DOI: 10.1016/j.immuni.2025.09.009

Resource: Trimmomatic (RRID:SCR_011848)

Curator: @scibot

SciCrunch record: RRID:SCR_011848

What is this?

Reviewer #3 (Public review):

Summary:

This manuscript investigates how mutations in the SARS-CoV-2 nucleocapsid protein (N) alter ribonucleoprotein (RNP) assembly, stability, and viral fitness. The authors focus on mutations such as P13L, G214C, and G215C, combining biophysical assays (SV-AUC, mass photometry, CD spectroscopy, EM), VLP formation, and reverse genetics. They propose that SARS-CoV-2 exploits "fuzzy complex" principles, where distributed weak interfaces in disordered regions allow both stability and plasticity, with measurable consequences for viral replication.

Strengths:

(1) The paper demonstrates a comprehensive integration of structural biophysics, peptide/protein assays, VLP systems, and reverse genetics.

(2) Identification of both de novo (P13L) and stabilizing (G214C/G215C) interfaces provides a mechanistic insight into RNP formation.

(3) Strong application of the "fuzzy complex" framework to viral assembly, showing how weak/disordered interactions support evolvability, is a significant conceptual advance in viral capsid assembly.

(4) Overall, the study provides a mechanistic context for mutations that have arisen in major SARS-CoV-2 variants (Omicron, Delta, Lambda) and a mechanistic basis for how mutations influence phenotype via altered biomolecular interactions.

Weaknesses:

(1) The arrangement of N dimers around LRS helices is presented in Figure 1C, but the text concedes that "the arrangement sketched in Figure 1C is not unique" (lines 144-146) and that AF3 modeling attempts yielded "only inconsistent results" (line 149).<br /> The authors should therefore present the models more cautiously as hypotheses instead. Additional alternative arrangements should be included in the Supplementary Information, so the readers do not over-interpret a single schematic model.

(2) Negative-stained EM fibrils (Figure 2A) and CD spectra (Figure 2B) are presented to argue that P13L promotes β-sheet self-association. However, the claim could benefit from more orthogonal validation of β-sheet self-association. Additional confirmation via FTIR spectra or ThT fluorescence could be used to further distinguish structured β-sheets from amorphous aggregation.

(3) In the main text, the authors alternate between emphasizing non-covalent effects ("a major effect of the cysteines already arises in reduced conditions without any covalent bonds," line 576) and highlighting "oxidized tetrameric N-proteins of N:G214C and N:G215C can be incorporated into RNPs". Therefore, the biological relevance of disulfide redox chemistry in viral assembly in vivo remains unclear. Discussing cellular redox plausibility and whether the authors' oxidizing conditions are meant as a mechanistic stress test rather than physiological mimicry could improve the interpretation of these results.

The paper could benefit if the authors provide a summary figure or table contrasting reduced vs. oxidized conditions for G214C/G215C mutants (self-association, oligomerization state, RNP stability). Explicitly discuss whether disulfides are likely to form in infected cells.

(4) VLP assays (Figure 7) show little enhancement for P13L or G215C alone, whereas Figure 8 shows that P13L provides clear fitness advantages. This discrepancy is acknowledged but not reconciled with any mechanistic or systematic rationale. The authors should consider emphasizing the limitations of VLP assays and the sources of the discrepancy with respect to Figure 8.

(5) Figures 5 and 6 are dense, and the several overlays make it hard to read. The authors should consider picking the most extreme results to make a point in the main Figure 5 and move the other overlays to the Supplementary. Additionally, annotating MP peaks directly with "2×, 4×, 6× subunits" can help non-experts.

(6) The paper has several names and shorthand notations for the mutants, making it hard to keep up. The authors could include a table that contains mutation keys, with each shorthand (Ancestral, Nο/No, Nλ, etc.) mapped onto exact N mutations (P13L, Δ31-33, R203K/G204R, G214C/G215C, etc.). They could then use the same glyphs (Latin vs Greek) consistently in text and figure labels.

(7) The EM fibrils (Figure 2A) and CD spectra (Figure 2B) were collected at mM peptide concentrations. These are far above physiological levels and may encourage non-specific aggregation. Similarly, the authors mention" ultra-weak binding energies that require mM concentrations to significantly populate oligomers". On the other hand, the experiments with full-length protein were performed at concentrations closer to biologically relevant concentrations in the micromolar range. While I appreciate the need to work at high concentrations to detect weak interactions, this raises questions about physiological relevance. Specifically:

a) Could some of the fibril/β-sheet features attributed to P13L (Figure 2A-C) reflect non-specific aggregation at high concentrations rather than bona fide self-association motifs that could play out in biologically relevant scenarios?

b) How do the authors justify extrapolating from the mM-range peptide behaviors to the crowded but far lower effective concentrations in cells?

The authors should consider adding a dedicated section (either in Methods or Discussion) justifying the use of high concentrations, with estimation of local concentrations in RNPs and how they compare to the in vitro ranges used here. For concentration-dependent phenomena discussed here, it is vital to ensure that the findings are not artefacts of non-physiological peptide aggregation..

Author response:

The following is the authors’ response to the original reviews

Recommendations for the Authors:

Reviewer #1:

We think that this manuscript brings an important contribution that will be of interest in the areas of statistical physicists, (microbiota) ecology, and (biological) data science. The evidence of their results is solid and the work improves the state-of-the-art in terms of methods. We have a few concerns that, in our opinion, the authors should address.

Major concerns:

(1) While the paper could be of interest for the broad audience of e-Life, the way it is written is accessible mainly to physicists. We encourage the authors to take the broad audience into account by i) explaining better the essence of what is being done at each step, ii) highlighting the relevance of the method compared to other methods, iii) discussing the ecological implications of the results.

Examples on how to approach i) include: Modify or expand Figure 1 so that non-familiar readers can understand the summary of the work (e.g. with cartoons representing communities, diseased states and bacterial interactions and their relationship with the inference method); in each section, summarize at the beginning the purpose of what is going to be addressed in this section, and summarize at the end what the section has achieved; in Figure 2, replace symbols by their meaning as much as possible-the same for Figure 1, at the very least in the figure caption.

Example on how to approach ii): Since the authors aim to establish a bridge between disordered systems and microbiome ecology, it could be useful to expand a bit the introduction on disordered systems for biologists/biophysicists. This could be done with an additional text box, which could also highlight the advantages of this approach in comparison to other techniques (e.g. model-free approaches can also classify healthy and diseased states).

Example on how to approach iii): The authors could discuss with more depth the ecological implications of their results. For example, do they have a hypothesis on why demographic and neutral effects could dominate in healthy patients?

We thank the reviewer for the observations. Following the suggestion in the revised version, each section outlines the goal of what will be addressed in that section, and summarizes what we have achieved at the end; We also updated Figure 1 and Figure 2.

(i) For figure 1, we expanded and hopefully made more clear how we conceptualize the problem, use the data, andestablish our method. In Figure 2, we enriched the y labels of each panel with the name associated with the order parameter.

(ii) We thank the reviewer for helping us improve the readability of the introductory part, thus providing moreinsights into disordered systems techniques for a broader audience. We have added a few explanations at the end of page 2 – to explain the advantages of such methodology compared to other strategies and models.

(iii) We thank the reviewer for raising the need for a more in-depth ecological discussion of our results. A simple wayto understand why neutral effects may dominate in healthy patients is the following. Neutrality implies that species differences are mainly shaped by stochastic processes such as demographic noise, with species treated as different realizations of the same underlying stochastic ecological dynamics. In our analysis, we observe that healthy individuals tend to exhibit highly similar microbial communities, suggesting that the compositional variability among their microbiomes is compatible—at least in part—with the fluctuations expected from demographic stochasticity alone. In contrast, patients with the disease display significantly more heterogeneous microbial compositions. The diversity and structure of their gut communities cannot be satisfactorily explained by neutral demographic fluctuations alone.

This discrepancy implies that additional deterministic forces—such as altered ecological interactions—are driving the divergence observed in dysbiotic states. In diseased individuals, the breakdown of such interactions leads to a structurally distinct regime that may correspond to a phase of marginal stability, as indicated by our theoretical modeling. This shift marks a transition from a community governed by neutrality and demographic noise to one dominated by non-neutral ecological forces (as depicted in Figure 4). We added these comments in the discussion section of the revised manuscript.

(2) Taking into account the broader audience, we invite the authors to edit the abstract, as it seems to jump from one ecological concept to another without explicitly communicating what is the link between these concepts. From the first two sentences, the motivation seems to be species diversity, but no mention of diversity comes after the second sentence. There is no proper introduction/definition of what macroecological states are. After that, the authors switch to healthy and unhealthy states, without previously introducing any link between gut microbiota states and the host’s health (which perhaps could be good in the first or second sentence, although other framings can be as valid). After that, interactions appear in the text and are related to instability, but the reader might not know whether this is surprising or if healthy/unhealthy states are generally related to stability.

We pointed out a few examples, but the authors could extend their revision on i), ii) and iii) beyond such specific comments. In our opinion, this would really benefit the paper.

In response to the reviewer’s concern about conceptual clarity and structure, we substantially revised the abstract to improve its accessibility and logical flow. In the revised abstract, we now clearly link species diversity to microbiome structure and function from the outset, addressing initial confusion. We provide a concise definition of ”macroecological states,” framing them as reproducible statistical patterns reflecting community-level properties. Additionally, the revised version explicitly connects gut microbiome states to host health earlier, resolving the previous abrupt shift in focus. Finally, we conclude by highlighting how disordered systems theory advances our understanding of microbiome stability and functioning, reinforcing the novelty and broader significance of our approach. Overall, the revised abstract better serves a broad interdisciplinary audience, including readers unfamiliar with the technicalities of disordered systems or microbial ecology, while preserving the scientific depth and accuracy of our work

(3) The connection with consumer-resource (CR) models is quite unusual. In Equation (12), why do the authors assume that the consumption term does not depend on R? This should be addressed, since this term is usually dependent on R in microbial ecology models.

In case this is helpful, it is known that the symmetric Lotka-Volterra model emerges from time-scale separation in the MacArthur model, where resources reproduce logistically and are consumed by other species (e.g., plants eaten by herbivores). Consumer-resource models form a broad category, while the MacArthur model is a specific case featuring logistic resource growth. For microbes, a more meaningful justification of the generalized Lotka-Volterra (GLV) model from a consumer-resource perspective involves the consumer-resource dynamics in a chemostat, where time-scale separation is assumed and higher-order interactions are neglected. See, for example: a) The classic paper by MacArthur: R. MacArthur. Species packing and competitive equilibrium for many species. Theoretical Population Biology, 1(1):1-11, 1970. b) Recent works on time-scale separation in chemostat consumer-resource models: Anna Posfai et al., PRL, 2017 Sireci et al., PNAS, 2023 Akshit Goyal et al., PRX-Life, 2025

We thank the reviewer for the observation. We apologize for the typo that appeared in the main text and that we promptly corrected. The Consumers-Resources model we had in mind is the classical case proposed by MacArthur, where resources are self-regulated according to a logistic growth mechanism, which leads to the generalized LotkaVolterra model we employ in our work.

Minor concerns:

(1) The title has a nice pun for statistical physicists, but we wonder if it can be a bit confusing for the broader audience of e-Life. Although we leave this to the author’s decision, we’d recommend considering changing the title, making it more explicit in communicating the main contribution/result of the work.

Following the reviewer’s suggestion, we have introduced an explanatory subtitle: “Linking Species Interactions to Dysbiosis through a Disordered Lotka-Volterra Framework”.

(2) Review the references - some preprints might have already been published: Pasqualini J. 2023, Sireci 2022, Wu 2021.

We thank the reviewer for pointing our attention to this inaccuracy. We updated the references to Pasqualini and Sireci papers. To our knowledge, Wu’s paper has appeared as an arXiv preprint only.

(3) Species do not generally exhibit identical carrying capacities (see Grilli, Nat. Commun., 2020; some taxa are generally more abundant than others. The authors could discuss whether the model, with the inferred parameters, can accurately reproduce the distribution of species’ mean abundances.

We thank the reviewer for this insightful comment. As discussed in the revised manuscript (lines 294–299), our current model does not accurately reproduce the empirical species abundance distribution (SAD). This limitation stems from the assumption of constant carrying capacities across species. While empirical observations (e.g., Grilli et al., Nat. Commun., 2020 [1]) show heterogeneous mean abundances often following power-law or log-normal distributions. However, our model assumes constant carrying capacity, resulting in SADs devoid of fat tails, which diverge from empirical data.

This simplification is implemented to maintain the analytical tractability of the disordered generalized Lotka-Volterra (dGLV) framework, a common approach also found in prior works such as Bunin (2017) and Barbier et al. (2018) [2, 3]. Introducing heterogeneity in carrying capacities, such as drawing them from a log-normal distribution, or switching to multiplicative (rather than demographic) noise, could indeed produce SADs that better align with empirical data. Nevertheless, implementing changes would significantly complicate the analytical treatment.

We acknowledge these directions as promising avenues for future research. They could help enhance the empirical realism of the model and its capacity to capture observed macroecological patterns while posing new theoretical challenges for disordered systems analysis

(4) A substantial number of cited works (Grilli, Nat. Commun., 2020; Zaoli & Grilli, Science Advances, 2021; Sireci et al., PNAS, 2023; Po-Yi Ho et al., eLife, 2022) suggest that environmental fluctuations play a crucial role in shaping microbiome composition and dynamics. Is the authors’ analysis consistent with this perspective? Do they expect their conclusions to remain robust if environmental fluctuations are introduced?

We thank the reviewer for stressing this point. The introduction of environmental fluctuations in the model formally violates detailed balance, thereby preventing the definition of an energy function. To date, no study has integrated random interactions together with both demographic and environmental noise within a unified analytical framework. This is certainly a highly promising direction that some of the authors are already exploring. However, given the inherently out-of-equilibrium nature of the system and the absence of a free energy, we would need to adopt a Dynamical Mean-Field Theory formalism and eventually analyze the corresponding stationary equations to be solved self-consistently. We added, however, a brief note in the Discussion section.

(5) The term “order parameters“ may not be intuitive for a biological audience. In any case, the authors should explicitly define each order parameter when first introduced.

We thank the reviewer for the comment. We introduced the names of the order parameters as soon as they are introduced, along with a brief explanation of their meaning that may be accessible to an audience with biological background.

(6) Line 242: Should ψU be ψD?

We thank the reviewer for the observation. We corrected the typo.

(7) Given that the authors are discussing healthy and diseased states and to avoid confusion, the authors could perhaps use another word for ’pathological’ when they refer to dynamical regimes (e.g., in Appendix 2: ’letting the system enter the pathological regime of unbounded growth’).

We thank the reviewer for the helpful comment. As suggested, we used the term “unphysical” instead of “pathological” where needed.

Reviewer #2:

(1) A technical point that I could not understand is how the authors deal with compositional data. One reason for my confusion is that the order parameters h and q0 are fixed n data to 1/S and 1/S2, and thus I do not see how they can be informative. Same for carrying capacity, why is it not 1 if considering relative abundance?

We thank the reviewer for raising this point. We acknowledge that the treatment of compositional data and the interpretation of order parameters h and q0 were not sufficiently clarified in the manuscript. Additionally, there was an imprecision in the text regarding the interpretation of these parameters.

As defined in revised Eq. (4) of the manuscript, h and q0 are to be averaged over the entire dataset, summing across samples α. Specifically, and , where S_α is the number of species present in sample α and is the average over samples. These parameters are therefore informative, as they encapsulate sample-level ecological diversity, and their variation reflects biological differences between healthy and diseased states. For instance, Pasqualini et al., 2024 [4] reported significant differences in these metrics between health conditions, thereby supporting their ecological relevance.

Regarding carrying capacities, we clarify that although we work with relative abundance data (i.e., compositional data), we do not fix the carrying capacity K to 1. Instead, we set K to the maximum value of xi (relative abundance) within each sample, to preserve compatibility with empirical data and allow for coexistence. While this remains a modeling assumption, it ensures better ecological realism within the constraints of the disordered GLV framework.

(2) Obviously I’m missing something, so it would be nice to clarify in simple terms the logic of the argument. I understand that Lagrange multipliers are going to be used in the model analysis, and there are a lot of technical arguments presented in the paper, but I would like a much more intuitive explanation about the way the data can be used to infer order parameters if those are fixed by definition in compositional data.

We thank the reviewer for the observation. The order parameters can be measured directly from the data, even in the presence of compositionality, as explained above. We can connect those parameters with the theory even for compositional data, because the only effect of adding the compositionality constraint is to shift the linear coefficient in the Hamiltonian, which corresponds to shifting the average interaction µ. However, the resulting phase diagram is mostly affected by the variance of the interactions σ2 (as µ is such that we are in the bounded phase).

(3) Another point that I did not understand comes from the fact that the authors claim that interaction variance is smaller in unhealthy microbiomes. Yet they also find that those are closer to instability, and are more driven by niche processes. I would have expected the opposite to be true, more variance in the interactions leading to instability (as in May’s original paper for instance). Is this apparent paradox explained by covariations in demographic stochasticity (T) and immigration rate (lambda)? If so, I think it would be very useful to comment on that.

As Altieri and coworkers showed in their PRL (2021) [5], the phase diagram of our model differs fundamentally from that of Biroli et al. (2018) [6]. In the latter, the intuitive rule – greater interaction variance yields greater instability – indeed holds. For the sake of clarity, we have attached below the resulting phase diagram obtained by Altieri et al.

The apparent paradox arises because the two phase diagrams are tuned by different parameters. Consequently, even at low temperature and with weak interaction variance, our system may sit nearer to the replica-symmetrybreaking (RSB) line.

Fig. 3 in the main text it is not a (σ,T) phase diagram where all other parameters are kept constant. Rather, it is a plot of the inferred σ and T parameters from the data (without showing the corresponding µ).

To capture the full, non-trivial influence of all parameters on stability, we studied the so-called “replicon eigenvalue” in the RS (i.e. single equilibrium) approximation. This leading eigenvalue measures how close a given set of inferred parameters – and hence a microbiome – is to the RSB threshold. For a visual representation of these findings, refer to Figure 4.

Author response image 1.

(4) What do the empirical SAD look like? It would be nice to see the actual data and how the theoretical SADs compare.

The empirical species abundance distributions (SADs) analyzed in our study are presented and discussed in detail in Pasqualini et al., 2024 [4]. Given the overlap in content, we chose not to reproduce these figures in the current manuscript to avoid redundancy.

As we also clarify in the revised text, the theoretical SAD is derived from the disordered generalized Lotka-Volterra (dGLV) model in the unique fixed point phase typically exhibit exponential tails. These distributions do not match the heavier-tailed patterns (e.g., log-normal or power-law-like) observed in empirical microbiome data. This discrepancy stems from the simplifying assumptions of the dGLV framework, including the use of constant carrying capacities and demographic noise.

In the revised manuscript, we have added a brief discussion in the revised manuscript to explicitly acknowledge this limitation and emphasize it as a direction for future refinement of the model, such as incorporating heterogeneous carrying capacities or exploring alternative noise structures.

(5) Some typos: often “niche” is written “nice”.

We thank the reviewer for this suggestion. After inspecting the text, we corrected the reported typos.

Reviewer #3:

Major comments:

(1) In the S3 text, the authors say that filtered metagenomic reads were processed using the software Kaiju. The description of the pipeline does not mention how core genes were selected, which is often a crucial step in determining the abundance of a species in a metagenomic sample. In addition, the senior author of this manuscript has published a version of Kaiju that leverages marker genes classification methods (deemed Core-Kaiju), but it was not used for either this manuscript or Pasqualini et al. (2014; Tovo et al., 2020). I am not suggesting that the data necessarily needs to be reprocessed, but it would be useful to know how core genes were chosen in Pasqualini et al. and why Core-Kaiju was not used (2014).

Prior to the current manuscript and the PLOS Computational Biology paper by Pasqualini et al. [4], we applied the core-Kaiju protocol to the same dataset used in both studies. However, this tool was originally developed and validated using general catalogs of culturable organisms, not specifically tuned for gut microbiomes. As a result, we have realized that in many samples Core Kajiu would filter only very few species (in some samples, the number of identified species was as low as 5–10), undermining the reliability of the analysis. Due to these limitations, we opted to use the standard Kaiju version in our work. We are actively developing an improved version of the core-Kaiju protocol that will overcome the discussed limitations and preliminary results (not shown here) indicate the robustness of the obtained patterns also in this case.

(2) My understanding of Pasqualini et al. was that diseased patients experienced larger fluctuations in abundance, while in this study, they had smaller fluctuations (Figure 3a; 2024). Is this a discrepancy between the two models or is there a more nuanced interpretation?

We thank the reviewer for the observation. This is only an apparent discrepancy, as the term fluctuation has different meanings in the two contexts. The fluctuations referred to by the reviewer correspond to a parameter of our theory—namely, noise in the interactions. Conversely, in Pasqualini et al. σ indicates environmental fluctuations. Nevertheless, there is no conceptual discrepancy in our results: in both studies, unhealthy microbiomes were found to be less stable. In fact, also in this study, notably Fig. 4, shows that unhealthy microbiomes lie closer to the RSB line, a phenomenon that is also associated with enhanced fluctuations.

(3) Line 38-41: It would be helpful to explicitly state what “interaction patterns” are being referenced here. The final sentence could also be clarified. Do microbiomes “host“ interactions or are they better described as a property (“have”, “harbor”). The word “host” may confuse some readers since it is often used to refer to the human host. I am also not sure what point is being made by “expected to govern natural ones”. There are interactions between members of a microbiome; experimental studies have characterized some of these interactions, which we expect to relate in some way to interactions in nature. Is this what the authors are saying?

Thanks. We agree that this sentence was not clear. Indeed, we are referring to pairwise species interactions and not to host-microbiome interactions. We have rewritten this part in the following way: In fact, recent work shows that the network-level properties of species-species interactions —for example, the sign balance, average strength, and connectivity of the inferred interaction matrix— shift systematically between healthy and dysbiotic gut communities (see for instance, [7, 8]). Pairwise species interactions have been quantified in simplified in-vitro consortia [9, 10]; we assume that the same classes of interactions also operate—albeit in a more complex form—in the native gut microbiome.

(4) Line 43: I appreciate that the authors separated neutral vs. logistic models here.

(5) Lines 51-75: The framing here is well-written and convincing. Network inference is an ongoing, active subject in ecology, and there is an unfortunate focus on inferring every individual interaction because ecologists with biology backgrounds are not trained to think about the problem in the language of statistical physics.

We thank the reviewer for these positive comments.

(6) Line 87: Perhaps I’m missing something obvious, but I don’t see how ρi sets the intrinsic timescale of the dynamics when its units are 1/(time*individuals), assuming the dimensions of ri are inverse time.

We thank the reviewer for the observation. We corrected this phrase in the main text.

(7) Lines 189-190: “as close as possible to the data” it would aid the reader if you specified the criteria meant by this statement.

We thank the reviewer for the observation. We removed the sentence, as it introduced some redundancy in our argument. In the subsequent text, the proposed method is exposed in details.

(8) Line 198: It would aid the reader if you provided some context for what the T - σ plane represents.

We thank the referee for the helpful indication. Indeed, we have better clarified the mutual role of the demographic noise amplitude and strength of the random interaction matrix, as theoretically predicted in the PRL (2021) by Altieri and coworkers [5]. Please, find an additional paragraph on page 6 of the resubmitted version.

(9) Line 217: Specifying what is meant by “internal modes“ would aid the typical life science reader.

We thank the reviewer for the suggestion. Recognizing that referring to “internal modes” to describe the SAD shape in that context might cause confusion, we replaced “internal modes“ with “peaks”.

(10) Line 219: Some additional justification and clarification are needed here, as some may think of “m“ as being biomass.

We added a sentence to better explain this concept. “In classical and quantum field theory, the particle-particle interaction embedded in the quadratic term is typically referred to as a mass source. In the context of this study, captures quadratic fluctuations of species abundances, as also appearing in the expression of the leading eigenvalue of the stability matrix.”

Minor comments:

(1) I commend the authors for removing metagenomic reads that mapped to the human genome in the preprocessing stage of their pipeline. This may seem like an obvious pre-processing step, but it is unfortunately not always implemented.

We thank the referee for pointing this potential issue. The data used in this work, as well as the bioinformatic workflow used to generate them has been described in detail in Pasqualini et al., 2024 [4]. As one of the main steps for preprocessing, we remove reads mapping to the human genome.

(2) Line 13: “Bacterial“ excludes archaea, and while you may not have many high-abundance archaea in your human gut data, this sentence does not specify the human gut. Usually, this exclusion is averted via the term “microbial“, though sometimes researchers raise objections to the term when the data does not include fungal members (e.g., all 16S studies).

We thank the reviewer for this suggestion. As to include archaeal organisms, we adopt the term “microbial“ instead of “bacterial“.

(3) Line 18: This manuscript is being submitted under the “Physics of Living Systems“ tract, but it may be useful to explicitly state in the Abstract that disordered systems are a useful approach for understanding large, complex communities for the benefit of life science researchers coming from a biology background.

Thank. We have modified the abstract following this suggestion.

(4) Line 68: Consider using “adapted“ or something similar instead of “mutated“ if there is no specific reason for that word choice.

We thank the reviewer for this suggestion, which was implemented in the text.

(5) Line 111: It would be useful to define annealed and quenched for a general life science audience.

We thank the reviewer for this suggestion. In the “Results” section, we have opted for “time-dependent disordered interactions” to reach a broader audience and avoid any jargon. Moreover, in the Discussion we added a detailed footnote: “In contrast to the quenched approximation, the annealed version assumes that the random couplings are not fixed but instead fluctuate over time, with their covariance governed by independent Ornstein–Uhlenbeck processes.”

(6) Line 124: Likewise for the replicon sector.

We thank the reviewer for the suggestion. We added a footnote on page 4, after the formula, to highlight the physical intuition behind the introduction of the replicon mode.

“The replicon eigenvalue refers to a particular type of fluctuation around the saddle-point (mean-field) solution within the replica framework. When the Hessian matrix of the replicated free energy is diagonalized, fluctuations are divided into three sectors: longitudinal, anomalous, and replicon. The replicon mode is the most sensitive to criticality signaling – by its vanishing trend – the emergence of many nearly-degenerate states. It essentially describes how ‘soft’ the system is to microscopic rearrangements in configuration space.”

(7) Figure 2: It would be helpful to include y-axis labels for each order parameter alongside the mathematical notation.

We thank the reviewer for this suggestion. Now the y-axis of Figure 2 includes, along the mathmetical symbol, the label of the represented quantities.

(8) Line 242: Subscript “U” is used to denote “Unhealthy” microbiomes, but “D” is used to denote “Diseased” in Figs. 2 and 3 (perhaps elsewhere as well).

We thank the reviewer for this observation. After checking the various subscripts in the text, coherently with figure 2 and 3, we homogenized our notation, adopting the subscript “D“ for symbols related to the diseased/unhealthy condition.

(9) Line 283: “not to“ should be “not due to“

We thank the reviewer for this suggestion. After inspecting the text, we corrected the reported error.

(10) Equations 23, 34: Extra “=“ on the RHS of the first line.

We consistently follow the same formatting across all the line breaks in the equations throughout the text.

We are thus resubmitting our paper, hoping to have satisfactorily addressed all referees’ concerns.

References

(1) Jacopo Grilli. Macroecological laws describe variation and diversity in microbial communities. Nature communications, 11(1):4743, 2020.

(2) Guy Bunin. Ecological communities with lotka-volterra dynamics. Physical Review E, 95(4):042414, 2017.

(3) Matthieu Barbier, Jean-Franc¸ois Arnoldi, Guy Bunin, and Michel Loreau. Generic assembly patterns in complex ecological communities. Proceedings of the National Academy of Sciences, 115(9):2156–2161, 2018.

(4) Jacopo Pasqualini, Sonia Facchin, Andrea Rinaldo, Amos Maritan, Edoardo Savarino, and Samir Suweis. Emergent ecological patterns and modelling of gut microbiomes in health and in disease. PLOS Computational Biology, 20(9):e1012482, 2024.

(5) Ada Altieri, Felix Roy, Chiara Cammarota, and Giulio Biroli. Properties of equilibria and glassy phases of the random lotka-volterra model with demographic noise. Physical Review Letters, 126(25):258301, 2021.

(6) Giulio Biroli, Guy Bunin, and Chiara Cammarota. Marginally stable equilibria in critical ecosystems. New Journal of Physics, 20(8):083051, 2018.

(7) Amir Bashan, Travis E Gibson, Jonathan Friedman, Vincent J Carey, Scott T Weiss, Elizabeth L Hohmann, and Yang-Yu Liu. Universality of human microbial dynamics. Nature, 534(7606):259–262, 2016.

(8) Marcello Seppi, Jacopo Pasqualini, Sonia Facchin, Edoardo Vincenzo Savarino, and Samir Suweis. Emergent functional organization of gut microbiomes in health and diseases. Biomolecules, 14(1):5, 2023.

(9) Jared Kehe, Anthony Ortiz, Anthony Kulesa, Jeff Gore, Paul C Blainey, and Jonathan Friedman. Positive interactions are common among culturable bacteria. Science advances, 7(45):eabi7159, 2021.

(10) Ophelia S Venturelli, Alex V Carr, Garth Fisher, Ryan H Hsu, Rebecca Lau, Benjamin P Bowen, Susan Hromada, Trent Northen, and Adam P Arkin. Deciphering microbial interactions in synthetic human gut microbiome communities. Molecular systems biology, 14(6):e8157, 2018.

Yo me sentí muy identificada con la parte donde dice que, si dejamos que la IA lo haga todo, terminaremos sin saber hacer nada por nosotros mismos, incluso a veces me pasa que uso ChatGPT o traductores para escribir algo rápido, pero después me doy cuenta de que mi propia capacidad para redactar o pensar argumentos se va oxidando. Creo que el ensayo nos recuerda que la práctica humana sigue siendo esencial.

Me parece muy interesante cómo el autor compara la escritura con la inteligencia artificial. Al principio parece una analogía exagerada, pero al final tiene mucho sentido que ambas son herramientas que cambian nuestra manera de pensar y sobre todo de aprender, lo que más me gustó es que no aterroriza el uso de la IA, sino que invita a usarla con conciencia, como una extensión del pensamiento humano y no como un reemplazo.

En el pasado, Sócrates, (Que en el pasado fue símbolo de los pensadores tradicionalistas), Critico una innovación, En este caso, la escritura o una nueva forma de conocimiento porque creía que corrompía las costumbres humanas o el pensamiento humano.

Al poner en paralelo con la inteligencia Artificial , estamos viviendo una situación parecida ya que la IA, Como aquella innovación antigua, Tiene demasiados críticos y muchos temores asociados pero en algún momento , se consolidara y transformará radicalmente nuestra forma de vivir y pensar, del mismo modo que ocurrió con la invención que Sócrates rechazaba.

Como toda gran innovación, aunque genere resistencia en el principio, termina cambiando al mundo.

como estudiante de cine, me hizo reflexionar, si en un futuro, vamos a dejar que la inteligencia artificial, nos construya las historias para trasmitirlas en imagenes, que no estaria del todo mal, pero hay cosas que la inteligencia artifiicial le hara falta y es, esperiencias vividas y un poco de vision, inplica una parte fundamental al momento de crear una historia, son perspectivas diferentes. Si dejamos que que la IA haga cine, nunca los seres humanos podran desarrolar una mentalidad artistica y crativa en la septima arte, a partir del CGi nos podra ahorra un poco el trabajo, pero la idea es utilizarla como herramienta no como solucion, lo relevante aca, es que nosotros como seres humanos siempre seamos autorores de nuestras propiias historias.

Dilan Alexander Ortiz

En esta parte del texto el autor dice algo que me parece muy interesante. Es verdad que la inteligencia artificial es una herramienta bastante útil, pero también ha hecho que muchas personas piensen menos o se reten menos a sí mismas. Aun así, no estoy del todo de acuerdo con la idea de que usarla todos los días nos haría perder nuestras habilidades. Para mí, saber usar bien la inteligencia artificial también es una habilidad importante. Si se utiliza como apoyo y no como sustituto, puede ayudarnos a trabajar con más eficiencia y a cometer menos errores, sobre todo en el ámbito laboral. Creo que, más que quitarnos capacidades, podría potenciarlas si aprendemos a usarla de la manera correcta

Esto lo interpreto como una reflexión del autor sobre el doble efecto que puede tener la inteligencia artificial en nuestra forma de pensar y aprender. Así como la escritura debilitó la memoria, la IA podría hacer que dependamos menos de nuestras propias habilidades cognitivas; sin embargo, también nos brinda la oportunidad de acceder a una cantidad de información y conocimiento mucho mayor de la que podríamos alcanzar por nosotros mismos. El autor parece querer mostrar que toda herramienta tecnológica implica una pérdida, pero también una ganancia, y que lo importante es encontrar un equilibrio entre aprovechar sus beneficios sin dejar de ejercitar nuestras capacidades humanas

Esto lo interpreto como una advertencia del autor, ya que al volvernos dependientes de la inteligencia artificial podríamos perder nuestra capacidad crítica y de aprendizaje, de la misma forma en que Sócrates pensaba que la escritura debilitaba la memoria.

La escritura debe seguir siendo una forma de arte, incluso en tiempos de inteligencia artificial. Aunque la IA pueda generar textos o ideas, carece de emociones, vivencias y conciencia, elementos esenciales para crear arte verdadero. Escribir no es solo comunicar, sino expresar lo que sentimos y pensamos, transformar nuestras experiencias en palabras con sentido humano. Por eso, debemos aprender a usar la tecnología como una herramienta de apoyo, sin dejar que sustituya nuestra voz ni nuestra creatividad. El equilibrio está en aprovechar lo que ofrece la IA, pero siempre aportando nuestro toque personal, crítico y sensible, porque solo así la escritura mantiene su esencia artística.

Claro que si, decir que Aristóteles fue de los últimos en “saberlo todo” tiene sentido si entendemos que ese “todo” era el conocimiento accesible en su mundo: en Atenas y el entorno helénico podía reunir y ordenar mucha información, pero fuera de ese horizonte había saberes (por ejemplo de China o América) que ni siquiera se imaginaban. Eso no le quita mérito; más bien nos recuerda que la amplitud del conocimiento siempre está limitada por las herramientas y las redes de su época, y que conviene admirar su logro sin olvidar la modestia intelectual.

La inteligencia artificial representa una nueva revolución tecnológica que, al igual que las anteriores, exige de nosotros una capacidad de adaptación inteligente y crítica. A lo largo de la historia, cada avance (desde la máquina de vapor hasta la era digital) generó miedo y resistencia, pero también impulsó transformaciones positivas cuando aprendimos a integrarlo sin perder nuestras capacidades humanas. La IA no debería verse como un reemplazo del pensamiento, sino como una extensión de nuestras posibilidades. El verdadero reto está en mantener el equilibrio: usar la tecnología para potenciar la creatividad y quizás la productividad, sin caer en la pasividad ni en la dependencia absoluta. Adaptarnos no significa rendirnos ante la máquina, sino aprender a convivir con ella, usándola con conciencia y criterio para seguir siendo los protagonistas de nuestro propio desarrollo.

Esa parte donde dice que "la escritura nos abrió la posibilidad de conocer mucho más allá de lo que puede guardar una memoria humana" me parece clave. Es el mejor ejemplo de que toda tecnología tiene un trade-off. Perdimos algo de memoria, pero ganamos el conocimiento colectivo de la humanidad. Con la IA pasa igual: el reto no es evitarla, sino usarla para expandir nuestra inteligencia sin dejar de ejercitar nuestro pensamiento crítico. Es encontrar ese punto medio entre la herramienta y nuestra autonomía.

Este fragmento me llamó mucho la atención porque refleja una idea fundamental sobre el papel de la inteligencia artificial en nuestra vida: la importancia de seguir desarrollando nuestras propias habilidades humanas. En una época en la que cada vez más tareas pueden automatizarse, este pensamiento nos invita a no perder de vista el valor del aprendizaje, la creatividad y el pensamiento crítico.

Me parece interesante que el texto no solo critique la dependencia tecnológica, sino que también resalte la necesidad de equilibrio. Aprender a usar la IA es importante, pero más importante aún es no dejar que reemplace nuestra capacidad de pensar y crear. Como estudiante, esto me hace reflexionar sobre cómo quiero usar la tecnología: no como una muleta, sino como una herramienta para potenciar mis propias habilidades.

Esta parte me deja pensando mucho. Siento que tiene algo profundamente cierto: cuando dejamos que una máquina piense o cree por nosotros, no solo perdemos una tarea, sino una parte de nosotros mismos. Me pasa a veces, cuando algo me sale mal y quiero buscar la solución rápida en internet o pedirle a una IA que lo haga, que me doy cuenta de lo fácil que es rendirse ante la comodidad. Pero también, de lo vacía que puede sentirse esa “facilidad”. Aprender algo nuevo, equivocarse, incluso frustrarse, tiene un valor que una máquina no puede darnos. Esa lucha, esa torpeza inicial, es donde realmente se forma el pensamiento crítico, donde se despierta la curiosidad. Si dejamos que la inteligencia artificial piense todo por nosotros, ¿en qué se convierte nuestra mente? Tal vez terminemos sabiendo más cosas, pero sintiendo menos. Y me parece que eso sería una pérdida demasiado grande, porque lo que nos hace humanos no es solo lo que sabemos, sino cómo llegamos a saberlo.

Este punto se me hace escencial para complementar mis comentarios anteriores, hemos pasado como sociedad tantos cambios que parecían difciles de superar o que pensabamos cambiarían nuestra manera de ver el mundo, y sí, el mundo ha cambiado radicalmente, pero hasta ahora esa exageración de pensar que cada cambio es el fin del mundo no nos ha llevado a nada, siempre nos terminamos acostumbrando o incluso encontramos la manera de usar estos cambios tan "extremos" a nuestro favor. Por supuesto hay muchos contras, es dificil adaptarse a algo tan nuevo y tan diferente como la inteligencia artifical, pero no va a ir a ningún lado y ya va siendo hora de buscar la manera de usarla a nuestro favor de manera sana y que no afecte nuestro progreso, no es buscar todas las respuestas sino apoyarse para ampliar nuestro conocimiento.

La inteligencia artificial es fascinante, hasta incluso ultimamente se ha vuelto indecifrable para el espectador, estoy de acuerdo con el autor, no es algo que cambiará completamente todo lo que conocemos como "sociedad", pero si se presta para muchos infortunios, no sé si considerarlo como solo una tecnologia más pero poco a poco siento que aprenderemos a vivir usando la inteligencia artifical en la cotidianidad.

Acá el autor hace una afirmación que siento podría ser muy interesante comentar, ya que claro, no podemos negar lo innegable, la inteligencia artificial es una herramienta muy útil pero que tambien ha contribuido en que la gente piense menos, o que al menos se rete menos. Lo que si me interesaría comentar de esta parte del texto es que el autor se refiere a que si la inteligencia artificial fuese usada todos los dias para todas las áreas nosotros nos quedaríamos sin habilidades, no concuerdo del todo, el uso correcto de la inteligencia artificial es una habilidad e incluso siento que si se llegara a usar en algunas áreas, no en todas, (como un apoyo) incrementaría la eficiencia y el porcentaje de error laboralmente.

El autor dice algo muy cierto: usar la IA tiene su precio. A veces sin darnos cuenta dejamos que piense por nosotros, y eso hace que no usemos tanto nuestra propia cabeza. No está mal apoyarse en ella, pero tampoco deberíamos dejarle todo el trabajo. Hay cosas que uno mismo tiene que pensar y resolver.

Esta parte me hace pensar en cómo la inteligencia artificial también puede crear una ilusión de saber. Podemos tener toda la información, pero si no la comprendemos de verdad, solo estamos repitiendo cosas que parecen correctas. Tal vez, como dice el autor, estamos llenándonos de conocimiento superficial sin llegar a entender profundamente lo que aprendemos.

el sugiere que la dependencia que hemos generado hacia la tecnología reduce nuestro esfuerzo intelectual y en base a este podemos generar una pregunta interesante : ¿La inteligencia artificial nos está volviendo más perezosos mentalmente, quitándonos las ganas de pensar y cuestionar, o nos está ayudando de forma positiva a mejorar nuestro método de aprendizaje?

Esta crítica de Sócrates hacia la escritura es muy similar al miedo que hoy existe frente a la inteligencia artificial: que al delegar funciones cognitivas en máquinas, perdamos nuestras propias capacidades (como la memoria o el razonamiento).

Esa parte me llamó mucho la atención porque siento que tiene razón. Hoy todo el mundo habla de la inteligencia artificial como si fuera algo que va a solucionarlo todo, pero en realidad todavía no sabemos si nos está ayudando tanto como creemos o si más bien nos está volviendo cómodos. A veces parece que dependemos demasiado de ella y se nos olvida pensar o crear por nuestra cuenta. Yo creo que, como con todo, lo importante es usarla con medida, sin dejar que nos quite lo que nos hace humanos.

allerdings weiß niemand, was uns nach dem Tod erwartet

agnostik ist ja sowas von langweilig... nach dem tod erwarten uns bakterien und pilze und würmer, die unseren kadaver in blumendünger verwandeln, und jeder der was anderes glaubt ist einfach nur ein idiot.

Author response:

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

Reviewer #1 (Public Review):

Summary:

The study explored the biomechanics of kangaroo hopping across both speed and animal size to try and explain the unique and remarkable energetics of kangaroo locomotion.

Strengths:

The study brings kangaroo locomotion biomechanics into the 21st century. It is a remarkably difficult project to accomplish. There is excellent attention to detail, supported by clear writing and figures.

