Reviewer #3 (Public review):

Summary:

The publication presents unique in-vivo images of the upper layer of the epidermis of glabrous skin when a flat object compresses or slides on the fingertip. The images are captured using OCT and show the strain that fingerprints experience during mechanical stimulation.

The most important finding is, in my opinion, that fingerprints undergo pure compression/tension without horizontal shear, suggesting that the shear stress caused by tangential load is transferred to the deeper tissues and ultimately to the mechanoreceptors (SA-I / RA-I).

Strengths:

Fascinating new insights into the mechanics of glabrous skin. To the best of my knowledge, this is the first experimental evidence of the mechanical deformation of fingerprints when subjected to dynamic mechanical stimulation. The OCT measurement allows unprecedented measurement of skin depth, whereas previous works were limited to tracking surface deformation.

The robust data analysis reveals the continuum mechanics underlying the deformation of the fingerprint ridges.

Weaknesses:

I do not see any major weaknesses. The work is mainly experimental and is rigorously executed.

Reviewer #1 (Public review):

Summary:

The authors analyse electron microscopy data of the nociceptive circuit in fly larvae at two developmental stages. They look for ways in which the connectivity of the circuit differs between these two stages, when neurons grow by a factor of about 5. They find that average synaptic weights do not change significantly, and that the density of synaptic inputs onto a dendrite is also unchanged despite the extreme change in size. Further, they find that synaptic weights become less variable and that synapses between pairs of neurons do not become more correlated over development. The second of these findings is evidence against many known long-term synaptic plasticity mechanisms having a significant effect on this circuit.<br /> They combine their first result with theoretical modelling to show that invariances in weight and density preserve neuronal responses despite scaling, and conclude that this is the mechanism by which the circuit can maintain useful function throughout development.

Strengths:

The paper carefully analyses a rich dataset of electron microscopy data and clearly highlights how the data support the authors' findings and not obvious alternative hypotheses. The overall finding, that this particular circuit can maintain stable responses using a local conservation of synaptic inputs, is quite striking.

Weaknesses:

The main weakness of this paper is in its argument that such a mechanism of input conservation might be a general developmental rule. The vast majority of literature on spine density in mammals finds that spine density increases early in development before falling again (Bourgeois & Rakic, J Neurosci 1993; Petanjek at el, PNAS 2011; Wildenberg et al, Nat Comms 2023). I find the analyses in this manuscript convincing, but the authors should more clearly highlight that this mechanism might be specific to insect nociceptive circuits. A further minor weakness is the fact that only staging data is available, where different individuals are imaged at different developmental stages. This is unavoidable and acknowledged in the manuscript, but it makes it harder to draw clear conclusions about plasticity mechanisms and specific changes in synaptic weight distributions.

Reviewer #2 (Public review):

Summary:

The authors utilize large volume electron microscopy ("connectomics") data to address how circuits remain stable during development. They focus on the development of the Drosophila nociceptive circuit between larval stages L1 and L3. Their analyses focus on changes to pre- and post-synaptic circuit partners (i.e., pre-synaptic axons and post-synaptic dendrites) and conduct a thorough analysis of eliminating likely changes to both that could balance circuits. Ultimately, they find that the change in axonal growth (i.e, cable length) is mismatched with dendritic growth, but that this is balanced by an increase in the synapse density of pre-synaptic axons.

Strengths:

The authors used connectomics, the gold standard for neural circuit tracing, to conduct their analyses, and thus their results are strongly supported by the quality of the data. They carefully eliminated several models for how pre- and post-synaptic changes could co-develop to preserve circuit stability until they identified a major driver in changes in the timing of axon development relative to dendritic development. I also admired their willingness to be transparent about the limitations of their studies, including a lack of analyses of changes to inhibitory inputs and a lack of dynamics in their data. Overall, it's difficult to argue their results are wrong, but they may be incomplete. That said, it's difficult to account for every variable, and they covered the more salient topics, and it's my opinion that this is an important contribution that moves the field forward while also being careful to note its limitations that could and should motivate future work.

