Reviewer #2 (Public review):

Summary:

The manuscript by Bai and colleagues investigates how Escherichia coli navigates and explores agar gels through chemotaxis and what parameters of bacterial swimming are tuned under selection pressure for rapid migration (i.e., reaching the edge of the agar plate quickly). Prior studies have examined related questions to a substantial degree. Examples include "Migration of Chemotactic Bacteria in Soft Agar: Role of Gel Concentration" (https://pmc.ncbi.nlm.nih.gov/articles/PMC3145277) and numerous other studies in this area (e.g., "Migration of bacteria in semi-solid agar" https://www.pnas.org/doi/10.1073/pnas.86.18.6973). From such studies has emerged the paradigm/model that reorientation (i.e., tumbling) is essential when bacteria navigate agar, which is considered a model for "complex" environments, because run-only bacteria become trapped in the agar matrix and are unable to migrate far. This new manuscript provides some evidence that this paradigm may be overly simplified or incomplete. As I understand it, the authors propose that migration is influenced to a greater extent by bias in the chemotactic run, where runs up attractant gradients are longer. The authors incorporate these data into a new model for chemotactic navigation and claim that this work establishes a general principle for how bacteria optimize active transport through complex environments.

I will first note to the editor and authors that I am not qualified to assess the detailed mathematics of the model, and my review therefore focuses on the biology and phenotypes described. Nevertheless, in my view, this manuscript, in its current form, has several important limitations. For each point, I provide suggestions for additional experiments that could strengthen the rigor of the work and clarify the claims.

Strengths:

A strength of this work is the use of microscopy and automated methods to characterize an extremely large number of bacterial cells, which strengthens the authors' claims. However, substantially greater detail on these approaches is needed for the analysis to be reproducible and to allow verification that the analyses were performed correctly.

Weaknesses:

Major concerns

(1) Claims are overly broad, and the experimental system is too artificial to support general conclusions about bacteria, chemotaxis, or evolution.

E. coli MG1655 is a longstanding model organism in the chemotaxis field, and agar chemotaxis assays are also widely used. However, the authors make very broad claims about how phenotypic changes observed during selection in 0.2% or 0.3% agar relate to bacterial chemotaxis and evolution more generally. In essence, the experimental foundation on which the authors build a complex theoretical framework is limited to a domesticated laboratory strain of E. coli and a highly artificial environment consisting of agar in a Petri dish. Although E. coli is well studied, its motility and taxis behaviors are not necessarily representative of bacteria across nature. In addition, natural environments are dynamic, and bacteria rarely experience stable gradients for extended periods, such as the 24-hour time-frame used here. The authors have also only focused on responses to attractant gradients with undefined complex growth media, and not assessed if this is also true for repellent gradients. This is important to consider because E. coli also generates repellent gradients (indole) that are not considered here. E. coli also generates AI-2, sensed as an attractant, that would be an opposing force for migration. For these reasons, it is not clear that the data and theory presented here generalize to diverse bacterial species, to natural environments, or to chemotaxis broadly.

The authors should acknowledge that further work is needed to generalise their findings by testing additional organisms, such as non-laboratory E. coli isolates, other enteric bacteria, and species with fundamentally different motility systems (e.g., Campylobacter jejuni). Further work could also expand beyond agar by examining chemotaxis in a biological matrix such as mucin, as well as testing responses to defined attractants and repellents.

(2) No genetic component is identified, so claims about evolution are not supported.

Evolution requires heritable genetic changes that produce phenotypes advantageous under a given selection pressure. The authors state that bacteria were selected for rapid migration and that this selection produced progressively more efficient migrators. However, no sequencing analyses of the evolved isolates were performed, no genetic changes were identified, and no mechanism underlying this phenotypic shift was described. Without identifying genetic alterations, they cannot substantiate the claim that evolution occurred. Whole-genome sequencing of the evolved isolates is necessary to determine whether specific mutations underlie the observed phenotypes.

(3) The predictive power of the model is not tested.

The authors develop a model with post-dictive capability, meaning the model reproduces behaviors similar to those observed in the data used to construct it. However, the manuscript does not demonstrate that the model has predictive power. Demonstrating predictive performance would substantially increase the value of the model. For example, the authors could perform an additional round of selection and predict the resulting bacterial behavior under a condition not used during model construction (such as a different agar concentration or predicting the behavior of different bacteria). Otherwise, the authors should tone down the claims.

(4) Limited novelty and impact of the environmental difference studied.

A central point of the manuscript is the difference between evolution in 0.2% versus 0.3% agar and how this difference relates to the proposed model. However, this represents a relatively minor change in the environment experienced by the bacteria. Developing an extensive theoretical framework and proposing that bacterial evolution is highly sensitive to these parameters based on this narrow experimental system may be premature. This would be addressed by the suggested broadening of experiments described above.

(5) The manuscript is too brief, and some data and methods are insufficiently described, particularly related to the machine learning analysis.

The manuscript addresses a complex topic, yet the main text, methods, and figures are very brief, which need not be the case. As a result, it is often difficult to understand exactly what was done and how the data support the authors' claims. More detailed descriptions of the experimental approaches and analyses are necessary.

One example is the machine learning approach used for cell tracking. This method is only briefly described, and no validation data are presented that would allow readers to evaluate whether the approach performs accurately. If the method is robust, it would be a powerful analytical tool, but the current description does not provide sufficient information to evaluate the reliability of the results. This issue is particularly important because the authors conclude that tumbles account for less than 3% of escape events, which contrasts with previous paradigms. Automated tracking methods can be susceptible to artifacts, and therefore, rigorous validation of the tracking pipeline, supported by appropriate figures and benchmark data, is essential.

Reviewer #2 (Public review):

Overall, we found the responses to be quite recalcitrant.

We have one remaining composite concern about the comparison between observed expression patterns with the new strains versus published data.

First, the authors only report patterns for one stage while it should be not too much effort to image the different life stages. However, since this is a revision, we are not formally requesting they do this.

Second, in the now provided Table (thank you) 'observed expression' (last column) is lacking for 9 of the 30 proteins, and for 6 of these the procedure was not successful. Why not report patterns for the other three? It is confusing also because on page 5, the authors say that "overall, 24 of 30 tags ...all of which were visible with fluorescence stereomicroscopy" - are we missing something? Also, they then said that they "obtained 6/9 of the originally failed tags"; why are the corresponding patterns not included in table 1, and are 9 proteins still labeled as "no" in the "success?" Column?

Third, we strongly feel that the response to our comments about expression patterns is not adequate. On page 5 the authors say that "all proteins were expected to be ubiquitously expressed" and that "scRNA-seq indicated that transcript abundance was ubiquitous and without strong tissue-specific enrichment with few exceptions". However, in their rebuttal, the authors now argue for tissue-specific expression for proteins with paralogs, turning around their own argument! Moreover, their Table indicates that many genes show tissue-enriched expression by RNA-seq while many of their tagged proteins exhibit ubiquitous expression.

Overall, this indicates that both the overall accomplishment of generating tagged protein strains and analyzing their expression is oversold.

Reviewer #2 (Public review):

Summary:

This paper starts with a large-scale yeast two-hybrid (Y2H) screen using Set1 (full-length and smaller parts) and other Set1C/COMPASS subunits as bait. There are hundreds possible interactions identified, but only a small number are given any follow-up. While it's useful to document all the possible interactions, the unfocused and preliminary nature of the results makes the paper feel scattered and incomplete.

Strengths:

The Y2H screen was very comprehensive, producing lots of interesting possible leads for further experiments.

Weaknesses:

Most interactions were not further tested, and even in the case of those that were, the experiments are often inconclusive or incomplete.

Reviewer #2 (Public review):

This is a very interesting study from Vandendoren and colleagues examining the role of PVN oxytocin neurons during thermoregulatory behaviors, in particular during thermoregulatory huddling. The findings are important and have implications for the thermoregulation field as well as the social/naturalistic behavior field. The findings are compelling and use a combination of state-of-the-art tools (photometry, optogenetics, automated behavior tracking, thermal imaging, and core body temperature measurement), often in combination with each other, to produce a rigorous and high-dimensional dataset.

Comments on revised version.

I appreciate the effort the authors have put into addressing all of my questions, and I have no remaining concerns.

Reviewer #2 (Public review):

Summary:

The manuscript "Canonical and phosphoribosyl ubiquitination coordinate to stabilize a proteinaceous structure surrounding the Legionella-containing vacuole" by Steinbach et al. is well written and presents strong evidence that satisfactorily supports the main hypothesis and research objectives. The authors have clearly demonstrated the presence of cloud-like, detergent-resistant GTPase Rab5 surrounding the LCV, and formation of the structure is dependent on the SidE family of effectors. The study provides insights into the relevant (associated with described phenotype) ubiquitination pathways. The findings advance our understanding of Legionella pneumophila vacuole remodeling during intracellular infection and open directions for future research to establish broader implications of this structure on Legionella pathogenesis.

Strengths:

The manuscript convincingly demonstrates the presence of a cloud-like, detergent-resistant GTPase Rab5 surrounding the LCV through elegant microscopy. The experimental evidence about the dependence of the observed phenotype on the SidE family of effectors is compelling and presented with strong scientific rigor. The introduction is well-written, and the discussion is thorough and satisfactory. The article is thought-provoking and shows preliminary evidence for ubiquitin-mediated protection and spatial organization of the LCV.

Reviewer #2 (Public review):

Summary:

Huang et al recorded anterior cingulate cortex activity in mice while they performed a shuttle escape task. The task utilized two auditory cues, each of which informed the mice to stay or escape depending on which side they were on, and incorrect responses were punished by shock administration. Analyses focused on ACC neurons that fired when mice crossed the shuttle box in either direction (A-->B or B-->A), coined "action state", or when mice crossed in one direction but not the other, coined "action content". The authors characterized these populations, and ACC firing changes mostly occurred around the time of shuttle crossing. This work will likely be of broad interest to those who are interested in neocortical neurophysiology broadly, anterior cingulate cortex specifically, and their contributions to learning about actions. The task is well-designed and provides a nice background for neurophysiological recordings. The authors leveraged these strengths in characterizing the neural populations that fire to shuttle crossings in both directions vs one direction.

Strengths:

The factorial design nicely controls for sensory coding and value coding, since the same stimulus can signal different actions and values.

The figures are well presented, labeled, and easy to read.

Additional analyses, such as the 2.5/7.5s windows and place-field analysis, are nice to see and indicate that the authors were careful in their neural analyses.

The n-trial + 1 analysis where ACC activity was higher on trials that preceded correct responses is a nice addition, since it shows that ACC activity predicts future behavior, well before it happens.

The authors identified ACC neurons that fire to shuttle crossings in one direction or to crossings in both directions. This is very clear in the spike rasters and population scaled color images. While other factors such as place fields, sensory input, and their integration can account for this activity, the authors discuss this and provide additional supplemental analyses.

Reviewer #2 (Public review):

Summary:

Learning in dynamic, stochastic environments is difficult, and neuromodulatory systems may shape where learning signals appear in the brain. Using fMRI from four probabilistic learning studies and a Bayesian ideal observer model, the authors examined latent variables driving learning, such as confidence and surprise. They found that brain activity related to confidence, and to a lesser degree surprise, is highly spatially invariant across tasks and modalities, suggesting a stable cortical organization. This invariant pattern aligns with PET-derived maps of receptors and transporters, implicating catecholamine and opioid systems, and supporting a neuromodulatory account of adaptive learning with receptor-level hypotheses.

Strengths:

(1) Elegant combination of computational modelling, functional magnetic resonance imaging (fMRI) and positron emission tomography (PET).

(2) The authors describe results of four separate experiments, with very similar results, in effect providing internal replications.

(3) Cross-validated results compared against a meaningful null model.

Weaknesses:

(1) Unclear rationale for using one-sided statistics (e.g., in Figure 3). One-sided tests appear to be invalid, given that the Introduction lacks a preregistered directional hypothesis at an operationalised level. This may have consequences for the following statement in the Discussion: "The associations between receptor architecture and functional topography were substantially weaker for the language network, which is not thought to rely strongly on neuromodulatory systems."

(2) Limited computational modelling. Since learning rates probably differ across subjects, I wonder if they have considered fitting the "volatility" instead of using the generative one. Would that give more meaningful fMRI maps, and better explained variance when correlating these to the PET-based predictors? I was also wondering how their surprise measure relates to "change-point probability" (e.g., Murphy et al., Nat Neurosci, 2021). Finally, I think it would be helpful to show average time courses of surprise and confidence time-locked to state changes.

(3) Lack of GLM validation. It would help to show that the model fits the data well. This is important given the many underlying assumptions (shape of the HRF, linear effects of variables, etc). For example, one could show average insula activity time-locked to state changes, as well as the model-predicted activity, and separately for three strata defined by how surprising the state change was (according to the ideal observer model). Related, the authors use a substantial number of predictors in their GLM, and the language in the Methods is a little casual. It would help to show part of a design matrix, and clearly describe the following: were (occasional) questions and responses modelled by separate stick functions? Which predictors (stimulus, questions, response) varied parametrically with which variables?

Reviewer #2 (Public review):

Summary:

This study by Mentch et al. uses naturalistic-movie fMRI and grayordinate-level stacked encoding models to test preregistered hypotheses about low/high-level and audio/visual feature encoding in autism and adolescence from openly available Healthy Brain Network data. Null results reported that autism was not linked to increased low-level encoding in primary sensory cortices. Exploratory analyses showed participants with autism showed reduced high-level visual encoding in social regions (pSTS, face areas), with the high-low feature shift tracking social responsiveness scale (SRS) scores. Age and laterality effects were also found.

Strengths:

(1) This study and hypotheses were preregistered.

(2) The study utilised proper variance partitioning, split-half noise ceilings, FD-threshold sensitivity analyses, and an explicit modelling framework that recovers known sensory hierarchies in the aggregated sample. The developmental sampling adds to the interest.

(3) The manuscript is written clearly, laying out the background and theories to be tested with encoding models. The analyses and reporting of results are clear.

Weaknesses:

(1) If I understand correctly, by only averaging the grayordinates that already passed a significance threshold, the resulting parcel value is guaranteed to look stronger than if all grayordinates had been included. This has been raised in neuroimaging (Kriegeskorte et al., 2009; Vul et al., 2009). Can the authors justify these choices?

(2) I assume that the phrase "temporally permuting the order of observations" on Page 22 means random shuffling of time points. The details of this exact permutation are not specified. Both the fMRI BOLD signal and movie features have strong temporal autocorrelation, and random shuffling will destroy this structure. This is important as grayordinate-level survivors will propagate to parcel pools. Circular shifting or phase randomization preserving the autocorrelation spectrum is appropriate.

(3) In the movie feature selection, the low-level visual model contains only two scalars: mean perceptual brightness and a single averaged value across 2,139 motion-energy filters. With only two low-level visual features, the low-level visual model potentially would underestimate low-level visual encoding. The H1.1 toward the null perhaps suggests to this. Principal components of the motion-energy outputs, as was done for the cochleagram, could be used.

(4) The pilot sample composition is not described. Features were selected based on their performance on an independent set of 54 pilot subjects. Please provide age, sex, and diagnostic composition of the pilot sample. The main point being whether the selected features were optimised for a population that differs from the subject studied.

(5) The authors acknowledge the lack of eye-tracking in theory study. I think this should be elaborated, especially why this modality is important for answering sensory and perceptual encoding. Face encoding may not be degraded, but just that faces are not being attended to.

(6) I think a more nuanced distinction about the representational nature of encoding-model R² should be mentioned, especially when the interpretation of findings is related to perceptual functioning (EPF theory). R² measures how well a feature set predicts brain activity, not perceptual function or cognitive integration.

(7) The literature also includes evidence for no Colavita effect, not just reverse Colavita in autism, and the framing should reflect this more even-handedly.

(8) The 0.2 mm per-volume threshold is quite strict. The 40%/60%/80% sensitivity analyses partially address this, but a brief justification for the choice of 0.2 mm would strengthen the Methods.

(9) Figure 1 seems confusing and would benefit from more information or text in the figure.

(10) Figure 2 supplement has caption A labelled twice; please correct.

(11) Acronyms. Please spell out MSI on first mention (page 2) and ISC/ISFC on first mention (page 4).

Reviewer #2 (Public review):

Summary:

The authors present a creative approach using visual anagrams matched on low-level image statistics to isolate animacy from low-level visual features and report consistent effects of animacy on visual working memory and attention. While this is a thoughtful design and is well executed across seven pre-registered experiments, it remains unclear whether the reported effect is truly driven by animacy, as opposed to broader differences in ensemble statistics or semantic structure across the "mixed animacy" versus "uniform animacy" conditions. As such, the interpretation of a "pure" animacy effect may be overstated.

Strengths:

(1) An important methodological advance in controlling low-level confounds that have historically complicated the study of animacy.

(2) The converging effects across multiple experiments, together with the pre-registered design, strengthen the reliability of the reported findings.

Weaknesses:

(1) Specificity of the animacy effect vs. category-level ensemble structure

The central claim is that animacy itself drives the observed effects. However, the key manipulation ("mixed animacy" versus "uniform animacy") also introduces differences in category-level ensemble structure. For example, in Experiments 1-2, cross-category change detection (e.g., dog to chair) may be easier not because of animacy per se, but because of a change in overall ensemble statistics (Brady & Alvarez, 2011, 2015). In addition, since each display contains five objects (two in one category and three in the other category), cross-category changes may also alter category balance in a way that further facilitates detection. In contrast, within-category changes preserve both ensemble structure and category composition, making them more difficult to detect.

Brady, T. F., & Alvarez, G. A. (2011). Hierarchical encoding in visual working memory: Ensemble statistics bias memory for individual items. Psychological Science.

Brady, T. F., & Alvarez, G. A. (2015). Contextual effects in visual working memory reveal hierarchically structured memory representations. Journal of Vision.

(2) Limited stimulus set and potential learning effects

The relatively small stimulus set (six anagram pairs) and repeated exposure raise the possibility of learning or familiarity effects. Does performance change over time? e.g., are there meaningful differences between early and late trials (e.g., first 10% vs. last 10%)? If such differences are present, they could suggest the development of task-specific strategies or increased efficiency with repeated exposure, rather than stable effects driven by the experimental manipulation itself.

(3) Role of semantics

Although the anagram paradigm effectively controls low-level visual features, it still relies on high-level semantics (e.g., "dog" vs. "boot"). These stimuli differ not only in animacy but also along other semantic dimensions such as natural versus manmade categories. From a semantic standpoint, it remains unclear whether the observed effects can be uniquely attributed to animacy or whether they reflect broader conceptual distinctions.

Reviewer #2 (Public review):

Summary:

Understanding the factors and mechanisms underlying the deleterious effects of distraction, and protection from distraction, in working memory is an important question that has a long and rich history in psychology and neuroscience, and continues to be highly relevant. In this study, the authors recorded the EEG while subjects viewed the initial presentation of two oriented-grating stimuli, aligned on either side of fixation along the horizontal meridian (memory array), followed by a 70%-valid cue, then one of three distractor conditions (overlapping cued item (40%), opposite cued item (40%), no distraction (20%)), followed by recall ("delayed estimation"). The behavioral and EEG results from this procedure are complemented with computational modeling with a two-tier bump-attractor model.

Weaknesses:

Interpretation of the results is complicated by several factors. One is the non-consideration of a considerable amount of extant research that is highly relevant to the question of interest (these include seminal studies from Gi-Yeul Bae and from Tatiana Pasternak). Relatedly, the manuscript emphasizes biasing effects of distractors to the exclusion of a conceptually distinct effect: degradation of representational precision. (For example, the actual focus of the study of Wimmer et al. (2014) that the manuscript cites with reference to bias is the degradation of precision; one only has to read the title of this paper to know this.) Also relatedly, the authors are aware of the possibility of misbinding (a.k.a. "swap") errors, in which subjects mistakenly recall a high-fidelity representation of a foil (in this case, the distractor) rather than the target, but they (1) fail to cite any of the extensive literature on this topic and (2) seem to erroneously attribute what their analyses would seem to identify as misbinding errors as "antagonistic bias" exerted by the distractor on the target item.

A second concern relates to the interpretation of patterns in the empirical results. In particular, Figure 1G is interpreted as displaying a pattern of repulsive bias exerted by the distractor on trials when the distractor appeared at the location opposite to the cued item. However, it is not clear that this is a repulsive bias. Rather, what the plot shows is that report error is attracted to "near" distractors with a positively signed offset but repelled by "near" distractors with a negatively signed offset. Stated another way, when one applies a model-free assessment of the influence of the distractor on the memorandum, there is no systematic bias: the AOC of positively signed offset values from 0 to +45 deg is roughly the same as the AOC negatively signed offset values from 0 to -45 deg. The same also seems to be true, albeit with a smaller magnitude, for trials featuring "stronger mnemonic neural representation" that are illustrated in Figure 2. And so it's unclear that the effect of the "Dist. Opp" distractor is indeed a repulsive bias, rather than a loss of precision.

The third primary concern is that the results from simulations from the two-tier bump-attractor modeling are difficult to interpret due to several poorly motivated and seemingly "hand-coded" assumptions. These include the (seemingly arbitrary) strengthening of HCVC feedback connections by 20% for cued vs. uncued items; and the choice to "transiently block[ed] feedforward connections from the VC to the HC during the maintenance epoch" as a consequence of cuing. There is frankly no evidence that the latter phenomenon actually happens in primate brains performing comparable tasks, including in papers (such as from Xu and from Rademaker) that are cited in this manuscript. The current consensus is that priority-related rotations of representational geometry are the scheme employed by mammalian nervous systems to control the otherwise deleterious effects of distraction.

Reviewer #2 (Public review):

The manuscript by Ono et al compares two prime editing strategies in zebrafish, one based on a nickase and the other on a nuclease, and evaluates their performance for introducing substitutions, short insertions, and transmission to the next generation. The study aims to clarify the relative strengths of these approaches and to extend their use for inserting short DNA sequences in vivo.

The study provides a useful and well-executed comparison of two editing strategies in a vertebrate model. In particular, the finding that the nuclease-based approach shows higher efficiency for short insertions is of practical interest for functional studies. The authors also present convincing evidence supporting their conclusions, including sequencing and phenotypic validation at selected loci. These results support the reliability of the approach in this system.

The overall conceptual advance remains somewhat limited, as the general strategy of delivering prime editing components in zebrafish has been described previously. The present study extends this work by comparing two editing modes and exploring insertion efficiency, which represents a useful but incremental advance.

Regarding the comparison between the two systems, the authors have made efforts to address concerns about generalizability by adding data from additional loci and by refining the scope of their conclusions. These additions strengthen the manuscript. However, the comparison is still based on a relatively small number of loci, and the conclusions may therefore remain somewhat context-dependent.

Overall, the authors largely achieve their stated aims of comparing two editing strategies and demonstrating their applicability in zebrafish. The data generally support the conclusions, particularly within the tested loci. The work provides practical value to the community, especially for researchers seeking efficient strategies for short sequence insertion in this model system, although its broader impact is somewhat limited by its incremental nature.

Reviewer #2 (Public review):

This article reports a cost-effectiveness comparison of intramural and extramural that NIH funded between 2009 and 2019. Using data obtained from NIH RePORTER, they linked total project costs to publication output, using robust validated metrics including Relative Citation Ratio (RCR), Approximate Potential to Translate (APT), and clinical citations. They find that after adjusting for confounders in regression and propensity-score analyses, extramural projects were generally more cost-effective, though intramural projects were more cost effective for generating clinical citations. They also describe differences in the topics of intramural- and extramural-funded publications, with intramural projects more likely to generate papers on viral infections and immunity or cancer metastases and survival, but less likely to generate papers on pregnancy and maternal health, brain connectivity and tasks, and adolescent experiences and depression. The authors aptly describe the different natures of the intramural and extramural funding models, including that extramural researchers spend much time writing grant applications and that the work described in extramural publications often receives funding from sources other than NIH grants.

Strengths:

The authors leveraged publicly available data (including RePORTER and the iCite repository) and used robust validated metrics (RCR, APT, clinical citations). They carefully considered a large number of confounders, including those related to the PI, and performed several well-described regression analyses.

Reviewer #2 (Public review):

Summary:

The goal of this proposal was to understand how two separate projection neurons from the medial prefrontal cortex, those innervating the basolateral amygdala (BLA) and nucleus accumbens (NAc), contribute to the encoding of emotional behaviors. The authors record the activity of these different neuron classes across three different behavioral environments. They propose that, although both populations are involved in emotional behavior, the two populations have diverging activity patterns in certain contexts. A subset of projections to the NAc appear particularly important for social behavior. They then attempt to link these changes to the emotional state of the animal and changes in synaptic connectivity.

Strengths:

The behavioral data builds on previous studies of these projection neurons supporting distinct roles in behavior and extend upon previous work by looking at the heterogeneity within different projection neurons across contexts, this is important to understand the "neural code" within the PFC that contributes to such behaviours and how it is relayed to other brain structures.

Weaknesses:

The diversity of neurons mediating these projections and their targeting within the BLA and NAc is not explored. These are not homogeneous structures and so one possibility is that some of the diversity within their findings may relate to targeting of different sub-structures within BLA or NAc or the diversity of projection neuron subtypes that mediate these pathways. This is an important future direction for this work but does not detract from the main finding as reported.

Reviewer #2 (Public review):

The authors have made several corrections to the original manuscript. For example, they revised the bootstrapping analysis to avoid arbitrarily inflating the degrees of freedom. However, most substantive concerns remain inadequately addressed.

(1) The primary issue is still the lack of baseline models against which to benchmark the predictive performance of the proposed DenseNet model. This concern was raised independently by two reviewers. Without such benchmarks, it is difficult to interpret the reported results in the context of prior work on MRI-based cognition prediction.

Notably, the authors state: "While we compared our model with the connectome predictive modeling (CPM) approach and observed better performance with our deep learning framework, we did not conduct a comprehensive benchmark across all available machine learning methods, nor was this the aim of the present study."

However, I could NOT find any discussion or results related to the CPM model in the manuscript. It is therefore unclear whether the DenseNet model was actually statistically compared with CPM, and, if so, how the comparison was conducted.

Note that the statement, "While Vieira et al. show that the majority (76%) of prior studies used linear modeling approaches, including CPM and penalized regressions, these models are often vulnerable to overfitting, especially when applied to high-dimensional fMRI data," is not entirely accurate. Linear models typically have far fewer parameters than deep-learning models and are therefore often less prone to overfitting. In fact, it is well established that deep-learning models are particularly susceptible to overfitting and usually require substantially larger sample sizes to achieve stable and reliable performance. Although deep-learning models may outperform shallower models once sufficient data are available and training is well controlled, this does not justify the authors' claim as stated. I therefore disagree with the argument put forward by the authors.

The authors further justify the absence of benchmarking by stating: "In this context, deep learning was employed as a flexible framework capable of modelling high-dimensional functional connectivity patterns across cognitive states, rather than as a claim of inherent methodological superiority. Thus, our goal was not to propose a universally superior prediction model, but rather to test how brain state influences predictive utility for WM and EM using a deep learning approach." However, most shallow models can likewise be applied across different brain states and cognitive targets. This rationale does not establish deep learning as a uniquely appropriate or necessary choice. If deep learning is indeed a better approach in this context, the authors should demonstrate this empirically through appropriate benchmarking against established baseline models.

(2) Additional analysis shows that "BCG is not significantly associated with cognition itself". This is the most perplexing result. This is like saying Brain Age Gap is not related to chronological Age. It is counterintuitive since the Brain Age Gap is calculated by chronological age minus actual age, and most research has shown a strong relationship between the Brain Age Gap and age.

If the brain cognition gap is not related to cognition, is it possible that the results found are mainly due to the predictive model not fitting well with another dataset? Regardless, the lack of association between BCG and cognition deserves a discussion.

(3) I still do not fully understand the rationale of the mediation analysis. The analysis and findings are still not related to aims 1 and 2, since DA and entropy are not part of the prediction models. But I appreciate the explanation that this part is related to the authors' previous work, and that the authors attempted to link to them somehow.