Weaknesses:

The authors oversell their findings, but the mystery still persists. 

The manuscript lacks a big-picture summary with pointers to how one might resolve the big question.

General Comments

This is a very impressive tour de force by an all-star collaborative team of researchers. The study represents a tremendous leap forward (pun intended) in terms of our understanding of kangaroo locomotion. Some might wonder why such an unusual species is of much interest. But, in my opinion, the classic study by Dawson and Taylor in 1973 of kangaroos launched the modern era of running biomechanics/energetics and applies to varying degrees to all animals that use bouncing gaits (running, trotting, galloping and of course hopping). The puzzling metabolic energetics findings of Dawson & Taylor (little if any increase in metabolic power despite increasing forward speed) remain a giant unsolved problem in comparative locomotor biomechanics and energetics. It is our "dark matter problem".

Thank you for the kind words.

This study is certainly a hop towards solving the problem. But, the title of the paper overpromises and the authors present little attempt to provide an overview of the remaining big issues. 

We have modified the title to reflect this comment.  “Postural adaptations may contribute to the unique locomotor energetics seen in hopping kangaroos”

The study clearly shows that the ankle and to a lesser extent the mtp joint are where the action is. They clearly show in great detail by how much and by what means the ankle joint tendons experience increased stress at faster forward speeds.

Since these were zoo animals, direct measures were not feasible, but the conclusion that the tendons are storing and returning more elastic energy per hop at faster speeds is solid. The conclusion that net muscle work per hop changes little from slow to fast forward speeds is also solid. 

Doing less muscle work can only be good if one is trying to minimize metabolic energy consumption. However, to achieve greater tendon stresses, there must be greater muscle forces. Unless one is willing to reject the premise of the cost of generating force hypothesis, that is an important issue to confront. Further, the present data support the Kram & Dawson finding of decreased contact times at faster forward speeds. Kram & Taylor and subsequent applications of (and challenges to) their approach supports the idea that shorter contact times (tc) require recruiting more expensive muscle fibers and hence greater metabolic costs. Therefore, I think that it is incumbent on the present authors to clarify that this study has still not tied up the metabolic energetics across speed problems and placed a bow atop the package. 

Fortunately, I am confident that the impressive collective brain power that comprises this author list can craft a paragraph or two that summarizes these ideas and points out how the group is now uniquely and enviably poised to explore the problem more using a dynamic SIMM model that incorporates muscle energetics (perhaps ala' Umberger et al.). Or perhaps they have other ideas about how they can really solve the problem.

You have raised important points, thank you for this feedback. We have added a limitations and considerations section to the discussion which highlights that there are still unanswered questions. Line 311-328

Considerations and limitations

“First, we believe it is more likely that the changes in moment arms and EMA can be attributed to speed rather than body mass, given the marked changes in joint angles and ankle height observed at faster hopping speeds. However, our sample included a relatively narrow range of body masses (13.7 to 26.6 kg) compared to the potential range (up to 80 kg), limiting our ability to entirely isolate the effects of speed from those of mass. Future work should examine a broader range of body sizes. Second, kangaroos studied here only hopped at relatively slow speeds, which bounds our estimates of EMA and tendon stress to a less critical region. As such, we were unable to assess tendon stress at fast speeds, where increased forces would reduce tendon safety factors closer to failure. A different experimental or modelling approach may be needed, as kangaroos in enclosures seem unwilling to hop faster over force plates. Finally, we did not determine whether the EMA of proximal hindlimb joints (which are more difficult to track via surface motion capture markers) remained constant with speed. Although the hip and knee contribute substantially less work than the ankle joint (Fig. 4), the majority of kangaroo skeletal muscle is located around these proximal joints. A change in EMA at the hip or knee could influence a larger muscle mass than at the ankle, potentially counteracting or enhancing energy savings in the ankle extensor muscle-tendon units. Further research is needed to understand how posture and muscles throughout the whole body contribute to kangaroo energetics.”

Additionally, we added a line “Peak GRF also naturally increased with speed together with shorter ground contact durations (Fig. 2b, Suppl. Fig 1b)” (line 238) to highlight that we are not proposing that changes in EMA alone explain the full increase in tendon stress. Both GRF and EMA contribute substantially (almost equally) to stress, and we now give more equal discussion to both. For instance, we now also evaluate how much each contributes: “If peak GRF were constant but EMA changed from the average value of a slow hop to a fast hop, then stress would increase 18%, whereas if EMA remained constant and GRF varied by the same principles, then stress would only increase by 12%. Thus, changing posture and decreasing ground contact duration both appear to influence tendon stress for kangaroos, at least for the range of speeds we examined” (Line 245-249)

We have added a paragraph in the discussion acknowledging that the cost of generating force problem is not resolved by our work, concluding that “This mechanism may help explain why hopping macropods do not follow the energetic trends observed in other species (Dawson and Taylor 1973, Baudinette et al. 1992, Kram and Dawson 1998), but it does not fully resolve the cost of generating force conundrum” Line 274-276.

I have a few issues with the other half of this study (i.e. animal size effects). I would enjoy reading a new paragraph by these authors in the Discussion that considers the evolutionary origins and implications of such small safety factors. Surely, it would need to be speculative, but that's OK.

We appreciate this comment from the reviewer, however could not extend the study to discuss animal size effects because, as we now note in the results: “The range of body masses may not be sufficient to detect an effect of mass on ankle moment in addition to the effect of speed.” Line 193

Reviewer #2 (Public Review):

Summary

This is a fascinating topic that has intrigued scientists for decades. I applaud the authors for trying to tackle this enigma. In this manuscript, the authors primarily measured hopping biomechanics data from kangaroos and performed inverse dynamics. 

While these biomechanical analyses were thorough and impressively incorporated collected anatomical data and an Opensim model, I'm afraid that they did not satisfactorily address how kangaroos can hop faster and not consume more metabolic energy, unique from other animals.  Noticeably, the authors did not collect metabolic data nor did they model metabolic rates using their modelling framework. Instead, they performed a somewhat traditional inverse dynamics analysis from multiple animals hopping at a self-selected speed.

In the current study, we aimed to provide a joint-level explanation for the increases of tendon stress that are likely linked to metabolic energy consumption.

We have now included a limitations section in the manuscript (See response to Rev 1). We plan to expand upon muscle level energetics in the future with a more detailed musculoskeletal model.

Within these analyses, the authors largely focused on ankle EMA, discussing its potential importance (because it affects tendon stress, which affects tendon strain energy, which affects muscle mechanics) on the metabolic cost of hopping. However, EMA was roughly estimated (CoP was fixed to the foot, not measured) and did not detectibly associate with hopping speed (see results Yet, the authors interpret their EMA findings as though it systematically related with speed to explain their theory on how metabolic cost is unique in kangaroos vs. other animals

As noted in our methods, EMA was not calculated from a fixed centre of pressure (CoP). We did fix the medial-lateral position, owing to the fact that both feet contacted the force plate together, but the anteroposterior movement of the CoP was recorded by the force plate and thus allowed to move. We report the movement (or lack of movement) in our results. The anterior-posterior axis is the most relevant to lengthening or shortening the distance of the ‘out-lever’ R, and thereby EMA. It is necessary to assume fixed medial-lateral position because a single force trace and CoP is recorded when two feet land on the force plate. The mediallateral forces on each foot cancel out so there is no overall medial-lateral movement if the forces are symmetrical (e.g. if the kangaroo is hopping in a straight path and one foot is not in front of the other). We only used symmetrical trials so that the anterior-posterior movement of the CoP would be reliable. We have now added additional details into the text to clarify this

Indeed, the relationship between R and speed (and therefore EMA and speed) was not significant. However, the significant change in ankle height with speed, combined with no systematic change in COP at midstance, demonstrates that R would be greater at faster speeds. If we consider the nonsignificant relationship between R and speed to indicate that there is no change in R, then these two results conflict. We could not find a flaw in our methods, so instead concluded that the nonsignificant relationship between R and speed may be due to a small change in R being undetectable in our data. Taking both results into account, we believe it is more likely that there is a non-detectable change in R, rather than no change in R with speed, but we presented both results for transparency. We have added an additional section into the results to make this clearer (Line 177-185) “If we consider the nonsignificant relationship between R (and EMA) and speed to indicate that there is no change in R, then it conflicts with the ankle height and CoP result. Taking both into account, we think it is more likely that there is a small, but important, change in R, rather than no change in R with speed. It may be undetectable because we expect small effect sizes compared to the measurement range and measurement error (Suppl. Fig. 3h), or be obscured by a similar change in R with body mass. R is highly dependent on the length of the metatarsal segment, which is longer in larger kangaroos (1 kg BM corresponded to ~1% longer segment, P<0.001, R²=0.449). If R does indeed increase with speed, both R and r will tend to decrease EMA at faster speeds.”

These speed vs. biomechanics relationships were limited by comparisons across different animals hopping at different speeds and could have been strengthened using repeated measures design

There is significant variation in speed within individuals, not just between individuals. The preferred speed of kangaroos is 2-4.5 m/s, but most individuals showed a wide speed range within this. Eight of our 16 kangaroos had a maximum speed that was 1-2m/s faster than their slowest trial. Repeated measures of these eight individuals comprises 78 out of the 100 trials.   It would be ideal to collect data across the full range of speeds for all individuals, but it is not feasible in this type of experimental setting. Interference with animals such as chasing is dangerous to kangaroos as they are prone to adverse reactions to stress. We have now added additional information about the chosen hopping speeds into the results and methods sections to clarify this “The kangaroos elected to hop between 1.99 and 4.48 m s^-1, with a range of speeds and number of trials for each individual (Suppl. Fig. 9).”  (Line 381-382)

There are also multiple inconsistencies between the authors' theory on how mechanics affect energetics and the cited literature, which leaves me somewhat confused and wanting more clarification and information on how mechanics and energetics relate

We thank the reviewer for this comment. Upon rereading we now understand the reviewers position, and have made substantial revisions to the introduction and discussion (See comments below) 

My apologies for the less-than-favorable review, I think that this is a neat biomechanics study - but am unsure if it adds much to the literature on the topic of kangaroo hopping energetics in its current form.

Again we thank the reviewer for their time and appreciate their efforts to strengthen our manuscript.

Reviewer #3 (Public Review):

Summary:

The goal of this study is to understand how, unlike other mammals, kangaroos are able to increase hopping speed without a concomitant increase in metabolic cost. They use a biomechanical analysis of kangaroo hopping data across a range of speeds to investigate how posture, effective mechanical advantage, and tendon stress vary with speed and mass. The main finding is that a change in posture leads to increasing effective mechanical advantage with speed, which ultimately increases tendon elastic energy storage and returns via greater tendon strain. Thus kangaroos may be able to conserve energy with increasing speed by flexing more, which increases tendon strain.

Strengths:

The approach and effort invested into collecting this valuable dataset of kangaroo locomotion is impressive. The dataset alone is a valuable contribution.

Thank you!

Weaknesses:

Despite these strengths, I have concerns regarding the strength of the results and the overall clarity of the paper and methods used (which likely influences how convincingly the main results come across).

(1) The paper seems to hinge on the finding that EMA decreases with increasing speed and that this contributes significantly to greater tendon strain estimated with increasing speed. It is very difficult to be convinced by this result for a number of reasons:

It appears that kangaroos hopped at their preferred speed. Thus the variability observed is across individuals not within. Is this large enough of a range (either within or across subjects) to make conclusions about the effect of speed, without results being susceptible to differences between subjects? 

Apologies, this was not clear in the manuscript. Kangaroos hopping at their preferred speed means we did not chase or startle them into high speeds to comply with ethics and enclosure limitations. Thus we did not record a wide range of speeds within the bounds of what kangaroos are capable of in the wild (up to 12 m/s), but for the range we did measure (~2-4.5 m/s), there is a large amount of variation in hopping speed within each individual kangaroo. Out of 16 individuals, eight individuals had a difference of 1-2m/s between their slowest and fastest trials, and these kangaroos accounted for 78 out of 100 trials. Of the remainder, six individuals had three for fewer trials each, and two individuals had highly repeatable speeds (3 out of 4, and 6 out of 7 trials were within 0.5 m/s). We have now removed the terminology “preferred speed” e.g line 115. We have added additional information about the chosen hopping speeds into the results and methods, including an appendix figure “The kangaroos elected to hop between 1.99 and 4.48 m s^-1, with a range of speeds and number of trials for each individual (Suppl. Fig. 9).” (Line 381-382)

In the literature cited, what was the range of speeds measured, and was it within or between subjects?

For other literature, to our knowledge the highest speed measured is ~9.5m/s (see supplementary Fig1b) and there were multiple measures for several individuals (see methods Kram & Dawson 1998). 

Assuming that there is a compelling relationship between EMA and velocity, how reasonable is it to extrapolate to the conclusion that this increases tendon strain and ultimately saves metabolic cost?  They correlate EMA with tendon strain, but this would still not suggest a causal relationship (incidentally the p-value for the correlation is not reported). 

The functions that underpin these results (e.g. moment = GRF*R) come from physical mechanics and geometry, rather than statistical correlations. Additionally, a p-value is not appropriate in the relationship between EMA and stress (rather than strain) because the relationship does not appear to be linear. We have made it clearer in the discussion that we are not proposing that entire change in stress is caused by changes in EMA, but that the increase in GRF that naturally occurs with speed will also explain some of the increase in stress, along with other potential mechanisms. The discussion has been extensively revised to reflect this. 

Tendon strain could be increasing with ground reaction force, independent of EMA. Even if there is a correlation between strain and EMA, is it not a mathematical necessity in their model that all else being equal, tendon stress will increase as ema decreases? I may be missing something, but nonetheless, it would be helpful for the authors to clarify the strength of the evidence supporting their conclusions.

Yes, GRF also contributes to the increase in tendon stress in the mechanism we propose (Suppl. Fig. 8), see the formulas in Fig 6, and we have made this clearer in the revised discussion (see above comment).  You are correct that mathematically stress is inversely proportional to EMA, which can be observed in Fig. 7a, and we did find that EMA decreases. 

The statistical approach is not well-described. It is not clear what the form of the statistical model used was and whether the analysis treated each trial individually or grouped trials by the kangaroo. There is also no mention of how many trials per kangaroo, or the range of speeds (or masses) tested. 

The methods include the statistical model with the variables that we used, as well as the kangaroo masses (13.7 to 26.6 kg, mean: 20.9 ± 3.4 kg). We did not have sufficient within individual sample size to use a linear mixed effect model including subject as a random factor, thus all trials were treated individually. We have included this information in the results section. 

We have now moved the range of speeds from the supplementary material to the results and figure captions. We have added information on the number of trials per kangaroo to the methods, and added Suppl. Fig. 9 showing the distribution of speeds per kangaroo.

We did not group the data e.g. by using an average speed per individual for all their trials, or by comparing fast to slow groups for statistical analysis (the latter was only for display purposes in our figures, which we have now made clearer in the methods statistics section). 

Related to this, there is no mention of how different speeds were obtained. It seems that kangaroos hopped at a self-selected pace, thus it appears that not much variation was observed. I appreciate the difficulty of conducting these experiments in a controlled manner, but this doesn’t exempt the authors from providing the details of their approach.

Apologies, this was not clear in the manuscript. Kangaroos hopping at their preferred speed means we did not chase or startle them into high speeds to comply with ethics and enclosure limitations. Thus we did not record a wide range of speeds within the bounds of what kangaroos are capable of in the wild (up to 12 m/s). We have now removed the terminology “preferred speed” e.g. line 115. We have added additional information about the chosen hopping speeds into the results and methods, including an appendix figure (see above comment). (Line 381-382)

Some figures (Figure 2 for example) present means for one of three speeds, yet the speeds are not reported (except in the legend) nor how these bins were determined, nor how many trials or kangaroos fit in each bin. A similar comment applies to the mass categories. It would be more convincing if the authors plotted the main metrics vs. speed to illustrate the significant trends they are reporting.

Thank you for this comment. The bins are used only for display purposes and not within the statistical analysis. We have clarified this in the revised manuscript: “The data was grouped into body mass (small 17.6±2.96 kg, medium 21.5±0.74 kg, large 24.0±1.46 kg) and speed (slow 2.52±0.25 m s^-1, medium 3.11±0.16 m s^-1, fast 3.79±0.27 m s^-1) subsets for display purposes only”. (Line 495-497)

(2) The significance of the effects of mass is not clear. The introduction and abstract suggest that the paper is focused on the effect of speed, yet the effects of mass are reported throughout as well, without a clear understanding of the significance. This weakness is further exaggerated by the fact that the details of the subject masses are not reported.

Indeed, the primary aim of our study was to explore the influence of speed, given the uncoupling of energy from hopping speed in kangaroos. We included mass to ensure that the effects of speed were not driven by body mass (i.e.: that larger kangaroos hopped faster). Subject masses were reported in the first paragraph of the methods, albeit some were estimated as outlined in the same paragraph.

(3) The paper needs to be significantly re-written to better incorporate the methods into the results section. Since the results come before the methods, some of the methods must necessarily be described such that the study can be understood at some level without turning to the dedicated methods section. As written, it is very difficult to understand the basis of the approach, analysis, and metrics without turning to the methods.

The methods after the discussion is a requirement of the journal. We have incorporated some methods in the results where necessary but not too repetitive or disruptive, e.g. Fig. 1 caption, and specifying we are only analysing EMA for the ankle joint

Reviewing Editor (Recommendations For The Authors):

Below is a list of specific recommendations that the authors could address to improve the eLife assessment:

(1) Based on the data presented and the fact that metabolic energy was not measured, the authors should temper their conclusions and statements throughout the manuscript regarding the link between speed and metabolic energy savings. We recommend adding text to the discussion summarizing the strengths and limitations of the evidence provided and suggesting future steps to more conclusively answer this mystery.

There is a significant body of work linking metabolic energy savings to measured increases in tendon stress in macropods. However, the purpose of this paper was to address the unanswered questions about why tendon stress increases. We found that stress did not only increase due to GRF increasing with speed as expected, but also due to novel postural changes which decreased EMA. In the revised manuscript, we have tempered our conclusions to make it clearer that it is not just EMA affecting stress, and added limitations throughout the manuscript (see response to Rev 1). 

(2) To provide stronger evidence of a link between speed, mechanics, and metabolic savings the authors can consider estimating metabolic energy expenditure from their OpenSIM model. This is one suggestion, but the authors likely have other, possibly better ideas. Such a model should also be able to explain why the metabolic rate increases with speed during uphill hopping.

Extending the model to provide direct metabolic cost estimates will be the goal of a future paper, however the models does not have detailed muscle characteristics to do this in the formulation presented here. It would be a very large undertaking which is beyond the scope of the current manuscript. As per the comment above, the results of this paper are not reliant on metabolic performance. 

(3) The authors attempt to relate the newly quantified hopping biomechanics to previously published metabolic data. However, all reviewers agree that the logic in many instances is not clear or contradictory. Could one potential explanation be that at slow speeds, forces and tendon strain are small, and thus muscle fascicle work is high? Then, with faster speeds, even though the cost of generating isometric force increases, this is offset by the reduction in the metabolic cost of muscular work. The paper could provide stronger support for their hypotheses with a much clearer explanation of how the kinematics relate to the mechanics and ultimately energy savings.

In response to the reviewers comments, we have substantially modified the discussion to provide clearer rationale.

(4) The methods and the effort expended to collect these data are impressive, but there are a number of underlying assumptions made that undermine the conclusions. This is due partly to the methods used, but also the paper's incomplete description of their methods. We provide a few examples below:

It would be helpful if the authors could speak to the effect of the limited speeds tested and between-animal comparisons on the ability to draw strong conclusions from the present dataset. ·

Throughout the discussion, the authors highlight the relationship between EMA and speed. However, this is misleading since there was no significant effect of speed on EMA. Speed only affected the muscle moment arm, r. At minimum, this should be clarified and the effect on EMA not be overstated. Additionally, the resulting implications on their ability to confidently say something about the effect of speed on muscle stress should be discussed. 

We have now provided additional details, (see responses above) to these concerns. For instance, we added a supplementary figure showing the speed distribution per individual. The primary reviewer concern (that each kangaroo travelled at a single speed) was due to a miscommunication around the terminology “preferred” which has now been corrected. 

We now elaborate in the results why we are not very concerned that EMA is insignificant. The statistical insignificance of EMA is ultimately due to the insignificance of the direct measurement of R, however, we now better explain in the results why we believe that this statistical insignificance is due to error/noise of the measurement which is relatively large compared to the effect size. Indirect indications of how R may increase with speed (via ankle height from the ground) are statistically significant. Lines 177-185. 

We consider this worth reporting because, for instance, an 18% change in EMA will be undetectable by measurement, but corresponds to an 18% change in tendon stress which is measurable and physiologically significant (safety factor would decrease from 2 to 1.67).  We presented both significant and insignificant results for transparency. 

We have also discussed this within a revised limitations section of the manuscript (Line 311328). 

Reviewer #1 (Recommendations For The Authors):

Title: I would cut the first half of the title. At least hedge it a bit. "Clues" instead of "Unlocking the secrets".

We have revised the title to: “Postural adaptations may contribute to the unique locomotor energetics seen in hopping kangaroos”

In my comments, ... typically indicates a stylistic change suggested to the text.

Overall, the paper covers speed and size. Unfortunately, the authors were not 100% consistent in the order of presenting size then speed, or speed then size. Just choose one and stick with it.

We have attempted to keep the order of presenting size and speed consistent, however there are several cases where this would reduce the readability of the manuscript and so in some cases this may vary. 

One must admit that there is a lot of vertical scatter in almost all of the plots. I understand that these animals were not in a lab on a treadmill at a controlled speed and the animals wear fur coats so marker placements vary/move etc. But the spread is quite striking, e.g. Figure 5a the span at one speed is almost 10x. Can the authors address this somewhere? Limitations section?

The variation seen likely results from attempting to display data in a 2D format, when it is in fact the result of multiple variables, including speed, mass, stride frequency and subject specific lengths. Slight variations in these would be expected to produce some noise around the mean, and I think it’s important to consider this while showing the more dominant effects. 

In many locations in the manuscript, the term "work" is used, but rarely if ever specified that this is the work "per hop". The big question revolves around the rate of metabolic energy consumption (i.e. energy per time or average metabolic power), one must not forget that hop frequency changes somewhat across speed, so work per hop is not the final calculation.

Thank you for this comment. We have now explicitly stated work per hop in figure captions and in the results (line 208). The change in stride frequency at this range of speeds is very small, particularly compared to the variance in stride frequency (Suppl. Fig. 1d), which is consistent with other researchers who found that stride frequency was constant or near constant in macropods at analogous speeds (e.g. Dawson and Taylor 1973, Baudinette et al. 1987). 

Line 61 ....is likely related.

Added “likely” (line 59)

Line 86 I think the Allen reference is incomplete. Wasn't it in J Exp Biology?

Thank you. Changed. 

Line 122 ... at faster speeds and in larger individuals.

Changed: “We hypothesised that (i) the hindlimb would be more crouched at faster speeds, primarily due to the distal hindlimb joints (ankle and metatarsophalangeal), independent of changes with body mass” (Line 121-122).

Line 124 I found this confusing. Try to re-word so that you explain you mean more work done by the tendons and less by the ankle musculature.

Amended: “changes in moment arms resulting from the change in posture would contribute to the increase in tendon stress with speed, and may thereby contribute to energetic savings by increasing the amount of positive and negative work done by the ankle without requiring additional muscle work” (Line 123)

Line 129 hopefully "braking" not "breaking"!

Thank you. Fixed. (Line 130)

Line 129 specify fore-aft horizontal force.

Added "fore-aft" to "negative fore-aft horizontal component" (Line 130-131)

Line 130 add something like "of course" or "naturally" since if there is zero fore-aft force, the GRF vector of course must be vertical. 

Added "naturally" (Line 132)

Line 138 clarify that this section is all stance phase. I don't recall reading any swing phase data.

Changed to: "Kangaroo hindlimb stance phase kinematics varied…" (Line 141)

Line 143 and elsewhere. I found the use of dorsiflexion and plantarflexion confusing. In Figure 3, I see the ankle never flexing more than 90 degrees. So, the ankle joint is always in something of a flexed position, though of course it flexes and extends during contact. I urge the authors to simplify to flextion/extension and drop the plantar/dorsi.

We have edited this section to describe both movements as greater extension (plantarflexion). (Line 147). We have further clarified this in the figure caption for figure 3.  

Line 147 ...changes were…

Fixed, line 150

Line 155 I'm a bit confused here. Are the authors calculating some sort of overall EMA or are they saying all of the individual joint EMAs all decreased?

Thank you, we clarified that it is at the ankle. Line 158

Line 158 since kangaroos hop and are thus positioned high and low throughout the stance phase, try to avoid using "high" and "low" for describing variables, e.g. GRF or other variables. Just use "greater/greatest" etc.

Thanks for this suggestion. We have changed "higher" into "greater" where appropriate throughout the manuscript e.g. line 161

Lines 162 and 168 same comment here about "r" and "R". Do you mean ankle or all joints?

Clarified that it is the gastrocnemius and plantaris r, and the R to the ankle. (Lines 164-165)

Line 173 really, ankle height?

Added: ankle height is "vertical distance from the ground". Line 177

Line 177 is this just the ankle r?

Added "of the ankle" line 158 and “Achilles” line 187 

Line 183 same idea, which tendon/tendons are you talking about here?

Added "Achilles" to be more clear (Line 187)

Line 195 substitute "converted" for "transferred".

Done (Line 210)

Line 223 why so vague? i.e. why use "may"? Believe in your data. ...stress was also modulated by changes....

Changed "may" to "is"

Line 229 smaller ankle EMA (especially since you earlier talked about ankle "height").

Changed “lower” to “smaller” Line 254

Line 2236 ...and return elastic energy…

Added "elastic" line 262

Line 244 IMPORTANT: Need to explain this better! I think you are saying that the net work at the ankle is staying the same across speed, BUT it is the tendons that are storing and returning that work, it's not that the muscles are doing a lot of negative/positive work.

Changed: “The consistent net work observed among all speeds suggests the ankle extensor muscle-tendon units are performing similar amounts of ankle work independent of speed, which would predominantly be done by the tendon.” Line 270-272)

Line 258-261 I think here is where you are over-selling the data/story. Although you do say "a" mechanism (and not "the" mechanism, you still need to deal with the cost of generating more force and generating that force faster.

We removed this sentence and replaced it with a discussion of the cost of generating force hypothesis, and alternative scenarios for the how force and metabolics could be uncoupled. 

Line 278 "the" tendon? Which tendon?

Added "Achilles"

Line 289. I don't think one can project into the past.

Changed “projected” to "estimated"

Line 303 no problem, but I've never seen a paper in biology where the authors admit they don't know what species they were studying!

Can’t be helped unfortunately. It is an old dataset and there aren’t photos of every kangaroo. Fortunately, from the grey and red kangaroos we can distinguish between, we know there are no discernible species effects on the data. 

Lines 304-306 I'm not clear here. Did you use vertical impulse (and aerial time) to calculate body weight? Or did you somehow use the braking/propulsive impulse to calculate mass? I would have just put some apples on the force plate and waited for them to stop for a snack.

Stationary weights were recorded for some kangaroos which did stand on the force plate long enough, but unfortunately not all of them were willing to do so. In those cases, yes, we used impulse from steady-speed trials to estimate mass. We cross-checked by estimated mass from segment lengths (as size and mass are correlated). This is outlined in the first paragraph of the methods.

Lines 367 & 401 When you use the word "scaled" do you mean you assumed geometric similarity?

No, rather than geometric scaling, we allowed scaling to individual dimensions by using the markers at midstance for measurements. We have amended the paragraph to clarify that the shape of the kangaroo changes and that mass distribution was preserved during the shape change (line 441-446) 

Lines 381-82 specify "joint work"

Added "joint work"  (Line 457)

Figure 1 is gorgeous. Why not add the CF equation to the left panel of the caption?

We decided to keep the information in the figure caption. “Total leg length was calculated as the sum of the segment lengths (solid black lines) in the hindlimb and compared to the pelvisto-toe distance (dashed line) to calculate the crouch factor”

Figure 2 specify Horizontal fore-aft.

Done

Figure 3g I'd prefer the same Min. Max Flexion vertical axis labels as you use for hip & knee.

While we appreciate the reviewer trying to increase the clarity of this figure, we have left it as plantar/dorsi flexion since these are recognised biomechanical terms. To avoid confusion, we have further defined these in the figure caption “For (f-g), increased plantarflexion represents a decrease in joint flexion, while increased dorsiflexion represents increased flexion of the joint.”

Figure 4. I like it and I think that you scaled all panels the same, i.e. 400 W is represented by the same vertical distance in all panels. But if that's true, please state so in the Caption. It's remarkable how little work occurs at the hip and knee despite the relatively huge muscles there.

Is it true that the y axes are all at the same scale. We have added this to the caption. 

Figure 5 Caption should specify "work per hop".

Added

Figure 7 is another beauty.

Thank you!

Supplementary Figure 3 is this all ANKLE? Please specify.

Clarified that it is the gastrocnemius and plantaris r, and the R to the ankle.

Reviewer #2 (Recommendations For The Authors):

To 'unlock the secrets of kangaroo locomotor energetics' I expected the authors to measure the secretive outcome variable, metabolic rate using laboratory measures. Rather, the authors relied on reviewing historic metabolic data and collecting biomechanics data across different animals, which limits the conclusions of this manuscript.

We have revised to the title to make it clearer that we are investigating a subset of the energetics problem, specifically posture. “Postural adaptations may contribute to the unique locomotor energetics seen in hopping kangaroos.” We have also substantially modified the discussion to temper the conclusions from the paper. 

After reading the hypothesis, why do the authors hypothesize about joint flexion and not EMA? Because the following hypothesis discusses the implications of moment arms on tendon stress, EMA predictions are more relevant (and much more discussed throughout the manuscript).

Ankle and MTP angles are the primary drivers of changes in r, R & thus, EMA. We used a two part hypothesis to capture this. We have rephased the hypotheses: “We hypothesised that (i) the hindlimb would be more crouched at faster speeds, primarily due to the distal hindlimb joints (ankle and metatarsophalangeal), independent of changes with body mass, and (ii) changes in moment arms resulting from the change in posture would contribute to the increase in tendon stress with speed, and may thereby contribute to energetic savings by increasing the amount of positive and negative work done by the ankle without requiring additional muscle work.”

If there were no detectable effects of speed on EMA, are kangaroos mechanically like other animals (Biewener Science 89 & JAP 04) who don't vary EMA across speeds? Despite no detectible effects, the authors state [lines 228-229] "we found larger and faster kangaroos were more crouched, leading to lower ankle EMA". Can the authors explain this inconsistency? Lines 236 "Kangaroos appear to use changes in posture and EMA". I interpret the paper as EMA does not change across speed.

Apologies, we did not sufficiently explain this originally. We now explain in the results our reasoning behind our belief that EMA and R may change with speed. “If we consider the nonsignificant relationship between R (and EMA) and speed to indicate that there is no change in R, then it conflicts with the ankle height and CoP result. Taking both into account, we think it is more likely that there is a small, but important, change in R, rather than no change in R with speed. It may be undetectable because we expect small effect sizes compared to the measurement range and measurement error (Suppl. Fig. 3h), or be obscured by a similar change in R with body mass. R is highly dependent on the length of the metatarsal segment, which is longer in larger kangaroos (1 kg BM corresponded to ~1% longer segment, P<0.001, R²=0.449). If R does indeed increase with speed, both R and r will tend to decrease EMA at faster speeds.” (Line 177-185)

Lines 335-339: "We assumed the force was applied along phalanx IV and that there was no medial or lateral movement of the centre of pressure (CoP)". I'm confused, did the authors not measure CoP location with respect to the kangaroo limb? If not, this simple estimation undermines primary results (EMA analyses).

We have changed "The anterior or posterior movement of the CoP was recorded by the force plate" to read: "The fore-aft movement of the CoP was recorded by the force plate within the motion capture coordinate system" (Line 406-407) and added more justification for fixing the CoP movement in the other axis: “It was necessary to assume the CoP was fixed in the mediallateral axis because when two feet land on the force plate, the lateral forces on each foot are not recorded, and indeed cancel if the forces are symmetrical (i.e. if the kangaroo is hopping in a straight path and one foot is not in front of the other). We only used symmetrical trials to ensure reliable measures of the anterior-posterior movement of the CoP.” (Line 408-413)

The introduction makes many assertions about the generalities of locomotion and the relationship between mechanics and energetics. I'm afraid that the authors are selectively choosing references without thoroughly evaluating alternative theories. For example, Taylor, Kram, & others have multiple papers suggesting that decreasing EMA and increasing muscle force (and active muscle volume) increase metabolic costs during terrestrial locomotion. Rather, the authors suggest that decreasing EMA and increasingly high muscle force at faster speeds don't affect energetics unless muscle work increases substantially (paragraph 2)? If I am following correctly, does this theory conflict with active muscle volume ideas that are peppered throughout this manuscript?

Yes, as you point out, the same mechanism does lead to different results in kangaroos vs humans, for instance, but this is not a contradiction. In all species, decreasing EMA will result in an increase in muscle force due to less efficient leverage (i.e. lower EMA) of the muscles, and the muscle-tendon unit will be required to produce more force to balance the joint moment. As a consequence, human muscles activate a greater volume in order for the muscle-tendon unit to increase muscle work and produce enough force. We are proposing that in kangaroos, the increase in work is done by the achilles tendon rather than the muscles. Previous research suggests that macropod ankle muscles contract isometrically or that the fibres do not shorten more at faster speeds i.e. muscle work does not increase with speed. Instead, the additional force seems to come from the tendon storing and subsequently returning more strain energy (indicated by higher stress). We found that the increase in tendon stress comes from higher ground force at faster speeds, and from it adopting a more crouched posture which increases the tendons’ stresses compared to an upright posture for a given speed (think of this as increasing the tendon’s stress capacity). We have substantially revised the discussion to highlight this.

Similarly, does increased gross or net tendon mechanical energy storage & return improve hopping energetics? Would more tendon stress and strain energy storage with a given hysteresis value also dissipate more mechanical energy, requiring leg muscles to produce more net work? Does net or gross muscle work drive metabolic energy consumption?

Based on the cost of generating force hypothesis, we think that gross muscle work would be linked to driving metabolic energy consumption. Our idea here is that the total body work is a product of the work done by the tendon and the muscle combined. If the tendon has the potential to do more work, then the total work can increase without muscle work needing to increase.

The results interpret speed effects on biomechanics, but each kangaroo was only collected at 1 speed. Are inter-animal comparisons enough to satisfy this investigation?

We have added a figure (Suppl Fig 9) to demonstrate the distribution of speed and number of trials per kangaroo. We have also removed "preferred" from the manuscript as this seems to cause confusion. Most kangaroos travelled at a range of “casual” speeds.

Abstract: Can the authors more fully connect the concept of tendon stress and low metabolic rates during hopping across speeds? Surely, tendon mechanics don't directly drive the metabolic cost of hopping, but they affect muscle mechanics to affect energetics.

Amended to: " This phenomenon may be related to greater elastic energy savings due to increasing tendon stress; however, the mechanisms which enable the rise in stress, without additional muscle work remain poorly understood." (Lines 25-27).

The topic sentence in lines 61-63 may be misleading. The ensuing paragraph does not substantiate the topic sentence stating that ankle MTUs decouple speeds and energetics.

We added "likely" to soften the statement. (Line 59)

Lines 84-86: In humans, does more limb flexion and worse EMA necessitate greater active muscle volume? What about muscle contractile dynamics - See recent papers by Sawicki & colleagues that include Hill-type muscle mechanics in active muscle volume estimates.

Added: “Smaller EMA requires greater muscle force to produce a given force on the ground, thereby demanding a greater volume of active muscle, and presumably greater metabolic rates than larger EMA for the same physiology”. (Line 80-82)

Lines 106: can you give the context of what normal tendon safety factors are?

Good idea. Added: "far lower than the typical safety factor of four to eight for mammalian tendons (Ker et al. 1988)." Line 106-107

I thought EMA was relatively stable across speeds as per Biewener [Science & JAP '04]. However the authors gave an example of an elephant to suggest that it is typically inversely related to speed. Can the authors please explain the disconnect and the most appropriate explanation in this paragraph?

Knee EMA in particular changed with speed in Biewener 2004. What is “typical” probably depends on the group of animals studied; e.g., cursorial quadrupedal mammals generally seem to maintain constant EMA, but other groups do not.

These cases are presented to show a range of consequences for changing EMA (usually with mass, but sometimes with speed). We have made several adjustments to the paragraph to make this clearer. Lines 85-93.

The results depend on the modeled internal moment arm (r). How confident are the authors in their little r prediction? Considering complications of joint mechanics in vivo including muscle bulging. Holzer et al. '20 Sci Rep demonstrated that different models of the human Achilles tendon moment arm predict vastly different relationships between the moment arm and joint angle.

Our values for r and EMA closely align with previous papers which measured/calculate these values in kangaroos, such as Kram 1998, and thus we are confident in our interpretation.  

This is a misleading results sentence: Small decreases in EMA correspond to a nontrivial increase in tendon stress, for instance, reducing EMA from 0.242 (mean minimum EMA of the slow group) to 0.206 (mean minimum EMA of the fast group) was associated with an ~18% increase in tendon stress. The authors could alternatively say that a ~15% decrease in EMA was associated with an ~18% increase in tendon stress, which seems pretty comparable.

Thank you for pointing this out, it is important that it is made clearer. Although the change in relative magnitude is approximately the same (as it should be), this does not detract from the importance. The "small decrease in EMA" is referring to the absolute values, particularly in respect to the measurement error/noise. The difference is small enough to have been undetectable with other methods used in previous studies. We have amended the sentence to clarify this.

It now reads: “Subtle decreases in EMA which may have been undetected in previous studies correspond to discernible increases in tendon stress. For instance, reducing EMA from 0.242 (mean minimum EMA of the slow group) to 0.206 (mean minimum EMA of the fast group) was associated with an increase in tendon stress from ~50 MPa to ~60 MPa, decreasing safety factor from 2 to 1.67 (where 1 indicates failure), which is both measurable and physiologically significant.” (Line 195-200)

Lines 243-245: "The consistent net work observed among all speeds suggests the ankle extensors are performing similar amounts of ankle work independent of speed." If this is true, and presumably there is greater limb work performed on the center of mass at faster speeds (Donelan, Kram, Kuo), do more proximal leg joints increase work and energy consumption at faster speeds?

The skin over the proximal leg joints (knee and hip) moves too much to get reliable measures of EMA from the ratio of moment arms. This will be pursued in future work when all muscles are incorporated in the model so knee and hip EMA can be determined from muscle force.

We have added limitations and considerations paragraph to the manuscript: “Finally, we did not determine whether the EMA of proximal hindlimb joints (which are more difficult to track via surface motion capture markers) remained constant with speed. Although the hip and knee contribute substantially less work than the ankle joint (Fig. 4), the majority of kangaroo skeletal muscle is located around these proximal joints. A change in EMA at the hip or knee could influence a larger muscle mass than at the ankle, potentially counteracting or enhancing energy savings in the ankle extensor muscle-tendon units. Further research is needed to understand how posture and muscles throughout the whole body contribute to kangaroo energetics.” (Line 321-328)

Lines 245-246: "Previous studies using sonomicrometry have shown that the muscles of tammar wallabies do not shorten considerably during hops, but rather act near-isometrically as a strut" Which muscles? All muscles? Extensors at a single joint?

Added "gastrocnemius and plantaris" Line 164-165

Lines 249-254: "The cost of generating force hypothesis suggests that faster movement speeds require greater rates of muscle force development, and in turn greater cross-bridge cycling rates, driving up metabolic costs (Taylor et al. 1980, Kram and Taylor 1990). The ability for the ankle extensor muscle fibres to remain isometric and produce similar amounts of work at all speeds may help explain why hopping macropods do not follow the energetic trends observed in quadrupedal species." These sentences confuse me. Kram & Taylor's cost of force-generating hypothesis assumes that producing the same average force over shorter contact times increases metabolic rate. How does 'similar muscle work' across all speeds explain the ability of macropods to use unique energetic trends in the cost of force-generating hypothesis context?

Thank you for highlighting this confusion. We have substantially revised the discussion clarify where the mechanisms presented deviate from the cost of generating force hypothesis. Lines 270-309

Reviewer #3 (Recommendations For The Authors):

In addition to the points described in the public review, I have additional, related, specific comments:

(1) Results: Please refer to the hypotheses in the results, and relate the the findings back to the hypotheses.

We now relate the findings back to the hypotheses 

Line 142 “In partial support of hypothesis (i), greater masses and faster speeds were associated with more crouched hindlimb postures (Fig. 3a,c).”.

Lines 205-206: “The increase in tendon stress with speed, facilitated in part by the change in moment arms by the shift in posture, may explain changes in ankle work (c.f. Hypothesis (ii)).” 

(2) Results: please provide the main statistical results either in-line or in a table in the main text.

We (the co-authors) have discussed this at length, and have agreed that the manuscript is far more readable in the format whereby most statistics lie within the supplementary tables, otherwise a reader is met with a wall of statistics. We only include values in the main text when the magnitude is relevant to the arguments presented in the results and discussion.

(3) Line 140: Describe how 'crouched' was defined.

We have now added a brief definition of ‘Crouch factor’ after the figure caption. (Line 143) (Fig. 3a,c; where crouch factor is the ratio of total limb length to pelvis to toe distance).

(4) Line 162: This seems to be a main finding and should be a figure in the main text not supplemental. Additionally, Supplementary Figures 3a and b do not show this finding convincingly There should be a figure plotting r vs speed and r vs mass.