Weaknesses:

I identified a few weaknesses that could benefit from revisions:

(1) I found parts of the text confusing, verging on misleading, specifically as it relates to other species. For example, in Line 93, the authors state that they have shown that synapses per unit dendrite length remain remarkably constant across species and brain regions. This was mentioned throughout the manuscript, and it wasn't clear to me whether this was referring to across development or in adults. If over-development, this contrasts with other recently published work of our own comparing synapse densities in the developing mouse and rhesus macaque. Whether they are different or the same is equally interesting and should be discussed more clearly. Related to this, it's not clear that mammalian circuits over development remain stable. For example, our work shows that the ratio of excitatory and inhibitory synapses changes quite a lot in developing mice and primates.

(2) I was not convinced by the use of axon-dendritic cable overlap. While axons and dendrites certainly need to be close together to make a synapse, I don't understand why this predicts they will connect. In connectomic data, axons pass by hundreds if not thousands of potential post-synaptic partners without making a synapse. Ultimately, the authors' data on changes in axon cable length between L1 and L3 would predict more overlap, but I found the use of overlap confusing and unnecessary, relative to the concreteness of their other analyses. I would suggest removing this from their analyses or providing a stronger argument for how overlap predicts connectivity.

(3) Figure 7. For non-computational neuroscientists, I think it would be tremendously helpful to include a table that outlines the metrics you used. The text states you constrained these models with your EM data, but it would be helpful to summarize the range of numerical data you used for each parameter.

(4) The most important finding to me was the asymmetry between axon and dendrite development. Perhaps beyond the scope of this work, it raises the question of whether there are privileged axons that uniquely increase their synapse density. Figure 5D alludes to this, where the fold change in cable length is not proportional to the change in synapse density. Could it be that over development, specific inputs become dominant while others prune their synapses, resulting in an overall balanced circuit, but dominance of specific partners changes? Either answer (i.e., yes, there are privileged circuits that emerge from L1 to L3, or no) would be very interesting and greatly elevate the significance of this work.

(5) Related to my comment #1, can the authors comment on whether these changes are unique to Drosophila nociceptive circuits? Do all circuits remain balanced over development in flies? Finally, could you clarify why L1 to L3 was chosen?

Reviewer #3 (Public review):

Summary:

Fritz et al. investigate the changes in synaptic connectivity between two different life stages of the Drosophila larva, L1 and L3. They focus on 3 types of nociceptive mechanosensory neurons and their connecting 6 downstream interneurons. Connectomic analysis reveals that connectivity and dendritic density are stable across development; however, axonal density, axodendritic overlap, and the number of synapses increase. Finally, using a modeling approach, they demonstrate that this conservation of most features enables stable output across life stages.

Strengths:

The authors analyse two different connectomes from fly larvae in two different life stages. By now, there are only very few such samples available; thus, this is a novel approach and will be helpful to guide further comparative connectomic studies in the future.

Weaknesses:

The authors analyze only a minimal circuit with 9 different cell types on each hemisphere; thus, their findings might be specialised for this specific nociceptive sensory to interneuron peripheral circuit. Also, more animals might need to be analyzed in different life stages to generalize these findings.

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the Drift and Diffusion Model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options in individuals with bulimia nervosa (BN) and healthy participants (2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has potential to improve the understanding of pathological food choices.

Weaknesses:

I thank the author for reviewing their manuscript.

However, I still have major concerns.

The authors say that they removed any causal claims in their revised version of the manuscript. The sentence before the last one of the abstract still says "bias for high-fat foods predicted more frequent subjective binge episodes over three months". This is a causal claim that I already highlighted in my previous review, specifically for that sentence (see my second sentence of my major point 2 of my previous review).

I also noticed that a comment that I added was not sent to the authors. In this comment I was highlighting that in Figure 2 of Galibri et al., I was uncertain about a difference between neutral and negative inductions of the average negative rating after the induction in the BN group (i.e. comparing the negative rating after negative induction in BN to the negative rating after neutral induction in BN). Figure 2 of Galibri et al. looks to me that:

(1) The BN participants were more negative before the induction when they came to the neutral session than when they came to the negative session. (2) The BN participants looked almost negatively similar (taking into account the error bars reported) after the induction in both sessions

These observations are of high importance because they may support the fact that BN patients were likely in a similar negative state to run the food decision task in both conditions (negative and neutral). Therefore, the lack of difference in food choices in BN patients is unsurprising and nothing could be concluded from the DDM analyses. Moreover, the strong negative ratings of BN patients in the neutral condition as compared to healthy participants together with almost similar negative ratings after the two inductions contradict the authors' last sentence of their abstract.