Reviewer #2 (Public review):

Summary:

Shapiro et al. set out to verify the American Beefalo Association's claim that Beefalo cattle possess 37.5% bison ancestry. They employ a comprehensive range of well-established population genomics methods to estimate ancestry in these hybrid populations, including PCA, ADMIXTURE, D and F statistics, and local ancestry inference. Their findings conclusively demonstrate that most Beefalo lack the claimed bison ancestry, with only 8 out of 47 samples showing any detectable bison ancestry, ranging from 2-18%.

Strengths:

The primary strength of this analysis lies in the comprehensive dataset available to the authors, which includes important foundational Beefalo individuals and various reference populations. The rigorous and multi-faceted methodological approach employs several well-established techniques in population genomics for detecting and measuring admixture. Each method used has a firm basis in the field, providing consistent and robust results. The authors' approach of using PCA to initially assess the data within a global context, followed by more specific analyses using ADMIXTURE and D-statistics, provides a clear and logical progression of evidence. The presentation of these results in figures is particularly effective, clearly illustrating the key findings of the study. Additionally, the examination of both autosomal and sex chromosome ancestry offers a more complete understanding of Beefalo genetic composition and the mechanics of bison-cattle hybridisation.

Weaknesses:

One limitation of this analysis is the relatively low coverage (~2x) of many Beefalo samples. However, the authors have taken steps to mitigate biases that may arise from this, and their downsampling experiment demonstrates that this level of coverage is appropriate for summarising species-level ancestry across Bos. Another potential weakness is the limited sampling of contemporary Beefalo populations, as the study focuses primarily on historical samples. The authors have justified this choice on the grounds that contemporary Beefalo breeding involves no further bison input, so founder-era individuals are the most informative samples for addressing the study's central question.

Appraisal:

The authors have clearly achieved their primary aim using a rigorous and comprehensive methodology. Their extensive dataset and multi-faceted analytical approach provide strong support for their conclusions. The study not only addresses its main research question but also reveals unexpected insights into Beefalo genetics, particularly the presence of zebu ancestry, predominantly from Brahman cattle.

Discussion:

This study is valuable for several reasons beyond its primary findings. First, it definitively addresses and refutes the claim of 37.5% bison ancestry in Beefalo, providing crucial information for those studying these interspecies hybrids and the viability of their offspring. Second, it reveals the unexpected presence of zebu ancestry, predominantly from Brahman cattle, in many Beefalo, raising intriguing questions about the breed's development and the potential role of zebu cattle in achieving desired traits. This finding suggests that the distinctive appearance of Beefalo may be due in part to zebu admixture rather than bison ancestry. Third, the study highlights the significant barriers to admixture between bison and cattle, both in controlled breeding programs and potentially in wild populations. This has important implications for conservation genetics and our understanding of gene flow between these species. Lastly, the study demonstrates the power of genomic analysis in verifying breed claims and understanding the complex history of domestic animal breeds. These findings open new avenues for research in bovine genomics, breed development, and the dynamics of interspecies hybridisation.

Comments on revised version:

Thanks for the responses, which address my comments in full. I have no further concerns.

Reviewer #2 (Public review):

Summary

The authors investigate how parallel olfactory pathways contribute to CO₂ valence processing in Drosophila. By combining multiple approaches, the study identifies LN23 as a previously unrecognized component of the CO₂ circuit and proposes a model in which distinct downstream pathways contribute to aversive and attractive behavioral responses. More broadly, the work aims to connect circuit organization with context-dependent sensory processing and behavioral valence.

Strengths

A major strength of the study is the integration of multiple complementary approaches spanning anatomy, circuit analysis, and behavior. This combination provides a rich and valuable framework for understanding how CO₂ information may be processed across different levels of the olfactory system. The identification of LN23 as an important component of the CO₂ pathway is particularly interesting and will likely be useful for future studies investigating olfactory processing, behavioral state modulation, and valence coding. The connectomic and anatomical analyses also provide a valuable resource for the community.

Another strength of the manuscript is its conceptual ambition. The work moves beyond a simple labeled-line view of olfactory processing and proposes that flexible behavioral responses may emerge from interactions between parallel downstream pathways and multimodal integration centers. The behavioral manipulations further support an important role for LN23 in CO₂-related behaviors.

Weaknesses

Several aspects of the conceptual interpretation would benefit from additional clarification or more cautious framing relative to the current experimental evidence. In particular, the distinction between atmospheric versus experimentally elevated CO₂ conditions, as well as the interpretation of chronic exposure in terms of habituation, remains somewhat unclear throughout the manuscript.

Some conclusions regarding valence coding and multimodal integration also appear more inferential than directly demonstrated experimentally, especially when moving from anatomical connectivity to functional interpretation.

Reviewer #2 (Public review):

This study uses monkey single-unit recordings to examine the role of the STN in combining noisy sensory information with reward bias during decision-making between saccade directions. Using multiple linear regressions and clustering approaches, the authors overall show that a highly heterogeneous activity in the STN reflects almost all aspects of the task, including choice direction, stimulus coherence, reward context and expectation, choice evaluation, and their interactions. The authors report in particular how three classes of neurons map to different decision processes evaluated via the fitting of a drift-diffusion model. Overall, the study provides evidence for functionally diverse and anatomically intermingled populations of STN neurons, supporting multiple roles in perceptual and reward-based decision-making.

This study follows up on work conducted in previous years by the same team and complements it. Extracellular recordings in monkeys trained to perform a complex decision-making task remain a remarkable achievement, particularly in brain structures that are difficult to target, such as the sub-thalamic nucleus. The authors conducted numerous analyses of STN activities, using sophisticated statistical approaches and functional computational modeling.

One criticism that I would still make in the revised version of the paper concerns the description of the behavior of the two monkeys which is still minimal, while acknowledging differences in their choice and RT performance that reflect "individual differences in sensitivity to motion stimulus and a common heuristic-based satisficing strategy". This sentence is not clear to me. Moreover, the potential consequences of these differences on neuronal activity are only considered in the cluster analysis done for each of the two animals separately and for which it turns out there is no notable difference.

Compared to the first version of the paper, the cluster analysis in this revised version yields three distinct populations instead of the previous four. While the authors suggest that these subpopulations play important roles in encoding different aspects of decision-making, the identification of three rather than four subpopulations seems to me an important update that warrants discussion.

Finally, I think it would have been interesting to identify the level of collinearity in the model proposed by the authors (equation 7). Indeed, one can expect significant collinearity between some of the proposed explanatory factors of neuronal activity, such as choice and coherence level, for example. Similarly, for the analysis relating neuron activity to decision evaluation signals (p 16), firing rates calculated using sliding averages with 1-ms steps are compared, but the method does not specify controls for multiple comparisons or for non-independent data.

Reviewer #2 (Public review):

Summary:

Many insects possess extremely sensitive olfactory systems that can detect chemical signals from distances of several kilometers. For decades, the arms race between bats and insects has served as a prime example of acoustic co-evolution. The auditory adaptations of insects to echolocation have been well documented. Cricket has a multi-sensory predator recognition system with keen olfactory, tactile, and auditory senses. However, whether crickets can use the scent of bats to avoid them remains unknown at present. The authors hypothesized that cricket prey (Loxoblemmus equestris) might eavesdrop on predator bat (Scotophilus kuhlii) VOCs as an early warning. L. equestris is one of the prey species of S. kuhlii, and the authors demonstrated that the body odor of the insectivorous bat S. kuhlii triggers robust avoidance and electrophysiological responses in the cricket L. equestris, and that a single compound, (-)-limonene, is sufficient to elicit this avoidance in the laboratory and suppress calling in the field. Overall, this paper has a complete chain of evidence and should be a highly praised study.

Comments:

(1) Olfactory eavesdropping can transcend the evolutionary divide between vertebrate predators and invertebrate prey, enabling invertebrates to trigger defensive avoidance behaviors in response to predator-derived volatile odors. This phenomenon is empirically well-documented and requires no excessive emphasis.

(2) Without quantitative analysis and without knowing the relative content of this key substance limonene, I don't quite understand how to determine the concentration of limonene standard for EAD, as well as the concentration in field experiments. How is the concentration of limonene determined in field spraying, and is this actually the case in the wild environment?

(3) Figures 1C and D should compare the GC-EAD response of L. equestris to the odor of bat body and the odor of bat nasal secretions. It should not be compared with the air control group. Figure 1D has the same problem.

Reviewer #2 (Public review):

Summary:

To investigate the detachment and reattachment kinetics of kinesin-1, 2 and 3 motors against loads oriented parallel to the microtubule, the authors used a DNA tensiometer approach comprising a DNA entropic spring attached to the microtubule on one end and a motor on the other. They found that for kinesin-1 and kinesin-2 the dissociation rates at stall were smaller than the detachment rates during unloaded runs. With regard to the complex reattachment kinetics found in the experiments, the authors argue that these findings were consistent with a weakly-bound 'slip' state preceding motor dissociation from the microtubule. The behavior of kinesin-3 was different and (by the definition of the authors) only showed prolonged "detachment" rates when disregarding some of the slip events. The authors performed stochastic simulations which recapitulate the load-dependent detachment and reattachment kinetics for all three motors. They argue that the presented results provide insight into how kinesin-1, -2 and -3 families transport cargo in complex cellular geometries and compete against dynein during bidirectional transport.

Strengths:

The present study is timely, as significant concerns have been raised previously about studying motor kinetics in optical (single-bead) traps where significant vertical forces are present. Moreover, the obtained data are of high quality and the experimental procedures are clearly described.

Reviewer #2 (Public review):

Summary:

Using E. coli K-12 as a model system, the authors investigated how phosphate (Pi) depletion induces polymyxin resistance in Enterobacteriaceae, which notably lack the canonical phospholipid remodeling pathways commonly associated with phosphate starvation responses. They demonstrated that low-phosphate conditions promote L-Ara4N modification of lipid A, thereby enhancing polymyxin resistance. Proteomic analyses revealed significant upregulation of the arn operon and ugd under phosphate-limited conditions, and promoter activity assays further confirmed that both promoters are strongly induced during Pi depletion. Through gene deletion experiments, the authors showed that arn expression is regulated by the PmrAB two-component system, whereas ugd is controlled by PhoBR under low-phosphate conditions. Using ICP-MS analysis, they further found that phosphate limitation increases cell-associated Fe levels, and that reducing Fe availability abolishes PmrAB-dependent activation of the arn operon. Finally, the study demonstrated that Mg supplementation and Fe chelation can suppress polymyxin resistance, highlighting the critical role of metal homeostasis in phosphate depletion-induced antimicrobial resistance.

Strengths:

Overall, I found this study to be well conducted, with convincing results that strongly support the proposed model. Through comprehensive genetic analyses and detailed characterization of metal ion homeostasis and membrane lipid modifications, the authors uncovered a novel regulatory connection among Mg²⁺, Fe³⁺, and the PmrAB pathway, a key driver of polymyxin resistance. These findings are highly interesting and have important implications for understanding the evolution of the Fe-sensing PmrAB system, as well as the broader role of nutrient availability in shaping antibiotic resistance.

Weaknesses:

I did not identify any particular weaknesses.

Reviewer #2 (Public review):

Synesthesia is a neurological condition where stimulation of one sensory channel leads to involuntary, automatic, and consistent experience of another, unrelated percept. For example, Sir Francis Galton (1880, Nature) famously described the robust tendency of some individual (synesthetes) to associate numerals with a distinct color. Ever since, synesthesia keeps attracting a broad interest in the cognitive neurosciences in light of its implications for the study of domains such as perception, consciousness, and brain connectivity, among others.

Strauch, Leenaars, and Rouw measured pupil size in a group of 16 grapheme-color synesthetes and two matched control groups. The participants were presented with gray digits - that is, visual stimuli having identical physical properties in terms of brightness. Each participant subsequently rated the corresponding evoked color and brightness: unlike controls, synesthetes did so in a very consistent and reliable fashion. Accordingly, this was also shown in their pupils: despite the same objective luminance, digits associated with brighter percepts caused their pupils to constrict and digits associated with darker percepts caused their pupils to dilate more than controls. These results highlight how crossmodal correspondences are deeply rooted in synesthetes, and puts forward pupillometry as a particularly appealing biomarker for some phenomenological experience (at least those grounded in "brightness").

Further strengths of the technique are its temporal resolution and its responsiveness to several constructs. Across several tasks, the authors show for example that responses to synesthetic light are somewhat slower than responses to real light (i.e., they are likely mediated), but at the same time faster than responses to mental imagery. The role of mental imagery can also be reasonably dismissed when considering the second feature of pupil size: its responsiveness to mental effort and cognitive load. The pupils tend to dilate with demanding, challenging tasks, and this was the case when control participants were asked to report the color of a digit for which they did not consistently experience a synesthetic association. The same task was, instead, seemingly effortless for synesthetes, again speaking in favor of the automaticity of number-color correspondences in their case.

Overall, the findings by Strauch, Leenaars, and Rouw are highly significant for the field and likely to be impactful. The strength of their evidence, when accounting for the relatively small sample size and the inherent variability of both phenomenology (color perception and subjective reporting) and physiology (pupil size), is adequate and sufficiently convincing.

Reviewer #2 (Public review):

Summary:

The study offers a thorough analysis of the prevalence of pain in women with polycystic ovary syndrome (PCOS) and its associations with health outcomes across various racial groups. Furthermore, the research investigates the prevalence of PCOS and pain among different racial demographics, as well as the increased risk of developing various conditions in comparison to individuals who have PCOS alone.

Strengths:

The study emphasizes pain as a significant comorbidity of PCOS, an area that is critically underexplored in existing literature. The findings regarding the increased prevalence of some of the diseases in the PCOS + pain group provide valuable direction for future research and clinical care. I believe physicians should incorporate pain score assessments into their clinical practice to improve patients' quality of life and raise awareness about pain management. If future research focuses on the mechanisms of pain, it would provide a better understanding of pain and allow for a focus on the underlying causes rather than just symptomatic management. The study also highlights the association between PCOS+pain and various comorbidities, such as obesity, hypertension, and type 2 diabetes, as well as conditions like infertility and ovarian cysts, offering a holistic view of the burden of PCOS.

Weaknesses:

Due to the nature of retrospective design, some data may not be readily available in the EHR system. Diagnosis of PCOS, pain is based on ICD codes, which may lead to misclassification and may not capture symptom severity or patient-reported experiences.

Reviewer #2 (Public review):

Since its original discovery, the mechanistic basis for TCT-mediated pathogenesis of Bordetella pertussis has been a moving target and difficult to uncouple from confounding variables. The current study provides some exciting data that suggest PGLYRP-1 modulates host responses upon 'activation' by TCT. While there are some strengths associated with the unbiased approaches and collective data to support the claims associated with TCT and PGLYRP-1's function in this system, caution should be used when interpreting and extrapolating some the information provided. While many of the initial concerns were addressed, one concern remains: using whole, intact PG sacculi from other species for comparative studies with a fragment of released PG (i.e., TCT).

Comments on revised version.

I have no further comments.

Reviewer #2 (Public review):

Summary:

The JAK-STAT pathway (JSP) exhibits cell-type-specific functional heterogeneity in breast cancer. This study investigates the JSP in breast cancer and its response to anti-PD‑1 immunotherapy. JSP displays distinct cell‑type heterogeneity: it promotes malignant phenotypes and immunosuppression in tumor cells, while enhancing cytotoxicity and reducing exhaustion in T cells. Elevated JSP expression correlates with improved immunotherapy responses, especially in triple‑negative breast cancer. These findings highlight the paradoxical roles of JSP, indicating that broad inhibition may compromise anti‑tumor immunity.

Strengths:

The major strengths of this study include the comprehensive characterization JSP heterogeneity across epithelial, tumor, and T cells in breast cancer. The identification of JSP and STAT4 as predictive biomarkers for immunotherapy response, particularly in triple‑negative breast cancer, provides clinically relevant insights for patient stratification.

Weaknesses:

The corresponding content has been revised.

Reviewer #2 (Public review):

Summary:

Savage et al. investigate the synchronization of retinal Ca2+ waves with developmental cell death, microglia activation, and vascular outgrowth. These developmental processes occur through a mechanism where apoptotic cells release ATP through Panx-1 channels to stimulate both Ca2+ retinal waves and microglia activation. Using scRNAseq, the authors classify autofluorescence cell clusters (ACCs) at the leading edge of vasculature outgrowth as Hmox-1+ microglia. From here, they show microglia engulfment of apoptotic RGCs, and the potential release of ATP may contribute to Ca2+ wave generation. The authors demonstrate these mechanisms through the use of two pharmacological agents to either block the ATP release from Panx-1 or block receptor binding to ATP. Furthermore, while previous studies have described the site of initiation of retinal Ca2+ waves as random, this study shows that the initiation of Ca2+ waves is biased to the leading edge of vascular growth in the developing retina. To do this, the authors use a combination of wide-field Ca2+ imaging and multi-electrode arrays to pinpoint the sites of Ca2+ wave initiation in the developing retina.

Strengths:

The authors use several techniques to interrogate these mechanisms, including single-cell RNAseq, wide-field Ca2+ imaging, and multi-electrode arrays. With these experiments, this manuscript proposes several novel ideas, such as ATP as the Ca2+ wave-initiating cue, and the localization of the Ca2+ wave initiation to the leading edge of vascular growth.

Weaknesses:

The main weakness of the manuscript is the overreliance on only two pharmacological agents to test the central hypotheses. These conclusions would be strengthened if, in addition to their pharmacological manipulations, they used genetic knockout models to perturb programmed cell death or ATP release (i.e., BAX-KO, Panx-1 KO).

Reviewer #2 (Public review):

Summary:

In this paper, the authors set out to better understand the genetic mechanisms underlying thermal adaptation in insects. They experimentally evolved diamondback moth (Plutella xylostella) populations - a pest species with a wide distribution - under both hot (12h:12h 32{degree sign}C/27{degree sign}C) and cold (15{degree sign}C/10{degree sign}C) thermal conditions, and conducted phenotypic assays and metabolic and transcriptomic profiling to analyze how populations changed to deal with this thermal stress compared to the nonevolved ancestral population (constant 26{degree sign}C). Phenotypic assays showed that evolved hot populations had increased survival at high temperatures (42-43{degree sign}C) while evolved cold populations had lower freezing points compared to the ancestral population. When measured at the constant 26{degree sign}C conditions, metabolic and transcriptomic profiles of 3rd instar larvae from the evolved population were distinctive from the ancestral population, with a set of overlapping metabolic and transcriptomic pathways that were significantly differentially expressed in both hot and cold evolved populations compared to the ancestral. The authors narrowed down this set of candidate genes further by focusing on genes with high expression levels overall, whose expression profile was correlated with differentially expressed metabolites, and that contained mutants in both hot and cold strains. From this set, they chose the PxSODC gene for further functional validation, as it has previously been shown to be involved in the response of insects to abiotic stress with its antioxidative role in cellular defense. At the constant 26{degree sign}C, this gene showed lower expression across development in evolved strains compared to the ancestral population, while it showed similar expression patterns under thermal stress. Knockdown of PxSODC resulted in decreased survival rates at high temperatures and higher freezing points compared to the ancestral population. Based on this validation, the authors hypothesize that the non-synonymous mutation in the PxSODC gene that they found in the cold and hot evolved populations might alter the conformation of the PxSODC protein, increasing enzyme capacity. Their experimental evolution experiment furthermore indicates the capacity of the pest species, the diamondback moth, to adapt to a wide range of temperatures, providing insights into its capacity for global dispersal.

Strengths:

(1) The authors did a tremendous amount of work to characterize the mechanisms underlying thermal adaptation in the diamondback moth, artificially selecting populations for three years in the lab and characterizing how they evolved as a result at different biological levels: from phenotypes in different life stages, to larval metabolites and gene transcription, to functionally validating how one of the resulting gene candidates influences the capacity to deal with thermal stress.

(2) The paper identifies and provides further evidence for candidate genetic mechanisms that might be particularly important for thermal adaptation in insects, including lipid metabolism, oxidoreductase activity, and DNA methylation. It is furthermore interesting that the authors found similar mechanisms to be involved in both the adaptation to cold and hot environments. Their functional validation of some of the genes involved in these mechanisms is very useful to understand how these genes might be causally involved in insect thermal adaptation.

(3) The paper also has applied value: the diamondback moth is a pest species with a wide distribution, so understanding its adaptive capacity to different thermal environments is important for predicting the prevalence and potential further range expansion of this species under future climate change.

Reviewer #2 (Public review):

Summary:

Cong et al. investigated the regulatory effects of ABHD6 on AMPARs. The authors performed adequate electrophysiology recordings to show the exact pattern of this regulation and covered major critical points.

Strengths:

The authors have performed high-quality ephys recordings and examined all potential regulatory aspects of ABHD6 on AMPARs. This is important to understand the AMPAR functions.

Weaknesses:

(1) The authors discussed CNIH-2 extensively from line 92-110 in the introduction, however, they did not perform related experiments. I suggest they move this part to the discussion where they also discussed the roles of CNIH.

(2) The authors need to report the "n" for all the experiments they have presented in this manuscript. How many cells were recorded in each condition? How many batches? This information has to be in all of the figure legends, but it is missing except Fig. 4.

(3) One question is what the physiological meanings of this regulatory effect are. The authors may consider adding some discussions.

(4) About statistics. The authors need to add more details and make sure their statistics sound. For example, they also need to check the equality of variances. In their Table EVs, where the P values are reported, the authors need to report which statistics they have used, one-way ANOVA, K-W test, or others, and the exact post-hoc test type for each comparison. For one-way ANOVA, report the F values simultaneously with the P values in all figure legends.

(5) Fig. 3J, the authors need to correct the label of the Y axis. It is shifted.

Comments on revised version.

In the revised manuscript, the authors have addressed all my concerns. The manuscript has been substantially strengthened by additional data and discussion.

Reviewer #2 (Public review):

In this manuscript, Burnsdon et al. aim to study PIK3CA-related overgrowth spectrum (PROS) by establishing a mosaic zebrafish model with overexpression of pik3ca carrying hotspot mutations, coupled with an mScarlet+ reporter. Using fluorescence microscopy, the authors demonstrated that overexpression of pik3ca with a number of hotspot mutations led to mesodermal and particularly vascular malformations in the zebrafish model. Interestingly, they found a paucity of mScarlet+ mutant cells in the vascular lesions, consistent with the finding of low PIK3CA mutation burden in PROS tissue. Such data suggest a non-cell-autonomous effect of PIK3CA mutation. Following this logic, the authors performed single-cell RNA-Sequencing on zebrafish overexpressing WT pik3ca and mutant pik3ca at 19 hpf, and demonstrated widespread transcriptomic perturbations across multiple lineages, including lineage frequencies, key cell pathways, and cell-cell interactions. Importantly, they demonstrate that mScarlet+ cells carrying mutant pik3ca cluster separately from other cell types, do not demonstrate clear lineage identity, and have a general downregulation in signaling components.

Overall, the conclusions in the manuscript are well-supported by the presented data. The imaging studies are particularly convincing. The transcriptomic analysis generated a list of potential pathways to further investigate and potentially target with future therapeutic interventions. Importantly, this study provides a valuable in vivo model of PROS that: 1) recapitulates key features of PROS (e.g., multiple mesodermal defects, paucity of mutation burden in lesions suggesting non-cell-autonomous interactions); 2) is scalable; and 3) offers direct visualization of lesion development, compatible with time-course live imaging. This model will be valuable to further understand PROS and potentially study other diseases where the PIK3CA pathway is altered (e.g., certain cancers).

The following are not necessarily weaknesses of the data, but rather suggestions where the manuscript could be further strengthened:

(1) The model recapitulates the variability of mesodermal lesions in PROS. It would be valuable to utilize this model to further study factors that are associated with the development of more severe lesions (e.g., by comparing samples with more severe lesions to those unaffected despite carrying the mutations, Figure 1F).

(2) ScRNA-seq analysis could be enriched with a comparison between cells overexpressing mutant pik3ca vs. those overexpressing WT pik3ca.

(3) In the scRNA-Seq analysis, it is curious that the C0 cluster, enriched with mScarlet+ cells, is found to have downregulated signaling interactions (Fig. 5C), yet exerts a widespread non-cell-autonomous effect. Meanwhile, there is also a noticeable loss of certain lineages (e.g., notochord, Figure 4E) and related cell-cell interactions (e.g., notochord-related interaction, Figure 5A). A deeper exploration of the basis of the non-cell-autonomous effect would be valuable.

(4) The scRNA-Seq analysis was performed at one time point (19 hpf). Additional analysis (not necessarily by scRNA-Seq) at other time points to study whether findings at 19 hpf are persistent throughout development or undergo dynamic changes (e.g., cell fate/state of mSc+ mutant cells) would be helpful.

(5) The scRNA-Seq analysis provides a valuable list of perturbed interactions that could be targeted by future therapeutic approaches. Validation of the scRNA-Seq findings with protein-level analysis, and studying the effect of targeting some of the pathways on the disease phenotype, would offer valuable data for the community.

Reviewer #2 (Public review):

Summary:

This manuscript presents an ambitious and technically challenging spatial-transcriptomic atlas of 26 gastruloids using seqFISH. The authors introduce quantitative metrics (mixing score, exposure index, L-metric / scL-metric, spatial L-metric, triplets) to characterize spatial organization at multiple scales. The dataset is valuable, and several analyses are original, particularly the rank-based L-metric family for mutual exclusivity.

Strengths:

The authors generate one of the most detailed spatial transcriptomic datasets of gastruloids to date. They propose creative computational metrics (L-metric/scL-metric) to quantify mutual exclusivity of gene expression without predefined thresholds, and they explore organizational principles from single-cell topology to cluster-level structure. Many observations align well with known gastruloid biology, such as posterior robustness and anterior variability. The writing is generally clear, and the figures are rich.

Weaknesses:

Several central claims rely on metrics whose computation and justification are insufficiently explained, making it difficult to assess how robust or interpretable the results are. Many choices in the analysis appear arbitrary or are insufficiently motivated (normalization schemes, choice of parameters such as the number of neighbors, the distance cutoffs, hierarchical clustering setup, and so on). The interpretations of spatial consistency, gene-program inference, and endothelial heterogeneity are plausible but might be stronger than the evidence currently supports.

The manuscript would benefit from stronger benchmarking, quantification of uncertainty, and explicit controls for known artifacts in spatial transcriptomics (e.g., spillover, 2D slicing, cell type assignment entropy). The biological insights are promising, but since several depend on methodological assumptions that have not yet been demonstrated to be stable, they would benefit from clearer methodological explanation.

The work is rich and could become a reference dataset. Then, clarifying and validating the quantitative methods will considerably strengthen the impact and reliability of the conclusions.

Reviewer #2 (Public review):

The study addresses the long-standing question in molecular biology and genetics: why has nature selected the current genetic code (SGC, or standard genetic code)? The authors have tested 'error minimization theory', one of the prevailing hypotheses to explain this. Their approach is to create a minimum genetic code (MGC) and its variants (3^9 theoretical possible codes). Using three parameters to quantify the effect of mutations (Polarity, volume, and hydropathy), they computationally test the cost of these genetic codes (3^9) by simulations. Finally, they test this cost experimentally using an in vitro translation system with 10 select genetic code variants with a range of costs (low to high). They use three randomly mutated reporter genes for this purpose - beta-galactosidase, luciferase, and mSG. They find no correlation between the cost of the genetic code and the reporters' output. Based on these observations, they suggest that error-minimization theory may not explain the current egocentric code.

The question they are asking is very exciting, and their approach is solid. The authors are very careful in their analyses and conclusions.

Reviewer #2 (Public review):

Summary:

The authors of this study are trying to resolve how cellular infection by enteropathogenic E. coli (EPEC) subverts cellular signaling pathways to promote infection and dampen immune responses. Specifically, alteration in calcium dynamics has been evidenced in the prior literature as a potential initiator of these adaptions, and this study provides ideas and mechanistic detail as to how cellular calcium dynamics may be subverted by pathogens.