The combination of r and R are represented in the EMA plot in the main text. The r and R plots are relegated to the supplementary because the main text is already very crowded.  Thank you for the suggestion for the figure plotting r and R versus speed, this is now included as Suppl. Fig. 3h

(5) Line 166: Supplementary Figure 3g does not show the range of dorsiflexion angles as a function of speed. It shows r vs dorsiflexion angle. Please correct.

Thanks for noticing this, it was supposed to reference Fig 3g rather than Suppl Fig 3g in the sentence regarding speed. We have fixed this, Line 170. 

We had added a reference to Suppl Fig 3 on Line 169 as this shows where the peak in r with ankle angle occurs (114.4 degrees).

(6) Line 184: Where are the statistical results for this statement?

The relationship between stress and EMA does not appear to be linear, thus we only present R^{^}2 for the power relationship rather than a p-value. 

(7) Line 192: The authors should explain how joint work and power relate/support the overall hypotheses. This section also refers to Figures 4 and 5 even though Figures 6 and 7 have already been described. Please reorganize.

We have added a sentence at the end of the work and power section to mention hypothesis (ii) and lead into the discussion where it is elaborated upon. 

“The increase in positive and negative ankle work may be due to the increase in tendon stress rather than additional muscle work.” Line 219-220 We have rearranged the figure order.

(8) The statistics are not reported in the main text, but in the supplementary tables. If a result is reported in the main text, please report either in-line or with a table in the main text.

We leave most statistics in the supplementary tables to preserve the readability of the manuscript. We only include values in the main text when the magnitude is relevant to the arguments raised in the results and discussion.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

This is a contribution to the field of developmental bioelectricity. How do changes of resting potential at the cell membrane affect downstream processes? Zhou et al. reported in 2015 that phosphatidylserine and K-Ras cluster upon plasma membrane depolarization and that voltage-dependent ERK activation occurs when constitutively active K-RasG12V mutants are overexpressed. In this paper, the authors advance the knowledge of this phenomenon by showing that membrane depolarization up-regulates mitosis and that this process is dependent on voltage-dependent activation of ERK. ERK activity's voltage-dependence is derived from changes in the dynamics of phosphatidylserine in the plasma membrane and not by extracellular calcium dynamics. This paper reports an interesting and important finding. It is somewhat derivative of Zhou et al., 2015. (https://www.science.org/doi/full/10.1126/science.aaa5619). The main novelty seems to be that they find quantitatively different conclusions upon conducting similar experiments, albeit with a different cell line (U2OS) than those used by Zhou et al. Sasaki et al. do show that increased K+ levels increase proliferation, which Zhou et al. did not look at. The data presented in this paper are a useful contribution to a field often lacking such data.

Strengths:

Bioelectricity is an important field for areas of cell, developmental, and evolutionary biology, as well as for biomedicine. Confirmation of ERK as a transduction mechanism and a characterization of the molecular details involved in the control of cell proliferation are interesting and impactful.

Weaknesses:

The authors lean heavily on the assumption that the Nernst equation is an accurate predictor of membrane potential based on K+ level. This is a large oversimplification that undermines the author's conclusions, most glaringly in Figure 2C. The author's conclusions should be weakened to reflect that the activity of voltage gated ion channels and homeostatic compensation are unaccounted for.

We appreciate the reviewer’s thoughtful comment regarding our reliance on the Nernst equation to estimate membrane potential. We agree that the Nernst equation is a simplification and does not account for the activity of other ions, voltage-gated channels, or homeostatic compensation mechanisms. To address this concern, we conducted electrophysiological experiments in which the membrane potential was directly controlled using the perforated patch-clamp technique (Fig. 3). Under these conditions, we also monitored the membrane potential and confirmed that there was negligible drift within 20 minutes of perfusion with 145 mM K^⁺ (only a 1–5 mV change). These results suggest that the influence of voltage-gated channels and homeostatic compensation is minimal in our experimental setup. We revised the manuscript to clarify these limitations and to present our conclusions more cautiously in light of this point.

“A potential limitation of extracellular K^⁺-based approaches is their reliance on the Nernst equation to estimate membrane potential, which oversimplifies the actual situation by neglecting voltage-gated ion channel activity and compensatory mechanisms. To directly address this concern, we measured membrane potential using the perforated patch-clamp technique and confirmed that the potential was stable during perfusion with 145 mM K^⁺ (only a 1–5 mV drift within 20 min). Moreover, we used a voltage clamp to precisely control the membrane potential and demonstrated that ERK activity was directly regulated by the voltage itself, excluding the influence of other secondary factors. An additional strength of electrophysiology is its ability to examine the effects of repolarization, which is difficult to assess with conventional perfusion-based methods owing to slow solution exchange.”

There are grammatical tense errors are made throughout the paper (ex line 99 "This kinetics should be these kinetics")

We thank the reviewer for pointing out the grammatical errors. We carefully revised the entire manuscript.

Line 71: Zhou et al. use BHK, N2A, PSA-3 cells, this paper uses U2OS (osteosarcoma) cells. Could that explain the differences in bioelectric properties that they describe? In general, there should be more discussion of the choice of cell line. Why were U2OS cells chosen? What are the implications of the fact that these are cancer cells, and bone cancer cells in particular? Does this paper provide specific insights for bone cancers? And crucially, how applicable are findings from these cells to other contexts?

We thank the reviewer for this valuable comment regarding the choice of cell line. We selected U2OS cells primarily because they are well suited for live-cell FRET imaging. We did not use BHK, N2A, or PSA-3 cells, and therefore it is difficult for us to provide a clear comparison with the specific bioelectric properties reported in Zhou et al. Nevertheless, we agree that cancer cell lines, including U2OS, may exhibit bioelectric properties that differ from those of non-cancerous cells. While this could be a potential limitation, we are inclined to consider voltage-dependent ERK activation to be a fundamental and generalizable phenomenon, not restricted to osteosarcoma cells. The key components of this pathway—phosphatidylserine, Ras, MAPK (including ERK)—are expressed in essentially all mammalian cells. In support of our view, we observed voltage-dependent ERK activation not only in U2OS cells but also in HeLa, HEK293, and A431 cells. These results strongly suggest that the mechanism we describe is not cell-type specific but rather a universal feature of mammalian cells. In the revised Discussion, we expanded our rationale to choose U2OS cells, while addressing the potential implications of using a cancer-derived cell line. 

“In this study, we primarily used U2OS cells because their flat morphology makes them suitable for live-cell FRET imaging. Although cancer cell lines, including U2OS, may display bioelectric properties that differ from those of noncancerous cells, our findings raise the possibility that voltage-dependent ERK activation is a fundamental and broadly applicable phenomenon rather than a feature specific to osteosarcoma cells. This conclusion is supported by the fact that essential components of this pathway, namely phosphatidylserine, Ras, and MAPK (including ERK), are ubiquitously expressed in mammalian cells. Consistent with this finding, we observed voltage-dependent ERK activation across multiple cell lines: U2OS, HeLa, HEK293, and A431 cells (Fig.S2). These observations indicate that the mechanism we describe is not cell-type-restricted, but rather a universal property of mammalian cells.”

Line 115: The authors use EGF to calibrate 'maximal' ERK stimulation. Is this level near saturation? Either way is fine, but it would be useful to clarify.

We thank the reviewer for raising this important point. The YFP/CFP ratio obtained after EGF stimulation is generally considered to represent saturation levels detectable by EKAREV imaging. However, we acknowledge that it remains uncertain whether 10 ng/mL EGF induces the absolute maximal ERK activity in all contexts. To clarify this point, we revised the manuscript (result) text as follows:

“To normalize variation among cells, cells were stimulated with EGF (10 ng/mL) at the end of the experiment, which presumably yielded a near-saturated YFP/CFP value (ERK activity). This value was used to determine the maximum ERK activity in each cell”

Line 121: Starting line 121 the authors say "Of note, U2OS cells expressed wild-type K-Ras but not an active mutant of K-Ras, which means voltage dependent ERK activation occurs not only in tumor cells but also in normal cells". Given that U2OS cells are bone sarcoma cells, is it appropriate to refer to these as 'normal' cells in contrast to 'tumor' cells?

We thank the reviewer for pointing this out. We agree that it is not appropriate to contrast U2OS cells with “normal” cells, since they are sarcoma-derived. To address this point, we revised the sentence to weaken the claim and avoid the misleading terminology.

“Importantly, as U2OS cells express wild-type K-Ras rather than an oncogenic mutant (16), our results raise the possibility that voltage-dependent ERK activation may also occur in non-transformed cells.”

Line 101: These normalizations seem reasonable, the conclusions sufficiently supported and the requisite assumptions clearly presented. Because the dish-to-dish and cell-to-cell variation may reflect biologically relevant phenomena it would be ideal if non-normalized data could be added in supplemental data where feasible.

We thank the reviewer for this helpful suggestion. As recommended, we added representative non-normalized data in the Supplemental Figure S1, which illustrates the non-normalized variation across cells and dishes.

Figure 2C is listed as Figure 2D in the text

There is no Figure 2F (Referenced in line 148)

We thank the reviewer for pointing out these errors. The incorrect figure citations were corrected.

Reviewer #2 (Public review):

Sasaki et al. use a combination of live-cell biosensors and patch-clamp electrophysiology to investigate the effect of membrane potential on the ERK MAPK signaling pathway, and probe associated effects on proliferation. This is an effect that has long been proposed, but a convincing demonstration has remained elusive, because it is difficult to perturb membrane potential without disturbing other aspects of cell physiology in complex ways. The time-resolved measurements here are a nice contribution to this question, and the perforated patch clamp experiments with an ERK biosensor are fantastic - they come closer to addressing the above difficulty of perturbing voltage than any prior work. It would have been difficult to obtain these observations with any other combination of tools.

However, there are still some concerns as detailed in specific comments below:

Specific comments:

(1) All the observations of ERK activation, by both high extracellular K+ and voltage clamp, could be explained by cell volume increase (more discussion in subsequent comments). There is a substantial literature on ERK activation by hypotonic cell swelling (e.g. https://doi.org/10.1042/bj3090013, https://doi.org/10.1002/j.1460-2075.1996.tb00938.x, among others). Here are some possible observations that could demonstrate that ERK activation by volume change is distinct from the effects reported here:

(i) Does hypotonic shock activate ERK in U2OS cells?

(ii) Can hypotonic shock activate ERK even after PS depletion, whereas extracellular K+ cannot?

(iii) Does high extracellular K+ change cell volume in U2OS cells, measured via an accurate method such as fluorescence exclusion microscopy?

(iv) It would be helpful to check the osmolality of all the extracellular solutions, even though they were nominally targeted to be iso-osmotic.

(2) Some more details about the experimental design and the results are needed from Figure 1:

(i) For how long are the cells serum-starved? From the Methods section, it seems like the G1 release in different K+ concentration is done without serum, is this correct? Is the prior thymidine treatment also performed in the absence of serum?

(ii) There is a question of whether depolarization constitutes a physiologically relevant mechanism to regulate proliferation, and how depolarization interacts with other extracellular signals that might be present in an in vivo context. Does depolarization only promote proliferation after extended serum starvation (in what is presumably a stressed cell state)? What fraction of total cells are observed to be mitotic (without normalization), and how does this compare to the proliferation of these cells growing in serum-supplemented media? Can K+ concentration tune proliferation rate even in serum-supplemented media?

(3) In Figure 2, there are some possible concerns with the perfusion experiment:

(i) Is the buffer static in the period before perfusion with high K+, or is it perfused? This is not clear from the Methods. If it is static, how does the ERK activity change when perfused with 5 mM K+? In other words, how much of the response is due to flow/media exchange versus change in K+ concentration?

(ii) Why do there appear to be population-average decreases in ERK activity in the period before perfusion with high K+ (especially in contrast to Fig. 3)? The imaging period does not seem frequent enough for photobleaching to be significant.

(4) Figure 3 contains important results on couplings between membrane potential and MAPK signaling. However, there are a few concerns:

(i) Does cell volume change upon voltage clamping? Previous authors have shown that depolarizing voltage clamp can cause cells to swell, at least in the whole-cell configuration: https://www.cell.com/biophysj/fulltext/S0006-3495(18)30441-7 . Could it be possible that the clamping protocol induces changes in ERK signaling due to changes in cell volume, and not by an independent mechanism?

(ii) Does the -80 mV clamp begin at time 0 minutes? If so, one might expect a transient decrease in sensor FRET ratio, depending on the original resting potential of the cells. Typical estimates for resting potential in HEK293 cells range from -40 mV to -15 mV, which would reach the range that induces an ERK response by depolarizing clamp in Fig. 3B. What are the resting potentials of the cells before they are clamped to -80 mV, and why do we not see this downward transient?

(5) The activation of ERK by perforated voltage clamp and by high extracellular K+ are each convincing, but it is unclear whether they need to act purely through the same mechanism - while additional extracellular K+ does depolarize the cell, it could also be affecting function of voltage-independent transporters and cell volume regulatory mechanisms on the timescales studied. To more strongly show this, the following should be done with the HEK cells where there is already voltage clamp data:

(i) Measure resting potential using the perforated patch in zero-current configuration in the high K+ medium. Ideally this should be done in the time window after high K+ addition where ERK activation is observed (10-20 minutes) to minimize the possibility of drift due to changes in transporter and channel activity due to post-translational regulation.

(ii) Measure YFP/CFP ratio of the HEK cells in the high K+ medium (in contrast to the U2OS cells from Fig. 2 where there is no patch data).

(iii) The assertion that high K+ is equivalent to changes in Vmem for ERK signaling would be supported if the YFP/CFP change from K+ addition is comparable to that induced by voltage clamp to the same potential. This would be particularly convincing if the experiment could be done with each of the 15 mM, 30 mM, and 145 mM conditions.

(6) Line 170: "ERK activity was reduced with a fast time course (within 1 minute) after repolarization to -80 mV." I don't see this in the data: in Fig. 3C, it looks like ERK remains elevated for > 10 min after the electrical stimulus has returned to -80 mV

Comments on revisions:

The authors have done a good job addressing the comments on the previous submission.

Reviewer #3 (Public review):

Summary:

This paper demonstrates that membrane depolarization induces a small increase in cell entry into mitosis. Based on previous work from another lab, the authors propose that ERK activation might be involved. They show convincingly using a combination of assays that ERK is activated by membrane depolarization. They show this is Ca2+ independent and is a result of activation of the whole K-Ras/ERK cascade which results from changed dynamics of phosphatidylserine in the plasma membrane that activates K-Ras. Although the activation of the Ras/ERK pathway by membrane depolarization is not new, linking it to an increase in cell proliferation is novel.

Strengths

A major strength of the study is the use of different techniques - live imaging with ERK reporters, as well as Western blotting to demonstrate ERK activation as well as different methods for inducing membrane depolarization. They also use a number of different cell lines. Via Western blotting the authors are also able to show that the whole MAPK cascade is activated.

Weaknesses

A weakness of the study is the data in Figure 1 showing that membrane depolarization results in an increase of cells entering mitosis. There are very few cells entering mitosis in their sample in any condition. This should be done with many more cells to increase the confidence in the results. The study also lacks a mechanistic link between ERK activation by membrane depolarization and increased cell proliferation.

The authors did achieve their aims with the caveat that the cell proliferation results could be strengthened. The results, for the most par,t support the conclusions.

This work suggests that alterations in membrane potential may have more physiological functions than action potential in the neural system as it has an effect on intracellular signalling and potentially cell proliferation.

In the revised manuscript, the authors have now addressed the issues with Figure 1, and the data presented are much clearer. They did also attempt to pinpoint when in the cell cycle ERK is having its activity, but unfortunately, this was not conclusive.

Reviewer #2 (Recommendations for the authors):

Small issues:

Fig. 1A. Please add a mark on the timeline showing when the K+ concentration is changed. Also, please add a time axis that matches the time axis in (C), so readers can know when in C the medium was changed.

1B caption: unclear what "the images were 20 min before and after cytokinesis" means, given that the images go from -30 min to +20 min. Maybe the authors mean, "the indicated times are measured relative to cytokinesis."

Thank you for bringing these points to our attention that can confuse readers. We revised the figure legend.

Line 214: nonoclusters --> nanoclusters

Line 475: 10 mm -> 10 ¥mum

Corrected.

Pero Sócrates tuvo razón en algo: la escritura sí atrofió nuestra memoria. No la de todos, por supuesto, pero sin duda relegó el acto de recordar a un segundo plano, tanto individual como colectivamente.

Me parece significativa esta frase porque hace una clara referencia a cómo la IA, hasta cierto punto, nos vuelve esclavos y perezosos. Con esto no me refiero a que la IA sea mala (como se menciona en el texto), sino que me llama la atención la manera en que, tanto ahora como antes, las personas prefieren un método más sencillo que uno complicado para lograr el objetivo que tienen en mente. Antes se trataba de recordar, lo cual se facilitó con la escritura; ahora, en cambio, hablamos de tareas más cotidianas: escribir, buscar la solución a un problema, pedir consejos, tomar decisiones, etc.

Como se menciona en la frase, no es una cuestión de bueno o malo, sino de que, como seres humanos, solemos buscar la facilidad en nuestras vidas. Inevitablemente, esto ha ocurrido, ocurre y seguirá ocurriendo a medida que sigamos evolucionando como sociedad.

Pero, a cambio, la escritura nos abrió la posibilidad de conocer mucho más allá de lo que puede guardar una memoria humana individual.

Esta frase es otra que me llamó mucho la atención debido a que refleja bien la realidad que tenemos en este momento con la IA. Si, es criticada por muchos, pero al mismo tiempo nos abrió puertas y caminos que antes no éramos capaces de visualizar.

La IA es una herramienta que debe usarse con responsabilidad y cabeza para no volvernos dependientes, pero reitero, no es que sea algo malo, solo debe tomarse como lo que es: una herramienta en la que uno puede apoyarse.

Pero Sócrates tuvo razón en algo: la escritura sí atrofió nuestra memoria. No la de todos, por supuesto, pero sin duda relegó el acto de recordar a un segundo plano, tanto individualmente, como colectivamente

Me parece significativa esta frase porque hace una clara referencia a cómo la IA, hasta cierto punto, nos vuelve esclavos y perezosos. Con esto no me refiero a que la IA sea mala (como se menciona en el texto), sino que me llama la atención la manera en que, tanto ahora como antes, las personas prefieren un método más sencillo que uno complicado para lograr el objetivo que tienen en mente. Antes se trataba de recordar, lo cual se facilitó con la escritura; ahora, en cambio, hablamos de tareas más cotidianas: escribir, buscar la solución a un problema, pedir consejos, tomar decisiones, etc.

Como se menciona en la frase, no es una cuestión de bueno o malo, sino de que, como seres humanos, solemos buscar la facilidad en nuestras vidas. Inevitablemente, esto ha ocurrido, ocurre y seguirá ocurriendo a medida que sigamos evolucionando como sociedad.

• Planning : what your are going to do
• Budget : How you are going to to it, quantifying the plan
• Control : planning + evaluation and feedback

seria benéfico aprofundar alguns aspetos práticos ligados à gestão da comunicação por e-mail. Por exemplo, o guia poderia incluir orientações sobre frequência de envio, boas práticas de design e segmentação, ou sobre a importância de políticas de consentimento e proteção de dados (em conformidade com a legislação europeia, como o RGPD).

Antes do quem somos, puxar DEPOIMENTOS (ocupando pouca altura)

• Colocar esteira lateral estilo slider minimalista para os depoimentos. Título: Veja o que clientes falam da Voob

• Logo depois colocar print de logos porem fazer slider de forma contínua e o titulo o escrito Credibilidade ficar colorido

Com apenas 1 áudio de distância, você tem tudo que precisa na palma da sua mão para vender mais: seu site de alta conversão, conectado com CRM simples e funcional.

Separar em 2 colunas sendo a da direita um design que humanize

Invés de ter a frase acima do headline colocar apenas BADGES de benefícios:

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(COLOCAR EMBAIXO DO SUBTITULO e alternando entre si para ocupar menos espaço)

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Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures. The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions. The manuscript has greatly improved after the revision.

Weaknesses:

Additional variance: In several experiments, a fixation cross preceded - at a variable interval - the onset of the background noise that aimed to reset the phase of an ongoing oscillation. There is the chance that the fixation cross also resets the phase, potentially adding variance to the data. In addition, the authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. There is good reason for doing so, but it means that correctly identified/discriminated targets will on average have a greater stimulus intensity. This may add variance to the data. These two aspects may potentially contribute to the observation of weak perceptual rhythmicity.

Figures: The text in Figures 4 and 6 is small. I think readers would benefit from a larger font size. Moreover, Figure 1A is not very intuitive. Perhaps it could be made clearer. The new Figure 5 was not discussed in the text. I wonder whether analyses with traditional t-tests could be placed in the supplements.

50% significant samples: The authors consider 50% of significant bootstrapped samples robust. For example: "This revealed that the above‐mentioned effects prevail for at least 50% of the simulated experiments, corroborating their robustness within the participant sample". Many of the effects have even lower than 50% of significant samples. It is a matter of opinion of what is robust or not, but I think combined with the overall variable nature of the effects in different frequency bands and ears etc. leaves more the impression that the effects are not very robust. I think the authors state it correctly in the last sentence of the first paragraph of the discussion: "At the same time the prevalence of significant effects in random samples of participants were mostly below 50%, raising questions as to the ubiquity of such effects." I think the authors should update the abstract in this regard to avoid that readers who only read the abstract get the wrong impression about the robustness of the effects. It is not clear to me if the same study (using the same conditions) was done in a different lab that the results would come out similarly to the results reported here.

Author response:

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

Reviewer #1 (Public review):

Summary:

This paper presents results from four independent experiments, each of which tests for rhythmicity in auditory perception. The authors report rhythmic fluctuations in discrimination performance at frequencies between 2 and 6 Hz. The exact frequency depends on the ear and experimental paradigm, although some frequencies seem to be more common than others.

Strengths:

The first sentence in the abstract describes the state of the art perfectly: "Numerous studies advocate for a rhythmic mode of perception; however, the evidence in the context of auditory perception remains inconsistent". This is precisely why the data from the present study is so valuable. This is probably the study with the highest sample size (total of > 100 in 4 experiments) in the field. The analysis is very thorough and transparent, due to the comparison of several statistical approaches and simulations of their sensitivity. Each of the experiments differs from the others in a clearly defined experimental parameter, and the authors test how this impacts auditory rhythmicity, measured in pitch discrimination performance (accuracy, sensitivity, bias) of a target presented at various delays after noise onset.

Weaknesses:

(1) The authors find that the frequency of auditory perception changes between experiments. I think they could exploit differences between experiments better to interpret and understand the obtained results. These differences are very well described in the Introduction, but don't seem to be used for the interpretation of results. For instance, what does it mean if perceptual frequency changes from between- to within-trial pitch discrimination? Why did the authors choose this experimental manipulation? Based on differences between experiments, is there any systematic pattern in the results that allows conclusions about the roles of different frequencies? I think the Discussion would benefit from an extension to cover this aspect.

We believe that interpreting these differences remains difficult and a precise, detailed (and possibly mechanistic) interpretation is beyond the goal of the present study. The main goal of this study was to explore the consistency and variability of effects across variations of the experimental design and samples of participants. Interpreting specific effects, e.g. at particular frequencies, would make sense mostly if differences between experiments have been confirmed in a separate reproduction. Still, we do provide specific arguments for why differences in the outcome between different experiments, e.g. with and without explicit trial initialization by the participants, could be expected. See lines 91ff in the introduction and 786ff in the discussion.

(2) The Results give the impression of clear-cut differences in relevant frequencies between experiments (e.g., 2 Hz in Experiment 1, 6 Hz in Exp 2, etc), but they might not be so different. For instance, a 6 Hz effect is also visible in Experiment 1, but it just does not reach conventional significance. The average across the three experiments is therefore very useful, and also seems to suggest that differences between experiments are not very pronounced (otherwise the average would not produce clear peaks in the spectrum). I suggest making this point clearer in the text.

We have revised the conclusions to note that the present data do not support clear cut differences between experiments. For this reason we also refrain from detailed interpretations of specific effects, as suggested by this reviewer in point 1 above.

(3) I struggle to understand the hypothesis that rhythmic sampling differs between ears. In most everyday scenarios, the same sounds arrive at both ears, and the time difference between the two is too small to play a role for the frequencies tested. If both ears operate at different frequencies, the effects of the rhythm on overall perception would then often cancel out. But if this is the case, why would the two ears have different rhythms to begin with? This could be described in more detail.

This hypothesis was not invented by us, but in essence put forward in previous work. The study by Ho et al. CurrBiol 2017 has reported rhythmic effects at different frequencies in the left and right ears, and we here tried to reproduce these effects. One could speculate about an ear-difference based on studies reporting a right-ear advantage in specific listening tasks, and the idea that different time scales of rhythmic brain activity may be specifically prevail in the left and right cortical hemispheres; hence it does not seem improbable that there could be rhythmic effects in both ears at different frequencies. We note this in the introduction, l. 65ff.

Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures.

The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions.

Weaknesses:

(1) Conceptional concerns:

The authors place their research in the context of a rhythmic mode of perception. They also discuss continuous vs rhythmic mode processing. Their study further follows a design that seems to be based on paradigms that assume a recent phase in neural oscillations that subsequently influence perception (e.g., Fiebelkorn et al.; Landau & Fries). In my view, these are different facets in the neural oscillation research space that require a bit more nuanced separation. Continuous mode processing is associated with vigilance tasks (work by Schroeder and Lakatos; reduction of low frequency oscillations and sustained gamma activity), whereas the authors of this study seem to link it to hearing tasks specifically (e.g., line 694). Rhythmic mode processing is associated with rhythmic stimulation by which neural oscillations entrain and influence perception (also, Schroeder and Lakatos; greater low-frequency fluctuations and more rhythmic gamma activity). The current study mirrors the continuous rather than the rhythmic mode (i.e., there was no rhythmic stimulation), but even the former seems not fully fitting, because trials are 1.8 s short and do not really reflect a vigilance task. Finally, previous paradigms on phase-resetting reflect more closely the design of the current study (i.e., different times of a target stimulus relative to the reset of an oscillation). This is the work by Fiebelkorn et al., Landau & Fries, and others, which do not seem to be cited here, which I find surprising. Moreover, the authors would want to discuss the role of the background noise in resetting the phase of an oscillation, and the role of the fixation cross also possibly resetting the phase of an oscillation. Regardless, the conceptional mixture of all these facets makes interpretations really challenging. The phase-reset nature of the paradigm is not (or not well) explained, and the discussion mixes the different concepts and approaches. I recommend that the authors frame their work more clearly in the context of these different concepts (affecting large portions of the manuscript).

Indeed, the paradigms used here and in many similar previous studies incorporate an aspect of phase-resetting, as the presentation of a background noisy may effectively reset ongoing auditory cortical processes. Studies trying to probe for rhythmicity in auditory perception in the absence any background noise have not shown any effect (Zoefel and Heil, 2013), perhaps because the necessary rhythmic processes along auditory pathways are only engaged when some sound is present. We now discuss these points, and also acknowledge the mentioned studies in the visual system; l. 57.

(2) Methodological concerns:

The authors use a relatively unorthodox approach to statistical testing. I understand that they try to capture and characterize the sensitivity of the different analysis approaches to rhythmic behavioral effects. However, it is a bit unclear what meaningful effects are in the study. For example, the bootstrapping approach that identifies the percentage of significant variations of sample selections is rather descriptive (Figures 5-7). The authors seem to suggest that 50% of the samples are meaningful (given the dashed line in the figure), even though this is rarely reached in any of the analyses. Perhaps >80% of samples should show a significant effect to be meaningful (at least to my subjective mind). To me, the low percentage rather suggests that there is not too much meaningful rhythmicity present. 

We note that there is no clear consensus on what fraction of experiments should be expected or how this way of quantifying effects should be precisely valued (l. 441ff). However, we now also clearly acknowledge in the discussion that the effective prevalence is not very high (l. 663).

I suggest that the authors also present more traditional, perhaps multi-level, analyses: Calculation of spectra, binning, or single-trial analysis for each participant and condition, and the respective calculation of the surrogate data analysis, and then comparison of the surrogate data to the original data on the second (participant) level using t-tests. I also thought the statistical approach undertaken here could have been a bit more clearly/didactically described as well.

We here realize that our description of the methods was possibly not fully clear. We do follow the strategy as suggested by this reviewer, but rather than comparing actual and surrogate data based on a parametric t-test, we compare these based on a non-parametric percentile-based approach. This has the advantage of not making specific (and possibly not-warranted) assumptions about the distribution of the data. We have revised the methods to clarify this, l. 332ff. 

The authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. In practice, this can be a disadvantage relative to keeping the intensity constant throughout, because, on average, correct trials will be associated with a higher intensity than incorrect trials, potentially making observations of perceptual rhythmicity more challenging. The authors would want to discuss this potential issue. Intensity adjustments could perhaps contribute to the observed rhythmicity effects. Perhaps the rhythmicity of the stimulus intensity could be analyzed as well. In any case, the adaptive procedure may add variance to the data.

We have added an analysis of task difficulty to the results (new section “Effects of adaptive task difficulty“) to address this. Overall we do not find systematic changes in task difficulty across participants for most of the experiments, but for sure one cannot rule out that this aspect of the design also affects the outcomes.  Importantly, we relied on an adaptive task difficulty to actually (or hopefully) reduce variance in the data, by keeping the task-difficulty around a certain level. Give the large number of trials collected, not using such an adaptive produce may result in performance levels around chance or near ceiling, which would make impossible to detect rhythmic variations in behavior. 

Additional methodological concerns relate to Figure 8. Figures 8A and C seem to indicate that a baseline correction for a very short time window was calculated (I could not find anything about this in the methods section). The data seem very variable and artificially constrained in the baseline time window. It was unclear what the reader might take from Figure 8.

This figure was intended mostly for illustration of the eye tracking data, but we agree that there is no specific key insight to be taken from this. We removed this. 

Motivation and discussion of eye-movement/pupillometry and motor activity: The dual task paradigm of Experiment 4 and the reasons for assessing eye metrics in the current study could have been better motivated. The experiment somehow does not fit in very well. There is recent evidence that eye movements decrease during effortful tasks (e.g., Contadini-Wright et al. 2023 J Neurosci; Herrmann & Ryan 2024 J Cog Neurosci), which appears to contradict the results presented in the current study. Moreover, by appealing to active sensing frameworks, the authors suggest that active movements can facilitate listening outcomes (line 677; they should provide a reference for this claim), but it is unclear how this would relate to eye movements. Certainly, a person may move their head closer to a sound source in the presence of competing sound to increase the signal-to-noise ratio, but this is not really the active movements that are measured here. A more detailed discussion may be important. The authors further frame the difference between Experiments 1 and 2 as being related to participants' motor activity. However, there are other factors that could explain differences between experiments. Self-paced trials give participants the opportunity to rest more (inter-trial durations were likely longer in Experiment 2), perhaps affecting attentional engagement. I think a more nuanced discussion may be warranted.

We expanded the motivation of why self-pacing trials may effectively alter how rhythmic processes affect perception, and now also allude to attention and expectation related effects (l. 786ff). Regarding eye movements we now discuss the results in the light of the previously mentioned studies, but again refrain from a very detailed and mechanistic interpretation (l. 782).

Discussion:

The main data in Figure 3 showed little rhythmicity. The authors seem to glance over this fact by simply stating that the same phase is not necessary for their statistical analysis. Previous work, however, showed rhythmicity in the across-participant average (e.g., Fiebelkorn's and similar work). Moreover, one would expect that some of the effects in the low-frequency band (e.g., 2-4 Hz) are somewhat similar across participants. Conduction delays in the auditory system are much smaller than the 0.25-0.5 s associated with 2-4 Hz. The authors would want to discuss why different participants would express so vastly different phases that the across-participant average does not show any rhythmicity, and what this would mean neurophysiologically.

We now discussion the assumptions and implications of similar or distinct phases of rhythmic processes within and between participants (l. 695ff). In particular we note that different origins of the underlying neurophysiological processes eventually may suggest that such assumptions are or a not warranted.  

An additional point that may require more nuanced discussion is related to the rhythmicity of response bias versus sensitivity. The authors could discuss what the rhythmicity of these different measures in different frequency bands means, with respect to underlying neural oscillations.

We expanded discussion to interpret what rhythmic changes in each of the behavioral metric could imply (l. 706ff).

Figures:

Much of the text in the figures seems really small. Perhaps the authors would want to ensure it is readable even for those with low vision abilities. Moreover, Figure 1A is not as intuitive as it could be and may perhaps be made clearer. I also suggest the authors discuss a bit more the potential monoaural vs binaural issues, because the perceptual rhythmicity is much slower than any conduction delays in the auditory system that could lead to interference.

We tried to improve the font sizes where possible, and discuss the potential monaural origins as suggested by other reviewers. 

Reviewer #3 (Public review):

Summary:

The finding of rhythmic activity in the brain has, for a long time, engendered the theory of rhythmic modes of perception, that humans might oscillate between improved and worse perception depending on states of our internal systems. However, experiments looking for such modes have resulted in conflicting findings, particularly in those where the stimulus itself is not rhythmic. This paper seeks to take a comprehensive look at the effect and various experimental parameters which might generate these competing findings: in particular, the presentation of the stimulus to one ear or the other, the relevance of motor involvement, attentional demands, and memory: each of which are revealed to effect the consistency of this rhythmicity.

The need the paper attempts to resolve is a critical one for the field. However, as presented, I remain unconvinced that the data would not be better interpreted as showing no consistent rhythmic mode effect. It lacks a conceptual framework to understand why effects might be consistent in each ear but at different frequencies and only for some tasks with slight variants, some affecting sensitivity and some affecting bias.

Strengths:

The paper is strong in its experimental protocol and its comprehensive analysis, which seeks to compare effects across several analysis types and slight experiment changes to investigate which parameters could affect the presence or absence of an effect of rhythmicity. The prescribed nature of its hypotheses and its manner of setting out to test them is very clear, which allows for a straightforward assessment of its results

Weaknesses:

There is a weakness throughout the paper in terms of establishing a conceptual framework both for the source of "rhythmic modes" and for the interpretation of the results. Before understanding the data on this matter, it would be useful to discuss why one would posit such a theory to begin with. From a perceptual side, rhythmic modes of processing in the absence of rhythmic stimuli would not appear to provide any benefit to processing. From a biological or homeostatic argument, it's unclear why we would expect such fluctuations to occur in such a narrow-band way when neither the stimulus nor the neurobiological circuits require it.

We believe that the framework for why there may be rhythmic activity along auditory pathways that shapes behavioral outcomes has been laid out in many previous studies, prominently here (Schroeder et al., 2008; Schroeder and Lakatos, 2009; Obleser and Kayser, 2019). Many of the relevant studies are cited in the introduction, which is already rather long given the many points covered in this study. 

Secondly, for the analysis to detect a "rhythmic mode", it must assume that the phase of fluctuations across an experiment (i.e., whether fluctuations are in an up-state or down-state at onset) is constant at stimulus onset, whereas most oscillations do not have such a total phase-reset as a result of input. Therefore, some theoretical positing of what kind of mechanism could generate this fluctuation is critical toward understanding whether the analysis is well-suited to the studied mechanism.

In line with this and previous comments (by reviewer 2) we have expanded the discussion to consider the issue of phase alignment (l. 695ff). 

Thirdly, an interpretation of why we should expect left and right ears to have distinct frequency ranges of fluctuations is required. There are a large number of statistical tests in this paper, and it's not clear how multiple comparisons are controlled for, apart from experiment 4 (which specifies B&H false discovery rate). As such, one critical method to identify whether the results are not the result of noise or sample-specific biases is the plausibility of the finding. On its face, maintaining distinct frequencies of perception in each ear does not fit an obvious conceptual framework.

Again this point was also noted by another reviewer and we expanded the introduction and discussion in this regard (l. 65ff).

Reviewer #1 (Recommendations for the authors):

(1) An update of the AR-surrogate method has recently been published (https://doi.org/10.1101/2024.08.22.609278). I appreciate that this is a lot of work, and it is of coursee up to the authors, but given the higher sensitivity of this method, it might be worth applying it to the four datasets described here.

Reading this article we note that our implementation of the AR-surrogate method was essentially as suggested here, and not as implemented by Brookshire. In fact we had not realized that Brookshire had apparently computed the spectrum based on the group-average data. As explained in the Methods section, as now clarified even better, we compute for each participant the actual spectrum of this participant’s data, and a set of surrogate spectra. We then perform a group-average of both to compute the p-value of the actual group-average based on the percentile of the distribution of surrogate averages. This send step differs from Harris & Beale, which used a one-sided t-test. The latter is most likely not appropriate in a strict statistical sense, but possibly more powerful for detecting true results compared to the percentile-based approach that we used (see l. 332ff).

(2) When results for the four experiments are reported, a reminder for the reader of how these experiments differ from each other would be useful.

We have added this in the Results section.

"considerable prevalence of differences around 4Hz, with dual‐task requirements leading to stronger rhythmicity in perceptual sensitivity". There is a striking similarity to recently published data (https://doi.org/10.1101/2024.08.10.607439 ) demonstrating a 4-Hz rhythm in auditory divided attention (rather than between modalities as in the present case). This could be a useful addition to the paragraph.

We have added a reference to this preprint, and additional previous work pointing in the same direction mentioned in there.  

(3) There are two typos in the Introduction: "related by different from the question", and below, there is one "presented" too much.

These have been fixed.

Reviewer #3 (Recommendations for the authors):

My major suggestion is that these results must be replicated in a new sample. I understand this is not simple to do and not always possible, but at this point, no effect is replicated from one experiment to the next, despite very small changes in protocol (especially experiment 1 vs 2). It's therefore very difficult to justify explaining the different effects as real as opposed to random effects of this particular sample. While the bootstrapping effects show the level of consistency of the effect within the sample studied, it can not be a substitute for a true replication of the results in a new sample.

We agree that only an independent replication can demonstrate the robustness of the results. We do consider experiment 1 a replication test of Ho et al. CurrBiol 2017, which results in different results than reported there. But more importantly, we consider the analysis of ‘reproducibility’ by simulating participant samples a key novelty of the present work, and want to emphasize this over the within-study replication of the same experiment.  In fact, in light of the present interpretation of the data, even a within-study replication would most likely not offer a clear-cut answer. 

As I said in the public review, the interpretation of the results, and of why perceptual cycles in arhythmic stimuli could be a plausible theory to begin with, is lacking. A conceptual framework would vastly improve the impact and understanding of the results.

We tried to strengthen the conceptual framework in the introduction. We believe that this is in large provided by previous work, and the aim of the present study was to explore the robustness of effects and not to suggest and discover novel effects. 

Minor comments:

(1) The authors adapt the difficulty as a function of performance, which seems to me a strange choice for an experiment that is analyzing the differences in performance across the experiment. Could you add a sentence to discuss the motivation for this choice?

We now mention the rationale in the Methods section and in a new section of the Results. There we also provide additional analyses on this parameter.

(2) The choice to plot the p-values as opposed to the values of the actual analysis feels ill-advised to me. It invites comparison across analyses that isn't necessarily fair. It would be more informative to plot the respective analysis outputs (spectral power, regression, or delta R2) and highlight the windows of significance and their overlap across analyses. In my opinion, this would be more fair and accurate depiction of the analyses as they are meant to be used.

We do disagree. As explained in the Methods (l. 374ff): “(Showing p-values) … allows presenting the results on a scale that can be directly compared between analysis approaches, metrics, frequencies and analyses focusing on individual ears or the combined data. Each approach has a different statistical sensitivity, and the underlying effect sizes (e.g. spectral power) vary with frequency for both the actual data and null distribution. As a result, the effect size reaching statistical significance varies with frequency, metrics and analyses.” 

The fact that the level of power (or R2 or whatever metric we consider) required to reach significance differs between analyses (one ear, both ears), metrics (d-prime, bias, RT) and between analyses approaches makes showing the results difficult, as we would need a separate panel for each of those. This would multiply the number of panels required e.g. for Figure 4 by 3, making it a figure with 81 axes. Also neither the original quantities of each analysis (e.g. spectral power) nor the p-values that we show constitute a proper measure of effect size in a statistical sense. In that sense, neither of these is truly ideal for comparing between analyses, metrics etc. 

We do agree thought that many readers may want to see the original quantification and thresholds for statistical significance. We now show these in an exemplary manner for the Binned analysis of Experiment 1, which provides a positive result and also is an attempt to replicate the findings by  Ho et al 2017. This is shown in new Figure 5. 

(3) Typo in line 555 (+ should be plus minus).

(4) Typo in line 572: "Comparison of 572 blocks with minus dual task those without"

(5) Typo in line 616: remove "one".

(6) Line 666 refers to effects in alpha band activity, but it's unclear what the relationship is to the authors' findings, which peak around 6 Hz, lower than alpha (~10 Hz).

(7) Line 688 typo, remove "amount of".

These points have been addressed.  

(8) Oculomotor effect that drives greater rhythmicity at 3-4 Hz. Did the authors analyze the eye movements to see if saccades were also occurring at this rate? It would be useful to know if the 3-4 Hz effect is driven by "internal circuitry" in the auditory system or by the typical rate of eye movement.

A preliminary analysis of eye movement data was in previous Figure 8, which was removed on the recommendation of another review.  This showed that the average saccade rate is about 0.01 saccade /per trial per time bin, amounting to on average less than one detected saccade per trial. Hence rhythmicity in saccades is unlikely to explain rhythmicity in behavioral data at the scale of 34Hz. We now note this in the Results.

Obleser J, Kayser C (2019) Neural Entrainment and Attentional Selection in the Listening Brain. Trends Cogn Sci 23:913-926.