I appreciate that the authors reproduced an analysis of their initial paper regarding the negative ratings (i.e. Table S1). It partly answers my aforementioned point but does not address the fact that BN may have been in a similar negative state in both conditions (neutral and negative) when running the food decision task: if BN patients were similarly negative after both induction (neutral and negative), nothing can be concluded from their differences in their results obtained from the DDM. As the authors put it, "not all loss-of-control eating occurs in the context of negative state", I add that far from all negative states lead to a loss-of-control eating in BN patients. This grounds all my aforementioned remarks and my remarks of my first review.

A solution for that is to run a paired t-test in BN patients only comparing the score after the induction in the two conditions (neutral and negative) reported in Figure 2 of their initial article.

I appreciate the analysis that the authors added with the restrictive subscale of the EDE-Q. That this analysis does not show any association with the parameters of interest does not show that there is a difference in the link between self reported restrictions and self reported binges. Only such a difference would allow us to claim that the results the authors report may be related to binges.

I appreciate the wording of the answer of the authors to my third point: "the results suggest that individuals whose task behavior is more reactive to negative affect tend to be the most symptomatic, but the results do not allow us to determine whether this reactivity causes the symptoms". This sentence is crystal clear and sums very well the limits of the associations the authors report with binge eating frequency. However, I do not see this sentence in the manuscript. I think the manuscript would benefit substantially from adding it.

Statistical analyses:

If I understood well the mixed models performed, analyses of supplementary tables S1 and S27 to S32 are considering all measures as independent which means that the considered score of each condition (neutral vs negative) and each time (before vs after induction) which have been rated by the same participants are independent. Such type of analyses does not take into account the potential correlation between the 4 scores of a given participant. As a consequence, results may lead to false positives that a linear mixed model does not address. The appropriate analysis would be to run adapted statistical tests pairing the data without running any mixed model.

Notes:

It is not because specific methods like correlating self reported measures over long periods with almost instantaneous behaviors (like tasks) have been used extensively in studies that these methods are adapted to answer a given scientific question. Measures aggregated over long periods miss the variations in instantaneous behaviors over these periods.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well-understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase the likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant, and the methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

Sample size was relatively small, and participants were all women with BN, which limits generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. These limitations are adequately noted in the discussion.

Reviewer #3 (Public review):

Summary:

The study uses the food choice task, a well-established method in eating disorder research, particularly in anorexia nervosa. However, it introduces a novel analytical approach-the diffusion decision model-to deconstruct food choices and assess the influence of negative affect on how and when tastiness and healthiness are considered in decision-making among individuals with bulimia nervosa and healthy controls.

Strengths:

The introduction provides a comprehensive review of the literature, and the study design appears robust. It incorporates separate sessions for neutral and negative affect conditions and counterbalances tastiness and healthiness ratings. The statistical methods are rigorous, employing multiple testing corrections.

A key finding-that negative affect induction biases individuals with bulimia nervosa toward prioritizing tastiness over healthiness-offers an intriguing perspective on how negative affect may drive binge eating behaviors.

Weaknesses:

A notable limitation is the absence of a sample size calculation, which, combined with the relatively small sample, may have contributed to null findings. Additionally, while the affect induction method is validated, it is less effective than alternatives such as image or film-based stimuli (Dana et al., 2020), potentially influencing the results.

Reviewer #1 (Public review):

This is a well-designed and very interesting study examining the impact of imprecise feedback on outcomes on decision-making. I think this is an important addition to the literature and the results here, which provide a computational account of several decision-making biases, are insightful and interesting.