Strengths:

The clear strengths of this paper relate to the new ideas inherent in the proposed hypothesis and their support from the experimental approaches used. Overall, the proposed work provides new ideas in this area, which will benefit from further investigation. Certainly, this is an interesting and challenging paradigm to pick apart mechanistically, and is important for improving treatments from intestinal infections. The authors have provided additional data to clarify and expand on concerns raised during the original review, and these additions are helpful.

Comments on revised version.

Thorough response to original review. No further comments.

Reviewer #2 (Public review):

Summary:

In this study, Zhang and colleagues examine the role of participant selection in creating and using functional templates to improve analyses using hyperalignment. Hyperalignment aligns participants' functional MRI data to a shared functional template, analogous to the anatomical templates used to bring anatomical MRI data into a shared space (e.g., MNI152). The question of appropriate template creation is especially pressing for population-level analyses, where a large number of demographic groups (e.g., different age ranges, clinical statuses) may be included in the same analysis. These different demographic groups may have differences in their functional organization that complicate the creation of a single study-specific functional template.

To provide an initial investigation of the potential effect of demographic-specific templates, the authors use the publicly available Cam-CAN dataset which contains participants from 18 to 87 years of age. They define a young adult (< 45 years of age) and an older adult group (> 65 years of age) from this dataset with approximately the same number of participants. They investigate whether "age-congruent" templates (i.e. defined in the same age group they are used) improve three analyses where hyperalignment has been previously shown to boost performance: inter-subject correlation, predicting individual connectomes, and predicting individual functional responses. Using the Cam-CAN derived older adult template, they then replicate the ISC analyses using the publicly available Dallas Lifespan Brain Study (DLBS).

Overall, the presented results are highly suggestive that age-congruent templates consistently improve performance, though the absolute effects are small.

Strengths:

The use of a separate validation sample-re-using the same template calculated with Cam-CAN-highlights the potential of developing independent templates for individual demographic groups and then distributing these for wider use, analogous to the MNI templates that are widely used throughout the field of neuroimaging. This suggests that the potential impact of this framework is significant.

Weaknesses:

In their revision, the authors have addressed the previously raised "weaknesses" by providing guidance for researchers interested in using age-specific hyperalignment templates in practice.

Impact:

Overall, this work is likely to encourage future development of age-specific functional templates in the imaging community.

Reviewer #2 (Public review):

Summary:

The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.

Strengths:

A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across several in vivo applications, including neurovascular imaging, blood flow measurements, optogenetic stimulation, and microglial dynamics.

Weaknesses:

In the in vivo demonstrations, the authors employ different Bessel beam configurations across experiments, but the beam parameters are not dynamically tuned during live imaging. A video example showing continuous or interactive tuning of the Bessel beam within a single in vivo imaging sequence would further highlight the practical advantages of this platform and strengthen the case for its potential applications. In addition, while excitation powers are reported, the manuscript does not place these values in the broader context of known photodamage thresholds for two-photon microscopy, which would be helpful to the readers. Denoising/image restoration are applied in one of the in vivo examples, but it is unclear why this step was used specifically for this dataset and whether it was necessary to achieve adequate SNR or primarily included as an additional demonstration.

Reviewer #2 (Public review):

Summary:

The authors investigated the effect of prolonged iron limitation (which does stop growth but does not lead to cell death) alters central metabolism in M. tuberculosis. The major tool they used is metabolomics combined with stable isotope tracing. They show that the Krebs cycle is still active, despite the fact that it is dependent on some iron-dependent enzymes. They show that carbon flux through the oxidative branch of the Krebs cycle is stalled, resulting in the accumulation of metabolites, such as malate and alpha-ketoglutarate that are partially secreted. Apparently, the carbon flux from glycolysis is partially diverted to the reductive branch of the Krebs cycle. This is not achieved by using the glyoxylate shunt but probably through the GABA shunt. This unprecedented split of the Krebs cycle and malate secretion allows a continuous flow of carbon through the core of carbon metabolism, overcoming the metabolic stalling triggered by iron starvation.

Strengths:

Novel insight in the central metabolism of a major pathogen and its adaptation to iron starvation. Carefully conducted experimentation. Paper ends with a clear and helpful model.

Weaknesses:

The authors show some surprising and important findings, but would need a little more effort to really substantiate this. Especially the role of the GABA shunt should be genetically tested, as they did for ICL and the glyoxylate shunt.

Also, the dataset 1 is not very convincing, it is only based on transcriptomics and shown with up or down, hardly a strong base for major conclusions. The very least you want is actual differences, preferable on the protein level, where it really counts....

Comments on the revised version:

In the revised version all these points were appropriately dealt with and discussed, although some of them textually and not experimentally, but for reasons that are logical.

Reviewer #2 (Public review):

Summary:

The authors design a custom Bayesian model to estimate the probabilities of access, use and use given access of insecticide-treated nets in six African countries, providing sub-national estimates and inferring the average duration of ITN use and access. An individual-based model was employed to simulate malaria epidemics and estimate the effectiveness of different ITN distribution strategies. The study finds that the mean probability of use or access did not reach 80% (a universal coverage formerly targeted by WHO) for any of the regions even for biennial campaigns, demonstrates that switching from triennial to biennial distribution campaigns increases population use by 7.9%, and evaluates the impact of employing more efficient ITNs on P. falciparum prevalence.

Strengths:

The authors developed a data-driven model that accounts for data collection imperfections and sources of uncertainty while differentiating between ITN use and access. They developed a methodology to infer the timing of mass campaign from publicly available data instead of assuming fixed dates. The probability of use given access allows determining the regions where ITN distribution is least effective. This work can help better inform future interventions by identifying regions where increasing mass campaign frequency or employing better ITNs are most effective. Finally, in addition to insights on ITN access and use for the six countries analyzed, the paper contributes with a methodological framework that can likely be extended to other countries.

Weaknesses:

Since the models employed are rather complex, the methodology description may be hard to follow for some readers. In addition, the models assume many hypotheses, including exponential decay of ITN use/access and narrow prior distributions. It is worth noting that, in the revised version of the manuscript, the authors justified the choice of exponential decay and narrow prior distributions, and made a significant effort to clarify the methodology and the model equations.

Comments on revised version:

I appreciate the improvements made to the text. The methodology description is much clearer now. I have no further suggestions.

Reviewer #2 (Public review):

Summary:

Programmed DNA elimination is increasingly recognised as an important phenomenon across many species, including in animals. Exactly how widespread is still unclear, and the function of PDE is even more mysterious in most species where it has been described. PDE has been discovered in several nematode species, and in this manuscript, the authors carry out a more extensive search for PDE. They find PDE in many species, indicating that it is widespread across the phylum.

Strengths:

The large number of species across many different clades provides good evidence that the phenomenon has evolved many times independently. The work will therefore prompt many further studies characterising individual species, and potentially linking the evolution of the phenomenon to other features of these species' ecological characteristics.

Weaknesses:

The major technical weakness of this project is the assay that is used to evaluate PDE. First, this assay is clearly insensitive, as the authors acknowledge, O. tipulae, which has PDE, does not appear in their screen. Second, the assay gives no information about breakpoints and only limited, non-quantitative information about how much DNA is eliminated. Thus, their data really is only a preliminary screen, which would need to be confirmed by genomic assays.

Reviewer #2 (Public review):

Summary:

The authors set out to test the extent to which differences in learning capacity and experience contribute to behavioural variation in a genetically identical population under identical environmental conditions.

Strengths:

The authors developed and used a scaled-up version of a simple two-choice behavioural paradigm, allowing them to test thousands of individuals across multiple genotypes. They then deployed clever and powerful statistical analysis methods and provided compelling evidence for a role of variability in learning in the expression of behavioural variation.

Weaknesses:

There are no major weaknesses, although some level of longitudinal analysis to strengthen the evidence for a strict definition of individuality would be a welcome extension of a future study. In addition, it would have been very interesting, although understandably beyond the current scope, to delineate a potential source of learning variability in the brain.

Reviewer #2 (Public review):

This manuscript presents a substantial technical advance for the genetic manipulation of Blastocystis by establishing an integrated workflow for stable episomal transgenesis, antibiotic selection, clonal recovery, and reporter-based imaging in the ST7-B subtype. The study is particularly valuable because it combines multiple previously fragmented approaches into a coherent and practically applicable toolkit, including endogenous regulatory elements, optimized electroporation conditions, selectable markers, and anaerobic compatible fluorescent reporters. This methodological work greatly expands the molecular toolbox and future studies focused on both basic and infection biology can now build on the ability to express and localize proteins in fixed as well as live cells.

The microscopy data are convincing and clearly demonstrate functional reporter expression and successful recovery of stable transgenic lines. Nevertheless, because this is primarily a methodological paper, the study would be further strengthened by the inclusion of Western blot validation of reporter expression and bicistronic constructs. In particular, biochemical analysis of the P2A-containing constructs would help assess the efficiency of ribosomal skipping and exclude the possible presence of uncleaved fusion proteins, thereby providing stronger support for the interpretation of the imaging data and the functionality of the expression system.

Reviewer #2 (Public review):

Summary:

Nian and colleagues comprehensively apply metabolomics, molecular, and genetic approaches to demonstrate that CLas hijacks the DA/DcDop2-miR-31a-AKH-JH signaling cascade to enhance lipid metabolism and fecundity in D. citri, while concurrently promoting its own replication.

Strengths:

These findings provide solid evidence of a mutualistic interaction between CLas proliferation and ovarian development in the insect host. This insight significantly advances our understanding of the molecular interplay between plant pathogens and vector insects and offers novel targets and strategies for HLB field management.

Weaknesses:

While the article investigates the involvement of dopamine signaling and specific microRNAs in enhancing fecundity and pathogen proliferation, it still needs to provide a detailed mechanistic understanding of these interactions. The precise molecular pathways and feedback mechanisms by which CLas manipulates dopamine signaling in Diaphorina citri remain unclear.

Reviewer #3 (Public review):

Summary:

Ioakeimidis and colleagues studied miscrostructural abnormalities in N=56 Huntington's disease (HD) patients compared to N=57 normative controls. The authors used a powerful MRI Connectom scanner and applied the SANDI model to estimate the soma size, neurite size, soma density, and extracellular fraction in key subcortical nuclei related to HD. In the striatum, they found decreased soma density and increased soma size, which also seemed to become more pronounced in advanced HD individuals in the final exploratory analyses. The authors conducted important analyses to find whether the SANDI measures correlate with clinical scores (i.e., QMotor) and whether the variance of the striatal volume is explained by the SANDI measures. They found a relationship of SANDI measures to both.

Strengths:

The study is both innovative and of high interest for the HD community. The authors provide a rich pool of statistical analyses and results which anticipate the questions that may emerge in the HD research community. Statistics are carefully chosen and image processing is done with state-of-the-art methods and tools. The sample size gives sufficient credibility to the findings. Altogether, I think this study sets a milestone in the attempts of the HD community to understand neuropathological processes with non-invasive methods, and extends the current knowledge of microstructural anomalies identified in HD with diffusion MRI. More importantly, the newly identified anomalies in soma size and soma density open new avenues for studying these biological effects further, and perhaps develop these biomarkers for use in clinical trials.

Weaknesses:

(1) An important question is whether the SANDI measures, which require an expensive scanner and elaborate processing, are better biomarkers than the more traditional DTI measures. Can the authors compare the effect size of FA/MD with SANDI measures. In some of the plots and tables, FA/MD seem to have comparable, if not higher, correlations with QMotor or CAP scores. On the same vein, it is unclear whether DTI measures were included in hierarchical stepwise regression. I wonder if the stepwise models may have picked up FA/MD instead of SANDI measures if they are given a chance. Overall, I hope the authors can discuss their findings also in this light of cost vs. benefit of adopting SANDI in future studies, which is an important topic for clinical trials.

(2) Similar to the above point, it is very important to consider how strong the biomarking signal is from SANDI measures compared to the good old striatal volume. Some plots seem to indicate that volumes still have the highest correlation with QMotor, and highest effect size in group comparisons. It would be helpful for the community to know where do the new SANDI measures stand compared to the most typically used volumes in terms of effect size.

(3) The diffusion measures are inevitably correlated to some degree. Please provide a correlation matrix in supplementary material including all DWI measures to enable readers to understand better how similar SANDI measures are between each other or vs. other DTI measures. Perhaps adding volumes to this correlation matrix may also be a good future reference.

(4) ISS stages:

(a) The online ISS calculator requires cut-offs derived from the longitudinal Freesurfer pipeline, while the authors do not have longitudinal data. Thus, the ISS classification might be inaccurate to some degree if the authors used the FS cross-sectional pipeline. Please review this issue and see if updated cut-offs should be used to classify participants.<br /> (b) Were there really no participants with ISS 0 among 56 HD individuals, please clarify in the manuscript?<br /> (c) A note on terminology that might be confusing to some readers. According to the creators of ISS, the ISS stages are created for research only, they are not used or applied in the clinic. On the other hand, the terms "premanifest" and "manifest" have a clinical meaning, typically based on the diagnostic confidence level. The assignment of ISS0-1 to premanifest and ISS2-3 to manifest may create some non-trivial confusion, if not opposition, in some segments the HD community. The authors can keep their current terminology but will need to at least clarify to the reader that this assignment is speculative, does not fully match the clinically-based categories, and should not be confused with similarly named groups in the previous literature.

Comments on revised version.

The authors have moved to address many points from reviewers. The manuscript had indeed become more objective, transparent, and to the point. The amount of information and analyses is large, which perhaps is inevitable when new methods are being tested for the first time in a neurodegenerative disease.

Reviewer #2 (Public review):

Summary

This study uses functional MRI to evaluate visual contrast sensitivity across the visual field at the level of the visual cortex, testing the method as a proof of principle in a small group of normally sighted individuals, modelling both normal vision and simulated vision loss, as well as a patient with independently verified vision loss. The results suggest a promising technique to measure vision objectively across the visual field and overcomes the requirement for careful fixation which is often challenging in those with low vision or sight loss.

Strengths

• Objective measure of central vision: The proposed method may provide a more comprehensive and objective assessment of residual visual function in individuals with sight loss. This may be particularly useful for those with central visual field loss without the requirement of stable fixation or subjective motor responses.

• More sensitive measure: The use of slope to calculate contrast sensitivity across a range of contrasts within the brain is clever and likely more sensitive than single threshold measurements or standard clinical measures of visual acuity using letter charts. Standard supra-threshold (high contrast) tests are not ideal for capturing residual vision or partial vision loss.

• Good agreement with standard atlas: The Benson atlas provides a good estimate of visual field maps within V1 based on anatomical landmarks, and the authors take steps to refine this informed by cortical magnification and V1 surface area (brain size) for each individual participant. This could allow the technique to be generalised without the need to collect lengthy individual mapping data from every participant.

• Within-subject reproducibility: The measurements appear to be sensitive and reproducible, particularly in those with normal vision, and are consistent with known features of visual sensitivity differences in different parts of the visual field.

• Potential tool to measure visual field sensitivity in controls: Even if the proposed methods are not ideal for widespread clinical translation, they do offer an exciting tool to test hypotheses about visual field differences in healthy controls. For example, there seems to be an increase in sensitivity on either side of the simulated ring scotoma (Fig 6 - perhaps due to the release of lateral inhibition?). Reliability measures suggest that individual differences are consistent in healthy controls (although not tested statistically, perhaps due to the small sample size?). Whether they reflect behaviourally meaningful differences in visual field sensitivity could be tested in individuals by comparing them to behavioural measures across the visual field.

• Potential tool to test novel treatments: The proposed techniques could be used to test within-subject changes in visual function in environments that are equipped to measure and analyse fMRI data, including clinical trials aimed at determining the success of novel treatments. Preliminary testing in healthy controls with eye movements also suggests that the method is suitable for testing low vision patients with unstable fixation (e.g., nystagmus), and the authors have modelled the effects of varying amounts and types of eye movements on functional outcome measures.

Weaknesses

• Questionable sensitivity to differences in patients. The variability in heat maps across healthy control participants is somewhat surprising, and it is uncertain whether they represent actual visual sensitivity differences or an artifact of the measurement technique, e.g., due to signal-to-noise differences introduced by local variations in brain anatomy. Thus, it is uncertain whether the substantial variance across controls will allow for a sufficiently stable baseline to detect meaningful differences in individual patients. Also, as the authors rightly point out, Benson atlas does not model differences along meridians, so that upper/lower field differences might not be detectable. However, the authors acknowledge that this is a pilot study, and further testing a wider range of scotoma types in patients and simulated in controls will only improve the methods. Furthermore, the ability to capture visual field representations in human visual cortex is also likely to improve with computational advances, making the use of atlases more feasible, obviating the need for individualised population receptive field mapping.

• Potential for clinical translation. Although it is a sensitive measure, functional MRI is costly, is not available in all clinical settings, requires significant post-processing analyses, and may be contraindicated in some individuals due to safety (e.g., metallic implants) or other concerns (e.g., claustrophobia). These could present significant barriers to widespread clinical translation, if this were the ultimate goal of the study.

• Limited range of spatial frequencies. The spatial frequencies tested were still quite low (0.3 and 3cpd) compared to measures such a visual acuity. Extending the measurements to higher spatial frequencies could allow better characterization of central vision, although necessarily for peripheral vision. However, this may depend on the typical visual abilities of the patient population of interest.

Appraisal and Impact:

The authors used appropriate and robust methods to assess and model known features of visual sensitivity differences across the visual field in sighted controls. In addition, the assessment technique successfully captured sensitivity changes due to simulated and actual partial field loss but was also fairly resilient to eye movements and fixation instability, typical of patients with sight loss. Although currently providing a proof of principle, the method is likely to improve with further testing and increasing normative sample sizes, and as computational methods continue to advance visual field map predictions. Although it may not be adopted widely as a standard clinical assessment technique due to the expense and other obstacles, it would provide a valuable tool in assessing clinical populations, for example in the context of clinical trials to assess suitability for treatment interventions or monitor treatment outcomes.

Reviewer #2 (Public review):

Summary:

This manuscript addresses a clear and widely relevant question: how ongoing fluctuations in alertness during wakefulness relate to large scale patterns of coordinated brain activity. The authors combine high field magnetic resonance imaging with simultaneous pupil measurements, and they compute an edgewise measure of arousal-related coupling for every pair of regions. Their main contribution is to show that arousal-related coupling is low dimensional and organized into seven reproducible "connectivity communities", each with characteristic network pair compositions. A secondary contribution is the observation that these communities exhibit systematic but community-specific hemispheric asymmetries, including a striking left/right dissociation within the ventral attention network, where the left side participates broadly across communities while the right side forms a more cohesive, segregated arousal responsive module. A final contribution is cross-context generalization: the same organizational structure and lateralization signatures are largely preserved during naturalistic movie watching.

Strengths:

(1) The paper moves beyond state contrasts and quantifies arousal related modulation continuously within wakefulness, directly addressing a gap highlighted in the Introduction.

(2) The hemispheric asymmetry result is not framed as a crude global dominance effect; the authors explicitly test and argue that the key signal lies in structured spatial heterogeneity rather than mean shifts.

(3) The cross-paradigm replication in movie watching is a strong design choice and supports the claim that the organizational motifs are not limited to unconstrained rest.

(4) Arousal effects on BOLD signals and on pupil size can have different delays. The authors have now tested lagged relationships (for example shifting the pupil series forward and backward) to show that the main community structure and lateralization results are not sensitive to an arbitrary temporal alignment.

(5) Time resolved connectivity results are now shown to be robust to changes in parameters.

Reviewer #2 (Public review):

In their manuscript "TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy", Yokoyama and colleagues investigate the role of Tgfbr2 expression by NK cells in the formation of tissue-resident uterine NK cells and subsequent importance in murine pregnancy. By transferring congenic splenic conventional NK cells into pregnant mice, they show conversion of circulating NK cells into uterine ivCD45 negative tissue-resident NK cells. When interfering with the formation of uterine trNK cells, spiral artery remodelling was impaired, fetal resorption rates were increased, and litter sizes were reduced.

Generally, this is a research topic of high interest, yet the manuscript is lacking detailed mechanistical insights and some questions remain open. At the current state, the data represent an interesting characterisation of the Tgfbr2-fl/fl Ncr1-Cre mice in pregnancy, but considering 1) the recent publication by the group (Ref#17) on the role of Eomes+ cNK cells during pregnancy, 2) the previously described role of Tgfbr2 and autocrine TGFb expression for uterine NK cell differentiation in virgin mice (also cited by the authors), and 3) the well-known relevance of uterine NK cells during pregnancy, additional experiments addressing the specific role of Tgfb during pregnancy would help to improve novelty and significance of the manuscript.

Comments on revised version:

In their revised version of the manuscript and their point-by-point response, the authors have very carefully addressed and discussed all of our concerns and suggestions.

Reviewer #2 (Public review):

Summary:

This study identified U2AF1/2 as a regulator of pre-mRNA splicing that either promotes or supresses the splicing of introns on different genes. The authors then focused on two genes PURPL and MALAT1 that U2AF1/2 can promote intron retention of specific introns, and characterized the biological implications of these introns regulated by U2AF1/2.

Strengths:

(1) The experiments in this manuscript are relatively rigorously designed and performed, often with validation checks such as verifying the knockout, verifying the treatment itself doesn't have an effect, etc.

(2) The experiments provided comprehensive support for the claims that these specific introns are important for the stability or nuclear localization of the RNA, as well as that U2AF1/2 suppresses the splicing of these introns.

(3) The writing of the manuscript is very clear and doesn't overstate the conclusions that can be drawn from the experiments.

Weaknesses:

I think one main weakness of this study is the lack of a deeper analysis of the mechanisms. Whether studying the mechanism is within the scope of this paper is probably debatable, but with the current experiment setup and data, I believe there are some analyses that can be relatively easily done to enhance the value or significance of this study. My detailed questions and suggestions are listed below:

(1) Line 194-195 and Figure 2A: How many RBPs are included in "other RBPs" in line 194? Does "other RBPs" only include PTBP1, PRPF8 and SRSF1 in Figure 2A, or do they include all the ~100 RBPs with HepG2 eCLIP data available on ENCODE? If U2AF1/2 have the highest occupancy around the intron 2 region among the ~100 RBPs, it would be nice to visualize it.

(2) Figure 2A and 2B: Why didn't U2AF2 show interaction with exon 2 and 3 in RNA-IP but showed enrichment over exon 2 and exon 3 regions in the eCLIP data?

(3) Figure 3C - 3F: Maybe I misinterpreted the experiments, but to my understanding, these experiments showed that the exogenous PURPL with intron 2 promoted cell proliferation compared to when the exogenous PURPL wasn't induced, but didn't compare to the effect of the same amount of PURPL with intron 2 removed. Wouldn't it be clearer to compare the effects of exogenous PURPL with intron 2 and exogenous PURPL without intron 2 to pinpoint whether the effect is related to intron 2? Without an intron 2 specific experiment, these current experiments don't seem to provide much added value than "PURPL promotes cell proliferation".

(4) It's not very clear what proportion of these introns are retained in the endogenous PURPL and MALAT1 in various tissues, cell types and conditions. I think it will be valuable to provide this background (either from previous research, public database or data from this study).

(5) Since U2AF1/2 have a wide range of targets as demonstrated by Figure 4A, I think it would be valuable to have some experiments that directly disrupt the interaction between U2AF1/2 and PURPL and MALAT1 and test the effect on splicing outcomes, such as by mutating the sequence that U2AF1/2 bind to. The section on the weak py-tract of PURPL touched upon this topic but focused more on how the weak py-tract causes the intron 2 retention in the background rather than how U2AF1/2 binding and action were affected by sequence mutations. I think experiments on disrupting the direct binding between U2AF1/2 on targets can provide valuable mechanistic insights.

(6) Across all the target genes of U2AF1/2, it might be feasible to do some systematic analysis to find what correlates with whether U2AF1/2 have a promoting or suppressing effect on intron splicing. For example, do genes with decreased IR after U2AF2 depletion systematically have a weak py-tract compared to genes with increased IR? This dataset can potentially provide many hypotheses for understanding the dual role of U2AF1/2.

Reviewer #2 (Public review):

The authors describe the first deep neurological characterization of WAC mutation in two vertebrate species (zebrafish and mouse). They examine these at various levels, guided by the work in humans that has associated a heterozygous WAC mutation with DeSantos Shinawi Syndrome (DESSH). Therefore, they investigate the animals for a variety of phenotypes, following a template for what is seen when characterizing a new mouse/fish model of a developmental disability gene. Investigations include analysis of skull and jaw for abnormalities(both species), MRI of brain structure(in mice), electrophysiology(mice), assessment of signaling pathways (by Western blot, in mice), cell counts (both, more in mice), transcriptomics (mice), and behavior (both).

Generally, this describes an important first characterization of the consequences of the mutation. Most of the studies appear well-conducted and reasonably powered, thus solid or convincing.

Reviewer #2 (Public review):

Summary:

The authors investigate the behavior of oncogenic cells in mammary and bronchial epithelia. They observe that individual oncogenic cells are preferentially excluded from the mammary epithelium, but they remain integrated in the bronchial epithelium. They also observe that clusters of oncogenic cells form a compact cluster in mammary epithelium, but they disperse in the bronchial epithelium. The authors demonstrate experimentally and in the vertex model simulations that the difference in observed behavior is due to the differential tension between the mutant and wild-type cells due to a differential expression of actin and myosin.

Strengths:

(1) Very detailed analysis of experiments to systematically characterize and quantify differences between mammary and bronchial epithelia.

(2) Detailed comparison between the experiments and vertex model simulations to identify the differential cell line tension between the oncogenic and wild-type cells as one of the key parameters that are responsible for the different behavior of oncogenic cells in mammary and bronchial epithelia.

Reviewer #2 (Public review):

Overall, this is a solid manuscript that delivers an important community resource. The execution is relatively simple, but the value is real, the work is rigorously performed, and the open dissemination through Zenodo, the F1000Research YCharOS Gateway and OGA is well executed. The effort invested in generating the knockout lines for validation experiments is a clear strength of the study. I have a number of comments that I think would strengthen the resource and the conclusions drawn from it.

Below, I list specific points.

(1) The rationale for the selection of these 33 genes is insufficient. The authors lean on the Nijs & Van Damme classification and on PubMed entry counts, but the number of PubMed entries is not a meaningful criterion for what constitutes an important ALS protein - some of the most disease-relevant genes are precisely those with fewer publications, while heavily cited genes such as CAV1 carry weak ALS-specific evidence. The authors should provide a more transparent and biologically motivated rationale for inclusion and exclusion (ClinGen evidence tier, replicated GWAS signals, large meta-analyses, ALSoD) and explain why specific risk genes outside this list were not part of ALS-RAP.

(2) "107 of 231 (46%) demonstrated specific target staining in IF." The criteria used to define "specific target staining" at the IF level are not stated. From the Galectin-1 example, the mosaic WT/KO strategy provides a binary readout, but for proteins with low expression, weak punctate staining or unusual subcellular distributions, a single threshold is unlikely to capture specificity uniformly across 231 antibodies.

(3) Several claims in the manuscript depend on differential protein abundance across cell types. As presented, these claims are supported by qualitative Western blot images only. They should be substantiated by quantification across multiple biological replicates.

(4) This manuscript represents a unique opportunity to address antibody recognition of splicing variants, which is something of of considerable value to the community. For each target, the predicted isoforms in Ensembl could be cross-referenced against the observed bands, and the pattern of bands compared across cell types could be informative about which isoforms each antibody captures. This would convert ambiguous "extra bands" into useful biological information and would substantially increase the value of the resource. I strongly encourage the authors to include this analysis.

(5) The iPSC-derived microglia receive a comprehensive QC panel (IBA1/PU.1 IF, CD45/CD11b flow, qRT-PCR for nine canonical markers; Figure S4), which allows the reader to assess culture purity. The other iPSC-derived lineages - motor neurons, dopaminergic neurons, oligodendrocytes and astrocytes - are validated by a single marker each in WB (Figure S3) without purity quantification. Given that several conclusions of the manuscript rest on the cell-type-specific detection of ALS-associated proteins, equivalent quality control should be performed for the other lineages so that the reader can evaluate the purity of each preparation.

(6) The robustness of the resource would be substantially increased by validating at least a subset of the targets in a second iPSC background, in at least some of the cell types analysed.