Schroeder CE, Lakatos P (2009) Low-frequency neuronal oscillations as instruments of sensory selection. Trends Neurosci 32:9-18.

Schroeder CE, Lakatos P, Kajikawa Y, Partan S, Puce A (2008) Neuronal oscillations and visual amplification of speech. Trends Cogn Sci 12:106-113.

Zoefel B, Heil P (2013) Detection of Near-Threshold Sounds is Independent of EEG Phase in Common Frequency Bands. Front Psychol 4:262.

https://wordart.com/create Crée un Nuage de mots sur le champ lexical de la technologie

The idea of a river sweating is peculiar. Sweating is the release of a liquid from the body (from the body’s sweat glands). It is a crucial bodily function for regulating temperature, and the cooling effect occurs when sweat evaporates from the skin, absorbing excess heat from the body to do so. If a river releases liquid—oil and tar—isn’t it just to mix with the rest of the water again? Actually—some light oils can evaporate in large bodies of water, if the water is warm and the surface area is large. But it is clearly heavy oils that are meant (like crude oil or motor oil), particularly with tar following. The river sweating here and human sweating remain the same in the most basic sense: both are a function to aid the source, a kind of homeostatic regulation. Contrastingly, the river sweating functions to rid of waste—explicitly material, but probably extending to moral, spiritual, emotional. Unfortunately, this effort is doomed from the start—in a horribly looping, muddled way in that it goes round and round with the oil and tar being supposedly somewhat separated from the rest of the water, or perhaps consolidated, just to mix in again. This process was doomed from the start, but the river wasn’t, with the arrival of the oil and tar. Their presence is obtrusively unnatural. This must be remembered as the following phrases wash over with their sense of predetermination/overarching control: “The barges drift / With the turning tide…sails…swing…The barges wash / Drifting logs…”

What jumped out to me as mirroring this futile process of mixing was the lines “Weialala leia / Wallala leialala”—repeated twice, with the third iteration being just “la la.” In looking back at some past annotations, I noted that Jeannie ’25 made a comment on how “Weialala leia” can be read as a wail. In fact, when attempting to read it out loud, it sounded as though I was reciting a string of jumbled duplications of the word “wail,” and as one, it definitely sounded as a wail. This word is what the letters form, mixed around—but as more and more is added distortion grows, not clarity. In Götterdämmerung, this wailing call is for Siegfried, to return the ring, and thus the Rhine-gold. While initially appearing successful as Siegried enters right after the first call, subsequent ones fall upon deaf ears—along with the rest of the words of the Rhine Daughters. It is almost as though between when the words leave their mouths and when they reach the ears of Siegried—or the reader—there is a warping. The Rhine Daughters’ final “La! la!” feels a dwindling final attempt with recognition of the futility—the “w” is gone—or perhaps marks an even greater warping. Eliot exaggerates this further, with his third repetition being just “la la.” All of this seems to me to very much connect to female voice in the poem—particularly the line “And still she cried, and still the world pursues, / ‘Jug Jug’ to dirty ears” in “A Game of Chess.”

• Atenção: A CLT é mais protetiva que o Código Civil quanto à prescrição de direitos do menor de 18 anos.

• No Código Civil, a prescrição somente não corre quanto aos absolutamente incapazes (< 16 anos - art. 198, I, CC)! Na CLT, a prescrição de direito trabalhista somente corre quando a pessoa completa 18 anos.

rodzaje odpowiedzialności dłużnika 1. osobista - odpowiada całym swoim majątkiem 2. rzeczowa - konieczność znoszenia zaspokojenia z konkretnego przedmiotu majątku

czasem są obie jednocześnie - np. hipoteka

istnienie długu, ale bez odpowiedzialności => wierzyciel ma prawo domagać się roszczenia, ale np. jeśli dłużnik powoła się na przedawnienie, to roszczenie traci zaskarżalność => dłużnik może spełnić świadczenie dobrowolnie, ale wierzyciel nie ma ochrony sądowej

podział uprawnień: 1. roszczenia - konkretna osoba uprawniona X może żądać od innego podmiotu spełnienia świadczenia na rzecz X 2. uprawnienia kształtujące - uprawniony X ma kompetencję do zmian/zakończenia stosunku prawnego przez jednostronną czynność prawną 3. zarzuty - uprawnienie do odmowy spełnienia roszczenia a) peremptoryjne (trwałe) - skutek: unicestwienie dochodzenia roszczenia w każdej możliwej chwili (np, przedawnienie) b) dylatoryjne (przejściowe) - skutek: ograniczenie możliwości dochodzenia roszczenia, ale tylko w określonym czasie

albo 1. sfera wolności osoby, której przysługuje prawo podmiotowe albo 2. ograniczenie wolności innych osób

Author response:

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

Reviewer #1 (Public review):

Summary:

This is an interesting study characterizing and engineering so-called bathy phytochromes, i.e., those that respond to near infrared (NIR) light in the ground state, for optogenetic control of bacterial gene expression. Previously, the authors have developed a structure-guided approach to functionally link several light-responsive protein domains to the signaling domain of the histidine kinase FixL, which ultimately controls gene expression. Here, the authors use the same strategy to link bathy phytochrome light-responsive domains to FixL, resulting in sensors of NIR light. Interestingly, they also link these bathy phytochrome light-sensing domains to signaling domains from the tetrathionate-sensing SHK TtrS and the toluene-sensing SHK TodS, demonstrating the generality of their protein engineering approach more broadly across bacterial two-component systems.

This is an exciting result that should inspire future bacterial sensor design. They go on to leverage this result to develop what is, to my knowledge, the first system for orthogonally controlling the expression of two separate genes in the same cell with NIR and Red light, a valuable contribution to the field.

Finally, the authors reveal new details of the pH-dependent photocycle of bathy phytochromes and demonstrate that their sensors work in the gut - and plant-relevant strains E. coli Nissle 1917 and A. tumefaciens.

Strengths:

(1) The experiments are well-founded, well-executed, and rigorous.

(2) The manuscript is clearly written.

(3) The sensors developed exhibit large responses to light, making them valuable tools for ontogenetic applications.

(4) This study is a valuable contribution to photobiology and optogenetics.

We thank the reviewer for the positive verdict on our manuscript.

Weaknesses:

(1) As the authors note, the sensors are relatively insensitive to NIR light due to the rapid dark reversion process in bathy phytochromes. Though NIR light is generally non-phototoxic, one would expect this characteristic to be a limitation in some downstream applications where light intensities are not high (e.g., in vivo).

We principally concur with this reviewer’s assessment that delivery of light (of any color) into living tissue can be severely limited by absorption, reflection, and scattering. That notwithstanding, at least two considerations suggest that in-vivo deployment of the pNIRusk setups we presently advance may be feasible.

First, while the pNIRusk setups are indeed less light-sensitive compared to, e.g., our earlier redlight-responsive pREDusk and pDERusk setups (see Meier et al. Nat Commun 2024), we note that the overall light fluences required for triggering them are in the range of tens of µW per cm₂. By contrast, optogenetic experiments in vivo, in particular in the neurosciences, often employ light area intensities on the order of mW per cm₂ and above. Put another way, compared to the optogenetic tools used in these experiments, the pNIRusk setups are actually quite sensitive to light.

Second, sensitivity to NIR light brings the advantage of superior tissue penetration, see data reported by Weissleder Nat Biotech 2001 and Ash et al. Lasers Med Sci 2017 (both papers are cited in our manuscript). Based on these data, the intensity of blue light (450 nm) therefore falls off 5-10 times more strongly with penetration depth than that of NIR light (800 nm).

We have added a brief treatment of these aspects in the Discussion section.

(2) Though they can be multiplexed with Red light sensors, these bathy phytochrome NIR sensors are more difficult to multiplex with other commonly used light sensors (e.g., blue) due to the broad light responsivity of the Pfr state. This challenge may be overcome by careful dosing of blue light, as the authors discuss, but other bacterial NIR sensing systems with less cross-talk may be preferred in some applications.

The reviewer is correct in noting that, at least to a certain extent, the pNIRusk systems also respond to blue light owing to their Soret absorbance bands (see Fig. 1). That said, we note two points:

First, a given photoreceptor that preferentially responds to certain wavelengths, e.g., 700 nm in the case of conventional bacterial phytochromes (BphP), generally absorbs shorter wavelengths to some degree as well. Absorption of these shorter wavelengths suffices for driving electronic and/or vibronic transitions of the chromophore to higher energy levels which often give rise to productive photochemistry and downstream signal transduction. Put another way, a certain response of sensory photoreceptors to shorter wavelengths is hence fully expected and indeed experimentally borne out, as for instance shown by Ochoa-Fernandez et al. in the so-called PULSE setup (Nat Meth 2020, doi: 10.1038/s41592-020-0868-y).

Second, known BphPs share similar Pr and Pfr absorbance spectra. We therefore expect other BphP-based optogenetic setups to also respond to blue light to some degree. Currently, there are insufficient data to gauge whether individual BphPs systematically differ in their relative sensitivity to blue compared to red or NIR light. Arguably, pertinent experiments may be an interesting subject for future study.

Reviewer #2 (Public review):

Summary:

In this manuscript, Meier et al. engineer a new class of light-regulated two-component systems. These systems are built using bathy-bacteriophytochromes that respond to near-infrared (NIR) light. Through a combination of genetic engineering and systematic linker optimization, the authors generate bacterial strains capable of selective and tunable gene expression in response to NIR stimulation. Overall, these results are an interesting expansion of the optogenetic toolkit into the NIR range. The cross-species functionality of the system, modularity, and orthogonality have the potential to make these tools useful for a range of applications.

Strengths:

(1) The authors introduce a novel class of near-infrared light-responsive two-component systems in bacteria, expanding the optogenetic toolbox into this spectral range.

(2) Through engineering and linker optimization, the authors achieve specific and tunable gene expression, with minimal cross-activation from red light in some cases.

(3) The authors show that the engineered systems function robustly in multiple bacterial strains, including laboratory E. coli, the probiotic E. coli Nissle 1917, and Agrobacterium tumefaciens.

(4) The combination of orthogonal two-component systems can allow for simultaneous and independent control of multiple gene expression pathways using different wavelengths of light.

(5) The authors explore the photophysical properties of the photosensors, investigating how environmental factors such as pH influence light sensitivity.

Weaknesses:

(1) The expression of multi-gene operons and fluorescent reporters could impose a metabolic burden. The authors should present data comparing optical density for growth curves of engineered strains versus the corresponding empty-vector control to provide insight into the burden and overall impact of the system on host viability and growth.

In response to this comment, we have recorded growth kinetics of bacteria harboring the pNIRusk-DsRed plasmids or empty vectors under both inducing (i.e., under NIR light) and noninducing conditions (i.e., darkness). We did not observe systematic differences in the growth kinetics between the different cultures, thus suggesting that under the conditions tested there is no adverse effect on cell viability.

We include the new data in Suppl. Fig. 5c-d and refer to them in the main text.

(2) The manuscript consistently presents normalized fluorescence values, but the method of normalization is not clear (Figure 2 caption describes normalizing to the maximal fluorescence, but the maximum fluorescence of what?). The authors should provide a more detailed explanation of how the raw fluorescence data were processed. In addition, or potentially in exchange for the current presentation, the authors should include the raw fluorescence values in supplementary materials to help readers assess the actual magnitude of the reported responses.

We appreciate this valid comment and have altered the representation of the fluorescence data. All values for a given fluorescent protein (i.e., either DsRed or YPet) across all systems are now normalized to a single reference value, thus enabling direct comparison between experiments.

(3) Related to the prior point, it would be useful to have a positive control for fluorescence that could be used to compare results across different figure panels.

As all data are now normalized to the same reference value, direct comparison across all figures is enabled.

(4) Real-time gene expression data are not presented in the current manuscript, but it would be helpful to include a time-course for some of the key designs to help readers assess the speed of response to NIR light.

In response to this comment, we include in the revised manuscript induction kinetics of bacterial cultures bearing pNIRusk upon transfer to inducing NIR-light conditions. To this end, aliquots were taken at discrete timepoints, transcriptionally and translationally arrested, and analyzed for optical density and DsRed reporter fluorescence after allowing for chromophore maturation.

We include the new data in Suppl. Fig. 5e and refer to them in the manuscript.

Moreover, we note that the experiments in Agrobacterium tumefaciens used a luciferase reporter thus enabling the continuous monitoring of the light-induced expression kinetics. These data (unchanged in revision) are to be found in Suppl. Fig. 9.

Reviewer #3 (Public review):

Summary:

This paper by Meier et al introduces a new optogenetic module for the regulation of bacterial gene expression based on "bathy-BphP" proteins. Their paper begins with a careful characterization of kinetics and pH dependence of a few family members, followed by extensive engineering to produce infrared-regulated transcriptional systems based on the authors' previous design of the pDusk and pDERusk systems, and closing with characterization of the systems in bacterial species relevant for biotechnology.

Strengths:

The paper is important from the perspective of fundamental protein characterization, since bathyBphPs are relatively poorly characterized compared to their phytochrome and cyanobacteriochrome cousins. It is also important from a technology development perspective: the optogenetic toolbox currently lacks infrared-stimulated transcriptional systems. Infrared light offers two major advantages: it can be multiplexed with additional tools, and it can penetrate into deep tissues with ease relative to the more widely used blue light-activated systems. The experiments are performed carefully, and the manuscript is well written.

Weaknesses:

My major criticism is that some information is difficult to obtain, and some data is presented with limited interpretation, making it difficult to obtain intuition for why certain responses are observed. For example, the changes in red/infrared responses across different figures and cellular contexts are reported but not rationalized. Extensive experiments with variable linker sequences were performed, but the rationale for linker choices was not clearly explained. These are minor weaknesses in an overall very strong paper.

We are grateful for the positive take on our manuscript.

Reviewer #1 (Recommendations for the authors):

(1) As eLife is a broad audience journal, please define the Soret and Q-bands (line 125).

We concur and have added labels in fig. 1a that designate the Soret and Q bands.

(2) The initial (0) Ac design in Figure 2b is activated by NIR and Red light, albeit modestly. The authors state that this construct shows "constant reporter fluorescence, largely independent of illumination" (line 167). This language should be changed to reflect the fact that this Ac construct responds to both of these wavelengths.

Agreed. We have amended the text accordingly.

(3) pNIRusk Ac 0 appears to show a greater light response than pNIRusk Av -5. However, the authors claim that the former is not light-responsive and the latter is. This conclusion should be explained or changed.

The assignment of pNIRusk Av-5 as light-responsive is based on the relative difference in reporter fluorescence between darkness and illumination with either red or NIR light. Although the overall fluorescence is much lower in Av-5 than for Av-0, the relative change upon illumination is much more pronounced. We add a statement to this effect to the text.

(4) The authors state that "when combining DmDERusk-Str-YPet with AvTod+21-DsRed expression rose under red and NIR light, respectively, whereas the joint application of both light colors induced both reporter genes" (lines 258-261). In contrast, Figure 3c shows that application of both wavelengths of light results in exclusive activation of YPet expression. It appears the description of the data is wrong and must be corrected. That said, this error does not impact their conclusion that two separate target genes can be independently activated by NIR and red light.

We thank the reviewer for catching this error which we have corrected in the revised manuscript.

(5) Line 278: I don't agree with the authors' blanket statement that the use of upconversion nanoparticles is a "grave" limitation for NIR-light mediated activation of bacterial gene expression in vivo. The authors should either expound on the severity of the limitation or use more moderate language.

We have replaced the word ‘grave’ by ‘potential’ and thereby toned down our wording.

Reviewer #2 (Recommendations for the authors):

(1) Please include a discussion on the expected depth penetration of different light wavelengths. This is most relevant in the context of the discussion about how these NIR systems could be used with living therapeutics.

Given the heterogeneity of biological tissue, it is challenging to state precise penetration depths for different wavelengths of light. That said, blue light for instance is typically attenuated by biological tissue around 5 to 10 times as strongly as near-infrared light is.

We have expanded the Discussion chapter to cover these aspects.

(2) It would be helpful for Figure 2C (or supplementary) to also include the response to blue light stimulation.

We agree and have acquired pertinent data for the blue-light response. The new data are included in an updated Fig. 2c. Data acquired at varying NIR-light intensities, originally included in Fig. 2c, have been moved to Suppl. Fig. 5a-b.

(3) In Figure 4A, data on the response of E. coli Nissle to blue and red light are missing. Including this would help identify whether the reduced sensitivity to non-NIR wavelengths observed in the E. coli lab strain is preserved in the probiotic background.

In response to this comment, we have acquired pertinent data on E. coli Nissle. While the results were overall similar to those in the laboratory strain, the response to blue and NIR light was yet lower in the Nissle bacteria which stands to benefit optogenetic applications.

We have updated Fig. 4a accordingly. For clarity, we only show the data for AvNIRusk in the main paper but have relegated the data on AcNIRusk to Suppl. Fig. 8. (Note that this has necessitated a renumbering of the subsequent Suppl. Figs.)

(4) On many of the figures, there are thin gray lines that appear between the panels that it would be nice to eliminate because, in some cases, they cut through words and numbers.

The grey lines likely arose from embedding the figures into the text document. In the typeset manuscript, which has become available on the eLife webpage in the meantime, there are no such lines. That said, we will carefully check throughout the submission/publishing/proofing process lest these lines reappear.

(5) Page 7, line 155: "As not least seen" typo or awkward phrasing.

We have restructured the sentence and thereby hopefully clarified the unclear phrasing.

(6) Page 7, line 167: It does not appear to be the case that the initial pNIRusk designs show constant fluorescence that is largely independent of illumination. AcNIRusk shows an almost twofold change from dark to NIR. Reword this to avoid confusion.

We concur with this comment, similar to reviewer #1’s remark, and have adjusted the text accordingly.

(7) Page 8, line 174: Related to the previous point, AvNIRusk has one design that is very minimally light switchable (-5), so stating that six light switchable designs have been identified is also confusing.

As stated in our response to reviewer #1 above, the assignment of AvNIRusk-5 as light-switchable is based on the relative fluorescence change upon illumination. We have added an explanation to the text.

(8) Page 10, line 228-229: I was not able to find the data showing that expression levels were higher for the DmTtr systems than the pREDusk and pNIRusk setups. This may be an issue related to the normalization point. It was not clear to me how to compare these values.

We apologize for the initially unclear representation of the data. In response to this reviewer’s general comments above, we have now normalized all fluorescence values to a single reference value, thus allowing their direct comparison.

(9) Page 12, line 264: "finer-grained expression control can be exerted..." Either show data or adjust the language so that it is clear this is a prediction.

True, we have replaced ‘can’ by ‘could’.

(10) Page 25, line 590: CmpX13 cells have a reference that is given later, but it should be added where it first appears.

Agreed, we have added the reference in the indicated place.

(11) Page 25, line 592: define LB/Kan.

We had already defined this abbreviation further up but, for clarity, we have added it again in the indicated position.

(12) Page 40, line 946: "normalized by" rather than "to".

We have implemented the requested change in the indicated and several other positions of the manuscript.

(13) Figures 2C, 3C, and similar plots in the supplementary material would benefit from having a legend for the colors.

We agree and have added pertinent legends to the corresponding main and supplementary figures.

(14) As a reader, I had some trouble following all the acronyms. This is at the author's discretion, but I would eliminate ones that are not strictly essential (e.g. MTP for microtiter plate; I was unable to identify what "MCS" meant; look for other opportunities to remove acronyms).

In the revised manuscript, we have defined the abbreviation ‘MCS’ (for ‘multiple-cloning site’) upon first occurrence. We have decided to retain the abbreviation ‘MTP’ in the text.

(15) Could the authors briefly speculate on why A. tumefaciens activation with red light might occur?

While we can but speculate as to the underlying reasons for the divergent red-light response in A. tumefaciens, we discuss possible scenarios below.

Commonly, two-component systems (TCS) exhibit highly cooperative and steep responses to signal. As a consequence, even small differences in the intracellular amounts of phosphorylated and unphosphorylated response regulator (RR) can give to significantly changed gene-expression output. Put another way, the gene-expression output need not scale linearly with the extent of RR phosphorylation but, rather, is expected to show nonlinear dependence with pronounced thresholding effects.

Differences in the pertinent RR levels can for instance arise from variations in the expression levels of the pNIRusk system components between E. coli and A. tumefaciens. Moreover, the two bacteria greatly differ in their two-component-system (TCS) repertoire. Although TCSs are commonly well insulated from each other, cross-talk with endogenous TCSs, even if limited, may cause changes in the levels of phosphorylated RR and hence gene-expression output. In a similar vein, the RR can also be phosphorylated and dephosphorylated non-enzymatically, e.g., by reaction with high-energy anhydrides (such as acetyl phosphate) and hydrolysis, respectively. Other potential origins for the divergent red-light response include differences in the strength of the promoters driving expression of the pNIRusk system components and the fluorescent/luminescent reporters, respectively.

(16) It would be helpful for the authors to briefly explain why they needed to switch to luminescence from fluorescence for the A. tumeraciens studies.

While there was no strict necessity to switch from the fluorescence-based system used in E. coli to a luminescence-based system in A. tumefaciens, we opted for luminescence based on prior experience with other Alphaproteobacteria (e.g., 10.1128/mSystems.00893-21), where luminescence offered significant advantages. Specifically, it provides essentially background-free signal detection and greater sensitivity for monitoring gene expression. In addition, as demonstrated in Suppl. Fig. 9c and d, the luminescence system enables real-time tracking of gene expression dynamics, which further supported its use in our experimental setup (see our response to reviewer #2’s general comments).

(17) This is a very minor comment that the authors can take or leave, but I got hung up on the word "implement" when it appeared a few times in the manuscript because I tended to read it as "put a plan into place" rather than its other meaning.

In the abstract, we have replaced one instance of the word ‘implement’ by ‘instrument’.

(18) The authors should include the relevant constructs on AddGene or another public strainsharing service.

We whole-heartedly subscribe to the idea of freely sharing research materials with fellow scientists. Therefore, we had already deposited the most relevant AvNIRusk in Addgene, even prior to the initial submission of the manuscript (accession number 235084). In the meantime, we have released the deposition, and the plasmid can be obtained from Addgene since May 15_th of this year.

Reviewer #3 (Recommendations for the authors):

Suggestion for improvement:

This paper relies heavily on variations in linker sequences to shift responses. I am familiar with prior work from the Moglich lab in which helical linkers were employed to shift responses in synthetic two-component systems, with interesting periodicity in responses with every 7 residues (as expected for an alpha helix) and inversion of responses at smaller linker shifts. There is no mention in this paper whether their current engineering follows a similar rationale, what types of linkers are employed (e.g. flexible vs helical), and whether there is an interpretation for how linker lengths alter responses. Can you explain what classes of linker sequences are used throughout Figures 2 and 3, and whether length or periodicity affects the outcome? This would be very helpful for readers who are new to this approach, or if the rationale here differs from the authors' prior work.

The PATCHY approach employed at present followed a closely similar rationale as in our previous studies. That is, linkers were extended/shortened and varied in their sequence by recombining different fragments of the natural linkers of the parental receptors, i.e., the bacteriophytochrome and the FixL sensor histidine kinase, respectively. We have added a statement to this effect in the text and a reference to Suppl. Fig. 3 which illustrates the principal approach.

Compared to our earlier studies, we isolated fewer receptor variants supporting light-regulated responses, despite covering a larger sequence space. Owing to the sparsity of the light-regulated variants, an interpretation of the linker properties and their correlation with light-regulated activity is challenging. Although doubtless unsatisfying from a mechanistic viewpoint, we therefore refrain from a pertinent discussion which would be premature and speculative at this point. As the reviewer raises a valid and important point, we have expanded the text by referring to our earlier studies and the observed dependence of functional properties on linker composition.

It is sometimes difficult to intuit or rationalize the differences in red/IR sensitivity across closely related variants. An important example appears in Figure 3C vs 3B. I think the AvTod+21 in 3B should be the equivalent to the DsRed response in the second column of 3C (AvTod+21 + DmDERusk), except, of course, that the bacteria in 3C carry an additional plasmid for the DERusk system. However, in 3B, the response to red light is substantial - ~50% as strong as that for IR, whereas in 3C, red light elicits no response at all. What is the difference? The reason this is important is that the AvTod+21 and DMDERusk represent the best "orthogonal" red and infrared light responses, but this is not at all obvious from 3B, where AvTod+21 still causes a substantial (and for orthogonality, undesirable) response under red light. Perhaps subtle differences in expression level due to plasmid changes cause these differences in light responses? Could the authors test how the expression level affects these responses? The paper would be greatly improved if observations of the diverse red/IR responses could be rationalized by some design criteria.

As noted above in our response to reviewer #2, we have now normalized all fluorescence readings to joint reference values, thus allowing a better comparison across experiments.

The reviewer is correct in noting that upon multiplexing, the individual plasmid systems support lower fluorescence levels than when used in isolation. We speculate that the combination of two plasmids may affect their copy numbers (despite the use of different resistance markers and origins of replications) and hence their performance. Likewise, the cellular metabolism may be affected when multiple plasmids are combined. These aspects may well account for the absent red-light response in AvTod+21 in the multiplexing experiments which is – indeed – unexpected. As, at present, we cannot provide a clear rationalization for this effect, we recommend verifying the performance of the plasmid setups when multiplexing.

The paper uses "red" and "infrared" to refer to ~624 nm and ~800 nm light, respectively. I wonder whether it might be possible to shift these peak wavelengths to obtain even better separation for the multiplexing experiments. Perhaps shifting the specific red wavelength could result in better separation between DERusk and AvTod systems, for example? Could the authors comment on this (maybe based on action spectra of their previously developed tools) or perhaps test a few additional stimulation wavelengths?

The choice of illumination wavelengths used in these experiments is dictated by the LED setups available for illumination of microtiter plates. On the one hand, we are using an SMD (surface-mount device) three-color LED with a fixed wavelength of the red channel around 624 nm (see Hennemann et al., 2018). On the other hand, we are deploying a custom-built device with LEDs emitting at around 800 nm (see Stüven et al., 2019 and this work). Adjusting these wavelengths is therefore challenging, although without doubt potentially interesting.

To address this reviewer comment, we have added a statement to the text that the excitation wavelengths may be varied to improve multiplexed applications.

Additional minor comments:

(1) Figure 2C: It would be very helpful to place a legend on the figure panel for what the colors indicate, since they are unique to this panel and non-intuitive.

This comment coincides with one by reviewer #2, and we have added pertinent legends to this and related supplementary figures.

(2) Figure 3C: it is not obvious which system uses DsRed and which uses YPet in each combination, since the text indicates that all combinations were cloned, and this is not clearly described in the legend. Is it always the first construct in the figure legend listed for DsRed and the second for YPet?

For clarification, we have revised the x-axis labels in Fig. 3C. (And yes, it is as this reviewer surmises: the first of the two constructs harbored DsRed and the second one YPet.)

Note de synthèse : Les formes de la violence et le témoignage

Ce document de synthèse explore les différentes formes et fonctions du témoignage face à la violence, en s'appuyant sur l'analyse de Didier Fassin dans "Les formes de la violence (8)".

Il met en lumière l'importance de l'attestation de la violence, les diverses figures du témoin, les défis de sa représentation, et l'émergence de nouvelles médiations technologiques pour révéler la vérité.

I. L'attestation de la violence : une urgence face à l'invisibilisation

La raison d'être la plus commune de l'écriture et de la représentation de la violence est de l'attester, une urgence d'autant plus grande que la réalité est invisibilisée. L'auteur cite deux exemples contemporains de cette invisibilisation et des tentatives d'attestation :

La violence coloniale française en Algérie : Malgré une loi de 2005 qui "oblige les programmes scolaires... à reconnaître le rôle positif de la présence française outre-mer", des travaux comme celui d'Alain Ruot (2024) dans "La première guerre en Algérie" rappellent les "spoliations de terre, les déplacements de population, les massacres de villageois, les enfumades de grottes, les centaines de milliers de morts surtout des civils" perpétrées par le corps expéditionnaire français.

L'expulsion des Palestiniens (la Nakba) : L'expulsion de "750 000 Palestiniens, soit environ la moitié de la population arabe de ce territoire", qui a entraîné la "destruction de villages et dans certains cas du meurtre de leurs habitants", a longtemps été ignorée.

Le film "Partition" (2025) de Dana Alan, prolongeant son ouvrage "Voices of the Nagba", vise à "restituer l'expérience de l'enagbactrale à travers les archives coloniales du mandat britannique" et les récits des Palestiniens.

Ces entreprises visent à attester ce que les nations ont "enfoui souvent dans les profondeurs de l'oubli".

Si les auteurs de violence peuvent avoir intérêt à la montrer pour "la jouissance de l'exercice de la force à la production d'un régime de terreur", ils ont souvent "un intérêt plus grand encore à la dissimuler, à la déguiser, à la nier" pour éviter la condamnation ou la sanction.

Dans ces cas, il est crucial pour les victimes, leurs proches, et les "entrepreneurs de justice" (avocats, militants des droits humains, chercheurs) d'apporter la preuve de la violence, ses circonstances et ses responsables.

"Attester la violence c'est donc combattre le déni, l'occultation, le mensonge, le révisionnisme historique. Attester la violence c'est emporter témoignage, c'est sans faire le témoin."

II. Les figures du témoin : entre objectivité et subjectivité S'appuyant sur Émile Benveniste, l'auteur distingue deux conceptions du témoin, principalement à travers le latin :

Testis : "celui qui assiste entière à une affaire où deux personnages sont intéressés ayant été présent au moment où les faits se sont produits".

Sa parole "peut être utilisé pour trancher un litige à condition qu'il soit établi qu'il n'était pas lui-même partie prenante". Le testis est extérieur à la scène, son observation est présumée objective.

Superstess : "décrit le témoin comme celui qui subsiste au-delà, témoin en même temps que survivant".

Son témoignage est autorisé par le fait d'avoir "vécu lui-même les faits notamment lorsqu'il s'implique un danger ou une épreuve et d'avoir survécu à ce péril".

Le superstess est la victime, son récit est nécessairement subjectif, mais non insoupçon.

Cette distinction est mise à l'épreuve par la littérature sur la Shoah.

A. Le défi du témoignage face à la dissimulation nazie

L'histoire de l'extermination des Juifs et des Roms n'est pas quelque chose dont les nazis se vantaient, mais qu'ils ont cherché à dissimuler, y compris "vis-à-vis du peuple allemand et vis-à-vis d'eux-mêmes".

Hannah Arendt, dans "Eichmann à Jérusalem", souligne l'usage d'un "langage codé" ou "règles de langage" qui étaient "dans le parler ordinaire... un mensonge", pour euphémiser les crimes : "solution finale", "traitement spécial", "évacuation".

L'effet de ce système de langage n'était pas "d'empêcher les gens de savoir ce qu'ils faisaient, mais de les empêcher de mettre leurs actes en rapport avec leur ancienne notion normale du meurtre et du mensonge, en somme de rendre mentalement acceptable ce qui aurait pu leur paraître moralement intolérable."

Pierre Vidal-Naquet ajoute que ce langage codé a facilité le négationnisme ultérieur.

Les nazis, conscients de ce qui allait se passer, avertissaient cyniquement les prisonniers : "De quelque façon que cette guerre se finisse, nous l'avons déjà gagné contre vous ; aucun d'entre vous ne restera pour porter témoignage.

Mais même si quelques-uns en réchappaient, le monde ne les croira pas, il n'y aura pas de certitude, car nous détruirons les preuves en vous détruisant." (Primo Levi, "Les naufragés et les rescapés").

Cette peur du non-crédit a hanté les survivants, qui ont souvent raconté un cauchemar récurrent où leurs proches ne les croyaient pas.

D'où l'importance vitale du témoignage, comme l'exprime Robert Antelme : "nous voulions parler, être entendu enfin".

B. La complexité du témoignage des survivants (Superstess/Testis)

Primo Levi, en écrivant "Si c'est un homme", cherchait à "attester" son expérience.

Cependant, il exprime une profonde gêne, estimant que "nous les survivants ne sommes pas les vrais témoins... car nous sommes ceux qui grâce à la prévarication, l'habileté ou la chance, n'ont pas touché le fond."

Les "musulmans" (ceux tellement affaiblis qu'ils étaient voués à mourir) sont les "témoins intégraux".

La réflexion de Levi met à l'épreuve la distinction testis/superstess :

• Il est un superstess incontestable, ayant survécu à l'impensable et décrivant l'insulte de la "démolition d'un homme".
• Mais il est aussi un testis, conscient de ne jamais pouvoir restituer l'expérience de ceux qui ont été dévorés, et pour qui il parle "à leur place, par délégation".

L'exemple d'Urbinec, l'enfant paralysé et mutique à Auschwitz, dont la "nécessité de parler jaillissait dans son regard avec une force explosive", et dont Primo Levi écrit "il témoigne à travers mes paroles", illustre cette réconciliation tragique des deux figures : "le superstès devenu testis sauve du néant la mémoire du petit garçon."

C. Diversité des styles et temporalités du témoignage

Les récits des survivants du génocide adoptent des styles et des temporalités variés :

• Témoignage immédiat : David Rousset ("L'univers concentrationnaire", 1946) rencontre un succès rapide malgré la réticence des sociétés européennes, peut-être grâce à une "forme de recherche esthétique" créant une distance "qui neutralise les émotions".

Son écriture est "austère et ironique", utilisant "des formules elliptiques et tranchantes, parfois caustiques et troublantes."

• Témoignage différé : Charlotte Delbo ("Aucun de nous ne reviendra", 1965), écrit un premier brouillon après sa sortie, puis le reprend 20 ans plus tard. Elle commence par la scène collective des arrivées de trains, utilisant des phrases courtes et des images fortes pour dire "l'inconcevable".

• Anti-mémoire : Imre Kertész ("Être et destin", 1985) adopte le regard "naïf déconcerté" d'un adolescent, décrivant la découverte progressive de l'horreur des camps, comme "l'odeur... doucâtre, en quelque sorte gluante" du crématorium.

Il décrit la "détérioration physique" sans pathos, et même un "désir sourd" de vivre au moment du "tri final des mourants".

• Méfiance et refus d'enfermement : Ruth Kluger ("Refus de témoigner. Une jeunesse", 1992) écrit pour exprimer sa méfiance face à la multiplication des témoignages et son refus d'être réduite à sa condition de déportée.

• L'expérience des victimes du nazisme est à la fois "spécifique" (partir d'un vécu individuel) et "indéterminée" (nécessité de trouver les mots et la forme face à "l'incommunicabilité abyssale").

Pour l'immense majorité des survivants, il faut "accepter de n'être ni superstès ni testice et donc se taire."

III. Autres figures du témoin et médiations

A. Auctor et Histor : l'autorité et la connaissance

Auctor (latin) : "celui qui augmente la confiance, le garant, la source et donc l'autorité" et "celui qui pousse à agir, l'instigateur, le créateur et donc l'auteur".

Le crédit est le fondement de son témoignage.

Histor (grec) : "celui qui sait, qui connaît... l'historien". L'enquête est le fondement de son témoignage.

Ces figures n'ont pas vécu les faits mais peuvent en être les garants. Les historiens contemporains "réunissent souvent les deux dimensions", bénéficiant du "crédit de leur discipline" et s'appuyant sur des "enquêtes menées dans des archives ou par des entretiens".

L'exemple de Jean Hatzfeld et son livre "Dans le nu de la vie" (2000) sur le génocide rwandais illustre l'auctor.

Il rassemble des récits de survivants, s'autorisant à les convaincre de parler malgré leur réticence.

Journaliste et écrivain, il utilise sa double autorité pour "attester ce qu'a été et ce qu'est encore... l'expérience de ces hommes, de ces femmes, de ces enfants qui ont vécu le massacre."

Bien que les récits soient rédigés à la première personne, ils sont "entièrement écrits par une troisième personne, l'auteur."

• L'histore est illustré par les chercheurs en sciences sociales qui restituent et interprètent les faits en s'appuyant sur des "archives nationales ou étrangères, des jugements rendus par des juridictions internationales, des articles de journaux locaux, des entretiens avec des personnes occupant des positions différentes, des observations de procès".

Les travaux de Mahmoud Mamdani ("When Victims Become Killers", 2001) interprètent le génocide rwandais à la lumière de l'histoire coloniale, distinguant le génocide conduit par les "settlers" (colons) et celui par les "natives" (indigènes).

Hélène Dumas ("Le génocide au village", 2014) se concentre sur la "mécanique microlocale des violences", montrant que le génocide est "une affaire de voisins et de parents" et que les génocidaires "éprouvent une jouissance dans la souffrance et l'humiliation de leurs victimes."

Beata Umubyeyi Mairesse ("Le convoi", 2024), une survivante du génocide rwandais, se distingue par sa réflexivité et son intégrité.

Elle est à la fois superstess, racontant sa survie, et testis, décrivant ce qu'elle a vu.

Elle se fait également historienne de son histoire, explorant des archives et conduisant des entretiens, mais "elle répugne à faire acte d'autorité," refusant d'être l'auctor.

B. Martous : le témoin-martyr

En grec ancien, "Martous" signifie le témoin, mais aussi, plus spécifiquement dans la Bible, le "témoin de Dieu", c'est-à-dire le martyr, celui qui "a accepté de mourir pour attester de sa croyance".

Giorgio Agamben ("Ce qui reste d'Auschwitz", 1998) note que le martyre chrétien a dû "justifier le scandale d'une mort insensée".

Le "shaï" arabe a un sens similaire, désignant à la fois le témoin et le martyr.

En Palestine, la figure du shaïd s'est développée comme "ciment de l'unité nationale".

Le shaïd peut être une victime tuée "sans l'avoir choisi" ou un combattant qui s'est exposé "volontairement pour la cause de son peuple".

Ce dédoublement transforme le sens du martyre, l'étendant du "sacrifice librement consenti à la mort subie", et du "strictement religieux au politique".

"Tout palestinien abattu ou exécuté par les Israéliens est un shaïd qui par sa mort dans un affrontement inégal atteste son appartenance à sa communauté et témoigne de la brutalisation de l'ennemi."

Pour les martyrs palestiniens, le sacrifice ou la mort est une réponse à une "vie impossible à quoi la mort viendrait tragiquement redonner du sens".

L'auteur cite la photojournaliste Fatima Assuna : "Quant à la mort qui est inévitable, si je meurs, je veux une mort retentissante, je ne veux pas être une simple brève dans un flash info ni un chiffre parmi d'autres, je veux une mort dont le monde entier entendra parler, une empreinte qui restera à jamais, des émotions, des images immortelles que ni le temps ni l'espace ne pourront enterrer."

IV. Les médiations technologiques du témoignage

Le témoignage ne s'exprime pas seulement par la parole, l'écrit ou le corps (dans le cas du martyr), mais aussi par des "médiations dans lesquelles les technologies peuvent être mobilisées".

L'exemple le plus innovant est Forensic Architecture (fondée en 2010 par Eyal Weizman), une agence qui développe des "techniques, méthodes et concepts pour conduire des investigations sur la violence d'État et la violence en entreprise".

• En combinant "l'imagerie spatiale par satellite, les caméras de surveillance, les enregistrements audio et vidéo, les témoignages individuels et collectifs", Forensic Architecture reconstitue en 3D des événements de violence qui ont été occultés.

Parmi les nombreux cas étudiés, on trouve le génocide des Herero et Nama, les massacres israéliens pendant la Nakba, l'assassinat d'otages en Colombie, le meurtre de Mark Duggan au Royaume-Uni, l'utilisation d'armes européennes au Yémen, et des événements en France (Adama Traoré, Zineb Reddouane).

Ces technologies permettent de "révéler de nombreuses violences, des crimes de guerre identifiés, des coupables reconnus, des versions officielles démenties, certaines vérités dites et la justice parfois rendue".

Elles "renforcent, enrichissent et parfois même remplacent le témoignage humain".

V. Conclusion : La complexité du témoignage pour faire exister la vérité

En résumé, l'auteur a esquissé cinq figures idéaltypiques du témoin :

• Le testis : présent au moment des faits, dont il peut raconter.
• Le superstess : survivant, qui peut transmettre ce qu'il a vécu.
• L'auctor : agent extérieur, qui apporte la crédibilité.
• L'histor : expert légitime, qui conduit une enquête.

• Le martous : victime sacrificielle, qui affirme la justesse de sa cause par son renoncement.

• Chacune de ces figures "engage des formes politiques et morales : la véracité du testis, l'authenticité du superstès, l'autorité de l'actor, la neutralité de l'histor, l'engagement du Martus."

Ces figures ne sont pas étanches et "se mêlent, se combinent, se déplacent, se complexifient" dans la réalité.

Au-delà de ces distinctions, "l'enjeu du témoignage c'est de faire exister une vérité et notamment... de la faire exister contre la dissimulation, l'invisibilisation, la dénégation".

C'est là toute l'importance de "celles et ceux qui ont pour projet de révéler la vérité ou tout au moins une part de la vérité à laquelle ils ont eu accès."

Author response:

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

Reviewer #1 (Public Review):

(1) The authors make fairly strong claims that "arousal-related fluctuations are isolated from neurons in the deep layers of the SC" (emphasis added). This conclusion is based on comparisons between a "slow drift axis", a low-dimensional representation of neuronal drift, and other measures of arousal (Figures 2C, 3) and motor output sensitivity (Figures 2B, 3B). However, the metrics used to compare the slow-drift axis and motor activity were computed during separate task epochs: the delay period (600-1100 ms) and a perisaccade epoch (25 ms before and after saccade initiation), respectively. As the authors reference, deep-layer SC neurons are typically active only around the time of a saccade. Therefore, it is not clear if the lack of arousal-related modulations reported for deep-layer SC neurons is because those neurons are truly insensitive to those modulations, or if the modulations were not apparent because they were assessed in an epoch in which the neurons were not active. A potentially more valuable comparison would be to calculate a slow-drift axis aligned to saccade onset. 