I do not believe I have substantive concerns related to the actual results presented; my concerns are more related to the framing of some of the work. My main concern is regarding the assertion that the results prove that non-normative and non-Bayesian learning is taking place. I agree with the authors that their results demonstrate that people will make decisions in ways that demonstrate deviations from what would be optimal for maximizing reward in their task under a strict application of Bayes rule. I also agree that they have built reinforcement learning models which do a good job of accounting for the observed behavior. However, the Bayesian models included are rather simple- per the author descriptions, applications of Bayes' rule with either fixed or learned credibility for the feedback agents. In contrast, several versions of the RL models are used, each modified to account for different possible biases. However more complex Bayes-based models exist, notably active inference but even the hierarchical gaussian filter. These formalisms are able to accommodate more complex behavior, such as affect and habits, which might make them more competitive with RL models. I think it is entirely fair to say that these results demonstrate deviations from an idealized and strict Bayesian context; however, the equivalence here of Bayesian and normative is I think misleading or at least requires better justification/explanation. This is because a great deal of work has been done to show that Bayes optimal models can generate behavior or other outcomes that are clearly not optimal to an observer within a given context (consider hallucinations for example) but which make sense in the context of how the model is constructed as well as the priors and desired states the model is given.

As such, I would recommend that the language be adjusted to carefully define what is meant by normative and Bayesian and to recognize that work that is clearly Bayesian could potentially still be competitive with RL models if implemented to model this task. An even better approach would be to directly use one of these more complex modelling approaches, such as active inference, as the comparator to the RL models, though I would understand if the authors would want this to be a subject for future work.

Abstract:

The abstract is lacking in some detail about the experiments done, but this may be a limitation of the required word count? If word count is not an issue, I would recommend adding details of the experiments done and the results. One comment is that there is an appeal to normative learning patterns, but this suggests that learning patterns have a fixed optimal nature, which may not be true in cases where the purpose of the learning (e.g. to confirm the feeling of safety of being in an in-group) may not be about learning accurately to maximize reward. This can be accommodated in a Bayesian framework by modelling priors and desired outcomes. As such the central premise that biased learning is inherently non-normative or non-Bayesian I think would require more justification. This is true in the introduction as well.

Introduction:

As noted above the conceptualization of Bayesian learning being equivalent to normative learning I think requires either further justification. Bayesian belief updating can be biased an non-optimal from an observer perspective, while being optimal within the agent doing the updating if the priors/desired outcomes are set up to advantage these "non-optimal" modes of decision making.

Results:

I wonder why the agent was presented before the choice - since the agent is only relevant to the feedback after the choice is made. I wonder if that might have induced any false association between the agent identity and the choice itself. This is by no means a critical point but would be interesting to get the authors' thoughts.

The finding that positive feedback increases learning is one that has been shown before and depends on valence, as the authors note. They expanded their reinforcement learning model to include valence; but they did not modify the Bayesian model in a similar manner. This lack of a valence or recency effect might also explain the failure of the Bayesian models in the preceding section where the contrast effect is discussed. It is not unreasonable to imagine that if humans do employ Bayesian reasoning that this reasoning system has had parameters tuned based on the real world, where recency of information does matter; affect has also been shown to be incorporable into Bayesian information processing (see the work by Hesp on affective charge and the large body of work by Ryan Smith). It may be that the Bayesian models chosen here require further complexity to capture the situation, just like some of the biases required updates to the RL models. This complexity, rather than being arbitrary, may be well justified by decision-making in the real world.

The methods mention several symptom scales- it would be interesting to have the results of these and any interesting correlations noted. It is possible that some of individual variability here could be related to these symptoms, which could introduce precision parameter changes in a Bayesian context and things like reward sensitivity changes in an RL context.

Discussion:

(For discussion, not a specific comment on this paper): One wonders also about participant beliefs about the experiment or the intent of the experimenters. I have often had participants tell me they were trying to "figure out" a task or find patterns even when this was not part of the experiment. This is not specific to this paper, but it may be relevant in the future to try and model participant beliefs about the experiment especially in the context of disinformation, when they might be primed to try and "figure things out".

