we do see F-actin bundling in our in vitro -F-actin polymerization experiment when both actin and ABD are in high concentration (data not shown). Under low concentration of ABD, there is not aggregation/bundling effect of F-actin.

      (4) Line 180: I think it's too premature to refer to the interaction as having "high specificity and affinity." We really don't know what else it's binding to.

      We have revised the text and reworded the sentence by removing "high specificity and affinity."

      (5) The authors should reconsider the color scheme used in the structural figures, particularly in Figures 2D and S4.

      Not sure the comments on the color scheme of the structure figures.

      (6) In Figure 3E, the WT curve fits the data poorly, possibly because the actin concentration exceeds the Kd of the interaction. It might fit better to a quadratic.

      We have performed quadratic fitting and replaced Figure 3E.

      (7) The authors propose that the individual helices of the Lfat1 ABD could be expressed on separate proteins and used to target multi-component biological complexes to F-actin by genetically fusing each component to a split alpha-helix. This is an intriguing idea, but it should be tested as a proof of concept to support its feasibility and potential utility.

      It is a good suggestion. We plan to thoroughly test the feasibility of this idea as one of our future directions.

      (8) The plot in Figure S2D appears cropped on the X-axis or was generated from a ~2× binned map rather than the deposited one (pixel size ~0.83 Å, plot suggests ~1.6 Å). The reported pixel size is inconsistent between the Methods and Table 1-please clarify whether 0.83 Å refers to super-resolution.

      Yes, 0.83 Å is super-resolution.  We have updated in the cryoEM table

      Reviewer #2:

      Weaknesses:

      (1) The authors should use biochemical reactions to analyze the KFAT of Llfat1 on one or two small GTPases shown to be modified by this effector in cellulo. Such reactions may allow them to determine the role of actin binding in its biochemical activity. This notion is particularly relevant in light of recent studies that actin is a co-factor for the activity of LnaB and Ceg14 (PMID: 39009586; PMID: 38776962; PMID: 40394005). In addition, the study should be discussed in the context of these recent findings on the role of actin in the activity of L. pneumophila effectors.

      We have new data showed that Actin binding does not affect Lfat1 enzymatic activity. (see response to Reviewer #1). We have added this new data as Figure S7 to the paper. Accordingly, we also revised the discussion by adding the following paragraph.

      “The discovery of Lfat1 as an F-actin–binding lysine fatty acyl transferase raised the intriguing question of whether its enzymatic activity depends on F-actin binding. Recent studies have shown that other Legionella effectors, such as LnaB and Ceg14, use actin as a co-factor to regulate their activities. For instance, LnaB binds monomeric G-actin to enhance its phosphoryl-AMPylase activity toward phosphorylated residues, resulting in unique ADPylation modifications in host proteins  (Fu et al, 2024; Wang et al, 2024). Similarly, Ceg14 is activated by host actin to convert ATP and dATP into adenosine and deoxyadenosine monophosphate, thereby modulating ATP levels in L. pneumophila–infected cells (He et al, 2025). However, this does not appear to be the case for Lfat1. We found that Lfat1 mutants defective in F-actin binding retained the ability to modify host small GTPases when expressed in cells (Figure S7). These findings suggest that, rather than serving as a co-factor, F-actin may serve to localize Lfat1 via its actin-binding domain (ABD), thereby confining its activity to regions enriched in F-actin and enabling spatial specificity in the modification of host targets.”

      (2) The development of the ABD domain of Llfat1 as an F-actin domain is a nice extension of the biochemical and structural experiments. The authors need to compare the new probe to those currently commonly used ones, such as Lifeact, in labeling of the actin cytoskeleton structure.

      We fully agree with the reviewer’s insightful suggestion. However, a direct comparison of the Lfat1 ABD domain with commonly used actin probes such as Lifeact, as well as evaluation of the split α-helix probe (as suggested by Reviewer #1), would require extensive and technically demanding experiments. These are important directions that we plan to pursue in future studies.

      For all other minors, we have made corrections/changes in our revised text and figures.