The reviewer makes an important point that the calculation of an axis can depend critically on the time window of neuronal response. We find when considering this that the slow drift axis is less sensitive to this issue because it is calculated on time-averaged activity over multiple trials. In previous work we found that slow drift calculated on the stimulus evoked response in V4 was very well aligned to slow drift calculated on pre-stimulus spontaneous activity (Cowley et al, Neuron, 2020, Supplemental Figure 3A and 3B). To address this issue in the present data, we compared the axis computed for an example session for neural activity during the delay period and neural activity aligned to saccade onset. As shown new Figure 2 – figure supplement 1 in the revised manuscript, we found a similar lack of arousal-related modulations for deep-layer SC neurons when slow drift was computed using the saccade epoch (25ms before to 25ms after the onset of the saccade). Figure 2 – figure supplement 1A shows loadings for the SC slow drift axis when it was computed using spiking responses during the delay period (as in the main manuscript analysis). In contrast, Figure 2 – figure supplement 1B shows loadings from the same session when the SC slow drift axis was computed using spiking responses during the saccade epoch. The plots are highly similar and in both cases the loadings were weaker for neurons recorded from channels at the bottom of the probe which have a higher motor index. Finally, we found that projections onto the SC slow drift axis for this session were strongly correlated when the slow drift axis was computed using spiking responses during the delay period and the saccade epoch (r = 0.66, p < 0.001, Figure 1C). Taken together, these results suggest that arousal-related modulations are less evident in deep-layer SC neurons irrespective of whether slow drift was computed during the delay or saccade epoch (see also Public Reviews, Reviewer 1, Point 2).

(2) More generally, arousal-related signals may persist throughout multiple different epochs of the task. It would be worthwhile to determine whether similar "slow-drift" dynamics are observed for baseline, sensory-evoked, and saccade-related activity. Although it may not be possible to examine pupil responses during a saccade, there may be systematic relationships between baseline and evoked responses. 

Similar to the point above, slow drift dynamics tend to be similar across different response epochs because they are averaged across many trials and seem to tap into responsivity trends that are robust across epochs. As shown in Author response image 1 below, and the Figure 2 – figure supplement 1 in the revised manuscript, similar dynamics were observed when the SC slow drift axis was computed using spiking responses during the baseline, delay, visual and saccade epochs. We did not investigate differences between baseline and evoked pupil responses in the current paper. However, these effects were characterized in one of our previous papers that focused exclusively on the relationship between slow drift and eye-related metrics (Johnston et al., 2022, Cereb. Cortex, Figure 6). In this previous work, we found a negative correlation between baseline and evoked pupil size. Both variables were significantly correlated with slow drift, the only difference being the sign of the correlation.

Author response image 1.

(A-C) Dynamics of slow drift for three example sessions when the SC slow drift axis was computed using spiking responses during the baseline, delay, visual and saccade epochs. Baseline = 100ms before the onset of the target stimulus; Delay = 600 to 1100ms after the offset of the target stimulus; Stim = 25ms to 125ms after the onset of the target stimulus; Sac = 25ms before to 25ms after the onset of the saccade.

Johnston R, Snyder AC, Khanna SB, Issar D, Smith MA (2022) The eyes reflect an internal cognitive state hidden in the population activity of cortical neurons. Cereb Cortex 32:3331–3346.

(3) The relationships between changes in SC activity and pupil size are quite small (Figures 2C & 5C). Although the distribution across sessions (Figure 2C) is greater than chance, they are nearly 1/4 of the size compared to the PFC-SC axis comparisons. Likewise, the distribution of r2 values relating pupil size and spiking activity directly (Figure 5) is quite low. We remain skeptical that these drifts are truly due to arousal and cannot be accounted for by other factors. For example, does the relationship persist if accounting for a very simple, monotonic (e.g., linear) drift in pupil size and overall firing rate over the course of an individual session? 

Firstly, it is important to note that the strength of the relationship between projections onto the SC slow drift axis and pupil size (r² = 0.06) is within the range reported by Joshi et al. (2016, Neuron, Figure 3). They investigated the median variance explained between the spiking responses of individual SC neurons and pupil size and found it to be approximately 0.02 across sessions. Secondly, our statistical approach of testing the actual distribution of r² values against a shuffled distribution was specifically designed to rule out the possibility that the relationship between SC spiking responses and pupil size occurred due to linear drifts. The shuffled distribution in Figure 2C of the main manuscript represents the variance that can be explained by one session’s slow drift correlated with another session’s pupil, which would contain effects that occurred due to linear drifts alone. That the actual proportion of variance explained was significantly greater than this distribution suggests that the relationship between projections onto the SC slow drift axis and pupil size reflects changes in arousal rather than other factors related to linear drifts.

Joshi S, Li Y, Kalwani RM, Gold JI (2016) Relationships between Pupil Diameter and Neuronal Activity in the Locus Coeruleus, Colliculi, and Cingulate Cortex. Neuron 89:221–234.

(4) It is not clear how the final analysis (Figure 6) contributes to the authors' conclusions. The authors perform PCA on: (i) residual spiking responses during the delay period binned according to pupil size, and (ii) spiking responses in the saccade epoch binned according to target location (i.e., the saccade tuning curve). The corresponding PCs are the spike-pupil axis and the saccade tuning axis, respectively. Unsurprisingly, the spikepupil axis that captures variance associated with arousal (and removes variance associated with saccade direction) was not correlated with a saccade-tuning axis that captures variance associated with saccade direction and omits arousal. Had these measures been related it would imply a unique association between a neuron's preferred saccade direction and pupil control- which seems unlikely. The separation of these axes thus seems trivial and does not provide evidence of a "mechanism...in the SC to prevent arousal-related signals interfering with the motor output." It remains unknown whether, for example, arousal-related signals may impact trial-by-trial changes in neuronal gain near the time of a saccade, or alter saccade dynamics such as acceleration, precision, and reaction time. 

The reviewer makes a good point, and we agree that more evidence is needed to determine if the separation of the pupil size axis and saccade tuning axis is the mechanism through which cognitive and arousal-related signals can be intermixed in the SC. In the revised manuscript (lines 679-682), we have raised this as a possible explanation that necessitates further study rather than stating definitively that it is the exact mechanism through which these signals are kept separate. Our analysis here is similar to the one from Smoulder et al (2024, Neuron, Fig. 2F), in which the interactions between reward signals and target tuning in M1 were examined (and found to be orthogonal). While we agree with the reviewer that it may seem “trivial” for these axes to be orthogonal, it does not have to be so. If, for example, neural tuning curves shifted with changes in pupil size through gain changes that revealed tuning or affected tuning curve shape, there could be projections of the pupil axis onto the target tuning axis. Thus, while we agree with the reviewer that it appears sensible for these two axes to be orthogonal, our result is nonetheless a novel finding. We have edited the text in our revised manuscript, however, to make sure the nuance of this point is conveyed to the reader.

Smoulder AL, Marino PJ, Oby ER, Snyder SE, Miyata H, Pavlovsky NP, Bishop WE, Yu BM, Chase SM, Batista AP. A neural basis of choking under pressure. Neuron. 2024 Oct 23;112(20):3424-33.

Reviewer #2 (Public Review):

(1) The greatest weakness in the present research is the fact that arousal is a functionally less important non-motoric variable. The authors themselves introduce the problem with a discussion of attention, which is without any doubt the most important cognitive process that needs to be functionally isolated from oculomotor processes. Given this introduction, one cannot help but wonder, why the authors did not design an experiment, in which spatial attention and oculomotor control are differentiated. Absent such an experiment, the authors should spend more time explaining the importance of arousal and how it could interfere with oculomotor behavior. 

Although attention does represent an important cognitive process, we did not design an experiment in which attention and oculomotor control are differentiated because attention does not appear to be related to slow drift. In our first paper that reported on this phenomenon, we investigated the effects of spatial attention on slow fluctuations in neural activity by cueing the monkeys to attend to a stimulus in the left or right visual field in a block-wise manner. Each block lasted ~20 minutes and we found that slow drift did not covary with the timing of cued blocks (see Figure 4A, Cowley et al., 2020, Neuron). Furthermore, there is a large body of work showing that arousal also impacts motor behavior leading to changes in a range of eye-related metrics (e.g., pupil size, microsaccade rate and saccadic reaction time - for review, see Di Stasi et al. 2013, Neurosci. Biobehav. Rev.). We also note that the terms attention and arousal are often used in nonspecific and overlapping ways in the literature, adding to some potential confusion here. Nonetheless, pupil-linked arousal is an important variable that impacts motor performance. This has now been stated clearly in the Introduction of the revised manuscript (lines 108-114) to address the reviewer’s concerns and highlight the importance of studying how precise fixation and eye movements are maintained even in the presence of signals related to ongoing changes in brain state. 

Cowley BR, Snyder AC, Acar K, Williamson RC, Yu BM, Smith MA (2020) Slow Drift of Neural Activity as a Signature of Impulsivity in Macaque Visual and Prefrontal Cortex. Neuron 108:551-567.e8.

(2) In this context, it is particularly puzzling that one actually would expect effects of arousal on oculomotor behavior. Specifically, saccade reaction time, accuracy, and speed could be influenced by arousal. The authors should include an analysis of such effects. They should also discuss the absence or presence of such effects and how they affect their other results. 

As described above, several studies across species have demonstrated that arousal impacts motor behavior e.g., saccade reaction time, saccade velocity and microsaccade rate (for review, see Di Stasi et al. 2013, Neurosci. Biobehav. Rev.). This has been clarified in the Introduction of the revised manuscript to address the reviewer's concerns (lines 108-114). Our prior work (Johnston et al, Cerebral Cortex, 2022) shows that slow drift impacts several types of oculomotor behavior. Overall, these studies highlight the impact of arousal on eye movements as a robust effect, and support the present investigation into arousal and oculomotor control signals. While we agree reaction time, accuracy, and speed all can be influenced by arousal depending on task demands, the present study is focused on the connection between slow fluctuations in neural activity, linked to arousal, and different subpopulations of SC neurons. 

Di Stasi LL, Catena A, Cañas JJ, Macknik SL, Martinez-Conde S (2013) Saccadic velocity as an arousal index in naturalistic tasks. Neurosci Biobehav Rev 37:968–975.

Johnston R, Snyder AC, Khanna SB, Issar D, Smith MA (2022) The eyes reflect an internal cognitive state hidden in the population activity of cortical neurons. Cereb Cortex 32:3331–3346.

(3) The authors use the analysis shown in Figure 6D to argue that across recording sessions the activity components capturing variance in pupil size and saccade tuning are uncorrelated. however, the distribution (green) seems to be non-uniform with a peak at very low and very high correlation specifically. The authors should test if such an interpretation is correct. If yes, where are the low and high correlations respectively? Are there potentially two functional areas in SC? 

We agree with the reviewer that our actual data distribution was non-uniform. We examined individual sessions with high and low variance explained and did not find notable differences. One source of this variation has to do with session length. Longer sessions in principle should have a chance distribution of variance explained closer to zero because they contained more time bins. Given that we had no specific hypothesis for a non-uniform distribution, we have simply displayed the full distribution of values in our figure and the statistical result of a comparison to a shuffled distribution.

Reviewer #3 (Public Review):

(1) However, I am concerned about two main points: First, the authors repeatedly say that the "output" layers of the SC are the ones with the highest motor indices. This might not necessarily be accurate. For example, current thresholds for evoking saccades are lowest in the intermediate layers, and Mohler & Wurtz 1972 suggested that the output of the SC might be in the intermediate layers. Also, even if it were true that the high motor index neurons are the output, they are very few in the authors' data (this is also true in a lot of other labs, where it is less likely to see purely motor neurons in the SC). So, this makes one wonder if the electrode channels were simply too deep and already out of the SC? In other words, it seems important to show distributions of encountered neurons (regardless of the motor index) across depth, in order to better know how to interpret the tails of the distributions in the motor index histogram and in the other panels of Figure Supplement 1. I elaborate more on these points in the detailed comments below. 

The reviewer makes a good point about the efferent signals from SC. It is true that electrical thresholds are often lowest in intermediate layers, though deep layers do project to the oculomotor nuclei (Sparks, 1986; Sparks & Hartwich-Young, 1989) and often intermediate and deep layers are considered to function together to control eye movements (Wurtz & Albano, 1980). As suggested by the reviewer, we have edited the text throughout the manuscript to say that slow drift was less evident in SC neurons with a higher motor index, as well as included the above references and points about the intermediate and deep layers (Lines 73-81). Aside from the question of which layers of the SC function as the “motor output”, the reviewer raises a separate and important question – are our deep recordings still in SC. Here, we can say definitively that they are. We removed neurons if they did not exhibit elevated (above baseline) firing rates during the visual or saccade epochs of the MGS task (see Methods section on “Exclusion criteria”). All included neurons possessed a visual, visuomotor or motor response, consistent with the response properties of neurons in the SC. In addition, we found a number of neurons well above the bottom of the probe with strong motor responses and minimal loadings onto the slow drift axis (see Figure 2 – figure supplement 1A), consistent with the reviewer’s comment that intermediate layer neurons are tuned for movement and play a role in saccade production.

Mohler CW, Wurtz RH. Organization of monkey superior colliculus: intermediate layer cells discharging before eye movements. Journal of neurophysiology. 1976 Jul 1;39(4):722-44.

Sparks DL. Translation of sensory signals into commands for control of saccadic eye movements: role of primate superior colliculus. Physiol Rev. 1986 Jan;66(1):118-71. doi: 10.1152/physrev.1986.66.1.118. PMID: 3511480.

Sparks DL, Hartwich-Young R. The deep layers of the superior colliculus. Reviews of oculomotor research. 1989 Jan 1;3:213-55.

Wurtz RH, Albano JE. Visual-motor function of the primate superior colliculus. Annu Rev Neurosci. 1980;3:189-226. doi: 10.1146/annurev.ne.03.030180.001201. PMID: 6774653.

(2) Second, the authors find that the SC cells with a low motor index are modulated by pupil diameter. However, this could be completely independent of an "arousal signal". These cells have substantial visual responses. If the pupil diameter changes, then their activity should be influenced since the monkey is watching a luminous display. So, in this regard, the fact that they do not see "an arousal signal" in most motor neurons (through the pupil diameter analyses) is not evidence that the arousal signal is filtered out from the motor neurons. It could simply be that these neurons simply do not get affected by the pupil diameter because they do not have visual sensitivity. So, even with the pupil data, it is still a bit tricky for me to interpret that arousal signals are excluded from the "output layers" of the SC. 

The reviewer makes an important point about the SC’s visual responses. Neurons with a low motor index are, conversely, likely to have a stronger visual response index. However, we do not believe that changes in luminance can explain why the correlation between SC spiking response and pupil size is weaker for neurons with a lower motor index. Firstly, the changes in pupil size observed in the current paper and our previous work are slow and occur on a timescale of minutes (Cowley et al., 2020, Neuron) and are correlated with eye movement measures such as reaction time and microsaccade rate (Johnston et al., 2022, Cerebral Cortex). This is in stark contrast to luminance-evoked changes in pupil size that occur on a timescale of less than a second. Secondly, as shown the new Figure 5 – figure supplement 1 in the revised manuscript, very similar results were found when SC spiking responses were correlated with pupil size during the baseline period, when only the fixation point was on the screen. Although the luminance of the small peripheral target stimulus can result in small luminance-evoked changes in pupil size, no changes in luminance occurred during the baseline period which was defined as 100ms before the onset of the target stimulus. In Figure 2 – figure supplement 1 and Author response image 1 above, we show that slow drift is the same whether calculated on the baseline response, delay period, or peri-saccadic epoch. Thus, the measurement of slow drift is insensitive to the precise timing of the selection of both the window for the spiking response and the window for the pupil measurement. If luminance were the explanation for the slow changes in firing observed in visually responsive SC neurons, it would require those neurons to exhibit robust, sustained tuned responses to the small changes in retinal illuminance induced by the relatively small fluctuations in pupil size we observed from minute to minute. We are aware of no reports of such behavior in visually-responsive neurons in SC. We have included these analyses and this reasoning in the revised manuscript on lines 478-495.

Reviewer#1 (Recommendations for the author):

(1) It would be useful to provide line numbers in subsequent manuscripts for reviewers.

Line numbers have been added in the revised version of the manuscript.

(2) Page #6; last sentence: "...even impact processing at the early to mid stages of the visuomotor transformation, without leading to unwanted changes in motor output." I do not believe the authors have provided evidence that arousal levels were not associated with changes in motor output.

As suggested by Reviewer 3 (see Public Reviews, Reviewer 3, Point 2), we have edited the text throughout the manuscript to say that slow drift was less evident in SC neurons with a higher motor index. This sentence in the revised manuscript now reads:

“This provides a potential mechanism through which signals related to cognition and arousal can exist in the SC, and even impact processing at the early to mid stages of the visuomotor transformation, without leading to unwanted changes in SC neurons that are linked to saccade execution.”

(3) Page #8; last paragraph: Although deep-layer SC neurons may not have been obtained during every recording session, a summary of the motor index scores observed along the probe across sessions would be useful to confirm their assumptions. 

See Author response image 2 below which shows the motor index of each recoded SC neuron on the x-axis and session number on the y-axis. The points are colored by to the squared factor loading which represents the variance explained between the response a neuron and the slow drift axis (see Figure 3B of the main manuscript). You can see from this plot that neurons with a stronger component loading (shown in teal to yellow) typically have a lower motor index whereas the opposite is true for neurons with a weaker component loading (shown in dark blue).

Author response image 2.

Scatter plot showing the motor index of each recorded neuron along with the session number in which it was recorded. The points are colored by to the squared factor loading for each neuron along the slow drift axis. Note that loadings above 0.5 (33 data points in total) have been thresholded at 0.5 so that we could effectively use the color range to show all of the slow drift axis loadings.

(4) Page #10; first paragraph: The authors should state the time window of the delay period used, since it may be distinct from the pupil analysis (first 200ms of delay). 

This has been stated in the revised version of the manuscript. The sentence now reads:

“We first asked if arousal-related fluctuations are present in the SC. As in previous studies that recorded from neurons in the cortex (Cowley et al., 2020), we found that the mean spiking responses of individual SC neurons during the delay period (chosen at random on each trial from a uniform distribution spanning 600-1100ms, see Methods) fluctuated over the course of a session while the monkeys performed the MGS task (Figure 2A, left).”

(5) Page #10; second paragraph: Extra period at the end of a sentence: " most variance in the data..". 

Fixed in the revised version of the manuscript.

(6) Page #12: "between projections onto the SC slow drift axis and mean pupil size during the first 200ms of the delay period when a task-related pupil response could be observed." What criteria was used to determine whether a task-related pupil response was observed? 

This was chosen based on the results of a previous study in our lab that used the same memory-guided saccade task to investigate the relationship between slow drift and changes in based and evoked pupil size (see Johnston et al., 2022, Cereb. Cortex, Figure 6B). The period was chosen based on plotting the average pupil size aligned on different trial epochs. As we show in Figure 5-figure supplement 3 above, the pupil interactions with slow drift did not depend on the particular time window of the pupil we chose.  

(7) Page #14; Figure 2A: The axes for the individual channels are strangely floating and quite different from all other figures. Please label the channel in the figure legend that was used as an example of the projected values onto the slow drift axis.

The figure has been changed in the revised version of the manuscript so that the tick mark denoting zero residual spikes per second is on the top layer of each plot. A scale bar was chosen instead of individual axes to reduce clutter in the figure as it was used to demonstrate how slow drift was computed. Residual spiking responses from all neurons were projected on the slow drift axis to generate the scatter plot in the bottom right-hand corner of Figure 2A. There is no single neuron to label.

(8) Page #16: "These results demonstrate that even though arousal-related fluctuations are present in the SC, they are isolated from deep-layer neurons that elicit a strong saccadic response and presumably reside closer to the motor output." In line with our major comments, lack of arousal-related activity during the delay period is meaningless for deep-layer SC neurons that are generally inactive during this time. It does not imply that there is no arousal signal! 

Addressed in Public Reviews, Reviewer 1, Point 1 & 2. We found a similar lack of arousal-related modulations reported for deep-layer SC neurons when slow drift was computed using the saccade epoch (Figure 1 above). In addition, similar dynamics were observed when the SC slow drift axis was computed using spiking responses during the baseline, delay, visual and saccade period (Figure 2).

(9) Page #18: "These findings provide additional support for the hypothesis that arousalrelated fluctuations are isolated from neurons in the deep layers of the SC." The same criticism from above applies.

Addressed in Public Reviews, Reviewer 1, Point 1 & 2.

(10) Page #20; paragraph 3: "Taken together, the findings outlined above..." Would be useful to be more specific when referring to "activity" ; e.g., "...these neurons did not exhibit large fluctuations in delay-period activity over time".

This sentence has been changed in the revised manuscript in light of the reviewer’s comments. It now reads:

“In addition to being more weakly correlated with pupil size, the spiking responses of these neurons did not exhibit large fluctuations over time (Figure 2), and when considering the neuronal population as a whole, explained less variance in the slow drift axis when it was computed using population activity in the SC (Figure 3) and PFC (Figure 4).”

Reviewer #3 (Recommendations for the author):

The paper is clear and well-written. However, I am concerned about two main points: 

(1) First, the authors repeatedly say that the "output" layers of the SC are the ones with the highest motor indices. This might not necessarily be accurate. For example, current thresholds for evoking saccades are lowest in the intermediate layers, and Mohler & Wurtz 1972 suggested that the output of the SC might be in the intermediate layers. Also, even if it were true that the high motor index neurons are the output, they are very few in the authors' data (this is also true in a lot of other labs, where it is less likely to see purely motor neurons in the SC). So, this makes one wonder if the electrode channels were simply too deep and already out of the SC. In other words, it seems important to show distributions of encountered neurons (regardless of motor index) across depth, in order to better know how to interpret the tails of the distributions in the motor index histogram and in the other panels of the figure supplement 1. I elaborate more on these points in the detailed comments below. 

Addressed in Public Reviews, Reviewer 3, Point 1.

(2) Second, the authors find that the SC cells with a low motor index are modulated by pupil diameter. However, this could be completely independent of an "arousal signal". These cells have substantial visual responses. If the pupil diameter changes, then their activity should be influenced since the monkey is watching a luminous display. So, in this regard, the fact that they do not see "an arousal signal" in most motor neurons (through the pupil diameter analyses) is not evidence that the arousal signal is filtered out from the motor neurons. It could simply be that these neurons simply do not get affected by the pupil diameter because they do not have visual sensitivity. So, even with the pupil data, it is still a bit tricky for me to interpret that arousal signals are excluded from the "output layers" of the SC. 

Addressed in Public Reviews, Reviewer 3, Point 2.

(3) I think that a remedy to the first point above is to change the text to make it a bit more descriptive and less interpretive. For example, just say that the slow drifts were less evident among the neurons with high motor index. 

We thank the reviewer for this suggestion (see Public Reviews, Reviewer 3, Point 1).

(4) For the second point, I think that it is important to consider the alternative caveat of different amounts of light entering the system. Changes in light level caused by pupil diameter variations can be quite large. 

We thank the reviewer for this suggestion (see Public Reviews, Reviewer 3, Point 2).

(5) Line 31: I'm a bit underwhelmed by this kind of statement. i.e. we already know that cognitive processes and brain states do alter eye movements, so why is it "critical" that high precision fixation and eye movements are maintained? And, isn't the next sentence already nulling this idea of criticality because it does show that the brain state alters the SC neurons? In fact, cognitive processes are already known to be most prevalent in the intermediate and deep layers of the SC. 

It seems clear that while cognitive state does affect eye movements, it is desirable to have some separation between cognitive state and eye movement control. Covert attention, for instance, is precisely a situation where eye movement control is maintained to avoid overt saccades to the attended stimulus, and yet there are clear indications of attention’s impact on microsaccades and fixation. We stand by our statement that an important goal of vision is to have precise fixation and movements of the eye, and yet at the same time the eyes are subject to numerous influences by cognitive state.

(6) Line 65: it is better to clarify that these are "functional layers" because there are actually more anatomical layers. 

We have edited this sentence in the revised version of the manuscript so that it now reads:

“The role of these projections in the visuomotor transformation depends on the functional layer of the SC in which they terminate”.

(7) Line 73: this makes it sound like only the deepest layers are topographically organized, which is not true. Also, as early as Mohler & Wurtz, 1972, it was suggested that the intermediate layers have the biggest impacts downstream of the SC. This is also consistent with electrical microstimulation current thresholds for evoking saccades from the SC. 

We have addressed the reviewers’ comments about the intermediate layers having the biggest impact downstream of the SC in Public Reviews, Reviewer 3, Point 1. Furthermore, line 73 has been changed in the revised manuscript so that it now reads:

“As is the case for neurons in the superficial and intermediate layers, they [SC motor neurons] form a topographically organized map of visual space (White et al. 2017; Robinson 1972; Katnani and Gandhi 2011)”.  

(8) Line 100: there is an analogous literature regarding the question of why unwanted muscle contractions do not happen. Specifically, in the context of why SC visual bursts do not automatically cause saccades (which is a similar problem to the ones you mention about cognitive signals interfering by generating unwanted eye movements), both Jagadisan & Gandhi, Curr Bio, 2022 and Baumann et al, PNAS, 2023 also showed that SC population activity not only has different temporal structure (Jagadisan & Gandhi) but also occupy different subspaces (Baumann et al) under these two different conditions (visual burst versus saccade burst). This is conceptually similar to the idea that you are mentioning here with respect to arousal. So, it is worth it to mention these studies here and again in the discussion. 

We are grateful to the reviewer for these suggestions and have included text in the Introduction (Lines 125-128) and Discussion (Lines 678-682) of the revised manuscript along with the references cited above.

(9) Line 147: as mentioned above, it is now generally accepted that there are quite a few "pure" motor neurons in the SC. This is consistent with what you find. E.g. Baumann et al., 2023. And, again see Mohler and Wurtz in the 1970's. So, I wonder how useful it is to go too much into this idea of the deeper motor neurons (e.g. the correlations in the other panels of the Figure 1 supplement). 

This is related to the reviewer’s comment that the output of the SC might be in the intermediate layers. This concern has been addressed in Public Reviews, Reviewer 3, Point 1.

(10) Figure 1 should say where the RF was for the shown spike rasters. i.e. were these the same saccade target across trials? And where was that location relative to the RF? It would help also in the text to say whether the saccade was always to the RF center or whether you were randomizing the target location. 

We centered the array of saccade targets using the microstimulation-evoked eye movement for SC (see Methods section “Memory-guided saccade task”) to find the evoked eccentricity, and then used saccade targets with equal spacing of 45 degrees starting at zero (rightward saccade target). We did not do extensive RF mapping beyond this microstimulation centering. In Figure 1, the spike rasters are shown for a target that was visually identified to be within the neuron’s RF based on assessing responses to all 8 target angles. We have added information about this to the figure caption.

(11) Line 218: but were there changes in the eye movement statistics? For example, the slow drift eye movements during fixation? Or even the microsaccades? 

Addressed in Public Reviews, Reviewer 2, Point 2.  

(12) Line 248: shuffling what exactly? I think that more explanation would be needed here. 

Addressed in Public Reviews, Reviewer 1, Point 3.  

(13) Line 263: but isn't this reflecting a sensory transient in the pupil diameter, since the target just disappeared? 

Addressed in Public Reviews, Reviewer 3, Point 2.  

(14) Line 271: I suspect that slow drift eye movements (in between microsaccades) would show higher correlations. Not sure how well you can analyze those with a video-based eye tracker. 

We agree that fixational drift would be a worthwhile metric, but it is not one we have focused on here and to our knowledge does require higher precision tracking. 

(15) Line 286: again, see above about similar demonstrations with respect to the visual and motor burst intervals, which clearly cause the same problem (even stronger) as the one studied here. 

See reply, including Figure 2.

(16) Line 330: again, I'm not sure deeper necessarily automatically means closer to the output. For example, current thresholds for evoked saccades grow higher as you go deeper. Maybe the authors can ask their colleague Neeraj Gandhi about this point specifically, just to be safe. Maybe the safest would be to remain descriptive about the data, and just say something like: arousal-related fluctuations were absent in our deepest recorded sites. 

Addressed in Public Reviews, Reviewer 3, Point 1.

(17) Line 332: likewise, statements like this one here would be qualified if the output was the intermediate layers......anyway if I understand what I read so far in the paper, the signal will be anyway orthogonal to the motor burst population subspace. So, maybe there's no need to emphasize that it goes away in the very deepest layers. 

See reply above, Public Reviews, Reviewer 1, Point 4.

(18) Figure 3A: related to the above, I think one issue could be that the deeper contacts might already be out of the SC. Maybe some cell count distribution from each channel should help in this regard. i.e. were you finding way fewer saccade-related neurons in the deepest channels (even though the few that you found were with high motor index)? If so, then wouldn't this just mean that the channel was too deep? I think there needs to be an analysis like this, to convince readers that the channels were still in the SC. Ideally, electrical stimulation current thresholds for evoking saccades at different depths would be tested, but I understand that this can be difficult at this stage. 

Addressed in Public Reviews, Reviewer 3, Point 1.

(19) I keep repeating this because in general, cognitive effects are stronger in the intermediate/deeper layers than in the superficial layers. If these interfere with eye movements like arousal, then why should arousal be different?

Few studies have investigated the effects of attention on “pure” movement SC neurons that only discharge during a saccade. One study, which we cited in Introduction (Ignashchenkova et al., 2004, Nat. Neurosci.), found significant differences in spiking responses between trials with and without attentional cueing for visual and visuomotor neurons. No significant difference was found for motor neurons, consistent with our hypothesis that signals related to cognition and arousal are kept separate from saccade-related signals in the SC.

(20) The problem with Figure 5 and its related text is that the neurons with low motor index are additionally visual. So, of course, they can be modulated if the pupil diameter changes!

Addressed in Public Reviews, Reviewer 3, Point 2.  

(21) I had a hard time understanding Figure 6. 

See reply above, Public Reviews, Reviewer 1, Point 4.

(22) Line 586: these cells have more visual responses and will be affected by the amount of light entering the eye. 

Addressed in Public Reviews, Reviewer 3, Point 2.

Synthèse sur la situation des enfants sans abri logés dans les écoles en France

Résumé

Le sans-abrisme infantile connaît une augmentation alarmante en France, avec une hausse de 133 % depuis 2020, exacerbée par l'inflation et la crise du logement.

Face à ce que le reportage décrit comme les "carences de l'État", des collectifs citoyens, notamment "Jamais sans toi" à Lyon, organisent l'occupation d'établissements scolaires pour offrir un abri nocturne à des familles à la rue.

Ce document de synthèse se penche sur ce phénomène à travers le témoignage d'une famille d'origine angolaise – une mère et ses enfants – hébergée dans une école lyonnaise.

Leur parcours met en lumière la précarité extrême, le traumatisme d'une tentative d'expulsion avortée, et l'impact psychologique profond sur les enfants.

La situation révèle une tension critique entre la solidarité citoyenne, incarnée par les enseignants et les parents d'élèves, et l'inaction des pouvoirs publics, qui non seulement échouent à proposer des solutions de logement pérennes, mais exercent également une pression administrative sur les acteurs de cette solidarité.

1. Le Phénomène du Sans-abrisme Infantile et la Réponse Citoyenne

Le reportage met en évidence une crise sociale majeure : l'explosion du nombre d'enfants sans domicile fixe en France.

• Expansion et Causes :

◦ Le sans-abrisme infantile a augmenté de 133 % depuis 2020.   

◦ Les facteurs identifiés sont l'inflation, la multiplication des expulsions locatives et la pénurie de logements sociaux.   

◦ Les solutions d'urgence, conçues pour être temporaires, "s'éternisent".

En 2023, les familles logées dans des écoles y sont restées en moyenne plus de six mois.

• L'Occupation des Écoles comme Palliatif :

◦ Face à cette situation, des collectifs citoyens comme "Jamais sans toi" à Lyon organisent l'occupation d'écoles pour héberger des familles.     ◦ Ampleur du phénomène à Lyon :      

▪ Actuellement, 17 écoles de la métropole lyonnaise accueillent 25 familles.       

▪ Depuis 2014, une soixantaine d'établissements ont servi de refuge à plus de 1000 enfants.   

◦ Ce mouvement n'est pas limité à Lyon ; des initiatives similaires existent à Strasbourg, Rennes et Paris.   

◦ Ce soutien repose sur la "générosité citoyenne" (parents d'élèves, professeurs, habitants) qui compense les défaillances de l'État.

2. Étude de Cas : Le Parcours d'une Famille Angolaise

Le reportage se concentre sur le témoignage poignant de Lucy (16 ans), Lina (12 ans) et leur mère, qui illustre la réalité humaine derrière les statistiques.

• De l'Angola à la Précarité en France :

◦ Arrivée en France lorsque Lucy avait 10 ans et Lina 5 ou 6 ans.   

◦ Premières expériences d'hébergement précaire : le 115 à Dijon dans une chambre partagée, puis un foyer à Digoin.   

◦ La journée, la famille devait quitter le 115 et trouver refuge dans des associations (Secours Populaire, églises) pour manger.   

◦ Lina décrit sa déception face à la réalité française, loin de l'image idéalisée des dessins animés :

« Un pays super bien, que tout se passait bien, qu'on avait une vie normale ».  

◦ Elle a également été victime de moqueries et de racisme à l'école en raison de sa langue et de ses cheveux.

• Le Traumatisme de l'Expulsion Manquée (OQTF) :

◦ Il y a deux ans, la famille a fait l'objet d'une Obligation de Quitter le Territoire Français (OQTF).  

◦ La police est intervenue en pleine nuit dans leur appartement. Lucy, alors âgée de 14 ans, décrit une scène de panique et de violence :

ses parents criant, son père menotté, et les enfants enfermés dans une chambre avec des policiers.   

◦ La famille a été conduite à Paris après 5 heures de route et placée dans un centre de détention pendant 4 heures.   

◦ À l'aéroport, leur vol pour l'Angola a été annulé. Les autorités les ont alors "abandonnés à l'aéroport", leur ordonnant simplement "de plus retourner où [ils] étaient".

• La Rupture Familiale et l'Errance :

◦ Après cet épisode, la famille est revenue à Lyon.

Le mariage des parents n'étant pas reconnu en France, leur séparation a suivi. La mère s'est retrouvée seule avec ses enfants.   

◦ Ils ont enchaîné les solutions d'hébergement temporaires :

un camping à Trévoux, un appartement à Bellecour, puis une association qui les a logés avec d'autres femmes, avant de trouver refuge dans l'école.

3. La Vie Quotidienne dans une Salle de Classe

L'école, bien qu'offrant un toit, impose des conditions de vie extrêmement contraignantes et précaires.

Aspect

Description

Logement

La famille dort sur des matelas gonflables dans une salle de classe. Les vêtements sont stockés dans les armoires de la classe et des valises.

Routine

Lever obligatoire entre 6h30 et 6h50.

La famille doit quitter les lieux avant 8h30 et ne peut revenir qu'après 18h00, une fois tous les élèves partis.

Discrétion

La nuit, il est interdit d'allumer les lumières pour ne pas attirer l'attention.

La famille utilise les lampes de poche des téléphones pour s'éclairer.

Insecurité

Des jeunes jouant dans la cour sont déjà montés et ont fouillé dans leurs affaires, profitant d'une porte laissée ouverte.

Perturbations

La vie de la famille est rythmée par la sonnerie de l'école, qui retentit "toutes les heures".

Lutte de la mère

Elle cherche activement du travail (nettoyage, restauration) et des formations gratuites, mais sa situation rend les démarches très difficiles.

4. Impacts Psychologiques et Sociaux sur les Enfants

La précarité et l'instabilité ont des conséquences profondes sur le bien-être et le développement des enfants.

• Le Poids du Secret et de la Honte :

◦ Lucy cache sa situation à la plupart de ses amies par peur du jugement :

« J'angoisse un peu, sachant que beaucoup de jeunes de mon âge [...] se permettent de juger tout simplement. »  

◦ Elle exprime un profond désir de normalité : « Des fois, je me dis que j'aimerais juste avoir une vie normale comme plein d'ados de mon âge. »  

◦ Lina exprime également la peur d'être mise à l'écart par ses camarades parce qu'elle vit dans une école.

• Aspirations et Résilience :

◦ Malgré les épreuves, Lucy est une bonne élève et aspire à devenir avocate.

Son ambition est directement liée à son vécu : « J'ai envie d'être avocate, de défendre les gens parce que je me dis que tout le monde a le droit à une deuxième chance. »   

◦ Face à la détresse, elle a développé une stratégie de contrôle émotionnel : « Quand c'est dur, bah je prends sur moi et puis je me dis ça va aller. »  

◦ Sa plus grande peur reste matérielle et existentielle : « J'ai peur de me retrouver à la rue. Ça me fait peur. »

5. La Solidarité Face à l'Inaction Institutionnelle

Le reportage oppose la solidarité active du terrain à la réponse passive, voire répressive, des institutions.

• Le Soutien du Corps Enseignant :

◦ Une enseignante de l'école s'est fortement impliquée, dormant sur place la première nuit pour rassurer l'équipe périscolaire.  

◦ Elle a accueilli la famille chez elle pendant les vacances de Noël, une période particulièrement symbolique car la famille avait passé le Noël précédent dehors.  

◦ Une cagnotte organisée par ses collègues a permis d'offrir des cadeaux et un repas de fête à la famille.

• La Pression de la Hiérarchie :

◦ Suite à l'occupation, l'enseignante et ses collègues ont été convoquées par l'inspectrice d'académie.   

◦ La rencontre est décrite comme "un bon remontage de bretelle", où elles se sont fait "engueuler".

L'inspectrice les a qualifiées d' "inconscientes", leur faisant porter "toute la responsabilité" sans reconnaître la vulnérabilité de la famille.

• L'Absence de Solutions Pérennes :

◦ Près d'un an après le début de l'occupation, "il n'y a aucune proposition de la mairie, de la métropole, aucune perspective, rien."   

◦ L'occupation de l'école a donc dû se poursuivre au-delà de l'année scolaire, mais avec des règles plus strictes :

la famille n'a plus le droit d'être dans le bâtiment pendant les heures de classe.

Roots are in capitals, and are not words in use at all, but serve as an elucidation of the words grouped together and a connection between them.

J.R.R. Tolkien's note in the Qenya Lexicon[1]

Sauron new

knew that he had been wrong - not everyone would want to use the ring for their own power and Glory

yes Frodo succumbed at the very very end but - he and Sam made it that far and - fate or Providence or the intervention of Uru? himself did the rest

some people are capable of selfless and purely good acts

it wasn't just just Sauron who fell - it was his entire worldview

hope and love and care and friendship - can triumph over evil - however powerful it may seem at the time

Modélisez ; tous les autres verbes de la listes sont conjugués à la 2ème personne du pluriel

attention, pas d'espace avant la virgule. il y a une espace après tous les signes de ponctuation, mais l'espace avant n'est que pour les signes de ponctuation doubles (; : ! ?)

https://hyp.is/go?url=https%3A%2F%2Fopenknowledgemaps.org%2Fsearch_api%2Fserver%2Fpaper_preview%2Fe848f2c418eac460c47698a7cffc71f1a59e4cb6b020dba2c862dbd0b96d10e6.PDF&group=world

escribir a partir de los antecendentes las secciones siguientes (que salen entonces de la secciones)

Résumé de la vidéo [00:00:14][^1^][1] - [00:28:20][^2^][2]:

Cette vidéo explore la managérialisation des associations et ses impacts.

Elle aborde les défis et propose des solutions pour renforcer le monde associatif face à cette tendance.

Temps forts:

• [00:00:14][^3^][3] Introduction et contexte
  • Accueil des participants
  • Présentation du webinaire
  • Objectifs de la série
• [00:03:27][^4^][4] Enjeux de la managérialisation
  • Définition et historique
  • Impact sur les associations
  • Comparaison avec d'autres modèles
• [00:07:03][^5^][5] Conséquences et critiques
  • Perte de dimension démocratique
  • Réduction des relations humaines
  • Exemples concrets et témoignages
• [00:15:01][^6^][6] Solutions et alternatives
  • Importance de la participation
  • Réappropriation des termes
  • Exemples de bonnes pratiques
• [00:22:00][^7^][7] Conclusion et perspectives
  • Invitation à l'action collective
  • Importance de la cohérence interne
  • Appel à la réflexion et à l'innovation

Résumé de la vidéo [00:28:22][^1^][1] - [00:54:06][^2^][2]:

Cette vidéo explore la gestion et la gouvernance des associations face à la managérialisation.

Elle met en lumière l'importance de la circulation de l'information, de l'intelligence collective, et de la délibération pour une gouvernance démocratique et efficace.

Points forts : + [00:28:22][^3^][3] Circulation de l'information * Importance de la diffusion de l'information * Mise en commun des connaissances * Héritage des sociétés savantes + [00:29:57][^4^][4] Intelligence collective * Animation et maïeutique * Création d'espaces de travail collaboratif * Qualité de l'animation + [00:31:02][^5^][5] Délibération et décision * Importance de la délibération pour de bonnes décisions * Définition de la démocratie par Paul Ricœur * Travail sur les contradictions + [00:35:02][^6^][6] Tensions et réussites * Identification des tensions dans la gouvernance * Conditions de réussite * Création d'une communauté apprenante + [00:39:02][^7^][7] Exemple pratique * Transformation de la gouvernance au sein du Réseau d'Échange et de Services aux Associations du Pays de Morlaix * Passage à un système de cercles thématiques * Participation et implication des salariés et bénévoles

Ces points forts couvrent les principaux aspects abordés dans la vidéo, offrant une vue d'ensemble des défis et des solutions pour une gouvernance associative efficace.