As a general comment, in the active inference literature, there has been discussion of state-dependent actions, or "habits", which are learned in order to help agents more rapidly make decisions, based on previous learning. It is also possible that what is being observed is that these habits are at play, and that they represent the cognitive biases. This is likely especially true given, as the authors note, the high cognitive load of the task. It is true that this would mean that full-force Bayesian inference is not being used in each trial, or in each experience an agent might have in the world, but this is likely adaptive on the longer timescale of things, considering resource requirements. I think in this case you could argue that we have a departure from "normative" learning, but that is not necessarily a departure from any possible Bayesian framework, since these biases could potentially be modified by the agent or eschewed in favor of more expensive full-on Bayesian learning when warranted. Indeed in their discussion on the strategy of amplifying credible news sources to drown out low-credibility sources, the authors hint to the possibility of longer term strategies that may produce optimal outcomes in some contexts, but which were not necessarily appropriate to this task. As such, the performance on this task- and the consideration of true departure from Bayesian processing- should be considered in this wider context. Another thing to consider is that Bayesian inference is occurring, but that priors present going in produce the biases, or these biases arise from another source, for example factoring in epistemic value over rewards when the actual reward is not large. This again would be covered under an active inference approach, depending on how the priors are tuned. Indeed, given the benefit of social cohesion in an evolutionary perspective, some of these "biases" may be the result of adaptation. For example, it might be better to amplify people's good qualities and minimize their bad qualities in order to make it easier to interact with them; this entails a cost (in this case, not adequately learning from feedback and potentially losing out sometimes), but may fulfill a greater imperative (improved cooperation on things that matter). Given the right priors/desired states, this could still be a Bayes-optimal inference at a social level and as such may be ingrained as a habit which requires effort to break at the individual level during a task such as this.

The authors note that this task does not relate to "emotional engagement" or "deep, identity-related, issues". While I agree that this is likely mostly true, it is also possible that just being told one is being lied to might elicit an emotional response that could bias responses, even if this is a weak response.

Comments on first revisions:

In their updated version the authors have made some edits to address my concerns regarding the framing of the 'normative' Bayesian model, clarifying that they utilized a simple Bayesian model which is intended to adhere in an idealized manner to the intended task structure, though further simulations would have been ideal.

The authors, however, did not take my recommendation to explore the symptoms in the symptom scales they collected as being a potential source of variability. They note that these were for hypothesis generation and were exploratory, fair enough, but this study is not small and there should have been sufficient sample size for a very reasonable analysis looking at symptom scores.

However, overall the toned-down claims and clarifications of intent are adequate responses to my previous review.

Comments on second revisions:

While I believe an exploration of symptom scores would have been a valuable addition, this is not required for the purpose of the paper, and as such, I have no further comments.

Reviewer #2 (Public review):

This important paper studies the problem of learning from feedback given by sources of varying credibility. The convincing combination of experiment and computational modeling helps to pin down properties of learning, while opening unresolved questions for future research.

Summary:

This paper studies the problem of learning from feedback given by sources of varying credibility. Two bandit-style experiments are conducted in which feedback is provided with uncertainty, but from known sources. Bayesian benchmarks are provided to assess normative facets of learning, and alternative credit assignment models are fit for comparison. Some aspects of normativity appear, in addition to possible deviations such as asymmetric updating from positive and negative outcomes.

Strengths:

The paper tackles an important topic, with a relatively clean cognitive perspective. The construction of the experiment enables the use of computational modeling. This helps to pinpoint quantitatively the properties of learning and formally evaluate their impact and importance. The analyses are generally sensible, and advanced parameter recovery analyses (including cross-fitting procedure) provide confidence in the model estimation and comparison. The authors have very thoroughly revised the paper in response to previous comments.

Weaknesses:

The authors acknowledge the potential for cognitive load and the interleaved task structure to play a meaningful role in the results, though leave this for future work. This is entirely reasonable, but remains a limitation in our ability to generalize the results. Broadly, some of the results obtained in cases where the extent of generalization is not always addressed and remains uncertain.