(7) The newly developed SGC scFv antibodies are arguably the most novel reagent contribution of this manuscript, yet they receive a single sentence in the body of the paper. A more thorough description is warranted.

(8) Accessibility of the resource through Zenodo is not straightforward - the reader currently has to navigate to individual antibody characterization reports one by one to extract recommendations for a given target. While the use of an established public repository is important for permanence, a dedicated ALS-RAP website with an interactive, searchable interface - filterable by target, application, host species and clonality - would meaningfully improve uptake. The relationship between such a portal and the existing OGA platform should also be clarified.

Reviewer #2 (Public review):

While the importance of asparagine in the differentiation and activation of CD8 T cells has been previously reported, its role in CD4 T cells remained unclear. Using culture media containing specific amino acids, the authors demonstrated that extracellular asparagine promotes CD4 T cell proliferation. Consistent with this, depletion of extracellular asparagine using PEG-AsnASE suppressed CD4 T cell activation. Proteomic analysis focusing on asparagine content revealed that, during the early phase of T cell activation, most asparagine incorporated into proteins is derived from extracellular sources. The authors further confirmed the importance of extracellular asparagine in vivo, demonstrating improved EAE pathology.

While the data are well organized and convincing, the mechanism by which asparagine deficiency leads to altered T cell differentiation remains unclear. It is also necessary to investigate the transporters involved in asparagine uptake. In particular, elucidating whether different T cell subsets utilize the same or distinct transport mechanisms would provide important insight into the immunoregulatory role of asparagine.

Comments on revised version:

The authors have addressed the previous concerns, and the manuscript has been significantly improved.

Reviewer #2 (Public review):

This study by Jonker et al., examines how the metabolic adaptations to the microenvironment by pancreatic ductal adenocarcinomas (PDAC) present vulnerabilities that could be used for therapeutic purposes. The evidence supporting the claims of the authors is mostly solid, and the multiplicity of models used, as well as the combination of in vitro and in vivo work are appreciated, but some conclusions would benefit from additional substantiation. This work would be of interest to biologists working on the impact of microenvironment and metabolism in cancer, and especially those investigating pancreatic cancer.

In this study, the authors use mostly "doublings per day" as an indicator of cell death, notably for figures 4 to 6. However, proliferative arrest (or a decrease in the proliferative rate) is not necessarily synonymous with cell death. It might be nice to complement these experiments with a true measure of cell death (e.g. PI uptake).

Reviewer #2 (Public review):

Summary:

This manuscript presents a comparative analysis of optomotor behavior in zebrafish and medaka larvae. Using multiple behavioral paradigms, the authors argue that the two species differ in both the spatial and temporal integration of visual motion. They further decompose turning behavior into large- and small-turn components and use a simple mechanistic model to capture several of the main response features. Overall, the study addresses an interesting question, and the comparative framework gives the work a clear conceptual appeal.

Strengths:

A major strength of the manuscript is the breadth of the behavioral analysis. The authors use several stimulus paradigms to probe spatial extent, temporal persistence, and response dynamics, which makes the cross-species comparison richer and more informative than a single-assay study. The decomposition into large and small turn components is also a useful feature of the work, as it provides a more structured account of where the species differences may arise. The modeling further helps organize the results and offers a useful framework for interpreting the behavioral differences.

Weaknesses:

The main limitations are in presentation and clarity rather than in the overall motivation or approach. In several places, it is difficult to determine exactly how some quantities are summarized statistically, and some figures and legends would benefit from clearer explanations. In addition, a few of the more specific interpretive claims would be strengthened by more explicit statistical framing and slightly clearer presentation. These issues appear addressable and do not detract from the overall interest of the study.

Reviewer #2 (Public review):

Summary:

This manuscript, "Modality-Specific and Amodal Language Processing by Single Neurons," presents an intracranial electrophysiology study investigating how language is represented in the human brain across spoken and written modalities. The authors analyze activity from over one thousand single neurons and local field potentials recorded in twenty-one neurosurgical patients while participants read and listened to sentences. Using encoding models based on temporal receptive fields, they examine whether neural responses track modality-specific features, such as phonological and orthographic information, as well as higher-level linguistic features. The results are interpreted as evidence for a dissociation between modality-specific processing in sensory regions and modality-independent ("amodal") representations in temporal and frontal cortices, supporting a two-stage model of language processing.

Strengths:

This study uses a rare and valuable dataset, combining single-neuron recordings with broader field potential measures in human participants. The large-scale recording, in terms of both neuron count and anatomical coverage across multiple regions and individuals, represents a significant technical achievement for intracranial research.

The use of encoding models to relate neural activity to multiple levels of linguistic representation is methodologically rigorous and provides a unified framework to compare phonological, orthographic, and higher-level features. This approach allows the authors to systematically test how different aspects of language are represented across neurons and regions.

Another key strength is the attempt to directly link concepts from Linguistics to neural data. By framing the results in terms of modality-specific versus amodal representations, the study engages with longstanding theoretical questions and offers a potential bridge between linguistic theory and systems neuroscience.

The manuscript is also very well written, and the data are presented clearly and effectively. The inclusion of raw data and raster plots is particularly valuable, as it allows readers to directly assess the neural responses and strengthens the transparency of the analyses.

Weaknesses:

Despite these strengths, the central claims of the paper are not fully supported by the analyses presented, and several key issues limit the strength of the conclusions.

A primary concern is the lack of clear reporting and statistical characterization of the proportion of neurons that significantly encode the tested linguistic features. While the paper presents illustrative examples and regional patterns of encoding, it does not systematically quantify how many neurons exhibit significant effects across conditions, nor does it provide formal statistical comparisons of these proportions across brain regions or feature types. As a result, it is difficult to determine whether the reported dissociations reflect robust population-level phenomena or relatively sparse subsets of neurons identified through model fitting. Figure 2H offers a visual depiction of the distribution of Brain-Score (a measure of model evaluation) across the fusiform gyrus and superior temporal gyrus, but it falls short of providing formal statistical testing or quantitative summaries, limiting its interpretability in supporting the authors' claims. Given that the authors employ temporal receptive field (TRF) analyses, the framework naturally allows for straightforward quantification of the proportion of neurons that significantly encode any linguistic features in the model, which could be reported by region as well as by stimulus condition (auditory vs. visual). Including such analyses would further strengthen the population-level interpretation of the results.

Relatedly, the interpretation of "amodal" neurons is not sufficiently substantiated. The classification of neurons as modality-independent relies on encoding model performance across conditions, but the statistical criteria for establishing cross-modal generalization are not always clearly defined or rigorously tested. Without explicit comparisons (e.g., testing whether the same neurons significantly encode features in both modalities above chance, and whether this exceeds what would be expected under appropriate null models), the claim of modality-independent representation remains somewhat underdetermined.

More generally, the reliance on encoding models introduces some interpretational ambiguity. Although the observed dissociation between fusiform and superior temporal regions is consistent with orthographic and phonological processing, respectively, the feature spaces used in the models are partially linked to lower-level sensory properties (e.g., visual form and acoustic features). The authors' single-neuron results suggest these effects reflect genuine linguistic selectivity, but the findings do not uniquely distinguish between linguistic and perceptual explanations. While fully disentangling these factors may be beyond the scope of the current study, the manuscript could benefit from a brief discussion acknowledging these correlations or clarifying how lower-level sensory contributions were considered.

Another limitation is that the proposed two-stage model of language processing is not directly tested against competing hypotheses. While the dissociation between modality-specific and amodal representations is consistent with this model, the authors note that higher-level features, such as syntax, may be encoded in a distributed or overlapping manner. These possibilities are not systematically tested, so the conclusions risk overinterpreting correlational patterns as evidence for a specific processing hierarchy. A more explicit discussion or quantitative consideration of these alternative accounts would strengthen the interpretation, while still allowing the two-stage model to be presented as a plausible framework.

Reviewer #2 (Public Review):

This paper is an interesting conceptual work where certain hotspot areas were found to induce unique gait patterns. These patterns differed from a classic change in speed or gait pattern from a walk to a gallop. From this, a hypothesis was formed that these areas could be important for possible alternative walking patterns seen, for example, during pathologies such as Parkinson's disease or perhaps related to stalking behaviors.

While I liked the work and found it interesting, it remains descriptive in that the actual behaviors observed can't be causally related to a particular behavior such as stalking or shuffling. If the necessity or sufficiency of this region was related to a specific hunting behavior, for example, its interest to the field would be greater.

Nevertheless, this paper does contribute to growing evidence that specific behaviors can be triggered by specific neuronal populations within the brainstem.

Reviewer #2 (Public review):

This manuscript aims to elucidate the mechanistic basis for the long-standing observation that DNA methylation and the histone variant H2A.Z occupy mutually exclusive genomic regions. The authors test two hypotheses: (i) that DNA methylation intrinsically destabilizes H2A.Z nucleosomes, thereby preventing H2A.Z retention, and (ii) that DNA methylation suppresses H2A.Z deposition by ATP-dependent chromatin-remodelling complexes. The revised manuscript addresses a number of previous concerns, and the manuscript has therefore improved accordingly. However, several limitations remain.

Comments on revisions:

The authors have addressed a number of my previous concerns, and the manuscript has improved accordingly. However, several limitations remain that, in my view, constrain the strength of the conclusions. In particular, the absence of a direct comparison with a canonical nucleosome assembled on the same DNA template. This control is essential to determine whether the observed effects are specific to H2A.Z or reflect more general properties of methylated DNA-nucleosome interactions. Notably, even within the authors' own data, there is a trend suggesting that methylated canonical H2A nucleosomes may also exhibit increased accessibility. Although this does not reach statistical significance, the authors themselves argue that subtle differences can be biologically meaningful; it is therefore plausible that extended digestion conditions (e.g., longer HinfI exposure) could reveal a significant effect. Unless a direct structural comparison with a canonical nucleosome is performed, the possibility that the reported phenomenon is not specific to H2A.Z remains. This is compounded by the reliance on a single restriction enzyme-based assay, which represents a limited experimental approach. Such an approach is insufficient to unequivocally support the central claim that DNA methylation increases accessibility of H2A.Z-containing nucleosomes. Additional orthogonal assays would be required to substantiate this conclusion. With respect to the cryo-EM analysis of methylated and unmethylated 601L H2A.Z nucleosomes, and in general, the authors still do not adequately consider the positional context of CpG methylation. Extensive literature demonstrates that the effects of DNA methylation on canonical nucleosome structure and stability are highly position-dependent. Without accounting for the location of methylated CpGs relative to key DNA-histone contact sites, the structural data remain difficult to interpret mechanistically. Overall, while the manuscript has improved, it remains a relatively limited study that draws broad mechanistic conclusions from a minimal experimental data.

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 feto-placental 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.

Comments on revised version:

The authors have adequately addressed all my previous comments.

Reviewer #2 (Public review):

Summary:

Muscle hypertrophy is a major regulator of human health and performance. Here, van der Pilj and colleagues assess the role of the giant elastic protein, titin, in regulating the longitudinal hypertrophy of diaphragm muscles following denervation. Interestingly, the authors find an early hypertrophic response, with 30% new serial sarcomeres added within 6 days, followed by subsequent muscle atrophy. Using RBM20 mutant mice, which express a more compliant titin, the authors discovered that this longitudinal hypertrophy is mediated via titin mechanosensing. Through an omics approach, it is suggested that the Muscle ankyrin proteins may regulate this approach. Genetic ablation of MARPs 1-3 blocks the hypertrophic response, although single knockouts are more variable, suggesting extensive complementation between these titin binding proteins. Finally, it is found through the administration of rapamycin that the mTOR signalling pathway plays a role in longitudinal hypertrophic growth.

Strengths:

This paper is well written and uses an impressive suite of genetic mouse models to address this interesting question of what drives longitudinal muscle growth.

Weaknesses:

While the findings are of interest, they lack sufficient mechanistic detail in the current state to separate cross-sectional versus longitudinal hypertrophy. The authors have excellent tools such as the RBM20 model to functionally dissect mTOR signalling to these processes. It is also unclear if this process is unique to the diaphragm or is conserved across other muscle groups during eccentric contractions.

Reviewer #2 (Public review):

Summary:

This work identifies a previously unknown way that red light can slow ageing. The authors show that red light lowers the level of a protein called SIRT4 in skin cells. Reducing SIRT4 boosts fatty acid use and increases a type of histone modification that keeps genes active. These changes help cells clear away signs of ageing, reduce inflammation, and restore normal metabolism. The findings open the possibility of developing new treatments that target SIRT4 to reverse age‑related decline.

Strengths:

The evidence is solid because the authors use several complementary methods. They test red light in both cultured cells and naturally aged mice, and they confirm the key role of SIRT4 by silencing its gene. Measurements of metabolism, protein changes, and ageing markers all point in the same direction. However, the exact way red light lowers SIRT4 levels is not fully explained, which leaves a minor gap. Overall, the conclusions are well supported and convincing.

Weaknesses:

The paper does not evolve to use the mechanistic discoveries of the manuscript to help our community to identify the mechanism of photobiomodulation, which is not known so far.

I would like to draw attention to a recently published paper by Herrera et al. (FEBS Letters 2025, doi:10.1002/1873-3468.70195), which shows that red light (660 nm) stimulates mitochondrial fatty acid oxidation in keratinocytes via AMPK‑dependent phosphorylation of ACC, without altering expression of electron transport chain complexes. I believe this paper is highly complementary to the current study.

Herrera et al. demonstrate that red light increases basal, ATP‑linked, and maximal oxygen consumption rates in keratinocytes specifically through enhanced fatty acid oxidation (inhibited by etomoxir). This independently validates the central finding of the current manuscript, i.e., red light boosts lipid metabolism, strengthening the robustness of this concept.

While the current manuscript focuses on the SIRT4‑MCD axis, Herrera et al. identify AMPK phosphorylation and ACC inhibition as key effectors. The authors can integrate and expand their discussion, since SIRT4 downregulation may converge on AMPK activation, or they may represent parallel, reinforcing mechanisms. This would enrich the mechanistic model and open new hypotheses.

The mechanism of photobiomodulation: Herrera et al. explicitly challenge the prevailing paradigm that red light acts solely via cytochrome c oxidase (by showing long‑lasting effects, unchanged OXPHOS protein levels, and no difference in permeabilised cells). The current finding (red light acts through SIRT4 downregulation, i.e., not direct enzymatic activation) aligns perfectly with Herrera´s critique.

Long‑term metabolic effects - Herrera et al. show that a single red light exposure elevates oxygen consumption for up to 2 days. The current study focuses on changes at 12‑24 h. Their data extend the time window and suggest that the metabolic reprogramming you describe may persist longer than currently discussed, which is clinically relevant.

Discussing Herrera et al.'s results would not only acknowledge independent, corroborating evidence but would also allow the authors to position their SIRT4‑centric mechanism within a broader, emerging understanding of red‑light photobiomodulation.

Reviewer #2 (Public review):

The authors investigate the impact of the deletion of the small GTPase regulator ARHGEF6 on the development and physiology of interneurons. Using public databases, they first show that ARHGEF6 is enriched in interneurons or in areas that give rise to them, both in development and adulthood, in humans and mice. Using a complete KO mouse previously reported, and using a GAD67-GFP reporter mice line, they show that in the adult mouse cortex and hippocampus, there is a notorious reduction GFP+ cells. These mice show increased apoptotic cells at different timepoints and areas of the brain during development. In the developing cortex of ARHGEF6-KO mice, there are fewer IN in all layers of the developing cortex, and cells present processes not correctly oriented. IN from the hippocampus in culture show reduced excitability and impaired neurite branching. The authors then established isogenic hiPSCs lines to study ARHGEF6 deletion in human cells and differentiated ventral forebrain neurons, to find interneuron-related and non-related phenotypes. Most importantly, human interneurons grown in organoids show reduced branching and altered growth cone morphology. The authors claim that the novel interneuron phenotypes found in these models can explain, in part, the human intellectual disabilities associated with mutations in this protein. The study is well conducted and opens new avenues of research not only for the role of small GTPases regulation in early nervous system development, but also for how interneuron deficiencies impact a wider range of intellectual disability syndromes found in humans.

However, most conclusions of the present version would be strengthened after considering the following comments:

Major comments

(1) The reported biological processes evaluated at different developmental stages may be directly or indirectly related to ARHGEF6 function itself. As a model of a hereditary disease, full organism gene deletion is valid, since the human patients suffer from that condition as well. However, to investigate the roles of a protein, complete deletions may not be very accurate since they can give rise to phenotypes that are only indirectly related to the protein function itself. Most conclusions of the present manuscript should either be discussed in this regard or add evidence for a direct role of the protein. One such evidence is typically performed with acute knockdowns in culture, or in developing brains by in utero electroporation. For example, Figure 1C shows that the principal excitatory neurons in the hippocampus do not express ARHGEF6. However, most electrophysiological and behavioral evidence of defects in ARHGEF6-KO mice arises from evaluating these cells (Remakers et al., 2012). I am not suggesting that either previous or actual evidence is wrong. But I believe readers would benefit from a clear distinction (or add caution notes) between a functional consequence of the deletion (that can be months away and in other cells than the actual molecular defect) and a true cell biological function of the protein under study. In favor of the authors, this is a concern with most conclusions derived from KO organisms.

(2) Figure 1E-G H I. All conclusions are made with a GAD67-GFP reporter, which is a very powerful and reliable tool for large-scale screening. All the conclusions of the paper would be strengthened if some immunohistochemical staining in the same areas of specific markers for interneurons would be added as supporting complementary evidence.

(3) Cell death in development: It is surprising that the high amount of TUNEL staining during development does not translate into gross histological changes in the adult brain (studied elsewhere). Can authors discuss possible explanations?

(4) Section 4 (Figures 2F-J) - The authors present this staining as an analysis of migration. Normally, migration studies are performed with a "pulse-chase" paradigm, where a single cohort is labeled and then followed over time (normally by in utero electroporation of a fluorescent protein). Tissue is then fixed at different time points, and migration can be followed. On the contrary, the evidence is from a single point, in an experimental setting in which all Gad67 IN are stained, and hence, one cannot imply a defect in migration. The differences between WT and ARHGEF6-KO are obvious and interesting; it is just that they cannot be solely attributed to a problem in migration.

Also, a true phenotype of migration in the current setting should have found that the cells that failed to migrate are accumulated in deeper layers. My impression is that the changes in IN per layer are easier explained by total cell number, rather than migration. Perhaps evaluating earlier timepoints could clarify this.

(5) It is known that ARHGEF6 deletion produces severe F-actin phenotypes in neurons. Have the authors confirmed in their hippocampal cultures GAD67 cells ALSO have these phenotypes? Stress fibers in somas, growth cones, and actin patches along neurites.

(6) Section 4. The authors present data for deficient migration of the GFP-labeled interneurons. Is it possible to assess, in the same sections, whether other cell types are also affected? Although the hypothesis that ARHGEF6 deletion will have an impact in IN is well rooted in expression data, by assessing other cell types, one can even include a positive control or evidence for a cell-autonomous phenotype.

(7) ARHGEDF6 deletion has an important impact on organoid development (size, shape, etc). Have the authors analysed whether these organoids produced fewer interneurons?

(8) In assembloids, the differences in migration parameters are very small between WT and ARHGEF6-KO, which reinforces that perhaps what is observed in the different layers of cortex during mouse development is likely not entirely due to migration, as concluded.

(9) To properly weigh the present evidence -interneuron deficits- using the ARHGEF6-KO model, authors should include a deeper discussion in light of much work that has been done using these mice. How does the finding of a diminished IN population in the brain of these mice explain the large amount of electrophysiological and behavioral evidence produced before with these animals? Perhaps the most important work to discuss these aspects is the initial ARHGEF6-KO report by Ramakers and colleagues (2012), but there are others.

Minor comments

(1) Figure 1A. It looks clear that the GE shows the highest expression of ARHGEF6; however, the reader needs the reference levels where the log2 expression is calculated. What are the reference levels?

(2) Have the authors compared the number of GAD67-eGFP cells in the hippocampal cultures between WT and ARHGEF6-KO mice?

(3) Section 3, as a caution note, authors should mention that it is not possible to know from the evidence provided which cells are dying.

(4) In the dorsal-ventral assembloids, it is expected that the ventral organoid would contain lots of GFP expression compared to the dorsal, but in the image shown (Figure 5A) both parts of the assembloid seem to have the same amount and distribution of GFP. How is that possible?

Reviewer #2 (Public review):

Summary:

This manuscript describes an investigation into the effect of diet and exercise interventions in WT and transgenic (male and female) mice who are exposed to either a high-fat or a low-fat diet. The outcome variables include MRI volume and brain morphology, as well as memory performance. First, this study measured the impact of genotype (WT vs 3xTgAD mice), then examined the impact of a high-fat or low-fat diet in each group, and finally examined the impact of a low-fat diet, exercise, or a combined low-fat diet and exercise intervention. This is an important study as it allows us to better understand how changes to lifestyle can affect neurocognitive function and potentially change a person's AD risk.

Strengths:

(1) The study uses a well-controlled longitudinal design, allowing the authors to track how diet and exercise interventions influence brain and behaviour over time.

(2) The integration of multiple levels of analysis (brain imaging, behaviour, and multivariate modelling) provides a rich and comprehensive assessment of intervention effects.

(3) The inclusion of both genotype and sex as key variables strengthens the relevance and interpretability of the findings, given known differences in risk and response across groups.

Weaknesses:

There are a lot of analyses in this paper, and I had a little bit of trouble distilling the major take-home messages. For example, I was left wondering:

(1) If the effect of genotype and the effect of the high-fat diet were consistent in the current study compared to the authors' previous work (e.g. Rollins et al., 2019). A more direct report on the consistency of these findings (maybe even an overlap map, if possible) would benefit the reader.

(2) How consistent/different are the volumetric and morphometric (DBM) results from each other? Especially in the regions of interest (hippocampus and cerebellum), are increases in volumes always related to "expansion" of a given region using DBM? Some of the similarities are reported in the results, but for transparency, a side-by-side table comparing the results across techniques for each effect of interest might provide more clarity.

(3) I was interested in the Partial Least Squares approach that the authors used to investigate how patterns of brain measures relate to the behavioral variables. Because they are presented mostly in the supplement (except for Figure 6E), it's difficult to map the LVs described onto the univariate contrasts in Figures 2-5. In general, greater clarity is needed regarding how the PLS-derived latent variables relate to the univariate findings, and whether the emphasis on LV3 reflects a principled selection or post hoc interpretation.

(4) If I understand the results correctly, there were only modest differences in behavior reported, and the patterns were somewhat inconsistent across sex and genotype. In fact, the authors report that the high-fat diet alone did not impair memory on the Morris Water maze (line 323). The discrepancy between robust neuroanatomical effects and relatively modest behavioural changes raises important questions about the functional significance of the observed structural alterations.

(5) On line 507, the authors state, "Notably, 3xTgAD mice already show smaller brain volumes at baseline, which may constrain the detectable impact of the diet." Is this true for the entire brain or just the hippocampus and cerebellum? Would a global reduction in brain volume due to the 3xTgAD AD model affect the interpretation of the intervention effects?

Reviewer #2 (Public review):

Summary:

The authors develop a miniaturized MR1 construct (SMART-MR1) in which the α1/α2 platform is stabilized by a synthetic domain, and show that it can bind ligands, engage a cognate TCR, and recapitulate native-like recognition by cryo-EM.

Strengths:

The work is well-written, technically strong and carefully executed. The authors combine biochemical, biophysical and structural approaches, including ITC, NMR and cryo-EM, to show that SMART-MR1 behaves in a manner closely resembling native MR1. The reduction in size and the demonstration of solution NMR are clear practical advantages for certain types of mechanistic studies.

Weaknesses:

The main limitation is that the manuscript does not clearly establish a practical advantage over existing MR1 formats, such as single-chain MR1-β2M or previously described stabilized constructs. The comparison is largely framed against native MR1, which risks overstating the problem, and on the basis of the data presented, it is unlikely that other researchers will adopt this system. In addition, the choice of the A-F7 TCR as a validation reagent may overestimate the generality of the approach, as this receptor is known to exhibit relatively broad ligand tolerance, including recognition of MR1 presenting vitamin B6 metabolites (PDB 9CGR) and structurally diverse synthetic ligands. The extent to which SMART-MR1 supports recognition by a broader range of MR1-restricted TCRs is not addressed.

Reviewer #2 (Public review):

Summary:

The authors aimed to evaluate whether integrating genomic (SNP) and transcriptomic information with machine learning can improve phenotypic prediction of polygenic traits across environments. The manuscript explored not only the predictability across models and predictor feature sets, but also attempted to identify meaningful genes and interactions underlying trait variation.

Strengths:

The main strength of the manuscript is its integration of SNP, transcriptomic, and phenotype datasets for 426 sorghum genotypes between Texas and Michigan. It provides a systematic comparison of predictor types (SNP versus transcriptomic abundance) and model strategies to integrate them.

Weaknesses:

(1) Experimental Design

The experimental design raises several concerns that should be clarified before strong biological conclusions are drawn from the transcriptomic analyses.

First, the transcriptomic sampling is not well aligned with the developmental stages most relevant to the phenotypes being modeled. Leaf tissue was collected at a single time point in each environment, whereas traits such as flowering time, biomass, tiller count, and panicle height arise from developmental processes occurring over extended and potentially distinct temporal windows. Consequently, the measured expression profiles are likely to reflect physiological states specific to the sampling dates (May 5-6 in Texas and June 22-24 in Michigan) rather than the regulatory processes underlying the target phenotypes.

Second, the phrase "haphazardly randomized" is questionable for a field experiment. It is unclear whether the design included formal randomization, blocking, row/column structure, or spatial correction. Without explicit accounting for spatial field heterogeneity, environmental variation within sites may confound genotype and transcriptomic effects.

Third, the Methods do not clearly describe biological replication for RNA-seq. If each genotype-by-environment combination were represented by a single transcriptomic sample, then within-genotype expression variance cannot be estimated. This is important because transcript abundance is highly sensitive to microenvironment, sampling time, tissue status, developmental stage, and technical variation. The absence of replication significantly weakens confidence in gene-level feature importance and gene-gene interaction claims.

Four, the analysis of expression differences across environments is based on a simple subtraction (TX - MI) followed by correlation with genetic similarity. This approach is not standard in transcriptomic analysis and does not account for variability, replication, or statistical uncertainty. Conventional methods for assessing differential expression and genotype-by-environment interactions rely on model-based frameworks that explicitly estimate variance components and test for interaction effects. Without such modeling, the observed expression differences may reflect noise or confounding factors rather than genotype-driven responses.

(2) SHAP contribution values

Although SHAP is a well-established framework for decomposing model predictions into feature-level contributions, its use in this manuscript raises several concerns regarding interpretation, statistical validity, and biological inference.

First, SHAP values quantify the contribution of features within the fitted model, conditional on the joint distribution of inputs and the model structure. They do not represent causal effects or direct biological importance. There is a difference where SHAP values are often in log-odds and the regression model uses absolute units. Without a fair evaluation of model fit, the interpretation of SHAP values needs to take a cautious step because a model could fit poorly when a feature shows very high SHAP values.

In genomic data, where features are highly correlated due to linkage disequilibrium and co-expression, SHAP values can distribute contribution values across correlated variables in ways that are not uniquely identifiable. As a result, features highlighted as "important" may reflect correlation structure rather than true functional relevance.

This correlative structure can be exacerbated in this manuscript because of the use of TPM-normalized transcript abundances as predictor variables without biological replicates. Assume the estimates of transcript abundances are robust, TPM values are compositional, with a constant-sum constraint that creates dependencies among all genes that induce negative correlations. This issue is particularly relevant for the interpretation of gene importance and interaction effects, where correlated predictors can lead to unstable and non-unique attributions. This biological interpretation of transcript-based features remains uncertain.

(3) Result interpretation

For example, in page 11, "plasticity SNP- and transcriptomic-based models generally outperformed single-environment models for traits with low cross-environment correlation, such as green-up (Fig. 2c, r = -0.13, p < 8.3 × 10⁻³) and tiller count (Fig. 2f, r = -0.08, p = 0.1) (Supplementary Fig. S1).", is too broad. For green-up, the Diff model appears much better than MI, but not clearly better than TX.