Résumé de la vidéo [00:54:11][^1^][1] - [01:19:33][^2^][2]:

Cette partie du webinaire traite de la gestion et de l'organisation des associations, en mettant l'accent sur la coprésidence et la participation collective.

Points forts : + [00:54:11][^3^][3] Introduction de la coprésidence * Modification des statuts en 2020 * Importance de la participation collective * Fonctionnement en commissions thématiques + [00:57:02][^4^][4] Formation et participation * Formation annuelle sur la gestion collective * Ouverture des chantiers de travail aux adhérents * Importance de la transparence et de la clarté + [01:00:00][^5^][5] Déplacements et cohésion * Budget pour les déplacements collectifs * Renforcement des liens entre membres * Importance de la convivialité et du plaisir + [01:03:09][^6^][6] Intégration de nouveaux membres * Augmentation du nombre de membres du CA * Processus d'intégration et d'accompagnement * Maintien de la transparence et de la confiance + [01:09:09][^7^][7] Réflexion sur le temps et la gouvernance * Importance de la gestion du temps * Opposition au néolibéralisme * Outils pratiques pour la gouvernance associative

Résumé de la vidéo [01:19:36][^1^][1] - [01:46:07][^2^][2]:

Cette vidéo traite de la managérialisation des associations et des défis liés à la gestion collective et à la formation continue des membres.

Temps forts: + [01:19:36][^3^][3] Partage d'expériences * Importance de partager les échecs * Encouragement à la discussion collective * Utilisation des retours d'expérience + [01:22:01][^4^][4] Formation continue * Formation des équipes salariées * Importance de la coopération * Nécessité de réexpliquer aux nouveaux membres + [01:27:03][^5^][5] Suivi des salariés * Organisation de réunions de médiation * Importance du bien-être au travail * Gestion des conflits internes + [01:33:00][^6^][6] Rôle du syndicalisme * Conditions de travail et temps de travail * Complémentarité entre engagement associatif et syndical * Importance de la démocratie interne + [01:38:00][^7^][7] Taille des associations * Impact de la taille sur la gestion * Importance de la volonté politique * Réflexion sur la géographie et l'échelle d'action

Résumé de la vidéo [01:46:09][^1^][1] - [01:58:34][^2^][2]:

Cette partie du webinaire aborde divers aspects de la gestion et de l'organisation des associations, en mettant l'accent sur les défis et les solutions possibles.

Temps forts: + [01:46:09][^3^][3] Questions sur la loi 3DS * Impact des certifications qualité * Partage de ressources et d'expertises * Importance de la loi pour les associations + [01:49:01][^4^][4] Réorganisation de la GD * Inclusion des salariés et bénéficiaires * Partenariat avec les financeurs * Protection des salariés uniques + [01:50:24][^5^][5] Participation des financeurs * Explication des projets aux financeurs * Importance de leur inclusion dans le CA * Délégation des responsabilités au sein de l'équipe + [01:53:06][^6^][6] Prévention des conflits d'intérêts * Retrait des élus des instances associatives * Importance de maintenir un lien fort avec les financeurs * Anticipation des changements législatifs + [01:55:00][^7^][7] Conclusion et perspectives * Recueil des expériences et des échecs * Construction d'une communauté apprenante * Invitation à partager des ressources et à poursuivre les échanges

La Prévention des Conflits d'Intérêts : Collectivités et Associations

Synthèse

Ce document de synthèse analyse les enjeux juridiques et pratiques liés à la prévention des conflits d'intérêts dans les relations entre les collectivités territoriales et les associations.

Basé sur les interventions d'experts juridiques et de formateurs d'élus, il met en lumière les risques pénaux encourus et propose des préconisations concrètes.

Les points critiques à retenir sont les suivants :

• 1. Le conflit d'intérêts n'est pas une infraction, mais un signal d'alerte. La situation devient délictuelle lorsqu'un élu ou un agent public, conscient de ce conflit, ne se déporte pas et participe à une décision, tombant ainsi sous le coup de la prise illégale d'intérêt, une infraction pénale sévèrement sanctionnée (jusqu'à 5 ans d'emprisonnement et 500 000 € d'amende).

• 1. La notion d'intérêt est extrêmement large. Elle couvre les intérêts matériels, mais aussi moraux ou familiaux. Il n'est pas nécessaire que l'élu se soit enrichi personnellement ou que la collectivité ait subi un préjudice ; la simple apparence d'une impartialité compromise peut suffire à caractériser l'infraction.
• 1. La règle pour les élus impliqués dans une association est le "déport général". Qu'ils soient membres du bureau à titre personnel ou en tant que représentants de la commune, ils doivent s'abstenir de toute participation à une délibération concernant cette association.

Ce déport doit être total :

• ◦ Absence de participation à l'instruction du dossier.
• ◦ Absence de participation aux débats.
• ◦ Absence de participation au vote.

• ◦ Sortie physique de la salle du conseil durant les débats et le vote.

• 1. Les élus locaux sous-estiment massivement ce risque. Les formations de terrain révèlent que la préoccupation principale des élus concerne les aspects techniques des subventions, tandis que le risque de conflit d'intérêts est souvent ignoré, en particulier dans les petites communes où les interférences entre mandats électifs et vie associative sont pourtant maximales.
• 1. Des outils et des bonnes pratiques existent pour sécuriser les processus.

La responsabilité première incombe à chaque élu, qui doit s'auto-évaluer en permanence.

Pour sécuriser les décisions, il est préconisé de voter les subventions au cas par cas, de systématiser la déclaration des conflits en début de séance et de s'appuyer sur des ressources externes comme la Haute Autorité pour la Transparence de la Vie Publique (HATVP) et le référent déontologue, désormais obligatoire pour toutes les communes.

1. Le Cadre Juridique et les Risques Pénaux

L'analyse juridique, menée par Luc Brunet de l'Observatoire SMAC, souligne la nécessité de distinguer deux notions fondamentales qui sont souvent confondues.

Définitions Fondamentales : Conflit d'Intérêts vs. Prise Illégale d'Intérêt

Le conflit d'intérêts est une situation, tandis que la prise illégale d'intérêt est une infraction pénale qui découle de la mauvaise gestion de cette situation. Caractéristique Conflit d'Intérêts Prise Illégale d'Intérêt Nature

Une situation d'interférence entre un intérêt public et des intérêts (publics ou privés) de nature à influencer ou paraître influencer l'exercice d'une fonction.

Une infraction pénale. Le fait de prendre, recevoir ou conserver, directement ou indirectement, un intérêt de nature à compromettre son impartialité.

Source Légale Loi du 11 octobre 2013

Article 432-12 du Code pénal

Sanction

Aucune (ce n'est pas une infraction). La situation doit être prévenue ou résolue.

Jusqu'à 5 ans d'emprisonnement et 500 000 € d'amende.

"Le conflit d'intérêts, c'est la vie. Nous avons tous des conflits d'intérêts. [...] Là où c'est pas normal [...] c'est quand on va se dire 'je vais surtout pas le dire que je suis en situation de conflit d'intérêt'. Et c'est là qu'on franchit la ligne jaune et qu'on passe [...] du côté du code pénal avec le délit de prise illégale d'intérêt." - Luc Brunet

Le Champ d'Application Vaste de la Prise Illégale d'Intérêt

Le délit de prise illégale d'intérêt est l'infraction numéro un pour laquelle les élus locaux sont poursuivis. Son champ d'application est particulièrement étendu :

• Tous les domaines : Contrairement au délit de favoritisme (limité à la commande publique), il s'applique à toutes les décisions d'une collectivité : urbanisme, recrutement, vente de biens, et notamment les subventions aux associations.

• Intérêt moral ou familial : L'intérêt n'est pas nécessairement matériel ou financier.

• Absence de préjudice requis : L'infraction est constituée même si la collectivité n'a subi aucun préjudice, voire si elle a bénéficié de l'opération.

• Intérêts indirects : Le délit couvre les intérêts pris par personne interposée (conjoint, ascendants, descendants, mais aussi amis proches).

La jurisprudence retient une vision très large : "l'infraction s'arrête où le soupçon s'arrête".

• La notion d'apparence : Il ne faut pas seulement ne pas être en conflit d'intérêts, mais aussi ne pas donner l'apparence de l'être.

La Doctrine de la Haute Autorité pour la Transparence de la Vie Publique (HATVP)

La HATVP a établi une doctrine pour clarifier les niveaux de risque. Pour les relations avec les associations, le risque est considéré comme large.

• Zone Rouge (Risque Large) : Concerne la participation d'un élu au sein d'un organisme de droit privé, comme une association, que ce soit à titre personnel ou comme représentant de la commune.

• Règle Appliquée : Le déport général. L'élu concerné doit s'abstenir de participer à toute délibération relative à cet organisme, y compris en l'absence d'enjeu financier direct. Adhérent ou Dirigeant : Une Distinction Cruciale ?

La question se pose de savoir si un simple adhérent est soumis aux mêmes règles qu'un membre du bureau (président, trésorier, etc.).

• Position de la HATVP (Avis du 3 mai 2022) : Le simple fait d'être adhérent ne justifie pas un déport systématique.

Cependant, une analyse au cas par cas doit être menée en fonction de la nature de l'association, de son nombre d'adhérents et de l'objet de la délibération.

• Conseil de Prudence : Face à l'incertitude de l'analyse au cas par cas, il est recommandé aux simples adhérents, par mesure de sécurité, de se déporter systématiquement lors du vote d'une subvention.

2. Règles Pratiques et Préconisations La prévention repose sur une démarche rigoureuse et transparente.

Les Quatre Étapes de la Prévention

• 1. Identifier les situations à risque : L'élu doit se poser les bonnes questions sur ses liens personnels, familiaux ou associatifs en rapport avec les dossiers de la collectivité.

• 2. Déclarer le conflit d'intérêts : Conformément à la Charte de l'élu local, l'élu doit faire connaître ses intérêts personnels avant le débat et le vote.

• 3. Se déporter complètement : Le déport ne se limite pas au non-vote. L'élu ne doit participer ni à l'instruction du dossier, ni aux débats qui précèdent le vote.

• 4. Ne pas influencer : L'élu doit s'abstenir de toute intervention, même informelle ("tirer les ficelles par derrière").

Jurisprudence : Des Exemples Concrets et Marquants Deux cas illustrent la sévérité avec laquelle la justice appréhende ce délit :

• Le maire de Plougastel-Daoulas : Des élus membres du bureau d'une association ad hoc n'ont pas participé au vote de la subvention, mais sont restés dans la salle.

Ce simple fait a été jugé suffisant pour caractériser une influence et a conduit à leur condamnation pour prise illégale d'intérêt.

• Une commune rurale de 250 habitants : Des élus, membres du bureau d'une association organisant la fête du village, ont participé au vote d'une subvention de 250 €.

Ils ont été condamnés pour prise illégale d'intérêt suite à la plainte d'un opposant politique.

Ces exemples démontrent que ni la bonne foi, ni la poursuite de l'intérêt général, ni le faible montant de la subvention ne constituent des protections contre une condamnation.

Préconisations pour Sécuriser les Délibérations

• Pas de vote global : Les subventions aux associations doivent être votées une par une, jamais en bloc.

• Sortir de la salle : L'élu concerné doit physiquement quitter la salle du conseil avant le début des débats et ne revenir qu'une fois le point de l'ordre du jour traité. Cette sortie doit être consignée au procès-verbal.

• Instaurer un "tour de table" déontologique : En début de chaque conseil, le maire peut demander à chaque élu de signaler d'éventuels conflits d'intérêts au regard de l'ordre du jour.

3. Le Témoignage du Terrain : Entre Méconnaissance et Difficultés d'Application

Le témoignage de Sophie Van migom, directrice d'un centre de formation pour élus, révèle un décalage important entre les exigences légales et la perception des élus sur le terrain.

Une Prise de Conscience Limitée chez les Élus

Lors des formations, les préoccupations des élus portent majoritairement sur des questions techniques (conventionnement, prêt de matériel, contrôle financier).

Le risque de conflit d'intérêts est très rarement abordé spontanément, en particulier par les élus des petites communes.

"Sur 90 participants, je n'ai que deux élus qui m'ont parlé de conflit d'intérêt. [...] Les élus des petites communes ne se posent pas la question, alors qu'il y a forcément des interférences entre leur mandat électif, leur vie familiale, leur vie associative." - Sophie Van migom

Les Conséquences Pratiques et les Défis Opérationnels

L'application stricte des règles de déport peut engendrer des difficultés de fonctionnement :

• Problèmes de quorum : Dans une commune de 620 habitants, la mise en place de règles de déport strictes a conduit à ce que la moitié du conseil municipal sorte de la salle, empêchant le quorum d'être atteint. La seule solution est de reconvoquer le conseil, ce qui retarde la décision.

• Paralysie de l'action des élus : Un élu engagé pour son expertise associative (ex: président de l'association des parents d'élèves devenu adjoint aux écoles) peut se retrouver dans l'incapacité d'agir sur les dossiers pour lesquels il a été élu.

Les Doubles Sanctions : Pénale et Administrative Le non-respect des règles de déport expose l'élu et la collectivité à un double risque :

1. Le risque pénal : L'élu est poursuivi pour prise illégale d'intérêt et le maire pour complicité.

2. Le risque administratif : La délibération elle-même est illégale.

Elle peut être annulée par le juge administratif suite à un recours d'un opposant, d'un contribuable ou du préfet. L'association pourrait alors être contrainte de rembourser la subvention perçue.

4. Outils et Bonnes Pratiques

La Responsabilité Personnelle de l'Élu

C'est à chaque élu d'évaluer sa propre situation, d'informer le maire et le conseil, et de prendre la décision de se déporter.

Cette réflexion doit être menée dès le début du mandat pour clarifier les limites de ses fonctions.

Les Aides à la Décision

Les élus ne sont pas seuls face à ces questionnements complexes. Ils peuvent solliciter :

• La Haute Autorité pour la Transparence de la Vie Publique (HATVP) : Il est possible de saisir la HATVP pour obtenir un avis confidentiel et rapide sur une situation personnelle.

• Le référent déontologue : Sa désignation est une obligation pour toutes les collectivités. Il offre un avis qui va au-delà du strict droit, en abordant les questions de probité et d'exemplarité.

Cas Spécifiques Abordés

• Agents de la collectivité : Ils sont également concernés par le délit.

S'ils sont en situation de conflit d'intérêts sur un dossier (ex: instruction d'un marché public pour l'entreprise d'un proche), ils doivent le signaler à leur hiérarchie pour que le dossier leur soit retiré.

• Subventions en nature : La mise à disposition de locaux, de matériel ou d'agents est considérée comme un avantage et suit exactement les mêmes règles de déport que les subventions financières.

• Associations "transparentes" : Une association qui n'est en réalité que le prolongement de la collectivité (ex: toutes les décisions sont prises par la commune) pose des problèmes juridiques majeurs.

Toutes les règles de la collectivité (comptabilité publique, marchés publics) s'appliquent alors à elle, créant un risque juridique élevé.

Medición de cohesión social longitudinal en Chile

Y en la presentación mencionar que es con ELSOC ... y qué es ELSOC, qué es COES, sentido del documento, etc etc. Es decir, falta redactar bien la presentación.

C'est à cause de l'être humain qui laisse le numérique intègre dans tous les domaines de la vie, qui nous aboutis à perde de plus en plus notre identité

on ne peut plus être indépendant d'eux

Utilisation de la psychologie pour nous faire attacher au numérique

La naturalisation de numérique qui devient une partie essentiel dans notre quotidien

on est dépendant de la nouvelle technologie et donc on perd en autonomie.

la conceptualizacion y medicion de cohesion social para AL del OCS

Relevaría aquí que, dada la relevancia de la cohesión social para mantener la estabilidad de la sociedad, el lazo etc, poder medirlo empiricamete es clave y ello es algo fragmentado a nivel internacional y no existe un esfuerzo como tal en Chile antes del OCS

Est-il usuel de désigner la péninsule par le seul adjectif « Indochinoise » ?

Pouvez-vous confirmer que le « fonds indo-chinois » est bien écrit avec cette orthographe et sans la majuscule dans le titre de ce document ?

Plus haut, à la note 9, la « Note sur le fonds Indochinois » est localisée sous la cote E317 ; dans la note précédente, la cote E178 est identifiée comme celle de la « Note à l'attention de Monsieur l'Administrateur général sur l'urgente nécessité ... ». La cote E178 est-elle correcte ?

« Monsieur l'Administrateur général » est habituellement écrit au long dans les autres documents, sans la majuscule à « général ». Pouvez-vous confirmer que l'honorifique est bien abbrévié dans ce document et que la majuscule est présente ?

Puisque la thèse est désormais soutenue, y a-t-il un lien où il serait possible de renvoyer le lectorat ? Elle pourrait minimalement être intégrée à la bibliographie.

Un peu de contexte supplémentaire serait nécessaire ici. Dans le passage précédent, on croit comprendre que la demande de stage de Phan Vô Kỵ à la bibliothèque nationale est refusée ; il est pourtant ici question de « retour à Sài Gòn » (depuis la France, croit-on comprendre). Vous citez également le dossier personnel d'employé de Phan Vô Kỵ à la BnF : y a-t-il donc finalement occupé un rôle ? Si c'est le cas, il serait bon de préciser lequel.

Y a-t-il une raison pour laquelle « Sud Viêtnam » est orthographié de deux manières différentes ?

On devrait lire « quels efforts nous avons faits », puisque « faits », participe passé de l'auxiliaire « avoir », s'accorde en nombre avec le complément d'objet direct « efforts », placé devant. Si le texte de la citation a été reproduit fidèlement, il faudrait l'accompagner de la mention [sic].

VALEASE était désigné par l'abbréviation seulement : elle a été résolue lors de la première occurrence.

This wikipedia page goes into an engraved tablet meant for Ea-nasir. It's a complaint from a customer about the bad quality of his goods. This was in the ancient mesipotamian culture and Ea-nasir was selling copper. They complained about the quality of the copper and the treatment they got from the delivery guy making it the oldest written complaint.

Adler, Mortimer J. 1940. “How to Mark a Book.” Saturday Review of Literature 6: 250–52. https://www.unz.com/print/SaturdayRev-1940jul06-00011/ (January 11, 2023).

Annotations: https://via.hypothes.is/https://docdrop.org/download_annotation_doc/Adler---1940---How-to-Mark-a-Book-fehef.pdf

Annotations alternate: https://jonudell.info/h/facet/?user=chrisaldrich&max=100&exactTagSearch=true&expanded=true&url=https%3A%2F%2Fdocdrop.org%2Fdownload_annotation_doc%2FAdler---1940---How-to-Mark-a-Book-fehef.pdf

Prior [.pdf copy]9https://stevenson.ucsc.edu/academics/stevenson-college-core-courses/how-to-mark-a-book-1.pdf): - Annotations https://hypothes.is/users/chrisaldrich?q=url%3Ahttps%3A%2F%2Fstevenson.ucsc.edu%2Facademics%2Fstevenson-college-core-courses%2Fhow-to-mark-a-book-1.pdf<br /> - Alternate annotation link https://jonudell.info/h/facet/?user=chrisaldrich&max=100&exactTagSearch=true&expanded=true&url=https%3A%2F%2Fstevenson.ucsc.edu%2Facademics%2Fstevenson-college-core-courses%2Fhow-to-mark-a-book-1.pdf

Summary

• Marking a book helps in increasing "the most efficient kind of reading."
• The marked (pun intended) difference between physical vs. intellectual ownership of books
• 3 types of book owners:
  • 1. collector of wood pulp and ink
  • 1. one whose read most and dipped into some
  • 1. one who's annotated and sucked the marrow out of them
• Active reading (annotating and staying awake) and engaging deeply, arguing with, and questioning the author is the point of reading.
• A historical record of your active reading allows you to continue the conversation you've had with the author and yourself. (p12)
• Adler's method of reading and marking:
  1. Underlining major points of importance
  2. Vertical lines for emphasis
  3. Marginal marks (stars, asterisks, etc.) (10-20 per book) to indicate the most important statements in conjunction with dogearing these pages for making it easier to find them subsequently
  4. Numbers in the margin to sequence arguments
  5. Page numbers in the margin for linking ideas across pages, ostensibly for juxtaposing them later
  6. Circling key words or phrases (unsaid here, but this is helpful for indexing as well as helping one to come to terms with the author)
  7. Marginal writing for synopsis of sections as well as questions raised by the text; use of endpapers for a personal index of ideas presented chronologically throughout the book
• Objections to marking books:
  • Using scratch pad (or index cards, which he doesn't mention specifically, but which could be implied) so as not to destroy a precious or rare physical copy (this is a repetition from earlier in the article)
  • Marking slows you down. This is part of the point! Slowing down makes you engage with the author and get more out of the text.
  • You can't loan books because they contain your important thoughts which you don't want to give away (and lose the historical record of your thinking). Solution: Simply require friends to buy their own copy.

/hyperpost/🌐/🎭/gyuri/do/web/snarf/📅/2025/10/2/Metaphysics.of.Adjacency.html

self

view

I found this page 6 years ago because I did a search for the phrase "metaphysics of adjacency" as the meaning/intent-full hash/name for what I've been out-tuiting towards at the time. That one post was the only one web page out of petazillions that had this three word combination on it I would call it today metanexialitic intenional tacit awareness

From the ones adjacents its the ones that are portals to the longest trails well worth blazing and expolring

all driven from future orientated awareness of possibilities

or even impossibiities that desrve to be rendered inevitable and present

How ridiculous LLMs look from this perspective?

not to mention bogus notions like the manyverse?

all its apparent plausibikity is lost if we consider new trails spanninn to the future starting at every moment

https://via.hypothes.is/https://hyperpost.peergos.me/%F0%9F%8E%AD/gyuri/do/web/snarf/%F0%9F%93%85/2025/10/2/Metaphysics.of.Adjacency.html

Author response:

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

Reviewer #1 (Public review): 

Summary:

The authors describe the degradation of an intrinsically disordered transcription factor (LMO2) via PROTACs (VHL and CRBN) in T-ALL cells. Given the challenges of drugging transcription factors, I find the work solid and a significant scientific contribution to the field. 

Strengths: 

(1) Validation of LMO2 degradation by starting with biodegraders, then progressing to chemical degrades. 

(2)interrogation of the biology and downstream pathways upon LMO2 degradation (collateral degradation §

(3) Cell line models that are dependent/overexpression of LMO2 vs LMO2 null cell lines. 

(4) CRBN and VHL-derived PROTACs were synthesized and evaluated. 

Weaknesses: 

(1) The conventional method used to characterize PROTACs in the literature is to calculate the DC50 and Dmax of the degraders, I did not find this information in the manuscript. 

As noted in the reply to referee’s point 4 below, our first generation compounds are not highly potent. The DC₅₀ values have been computed specifically using Western blot reflected in the data shown in Fig. 2. The revised version Supplementary Fig. S3 shows these quantified Western blot data from a time course of treating KOPT-K1 cells with either Abd-CRBN and Abd-VHL, where the 24 hour blot data are shown in Figure 2, G and E, and the quantified data from each 24 hour treatment are quantified in Supplementary Fig. S3). With these data, the DC₅₀ values 9 μM for Abd-CRBN and 15 μM Abd-VHL), included in in the main text and the Supplementary Fig. S3 figure legend.

In addition, the loss of signal of the LMO2-Rluc reporter protein from PROTAC treated cells shown in Fig. 2M has been used to calculate a half-point of degradation; although strictly not DC₅₀, as it measures a reporter protein, this yielded values are 10 μM for Abd-CRBN and 9 μM Abd-VHL. 

(2) The proteomics data is not very convincing, and it is not clear why LMO2 does not show in the volcano plot (were higher concentrations of the PROTAC tested? and why only VHL was tested and not CRBN-based PROTAC?).

Due to the relatively small size of the LMO2 protein, it is challenging to produce enough unique peptides for reliable identification, especially to distinguish some proteins in the LMO2 complex.  

(3) The correlation between degradation potency and cell growth is not well-established (compare Figure 4C: P12-Ichikawa blots show great degradation at 24 and 48 hrs, but it is unclear if the cell growth in this cell line is any better than in PF-382 or MOLT-16) - Can the authors comment on the correlation between degradation and cell growth?  

In this study (Fig. 4) we did not aim to compare the effect of LMO2 loss on cell growth among LMO2 positive cells. Rather, we aimed to evaluate the LMO2 importance for cell growth in LMO2-expressing T-ALL cells compared to non-expressing cells and to correlate the loss of the protein with this effect on the cell growth. In addition, the treatment of cells with the LMO2 compounds did now show an effect to LMO2 negative cells until at least 48 hours of treatment indicating that low toxicity of our PROTAC compounds and providing correlation between LMO2 loss and cell growth. 

(4) The PROTACs are not very potent (double-digit micromolar range?) - can the authors elaborate on any challenges in the optimization of the degradation potency? 

The Abd methodology to use intracellular domain antibodies to screen for compounds that bind to intrinsically disordered proteins such as the LMO2 transcription factors offers a tractable approach to hard drug targets but, in so doing, creates challenging factors to improve the potency that are not the same as those targets for which structural data are available. LMO2 is an intrinsically disordered protein, for which soluble recombinant protein is not readily available to identify the binding pocket of compounds. The potency has so far been optimized solely based on the different moieties substituted in cell-based SAR studies (http://advances.sciencemag.org/cgi/content/full/7/15/eabg1950/DC1) and all new compounds were tested with BRET assays. Thus, currently optimization of the degradation potency (including properties such as improved solubility) for the LMO2-binding compounds relies on chemical modification the three areas of the compounds indicated in Fig. 2 B,C.  

(5) The authors mentioned trying six iDAb-E3 ligase proteins; I would recommend listing the E3 ligases tried and commenting on the results in the main text. 

The six chimaeric iDAb-E3 ligase proteins involved one anti-LMO2 iDAb and three different E3 ligase where either fused at the N- or the C-terminus of the VH (giving six protein formats). These six fusion proteins were described in the text referring to the degrader studies described in Supplementary Fig. 1. 

Reviewer #2 (Public review): 

Summary: 

Sereesongsaeng et al. aimed to develop degraders for LMO2, an intrinsically disordered transcription factor activated by chromosomal translocation in T-ALL. The authors first focused on developing biodegraders, which are fusions of an anti-LMO2 intracellular domain antibody (iDAb) with cereblon. Following demonstrations of degradation and collateral degradation of associated proteins with biodegraders, the authors proceeded to develop PROTACs using antibody paratopes (Abd) that recruit VHL (Abd-VHL) or cereblon (Abd-CRBN). The authors show dose-dependent degradation of LMO2 in LMO2+ T-ALL cell lines, as well as concomitant dose-dependent degradation of associated bHLH proteins in the DNA-binding complex. LMO2 degradation via Abd-VHL was also determined to inhibit proliferation and induce apoptosis in LMO2+ T-ALL cell lines. 

Strengths: 

The topic of degrader development for intrinsically disordered proteins is of high interest, and the authors aimed to tackle a difficult drug target. The authors evaluated methods, including the development of biodegraders, as well as PROTACs that recruit two different E3 ligases. The study includes important chemical control experiments, as well as proteomic profiling to evaluate selectivity. 

Weaknesses: 

The overall degradation is relatively weak, and the mechanism of potential collateral degradation is not thoroughly evaluated

The purpose of the study was to evaluate effects of LMO2 degraders. The mechanism of the observed collateral degradation could not be investigated directly within the scope of our study. In the main text, discussed two possible, not exclusive, explanations. One being that our work (and previously published, cited work) indicates that the DNA-binding bHLH proteins have relatively short half file (Supplementary Fig. S12) and may therefore be subject to normal turnover when the LMO2, which is in the complex, turns over. Further, the known structure of the LMO2-bHLH interactions (from Omari et al, doi: 10.1016/j.celrep.2013.06.008) was also examined for the location of lysines in the TAL1 & E47 partners (Supplementary Fig. S11). It is possible that their local association with the LMO2-E3-ligase complex created by the PROTAC interaction, could cause their concurrent degradation. Mutagenesis and structural analysis would be needed to establish this point.

In addition, experiments comparing the authors' prior work with their anti-LMO2 iDAb or Abl-L are lacking, which would improve our understanding of the potential advantages of a degrader strategy for LMO2.  

A major motivation behind developing the Antibody-derived (Abd) method to select compounds, which are surrogates of the antibody paratope, is because using iDAbs directly as inhibitors requires the development of delivery technologies for these macromolecules, as protein directly or as vectors or mRNA for their expression. Ultimately, high affinity anti-LMO2 iDAbs should directly be used as tractable inhibitors when delivery methods redeveloped. In the meantime, Abd compounds were envisaged as being surrogates suitable for development into reagents, and potentially drugs, by medicinal chemistry. We evaluated selected first generation LMO2-binding Abd compounds previously, finding their ability to interfere with LMO2-iDAb BRET signal to EC_max about 50% but these compounds do not have potency to have an effect on the interaction of LMO2 with a non-mutated iDAb (nM affinity). These data indicated that efficacy improvement for the PROTACs was needed. In addition, in the current study, we observed viability effects in T-ALL lines at high concentrations (20 μM) irrespective of LMO2 expression (Supplementary Fig. S 2A, B) These data indicated that efficacy improvement was needed and potentially converting the degraders (PROTACs) would add to in-cell potency. By adding the E3 ligase ligands, we found the toxicity of non-LMO2 expressing Jurkat was significantly reduced (Supplementary Fig. S 2E, F). 

Reviewer #2 (Recommendations for the authors): 

Suggestions for additional experiments: 

(1) The data presented is primarily focused on demonstrating targeted degradation of LMO2, with a focus on phenotypes such as proliferation and apoptosis. In this manuscript, there are limited comparative evaluations of anti-LMO2 iDAb or Abl-L to show the potential benefits of a degrader approach to their previously described work, as well as why targeted degradation is in fact, advantageous. For example, the authors' previous work has shown that anti-LMO2 iDAb inhibits tumor growth in a mouse transplantation model. Comparisons in vitro would be supportive of the importance of continued degrader optimization/development.  

we have previously shown that an anti-LMO2 scFv inhibits tumour growth in a mouse model but this work used an expressed scFv antibody that binds to LMO2 in nM range. The Abd compounds are much lower potency that the antibody and, because recombinant LMO2 is difficult to work with, we could only evaluate interactions of compounds with LMO2 in cell-based assays like BRET (LMO2-iDAb BRET). In this cell-based assay, the first generation Abd compounds do not have sufficient potency to block LMO2-iDAb interaction unless the affinity of the iDAb is reduced to sub-μM. The justification for proceeding on the degrader process rather than just using the protein-protein interaction (PPI) inhibition was based largely around the low potency of the first generation PPI compounds in cell assays and that incorporation protein degradation with PPI inhibition would enhance the efficacy.

In addition, the viability experiments are also very short-term; is there a reason why the authors did not carry out these experiments for 3-5 days to fully understand the impacts on proliferation? 

In Supplementary Fig. S5, we did show assays up to 3 days. In KOPT-K1 (LMO2+), the LMO2 levels were reduced during the time course of this assay (from a single compound dose at time zero) (Supplementary Fig S 5A, B). We also show CellTitreGlo assays up to 3 days and, with these second generation compounds, we observed sustained effects on KOPT-K1 (LMO2+) but low non-DMSO toxicity in Jurkat (LMO2-) (revised version Supplementary (Fig S5 C, D).

(2) The potential mechanism of collateral degradation is interesting and important in evaluating the on-target responses and consequences of degrading LMO2. At this time, the data supporting collateral degradation is limited and would be strengthened by showing that it is not due to a change in mRNA levels and not due to complex dissociation. Overall, the kinetics and depth of loss of complex members such as E47 in Figure 3 appear more substantial than LMO2 itself, and as presented, collateral degradation is not effectively demonstrated. In addition, to aid in the readers' assessments, additional background and references around the roles of TAL1 and E47 would be helpful. For example, structurally, where do they (and other associated proteins that are not degraded) fit in the complex? 

We have responded above in relation to the Public Review Comments and note that a structure of the complex was in submitted version (now revised version Supplementary Fig. S11). 

(3) In Figure 1A, the blots show decreased levels of endogenous CRBN with iDAB-CRBN. Is this a known consequence of this approach in these cell lines? Does the partial recovery of endogenous CRBN in KOPTK1 cells have any indication of iDAB-CRBN levels? 

We cannot be sure why the endogenous level of CRBN decreases in doxycycline treated cells. It has been shown (DOI:10.1371/journal.pone.0064561) that doxycycline used in the inducible expression system (and its derivatives), such as the lentivirus we used, has an effect to gene expression patterns, which can be increase or decrease expression. Although the published study did not examine CRBN expression, the effect might explain the CRBN expression decrease on doxycycline addition and remains the same level after that. 

(4) In Figure S7, the authors do not fully explain the results and why there is minimal rescue with epoxomicin (S7A) or MLN4924 (S7J). This could indicate an alternative mechanism of degradation and loss at play, given the lack of rescue. Can the authors comment on this discrepancy, and have they looked autophagy inhibitor or other agents to achieve the chemical rescue? 

In the experiments such as in revised version Supplementary Fig. S6, we used KOPT-K1 cells with a single concentration of the inhibitors and the cells may less susceptible to the epoxomicin (0.8 μM) but lenalidomide and free thalidomide restored the LMO2 levels fully. In the main text Fig. 3D, we also showed that including epoxomicin and thalidomide with the Abd-CRBN in KOPT-K1 and CCRF-CEM restore LMO2 levels, supporting the conclusion that the main mechanism of degradation is through ubiquitination proteosomal route.

(5) For the proteomics data, it would be helpful to have the proteins in yellow highlighted to have them noted in 5D and 5E. In addition, can the authors comment on why LMO2 or their collateral targets are not confirmed in the table? Furthermore, 5C is difficult to interpret; if there are no significantly changing proteins in the Jurkat cells, why are there pathways that are identified? 

As mentioned in reply to referee 1, due to the relatively small size of the LMO2 protein, it is challenging to produce enough unique peptides for reliable identification, especially to distinguish some proteins in the LMO2 complex where expression levels are low.

Gyuri Web Snarf 2025-10-2

exp - lore - eriment - perience

the info-morphic-communication flow

Let the spice of Life flow

Reviewer #3 (Public review):

Summary:

Through micro-electroencephalography, Hight and colleagues studied how the auditory cortex in its ensemble responds to cochlear implant stimulation compared to the classic pure tones. Taking advantage of a double-implanted rat model (Micro-ECoG and Cochlear Implant), they tracked and analyzed changes happening in the temporal and spatial aspects of the cortical evoked responses in both normal hearing and cochlear-implanted animals. After establishing that single-trial responses were sufficient to encode the stimuli's properties, the authors then explored several decoder architectures to study the cortex's ability to encode each stimulus modality in a similar or different manner. They conclude that a) intracranial EEG evoked responses can be accurately recorded and did not differed between normal hearing and cochlear-implanted rats; b) Although coarsely spatially organized, CI-evoked responses had higher trial-by-trial variability than pure tones; c) Stimulus identity is independently represented by temporal and spatial aspect of cortical representations and can be accurately decoded by various means from single trials; d) and that Pure tones trained decoder can't decode CI-stimulus identity accurately.

Strength:

The model combining micro-eCoG and cochlear implantation and the methodology to extract both the Event Related Potentials (ERPs) and High-Gammas (HGs) is very well designed and appropriately analyzed. Likewise, the PCA-LDA and TCA-LDA are powerful tools that take full advantage of the information provided by the cortical ensembles.

The overall structure of the paper, with a paced and exhaustive progress through each step and evolution of the decoder, is very appreciable and easy to follow. The exploration of single-trial encoding and stimulus identity through temporal and spatial domains is providing new avenues to characterize the cortical responses to CI stimulations and their central representation. The fact that single trials suffice to decode the stimulus identity regardless of their modality is of great interest and noteworthy. Although the authors confirm that iEEG remains difficult to transpose in the clinic, the insights provided by the study confirm the potential benefit of using central decoders to help in clinic settings.

Weaknesses:

The conclusion of the paper, especially the concept of distinct cortical encoding for each modality, is unfortunately partially supported by the results, as the authors did not adequately consider fundamental limitations of CI-related stimulation.

First, the reviewer assumed that the authors stimulated in a Monopolar mode, which, albeit being clinically relevant, notoriously generates a high current spread in rodent models. Second, comparing the averaged BF maps for iEEG (Figure 2A, C), BFs ranged from 4 to 16kHz with a predominance of 4kHz BFs. The lack of BFs at higher frequencies hints at a potential location mismatch between the frequency range sampled at the level of the cortex (low to medium frequencies) and the frequency range covered by the CI inserted mostly in the first turn-and-a-half of the cochlea (high to medium frequencies). Looking at Figure 2F (and to some extent 2A), most of the CI electrodes elicited responses around the 4kHz regions, and averaged maps show a predominance of CI-3-4 across the cortex (Figure 2C, H) from areas with 4kHz BF to areas with 16kHz BF. It is doubtful that CI-3-4 are located near the 4kHz region based on Müller's work (1991) on the frequency representation in the rat cochlea.

Taken together with the Pearsons correlations being flat, the decoder examples showing a strong ability to identify CI-4 and 3 and the Fig-8D, E presenting a strong prediction of 4kHz and 8kHz for all the CI electrodes when using a pure tone trained decoder, it is possible that current spread ended stimulating indistinctly higher turns of the cochlea or even the modiolus in a non-specific manner, greatly reducing (or smearing) the place-coding/frequency resolution of each electrode, which in turn could explain the coarse topographic (or coarsely tonotopic according to the manuscript) organization of the cortical responses. Thus, the conclusion that there are distinct encodings for each modality is biased, as it might not account for monopolar smearing. To that end, and since it is the study's main message and title, it would have benefited from having a subgroup of animals using bipolar stimulations (or any focused strategy since they provide reduced current spread) to compare the spatial organization of iEEG responses and the performances of the different decoders to dismiss current spread and strengthen their conclusion.

Nevertheless, the reviewer wants to reiterate that the study proposed by Hight et al. is well constructed, relevant to the field, and that the overall proposal of improving patient performances and helping their adaptation in the first months of CI use by studying central responses should be pursued as it might help establish new guidelines or create new clinical tools.

Reviewer #1 (Public review):

In this manuscript, Clausner and colleagues use simultaneous EEG and fMRI recordings to clarify how visual brain rhythms emerge across layers of early visual cortex. They report that gamma activity correlates positively with feature-specific fMRI signals in superficial and deep layers. By contrast, alpha activity generally correlated negatively with fMRI signals, with two higher frequencies within the alpha reflecting feature-specific fMRI signals. This feature-specific alpha code indicates an active role of alpha oscillations in visual feature coding, providing compelling evidence that the functions of alpha oscillations go beyond cortical idling or feature-unspecific suppression.

The study is very interesting and timely. Methodologically, it is state-of-the-art. The findings on a more active role of alpha activity that goes beyond the classical idling or suppression accounts are in line with recent findings and theories. In sum, this paper makes a very nice contribution. I still have a few comments that I outline below, regarding the data visualization, some methodological aspects, and a couple of theoretical points.

(1) The authors put a lot of effort into the figure design. For instance, I really like Figure 1, which conveys a lot of information in a nice way. Figures 3 and 4, however, seem overengineered, and it takes a lot of time to distill the contents from them. The fact that they have a supplementary figure explaining the composition of these figures already indicates that the authors realized this is not particularly intuitive. First of all, the ordering of the conditions is not really intuitive. Second, the indication of significance through saturation does not really work; I have a hard time discerning the more and less saturated colors. And finally, the white dots do not really help either. I don't fully understand why they are placed where they are placed (e.g., in Figure 3). My suggestion would be to get rid of one of the factors (I think the voxel selection threshold could go: the authors could run with one of the stricter ones, and the rest could go into the supplement?) and then turn this into a few line plots. That would be so much easier to digest.

(2) The division between high- and low-frequency alpha in the feature-specific signal correspondence is very interesting. I am wondering whether there is an opposite effect in the feature-unspecific signal correspondence. Would the high-frequency alpha show less of a feature-unspecific correlation with the BOLD?

(3) In the discussion (line 330 onwards), the authors mention that low-frequency alpha is predominantly related to superficial layers, referencing Figure 4A. I have a hard time appreciating this pattern there. Can the authors provide some more information on where to look?

(4) How did the authors deal with the signal-to-noise ratio (SNR) across layers, where the presence of larger drain veins typically increases BOLD (and thereby SNR) in superficial layers? This may explain the pattern of feature-unspecific effects in the alpha (Figure 3). Can the authors perform some type of SNR estimate (e.g., split-half reliability of voxel activations or similar) across layers to check whether SNR plays a role in this general pattern?

(5) The GLM used for modelling the fMRI data included lots of regressors, and the scanning was intermittent. How much data was available in the end for sensibly estimating the baseline? This was not really clear to me from the methods (or I might have missed it). This seems relevant here, as the sign of the beta estimates plays a major role in interpreting the results here.

(6) Some recent research suggests that gamma activity, much in contrast to the prevailing view of the mechanism for feedforward information propagation, relates to the feedback process (e.g., Vinck et al., 2025, TiCS). This view kind of fits with the localization of gamma to the deep layer here?

(7) Another recent review (Stecher et al., 2025, TiNS) discusses feature-specific codes in visual alpha rhythms quite a bit, and it might be worth discussing how your results align with the results reported there.