Reviewer #3 (Public review):

Summary

This paper investigates how disinformation affects reward learning processes in the context of a two-armed bandit task, where feedback is provided by agents with varying reliability (with lying probability explicitly instructed). They find that people learn more from credible sources, but also deviate systematically from optimal Bayesian learning: They learned from uninformative random feedback and updated too quickly from fully credible feedback (especially following low-credibility feedback). People also appeared to learn more from positive feedback and there is tentative evidence that this bias is exacerbated for less credible feedback.

Overall, this study highlights how misinformation could distort basic reward learning processes, without appeal to higher order social constructs like identity.

Strengths - The experimental design is simple and well-controlled; in particular, it isolates basic learning processes by abstracting away from social context - Modeling and statistics meet or exceed standards of rigor - Limitations are acknowledged where appropriate, especially those regarding external validity and challenges in dissociating positivity bias from perseveration - The comparison model, Bayes with biased credibility estimates, is strong; deviations are much more compelling than e.g. a purely optimal model - The conclusions are of substantial interest from both a theoretical and applied perspective

Weaknesses

The authors have done a great job addressing my concerns with the two previous submission. The one issue that they were not able to truly address is the challenge of dissociating positivity bias from perseveration; this challenge weakens evidence for the conclusion that less credible feedback yields a stronger positivity bias. However, the authors have clearly acknowledged this limitation and tempered their conclusions accordingly. Furthermore, the supplementary analyses on this point are suggestive (if not fully conclusive) and do a better job of at least trying to address the confound than most work on positivity/confirmation bias.

I include my previous review describing the challenge in more detail for reference. I encourage interested readers to see the author response as well. It has convinced me that this weakness is not a reflection of the work, but is instead a fundamental challenge for research on positivity bias.

Absolute or relative positivity bias?

The conclusion of greater positivity bias for lower credible feedback (Fig 5) hinges on the specific way in which positivity bias is defined. Specifically, we only see the effect when normalizing the difference in sensitivity to positive vs. negative feedback by the sum. I appreciate that the authors present both and add the caveat whenever they mention the conclusion. However, without an argument that the relative definition is more appropriate, the fact of the matter is that the evidence is equivocal.

There is also a good reason to think that the absolute definition is more appropriate. As expected, participants learn more from credible feedback. Thus, normalizing by average learning (as in the relative definition) amounts to dividing the absolute difference by increasingly large numbers for more credible feedback. If there is a fixed absolute positivity bias (or something that looks like it), the relative bias will necessarily be lower for more credible feedback. In fact, the authors own results demonstrate this phenomenon (see below). A reduction in relative bias thus provides weak evidence for the claim.

It is interesting that the discovery study shows evidence of a drop in absolute bias. However, for me, this just raises questions. Why is there a difference? Was one just a fluke? If so, which one?

Positivity bias or perseveration?

Positivity bias and perseveration will both predict a stronger relationship between positive (vs. negative) feedback and future choice. They can thus be confused for each other when inferred from choice data. This potentially calls into question all the results on positivity bias.

The authors clearly identify this concern in the text and go to considerable lengths to rule it out. However, the new results (in revision 1) show that a perseveration-only model can in fact account for the qualitative pattern in the human data (the CA parameters). This contradicts the current conclusion:

Critically, however, these analyses also confirmed that perseveration cannot account for our main finding of increased positivity bias, relative to the overall extent of CA, for low-credibility feedback.

Figure 24c shows that the credibility-CA model does in fact show stronger positivity bias for less credible feedback. The model distribution for credibility 1 is visibly lower than for credibilities 0.5 and 0.75.

The authors need to be clear that it is the magnitude of the effect that the perseveration-only model cannot account for. Furthermore, they should additionally clarify that this is true only for models fit to data; it is possible that the credibility-CA model could capture the full size of the effect with different parameters (which could fit best if the model was implemented slightly differently).

The authors could make the new analyses somewhat stronger by using parameters optimized to capture just the pattern in CA parameters (for example by MSE). This would show that the models are in principle incapable of capturing the effect. However, this would be a marginal improvement because the conclusion would still rest on a quantitative difference that depends on specific modeling assumptions.