And, same page 11, "...Diffexp was more predictive than SNPs for trait plasticity in biomass, flowering time, and tiller count..." only holds true for biomass, not flowering time, or tiller count.

The aspect of "complementary information" between SNP and transcriptomic models in page 12 is stronger than what is supported by Figure 2. Figure 2 shows different predictive performance, but it does not by itself demonstrate complementarity. Establishing complementarity requires evidence that combining SNP+T improves prediction consistently or captures distinct, non-overlapping signals. Yet the preceding section says SNP+T outperformed either single data type in only 15% of cases, with modest gains. This is confusing. Also, there was not G+T in Figure 2; it is SNP+T.

Reviewer #2 (Public review):

Summary:

This review is valuable in principle because circadian rhythms in zebrafish are unexplored and therefore this degree is valuable in principle. There are a number of significant weaknesses that should be addressed for it to have an impact. First, while the review covers a broad range of topics in chronobiology, it does not put them in context. Placing zebrafish work in the context of other model organisms that are better understood and other fish species would broaden the appeal. The review could also expand to a discussion of sleep, where the understanding in zebrafish is much more advanced. Critically, providing a novel framework, identifying new areas of opportunity and limitations of the system would expand the interest to non-zebrafish research groups. In addition, there are a number of misstatements/mis-citations that are critical to correct. Therefore, I find this review potentially impactful, but its current form is likely to limit its impact.

Strengths:

Focusing on decentralized photo sensing is a strength because it is relatively unique to zebrafish.

The breadth of discussion in zebrafish is a strength.

Weaknesses:

It might be helpful to reorganize the review with an introduction on what is known in other better studied systems to be highly conserved, then to focus in on the components of zebrafish that are discussed here.

A weakness is the lack of integration with other model organisms and other fish systems. Therefore, the narrow focus on zebrafish is unlikely to appeal to broader audiences.

It's surprising that there is not more discussion of sleep, which has been studied in detail, and its relationship to the clock.

Discussions of limitations of the model, including adult vs larval analysis and challenges performing long-term behavioral analysis in fish, would be valuable.

Reviewer #3 (Public review):

Sheidaei et al. report how chromosomes are favourably positioned to facilitate kinetochore-microtubule interactions during early mitosis. Studying kinetochore capture during early prophase is extremely difficult due to kinetochore crowding, but the team has taken up the challenge by classifying types of kinetochore movements, carefully marking kinetochore positions in early mitosis, and linking these to map their fate/next positions over time. The work is an excellent addition to the chromosome segregation field, as most of the literature has thus far focused on tracking kinetochores at slightly later stages of mitosis. The authors show that PANEM facilitates chromosome positioning toward the interior of the newly forming spindle, which in turn promotes chromosome congression. In the absence of PANEM, chromosomes end up in unfavourable locations and fail to form proper kinetochore-microtubule interactions. The work highlights the perinuclear actomyosin network in early mitosis (PANEM) as a key spatial and temporal element of chromosome congression, a step that precedes the segregation process.

Comments on revised version:

The authors' revisions have brought clarity to the description of movements in many of the figures. The manuscript ties a fundamental process to differences in cancer cell lines.

The work extends their published discovery that an actomyosin network forms on the cytoplasmic side of the nuclear envelope during prophase. The current manuscript explains how this network facilitates chromosome capture and congression by tracking the motions of individual kinetochores during early mitosis. The findings are broadly useful for the cell division and cytoskeletal fields.

Reviewer #2 (Public review):

Summary:

Here, the authors performed a phylogenetic analysis of mitochondrial ATP/ADP carrier (AAC) proteins. They also performed a structure-based screen for remote homologs, seeking to reveal their evolutionary origins. The authors claim that AACs are found at the root of their family tree, and through a structure-based homolog search protocol, identify putative prokaryotic homologs.

The proposed evolutionary history of AACs is bold and complicated, but the phylogenetic methodology and the way in which the tree is interpreted are incomplete and unconvincing. Further, the structure-based search strategy uses very relaxed cutoffs for fold similarity, which may be fine, but it does not clearly justify this decision. This is potentially very problematic, as I did not find the quantitative or qualitative assessments of fold similarity particularly compelling.

In summary, the authors have presented a bold and extremely interesting hypothesis for the evolution of these proteins, but there is insufficient support for their claims.

Strengths:

(1) The authors are presenting a very interesting hypothesis about the birth of these proteins, including that they may have undergone a radical rearrangement in their sequence at some point in evolution.

(2) The paper makes use of appropriate tools for structure-based homolog identification.

(3) Identification of a conserved sequence motif in these twilight zone proteins would be a rare and interesting occurrence, and could be consistent with their proposed homology.

Weaknesses:

(1) The phylogenetic analysis and its interpretations are incomplete. The authors regularly refer to the root of the tree, and its placement is given central importance. However, the methodology by which they selected the root is unexplained. This is notable, as the proposed root is curious and quite confusing. It implies that (at least) yeast and Paramecium AACs are independently paraphyletic. While certainly not impossible, this evokes quite a complicated evolutionary history. The taxonomy of this gene family, when rooted this way, does not seem to echo the phylogeny of species, suggesting an extremely complex history of duplication/loss and horizontal gene transfer, none of which the authors discuss in detail. Perhaps more clearly and specifically: I'm very surprised by the branching order at the root, where there are three independent branches of fungal proteins, followed by the excavate proteins in a monophyletic clade, followed by several independent branches of the Paramecium proteins. I very much expect incomplete lineage sorting at this evolutionary depth, but this seems extreme to the point that I question if it is accurately placed. More directly: this very much looks like an unrooted tree, presented radially.

(2) The Bayesian and ML trees seem quite incongruent, but this is not discussed. In fact, the text states that they "exhibit a similar tree topology." This is admittedly very difficult to assess without very carefully going over the tree, branch by branch, but there are nevertheless differences, the most obvious being paraphyly vs monophyly of taxon-specific AAC clades. Do the authors have any comments on this, and can they show some sort of consensus tree? How does this affect their interpretation?

(3) Presenting branch support as similarly-sized points makes it nearly impossible to actually judge the strength of support.

(4) The use of structure for remote homology detection is becoming increasingly popular, and in my opinion, is very powerful. But it is still much too early to be taken for granted. The methodology must be justified. Most importantly, the authors have not clearly described why they chose these quantitative cutoffs (I'm mostly thinking of the Dali Z-score cutoff, which here seems very low for a transmembrane protein of this size, as the Z-score is very dependent on alignment length). The authors reference categories defined by tool authors, but why a Z-score of 3, specifically? The same goes for TM scores. There are not yet any defined best practices, to my knowledge, so the authors should independently validate/justify their approach in some way and/or cite and discuss relevant literature (there have been a growing number of these screens using similar approaches in recent years).

(5) The proposed homologs have very little quantitative structural similarity to the query structure, or to each other, as shown in Figure 3 (and hence my concerns about the methodology). Also, I did not find the structural alignments in the supplement or Figure 4 to be qualitatively compelling. They simply appear too different, and I cannot discard this qualitative assessment because the quantitative similarities are likewise very weak. It's not clear to me if this is because the folds are in fact different, or if my view of them is a presentation issue (perhaps it could be improved by visualizing more angles, or more carefully cartooning the similarities and differences).

(6) The authors point out that the alpha-helices are ordered differently in YihY and CysZ, and that their membrane orientation is flipped. Taken at face value, I would view this as evidence against homology. This could perhaps be more reasonably explained as convergent global fold similarity resulting from different underlying structures. However, the authors imply that this may be the result of the transposition of the sequences encoding these alpha helices, yet there is no convincing description or argument concerning when and how this could have occurred. I think this would be a deeply interesting phenomenon, but there is insufficient evidence and discussion to seriously consider whether or not it is homology or convergence.

(7) Following up on comment #5, the authors did perform a very interesting in silico experiment by transposing sequences to reorder the helices. They then note that structural similarity improved. This is very, very interesting, but without other evidence of homology between the transposed alpha helices, I do not think this disproves alternative hypotheses. Does any such evidence exist?

(8) The authors show in Figure 5E-F that sequence transposition flips the membrane orientation, such that YihY and CysZ have extracellular termini (which you would expect from homologs, I suppose). But it is just cartooned and not discussed. Is this computationally or experimentally supported?

(9) The putative presence of a conserved motif would be a very compelling piece of evidence consistent with homology. However, it is not clear to me in the text which proteins actually have the repeats - is it truly just CysZ? What does this mean for YihY? Further, what specifically is being proposed to be homologous? Is SLC25 repeat 2 proposed to be homologous to CysZ repeat 2 (and the same for 3 to 3)? If so, this would seem to have implications for the transposition hypothesis. The helix nomenclature (e.g., H1-6) suggests homology across the proteins (i.e, H1 is homologous to H1); however, wouldn't the presence of these conserved domains instead, for example, suggest homology between SLC H3 and CysZ H2? The authors' conclusions are not clear, and it is difficult to interpret what the implications are for assessing homology.

(10) The sequence retrieval methods are incomplete, so it is impossible to reproduce the searches or to judge their accuracy and scope. What were the E-value cutoffs and other settings used in the searches?

(11) The phylogenetic methods are incomplete. What substitution models were used, and how were they chosen? What branch support method was used? What were the stop conditions of the Bayesian analysis (e.g. did the authors monitor for convergence, and how)? How much of the Bayesian analysis was considered burn-in, if any? And echoing points 1 & 2 above, how were these phylogenies rooted?

(12) Throughout, there is a distinct lack of careful, evolutionarily informative language.

(i) In reference to the phylogeny, the authors frequently refer to "grouping," but it's not entirely clear what this means. Referring to clades and their branching order would be more informative.

(ii) The authors refer to the excavate branch as the "most ancient." Whether or not excavates most closely resemble LECA is somewhat irrelevant, because the branch itself is not the most ancient - it is equally as ancient as its sister branch, which may be all other eukaryotes.

(iii) Likewise, the authors refer to bacterial proteins as "the evolutionary ancestor of mitochondrial AACs," and state that "AAC emerged from the conserved sulfat transporter CysZ." But extant bacteria are not the ancestors of mitochondria - nor are extant proteins descended from other extant proteins. They are, perhaps more accurately, cousins.

(iv) The authors refer to AACs as "evolutionarily founder member of the SLC25 carrier family," but I'm not sure that has a clear evolutionary meaning, unless the authors mean to say that the common ancestor was more AAC-like than anything-else-like. Even if the rooting is accurate, a basal branch does not necessarily reflect the ancestral state.

Reviewer #2 (Public review):

Summary:

Overall, this is an excellent paper, making use of a newly developed system for monitoring the behaviour of chromatophores in the skin of (mostly) free swimming bobtail squid and European cuttlefish. The manuscript is very well written, clearly presented and very well structured. The central finding, that individual chromatophores are connected to multiple motor neurones, is not new. Novelty instead comes from the ability to measure the actuation of chromatophore sections across wide areas of skin in free-swimming animals, showing the diversity of local motor units and reinforcing the notion that individual chromatophores are not necessarily the individual units of colour change, but rather local motor units that cover multiple neighbour and near neighbour chromatophore muscles. This is an excellent finding and one that will shape our understanding of the neural control of cephalopod skin colour. I have a number of minor points below that the authors will need to address before acceptance.

Strengths:

The methodological approach to collecting large amounts of data about local variations in the expansion of sections of chromatophores is exciting, and the analysis pipeline for clustering sections of chromatophores whose spontaneous activity correlated over time is powerful and exciting.

Comments on revisions:

All concerns have been addressed in the revised version of the manuscript.

Reviewer #3 (Public review):

Summary:

Large Language Models have revolutionized Artificial Intelligence and can now match or surpass human language abilities on many tasks. This has fuelled interest in cognitive neuroscience in exposing representational similarities between Language Models and brain recordings of language comprehension. The current study breaks from this mold by: (1) Systematically identifying sentence structures for which brain and Large Language Model representations diverge. (2) Accounting for such sentence structures using a model structured by semantic roles. As such the study may now fuel interest in characterizing how Large Language Models and brain representations differ, which may prompt new more brain like language models.

Strengths:

* This study presents a bold challenge to a literature trend that has touted similarities between Transformer models and human cognition based on representational correlations with brain activity. This challenge is substantiated by identifying sentences for which brain and model representations of sentences diverge.

* This study conducts a rigorous pre-registered analysis of a comprehensive selection of the state-of-the-art Large Language Models, on a controlled sentence comprehension fMRI dataset. The analysis is conducted within a Representation Similarity framework to support similarity comparisons between graph structures and brain activity without needing to vectorize graphs. Transformer models are predicted and shown to diverge from brain representations on subsets of sentences with similar word-level content but different sentence structures.

* The study introduces a 7T fMRI sentence comprehension dataset and accompanying human sentence similarity ratings which may be a fruitful resource for developing more human-like language models. Unlike other model-based sentence datasets, the relation between grammatical structure and word-level content is controlled, and subsets of sentences for which models and brains diverge are identified.

Weaknesses:

* The interpretation of findings is nuanced. Although Transformers underperform as brain models on the critical subsets of controlled sentences, a Transformer outperforms all other models when evaluated on the union of all sentences when both word-level content and structure vary. Transformers also yield equivalent or better models of human behavioral data. Thus, although Transformers have demonstrable flaws as human models which are pinpointed here, in the general case (some) Transformers are more human-like than the other models considered.

* There may be confounds between the critical sentence structure manipulations and visual processing. This is inconvenient because activation in brain regions that process semantics tends to partially correlate with low-level representations of sentence surface features encoded in visual cortex. Although the study commendably controls for confounds associated with sentence length, correlations with the key sentence structure models are most salient in visual cortex and diminish in other brain networks when V1-V4 activation is controlled for.

* Sentence similarity computations are emphasized as the basis for unifying comparative analyses of graph structures and vector data. A strength of this approach is that correlation is not always the ideal similarity metric. However, a weakness is that similarity computations are not unified across models. This has practical consequences because different similarity metrics applied to the same model produce positive or negative correlations with brain data and repeating analyses with a different representational dissimilarity measure seems to produce some anomalous results.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigate how cytosolic acetyl-CoA metabolism influences replicative aging in budding yeast. They propose that acetyl-CoA regulates aging through three major pathways: (1) mitochondrial transport to support mitochondrial function, (2) fatty acid synthesis, and (3) global protein acetylation. The data show that AMPK activation promotes mitochondrial import of acetyl-CoA and partially mitigates mitochondrial decline in a subset of aging cells.

Furthermore, the engineered A2A strain, which enhances mitochondrial acetyl-CoA utilization while relieving inhibition of fatty acid synthesis, increases the proportion of cells exhibiting a "low senescence" phenotype.

Overall, this is a thoughtful and potentially impactful study that advances our understanding of metabolic control of aging. Addressing the points below, particularly by refining interpretations and, where feasible, incorporating additional analyses, will further strengthen the manuscript and its conclusions.

Strengths:

The study has several notable strengths. It addresses an important question by shifting the focus from lifespan to preservation of late-life fitness, which is highly relevant to aging biology. The work integrates metabolic, genetic, and functional analyses to link cytosolic acetyl-CoA flux with distinct aging outcomes, and the engineering of the A2A strain provides a clear and elegant demonstration of how coordinated pathway modulation can improve cellular fitness.

Weaknesses:

(1) While the manuscript focuses on mitochondrial transport and fatty acid synthesis, cytosolic acetyl-CoA is also a key regulator of histone acetylation and chromatin silencing. It would strengthen the study to consider whether acetyl-CoA depletion contributes to improved fitness through enhanced rDNA silencing. Given the well-established role of rDNA instability in yeast aging, additional experiments examining rDNA silencing and stability would be valuable. For example, monitoring rDNA copy number changes (not necessarily ERCs) under AMPK activation, oleic acid supplementation, and in the A2A strain, similar to approaches used in the authors' prior work, would help clarify whether chromatin regulation contributes to the observed phenotypes.

(2) The current data do not fully distinguish whether AMPK activation and oleic acid supplementation act on distinct subpopulations of aging cells. An alternative explanation is that oleic acid supplementation enhances mitochondrial function and acts additively with AMPK activation, thereby increasing the fraction of cells in the "low senescence" state. Since this distinction is not central to the main conclusions, I suggest softening the language around subpopulation specificity. Emphasizing instead that the A2A strain coordinately modulates multiple branches of acetyl-CoA metabolism to improve late-life fitness would maintain the strength of the central message without overinterpretation.

(3) The manuscript proposes that lipid starvation and excess acetyl-CoA are major drivers of senescence in distinct subpopulations of wild-type aging cells. This conclusion is not yet fully supported by the presented data. Direct measurements of age-dependent divergence in acetyl-CoA and fatty acid levels at the single-cell level would be needed to substantiate this model. Based on the current evidence, a more conservative interpretation would be that aging cells exhibit differential sensitivity to perturbations in acetyl-CoA and lipid metabolism. Accordingly, I recommend revising the statement in the Abstract ("We further implicate lipid starvation and excess acetyl coenzyme A availability as major drivers of senescence...") and the corresponding discussion text to better align with the data.

Reviewer #2 (Public review):

Summary:

Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.

Strengths:

The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.

Reviewer #2 (Public review):

This study explores the dynamic association between malate dehydrogenase (MDH1) and citrate synthase (CIT1) in Saccharomyces cerevisiae, with the aim of linking this interaction to respiratory metabolism. Utilizing a NanoBiT split-luciferase system, the authors monitor protein-protein interactions in vivo under various metabolic conditions.

Major Concerns:

(1) NanoBiT Signal May Reflect Protein Abundance Rather Than Interaction Strength<br /> In Figure 1C, the authors report increased MDH1-CIT1 interaction under respiratory (acetate) conditions and decreased interaction during fermentation (glucose), as indicated by NanoBiT luminescence. However, this signal appears to correlate strongly with the expression levels of MDH1 and CIT1, raising the possibility that the observed luminescence reflects protein abundance rather than specific interaction dynamics. To resolve this, NanoBiT signals should be normalized to the expression levels of both proteins to distinguish between abundance-driven and interaction-driven changes.

(2) Lack of Causal Evidence<br /> The study presents a series of metabolic perturbation experiments (e.g., arsenite, AOA, antimycin A, malonate) and correlates changes in metabolite levels with NanoBiT signals. However, these data are correlative and do not establish a functional role for the MDH1-CIT1 interaction in metabolic regulation. To demonstrate causality, the authors should implement approaches to specifically disrupt the MDH1-CIT1 interaction. One strategy could involve using a 15-residue peptide (Pept1) derived from the Pro354-Pro366 region of CIT1, previously shown to mediate the interaction or introducing the cit1Δ3 (Arg362Glu) mutation, which perturbs binding. Metabolic flux analysis using ^13C-labeled glucose and mitochondrial respiration assays (e.g., Seahorse) could then assess functional consequences.

(3) Absence of Protein Expression Controls Under Perturbation Conditions<br /> In experiments involving acetate, arsenite, AOA, antimycin A, and malonate, the authors infer changes in MDH1-CIT1 association based solely on NanoBiT signals. However, no accompanying data are provided on MDH1 and CIT1 protein levels under these conditions. This omission weakens the conclusions, as altered expression rather than interaction strength could underlie the observed luminescence changes. Immunoblotting or quantitative proteomics should be used to confirm constant protein expression across conditions.

Conclusion:

Although the central question is compelling and the use of NanoBiT in live cells is a strength, the manuscript requires additional experimental rigor. Specifically, normalization of interaction signals, introduction of causative perturbations, and validation of protein expression are essential to substantiate the study's claims.

Comments on revised version:

The manuscript is much improved.

Reviewer #2 (Public review):

Summary:

Castanheira et al. investigate the role of spatial attention for planning during three maze navigation experiments (one new experiment and two existing datasets). Effective planning in complex situations requires the construction of simplified representations of the task at hand. The authors find that these mental representations (as assessed by conscious awareness) of a given stimulus are influenced by (spatially) surrounding stimuli. Individual participants varied in the degree to which attention influenced their task representations, and this attentional effect correlated with the sparsity of representations (as measured by the range of awareness reports across all stimuli). Spatially grouping task-relevant information on either the left or right side of the maze led to mental representations more similar to optimal representations predicted by the value-guided construal (VGC) model - a normative model describing a theoretical approach to simplifying complex task information. Finally, the authors propose an update to this model, incorporating an attentional spotlight component; the revised descriptive model predicts empirical task representations better than the original (normative) VGC model.

Strengths:

The novelty of this study lies in the proposal and investigation of a cognitive mechanism through which a normative model like value-guided construal can enable human planning. After proposing attention as this mechanism, the authors make concrete hypotheses about mismatches between the VGC predictions and real human behavior, which are experimentally validated. Thus, not only does this study describe a possible mechanism for simplification of task information for planning, but the authors also propose a descriptive model, revising VGC to incorporate this attentional component.

A strength of this paper is the variety of investigative approaches: analysis of existing data, novel experiment, and a computational approach to predict experimental findings from a theoretical model. Analyzing pre-existing datasets increases the size of the participant cohort and strengthens the authors' conclusions. Meanwhile, comparing the predictions of the existing normative model and the authors' own refined model is a clever approach to substantiate their claims. In addition, the authors describe several crucial controls, which are key to the interpretability of their results. In particular, the eye tracking results were critical.

In summary, this paper constitutes an important step toward a more complete understanding of the human ability to plan.

Comments on revised version:

I am overall happy with the revision and agree that the authors have addressed most of the comments.

Reviewer #2 (Public review):

Summary:

Castanheira et al. investigate the role of spatial attention for planning during three maze navigation experiments (one new experiment and two existing datasets). Effective planning in complex situations requires the construction of simplified representations of the task at hand. The authors find that these mental representations (as assessed by conscious awareness) of a given stimulus are influenced by (spatially) surrounding stimuli. Individual participants varied in the degree to which attention influenced their task representations, and this attentional effect correlated with the sparsity of representations (as measured by the range of awareness reports across all stimuli). Spatially grouping task-relevant information on either the left or right side of the maze led to mental representations more similar to optimal representations predicted by the value-guided construal (VGC) model - a normative model describing a theoretical approach to simplifying complex task information. Finally, the authors propose an update to this model, incorporating an attentional spotlight component; the revised descriptive model predicts empirical task representations better than the original (normative) VGC model.

Strengths:

The novelty of this study lies in the proposal and investigation of a cognitive mechanism through which a normative model like value-guided construal can enable human planning. After proposing attention as this mechanism, the authors make concrete hypotheses about mismatches between the VGC predictions and real human behavior, which are experimentally validated. Thus, not only does this study describe a possible mechanism for simplification of task information for planning, but the authors also propose a descriptive model, revising VGC to incorporate this attentional component.

A strength of this paper is the variety of investigative approaches: analysis of existing data, novel experiment, and a computational approach to predict experimental findings from a theoretical model. Analyzing pre-existing datasets increases the size of the participant cohort and strengthens the authors' conclusions. Meanwhile, comparing the predictions of the existing normative model and the authors' own refined model is a clever approach to substantiate their claims. In addition, the authors describe several crucial controls, which are key to the interpretability of their results. In particular, the eye tracking results were critical.

In summary, this paper constitutes an important step toward a more complete understanding of the human ability to plan.

Weaknesses:

(1) There is a critical conceptual gap in the study and its interpretation, mainly due to the reliance on a self-report metric of awareness (rather than an objective measure of behavioral performance).

a. Awareness is tested by a 9-point self-report scale. It is currently unclear why awareness of task-irrelevant obstacles in this task would necessarily compromise optimal planning. There is no indication of whether self-reported awareness affects performance (e.g., navigation path distance, time to complete the maze, number of errors). Such behavioral evidence of planning would be more compelling.

b. Relatedly, it would have been more convincing to have an objective measure of awareness, for instance, how the presence or absence of a "task-irrelevant" obstacle affects performance (e.g., change navigation path distance or time to complete the maze), or whether participants can accurately recall the location of obstacles.

c. Consequently, I'm not sure that we can conclude that the spatial context does impact participants' ability to plan spatial navigation or to "incorporate task-relevant information into their construal". We know that the spatial context affects subjective (self-reported) awareness, but the authors do not present evidence that spatial context affects behavioral performance.

d. Another concern that may complicate interpretation is the following: Figure 3c shows improved VGC model predictions (steeper slope) for mazes with greater lateralization. However, there are notable outliers in these plots, where a high lateralization index does not correspond to good model performance. There is currently no discussion/explanation of these cases.

(2) I noticed an issue with clarity regarding task-relevance. It is currently not fully clear which obstacles are "task irrelevant". Also, the term is used inconsistently, sometimes conflating with "awareness". For example, in the "Attentional spotlight model of task representations" section, the authors state that "task-relevant information becomes less relevant when surrounded by task-irrelevant information". But they really mean that participants become less aware of those task-relevant obstacles. I assume task-relevance is an objective characteristic related to maze organization, not to a participant's construal. Indeed, the following paragraph provides evidence of model predictions of awareness.

(3) The behavioral paradigm has some distinct disadvantages, and the validity of the task is not backed up by behavioral data.

a. I understand the need for central fixation, but it also makes the task less naturalistic.

b. The task with its top-down grid view does not seem to mimic real human navigation. Though this grid may be similar to mental maps we form for navigation, the sensory stimuli corresponding to possible paths and to spatial context during real-life navigation are very different.

c. Behavioral performance is not reported, so it is unknown whether participants are able to properly complete the task. The task seems pretty difficult to navigate, especially when the obstacles disappear, and in combination with the central fixation.

d. There is no discussion of whether/how this navigation task generalizes to other forms of planning.

Reviewer #2 (Public review):

Summary:

The authors aiming in developing a neural mass model characterized by few collective variables mimicking the dynamics of a network of Hodgkin - Huxley neurons encompassing ion-exchange mechanisms. They describe in details the derivation of the mean-field model , then they compare experimental results obtained for the hippocampus of a mice with the neural network simulations and the mean-field results. Furthermore, they report a bifurcation analysis of the developed model and simulation of a small network containing various coupled neural masses, somehow moving towards the simulation of an entire connectome.

Strengths:

The author attempts to develop a mean-field model for a globally coupled network of heterogeneous Hodgkin-Huxley neurons with explicit ion exchange mechanism between the cell interior and exterior.

Weaknesses:

(1) They do not employ the reduction methodology more suited for the single neuron model they consider.<br /> (2) Their derivation of the neural mass model is based on several assumptions, and not all well justified.<br /> (3) Their formulation of the mean-field derivation is unnecessary complicated, it can be strongly simplified by following previously published approaches to derive biologically realistic neural masses.<br /> (4) Their model seems to work only for highly synchronized situations and not for the standard asynchronous evolution usually observed in neural circuits.

General Statements:

The authors honestly declared the many limitations of their approach, once assumed this the results of the mean-field are somehow inconsistent with the neural network simulations as expected.

The authors suggest to employ this model for the simulations on the whole connectome to follow seizure propagation, however I believe that a simpler model, as the Epileptor, remains superior in this respect to this model. That indeed includes biophysical parameters but their correspondence with the ones employed in the network dynamics remain elusive, due to the many assumptions required to derive this mean field model. Furthermore it is more complicated than the Epileptor, I do not think that the present model will be largely employed by the community.

Comments on revisions:

The authors have corrected mistakes present in the manuscript and put a correct list of references.

However, they refuse

(1) To simplify the formulation of the model, the model contains unnecessary complications, as I have clearly written in my report, the authors agree, but they do not want to change the formulation;

(2) To derive the mean field model in a simpler way, as possible, and as I asked many times in my Referee report, this would help the readers to understand the important aspect of the derivation, without not needed and confusing complicated formulations;

(3) To compare direct simulations of the network with neural mass results in sub-section "Bifurcation analysis: emergent network states and multistability" to show bistability, as I asked.

As a matter of fact the performed modifications do not solve my previous doubts on the validity of the results reported in the manuscript.

Therefore, my previous assessments remain valid.