Reviewer #3 (Public review):

Summary:

Clausner et al. investigate the relationship between cortical oscillations in the alpha and gamma bands and the feature-specific and feature-unspecific BOLD signals across cortical layers. Using a well-designed stimulus and GLM, they show a method by which different BOLD signals can be differentiated and investigated alongside multiple cortical oscillatory frequencies. In addition to the previously reported positive relationship between gamma and BOLD signals in superficial layers, they show a relationship between gamma and feature-specific BOLD in the deeper layers. Alpha-band power is shown to have a negative relationship with the negative BOLD response for both feature-specific and feature-unspecific contrasts. When separated into lower (8-10Hz) and upper (11-13Hz) alpha oscillations, they show that higher frequency alpha showed a significantly stronger negative relationship with congruency, and can therefore be interpreted as more feature-specific than lower frequency alpha.

Strengths:

The use of interleaved EEG-fMRI has provided a rich dataset that can be used to evaluate the relationship of cortical layer BOLD signals with multiple EEG frequencies. The EEG data were of sufficient quality to see the modulation of both alpha-band and gamma-band oscillations in the group mean VE-channel TFS. The good EEG data quality is backed up with a highly technical analysis pipeline that ultimately enables the interpretation of the cortical layer relationship of the BOLD signal with a range of frequencies in the alpha and gamma bands. The stimulus design allowed for the generation of multiple contrasts for the BOLD signal and the alpha/gamma oscillations in the GLM analysis. Feature-specific and unspecific BOLD contrasts are used with congruently or incongruently selected EEG power regressors to delineate between local and global alpha modulations. A transparent approach is used for the selection of voxels contributing to the final layer profiles, for which statistical analysis is comprehensive but uses an alternative statistical test, which I have not seen in previous layer-fMRI literature.

A significant negative relationship between alpha-band power and the BOLD signal was seen in congruently (EEGco) selected voxels (predominantly in superficial layers) and in feature-contrast (EEGco-inco) selected (superficial and deep layers). When separated into lower (8-10Hz) and upper (11-13Hz) alpha oscillations, they show that higher frequency alpha showed a significantly stronger negative relationship with congruency than lower frequency alpha. This is interpreted as a frequency dissociation in the alpha-BOLD relationship, with upper frequency alpha being feature-specific and lower frequency alpha corresponding to general modulation. These results are a valuable addition to the current literature and improve our current understanding of the role of cortical alpha oscillations.

There is not much work in the literature on the relationship between alpha power and the negative BOLD response (NBR), so the data provided here are particularly valuable. The negative relationship between the NBR and alpha power shown here suggests that there is a reduction in alpha power, linked to locally reduced BOLD activity, which is in line with the previously hypothesized inhibitory nature of alpha.

Weaknesses:

It is not entirely clear how the draining vein effect seen in GE-BOLD layer-fMRI data has been accounted for in the analysis. For the contrast of congruent-incongruent, it is assumed that the underlying draining effect will be the same for both conditions, and so should be cancelled out. However, for the other contrasts, it is unclear how the final layer profiles aren't confounded by the bias in BOLD signal towards the superficial layers. Many of the profiles in Figure 3 and Figure 4A show an increased negative correlation between alpha power and the BOLD signal towards the superficial layers.

When investigating if high alpha (8-10 Hz) and low alpha (11-13 Hz) are two different sources of alpha, it would be beneficial to show if this effect is only seen at the group level or can be seen in any single subjects. Inter-subject variability in peak alpha power could result in some subjects having a single low alpha peak and some a single high alpha peak rather than two peaks from different sources.

The figure layout used to present the main findings throughout is an innovative way to present so much information, but it is difficult to decipher the main findings described in the text. The readability would be improved if the example (Appendix 0 - Figure 1) in the supplementary material is included as a second panel inside Figure 3, or, if this is not possible, the example (Appendix 0 - Figure 1) should be clearly referred to in the figure caption.

Reviewer #2 (Public review):

Summary:

This paper addresses an interesting issue: how is the search for a visual target affected by its orientation (and the viewer's) relative to other items in the scene and gravity? The paper describes a series of visual search tasks, using recognizable targets (e.g., a cat) positioned within a natural scene. Reaction times and accuracy at determining whether the target was present or absent, trial-to-trial, were measured as the target's orientation, that of the context, and of the viewer themselves (via rotation in a flight simulator) were manipulated. The paper concludes that search is substantially affected by these manipulations, primarily by the reference frame of gravity, then visual context, followed by the egocentric reference frame.

Strengths:

This work is on an interesting topic, and benefits from using natural stimuli in VR / flight simulator to change participants' POV and body position.

Weaknesses:

There are several areas of weakness that I feel should be addressed.

(1) The literature review/introduction seems to be lacking in some areas. The authors, when contemplating the behavioral consequences of searching for a 'rotated' target, immediately frame the problem as one of rotation, per se (i.e., contrasting only rotation-based explanations; "what rotates and in which 'reference frame[s]' in order to allow for successful search?"). For a reader not already committed to this framing, many natural questions arise that are worth addressing.

1a) Why do we need to appeal to rotation at all as opposed to, say, familiarity? A rotated cat is less familiar than a typically oriented one. This is a long-standing literature (e.g., Wang, Cavanagh, and Green (1994)), of course, with a lot to unpack.

1b) What are the triggers for the 'corrective' rotation that presumably brings reference frames back into alignment? What if the rotation had not been so obvious (i.e. for a target that may not have a typical orientation, like a hand, or a ball, or a learned, nonsense object?) or the background had not had such clear orientation (like a cluttered non-naturalistic background of or a naturalistic backdrop, but viewed from an unfamiliar POV (e.g., from above) or a naturalistic background, but not all of the elements were rotated)? What, ultimately, is rotated? The entire visual field? Does that mean that searching for multiple targets at different angles of rotation would interfere with one another?

1c) Relatedly, what is the process by which the visual system comes to know the 'correct' rotation? (Or, alternatively, is 'triggered to realize' that there is a rotation in play?) Is this something that needs to be learned? Is it only learned developmentally, through exposure to gravity? Could it be learned in the context of an experiment that starts with unfamiliar stimuli?

1d) Why the appeal to natural images? I appreciate any time a study can be moved from potentially too stripped-down laboratory conditions to more naturalistic ones, but is this necessary in the present case? Would the pattern of results have been different if these were typical laboratory 'visual search' displays of disconnected object arrays?

1e) How should we reconcile rotation-based theories of 'rotated-object' search with visual search results from zero gravity environments (e.g., for a review, see Leone (1998))?

1f) How should we reconcile the current manipulations with other viewpoint-perspective manipulations (e.g., Zhang & Pan (2022))?

(2) The presentation/interpretation of results would benefit from more elaboration and justification.

2a) All of the current interpretations rely on just the RT data. First, the RT results should also be presented in natural units (i.e., seconds/ms), not normalized. As well, results should be shown as violin plots or something similar that captures distribution - a lot of important information is lost when just presenting one 'average' dot across participants. More fundamentally, I think we need to have a better accounting for performance (percent correct or d') to help contextualize the RT results. We should at least be offered some visualization (Heitz, 2014) of the speed accuracy trade-off for each of the conditions. Following this, the authors should more critically evaluate how any substantial SAT trends could affect the interpretation of results.

2b) Unless I am missing something, the interpretation of the pattern of results (both qualitatively and quantitatively in their 'relative weight' analysis) relies on how they draw their contrasts. For instance, the authors contrast the two 'gravitational' conditions (target 0 deg versus target 90 deg) as if this were a change in a single variable/factor. But there are other ways to understand these manipulations that would affect contrasts. For instance, if one considers whether the target was 'consistent' (i.e., typically oriented) with respect to the context, egocentric, and gravitational frames, then the 'gravitational 0 deg' condition is consistent with context, egocentric view, but inconsistent with gravity. And, the 'gravitational 90 deg' condition, then, is inconsistent with context, egocentric view, but consistent with gravity. Seen this way, this is not a change in one variable, but three. The same is true of the baseline 0 deg versus baseline 90 deg condition, where again we have a change in all three target-consistency variables. The 'one variable' manipulations then would be: 1) baseline 0 versus visual context 0 (i.e., a change only in the context variable); 2) baseline 0 versus egocentric 0 (a change only in the egocentric variable); and 3) baseline 0 versus gravitational 0 (a change only in the gravitational variable). Other contrasts (e.g., gravitational 90 versus context 90) would showcase a change in two variables (in this case, a change in both context and gravity). My larger point is, again, unless I am really missing something, that the choice of how to contrast the manipulations will affect the 'pattern' of results and thereby the interpretation. If the authors agree, this needs to be acknowledged, plausible alternative schemes discussed, and the ultimate choice of scheme defended as the most valid.

2c) Even with this 'relative weight' interpretation, there are still some patterns of results that seem hard to account for. Primarily, the egocentric condition seems hard to account for under any scheme, and the authors need to spend more time discussing/reconciling those results.

2d) Some results are just deeply counterintuitive, and so the reader will crave further discussion. Most saliently for me, based on the results of Experiment 2 (specifically, the fact that gravitational 90 had better performance than gravitational 0), designers of cockpits should have all gauges/displays rotate counter to the airplane so that they are always consistent with gravity, not the pilot. Is this indeed a fair implication of the results?

2e) I really craved some 'control conditions' here to help frame the current results. In keeping with the rhetorical questions posed above in 1a/b/c/d, if/when the authors engage with revisions to this paper, I would encourage the inclusion of at least some new empirical results. For me the most critical would be to repeat some core conditions, but with a symmetric target (e.g. a ball) since that would seem to be the only way (given the current design) to tease out nuisance confounding factors such as, say, the general effect of performing search while sideways (put another way, the authors would have to assume here that search (non-normalized RT's and search performance) for a ball-target in the baseline condition would be identical to that in the gravitational condition.)

Files modified after 30/09/2025

/hyperpost/🌐/🎭/gyuri/📓/2025/10/list-files.html

Author response:

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

Reviewer #1 (Public review):

The authors present MerQuaCo, a computational tool that fills a critical gap in the field of spatial transcriptomics: the absence of standardized quality control (QC) tools for image-based datasets. Spatial transcriptomics is an emerging field where datasets are often imperfect, and current practices lack systematic methods to quantify and address these imperfections. MerQuaCo offers an objective and reproducible framework to evaluate issues like data loss, transcript detection variability, and efficiency differences across imaging planes.

Strengths:

(1) The study draws on an impressive dataset comprising 641 mouse brain sections collected on the Vizgen MERSCOPE platform over two years. This scale ensures that the documented imperfections are not isolated or anecdotal but represent systemic challenges in spatial transcriptomics. The variability observed across this large dataset underscores the importance of using sufficiently large sample sizes when benchmarking different image-based spatial technologies. Smaller datasets risk producing misleading results by over-representing unusually successful or unsuccessful experiments. This comprehensive dataset not only highlights systemic challenges in spatial transcriptomics but also provides a robust foundation for evaluating MerQuaCo's metrics. The study sets a valuable precedent for future quality assessment and benchmarking efforts as the field continues to evolve.

(2) MerQuaCo introduces thoughtful metrics and filters that address a wide range of quality control needs. These include pixel classification, transcript density, and detection efficiency across both x-y axes (periodicity) and z-planes (p6/p0 ratio). The tool also effectively quantifies data loss due to dropped images, providing tangible metrics for researchers to evaluate and standardize their data. Additionally, the authors' decision to include examples of imperfections detectable by visual inspection but not flagged by MerQuaCo reflects a transparent and balanced assessment of the tool's current capabilities.

Weaknesses:

(1) The study focuses on cell-type label changes as the main downstream impact of imperfections. Broadening the scope to explore expression response changes of downstream analyses would offer a more complete picture of the biological consequences of these imperfections and enhance the utility of the tool.

Here, we focused on the consequences of imperfections on cell-type labels, one common use for spatial transcriptomics datasets. Spatial datasets are used for so many other purposes that there are almost endless ways in which imperfections could impact downstream analyses. It is difficult to see how we might broaden the scope to include more downstream effects, while providing enough analysis to derive meaningful conclusions, all within the scope of a single paper. Existing studies bring some insight into the impact of imperfections and we expect future studies will extend our understanding of consequences in other biological contexts.

(2) While the manuscript identifies and quantifies imperfections effectively, it does not propose post-imaging data processing solutions to correct these issues, aside from the exclusion of problematic sections or transcript species. While this is understandable given the study is aimed at the highest quality atlas effort, many researchers don't need that level of quality to compare groups. It would be important to include discussion points as to how those cut-offs should be decided for a specific study.

Studies differ greatly in their aims and, as a result, the impact of imperfections in the underlying data will differ also, preventing us from offering meaningful guidance on how cut-offs might best be identified. Rather, our aim with MerQuaCo was to provide researchers with tools to generate information on their spatial datasets, to facilitate downstream decisions on data inclusion and cut-offs.

(3) Although the authors demonstrate the applicability of MerQuaCo on a large MERFISH dataset, and the limited number of sections from other platforms, it would be helpful to describe its limitations in its generalizability.

In figure 9, we addressed the limitations and generalizability of MerQuaCo as best we could with the available datasets. Gaining deep insight into the limitations and generalizability of MerQuaCo would require application to multiple large datasets and, to the best of our knowledge, these datasets are not available.

Reviewer #2 (Public review):

The authors present MerQuaCo, a computational tool for quality control in image-based spatial transcriptomic, especially MERSCOPE. They assessed MerQuaCo on 641 slides that are produced in their institute in terms of the ratio of imperfection, transcript density, and variations of quality by different planes (x-axis).

Strengths:

This looks to be a valuable work that can be a good guideline of quality control in future spatial transcriptomics. A well-controlled spatial transcriptomics dataset is also important for the downstream analysis.

Weaknesses:

The results section needs to be more structured.

We have split the ‘Transcript density’ subsection of the results into 3 new subsections.

Reviewer #3 (Public review):

MerQuaCo is an open-source computational tool developed for quality control in imagebased spatial transcriptomics data, with a primary focus on data generated by the Vizgen MERSCOPE platform. The authors analyzed a substantial dataset of 641 freshfrozen adult mouse brain sections to identify and quantify common imperfections, aiming to replace manual quality assessment with an automated, objective approach, providing standardized data integrity measures for spatial transcriptomics experiments.

Strengths:

The manuscript's strengths lie in its timely utility, rigorous empirical validation, and practical contributions to methodology and biological discovery in spatial transcriptomics.

Weaknesses:

While MerQuaCo demonstrates utility in large datasets and cross-platform potential, its generalizability and validation require expansion, particularly for non-MERSCOPE platforms and real-world biological impact.

We agree that there is value in expanding our analyses to non-Merscope platforms, to tissues other than brain, and to analyses other than cell typing. The limiting factor in all these directions is the availability of large enough datasets to probe the limits of MerQuaCo. We look forward to a future in which more datasets are available and it’s possible to extend our analyses

Reviewer #1(Recommendation for the Author):

(1) To better capture the downstream impacts of imperfections, consider extending the analysis to additional metrics, such as specificity variation across cell types, gene coexpression, or spatial gene patterning. This would deepen insights into how these imperfections shape biological interpretations and further demonstrate the versatility of MerQuaCo.

These are compelling ideas, but we are unable to study so many possible downstream impacts in sufficient depth in a single study. Insights into these topics will likely come from future studies.

(2) In Figure 7 legend, panel label (D) is repeated thus panels E-F are mislabelled. 

We have corrected this error.

(3) Ensure that the image quality is high for the figures. 

We will upload Illustrator files, ensuring that images are at full resolution.

Reviewer #2 (Recommendation for the Author):

(1) A result subsection "Transcript density" looks too long. Please provide a subsection heading for each figure. 

We have split this section into 3 with new subheadings.

(2) The result subsection title "Transcript density" sounds ambiguous. Please provide a detailed title describing what information this subsection contains. 

We have renamed this section ‘Differences in transcript density between MERSCOPE experiments’.

Minor: 

(1) There is no explanation of the black and grey bars in Figure 2A.

We have added information to the figure legend, identifying the datasets underlying the grey and black bars.

(2) In the abstract, the phrase "High-dimension" should be "High-dimensional". 

We have changed ‘high-dimension’ to ‘high-dimensional’.

(3) In the abstract, "Spatial results" is an unclear expression. What does it stand for? 

We have replaced the term ‘spatial results’ with ‘the outputs of spatial transcriptomics platforms’.

Reviewer #3 (Recommendation for the Author):

(1) While the tool claims broad applicability, validation is heavily centered on MERSCOPE data, with limited testing on other platforms. The authors should expand validation to include more diverse platforms and add a small analysis of non-brain tissue. If broader validation isn't feasible, modify the title and abstract to reflect the focus on the mouse brain explicitly.

We agree that expansion to other platforms is desirable, but to the best of our knowledge sufficient datasets from other platforms are not available. In the abstract, we state that ‘… we describe imperfections in a dataset of 641 fresh-frozen adult mouse brain sections collected using the Vizgen MERSCOPE.’

(2) The impact of data imperfections on downstream analysis needs a more comprehensive evaluation. The authors should expand beyond cluster label changes to include a) differential expression analysis with simulated imperfections, b) impact on spatial statistics and pattern detection, and c) effects on cell-cell interactions. 

Each of these ideas could support a substantial study. We are unable to do them justice in the limited space available as an addition to the current study.

(3) The pixel classification workflow and validation process need more detailed documentation. 

The methods and results together describe the workflow and validation in depth. We are unclear what details are missing.

(4) The manuscript lacks comparison to existing. QC pipelines such as Squidpy and Giotto. The authors should benchmark MerQuaCo against them and provide integration options with popular spatial analysis tools with clear documentation.

To the best of our knowledge, Squidpy and Giotto lack QC benchmarks, certainly of the parameters characterized by MerQuaCo. Direct comparison isn’t possible.

DOI: 10.1002/jnr.24803

Resource: Caenorhabditis Genetics Center (RRID:SCR_007341)

Curator: @sonofthor

SciCrunch record: RRID:SCR_007341

What is this?

Synthèse sur le rôle de l'alcool dans la société

Résumé

Ce document de synthèse analyse le rôle complexe et paradoxal de l'alcool dans la société, en se basant sur des perspectives historiques, socioculturelles, scientifiques et politiques.

L'alcool est présenté comme une substance à double tranchant : d'une part, un puissant lubrifiant social et un pilier de rituels culturels et de moments de convivialité, profondément ancré dans l'histoire de l'humanité depuis des millénaires.

D'autre part, il est une force destructrice majeure, responsable de 2 200 décès par jour en Europe selon l'OMS, lié à plus de 200 maladies, et engendrant des coûts sociétaux colossaux, estimés à 57 milliards d'euros par an rien qu'en Allemagne.

Le document met en lumière l'ambivalence fondamentale de la société face à l'alcool, oscillant entre sa célébration dans les rituels et la stigmatisation de la dépendance individuelle.

Les tentatives historiques et modernes de régulation se sont souvent heurtées à une forte résistance populaire, illustrant la difficulté de gérer une substance si intimement liée au plaisir, à l'identité et à la cohésion sociale.

En définitive, les politiques les plus efficaces pour réduire les méfaits de l'alcool, à savoir l'augmentation des prix et la limitation de l'accès, se heurtent à cette acceptation culturelle profondément enracinée.

1. Le Paradoxe Fondamental de l'Alcool : Plaisir et Destruction

L'alcool occupe une place centrale et ambivalente dans la société, incarnant à la fois le plaisir et le danger.

Cette dualité est au cœur de notre rapport à cette substance.

• Le Côté Positif : L'alcool est associé à des sensations agréables, comme une "douce sensation de chaleur dans le ventre", et à des contextes plaisants.

Il est perçu comme un facilitateur de convivialité, pouvant donner lieu à des "conversations intéressantes" et favoriser le sentiment d'appartenance.

Une citation résume bien ce paradoxe :

"je dis toujours que j'ai passé certaines des meilleures nuits de ma vie avec de l'alcool et aussi certaines des pires."

• Le Côté Sombre : Son pouvoir destructeur est immense.

◦ Mortalité : L'OMS estime qu'environ 2 200 personnes meurent chaque jour en Europe à cause de l'alcool.      ◦ Maladies : Des études récentes lient une consommation régulière d'alcool à plus de 200 maladies.   

◦ Dépendance : L'alcool est la troisième substance la plus addictive en Allemagne, après le tabac et les médicaments.

En France, une personne sur dix a un problème avec l'alcool.   

◦ Conséquences Sociales : Il mène à la solitude, l'anxiété, la dépression et la dépendance.

Bien que la consommation globale soit en baisse en Europe, elle reste significative.

En Allemagne, elle est passée de 141 L à 115 L de boisson alcoolisée par an et par habitant depuis 2008, ce qui équivaut encore à "une bière par jour".

2. Une Perspective Historique : Un Compagnon de l'Humanité

La relation de l'humanité avec l'alcool est millénaire, suggérant qu'il a pu jouer un rôle dans notre évolution et le développement de nos civilisations.

• Origines Ancestrales : Des indices suggèrent que l'alcool est "aussi vieux que l'humanité".

◦ Des archéologues ont découvert en Chine des récipients contenant des restes de vin vieux de 9 000 ans.   

◦ En Géorgie, la consommation d'alcool remonte à au moins 8 000 ans.  

◦ La découverte est probablement fortuite, issue de fruits fermentés naturellement.

• Avantages Historiques :

◦ Source d'Énergie : 1 gramme d'alcool contient 7 calories, soit presque le double des protéines ou des glucides.  

◦ Sécurité Sanitaire : L'alcool dissout la membrane des germes, rendant les boissons fermentées (bière, vin) plus sûres à consommer que l'eau potentiellement contaminée.  

◦ Moyen de Paiement : La bière était utilisée comme une quasi-monnaie.

Un bulletin de paie en argile de Mésopotamie, vieux de 5 000 ans, indique des unités de bière.

En Égypte, les ouvriers des pyramides étaient rémunérés en bière.

• Consommation Massive : Au Moyen Âge en Europe, des chercheurs estiment la consommation à 3 litres de boisson alcoolisée par jour et par habitant, y compris pour les enfants.

3. Le Rôle Socioculturel : Ciment des Relations Humaines

L'alcool est omniprésent dans les structures sociales, agissant comme un "lubrifiant social" et un marqueur des moments importants.

• Cohésion Sociale :

◦ Il favorise le "sentiment d'appartenance" en créant une expérience collective.   

◦ Une expérience a montré qu'un groupe consommant un peu de vodka "interagissait davantage, riait beaucoup plus et passait globalement un moment plus agréable".  

◦ Des études indiquent que les personnes qui fréquentent régulièrement les bars avec modération sont mieux intégrées socialement.

• Rituels et Célébrations : L'alcool sert à marquer la frontière entre le "quotidien et la normalité de l'exceptionnel".

◦ Il est présent à chaque étape de la vie : naissance ("mouiller la tête"), mariages (champagne), enterrements.   

◦ Même dans un contexte religieux, le vin est utilisé pour représenter le sang du Christ.  

◦ Utiliser une boisson plus chère et exceptionnelle comme le champagne pour un anniversaire est une façon de "marquer un moment solennel".

• Influence sur le Développement Sociétal :

◦ Sédentarisation : Une théorie postule que la production de bière sur des sites comme Göbekli Tepe (il y a 12 000 ans) a pu renforcer la cohésion sociale et inciter les groupes humains à se sédentariser.    

◦ Infrastructures : La production d'alcool a influencé le développement des moyens de transport (fûts), des espaces de stockage et des bâtiments (brasseries).

• Variations Culturelles : Les coutumes de consommation varient :

◦ Norvège : Sobriété la semaine, forte consommation le week-end.  

◦ France/Italie : Un verre de vin au déjeuner.

4. Impacts sur la Santé et Mécanismes d'Action

D'un point de vue chimique et biologique, les effets de l'alcool sur le corps expliquent à la fois son attrait et sa dangerosité.

• La Molécule d'Éthanol : Petite molécule (deux atomes de carbone, six d'hydrogène, un d'oxygène), elle traverse facilement la barrière hémato-encéphalique pour agir sur le cerveau.

• Action sur les Neurotransmetteurs : L'alcool influence trois systèmes principaux : | Système | Effet Principal | Conséquence | | :--- | :--- | :--- | | GABA | Anxiolytique | Sensation de détente, réduction de l'anxiété | | Glutamate | Augmente la vigilance | Stimulation de la présence et de l'attention | | Dopamine | Rend heureux | Sensation de plaisir, voire d'euphorie |

• Toxicité Métabolique :

◦ Le foie transforme l'alcool en acétaldéhïde, qui est un "poison".   

◦ Cette substance circule dans le sang et atteint tous les organes (cerveau, peau, etc.).  

◦ Dommages Spécifiques : L'alcool peut provoquer des gastrites (attaque des muqueuses de l'estomac), endommager le foie, entraîner une atrophie du cervelet et être toxique pour le pancréas.  

◦ Risque de Cancer : La consommation régulière d'alcool augmente le risque de tumeurs et de cancer.

5. Dépendance, Coûts et Ambivalence Sociétale

La société entretient une relation contradictoire avec l'alcool, le célébrant tout en laissant les individus gérer seuls ses conséquences les plus graves.

• La Dépendance :

◦ La plus grande difficulté est le déni : "plus les gens sont dépendants, moins ils se rendent compte qu'ils le sont."   

◦ La dépendance isole l'individu, produisant l'effet inverse du sentiment d'appartenance initialement recherché.

• Coûts Économiques :

◦ Selon l'annuaire des addictions, l'alcool coûte 57 milliards d'euros par an en Allemagne.  

◦ Ces coûts incluent les délits, la violence, la conduite en état d'ivresse, les arrêts maladie et les traitements.

• L'Hypocrisie Sociale :

◦ La société vend l'alcool comme "quelque chose de positif associé à des fêtes", mais "ceux qui ne savent pas gérer leur consommation sont livrés à eux-mêmes".

La responsabilité est individualisée.   

◦ Cette ambivalence se reflète dans les politiques publiques : en 2024, la Société allemande de nutrition a recommandé "zéro alcool", tandis que 30 % du budget de prévention des addictions était supprimé.  

◦ La publicité pour l'alcool reste peu réglementée et la "consommation accompagnée" (dès 14 ans) est autorisée en Allemagne.

6. Les Tentatives de Régulation et la Résistance Populaire

L'histoire montre que les tentatives de contrôle de la consommation d'alcool par les autorités se sont souvent soldées par des échecs face à la pression sociale.

• Le Cas de la Bavière (1844) : Le roi Louis Ier a tenté d'augmenter le prix de la bière.

La mesure a provoqué de tels "remous au sein de la population" qu'elle a été annulée après seulement quatre jours.

L'alcool est perçu comme un "dernier bastion qui nous permet de nous distinguer en tant qu'être humain".

• La Campagne de Gorbatchev (années 1980) : Mikhaïl Gorbatchev a lancé une campagne anti-alcool en URSS pour améliorer la santé publique.

◦ Résultats sanitaires : La mortalité a considérablement diminué durant cette période.   

◦ Échec politique : La campagne a été un "désastre" pour Gorbatchev, contribuant à sa chute. L'ironie veut qu'il ait cédé le pouvoir à Boris Eltsine, "notoirement alcoolique".

• La Prohibition aux États-Unis : Bien qu'elle ait généré un marché noir, la prohibition a entraîné une baisse considérable de la consommation d'alcool et des maladies et décès qui y sont liés.

• L'Ambivalence de l'Église : L'Église chrétienne a prêché la modération ("l'idéal chrétien de la juste mesure") tout en intégrant le vin dans ses rites les plus sacrés (la Cène, les noces de Cana), illustrant une "hypocrisie généralisée vis-à-vis de l'alcool".

7. Vers des Politiques Efficaces ?

Le document suggère que les campagnes de sensibilisation actuelles sont largement inefficaces et que des mesures plus structurelles sont nécessaires pour réduire les méfaits de l'alcool.

• Inefficacité des Campagnes : Les campagnes de sensibilisation sont jugées peu efficaces ; elles servent surtout à "donner bonne conscience".

• Les Deux Leviersefficaces : Pour réduire la consommation, deux mesures sont jugées primordiales :

1. Limiter l'accès à l'alcool.    2. Augmenter son prix.

• L'Exemple du Tabac : Le Royaume-Uni est cité en exemple.

Avec un paquet de cigarettes à 16 €, le taux de fumeurs est de 11,9 %, contre 24,5 % en France et 20,1 % en Allemagne, où les prix sont plus bas.

• La Question de la Fiscalité : Il est noté que l'alcool est "très bon marché" dans de nombreuses régions d'Europe. Par exemple, la taxe minimale sur le vin fixée au sein de l'UE est de 0 €.

8. Conclusion : Accepter une Réalité Humaine et Complexe

L'attrait pour l'alcool, malgré ses dangers connus, semble être une caractéristique profondément humaine, liée à une "dimension autodestructrice" ou à un "désir d'échapper à la réalité de la vie".

Les individus réagissent souvent avec colère aux avertissements, les percevant comme une forme d'infantilisation.

La conclusion suggère qu'il est peut-être impossible d'apprécier l'alcool "sans la double morale qui l'accompagne".

La première étape serait de reconnaître pleinement le paradoxe de l'alcool, ses avantages et ses inconvénients, afin d'apprendre à vivre avec cette substance complexe qui ne semble pas prête de disparaître de nos sociétés.

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L'Éducation comme Instrument de Pouvoir : Une Analyse Historique

https://www.youtube.com/watch?v=JCKbqhfFKy8

Résumé

Ce document de synthèse analyse le rôle historique de l'éducation, démontrant qu'au-delà de son idéal d'épanouissement personnel et de service du bien commun, elle a principalement été un instrument stratégique utilisé par les élites pour asseoir et maintenir leur pouvoir.

L'analyse, qui s'étend de la Sparte antique à l'époque contemporaine, révèle un schéma récurrent :

la mise en place de systèmes d'instruction publique est souvent une réponse directe aux troubles sociaux et vise à former des citoyens obéissants, à consolider des empires et à imposer des normes culturelles.

Des cas d'étude allant de la Prusse, pionnière de l'école obligatoire pour mater les révoltes paysannes, à la Chine impériale, utilisant des examens méritocratiques pour briser le pouvoir de la noblesse, illustrent cette thèse.

L'exemple tragique des pensionnats autochtones au Canada expose la forme la plus extrême de cette instrumentalisation, où l'éducation devient une arme de domination culturelle et d'éradication.

En conclusion, l'histoire révèle une tension fondamentale entre une éducation visant l'autonomie et la pensée critique, et une formation axée sur la performance, l'obéissance et la consolidation du statu quo.

1. Introduction : Le Droit à l'Éducation et ses Desseins Cachés

L'idéal moderne de l'éducation, tel que conçu par Platon comme une sortie de "la caverne de notre propre ignorance" et consacré par l'article 26 de la Déclaration universelle des droits de l'homme de 1948, postule l'instruction comme un droit fondamental au service de l'intérêt général.

Des études américaines corrèlent même un diplôme universitaire à une espérance de vie accrue de près de neuf ans.

Cependant, un examen historique approfondi soulève une question essentielle : l'éducation a-t-elle toujours poursuivi cet objectif d'émancipation ?

L'histoire suggère que l'instruction publique a souvent été un outil au service d'intérêts politiques et de stratégies de pouvoir bien définies.

2. Les Origines du Contrôle Social par l'Instruction

Loin d'être une invention des démocraties modernes, l'instruction publique obligatoire trouve ses racines dans des régimes autocratiques qui y ont vu un moyen efficace de garantir l'ordre social et la stabilité de leur pouvoir.

Sparte : Former le Guerrier-Citoyen Obéissant

Le premier exemple d'un système éducatif public structuré ne se trouve pas dans l'Athènes démocratique, mais dans la dictature militaire et esclavagiste de Sparte.

• Contexte de Domination : La société spartiate était composée d'une minorité de citoyens libres (les Spartiates) dominant une très large population d'hilotes, des serfs autochtones.

Le rapport était estimé à sept hilotes pour un Spartiate.

• L'Agogé, un Outil de Contrôle : Pour maintenir le contrôle sur cette population asservie et supérieure en nombre, Sparte a mis en place l'agogé.

Il s'agissait d'un système éducatif public et obligatoire pour les garçons spartiates dès l'âge de 7 ans, conçu comme un camp d'entraînement militaire visant à former des "guerriers surhumains".

• Objectifs Pédagogiques : L'accent était mis sur l'endurance, l'obéissance et la suppression de toute faiblesse, comme en témoigne le "concours de flagellation".

• Une Alphabétisation Stratégique : Bien que les hilotes en soient exclus, le programme incluait l'alphabétisation.

L'objectif n'était pas l'épanouissement intellectuel, mais une compétence militaire :

"Si un spartiate est envoyé en mission d'espionnage et qu'il intercepte un message écrit, il doit être capable de le lire."

• Conclusion : L'éducation spartiate n'avait pas pour but le développement personnel mais la formation de citoyens-soldats obéissants, un instrument essentiel à la survie du pouvoir en place.

L'Empire Carolingien : Unifier pour Mieux Régner

Après la chute de l'Empire romain d'Occident, Charlemagne initia la première grande expansion de l'éducation en Europe.

Son projet, loin d'être purement altruiste, était une manœuvre calculée pour consolider son vaste empire.

• Besoin Administratif : Pour contrôler son territoire, Charlemagne avait besoin d'une administration solide et unifiée.

L'école de la cour servait de "vivier de futur haut fonctionnaire".

• Unification Religieuse et Culturelle : Le pouvoir de l'empereur reposant sur Dieu, il était crucial de diffuser un christianisme uniformisé.

La réforme éducative visait à améliorer le niveau des ecclésiastiques et à standardiser la liturgie dans tout l'empire.

• Harmonisation de l'Écriture : Pour une administration efficace, une écriture commune était nécessaire.

La "minuscule Caroline" fut développée à cette fin, unifiant la communication écrite.

Cette police est l'ancêtre directe de la police de caractères Times New Roman.

• Conclusion : Pour Charlemagne, l'éducation n'était pas une fin en soi, mais un "instrument nécessaire pour maintenir la cohésion de l'empire".

La Prusse : L'École Obligatoire comme Rempart contre les Révoltes

C'est en Prusse, en 1763, que Frédéric II promulgua la loi créant le premier système d'enseignement primaire obligatoire au monde.

L'analyse de la politologue Agustina Paglayan révèle que cette initiative, loin d'être un progrès démocratique, était une stratégie de contrôle social.

• Le Paradoxe des Autocraties : Paglayan souligne que "ce ne sont pas les démocraties qui ont conduit à la création d'un enseignement primaire dans le monde occidental.

Celui-ci s'est surtout développé et étendu avant que les pays ne deviennent démocratiques."

• L'Éducation en Réponse aux Crises : Un schéma récurrent a été identifié :

la plupart des lois sur la scolarité obligatoire ont été adoptées juste après des révoltes populaires.

◦ Prusse (milieu du 18e siècle) : La loi est promulguée suite à des rebellions paysannes.    ◦ Massachusetts (années 1780) :

La première loi américaine sur la scolarité obligatoire répond à la révolte de Shays.  

◦ France (1833) : La loi suit la révolution de Juillet.   

◦ Pérou (2000) : La scolarité est imposée dans les anciennes zones rebelles après une guerre civile de 20 ans.

• Objectif : l'Endoctrinement : Face à la peur des masses, les élites politiques ont utilisé l'école primaire pour "enseigner aux enfants que le statut quo est acceptable et qu'il n'y a aucune raison de se rebeller".

L'enfance est ciblée car c'est la période où "les valeurs morales et les comportements politiques se façonnaient le mieux".

• L'École comme "Prison de Jour" : Reprenant les idées de Michel Foucault, le document décrit l'école comme une institution disciplinaire.

Les enseignants agissent comme des gardiens, inculquant la ponctualité, l'immobilité, la sagesse et la soumission.

Le but est de "créer une machine sociale bien huilée".

• Le Modèle Humboldtien : Une vision alternative fut proposée par le Prussien Wilhelm von Humboldt, pour qui l'éducation devait viser l'épanouissement personnel de chaque individu, "quel que soit leur origine sociale".

Cependant, après la défaite de Napoléon, ses idées jugées "dangereuses" furent écartées au profit d'un retour à l'"obéissance aveugle".

3. L'Éducation comme Outil de Sélection et de Pouvoir

Au-delà de l'inculcation de l'obéissance, l'éducation a aussi servi à structurer les hiérarchies du pouvoir, comme le montre l'exemple de la Chine impériale.

La Chine Impériale et le Système des Examens (Keju)

Pendant plus de 1000 ans, la Chine a utilisé un système d'examens (le Keju, institué au 7e siècle) pour attribuer les postes de la fonction publique.

• Une Méritocratie de Façade : En apparence, le système était basé sur le mérite.

Les candidats, parfois près d'un million pour environ 400 postes de finalistes, devaient mémoriser des classiques confucéens comptant jusqu'à 400 000 caractères.

• Un Objectif Politique : L'objectif réel de l'empereur était de limiter l'emprise des familles nobles qui contrôlaient traditionnellement l'administration.

En instituant un système basé sur des examens, il étendait son propre pouvoir en créant une bureaucratie qui lui était directement redevable.

• Influence Globale : Ce modèle, basé sur le mérite pour contrer le népotisme, a inspiré des réformes similaires jusqu'en Angleterre au milieu du 19e siècle.

4. L'Éducation comme Arme de Domination Culturelle

Le cas des pensionnats pour autochtones au Canada représente l'utilisation la plus sinistre de l'éducation, où elle est détournée pour devenir un outil d'éradication culturelle.

Le Témoignage de Gary Godfriitson (Peuple Sir Weepom)

Gary Godfriitson, gardien du savoir de la communauté Sir Weepom, décrit le système éducatif autochtone traditionnel comme étant basé sur "une étude attentive des enfants" pour découvrir leurs talents individuels et leur assigner des mentors experts.

Ce système, jugé "rétrograde" par les colons européens, fut systématiquement démantelé.

• Les Pensionnats : Des écoles spéciales, ou pensionnats, furent créées avec pour objectif de "détruire les cultures autochtones du Canada".

Gary Godfriitson, entré à 5 ans, se souvient : "Nous avons appris à nous taire. Nous avons appris que nous n'avions pas de voix dans ces pensionnats."

• Un Système d'Abus : Les enfants étaient soumis à un régime de discipline stricte, de prières constantes et de travail forcé ("un camp de travail pour enfants").

Ils subissaient "toutes sortes de violences (...) sexuel, physique, émotionnel".

• Bilan Tragique : Environ 150 000 enfants autochtones sont passés par ces établissements.

Un institut de recherche canadien estime qu'au moins 4 100 d'entre eux y sont morts de maladie, de négligence, de mauvais traitements ou en tentant de fuir.

• Un Projet Colonial Global : Ces pensionnats n'étaient pas une exception mais "l'un des outils majeurs pour la domination culturelle et soumettre l'autre".

5. Conclusion : Quelle Finalité pour l'Éducation de Demain ?

L'histoire démontre que l'éducation a trop rarement été "vouée au seul bien commun".

Elle a plus souvent servi à "garantir le pouvoir, à orienter les carrières et à imposer des normes".

Aujourd'hui, une tension persiste entre deux modèles :

1. L'Éducation comme Formation : Un modèle axé sur la performance, la fonctionnalisation et la monétisation des connaissances, qui forme des individus adaptés à une "machine sociale bien huilée".

2. L'Éducation comme Épanouissement : Le modèle de Humboldt, qui privilégie le développement personnel, la recherche de la connaissance et du sens, et qui promeut la pensée critique et la créativité comme compétences fondamentales.

La question finale demeure : "Quelle formule souhaitons-nous pour l'avenir ?

Une éducation qui nous dicte ce que nous devons savoir ou une éducation qui nous aide à découvrir qui nous voulons vraiment être ?"

• ADI 6595
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. RICARDO LEWANDOWSKI
• Julgamento: 23/05/2022
• Publicação: 05/08/2022

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. LEI FEDERAL 13.967/2019. VEDAÇÃO DE MEDIDA PRIVATIVA E RESTRITIVA DE LIBERDADE. NORMA QUE VERSA SOBRE REGIME JURÍDICO DE POLICIAIS MILITARES E CORPOS DE BOMBEIRO MILITARES. INICIATIVA LEGISLATIVA PRIVATIVA DO CHEFE DO PODER EXECUTIVO ESTADUAL. INCONSTITUCIONALIDADE FORMAL. PRECEDENTES. PRINCÍPIOS DA HIERARQUIA E DISCIPLINA INFORMADORES DA VIDA CASTRENSE. NÃO CABIMENTO DE HABEAS CORPUS CONTRA PRISÕES ADMINISTRATIVAS DE MILITARES. PREVISÃO EXPRESSA DOS ARTS. 5º, LXI, E 142, § 2º, DA CF. INCONSTITUCIONALIDADE MATERIAL. AÇÃO DIRETA JULGADA PROCEDENTE.

• I - A iniciativa legislativa para estabelecer normas sobre o regime jurídico dos integrantes das Forças Armadas é privativa do Presidente da República, a teor do 61, § 1º, II, f, da Constituição Federal.

• II – De outra parte, a Lei Maior, no art. 22, XXI, outorga à União a competência para legislar acerca de “normas gerais de organização, efetivos, material bélico, garantias, convocação e mobilização das polícias militares e corpos de bombeiros militares”.

• III – Tal competência, porém, “há que ser interpretada restritivamente, dentro de princípios básicos da organização federativa: ela só se justifica em termos da imbricação dos prismas gerais da estruturação das polícias militares com o seu papel de ´forças auxiliares e reserva do Exército´”(ACO 3.396/DF, Rel. Min. Alexandre de Moraes).