New simulations clearly demonstrate the confound in relative bias

Figure 24 also speaks to the relative vs. absolute question. The model without positivity bias shows a slightly stronger absolute "positivity bias" for the most credible feedback, but a weaker relative bias. This is exactly in line with the logic laid out above. In standard bandit tasks, perseveration can be quite well-captured by a fixed absolute positivity bias, which is roughly what we see in the simulations (I'm not sure what to make of the slight increase; perhaps a useful lead for the authors). However, when we divide by average credit assignment, we now see a reduction. This clearly demonstrates that a reduction in relative bias can emerge without any true differences in positivity bias.

Given everything above, I think it is unlikely that the present data can provide even "solid" evidence for the claim that positivity bias is greater with less credible feedback. This confound could be quickly ruled out, however, by a study in which feedback is sometimes provided in the absence of a choice. This would empirically isolate positivity bias from choice-related effects, including perseveration.

Comments on revisions:

Great work on this. The new paper is very interesting as well. I'm delighted to see that the excessive amount of time I spent on this review has had a concrete impact.

Reviewer #1 (Public review):

Summary:

The authors provide a compelling case that the unique variance explained by LLMs is different (and later) than the unique variance explained by DNNs. This characterises when, and to some extent where, these differences occur, and for LLMs, why. The authors also probe what in the sentences is driving the brain alignment.

Strengths:

(1) The study is timely.

(2) There is a robust dataset and results.

(3) There is compelling separation between unique responses related to LLMs and DNNs.

(4) The paper is well-written.

Weaknesses:

The authors could explore more of what the overlap between the LLM and DNN means, and in general, how this relates to untrained networks.

Reviewer #2 (Public review):

Summary:

This study provides an investigation into the temporal dynamics of visuo-semantic processing in the human brain, leveraging both deep neural networks (DNNs) and large language models (LLMs). By developing encoding models based on vision DNNs, LLMs, and their fusion, the authors demonstrate that vision DNNs preferentially account for early, broadband EEG responses, while LLMs capture later, low-frequency signals and more detailed visuo-semantic information. It is shown that the parietal cortex shows responses during visuo-semantic processing that can be partially accounted for by language features, highlighting the role of higher-level areas in encoding abstract semantic information.

Strengths:

The study leverages a very large EEG dataset with tens of thousands of stimulus presentations, which provides an unusually strong foundation for benchmarking a variety of vision DNNs and LLMs. This scale not only increases statistical power but also allows robust comparison across model architectures, ensuring that the conclusions are not idiosyncratic to a particular dataset or stimulus set.

By using high-density EEG, the authors are able to capture the fine-grained temporal dynamics of visuo-semantic processing, going beyond the coarse temporal resolution of fMRI-based studies. This enables the authors to disentangle early perceptual encoding from later semantic integration, and to characterize how different model types map onto these stages of brain activity. The temporal dimension provides a particularly valuable complement to previous fMRI-based model-to-brain alignment studies.

The encoding models convincingly show that vision DNNs and LLMs play complementary roles in predicting neural responses. The vision DNNs explain earlier broadband responses related to perceptual processing, while LLMs capture later, lower-frequency signals that reflect higher-order semantic integration. This dual contribution provides new mechanistic insights into how visual and semantic information unfold over time in the brain, and highlights the utility of combining unimodal models rather than relying on multimodal networks alone.

Weaknesses:

(1) The experimental design is insufficiently described, particularly regarding whether participants were engaged in a behavioral task or simply passively viewing images. Task demands are known to strongly influence neural coding and representations, and without this information, it is difficult to interpret the nature of the EEG responses reported.

(2) The description of the encoding model lacks precision and formalization. It is not entirely clear what exactly is being predicted, how the model weights are structured across time points, or the dimensionality of the inputs and outputs. A more formal mathematical formulation would improve clarity and reproducibility.

(3) The selected vision DNNs (CORnet-S, ResNet, AlexNet, MoCo) have substantially lower ImageNet classification accuracies than current state-of-the-art models, with gaps of at least 10%. Referring to these models collectively as "vision DNNs" may overstate their representational adequacy. This performance gap raises concerns about whether the chosen models can fully capture the visual and semantic features needed for comparison with brain data. Clarification of the rationale for choosing these particular networks, and discussion of how this limitation might affect the conclusions, is needed.