Reviewer #2 (Public review):

Summary:

This manuscript presents an elegant and innovative imaging approach to visualize DNase activity at the interface between macrophages and extracellular substrates. The platform is technically strong and enables the study of localized DNA degradation with high spatial resolution. The work is of clear interest and provides a useful framework to investigate how immune cells process extracellular DNA. However, several aspects of the mechanistic interpretation and conceptual framing would benefit from clarification.

Strengths:

(1) The study introduces a creative and well-designed imaging platform that allows visualization of localized DNase activity at cell-substrate interfaces.

(2) The approach is technically robust and represents a valuable tool that could be broadly useful to the field.

(3) The experiments are thoughtfully designed and address an important question regarding how immune cells interact with extracellular DNA.

(4) The work opens interesting avenues for studying DNA processing in contexts such as infection and inflammation.

Weaknesses:

While the experimental approach is strong, several key conclusions rely on interpretations that would benefit from further clarification:

(1) First, the conclusion that DNaseX is recruited to phagocytic cups from the "cytoplasm" appears conceptually imprecise. Given that DNaseX is a membrane-anchored protein, it is unlikely to exist as a freely soluble cytoplasmic pool. A more plausible interpretation is that DNaseX is supplied from intracellular membrane compartments. This interpretation would also be more consistent with the data showing dependence on a membrane anchor.

(2) Second, the interpretation that actin polymerization is not required for DNaseX recruitment raises concerns. Phagocytic cup formation is known to depend strongly on actin dynamics, and it is therefore unclear whether the structures observed under actin inhibition represent fully formed functional cups or partial cell-substrate contacts. This distinction is important for interpreting recruitment versus activity, particularly since enzymatic activity is reduced under these conditions.

(3) Third, the identification of DNaseX as the main nuclease responsible for the observed activity is not fully resolved. The conclusions rely primarily on gene silencing and staining approaches, but the specificity of these strategies relative to other nucleases is not addressed. It therefore remains possible that additional enzymes contribute to the observed activity.

(4) Finally, the interpretation of the biofilm experiments may be overstated. While the data clearly show localized DNA degradation in contact with macrophages, it is not fully established that this process depends specifically on phagocytic cup structures. An alternative explanation is that membrane-associated DNase activity more generally mediates this effect. In addition, the physiological relevance of this mechanism would benefit from further discussion.

Overall, the study is technically strong and introduces a valuable methodology, but several central conclusions are only partially supported by the current data and would benefit from more cautious interpretation and clearer conceptual framing.

Reviewer #2 (Public review):

In this study, the authors address the molecular mechanism underlying the transcriptional changes during erythroid differentiation from hematopoietic progenitor cells. The authors combine single-molecule live cell imaging and CUT&RUN to analyze the chromatin binding properties of the GATA2 transcription factor prior to and after initiation of differentiation into the erythroid cell lineage. Using three distinct cellular systems, the authors demonstrate that the chromatin binding of GATA2 is transiently increased early in the differentiation process, as evidenced by increased chromatin binding residence time and the emergence of new genomic binding sites identified by CUT&RUN. The strength of the study lies in the combination of single-molecule imaging, which reports on binding dynamics but is agnostic of the binding site, with CUT&RUN, which reports on the binding sites but does not provide dynamic information. The authors clearly demonstrate that chromatin binding of GATA2 is altered early in the differentiation process and is later displaced as cells switch to expression of GATA1, which has been previously observed. The use of three distinct cell lines, in particular the GATA2-SNAP mouse model, is a strength in principle; however, the results are not fully consistent between the different cell systems. A key difference is that the G1E-ER4 and HPC7 cell line models express HaloTagged GATA2 in addition to the endogenous GATA2 protein. The authors go through great lengths to control GATA2-HaloTag expression levels, but they use polyclonal cell lines and do not analyze expression levels of the GATA2-HaloTag transgene, which is a key variable in interpreting their experimental results. Finally, a key variable determined in their single-molecule analysis is the number of binding events observed during the distinct differentiation changes. The number of binding events observed is influenced by the expression level of the tagged protein, which in turn is controlled by the Shield-1 ligand, and the fraction of molecules labeled with the HaloTag ligand. Since transgene protein levels and the labeling efficiency were not determined, it is hard to assess how reliable the measurements of the number of binding events are across all cell lines.

To address the weaknesses summarized above the authors could take the following steps:

(1) Determine the expression levels of the GATA2-HaloTag transgene over the course of differentiation under the conditions used for single-molecule imaging. This will not only allow them to determine the expression of the transgene but also the endogenous untagged protein with which the GATA2-HaloTag fusion proteins compete for binding sites.

(2) To determine the fraction of molecules labeled during imaging, the authors could carry out a titration of the HaloTag ligand and compare the amount of labeled protein under single-molecule imaging conditions to that of saturating labeling of the HaloTag. This approach will ensure that the number of labeled molecules per cell is comparable across experimental conditions and allow the authors to draw more solid conclusions regarding the number of binding events.

(3) The analysis of residence times using single-molecule imaging requires robust single-particle tracking without gaps or interruptions of trajectories. The authors should show images of their particle trajectories to demonstrate that their tracking is robust. Or even better, movies superimposing the trajectories onto the imaging data.

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important and timely question in the molecular simulation of biomolecular condensates. Most residue-level coarse-grained models used for IDP phase separation employ implicit solvent and represent effective interactions through relatively simple pairwise potentials. While these models have been very useful, they usually do not explicitly distinguish direct contacts from solvent-separated interactions, nor do they include an energetic barrier associated with water removal. This manuscript attempts to address that limitation by introducing desolvation-inspired terms into coarse-grained models and examining their consequences for phase behavior, chain conformations, dense-phase packing, and dynamics.

Strengths:

The central idea is physically well motivated. Using a simple homopolymer model, the authors show that increasing the desolvation barrier suppresses phase separation, whereas stabilizing solvent-separated contacts enhances phase separation. They further show that solvent-separated interactions can reduce dense-phase over-compaction, which is a meaningful result given the known challenges in obtaining both accurate single-chain dimensions and realistic dense-phase properties from the same coarse-grained model. The finding that desolvation-like terms can reshape dense-phase packing without simply rescaling the overall interaction strength is interesting and could be useful for future model development. I also found the attempt to connect conformational changes across dilute and dense phases with thermal distance from the critical point to be intriguing. The dynamic analysis, including the FRAP-like simulations and the discussion of kinetic arrest during coarsening, adds another useful dimension to the work.

Weaknesses:

At the same time, there are several places where the manuscript would benefit from more careful framing. First, the desolvation terms are still effective coarse-grained parameters rather than a direct representation of water molecules. The language sometimes gives the impression that desolvation is being treated explicitly, whereas the model introduces desolvation-inspired effective interactions into an implicit-solvent framework. Second, the conformational analysis is interesting, but the broader context of prior work on dilute-to-dense phase conformational reorganization of IDPs could be more clearly discussed. This would help clarify what is new in the present work, whether it is the conformational change itself, its dependence on desolvation terms, or the proposed scaling with distance from the critical point. Third, the dynamic results are potentially useful, but the manuscript should more clearly articulate what is nontrivial beyond the expected slowing of local rearrangements by an added barrier in the potential.

Overall, I think this is a useful and potentially important contribution.

Reviewer #2 (Public review):

Summary:

Hann and colleagues introduce a gaze-based analytical framework designed to capture, on a trial-by-trial basis, how people form and revise their predictions during implicit probabilistic sequence learning. Using an eye-tracking adaptation of an alternating sequence task, they record the first anticipatory saccade during the response-stimulus interval and classify each such saccade along two dimensions: whether it was directed toward a high- or low-probability upcoming stimulus (the learning-dependent vs. not-learning-dependent distinction), and whether the anticipated location coincided with the stimulus that actually appeared. A complementary iterative-updating metric codes whether a participant's prediction for a given three-element context is repeated or revised on successive encounters of that context.

On the basis of these measures, the authors report that errors congruent with the inferred regularity - which they interpret as reflecting environmental noise - become progressively more frequent than errors reflecting an inaccurate internal model; that participants show a pronounced tendency to repeat their previous prediction rather than revise it; and that updates depend more on whether a prior belief is congruent with the task's statistical structure than on whether the previous prediction was confirmed. They interpret these results as evidence that statistical learning is less error-driven and more repetition-based (Hebbian in character) than is typically assumed.

Strengths:

The methodological ambition of the work is considerable, and the paper makes several contributions that are likely to be useful to the implicit-learning and predictive-processing communities. Using the first anticipatory saccade as a pre-response behavioral readout of prediction is conceptually well-motivated: it provides a trial-by-trial index of predictive orienting at a temporal resolution that manual reaction times cannot deliver, and it does so before the outcome of the trial is known. The explicit distinction between errors arising because the task's outcome is stochastic - that is, predictions congruent with the statistical structure but unconfirmed by the stochastic sample - and errors arising because the internal model is inaccurate is a theoretically meaningful move: predictive-coding and Bayesian accounts have long argued that these two sources of surprise should carry different weight for model revision, and the authors offer a behavioral operationalization of that distinction. The analytical pipeline is not tied to the specific paradigm used here and could be applied to other probabilistic sequence-learning tasks, which gives it broader methodological utility than a single-paradigm report. Finally, the demonstration that learners maintain their prior across successive occurrences of the same context, even when it has been disconfirmed by the most recent outcome, is a robust behavioral observation that speaks directly to an unresolved debate about whether statistical learning is dominantly error-driven.

Weaknesses:

The framework and the core behavioral observations are valuable, but several inferential steps - from the gaze signal to the cognitive constructs the authors invoke - are not fully supported by the present design, and these gaps affect how readers should interpret the stronger theoretical conclusions.

The "process-pure" framing conflates sensitivity with construct purity. The authors repeatedly describe the eye-tracking measure as providing a more process-pure index of statistical learning than manual-response paradigms. Anticipatory saccades are themselves a learned motor behavior - the oculomotor system is among the most plastic motor outputs the primate brain generates, and sequence learning in the saccadic system is well-documented. The present design does not dissociate learning of the statistical structure from learning of the oculomotor sequence that expresses it, so the measure is not, on its face, free from the motor-learning confound that the authors criticize in button-press paradigms. The framing should be read as aspirational rather than as demonstrated by the present data.

The oculomotor reaction-time data do not show the canonical signature of statistical learning. Reaction times for low-probability trials rise across epochs while those for high-probability trials remain approximately flat (Figure 5). The emerging difference between the two trial types, therefore, appears to be driven by a slowing of responses to low-probability stimuli rather than by a facilitation of responses to high-probability ones, and the authors do not rule out the alternative interpretations that this pattern reflects fatigue, a motor floor effect, or inhibition of unexpected locations. Because no fixation constraint is imposed during the response-stimulus interval, pre-stimulus gaze drift toward the anticipated location will artifactually reduce reaction time on precisely those trials the authors wish to treat as learning-driven; the fact that measured reaction times remain well above zero even on trials classified as correct anticipations is itself evidence that this contamination is present. The oculomotor reaction-time data, therefore, do not provide as clean a verification of learning as the manuscript implies.

The correct/error labeling of anticipatory saccades incorporates information that the participant did not have. Because the first saccade occurs during the response-stimulus interval - that is, before the upcoming stimulus is revealed - the participant's internal predictive state is identical whether the trial is subsequently classified as a learning-dependent correct response or a learning-dependent error. Any difference in the epochwise frequency of these two categories must therefore be driven, at least in part, by the external stochastic structure of the task rather than by a difference in the predictive process itself. In particular, the observation that learning-dependent errors are the most frequent saccade type (Figure 7) is predicted by the prior probabilities of the outcomes alone, given a high-probability prediction, without appeal to any difference in predictive state. Readers should recognize that the theoretically meaningful contrast is between learning-dependent and not-learning-dependent anticipations (two categories), and that the four-way split risks confounding predictive state with outcome stochasticity.

The iterative-updating metric does not distinguish prior revision from alternative processes. The binary update / no-update code, computed across non-contiguous occurrences of the same three-element context, does not discriminate between a genuine update of the internal model, simple episodic retrieval of a previously encountered triplet, and oculomotor perseveration. Without a formal generative model to anchor the interpretation, the central theoretical claim - that statistical learning is less error-driven than commonly assumed - is underdetermined by the data. The repetition pattern the authors observe is equally consistent with an error-driven model equipped with a low learning rate in a stable environment, an interpretation the authors themselves acknowledge in the Discussion. Adjudicating between these possibilities requires comparison against explicit computational models, which the present manuscript does not provide.

Data loss and the absence of fixation control. An interpretable saccade is detected on fewer than half of all trials (48.76%; line 889), and the manuscript does not report the distribution of saccade counts per interval, the per-condition trial counts after all exclusions, or the decomposition of the 20% missing-data threshold into its underlying causes. Given that the entire inferential apparatus rests on this subset of trials, the degree of data loss is a relevant context for the reader. Separately, no fixation constraint is imposed between trials: the participant's starting gaze position at the onset of each response-stimulus interval is whatever position was reached at the end of the preceding response, and this starting position carries trial-history information correlated with the upcoming stimulus. This leaves open the possibility that what is classified as predictive orienting partly reflects the mechanical consequences of where the eye happened to be at the end of the previous trial. The authors defend the absence of a fixation cross on the grounds that it would transform the transitional structure of the task, but this is an empirical claim presented without a supporting citation.

Heterogeneity within the high-probability condition is not addressed. The two routes to a high-probability triplet in the design - pattern-random-pattern (50% of trials) and random-pattern-random (12.5%) - differ both in their base rate and in the reliability of the contextual cue they provide. Collapsing across these subtypes is an analytical choice that may conceal heterogeneity in the underlying learning process.

Appraisal: Do the results support the authors' conclusions?

The framework succeeds in providing a trial-by-trial behavioral readout of predictive orienting that is more fine-grained than conventional reaction-time measures, and the behavioral dissociation between errors congruent with the regularity and errors reflecting an inaccurate internal model is a genuine empirical contribution. The conclusions about the mechanistic nature of statistical learning should be read as motivating hypotheses for future modeling work rather than as settled empirical claims.

Impact and utility:

The analytical framework introduced here is likely to be useful to researchers working on implicit learning, predictive processing, and Bayesian models of perception and cognition. The measure of predictive orienting and the iterative-updating code could be adapted to a range of probabilistic learning paradigms, and the behavioral dissociation between noise-driven and model-mismatch errors fills a methodological gap that the field has long acknowledged. The authors share their data and code openly, which will facilitate reuse. The most durable contribution of the paper is methodological; the theoretical claims about the nature of statistical learning will require additional computational modeling before they can be regarded as established.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors leverage a high-powered 7T fMRI dataset of subjects viewing naturalistic audiovisual movies to elucidate the topographic organization of the human auditory cortex. By applying a nonlinear pRF model, they successfully map tonotopic gradients extending beyond the auditory core into the STG and STS areas. A primary finding is a medial-to-lateral gradient of increasing response compressivity, which the authors claim mirrors the hierarchical cascade architecture of the visual system. Furthermore, the modeling reveals that regions exhibiting high speech selectivity predominantly occupy the low-frequency portions of non-primary tonotopic maps. The authors argue that this architecture reflects an efficient coding mechanism where the cortex magnifies specific spectral features to facilitate the transition from acoustic encoding to flexible speech representation.

Overall, the study presents concise analyses and compelling high-resolution results that advance our understanding of auditory cortical organization. However, the manuscript currently exhibits several significant theoretical and methodological gaps that temper its broader claims. Most notably, the authors' reliance on a spatial, retinotopic-like analogy overlooks the fundamentally temporal nature of audition. Decoding continuous, natural speech relies heavily on dynamic, full-spectrum temporal integration and contextual recurrent computations, which are difficult to reconcile with the purely static, low-frequency spatial tuning observed here.

Strengths:

(1) The utilization of ultra-high-field 7T functional imaging combined with large-scale, naturalistic continuous stimuli provides an excellent signal-to-noise ratio and captures cortical responses under ecologically valid conditions.

(2) The application of a non-linear pRF encoding model provides a robust, quantitative method for parameterizing and mapping tonotopic features across the cortex, moving beyond simple contrast-based parcellations.

(3) The manuscript effectively demonstrates the relationship between category selectivity (e.g., speech) and underlying tonotopy, drawing an elegant and structurally useful analogy to the well-established relationship between category selectivity and retinotopy in the visual cortex.

Weaknesses:

(1) While the PCA mapping of the functional and structural parameter space is visually compelling, the robustness of this representational geometry across varying acoustic contexts remains ambiguous. Because the model relies on the specific statistical regularities of a single naturalistic audiovisual stimulus set, it is unclear if this low-dimensional structure would hold when tested against isolated speech sounds, environmental noise, or spectrally matched non-speech control stimuli.

(2) The methodological descriptions currently lack the computational precision required for replication and deep evaluation. I would suggest that the exact mathematical formulation of the encoding model be fully specified in the Methods section. This should include an explicit definition of the objective function, a clear accounting of all terms and hyperparameters utilized during the fitting process, and the exact dimensionalities of both the input feature space and the resulting parameter space.

(3) There is a critical theoretical disconnect between the observed static, low-frequency tuning in the STG and the known acoustic requirements for continuous speech perception. Speech is a full-spectrum signal; while fundamental frequencies and formants dominate the lower spectrum (which is vital for processing dynamic pitch contours), high-frequency bands (>1 kHz) carry indispensable phonetic information, such as the rapid spectrotemporal dynamics of consonants, especially fricatives. If the speech-responsive cortex is primarily and statically tuned to a low-frequency spectrum, it is unclear how the dynamic, high-frequency spectral information required for semantic decoding is represented. A rich body of electrophysiological literature documents diverse spectrogram coding in the STG. For example, Mesgarani et al. (Science, 2014) demonstrated using spectrotemporal receptive field models that neural populations in the STG are tuned to both low and high-frequency spectrograms well above 1 kHz. The authors must address this discrepancy and attempt to reconcile their static tonotopic findings with the existing literature on dynamic speech encoding.

(4) While drawing parallels between visual and auditory processing hierarchies is conceptually attractive, the modalities face fundamentally different computational challenges. Vision is largely resolved in space, making a retinotopic spatial coding strategy ecologically and computationally sound. Audition, however, evolves continuously in time. Complex temporal structure, continuous temporal integration, and contextual recurrent computations are paramount for auditory processing, particularly for speech comprehension. In this sense, a purely spatial or tonotopic coding framework is insufficient to fully explain the complex temporal processing dynamics required in the higher-order auditory domain.

Reviewer #2 (Public review):

Summary:

The authors study cardiac deceleration during threat responses in Drosophila. Particularly, it focuses on identifying the neuronal control of this deceleration. Using behavioral and cardiac tracking and analysis, genetics, and calcium imaging, they identify two pairs of dopaminergic neurons involved in cardiac deceleration during air puff responses

Strengths:

The study is overall well done, and the paper is clearly written. Particularly, the work on identifying the two pairs of dopaminergic neurons involved in cardiac deceleration using a series of drivers and generating new ones is rigorous and extensive. Finally, the authors manipulate the heartbeat to investigate how it influences threat responses

Weaknesses:

There are, however, several points that need to be clarified, as some claims are not entirely supported by evidence.

The authors, for example, claim that dopaminergic neurons are responsible for cardiac deceleration (during the air puff, lines 182-3, page 9). However, based on the work in this study, it seems that other neurons could be involved in this control as well. In addition to dopaminergic neurons, the authors test serotonergic and octopaminergic neurons, which, based on silencing experiments, also show an implication in heart-beat deceleration. Furthermore, because they find that dopaminergic neurons are the only ones that, upon thermogenetic activation, lead to lower heart beat frequency, they conclude that the dopaminergic neurons are responsible for air -puff induced cardiac deceleration.

However, these activation experiments are done in a different context than the air puff experiments (at a higher temperature, which could have an effect on the heartbeat changes upon activation of different neuron groups), and because silencing of other monoaminergic neuron types during the air puff also resulted in less cardiac deceleration, one cannot exclude the implication of octopaminergic or serotonergic neurons in air-puff-induced deceleration.

Activation experiments without high temperatures (using, for example, optogenetics) and/or in the presence of the air puff would be important to determine that the dopaminergic neurons are the main type of monoaminergic neurons involved in air-puff-induced cardiac deceleration. Otherwise, the related claims should be rephrased in a way that clearly doesn't exclude a possible implication of other monoaminergic neurons.

Regarding the interactions between the cardiac deceleration and locomotion, the authors propose, based on the results, that the optogenetic cardiac deceleration is sufficient to induce an increase in locomotion, and that it is the decrease in heartbeat that would be responsible via interoceptive pathways to trigger an increase in locomotion. In the model they propose, the DA-WED neurons would induce a decrease in heartbeat that, in turn, would trigger an increase in locomotion. There is not enough proof that cardiac deceleration is the one that triggers an increase in locomotion during air puff responses. As the authors themselves state, the experiments that would demonstrate this would involve preventing cardiac deceleration while optogenetically activating DA-WED. It can therefore not be excluded that the DA-WED neurons trigger an increase in locomotion that is possibly modulated by the cardiac activity. Both alternatives should be considered (models in Figures 4 and 5).

Reviewer #2 (Public review):

This study addresses an important question regarding exercise-induced modulation of pain in women, but the conclusions appear to be based on relatively limited and selective evidence. The authors report an interaction between exercise intensity and stimulus intensity, which they interpret as evidence for exercise-induced hypoalgesia and conclude that fitness, but not sex, modulates this effect. However, this main result relies on a relatively small interaction that emerges only under specific conditions, with inconsistent findings across pain modalities and stimulus intensities, and an analysis approach that does not fully exploit the continuous pain ratings collected. The lack of a baseline condition further limits the interpretability of the findings as reflecting hypoalgesia, and overall, the data provide a rather constrained basis for drawing broader conclusions.

Strengths:

(1) The focus on women is important and timely, particularly given the ambiguity in prior findings and the historical bias toward male-dominated samples.

(2) The attempt to revisit previous findings in a new cohort is valuable in principle.

Weaknesses:

(1) The core interpretation may not be fully supported by the data

The central claim-that the results demonstrate exercise-induced hypoalgesia and its dependence on fitness but not sex-does not appear to be fully supported by the evidence presented.

1.1 Lack of baseline condition

The absence of a no-exercise baseline substantially limits interpretation. The study compares high- and low-intensity exercise, but without a baseline, it is not possible to determine whether either condition produces hypoalgesia or hyperalgesia relative to calibration. The observed HI-LI difference, therefore, reflects only a relative contrast between exercise intensities, not an absolute reduction in pain. As a result, attributing the findings to "hypoalgesia" may be difficult to justify fully.

1.2 Lack of internal replication across conditions

The reported effect is highly specific and does not clearly generalise across the experimental design. It emerges significantly only for heat pain at the highest stimulus intensity, with no clear effects for other intensities and for pressure pain. Moreover, the main statistical result is a relatively small interaction effect with a modest p value, which translates into a difference of approximately 6-8 VAS units on a 150 scale. This combination-a small effect size, limited statistical strength, and restriction to a single condition-substantially weakens the evidence for a robust or generalisable effect.

1.3 Deviations from the original study and selective use of data

Although framed as a follow-up to previous work, the current study introduces substantial methodological changes, particularly in the acquisition and scaling of pain ratings (continuous vs post-hoc ratings, modified VAS with sub-threshold range). Despite collecting rich continuous data, the analysis focuses on peak responses to approximate the previous study. While this may aid comparability, it results in a strong emphasis on a single data point (highest intensity), rather than leveraging the full dataset. This limits both interpretability and comparability.

1.4 Over-reliance on null results regarding sex differences

The conclusion that fitness, but not sex, modulates exercise-induced pain may not be directly supported by the data presented. The current study includes only highly fit women, and comparisons with men or less-fit women rely on non-significant differences in a previous cohort. The absence of a significant difference does not provide evidence for equivalence, and no formal statistical support for a null effect is provided. As such, conclusions about the absence of sex differences would unfortunately benefit from more cautious interpretation.

(2) Limited sample and lack of diversity

The dataset is narrow in scope, comprising a small sample (N = 21) of healthy, highly fit women. Key demographic characteristics (e.g. age range, BMI distribution) are not fully presented, explored or discussed. This limits generalisability and makes it difficult to draw broader conclusions about exercise-induced pain modulation in women, as the main focus of the study.

(3) Methodological choices limit the interpretability of the data

Several methodological decisions would benefit from stronger justification:

3.1 The use of a non-standard VAS scale (0-150 with a fixed pain threshold at 50) is unconventional and may influence how participants report pain, while limiting comparability with related literature.

3.2 Participants explicitly reported expecting exercise to reduce pain, introducing a potential confound that is not presently addressed.

3.3 A more comprehensive use of the full time series of pain ratings would provide a stronger and more transparent basis for interpretation of the present findings.

Reviewer #2 (Public review):

In the manuscript "Cancer cells differentially modulate mitochondrial respiration to alter redox state and enable biomass synthesis in nutrient-limited environments", Chang et al investigate how cancer cells respond to the limitation of certain environmental nutrients by regulating the cellular NAD+/NADH ratio. They focus on serine and lipid metabolism, pathways known to be controlled by the NAD+/NADH ratio, and propose that changes in mitochondrial respiration in response to deprivation of these nutrients can influence the NAD+/NADH ratio, thereby impacting biomass synthesis.

While the study is descriptive in nature and does not investigate specific molecular mechanisms that explain the crosstalk between nutrient availability and mitochondrial redox changes, the experimental component is robust, and the conclusions are well supported by the results. Some suggestions could further refine the conclusions and enhance the quality of the manuscript.

Comments on revised version:

The authors have provided a very comprehensive response. Their updated paper has improved, and the critiques have been mitigated.

Reviewer #2 (Public review):

Original Review:

The manuscript by Eroglu and Hobert presents a set of strains each harboring up to three fluorescently tagged endogenous proteins. While there is technically nothing wrong with the method and the images are beautiful, we struggled to appreciate the advance of this work - who is this paper for?

As a technical method, the advance is minimal since the first author had already demonstrated that three mutations (fluorophore insertion and co-CRISPR marker) could be introduced simultaneously.

As a pilot for creating genome-scale resources, it is not clear whether three different fluorophores in one animal, while elegantly designed and implemented, will be desired by the broader community.

Finally, the interpretation of the patterns observed in the created lines leaves much to be desired. A Table with all the observations must be included and can replace the tedious (and often wrong) descriptions of the observations with the different lines. It would be too much to point out every mistaken expectation of protein expression. Two examples include:

The expectation that ACDH-10 is enriched in the intestine and epidermal tissues (hypodermis) is naïve - there are multiple paralogs of this protein (look at WormPaths or WormFlux) that may share functions in different tissues. There is also no reason to assume that fatty acid metabolism does not occur in other tissues (including the germline). Finally, there are no published studies about this enzyme, so we really don't know for sure what it's doing.

The expectation that HXK-1 is ubiquitously expressed is similarly naïve. There are three paralogous enzymes that are all associated with the same reaction, and we have shown that these three function redundantly in vivo, perhaps in different tissues (PMID: 40011787). Moreover, single cell RNA-seq data (PMID: 38816550) also shows enrichment of hxk-1 in gonadal sheath cells.

The table should have at least the following information: gene/protein name - Wormbase ID - TPM levels of single cell data assigned to tissues for L2, L4 and adult (all published) - tissues in which expression is observed in the lines presented by the authors.

Other points:

(1) We would encourage the authors to provide systematic validation of the reported insertions. The manuscript reports that 24 of 30 tags were isolated and visible but does not clearly state whether each isolated line was confirmed by sequence‑level validation to be correctly in‑frame and free of unintended mutations at the target locus.

(2) The manuscript presents aggregated success counts (e.g., 8/10 mTagBFP2 tags, 9/10 mStayGold, 7/10 mScarlet3) and useful narrative descriptions of injection outcomes. We suggest also to include per‑locus success rates.

(3) For pools that required re‑injection after initial failures, we would like to see a description of the specific changes that were made to the injection mixes or procedures (e.g., new repair template prep, different Cas9 reagent lot, guide redesign). This will be useful troubleshooting information for others.

(4) The authors states that the fluorophore sequences are codon-optimized for C. elegans. We suggest they provide the exact donor/tag sequences used specifically state whether the fluorophore sequences contain any synthetic/artificial introns or other sequence modifications (e.g., silent PAM‑disrupting mutations) were included in the donor templates.