• IV – Por isso, quando se trata de regular o regime jurídico de servidores militares estaduais, a jurisprudência do Supremo Tribunal Federal é pacífica no sentido de assentar que a <u>iniciativa é privativa do Chefe do Executivo estadual</u>, por força do princípio da simetria.

• V – Nesse sentido, o § 6º do art. 144 da CF é expresso ao consignar que “as polícias militares e os corpos de bombeiros militares, forças auxiliares e reserva do Exército subordinam-se, juntamente com as polícias civis e as polícias penais estaduais e distrital, aos Governadores dos Estados, do Distrito Federal e dos Territórios”.

• VI - As polícias militares e os corpos de bombeiros militares constituem forças auxiliares e reserva do Exército, sendo responsáveis, segundo o art. 144 da CF - juntamente com as polícias de natureza civil - pela preservação da ordem pública e da incolumidade das pessoas e do patrimônio, inclusive mediante o uso da força, se necessário.

• VII – Consideradas as especificidades das respectivas carreiras, os servidores militares submetem-se a regime jurídico diferenciado, cujos valores estruturantes repousam, conforme os arts. 42 e 142, da CF, na hierarquia e disciplina, precisamente para que possam desempenhar, de forma expedita e rigorosa, o delicado múnus público que lhes é cometido.

• VIII – Não por outra razão, a própria Constituição Federal, de maneira clara e inequívoca, estabelece, em seu art. 142, § 2º, que “[n]ão caberá habeas corpus em relação a punições disciplinares militares”.

• IX- Tal preceito deita raízes no art. 5º, LXI, da CF, com a seguinte dicção: “ninguém será preso senão em flagrante delito ou por ordem escrita e fundamentada de autoridade judiciária competente, “salvo nos casos de transgressão militar ou crime propriamente militar, definidos em lei”.

• X – Por tais motivos, a presente ação direta é julgada procedente para declarar a inconstitucionalidade <u>formal</u> e <u>material</u> da Lei Federal 13.967/2019.

Ya

intressant

Eficiência não se confunde com eficácia nem com efetividade. - Eficiência consiste em realizar algo da melhor maneira possível com celeridade e o mínimo de desperdício. Relaciona-se com os meios e procedimentos para atingir um resultado. - Eficácia é o atingimento do objetivo ou da meta a que se propôs. Relaciona-se com o resultado. Veja que uma atividade pode ser eficiente, mas não alcançar eficácia ou vice-versa. - Já efetividade é a conjugação de eficiência e eficácia, transformando uma realidade externa por meio de uma boa atuação.

TCU - ACÓRDÃO 14/2025 - PRIMEIRA CÂMARA

TOMADA DE CONTAS ESPECIAL. CONTRATO DE REPASSE. OBRAS NÃO CONCLUÍDAS. NÃO ATINGIMENTO DO OBJETO PACTUADO. IMPRESTABILIDADE DAS PARCELAS EXECUTADAS. CITAÇÃO. REJEIÇÃO DAS ALEGAÇÕES DE DEFESA. CONTAS IRREGULARES. DÉBITO E MULTA.

[...] 15.Nessa circunstância, o responsável descumpriu, a um só tempo, dois princípios, quais sejam: o princípio da eficiência (art. 37, caput, da Constituição Federal), na medida em que deixou de dar andamento às obras, ocasionando a inutilidade da parte executada; e o princípio da continuidade administrativa, pois descumpriu com sua obrigação de encerrar a execução de empreendimento iniciado na gestão anterior, sempre visando ao interesse público.

Os casos de contratação por tempo determinado deve ser prevista em <u>lei ordinária</u>. Não há razão para se exigir lei complementar para regulamentação da contratação por tempo determinado. Nesse sentido:

• Informativo 1162
• ADI 7057 / CE
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. DIAS TOFFOLI
• Julgamento: 06/12/2024 (Virtual)
• Ramo do Direito: Administrativo
• Matéria: Agente Público; Contratação Temporária; Requisitos; Agente Socioeducativo; Regulamentação; Lei Complementar Estadual

Contratação temporária em âmbito estadual e sua regulamentação por lei complementar

Resumo - É inconstitucional — pois viola o princípio da simetria e o princípio democrático — norma de Constituição estadual que <u>exige a edição de lei complementar</u> para a regulamentação dos casos de contratação por tempo determinado para atender a necessidade temporária de excepcional interesse público.

• São inconstitucionais — pois não observam o princípio do concurso público (CF/1988, art. 37, II) nem os requisitos para a contratação temporária (CF/1988, art. 37, IX) — as Leis Complementares cearenses nº 163/2016, nº 169/2016 e nº 228/2020, que autorizam, por tempo determinado e para atender a necessidade temporária e de excepcional interesse público, a admissão de profissionais para a execução de atividades técnicas especializadas no âmbito do Sistema Estadual de Atendimento Socioeducativo.

• Ao tratar do instituto da contratação temporária, a Constituição Federal não determinou que sua regulamentação fosse realizada por meio de lei complementar (1).

• De acordo com a jurisprudência desta Corte (2), exigir lei complementar em situações para as quais a Constituição Federal não a previu restringe o arranjo democrático-representativo por ela estabelecido.

• Para que se considere válida a contratação temporária, exige-se que: i) os casos excepcionais estejam previstos em lei; ii) o prazo de contratação seja predeterminado; iii) a necessidade seja temporária; iv) o interesse público seja excepcional; v) a necessidade de contratação seja indispensável, sendo vedada a contratação para os <u>serviços ordinários permanentes</u> do Estado, e que <u>devam estar sob o espectro das contingências normais</u> da Administração (3).

• Na espécie, as Leis Complementares cearenses nº 163/2016 e nº 169/2016, embora estabeleçam prazo predeterminado para a contratação, visando realização de um objetivo público de grande relevância, não tratam de situação excepcional, porquanto a busca pelo aprimoramento dos serviços para melhor servir à sociedade é inerente à Administração Pública.

• Ademais, os anexos dessas normas demonstram tratar-se de diversas funções da estrutura administrativa do Sistema Estadual de Atendimento Socioeducativo que deveriam ter sido preenchidas por detentores de cargos públicos, tendo em vista a natureza ordinária e permanente das atividades.

• Por sua vez, a Lei Complementar nº 228/2020, editada no contexto da pandemia da Covid-19, apontou que a necessidade temporária da contratação compreenderia o período necessário à realização de concurso público para o provimento de cargos efetivos. Entretanto, o certame somente foi lançado em abril de 2024, quase oito anos após a criação, pela Lei estadual nº 16.040/2016, da Superintendência do Sistema Estadual de Atendimento Socioeducativo. A perpetuação dessas contratações pretensamente de caráter temporário evidencia a inércia administrativa em regularizar a estrutura de pessoal daquela superintendência.

• Com base nesses e em outros entendimentos, o Plenário, por maioria, julgou parcialmente procedente ação para: (i) declarar a inconstitucionalidade da expressão “complementar” do art. 154, inciso XIV, da Constituição do Estado do Ceará, com efeito ex nunc, para que a decisão, no ponto, produza efeitos a partir da publicação da ata deste julgamento; e (ii) declarar a inconstitucionalidade das Leis Complementares estaduais nº 163/2016; nº 169/2016; e nº 228/2020, garantindo-se a vigência das contratações temporárias celebradas com base nos citados diplomas, até que expirem os prazos de duração, após o que deverá o Estado do Ceará preencher os quadros de seu Sistema Estadual de Atendimento Socioeducativo com servidores aprovados em concurso público.

Somente se houver expresso assentimento dos entes envolvidos, poderá haver amortização de dívidas com sentenças transitadas em julgado. Nesse sentido:

• RE 657686
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. LUIZ FUX
• Julgamento: 23/10/2014
• Publicação: 05/12/2014

RECURSO EXTRAORDINÁRIO COM REPERCUSSÃO GERAL. DIREITO CONSTITUCIONAL. REGIME DE EXECUÇÃO PECUNIÁRIA DA FAZENDA PÚBLICA. COMPENSAÇÃO DE DÉBITOS PERANTE A FAZENDA PÚBLICA COM CRÉDITOS SUJEITOS A REQUISIÇÃO DE PEQUENO VALOR. IMPOSSIBILIDADE. JULGAMENTO DAS ADI’S 4357 E 4425 PELO PLENÁRIO DO SUPREMO TRIBUNAL FEDERAL. EMENDA CONSTITUCIONAL Nº 62/2009. INCONSTITUCIONALIDADE DA SISTEMÁTICA DE COMPENSAÇÃO EM PROVEITO EXCLUSIVO DA FAZENDA PÚBLICA. EMBARAÇO À EFETIVIDADE DA JURISDIÇÃO (CRFB, ART. 5º, XXXV), DESRESPEITO À COISA JULGADA MATERIAL (CRFB, ART. 5º XXXVI), OFENSA À SEPARAÇÃO DOS PODERES (CRFB, ART. 2º) E ULTRAJE À ISONOMIA ENTRE O ESTADO E O PARTICULAR (CRFB, ART. 1º, CAPUT, C/C ART. 5º, CAPUT). ENTENDIMENTO QUE SE APLICA NA MESMA EXTENSÃO ÀS REQUISIÇÕES DE PEQUENO VALOR. RECURSO EXTRAORDINÁRIO A QUE SE NEGA PROVIMENTO. - 1. A compensação de tributos devidos à Fazenda Pública com créditos decorrentes de decisão judicial caracteriza pretensão assentada em norma considerada inconstitucional (art. 100, §§ 9º e 10, da Constituição da República, com redação conferida pela EC nº 62/2009).

• 2. O Plenário do Supremo Tribunal Federal, ao julgar as ADIs nº 4.357 e 4.425, assentou a inconstitucionalidade dos §§ 9º e 10 do art. 100 da Constituição da República, com redação conferida pela EC nº 62/2009, forte no argumento de que a compensação dos débitos da Fazenda Pública inscritos em precatórios embaraça a efetividade da jurisdição (CRFB, art. 5º, XXXV), desrespeita a coisa julgada material (CRFB, art. 5º, XXXVI), vulnera a Separação dos Poderes (CRFB, art. 2º) e ofende a isonomia entre o Poder Público e o particular (CRFB, art. 5º, caput), cânone essencial do Estado Democrático de Direito (CRFB, art. 1º, caput).

• 3. Destarte, não se revela constitucionalmente possível a compensação unilateral de débitos em proveito exclusivo da Fazenda Pública mesmo que os valores envolvidos estejam sujeitos ao regime de pagamento por requisição de pequeno valor (RPV).

• 4. Recurso extraordinário a que se nega provimento.

Tema 511

• Compensação de débitos tributários com requisições de pequeno valor - RPV.

Tese - É constitucionalmente vedada a compensação unilateral de débitos em proveito exclusivo da Fazenda Pública ainda que os valores envolvidos não estejam sujeitos ao regime de precatórios, <u>mas apenas à sistemática da requisição de pequeno valor</u>.

• ADI 7064
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. LUIZ FUX
• Julgamento: 01/12/2023
• Publicação: 19/12/2023

39. O estabelecimento de uma comissão de controle externo junto ao Poder Legislativo para avaliação dos precatórios expedidos pelo Poder Judiciário, conforme art. 6º da EC 114/21 destoa do sistema de separação de poderes posto na Constituição Federal. O dispositivo havido da Emenda Constitucional 114/21 subverte a ordem de atribuições, impondo um controle sobre a atividade tanto do Poder Executivo, condenado em demandas judiciais, quanto do Poder Judiciário, que julga o melhor direito e condena o Estado a pagar o cidadão.

• Conforme entendimento jurisprudencial do STF, o crédito preferencial transmudar-se-á para superpreferencial acaso o titular complete 60 anos no decurso do prazo para pagamento do precatório.
• A redação anterior do § 2º contemplava o privilégio da superpreferência apenas quando, na data de emissão do precatório, o titular tivesse mais de 60 anos.

Nesse sentido:

• No tocante ao art. 100, § 2º, da CF [“Os débitos de natureza alimentícia cujos titulares tenham 60 (sessenta) anos de idade ou mais na data de expedição do precatório, ou sejam portadores de doença grave, definidos na forma da lei, serão pagos com preferência sobre todos os demais débitos, até o valor equivalente ao triplo do fixado em lei para fins do disposto no § 3º deste artigo, admitido o fracionamento para essa finalidade, sendo que o restante será pago na ordem cronológica de apresentação do precatório”], assinalou-se que a emenda, em primeira análise, criara benefício anteriormente inexistente para os idosos e para os portadores de deficiência, em reverência aos princípios da dignidade da pessoa humana, da razoabilidade e da proporcionalidade. Entretanto, relativamente à expressão “na data da expedição do precatório”, entendeu-se haver transgressão ao princípio da igualdade, porquanto <u>a preferência deveria ser estendida a todos credores que completassem 60 anos de idade na pendência de pagamento de precatório de natureza alimentícia</u>. ADI 4425**

65% da parcela a ser destinado aos municípios: - Vinculado ao valor adicionado em operações realizadas nos territórios municipais.

• ARE 1523820 AgR-terceiro
• Órgão julgador: Primeira Turma
• Relator(a): Min. CRISTIANO ZANIN
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 25/06/2025
• Publicação: 18/07/2025

AGRAVO INTERNO. RECURSO EXTRAORDINÁRIO COM AGRAVO. PODER GERAL DE CAUTELA DOS TRIBUNAIS DE CONTAS. ART. 71, IX E ART. 75 DA CONSTITUIÇÃO FEDERAL. TEORIA DOS PODERES IMPLÍCITOS. COMPETÊNCIA PARA DETERMINAR À AUTORIDADE ADMINISTRATIVA QUE PROMOVA ANULAÇÃO DE CONTRATO E, SE FOR O CASO, DA LICITAÇÃO DE QUE SE ORIGINOU. PRECEDENTES. AGRAVO INTERNO A QUE SE DÁ PROVIMENTO. - 1. O Plenário do SUPREMO TRIBUNAL FEDERAL já se pronunciou no sentido da validade do <u>poder geral de cautela</u> dos Tribunais de Contas dos Estados (SS 5658 AgR, Rel. Min. LUÍS ROBERTO BARROSO (Presidente), Tribunal Pleno, DJe 04-04-2024).

• 2. Incorporou-se em nosso ordenamento jurídico a pacífica doutrina constitucional norte-americana sobre a teoria dos poderes implícitos - inherent powers -, pela qual no exercício de sua missão constitucional enumerada, o órgão executivo deveria dispor de todas as funções necessárias, ainda que implícitas, <u>desde que não expressamente limitadas</u> (Myers v. Estados Unidos US 272 52, 118), consagrando-se, dessa forma, e entre nós aplicável ao Tribunal de Contas da União e, por simetria, aos Tribunais de Contas dos Estados, o reconhecimento de competências genéricas implícitas que possibilitem o exercício de sua missão constitucional, apenas sujeitas às proibições e limites estruturais da Constituição Federal.

• 1. O “Tribunal de Contas da União – embora não tenha poder para anular ou sustar contratos administrativos – tem competência, conforme o art. 71, IX, para determinar à autoridade administrativa que promova a anulação do contrato e, se for o caso, da licitação de que se originou” (MS 23.550, Red. p/o acórdão o Min. Sepúlveda Pertence). Igual competência é atribuída ao Tribunal de Contas do Estado do Ceará, na forma do art. 75 da Constituição.

• 4. Não configura ilegalidade ou abuso de poder o ato do Tribunal de Contas que aplique medidas cautelares, porque relacionada com a competência constitucional implícita para cumprimento de suas atribuições.

• 5. Agravo Interno a que se dá provimento.

Observação - Acórdão(s) citado(s): (TRIBUNAL DE CONTAS, PRERROGATIVA, PODER GERAL DE CAUTELA, TEORIA DOS PODERES IMPLÍCITOS) MS 23550 (1ªT), MS 24510 (TP), SS 5658 AgR (TP). (FUNDAMENTAÇÃO, DECISÃO JUDICIAL) AI 791292 QO-RG (TP). (RE, DEVIDO PROCESSO LEGAL, AMPLA DEFESA, CONTRADITÓRIO, LIMITES DA COISA JULGADA) ARE 748371 RG (TP). (RE, INDEFERIMENTO, PRODUÇÃO DE PROVA, OFENSA INDIRETA) ARE 639228 RG (TP). (RE, PRINCÍPIO DA INAFASTABILIDADE DA JURISDIÇÃO, OFENSA INDIRETA, REEXAME, FATO, PROVA) RE 956302 RG (TP). - Decisão estrangeira citada: Caso Myers vs. Estados Unidos, US 272 52, 118, da Suprema Corte dos Estados Unidos. Número de páginas: 16. Análise: 11/08/2025, MAV.

Partes AGTE.(S) : TRIBUNAL DE CONTAS DO CEARA PROC.(A/S)(ES) : PROCURADOR-GERAL DO TRIBUNAL DE CONTAS DO ESTADO DO CEARÁ PROC.(A/S)(ES) : LÍLIAN DE CASTRO E SILVA MENEZES DO VALE (15518/CE) PROC.(A/S)(ES) : MAYSA CORTEZ CORTEZ (32431/CE) PROC.(A/S)(ES) : TALLITA FALKENSTINS GOIS MENDES CORDEIRO (31661/CE)

• MS 35920 ED
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 30/10/2023
• Publicação: 14/11/2023

EMBARGOS DE DECLARAÇÃO NO MANDADO DE SEGURANÇA. TRIBUNAL DE CONTAS DA UNIÃO. TOMADA DE CONTAS ESPECIAL. DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA. POSSIBILIDADE. REQUISITOS LEGAIS OBSERVADOS. AUSÊNCIA DE DIREITO LÍQUIDO E CERTO. DENEGAÇÃO DA SEGURANÇA. AUSÊNCIA DE OMISSÃO, CONTRADIÇÃO, OBSCURIDADE OU ERRO MATERIAL.

• 1. Os embargos de declaração são cabíveis para sanar a ocorrência de omissão, contradição, obscuridade ou erro material, hipóteses não verificadas no caso dos autos.
• 2. É legal e constitucionalmente fundada a <u>desconsideração da pessoa jurídica</u> pelo TCU, de modo a alcançar o patrimônio de pessoas físicas ou jurídicas envolvidas na prática de atos lesivos ao erário público, observados o contraditório e a ampla defesa.
• 3. TCU assentou a existência de indícios de que a impetrante praticou condutas ilegais que consubstanciariam a desconsideração da personalidade jurídica.
• 4. Feito se encontra em etapa preambular cabendo o exercício exauriente do direito de defesa pelos entes integrados à relação processual, em procedimento sujeito a controle jurisdicional.
• 5. Embargos de declaração rejeitados.

Observação - Acórdão(s) citado(s): (TRIBUNAL DE CONTAS, DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA) MS 35506 (1ªT). (TEORIA DO ATO ULTRA VIRES) MS 35555 AgR (2ªT). Número de páginas: 16. Análise: 15/04/2024, SOF.

Indexação TRAMITAÇÃO, TRIBUNAL DE CONTAS DA UNIÃO (TCU), POSSIBILIDADE, APLICAÇÃO, TEORIA DO ATO ULTRA VIRES, RESPONSABILIZAÇÃO, FORMA DIRETA, PESSOA FÍSICA, RESPONSABILIDADE, COMETIMENTO, ATO ILÍCITO. DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA, RESERVA DE JURISDIÇÃO. DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA, AUSÊNCIA

Outras ocorrências Partes (1)

• ARE 1306779 AgR
• Órgão julgador: Segunda Turma
• Relator(a): Min. EDSON FACHIN
• Julgamento: 03/05/2023
• Publicação: 09/05/2023
• 2. Ademais, este Supremo Tribunal Federal já assentou a <u>plena possibilidade</u> de a Corte de Contas, no cumprimento de seu mister constitucional, decretar a indisponibilidade de bens e de outras medidas assecuratórias do interesse público, diante de circunstâncias graves que justifiquem a necessidade de proteção efetiva do patrimônio público.
• 1. O Plenário também já afirmou a plena possibilidade de que o TCU, orientação que também se aplica às Cortes de Contas Estaduais, determine a aplicação de medidas cautelares, como verdadeira competência constitucional implícita para cumprimento de suas atribuições, nos termos do artigo 71 da Carta Magna.

Trauma complexe = + que neuroscience ou application rigide de protocole.

au delà de la technique, l'importance des compétences du clinicien.

explication

limite.

honorer la singularité

au delà du DSM et de la CIM, ce serait quoi d'autre ?

travailler AVEC la complexité

coeur

2 risque

nuance

yes

"réduction du réel au mesurable"

O prazo decadencial para revogar doação é de 1 ano, contado da data de conhecimento do fato pelo doador.

Recebeu imóvel indevidamente: - Se há alienação de boa-fé = responde o alienante pela quantia recebida; - Se há alienação de má-fé = responde pelo valor do imóvel + perdas e danos.

Hart, Keith, Jean-Louis Laville, and Antonio David Cattani, eds. 2010. The Human Economy: A Citizen’s Guide. 1st ed. Malden, MA; Cambridge, UK: Polity. https://amzn.to/4q7hwTi (October 11, 2025).

Annotations: urn:x-pdf:c0c4b707a9de803a95b50ebebe19c70c

Alternate annotation link: urn:x-pdf:c0c4b707a9de803a95b50ebebe19c70c

Em decorrência dos postulados da segurança jurídica, a nova interpretação dada pela Administração deve ter efeitos prospectivos. Nessa linha, também a LINDB estabelece nos arts. 24 e 30:

Art. 24. A revisão, nas esferas administrativa, controladora ou judicial, quanto à validade de ato, contrato, ajuste, processo ou norma administrativa cuja produção já se houver completado levará em conta as orientações gerais da época, sendo vedado que, com base em mudança posterior de orientação geral, se declarem inválidas situações plenamente constituídas. <br /> - Parágrafo único. Consideram-se orientações gerais as interpretações e especificações contidas em atos públicos de caráter geral ou em jurisprudência judicial ou administrativa majoritária, e ainda as adotadas por prática administrativa reiterada e de amplo conhecimento público.

Art. 30. As autoridades públicas devem atuar para aumentar a segurança jurídica na aplicação das normas, inclusive por meio de regulamentos, súmulas administrativas e respostas a consultas.

• Perceba que, apesar da abrangência dos legitimados a promover a desapropriação, o rol não inclui a possibilidade de execução dos atos expropriatórios pelos contratados em regime semi-integrado.

• No entanto, parece ser hipótese de "esquecimento" do legislador, já que seria ilógico excluir essa possibidade à contratação semi-integrada, ao passo que é autorizada para o empreiteiro (que tem menos responsabilidades contratuais) ou mesmo aos autorizatários.

• Ampla gama de legitimados para a execução da desapropriação, haja vista a possibilidade de até mesmo permissionários, arrendatários e autorizatários promoverem a desapropriação prevista em lei ou em contrato

• A correção monetária dos valores contratuais deve estar vinculada à data-base do orçamento realizado pela Administração Pública, o que é completamente razoável ao se considerar que correção monetária não é acréscimo de valores ou sanção pecuniária.

• Com isso, se o que se busca é atualização de valores em vista do fenômeno econômico da inflação, a qual corroí com o tempo o poder de compra, deve se ter, por termo inicial, a data em que os valores foram constatados para os fins de correção monetária.

• Do contrário, acaso a vinculação da correção monetária fosse a data da assinatura do contrato, não haveria a devida compensação de valores em decorrência da inflação, sobretudo porque são momentos completamente distintos a realização do orçamento e da assinatura do contrato, podendo se distanciarem por meses ou mesmo mais de ano. Acaso não a data do orçamento, estar-se-ia diante de possível distorção monetária dos valores contratuais, principalmente aqueles de grande vulto.

JURISPRUDÊNCIA EM TESES - EDIÇÃO Nº 160 - DIREITO DO CONSUMIDOR - IV

• 8) O Código de Defesa do Consumidor - CDC, em regra, é inaplicável aos contratos administrativos, tendo em vista as prerrogativas já asseguradas pela lei à administração pública.

• 9) Em situações <u>excepcionais</u>, a administração pública pode ser considerada consumidora de serviços (art. 2º do CDC) por ser possível reconhecer sua vulnerabilidade, <u>mesmo em relações contratuais regidas, preponderantemente, por normas de direito público</u>, e por se aplicarem aos contratos administrativos, de forma supletiva, as normas de direito privado (art. 54 da Lei n. 8.666/1993).

• 10) O Código de Defesa do Consumidor é inaplicável a contrato acessório de contrato administrativo, pois não se origina de uma relação de consumo.

ADITAMENTO DA INICIAL

• Petição inicial. Ilegitimidade ativa para a causa. Correção. É lícito, em ação direta de inconstitucionalidade, aditamento à petição inicial <u>anterior à requisição das informações</u>. [ADI 3.103, rel. min. Cezar Peluso, j. em 1-6-2006, P, DJ de 25-8-2006.]

• "Pedido de aditamento da inicial após inclusão em pauta da ação para julgamento final pelo rito do artigo 12 da Lei 9.868/1999. Admissão do aditamento, tendo em vista a irrelevância das alterações promovidas no texto normativo impugnado. Admitido o aditamento, necessária é a abertura de prazo para a manifestação dos requeridos." (ADI 3.434-MC, rel. min. Joaquim Barbosa, j. 23-8-2006, P, DJ de 28-9-2007.)

• "A renumeração do preceito constitucional estadual impugnado, mantido na íntegra o texto original, não implica a prejudicialidade da ação direta, <u>desde que promovido o aditamento à petição inicial</u>. Precedente: ADI 1.874, Relator o Ministro Maurício Corrêa, DJ de 7-2-03." (ADI 246, rel. min. Eros Grau, j. 16-12-2004, P, DJ de 29-4-2005.) No mesmo sentido: ADI 3.832, rel. min. Cármen Lúcia, decisão monocrática, j. 22-6-2010, DJE de 29-6-2010.

PERDA DE OBJETO

• A jurisprudência desta Suprema Corte é firme no sentido de que a revogação expressa ou tácita da norma impugnada, bem como sua alteração substancial, após o ajuizamento da ação direta de inconstitucionalidade <u>acarreta a perda superveniente do seu objeto</u>, independentemente da existência de efeitos residuais concretos dela decorrentes. Vocação dessa espécie de ação constitucional a assegurar a higidez da ordem jurídica vigente. [ADI 3.557, rel. min. Rosa Weber, j. 18-12-2021, P, DJE de 28-1-2022.]

• Há jurisprudência consolidada no STF no sentido de que a revogação da norma cuja constitucionalidade é questionada por meio de ação direta enseja a perda superveniente do objeto da ação. Nesse sentido: ADI 709, rel. min. Paulo Brossard, DJ de 20-5-1994; ADI 1.442, rel. min. Celso de Mello, DJ de 29-4-2005; ADI 4.620 AgR, rel. min. Dias Toffoli, DJe de 1-8-2012.

• Excepcionam-se desse entendimento os casos em que há indícios de fraude à jurisdição da Corte, como, a título de ilustração, quando a norma é revogada com o propósito de evitar a declaração da sua inconstitucionalidade. Nessa linha: ADI 3.306, rel. min. Gilmar Mendes, DJe de 7-6-2011.
• Excepcionam-se, ainda, as ações diretas que tenham por objeto leis de eficácia temporária, quando: (i) houve impugnação em tempo adequado, (ii) a ação foi incluída em pauta e (iii) seu julgamento foi iniciado antes do exaurimento da eficácia. Nesse sentido: ADI 5.287, rel. min. Luiz Fux, DJe de 12-9-2016; ADI 4.426, rel. min. Dias Toffoli, DJe de 17-5-2011; ADI 3.146/DF, rel. min. Joaquim Barbosa, DJ de 19-12-2006.

• Com maior razão, a prejudicialidade da ação direta também deve ser afastada nas ações cujo mérito já foi decidido, em especial se a revogação da lei só veio a ser arguida posteriormente, em sede de embargos de declaração. Nessa última hipótese, é preciso não apenas impossibilitar a fraude à jurisdição da Corte e minimizar os ônus decorrentes da demora na prestação da tutela jurisdicional, mas igualmente preservar o trabalho já efetuado pelo Tribunal, bem como evitar que a constatação da efetiva violação à ordem constitucional se torne inócua. [ADI 951 ED, rel. min. Roberto Barroso, j. 27-10-2016, P, DJE de 21-6-2017.]

• "Inicialmente, considero que a remuneração do art. (...), sem mudança do texto impugnado, não leva à alteração substancial do objeto do controle concentrado de constitucionalidade, de modo a persistir o interesse e a competência desta Corte para julgar a ação direta de inconstitucionalidade." (ADI 238, voto do rel. min. Joaquim Barbosa, j. 24-2-2010, P, DJE de 9-4-2010.)

Regra Geral: A Não Submissão à Preclusão - A principal regra extraída é que as matérias de ordem pública, em princípio, não precluem, podendo ser analisadas a qualquer momento nas instâncias ordinárias.

• A inépcia da petição inicial, por ser matéria de ordem pública, "pode ser suscitada e examinada a qualquer tempo nas <u>instâncias ordinárias</u>, não se submetendo à preclusão" (AgInt no AREsp 2.270.272). Da mesma forma, a conformidade do valor executado com o título judicial é matéria de ordem pública e "não há preclusão pro judicato na atividade probatória para o julgador" que busca aferir a exatidão dos cálculos (AgInt no AREsp 2.405.050).

Exceções Reconhecidas: Quando a Matéria de Ordem Pública Preclui

Apesar da regra geral, a jurisprudência do STJ estabelece situações claras em que a preclusão atinge, sim, as matérias de ordem pública.

1. Preclusão pro judicato (Quando a matéria já foi decidida no processo) - A exceção mais recorrente é a que impede o juiz ou o tribunal de reexaminar uma questão de ordem pública que já tenha sido objeto de decisão anterior no mesmo processo, sem que houvesse recurso oportuno da parte.

• Matérias de ordem pública "se submetem à preclusão pro judicato nas hipóteses [...] em que a questão [...] já tenha sido examinada e decidida, sem que, contra a conclusão plasmada no respectivo decisum, tenha havido insurgência da parte contrária" (AgInt no REsp 1.535.655). Essa preclusão "impede a revisão de matérias decididas no processo, inclusive as de ordem pública, que não tenham sido impugnadas pelo recurso cabível no momento próprio" (EDcl no REsp 1.708.238).

2. Preclusão para as Partes (Consumativa e Temporal)

• Se a parte interessada não alega a nulidade ou a questão de ordem pública na primeira oportunidade que tem para falar nos autos após a sua ocorrência, ou se a questão já foi decidida, opera-se a preclusão para a parte.

• A nulidade "não foi oportunamente alegada nos embargos de declaração [...], o recorrente não levantou a nulidade na <u>primeira oportunidade</u> após a ocorrência do vício, restando configurada a preclusão da matéria, nos termos do art. 278 do CPC/2015" (REsp 1.809.204). Ademais, "estão sujeitas à preclusão as matérias não impugnadas no momento oportuno, <u>inclusive as de ordem pública</u>" (EDcl no AgInt no REsp 2.129.882). A "preclusão consumativa impede a rediscussão de questões já decididas, inclusive as de ordem pública" (AgInt no AREsp 2.302.911).

3. Preclusão nas Instâncias Superiores (Ausência de Prequestionamento) - Para que uma matéria de ordem pública seja analisada em Recurso Especial (STJ), ela precisa ter sido previamente debatida e decidida pelo tribunal de origem. A ausência desse debate gera a preclusão da análise na instância superior.

• "o acesso à via extraordinária depende do indispensável prequestionamento da matéria perante o Tribunal a quo, requisito constitucional exigido inclusive para as matérias de ordem pública" (REsp 1.809.204 e Informações Complementares à Ementa do REsp 1.809.209).

4. Preclusão Máxima (Coisa Julgada) - Após o trânsito em julgado de uma decisão, as questões decididas, ainda que de ordem pública, estabilizam-se, não podendo ser rediscutidas, em respeito à segurança jurídica.

• "A preclusão da matéria e o respeito à coisa julgada impedem a análise de pedido de desclassificação de conduta após o trânsito em julgado, em observância ao princípio da segurança jurídica" (AgRg no RHC 953.536).

1. Deve ser aplicada a técnica de julgamento ampliado nos embargos de declaração toda vez que o voto divergente possua aptidão para alterar o resultado unânime do acórdão de apelação. (REsp 1.910.317-PE)
2. A técnica de ampliação do colegiado, prevista no art. 942 do CPC/2015, aplica-se também ao julgamento de apelação interposta contra sentença proferida em mandado de segurança. (REsp 1.868.072-RS)

• Perceba que, nos casos de ação rescisória e decisão interlocutória de mérito, o julgamento ampliado tem como objetivo prestigiar a decisão do juízo de origem, desde que não haja unanimidade.

• Isso porque o julgamento ampliado somente terá vez se, na ação rescisória, a votação for no sentido de rescindir a sentença; na decisão interlocutória de mérito, se houve reforma da decisão de origem.

• Do contrário - havendo manutenção da sentença ou da decisão interlocutória de mérito - não haverá obrigação de julgamento ampliado.

JURISPRUDÊNCIA EM TESES - EDIÇÃO 189 - EMBARGOS DE DECLARAÇÃO I

• 1) Os embargos de declaração não podem ser utilizados para adequar a decisão ao entendimento da parte embargante, acolher pretensões que refletem mero inconformismo ou rediscutir matéria já decidida.

• 2) A contradição que autoriza a oposição de embargos de declaração é a interna, caraterizada pela existência de proposições inconciliáveis entre si.

• 3) Não é necessário ratificar o recurso especial interposto na pendência do julgamento dos embargos de declaração, quando inalterado o resultado anterior. (Súmula n. 579/STJ)

• 4) Não compete ao Superior Tribunal de Justiça - STJ, ainda que para fim de prequestionamento, examinar dispositivos constitucionais em embargos de declaração, sob pena de usurpação da competência do Supremo Tribunal Federal - STF.

• 5) A oposição de embargos de declaração com notório propósito de prequestionamento não possui caráter protelatório, assim, deve ser afastada a aplicação da multa prevista no art. 1.026, § 2º, do Código de Processo Civil, nos termos da Súmula n. 98/STJ.

• 6) Os embargos de declaração devem ser apreciados pelo órgão julgador da decisão embargada, independentemente da alteração de sua composição, o que não ofende o princípio do juiz natural nem excepciona o princípio da identidade física do juiz.

• 7) Admite-se, excepcionalmente, a oposição de embargos de declaração para obter a juntada de notas taquigráficas aos autos quando indispensáveis à compreensão do acórdão ou ao exercício da ampla defesa.

• 8) É possível a imposição cumulativa de multa por oposição de embargos de declaração protelatórios com multa por litigância de má-fé, pois possuem naturezas distintas.

• 9) Em observância aos princípios da fungibilidade recursal e da instrumentalidade das formas, é admitida a conversão de embargos de declaração em agravo interno quando a pretensão declaratória possui manifesto caráter infringente.

• 10) Não é cabível o recebimento de embargos declaratórios como pedido de reconsideração nem deste como aqueles.

JURISPRUDÊNCIA EM TESES - EDIÇÃO 190 -EMBARGOS DE DECLARAÇÃO II

• 1) Na hipótese de concessão de efeito infringente aos embargos de declaração, é necessária intimação prévia do embargado para apresentar impugnação, sob pena de nulidade de julgamento e violação aos princípios do contraditório e da ampla defesa.

• 2) Os embargos de declaração, quando opostos contra decisão de inadmissibilidade do recurso especial proferida na instância ordinária, não interrompem o prazo para a interposição do agravo previsto no art. 1.042 do CPC, único recurso cabível, salvo quando a decisão for tão genérica que impossibilite ao recorrente aferir os motivos pelos quais teve seu recurso negado, de modo a inviabilizar a interposição do agravo.

• 3) Deve-se aplicar a técnica do julgamento ampliado, prevista no art. 942 do CPC, aos embargos de declaração quando o voto divergente puder alterar o resultado unânime do acórdão de apelação.

• 4) Os segundos embargos de declaração estão restritos ao argumento da existência de vícios no acórdão proferido nos primeiros aclaratórios, pois, em virtude da preclusão consumativa, é descabida a discussão acerca da decisão anteriormente embargada.

• 5) Não é possível, em embargos de declaração, adaptar o entendimento do acórdão embargado em razão de posterior mudança jurisprudencial.

• 6) São cabíveis embargos de declaração para, em caráter excepcional, adequar o acórdão embargado à orientação firmada no âmbito de repercussão geral reconhecida pelo Supremo Tribunal Federal e de recurso julgado sob o rito dos repetitivos.

• 7) Embargos de declaração que visam rediscutir matéria já apreciada e decidida pela Corte de origem em conformidade com súmula do STJ ou STF ou, ainda, precedente julgado pelo rito dos recursos repetitivos são considerados protelatórios.

• 8) O julgamento colegiado dos embargos de declaração opostos à decisão monocrática de relator, sem a interposição de agravo interno, não acarreta o exaurimento da instância para efeito de interposição de recurso especial.

• 9) O julgamento monocrático dos embargos de declaração opostos ao acórdão do Tribunal de origem, sem a interposição do agravo interno, não acarreta o exaurimento da instância para efeito de interposição de recurso especial.

• 10) É possível o julgamento monocrático pelo relator de embargos de declaração opostos contra decisão colegiada.

JURISPRUDÊNCIA EM TESES - EDIÇÃO 191 - EMBARGOS DE DECLARAÇÃO III

• 1) Não é cabível a majoração dos honorários recursais no julgamento de embargos de declaração.

• 2) Não são cabíveis embargos de declaração contra despacho que determina a intimação da parte para regularizar o preparo recursal, pois tal ato não possui natureza decisória.

• 3) A ausência de manifestação sobre o mérito de recurso que não ultrapassou o juízo de admissibilidade não caracteriza omissão apta a autorizar a oposição de embargos de declaração.

• 4) É desnecessária a intimação para complementar as razões recursais a que se refere o art. 1.024, § 3º, do CPC, quando os embargos de declaração recebidos como agravo regimental impugnam especificamente os fundamentos da decisão monocrática.

• 5) O julgamento dos embargos de declaração independe de inclusão em pauta e intimação da data da sessão de julgamento, mediante publicação na imprensa oficial, pois o feito é apresentado em mesa e não cabe sustentação oral.

• 6) Diante da reiterada oposição de embargos de declaração meramente protelatórios, deve ser determinada a baixa dos autos à origem, independentemente da publicação do acórdão recorrido e da certificação do trânsito em julgado.

• 7) Na hipótese de concessão de efeito suspensivo aos embargos de declaração para interposição de outros recursos, tem-se que este suspende o prazo apenas quanto ao respectivo acórdão embargado, assim, não têm efeitos ultraprocessuais para suspender o prazo em relação a decisões em outros incidentes processuais.

• 8) Os embargos de declaração opostos por uma das partes não interrompem ou suspendem o prazo que a outra dispõe para embargar a mesma decisão, pois o prazo para recorrer é comum entre elas.

JURISPRUDÊNCIA EM TESES - EDIÇÃO 192 - EMBARGOS DE DECLARAÇÃO IV

• 1) É vedado, em embargos de declaração, ampliar as questões veiculadas no recurso para incluir teses que não foram anteriormente suscitadas, ainda que se trate de matéria de ordem pública, por configurar inovação recursal e revelar falta de prequestionamento, pois o cabimento dessa espécie recursal restringe-se às hipóteses em que existe vício no julgado.

• 2) A ausência de indicação, nas razões dos embargos declaratórios, da presença de quaisquer dos vícios de cabimento do recurso, implica o não conhecimento dos aclaratórios por fundamentação recursal deficiente. (Súmula n. 284 do STF).

• 3) O erro material sanável nos embargos de declaração é aquele evidente, conhecível de plano, que prescinde da análise do mérito, ou que diz respeito a incorreções internas do próprio julgado.

• 4) A oposição de embargos declaratórios intempestivos não interrompe nem suspende o prazo para a interposição de novos recursos.

• 5) Reconhecida a intempestividade do agravo, não se conhece dos embargos de declaração posteriormente opostos que não se insurgem contra referido óbice recursal.

• 6) Nos casos em que o órgão colegiado julga matéria submetida à sistemática da repercussão geral, admite-se, excepcionalmente, a oposição de embargos de declaração para atribuir-lhes efeitos modificativos, anular o acórdão embargado e determinar a devolução dos autos ao Tribunal de origem para exercer juízo de conformação após o julgamento do paradigma.

• 7) Não são admissíveis os segundos embargos de declaração opostos pela mesma parte, contra a mesma decisão, em razão da preclusão consumativa e do princípio da unirrecorribilidade.

• 8) É possível o conhecimento dos embargos de declaração, independentemente do depósito prévio da multa prevista no art. 1.021, § 4º, do CPC, quando o recurso questiona a própria aplicação da penalidade, quanto à sua base de cálculo.

• Julgando-se improcedente os embargos à execução, a ação executiva poderá ter continuidade independentemente de interposição de apelação, visto a exceção criada pelo § 1º.

• No entanto, se há julgamento de procedência dos embargos à execução, a ação executiva não será extinta automaticamente; visto que, em caso de apelação, haverá sim efeito suspensivo.

O executado somente terá legitimidade passiva em Embargos de Terceiro quando houver indicação sua de bens à penhora. Do contrário, não há legitimidade para compor o polo passivo da ação conjuntamente com o exequente.

• A vedação de transferências voluntárias somente se aplica quando o ente não cumpre com a responsabilidade da gestão fiscal no que se refere à instituição, previsão e arrecadação dos <u>impostos</u> somente.

• Dito isso, não existe razão para a vedação de transferências voluntárias na hipótese de não instituição de taxas ou qualquer outro tributo.