(5) Page 3: Include a reference for "The C. elegans genome encodes around 20,000 genes"

We hope these comments are useful.

Comments on Revised Version:

Overall, we found the responses to be quite recalcitrant.

We have one remaining composite concern about the comparison between observed expression patterns with the new strains versus published data.

First, the authors only report patterns for one stage while it should be not too much effort to image the different life stages. However, since this is a revision, we are not formally requesting they do this.

Second, in the now provided Table (thank you) 'observed expression' (last column) is lacking for 9 of the 30 proteins, and for 6 of these the procedure was not successful. Why not report patterns for the other three? It is confusing also because on page 5, the authors say that "overall, 24 of 30 tags ...all of which were visible with fluorescence stereomicroscopy" - are we missing something? Also, they then said that they "obtained 6/9 of the originally failed tags"; why are the corresponding patterns not included in table 1, and are 9 proteins still labeled as "no" in the "success?" Column?

Third, we strongly feel that the response to our comments about expression patterns is not adequate. On page 5 the authors say that "all proteins were expected to be ubiquitously expressed" and that "scRNA-seq indicated that transcript abundance was ubiquitous and without strong tissue-specific enrichment with few exceptions". However, in their rebuttal, the authors now argue for tissue-specific expression for proteins with paralogs, turning around their own argument! Moreover, their Table indicates that many genes show tissue-enriched expression by RNA-seq while many of their tagged proteins exhibit ubiquitous expression.

Overall, this indicates that both the overall accomplishment of generating tagged protein strains and analyzing their expression is oversold.

Reviewer #2 (Public review):

Summary:

The authors aimed to determine Molidustat targets and the potential utility of these findings. They clearly demonstrate that Molidustat interferes with GSTP1 and some other proteins on top of PHD2. They also demonstrate that PHD2 deletion is not sufficient to recapitulate Molidustat effects in cells and proteomes. Finally, they demonstrate synthetic lethality in organoids for Molidustat and APC deletion.

Strengths:

The data on Molidustat proteomes, GSTP1 binding, inhibition and metabolic health of organoids is really clear. All biochemical, docking and omic data are really strong. The potential impact of these findings could be the use of Molidustat in APC null tumours and awareness of potential off-target effects.

Reviewer #2 (Public review):

Synesthesia is a neurological condition where stimulation of one sensory channel leads to involuntary, automatic, and consistent experience of another, unrelated percept. For example, Sir Francis Galton (1880, Nature) famously described the robust tendency of some individual (synesthetes) to associate numerals with a distinct color. Ever since, synesthesia keeps attracting a broad interest in the cognitive neurosciences in light of its implications for the study of domains such as perception, consciousness, and brain connectivity, among others.

Strauch, Leenaars, and Rouw measured pupil size in a group of 16 grapheme-color synesthetes and two matched control groups. The participants were presented with gray digits - that is, visual stimuli having identical physical properties in terms of brightness. Each participant subsequently rated the corresponding evoked color and brightness: unlike controls, synesthetes did so in a very consistent and reliable fashion. Accordingly, this was also shown in their pupils: despite the same objective luminance, digits associated with brighter percepts caused their pupils to constrict and digits associated with darker percepts caused their pupils to dilate more than controls. These results highlight how crossmodal correspondences are deeply rooted in synesthetes, and puts forward pupillometry as a particularly appealing biomarker for some phenomenological experience (at least those grounded in "brightness").

Further strengths of the technique are its temporal resolution and its responsiveness to several constructs. Across several tasks, the authors show for example that responses to synesthetic light are somewhat slower than responses to real light (i.e., they are likely mediated), but at the same time faster than responses to mental imagery. The role of mental imagery can also be reasonably dismissed when considering the second feature of pupil size: its responsiveness to mental effort and cognitive load. The pupils tend to dilate with demanding, challenging tasks, and this was the case when control participants were asked to report the color of a digit for which they did not consistently experience a synesthetic association. The same task was, instead, seemingly effortless for synesthetes, again speaking in favor of the automaticity of number-color correspondences in their case.

Overall, the findings by Strauch, Leenaars, and Rouw are highly significant for the field and likely to be impactful. The strength of their evidence, when accounting for the relatively small sample size and the inherent variability of both phenomenology (color perception and subjective reporting) and physiology (pupil size), is adequate and sufficiently convincing.

Comments on revisions:

I thank the authors for addressing all my comments in a satisfactory way. I think that the paper has improved, especially in terms of transparency of the reporting and clarity of the results.

Reviewer #2 (Public review):

Summary:

Chang et al. attempted to analyze a large number of ribo-seq datasets through a standardized pipeline, identifying novel non-canonical ORFs and elucidating their evolutionary and expression characteristics.

Strengths:

(1) The datasets analyzed by the authors are sufficiently comprehensive, and the use of standardized pipelines ensures excellent analytical consistency.

(2) Their analyses of ORF evolution and co-expression further deepen our understanding of these ORFs.

Weaknesses:

(1) The authors primarily conducted analyses through bioinformatics, lacking sufficient wet-lab experimental evidence.

(2) Some analytical methods and standards were not clearly presented in the manuscript.

Reviewer #2 (Public review):

Summary:

In this manuscript, Meijer and colleagues investigated the effects of inactivation (conditional silencing) of cortical layer 6b neurons on sleep-wake states and EEG spectral power under the following three conditions: during natural sleep-wake states, after sleep deprivation, or after intracerebroventricular administration of orexin A and B. The authors report that silencing of L6b neurons did not have a significant effect on the total time spent in sleep-wake states, duration or number of state epochs, or the response to sleep deprivation. However, silencing of L6b neurons did slow down theta-frequency (6-9 Hz) during wake and REM sleep, and reduced the total EEG power during NREM sleep. Infusion of orexin A in the mice in which cortical layer 6b neurons were inactivated produced an increase in wakefulness. A similar effect was observed after infusion of orexin A in the mice in which these neurons were not silenced, but the effect (i.e., increase in wakefulness) was of a smaller magnitude. Silencing of cortical layer 6b neurons attenuated the effect of orexin B in increasing theta activity, as was observed in the control mice. The authors conclude that the cortical neurons in layer 6b play an essential role in state-dependent dynamics of brain activity, vigilance state control and sleep regulation.

Strengths:

- A focus on cortical layer 6b neurons, which is an understudied neuronal population, especially in the context of brain and behavioral state transitions.

- The authors used a well-established mouse model to study the effect of inactivation of cortical layer 6b neurons.

Weaknesses:

- Although the authors used a highly selective approach to silence layer 6b neurons, the observed changes in EEG oscillations cannot be solely attributed to layer 6b neurons because of the ICV route for orexin administration.

- The rationale for using only male rats is not provided.

Comments on revised version:

The authors have addressed my concerns.

Reviewer #2 (Public review):

Summary:

The authors investigate how ELF3, a disordered scaffolding protein in the plant circadian Evening Complex, responds to temperature by forming reversible nuclear condensates. They focus on the C-terminal prion-like domain and on a variable polyglutamine tract within it, asking how the tract length and surrounding sequence context tune temperature-responsive structural and condensation behavior. Using a tiered set of computational approaches, including sequence heuristics, hierarchical chain-growth ensembles, all-atom enhanced-sampling simulations, and coarse-grained condensate simulations of 100 monomers, they characterize wild-type, polyQ deletion, polyQ expansion, and an aromatic-disrupting F527A variant. In the revised manuscript, the central claim has been reframed so that polyQ length is now described as tuning condensate material properties rather than driving temperature-sensitive phase separation, with temperature-responsive condensation attributed primarily to a sticker-rich aromatic contact network.

Strengths:

The biological question is important and timely, and the multiscale computational strategy provides a fresh view of an intrinsically disordered protein and its variants. The all-atom enhanced sampling analyses identify a temperature-dependent long-range aromatic contact involving F527 and a methionine-tyrosine coordination motif, which are concrete and mechanistically interesting observations beyond what coarse-grained or sequence-only methods could provide. In response to the previous round of review the authors have added replicate averaged statistics with error bars on the new condensate analyses, introduced new dynamics observables including effective diffusivity, an anomalous diffusion exponent, the self van Hove function, shape anisotropy, per chain radius of gyration in the condensed phase, and a condensate lifetime, provided cluster size time series for transparency, justified the choice of polyQ tract lengths against published Arabidopsis polymorphisms, expanded the Methods with explicit formulas for the new analyses, and included a split half convergence check for the all atom ensembles. The reframing toward a sticker spacer interpretation is consistent with recent experimental work and represents a more cautious and defensible reading of the data.

Weaknesses:

Despite these substantive additions, several core concerns from the previous review remain only partially addressed, and, on close reading, the new supplementary analyses do not robustly support the reframed claim that polyQ length tunes condensate material properties. Error bars and replicate-averaged statistics were added to the new condensate panels, but the helical propensity and per-residue analyses throughout the rest of the manuscript still show only a single curve per temperature, so variability for these key observables remains unreported. Several of the newly added dynamics observables show that the variants are essentially indistinguishable within the reported uncertainty: the self van Hove distributions, the shape anisotropy distributions, and the per chain radius of gyration distributions in the condensed phase overlap almost entirely across variants, and the anomalous diffusion exponent has between replica spreads at low temperature that exceed the variant to variant differences, with variant orderings that change with temperature. The variant-dependent signal that does survive, namely a drop in condensate lifetime for the polyQ expansion and the aromatic mutant at the highest temperature studied, rests on a single temperature point, with replicate spreads spanning most of the metric's dynamic range.

The cluster size time series at higher temperatures shows the dominant cluster oscillating over a wide range across replicas, indicating intermittent dissolution and incomplete convergence in the very temperature regime where the variant-specific claims are made. The only convergence test provided is a split-half radius-of-gyration analysis for the all-atom ensembles, with no slab-geometry or coexistence-density check for the coarse-grained condensate simulations. The polyQ deletion variant forms dominant clusters comparable in size to wild type at low and intermediate temperatures, which on its own argues that variable polyQ presence is not a primary determinant of clustering and supports the earlier concern that the temperature sensitive behavior is dominated by generic chain length and aromatic sticker effects rather than polyQ specific sequence effects, a concern that the reframing softens but does not resolve. Statistical significance is not assessed anywhere, and with three replicas and largely overlapping error bars, claims of variant-specific differences would benefit from explicit statistical tests. Minor quality control issues are also visible in the supplementary material, including a mislabeling of the aromatic mutant in two analysis panels and an inconsistent trajectory length for one variant at one temperature.

Additional Context for Readers:

Readers should interpret the molecular mechanism proposed here with caution. The reframing from polyQ length driving temperature-sensitive phase separation to polyQ length tuning of condensate material properties is more scientifically measured and aligns with recent experimental work, but several of the supplementary observables introduced to support this revised claim indicate that the variants studied are statistically indistinguishable within the reported replicate uncertainty. The most robust observation in the revised work is that the prion-like domain undergoes a temperature-responsive break of an aromatic contact in all-atom simulations and that aromatic sticker contacts dominate inter-protein interactions in coarse-grained condensate simulations. The mechanistic role of the polyQ tract, beyond generic chain length and hydration effects, remains, as in the original submission, not clearly established by the simulations presented. Independent experimental validation of the proposed aromatic contact and of the predicted material-state differences between polyQ variants will be needed to establish the molecular mechanism, and improved condensate convergence tests, uniformly reported error bars across all simulation-derived figures, and explicit statistical tests of variant-versus-variant differences would substantially strengthen confidence in the conclusions.

Reviewer #2 (Public review):

Summary:

This study introduces a method that combines physical expansion of cells, imaging-guided isolation of defined regions, and protein identification to enable compartment-resolved analysis of protein composition at the subcellular scale. The authors aim to address a central limitation in existing approaches, namely the loss of spatial information during sample preparation or the indirect nature of proximity-based labeling methods. Using several cellular compartments as examples, they demonstrate that their approach can recover compartment-enriched protein sets and identify candidate proteins with previously unassigned localization.

Strengths:

A major strength of this work is the conceptual simplicity and accessibility of the approach. By combining established techniques in a modular way, the method avoids the need for genetic manipulation or specialized labeling strategies, making it broadly adaptable across experimental systems. The ability to directly select regions of interest based on imaging represents a clear advantage over indirect enrichment strategies and allows flexible targeting of both membrane-bound and non-membrane-bound compartments.

The experimental design is also a strong aspect of the study. The use of complementary comparison strategies-analyzing isolated compartments alongside matched "subtracted" controls-provides an internal framework for assessing enrichment and depletion, increasing confidence in spatial assignment. The application of the method across multiple organelles of different sizes and properties demonstrates versatility, and the reported specificity for several compartments is encouraging. In particular, the ability to profile small and biochemically challenging structures highlights a potentially important niche for the approach.

Weaknesses:

Despite these strengths, several methodological limitations constrain the interpretation of the results. The most important relates to spatial accuracy in three dimensions. While lateral resolution is improved through physical expansion, the lack of depth resolution introduces uncertainty regarding contributions from structures above and below the selected region. Although the authors argue that this does not substantially affect specificity, the current evidence is largely indirect, and a more rigorous quantification of potential contamination would strengthen this conclusion.<br /> Quantitative interpretation also remains challenging. Because the measurements reflect total protein abundance rather than local concentration, differences in compartment size and protein density can influence enrichment values, particularly for small structures embedded within larger volumes. This issue is evident in the analysis of smaller compartments and complicates direct comparison across conditions. Additional normalization or modeling would help clarify how to interpret these measurements.

Another limitation concerns variability in the expansion process and its downstream consequences. Differences in expansion factor across samples may affect the definition of regions of interest and introduce variability in sampling, yet the impact of this variability is not fully explored. Similarly, the use of a modified chemical treatment to preserve proteins for downstream analysis is central to the workflow but is not extensively validated with respect to preservation of spatial organization.

While the identification of previously unannotated proteins is an appealing aspect of the study, validation is limited to a small number of examples, and broader support from independent datasets or literature context is lacking. In addition, the study primarily focuses on steady-state measurements in a single cell type, and therefore does not yet demonstrate the ability of the method to capture dynamic or condition-dependent changes in protein localization.

Finally, the positioning of the method relative to existing approaches could be more clearly articulated. Although qualitative comparisons are provided, a more systematic and quantitative benchmarking against alternative strategies would help readers better understand the specific advantages and trade-offs.

Reviewer #2 (Public review):

Summary

The paper investigates whether the real-time physical experience of the body shapes high-level physical reasoning. Participants played a set of computerized tool-use reasoning games (the Virtual Tools paradigm) in which they must use knowledge of physical laws - including gravity, collisions, and inertia - to guide a ball into a target area. In Study 1, participants played the games under terrestrial gravity while receiving either Galvanic Vestibular Stimulation (GVS), which introduces noise into the vestibular organ and disrupts gravitational signalling, or a Sham condition with matched skin sensation. In Study 2, a separate cohort played the same games redesigned under hypogravity (0.5 g - half Earth g) or hypergravity (2 g - double Earth g), again with concurrent GVS or Sham stimulation. Performance was assessed through success rate, number of attempts, and time per attempt; strategy was assessed through the spatial distance between successive tool placements and the frequency of tool switching across attempts. A post-hoc gravity-weighted index (GWI) was computed to compare the effect of vestibular perturbation across the two studies. The main finding is that GVS impairs performance in gravity-dependent games under terrestrial gravity, yet the same perturbation appears to be neutral or even beneficial when the game environment involves non-terrestrial gravity - a result the authors interpret as evidence for an adaptable, body-grounded internal model of physics.

Strengths

One of the most notable strengths of this work is its conceptual positioning at the intersection of embodied cognition and physical reasoning. Rather than treating the human body either as an abstract information-processing device or as a purely biomechanical system, the authors take seriously the idea that cognition is scaffolded by ongoing sensorimotor state - and they test this idea with a paradigm that is both tractable and theoretically motivated. The use of the Virtual Tools paradigm is well-suited to this goal: the games vary systematically in their reliance on gravitational predictions, allowing selective impairment (rather than general disruption) to serve as a signature of embodied physical reasoning.

The dual-study design is another strength. Testing the same vestibular perturbation under terrestrial and altered game-gravity conditions, and observing a reversal in its effect depending on context, provides a form of internal control that is conceptually compelling. The additional clustering analyses (Dirichlet Process Gaussian Mixture Model and leave-one-out kernel density classification) strengthen the strategy results beyond raw distance measures, confirming that GVS systematically shifts participants' spatial exploration strategies.

The paper is also clearly written and engages meaningfully with relevant theoretical frameworks - predictive coding, embodied cognition, and stochastic resonance - making it accessible and stimulating for a broad audience.

Weaknesses

(1) Absence of multiple-comparisons correction. A large number of game-level pairwise t-tests are conducted in both studies (upward of twenty per study) without correction for familywise error rate. The game-level effects that anchor the main narrative - in Study 1 alone: Remove, GoalMove, Spiky, Falling_A, Shafts_B, Gap, and Chaining - arise from an uncorrected pool of comparisons. The probability that some of these constitute false positives is non-trivial. The authors should apply a correction (e.g., Benjamini-Hochberg) or at a minimum discuss this limitation explicitly.

(2) The facilitation claim rests on a post-hoc and arbitrarily parameterized index. The gravity-weighted index (GWI), which drives the central cross-study comparison, uses integer coefficients (1, 2, 3) to weight games by gravity dependency level. These coefficients are entirely arbitrary and bear no principled relationship to the actual gravitational magnitudes used in the study. Why not use the gravity dependency ratings themselves, or the empirically estimated gravity impact scores from the computational modelling mentioned in the Methods? The choice of weights should be either principled or tested across a range of values to demonstrate robustness. Furthermore, the notation in equation (1) as currently typeset reads as "Gravity minus Weighted Index" rather than "Gravity-Weighted Index"; this should be corrected.

(3) The "facilitation" interpretation exceeds what the data in Study 2 directly support. Across all games in Study 2, GVS versus Sham differences in absolute performance are non-significant in all directions. The facilitation claim derives entirely from the GWI being higher in Study 2 than in Study 1 - a between-subjects comparison involving different participant groups and a non-pre-registered metric. The language of "facilitation" should be tempered accordingly, or the authors should provide additional analyses to support this framing.

(4) Gravitational manipulation is visual only, and the vestibular system is only one component of the gravity-sensing network. Gravity perception results, as the authors very well know, from a distributed multisensory integration process that involves, in addition to the vestibular system, visual, proprioceptive, and visceral inputs. The present paradigm manipulates gravitational context solely through visual cues and targets the vestibular system through GVS - a point the authors acknowledge but do not discuss in sufficient depth. It is important to distinguish clearly between real gravitational alterations (as achieved in parabolic flight or centrifuge environments, where the entire body is physically subjected to a different gravitational vector) and virtually altered gravity, where only one sensory modality is targeted while others remain anchored to 1 g. The scope of the conclusions should reflect this distinction.

(5) The choice of 0.5 g and 2 g may lack sensitivity. Combining the two altered-gravity conditions in Study 2, because no significant effect of hypo versus hypergravity was found, is statistically pragmatic but conceptually unsatisfying. There is evidence in the space physiology literature that gravitational processing is not linearly symmetric around 1 g: threshold effects exist below and above terrestrial gravity that may not be captured by modest deviations (half and double g) - see refs below. It is worth discussing whether the absence of a hypo/hyper distinction in Study 2 reflects a genuine equivalence or a lack of sensitivity, and whether more extreme conditions (e.g., near-zero g or 4-5 g) might reveal different processing regimes. Whether 0.5 g and 2 g were sufficient to saturate the system or merely insufficient to perturb it remains an open question with direct implications for the interpretation of the null GWI effects on strategy measures.

Lee SMC, Ribeiro LC, Martin DS, Zwart SR, Feiveson AH, Laurie SS, Macias BR, Crucian BE, Krieger S, Weber D, Grune T, Platts SH, Smith SM, and Stenger MB. Arterial structure and function during and after long-duration spaceflight. J Appl Physiol (1985) 129: 108-123, 2020.

de Winkel KN, Clément G, Groen EL, and Werkhoven PJ. The perception of verticality in lunar and Martian gravity conditions. Neurosci Lett 529: 7-11, 2012.

Clément G, Moore ST, Raphan T, and Cohen B. Perception of tilt (somatogravic illusion) in response to sustained linear acceleration during spaceflight. Exp Brain Res 138: 410-418, 2001.

Benson AJ, Kass JR, and Vogel H. European vestibular experiments on the Spacelab-1 mission: 4. Thresholds of perception of whole-body linear oscillation. Exp Brain Res 64: 264-271, 1986.

(6) High-level reasoning is not defined with sufficient precision. The term "high-level reasoning" appears from the title onward and in the heading of the Study 1 results section (line 138), but it is never formally defined. The reader needs a clearer account of what distinguishes high-level physical reasoning from low-level sensorimotor prediction, and where the games used here fall along that continuum. What specific physical competencies - ballistic trajectories, free-fall predictions, collision dynamics, frictional forces, inertial effects - are required across the game set? When describing the subset of games that drive key effects, this information is critical for evaluating whether effects are specific to gravity reasoning or to some other physical concept.

(7) Performance measures are disconnected from underlying kinematics. The performance measures (success rate, number of attempts, time per attempt) are coarse, high-level summaries. Time per attempt is used as a proxy for performance efficiency, yet participants received no instructions regarding speed, and different individuals may have adopted systematically different speed-accuracy trade-offs. It would be valuable to know whether time per attempt correlates with attempt number within a given game (which would indicate within-game learning) and whether mouse movement data - trajectory, velocity, hesitation - were recorded and could be analysed to provide more mechanistic insight into strategy formation.

Reviewer #2 (Public review):

Summary:

In this manuscript, Fukui et al. re-examined the ATP hydrolysis mechanism in GHKL ATPases, revealing a cooperative role of two conserved acidic residues rather than one. The authors have used a range of biochemical and structural techniques on various mutants from different members of the GHKL ATPase family to test and validate their proposed mechanism.

Through a detailed re-analysis of their previously published structure of the aqMutL NTD (ATPase domain) in complex with AMPPCP, they identified Glu29 and Glu32 as interacting with nucleophilic water for the catalysis. The authors carefully dissected the respective roles of these two acidic residues with a series of site-directed mutations. Mutations at Glu29 impaired ATPase activity without affecting protein secondary structure or ATP binding in the case of the E29Q mutant. Moreover, mutations at Glu32 did not affect secondary structure (except for E32G) but reduced ATPase activity. Activity was abolished when both residues (E29Q/E32Q) were mutated.

The authors extended their study to another GHKL ATPase, aqGyrB. Their findings further supported the cooperative function of the corresponding acidic residues in aqGyrB (Glu48 and Asp51) during ATP hydrolysis. Mutation of these residues partially impaired ATP hydrolysis without affecting protein secondary structure. ATPase activity was completely lost in the double mutant E48Q/D51M. While the E48Q mutant retained the ability to bind ATP, the E48A mutant did not. High-resolution structures of the WT and E48A, E48Q, D51A, and D51N mutants of the aqGyrB NTD demonstrated that nucleophilic water positioning depended on these residues. E48 played a dominant role in water positioning and is critical for stabilising ATP lid formation and associated conformational changes, whereas D51 contributed cooperatively to catalysis.

The authors investigated the functional impact of mutating the corresponding residues in the human MutL homologs PMS2 and MLH1. Clinical variants consistently exhibited reduced or abolished ATPase activity, providing a potential molecular basis for Lynch syndrome through impaired DNA mismatch repair.

Lastly, through evolutionary analysis, the authors inferred that the second acidic residue was likely present in the common ancestor of MutL, GyrB, and MORC proteins, but was lost in the case of Hsp90.

Strengths:

(1) This study contains a detailed structural and biochemical analysis of a biologically important set of GHKL ATPases. The authors identify a second acidic residue that is conserved and contributes to catalysis in a large subset of GHKL ATPases. An updated and extended mechanistic model of ATP hydrolysis by this class of enzymes is proposed, which involves cooperative and partially overlapping roles for the catalytic residue pair. This revised mechanistic model is invaluable for the interpretation of clinical variants of GHKL ATPases such as PMS2 and MLH1.

(2) The work described was performed to an excellent and rigorous technical standard. The structural and biochemical data are sound. The evidence supporting the claims is compelling.

Weaknesses:

(1) The identification in this study of a second acidic residue contributing to catalysis but not absolutely essential for catalysis is a useful finding. However, given that many structures of GHLK ATPases have been determined with different nucleotide analogs bound and that the essential role of the first acidic residue is well established, the importance and scope of the advances described here remain focused within the field of study of GHKL ATPases.

(2) The authors assessed the consequences of variants in the human MutL homologs PMS2 and MLH1, but various other human GHKL ATPases contain clinically relevant variants, some of which have stronger disease associations than the mutations examined in this study. A broader analysis of the effect (or likely effect) of disease-linked mutations in GHKL ATPases would have strengthened this study.

(3) In MLH1, the E37K mutation completely abolishes ATPase activity, but the corresponding mutations in aqMutL, aqGyrB, and PMS2 do not. It remains unclear why E37K in MLH1 leads to complete loss of activity, as the authors propose that water molecule positioning via the first acidic residue, as well as ATP lid stabilisation and associated conformational changes, should still be possible.

(4) The authors do not examine ATP binding in the E32 mutants of aqMutL NTD and the D51 mutants of aqGyrB, or AMPPNP binding of the NLH1 and PMS2 mutants. Hence, the relative contributions of the acidic residues to ATP binding and hydrolysis remain partially unclear.

(5) The ATPase assays for PMS2 and MLH1 (Figure 7 and Table 1) were performed with purification/solubility tags still present. Hence, it cannot be ruled out that these tags influence the measured activities.

(6) The authors suggest that the two-acidic-residue mechanism proposed in this study could be shared among several GHKL ATPase families, yet they also state that the hydrogen-bonding network was not observed in MutL and MORC family proteins. This raises doubt about how conserved the mechanism is, e.g., in MutL and MORC proteins.

Reviewer #2 (Public review):

Summary:

This is an extremely interesting mouse study, trying to understand how sepsis is tolerated during obesity/NAFLD. The researchers combine a well-established model of NASH (Choline-deficiency with High Fat Diet) with a sepsis model (IP injection of 10mg/kg LPS), leading to dramatic mortality in mice. Using this model, they characterize the complex contributions of immune cells. Specifically, they find that NK-cells and Neutrophils contribute the most to mortality in this model due to IFNG and PD-L1+ Neutrophils.

Strengths:

The biggest strength of the manuscript is how clear the primary phenotypes/endpoints of their model are. Within 6 hours of LPS injection, there is a stark elevation of liver inflammation and damage, which is exacerbated by a High Fat/CholineDeficient diet (HFCD). And after 1 day, almost all of the mice die. Using these endpoints, the authors were able to identify which cells were critical for mortality in the model and the specific mediators involved.

Comments on revisions:

I have no further comments.

Reviewer #2 (Public review):

Schwarze et al. investigated whether synaptic efficacy is brain-region specific. To this end, they compared synaptic connections established by layer 5 (L5) neocortical pyramidal cells and between L5 and L2/3 pyramidal cells. In order to identify the mechanism of this brain region specificity, the authors employed several experimental approaches, including paired electrophysiological recordings, extracellular stimulation, low- and high-affinity intracellular calcium chelators (EGTA and BAPTA), multiple probability fluctuation analysis (MPFA), and intracellular measurements of calcium transients as well as computational modelling. The findings of the present study indicate that synaptic connections in the primary somatosensory cortex (S1) are significantly stronger and more reliable than those in the prefrontal cortex (PFC).

The study is timely, and the topic is of significant interest to the neuroscience community. Despite the extensive research that has been carried out on the neuroanatomy and receptor distribution of different brain regions, comparatively little attention has been paid to differences in synaptic physiology. The authors' approach is characterised by its elegance and comprehensive nature, and the conclusions drawn are compelling. Nevertheless, there are a number of unresolved issues.

Major points:

(1) The authors state that data from the S1 cortex were obtained in a previous study. In the context of an explicitly comparative study (PFC vs. S1cortex), it would have been advantageous for the authors to perform a subset of experiments in which both cortices were obtained from a single animal. This is a feasible undertaking, given the spatial separation of the PFC and S1 cortex.

(2) Figure 1A is somewhat misleading because it could suggest that the authors have performed dual recordings in identified PFC pyramidal cells.

(3) PFC and S1 cortex in rodents differ markedly in their morphological organisation. For example, in all sensory cortices, layer 4 is very pronounced; however, in the PFC of rodent,s no clear layer 4 can be found. On the other hand, PFC shows a clear separation of layers 2 and 3, which is not visible inthe S1 cortex. Furthermore, PFC pyramidal cells in layers 2, 3, and 5 exhibit significant heterogeneity, diverging considerably from those found in layers 5a and 5b of S1 cortex. Thus, there is no clear correlation between L5 pyramidal cells in the PFC and the S1 cortex. In order to achieve a meaningful comparison of the data obtained in PFC and S1 cortex, it is necessary for the authors to determine whether the record is from similar pyramidal cell populations.

(3) In addition, PFC pyramidal cells in layer 2, 3 and 5 are highly heterogeneous and differ markedly from those in layer 5a and 5b of S1 cortex. To achieve a meaningful comparison of the data obtained in the PFC and the S1 cortex, the authors need to determine whether the record from similar pyramidal cell populations.

(4) For the S1 cortex, in rats it has been found that L5 synaptic connection between pairs of L5a pyramidal cells and pairs of L5b pyramidal cells differ markedly with respect to mean EPSP amplitude, latency and coefficient of variation (cv, a surrogate measure for the synaptic release probability) (cf. Markram et al., 1997; Frick et al., 2008). It is therefore likely that PFC and S1 pre- and postsynaptic pyramidal cells are not only morphologically and electrophysiological distinct but also with respect to their synaptic properties. At least, the authors need to discuss these confounding issues and preferentially address them experimentally. For example, it would be helpful to demonstrate that paired recordings were made from the same pyramidal cell types, perhaps by documenting their morphology and/or firing patterns. In addition, they should discuss the marked difference in EPSP amplitude and putative release probability between their data and the earlier studies.

(5) In order to perform multiple probability fluctuation analysis (MPFA), a parabolic fit with a mere three points is inadequate, particularly because 2 mM and 5 mM Ca2+ are close to the peak of the variance-to-mean parabola, and only 1 mM Ca2+ is on its initial linear part. A more meaningful result would have been obtained with an additional Ca2+ concentration between 1.0 and 2.0 mM, as these are closer to the physiological range. In this context, the authors should have quoted the more recent and more detailed paper by the Silver group (Saviane and Silver, 2006; Lanore and Silver, 2016) and not just the Clements and Silver review paper.

(6) Methods: The authors should clarify whether their paired recordings from L5 pyramidal cells involved whole-cell recordings from both pre- and postsynaptic neurons. From Figure 1B, it appears as if the presynaptic neurons were not recorded in whole cell mode but rather stimulated in cell-attached mode. This is also reflected in the artefact visible in the current trace recorded in the postsynaptic neuron. The authors should explicitly state their methodological approach and mention how reliable the timing of the presynaptic action potential was under these circumstances. The same holds true for the extracellular stimulation protocol. A significantly more detailed description of the experimental protocol is necessary here.

(7) Methods: The authors use Student's t-test for data comparison. The authors should verify that the data distribution was indeed normal, e.g. by using a Shapiro-Wilk test. If this is not the case, non-parametric tests should be used.

Reviewer #2 (Public review):

Summary:

The authors set out to test whether a defined set of small molecules can lessen damaging effects caused by venoms from several Bothrops species, and whether these effects are consistent enough to suggest a broadly applicable approach. They present a cross-venom dataset spanning in-vitro activity readouts and blood-based functional outcomes, and include a chicken embryo model to explore whether venom inhibition can translate into improved survival. The central message is that certain small molecules can reduce specific venom-driven effects across multiple samples, providing a comparative resource for the field and a basis for prioritizing future validation.

Strengths:

The main value of this work is the breadth and structure of the dataset, which places multiple venoms and multiple readouts into a single, comparable framework that should be useful for readers evaluating patterns across samples. The experimental flow is generally coherent, moving from activity measurements to functional outcomes and then to an in-vivo test, which helps the reader understand how the authors link mechanism-oriented assays to more integrated endpoints. The manuscript also provides practical information for the community by highlighting which readouts appear most consistently affected across venoms, which can help guide hypothesis generation and study design in follow-up work.

Comments on revisions:

I would like to thank the authors for answering my questions. The manuscript has gained in quality, knowing the limitations that are now better stated in the manuscript.

Reviewer #2 (Public review):

Summary:

Based on a detailed dataset, the authors present a novel Bayesian approach to classify malaria cases as either imported or locally acquired.

Strengths:

The proposed Bayesian approach for case classification is simple, well justified, and allows the integration of parasite genomics, travel history, and epidemiological data.

Weakness:

While the authors aim to classify cases as imported or locally acquired, the method does not quantify the contribution of each case type to overall transmission, which the authors leave for future study.

Reviewer #2 (Public review):

Summary:

This work introduces a novel framework to systematically learn the latent dimensions of single-cell data, grounded in the theory of the Riemannian manifold. The authors demonstrate how this framework can be applied to various important tasks, such as estimating intrinsic dimensionalities, annotating cell types, etc. They did a great job of tackling an important but not yet established problem in the field and approaching it with a theoretically sound and novel approach. I think after a more rigorous and comprehensive validation, this work could be impactful.

Strengths:

- Dimensionality reduction is a routine step in analyzing many high-dimensional data, such as molecular data. While the downstream analysis results depend heavily on this step, existing methods rely on strong assumptions and are sometimes heuristic. The authors present a novel, theoretically grounded approach to address this important problem.

- The authors demonstrated its usability in downstream analysis in a comprehensive manner. Especially, they show evidence suggesting novel T-cell subpopulations.

- I commend the authors for releasing and maintaining their software well with comprehensive documentation. This significantly increases the usability and accessibility of the method.

Weaknesses:

- The paper lacks experiments that validate the results. It would be beneficial to see additional evaluation settings with better-established ground truths to more strongly demonstrate the method's effectiveness.

- Batch effects are prevalent in single-cell data. The paper does not adequately address how the proposed method handles this issue.

Reviewer #2 (Public review):

Summary:

Ding et al. examine the role of TIE1 in cardiac chamber morphogenesis using genetic mouse models targeting Tie1, Tek, or both, and analyzing endocardial cell-mediated chamber formation across multiple embryonic developmental and postnatal stages, supported by analysis of published single-cell datasets and new bulk RNA seq analyses of murine cardiac tissue. The authors find that Tie1 and Tek expression is higher in atrial than ventricular endocardial cells. Notably, endothelial Tie1 is required for atrial trabeculation at E12.5, but is less critical in ventricular trabeculation. TIE1 also acts synergistically with TIE2 during atrial trabeculation. While Tie1 deficiency alone does not cause defects at E10.5, combined heterozygous deletion of Tek disrupts both atrial and ventricular development at E10.5. This synergy is further supported by analyses at later embryonic stages and in postnatal hearts.

Strengths:

The study is well-designed, clearly written, and supported by high-quality figures. The performed experiments demonstrate a previously unrecognized role for Tie1 in cardiac development and identify synergistic control of cardiac morphogenesis by Tie1 and Tie2. This synergy is consistent with the previously identified roles of Tie1 and Tek in venous development and with Tie1 involvement in angiopoietin-dependent postnatal vascular and lymphatic remodeling. Together, these findings support a role for Tie1 as a contributor to Ang1-Tie2 signaling during heart development.

Weaknesses:

The manuscript does not include direct mechanistic studies; however, RNA seq analysis of atria and ventricles showed reduced expression of Tek, Dll1, and Notch1 upon Tie1 deficiency in developing hearts. Although previously reported mechanisms, such as TIE1-TIE2 heterodimer formation and effects on endothelial junctions, migration, or survival are discussed, no direct mechanistic experiments are performed. Addressing some of these mechanisms would have clarified the basis of Tie1-Tie2 synergy. As two distinct Tie1 models are used, including one targeting the kinase domain, the authors should state whether phenotypes differed or were similar between models.

Reviewer #2 (Public review):

Summary:

In this work, the authors review the study of the neural correlates of consciousness (NCCs). They discuss several of the difficulties that researchers must face when studying NCCs, and argue that several of these difficulties can be alleviated by using intracranial recordings in humans.

They describe what constitutes an NCC, and the difficulties to distinguish between an NCC proper from the prerequisites and consequences of conscious processing.

They also describe the two main types of experimental designs used to study NCCs. These are the contrastive approach (with its report and non-report variants), and the supraliminal approach, each with their own merits and pitfalls.

They discuss the limitations of non-invasive methods, such as fMRI, EEG and MEG, as well as the limitations of the use of invasive recordings in non-human animals.

After setting the stage in this way, the authors provide an extensive review on the knowledge acquired by using invasive recordings in humans. This included population level measurements in vision and in other sensory modalities, as well as single neuron level studies. The authors also discuss studies of subcortical NCCs.

The second half of this work discusses the theoretical insights gained through the use of intracranial recordings, as well as their limitations, and a perspective for future work.

Strengths:

This work offers an impressive review, which will serve as a useful reference document, both for newcomers to the study of NCC as for experienced researchers. The inclusion of non-visual and subcortical NCCs is of particular merit, as these have been understudied.

Besides serving as a review, this work includes a perspective, exploring several directions to pursue for the progress of the field.

Weaknesses:

No major weaknesses.

Appraisal of whether the authors achieved their aims:

In this work, the authors have gathered an impressive review, and have discussed several important problems in the field of study of NCCs, as well as provided a perspective on how the field could move forward.

Discussion of the likely impact of the work on the field:

This work has the potential of becoming a must read for anyone working in the field of consciousness research.

Comment on revised version:

The authors have addressed all my concerns. Once again, my compliments for a nice piece of work.

Reviewer #2 (Public review):

Summary:

In this work, the authors review the study of the neural correlates of consciousness (NCCs). They discuss several of the difficulties that researchers must face when studying NCCs, and argue that several of these difficulties can be alleviated by using intracranial recordings in humans.

They describe what constitutes an NCC, and the difficulties to distinguish between an NCC proper from the prerequisites and consequences of conscious processing.

They also describe the two main types of experimental designs used to study NCCs. These are the contrastive approach (with its report and non-report variants), and the supraliminal approach, each with its own merits and pitfalls.

They discuss the limitations of non-invasive methods, such as fMRI, EEG and MEG, as well as the limitations of the use of invasive recordings in non-human animals.

After setting the stage in this way, the authors provide an extensive review of the knowledge acquired by using invasive recordings in humans. This included population-level measurements in vision and in other sensory modalities, as well as single-neuron level studies. The authors also discuss studies of subcortical NCCs.

The second half of this work discusses the theoretical insights gained through the use of intracranial recordings, as well as their limitations, and a perspective for future work.

Strengths:

This work offers an impressive review, which will serve as a useful reference document, both for newcomers to the study of NCC and for experienced researchers. The inclusion of non-visual and subcortical NCCs is of particular merit, as these have been understudied.

Besides serving as a review, this work includes a perspective, exploring several directions to pursue for the progress of the field.

Weaknesses:

The intention of the authors is to argue how some of the problems faced when studying NCCs are alleviated by the use of intracranial recordings in humans. But in some cases, the link between the problems related to the study of NCCs and the advantages of intracranial recordings over non-invasive methods is not clear.

For example, the authors explain the difficulties in distinguishing between true NCCs from their prerequisites and consequences. This constitutes a difficult conceptual problems that plague all recording techniques. The authors don't provide a convincing explanation of how intracranial recordings offer advantages over EEG or MEG when dealing with these problems.

For example, the authors explain how the use of non-report designs to rule out post-perceptual processing relies on null results, which, according to them, are harder to interpret given the low resolution of non-invasive methods. But the interpretation of null results is actually more complicated in the case of intracranial recordings. As the coverage achieved by the electrodes is sparse, if a null result is attested, it remains possible that a true effect was present in a nearby patch of cortex out of coverage.

The authors argue that the spatial resolution of intracranial recordings is better than that of EEG and MEG. While this is technically true (especially compared to EEG), the true spatial scale of the NCCs is unknown. If NCCs' span is in the mm range, then the additional spatial resolution of intracranial recordings might not be an advantage.

Another factor that should be taken into consideration when assessing the spatial resolution of intracranial recordings is that while the listening zone of individual intracranial contacts is small, coverage is sparse and defined by clinical criteria (something that the authors discuss). In practice, the activity recorded by contacts is usually attributed to anatomically defined ROIs with a scale in the cm range. Given the sparse and uneven (across regions and patients) coverage afforded by intracranial recordings, the advantage of intracranial recordings in terms of spatial resolution is overstated.

Appraisal of whether the authors achieved their aims:

In this work, the authors have gathered an impressive review and have discussed several important problems in the field of study of NCCs, as well as provided a perspective on how the field could move forward.

What is less clear is how the use of intracranial recordings per se holds potential to overcome problems such as the distinction between true NCCs and the prerequisites and consequences of conscious processing.

Discussion of the likely impact of the work on the field:

This work has the potential of becoming a must-read for anyone working in the field of consciousness research.

Reviewer #2 (Public review):

The data show that BDNF regulates the PD-associated kinase LRRK2, they place LRRK2 within well-described BDNF pathways biochemically, and they show that LRRK2 can play a role mediating BDNF-driven synaptic outcomes at excitatory synapses. The chief strength is that the data provide a potential focal point for multiple observations that have been made across many labs. The findings will be of broad interest because LRRK2 has emerged as a protein that is likely to be part of Parkinson's pathology and its normal and pathological actions remain poorly understood.

A major strength of the study is the multiple approaches that were used (biochemistry, bioinformatics, light and electron microscopy and electrophysiology) across different experimental models (cells, primary neurons, human neurons, mice) to identify and examine the impact of BDNF on LRRK2 signaling and functions. Noteworthy is also the employment of LRRK2KO preparations to validate outcomes and to place LRRK2 actions up or downstream.

The demonstration that LRRK2 and drebrin interact directly is important and suggests that other interacting proteins identified biochemically and bioinformatically in the paper will be important to pursue.

Reviewer #2 (Public review):

Summary:

This study investigates the role of the enzyme Alcohol Dehydrogenase 5 (ADH5) in brown adipose tissue (BAT) during aging. BAT is crucial for thermogenesis and energy balance, but its function and mass diminish with age, contributing to metabolic dysfunction and age-related diseases. ADH5, also known as S-nitrosoglutathione reductase, regulates nitric oxide (NO) signaling by removing damaging S-nitrosylation modifications from proteins. The authors show that aging in mice leads to increased protein S-nitrosylation associated with a combination of increased Nos2 expression and reduced ADH5 expression in BAT, resulting in impaired metabolic and cognitive functions. Deletion of ADH5 in BAT accelerates tissue senescence and systemic metabolic decline. Mechanistically, aging suppresses ADH5 via downregulation of heat shock factor 1 (HSF1), a master regulator of protein homeostasis. Importantly, pharmacologically boosting HSF1 improves BAT function and mitigates both metabolic and cognitive declines in aged mice. The findings highlight a critical HSF1-ADH5 pathway in BAT that protects against aging-related dysfunction, suggesting that targeting this pathway may offer new therapeutic strategies for improving metabolic health and cognition during aging.

Strengths:

This research provides insight into the interplay between redox biology, proteostasis, and metabolic decline in aging. By showing that age regulates genes that control SNO status in BAT and further developing a therapy to target ADH5 in BAT to prevent age related decline, the authors have identified a putative mechanism to combat age related decline in BAT function.

Weaknesses:

None identified.

Comments on revised version:

Congratulations to the authors for this interesting manuscript. I don't want to pat myself on the back, but I found the increased Nos2 expression in Figure 1C of the revised manuscript very satisfying, as it reinforces the shift in the regulation of SNO status that happens in BAT with aging. I appreciate the authors addressing this suggestion.

Reviewer #2 (Public review):

Using the MCF10 breast cancer progression sequence, the authors combined high-resolution Micro-C chromatin conformation capture with RNA-seq and ChIP-seq to depict the sequential reorganization of compartments, topologically associated domains (TADs), and long-range loops in benign, pre-tumor, and metastatic states, and coupled these three-dimensional changes with gene expression and enhancer activity. Four main findings were: (i) chromatin structure was largely quiescent, still limiting gene output differentiation, with upregulated sites being most significantly affected; (ii) enhancer-promoter contact strength covariated with transcriptional amplitude; (iii) 127 genes gained expression with increasing chromatin contact; and (iv) progression-related genes acquired altered histone markers in distal enhancers, which remained connected by stable loops. These conclusions are widely accepted and provide strong justification for the publication of this paper.

Reviewer #2 (Public review):

Summary:

Vig's lab delineates a critical role for STX11 in CRAC channel function, particularly in the context of the fatal immune disorder familial hemophagocytic lymphohistiocytosis type 4 (FHL4). They demonstrate that Syntaxin 11 directly binds and regulates Orai1, and that STX11 depletion abolishes CRAC currents and downstream signaling. Loss of STX11 reduces IL2 gene expression and impairs degranulation, both of which are rescued by the constitutively active Orai1 mutant H134S, whereas a gain‑of‑function mutant targeting the C‑terminus fails to restore these defects. The authors conclude that STX11 primes Orai1 for optimal local assembly that is independent of STIM1 yet required for CRAC channel gating.

Strengths:

This study is firmly grounded in disease biology and demonstrates that STX11 downregulation leads to profound functional defects. Using a comprehensive suite of methods and analyses, the authors interrogate the co-regulation of STX11 and Orai1 and present a near-complete view of STX11's modulatory role in CRAC channel function and downstream signaling pathways. The figures are clear, and the statistical analyses are rigorous and convincing.

Weaknesses:

The authors conclude that Syntaxin 11 directly binds Orai1. This conclusion is well supported by a multifaceted approach, including co-immunoprecipitation (co-IP), molecular dynamics simulations, co-localization/FRET assays, and targeted mutational analysis-all of which are thoroughly executed. While the interaction appears reasonably strong in co-IP experiments, the STX11-Orai1 interaction is comparatively weaker in pull-down assays, which the authors attribute to instability of the purified His-STX11 protein. A remaining gap is direct evidence of interaction in live cells; this is understandably challenging given that fluorescent tagging of STX11 is not feasible. Fully resolving this question lies beyond the scope of the present study and will require more advanced approaches to capture STX11 binding dynamics.

Reviewer #2 (Public review):

In this manuscript, the authors implement a three-step genetic programme in E. coli that converts an initially homogeneous population into spatially structured sender, receiver, and "matured" receiver colonies on agar without externally supplied positional information. They combine a TetR/LacI toggle switch for symmetry breaking, LuxI/LuxR quorum sensing for a paracrine signalling step, and CinI/CinR for an autocrine signalling-like maturation step, and complement the experiments with a mathematical model that qualitatively reproduces pattern formation over a range of initial conditions.

While the article has many strengths such as a clear conceptual framing using Waddington landscapes, a modular and carefully optimised circuit design, thorough experimental characterisation of the toggle and quorum-sensing modules, integration of spatial modelling with experiments, and generally clear writing and figures, I think it will benefit the article to clarify the definition and stability of "differentiated" states, clarify several quantitative and modelling aspects, better explain how fitted curves and promoter engineering were done, and improve some figure design and wording to avoid ambiguity.

Detailed comments below:

(1) P5-8 / and more generally: A major concern is that producing a reporter output is not, by itself, differentiation. For a state to be credibly called "differentiated", it should be stable (self-maintained) over relevant timescales, ideally in the absence of the inducing context. As written, the manuscript sometimes seems to equate cell type with reporter expression. I strongly suggest adding a short subsection explicitly defining state versus output, and for each claimed state, stating whether it is stable/bistable or unstable/reversible, with evidence. Concretely, the authors should enumerate:<br /> a) Toggle-derived sender versus receiver: stable? under what conditions (inducer ranges, hysteresis window)?<br /> b) Paracrine-induced "red" receivers: is this a stable differentiated state, or a context-dependent induction requiring proximity to senders?<br /> c) "Mature" (yellow) state: does it persist after removal from the spatial signal field? If not, it should be described as an induced output programme rather than a mature lineage state.

At present, later sections (and the "maturation" language) risk over-stating what is demonstrated.

(2) Figure 2d: It is unclear whether this panel is intended to be qualitative (schematic/illustrative) or generated from quantitative data. The legend should explicitly state the origin (e.g., representative image, averaged data, simulation output, schematic) and, if quantitative, what was measured, how many replicates, and how the visualisation was constructed.

(3) Figure 2e: The cross-sectional line is described as meant to be comparable, yet the leftmost plot appears to have a different slope from the others. The authors should explain whether this reflects a different scaling/normalisation, a different underlying dataset/condition, or simply a plotting artefact. If these are fitted trends, report the fit function (see also the comment on fitted lines below).

(4) Around P7-8: (saddle/separatrix description): When describing the saddle or separatrix between the two valleys, it would be helpful to briefly connect this more directly to a quantitative dynamical-systems perspective: for instance, the intersection of nullclines and how nullcline geometry changes under IPTG/aTc induction. This will make the landscape picture more complete for readers familiar with the original genetic toggle switch work (Garder et al., 2000).

(5) P9, lines 157-159: The current phrasing ("in absence of noise, the system would be fully deterministic... in living cells, however, stochastic bursts... change the trajectory") risks conflating predicting population-level percentages with predicting colony-level trajectories. It would help to clearly separate (i) the ability to predict the overall fraction of ON/OFF (green/blue) colonies from inducer conditions (which is largely deterministic at the population level) from (ii) the intrinsically stochastic choice of state made by any given founder cell and its colony.

(6) P11, lines 193-195 (promoter engineering): The main text currently only refers to screening variants and choosing pLux76; I suggest briefly stating in the main text (not only in the supplement) what was changed (for example, promoter box variants, core promoter strength modifications) and what design criteria were used (reduced leakiness, increased dynamic range).

(7) Use of fitted lines (Figures 2, 4, 5, 7): Wherever fitted curves are overlaid on data, the asuthors should indicate in the figure legend the explicit form of the fit as well as the fit equation/ parameters. As a reader, it is difficult to interpret what is empirical smoothing versus what is a mechanistic functional form.

(8) P13, lines 232-235: The comparison between induction directly with C6-HSL and induction from sender colonies is qualitative ("significantly smaller range"). The authors should provide distances (for example, in mm) for the induction range in each case and, if possible, approximate total HSL amounts or concentrations, so that the reader can appreciate the magnitude of the difference.

(9) P13, lines 259-262: The authors model the transition to the stationary phase via a monotonically decreasing sigmoid in time for biosynthetic capacity. What is the rationale or literature basis for this approach to model entry into the stationary phase? The authors should cite prior work and clarify why this form is appropriate here, versus alternatives (nutrient diffusion limitation, logistic growth with resource depletion, etc.).

(10) Figure 6c: Are the areas of the plate shown in each column the same field of view across conditions/time, or are these simply representative regions selected per condition (possibly from different plates)? The caption/legend should clarify whether these are matched locations and how images were chosen.

(11) Figure 7a: The combination of solid, dashed, and dash-dot arrows/lines is visually hard to read. I suggest replacing the dash-dot line with a fully dotted line or using different colours (if consistent with journal style) to improve readability.

(12) Figure 7e and similar analyses: The authors should explain in the Methods and/or captions how "distance from sender colonies" is computed when multiple senders exist. Is the distance always measured to the nearest sender, and how are cases handled where a receiver is in the overlapping influence of several senders? This clarification is important for interpreting the fitted curves.

Reviewer #2 (Public review):

Summary:

This study presents an analysis of the metabolism of Drosophila larval immune cells during development and activation. The authors compared the utilization of glycolysis and oxidative phosphorylation for energy metabolism. Although this topic has been widely discussed and well-studied in immune cell research, particularly in mammals, it has received little attention in insects. The authors demonstrated that quiescent and activated larval Drosophila immune cells predominantly use mitochondrial oxidative phosphorylation to produce energy. This finding is significant for the emerging field of insect immunometabolism research and is interesting in comparison to mammalian immunity, where immune cell activation is often associated with a shift toward greater reliance on glycolysis.

Strengths:

Using the Agilent Seahorse system, the authors developed and fine-tuned a method to measure the energy metabolism of Drosophila immune cells, obtaining high-quality, robust data. Through genetic manipulations targeting immune cells specifically, they analyzed metabolic changes in cells with different activations, going beyond developmental changes. They convincingly demonstrated ATP production, primarily in the mitochondria of immune cells, at various developmental stages and in various activated states. The results presented mostly support the conclusions drawn. This methodology and its results are valuable for further studies of insect immunometabolism. In a broader context, they are also valuable for comparing the metabolism of immune cells across different animal groups.

Weaknesses:

The genetic manipulations used were suitable for obtaining immune cells of various types and activation states, such as proliferation, differentiation, and immune activation. However, this method has limitations: the mixture of different cell types was always analyzed, and the specific type of interest was often a minority cell population. Had the other cells remained in their initial control state, the observed change in metabolism could have been primarily attributed to the desired cell type. However, the remaining cells that did not transform into the desired type were also usually influenced or activated in some way, making it difficult to determine to which group the observed change should be attributed. For example, consider the induction of lamellocyte differentiation using Hml>Hop[tum]. There are approximately 1,000 lamellocytes per larva, but according to Supplementary Figure 4, there are still about 5,000 Hml+ cells, and even these cells have activated Jak/Stat signaling. Therefore, it can be assumed that they are also activated. After a real infection, the proportion of lamellocytes is greater, but the remaining plasmatocytes are also activated. The authors should mention these limitations more clearly. However, as the authors correctly note, solving this problem will require single-cell approaches, which current technologies still limit. I see this as a problem when interpreting the proliferation effect. The crucial question is what percentage of the analyzed cells induced by Hml>Ras[V12] were actually in the division stage. Not all hemocytes are Hml+, so not all are induced. Of those that are induced, how many are in the division stage at the time of analysis? Meanwhile, those that were not dividing at that moment also had activated Ras, which triggers many processes besides division. Information on what percentage of the analyzed cells were dividing is missing. This information is important because the finding that dividing Drosophila immune cells primarily use mitochondria and oxidative phosphorylation to produce ATP contrasts with the debated significance of the Warburg effect in dividing mammalian cells. This finding would be significant, but unfortunately, it is not robustly supported by the presented data.

Reviewer #3 (Public review):

Summary and strengths:

In this manuscript, Grimes presents an extension of Ellipse of Insignificant (EOI) and Region of Attainable Redaction (ROAR) metrics to meta-analysis setting as metrics for fragility and robustness evaluation of meta-analysis. The author applies these metrics to three meta-analyses of Vitamin D and cancer mortality, finding substantial fragility in their conclusions. Overall, I think extension/adaption is a conceptually valuable addition to meta-analysis evaluation, and the manuscript is generally well-written.

Reviewer #2 (Public review):

This study has developed a single-step method to assemble active bacterial ribosomes under near-physiological conditions by using the GTPase factors EngA and ObgE. These factors eliminate the need for the traditional, harsh manipulations of temperature and magnesium levels. This integration is an important step toward the bottom-up construction of synthetic cells.

Reviewer #3 (Public review):

This is a technically sophisticated study that integrates coarse-grained modeling with live-cell imaging to address an important and timely question regarding HIV-1 capsid inhibition by lenacapavir.

In summary, in my view, the manuscript represents a solid contribution to the field.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the role of EV-D68 proteases 2A and 3C in nuclear pore complex (NPC) dysfunction and their contribution to motor neuron toxicity. The authors demonstrate that both proteases cleave only a limited number of nucleoporins, with 2A^pro showing the strongest impact by inhibiting nuclear import and export of proteins and disrupting NPC permeability without affecting RNA export. Importantly, treatment with the 2A^pro inhibitor telaprevir reduced neuronal cell death in a dose-dependent manner, achieving neuroprotection at concentrations below those required to inhibit viral replication. The study addresses a relevant mechanism underlying EV-D68-induced neuropathology and explores a potential therapeutic intervention.