(4) The analytic framework treats "vision" and "language" as strictly separate representational domains. However, semantics are known to emerge in many state-of-the-art visual models, with different layers spanning a gradient from low-level visual features to higher-level semantic representations. Some visual layers may be closer to LLM-derived representations than others. By not examining this finer-grained representational structure within vision DNNs, the study may oversimplify the distinction between vision- and language-based contributions.

(5) The study uses static images, which restricts the scope of the findings to relatively constrained visual semantics. This limitation may explain why nouns and adjectives improved predictions over vision DNNs, but verbs did not. Verbs often require dynamic information about actions or events, which static images cannot convey.

Reviewer #3 (Public review):

Summary:

Rong et al., compare EEG image responses from a large-scale dataset to state-of-the-art vision and language models, as well as their fusion. They find that the fusion of models provides the best predictivity, with early contribution from vision models and later predictivity from language models. The paper has several strengths: high temporal resolution data (though at the expense of spatial resolution), detailed comparison of alignment (and differences) between vision and language model embeddings, and comparison of "fusion" of different DNN models.

Despite the paper's strengths, it is not clear what is at stake with these findings or how they advance our knowledge beyond other recent studies showing vision versus language model predictions of visual cortex responses with fMRI.

Strengths:

The authors use a large-scale EEG dataset and a comprehensive modeling approach. The methods are sound and involve multiple model comparisons. In particular, the disentangling of vision and language model features is something that has been largely ignored in prior related studies.

Weaknesses:

(1) The authors state their main hypothesis (lines 48-51) that human neural responses to visual stimulation are better modelled by combining representations from a vision DNN and an LLM than by the representations from either of the two components alone, and that the vision DNN and LLM components would uniquely predict earlier and later stages of visual processing, respectively.

While they confirm this hypothesis in largely compelling ways, it is not clear whether these results tell us something about the brain beyond how to build the most predictive model.

In particular, why do language models offer advantages over vision models, and what does this tell us about human visual processing? In several places, the discussion of advantages for the language model felt somewhat trivial and did not seem to advance our understanding of human vision, e.g., "responses for visual stimulation encode detailed information about objects and their properties" (lines 266-270) and "LLM representations capture detailed visuo-semantic information about the stimulus images" (line 293).

(2) It is not clear what the high temporal resolution EEG data tell us that the whole-brain fMRI data do not. The latency results seem to be largely in line with fMRI findings, where the early visual cortex is better predicted by vision models, and the language model is better in later/more anterior regions. In addition, it would help to discuss whether the EEG signals are likely to be restricted to the visual cortex, or could the LLM predictivity explain downstream processing captured by whole-brain EEG signals?

Relatedly, it would help the authors to expand on the implications of the frequency analysis.

(3) While the authors test many combinations of vision and language models and show their "fusion" advantages are largely robust to these changes, it is still hard to ignore the vast differences between vision and language models, in terms of architecture and how they are trained. Two studies (Wang et al., 2023, and Conwell et al., 2024) have now shown that when properly controlling for architecture and dataset, there is little to no advantage of language alignment in predicting visual cortex responses. It would help for the authors to both discuss this aspect of the prior literature and to try to address the implications for their own findings (related to pt 1 about what, if anything, is "special" about language models).

(4) Model features - it would help to state the dimensionality of the input embeddings for each model and how much variance is explained and preserved after the PCA step? I wonder how sensitive the findings are to this choice of dimensionality reduction, and whether an approach that finds the optimal model layer (in a cross-validated way) would show less of a difference between vision/language models (I realize this is not feasible with models like GPT-3).

(5) To better understand the fusion advantage, it would help to look at the results, look for a pair of vision models and a pair of language models. Can a similar advantage be found by combining models from the same modality?

Reviewer #1 (Public review):

Summary:

Rahmani et al. utilize the TurboID method to characterize global proteome changes in the worm's nervous system induced by a salt-based associative learning paradigm. Altogether, they uncover 706 proteins tagged by the TurboID method in worms that underwent the memory-inducing protocol. Next, the authors conduct a gene enrichment analysis that implicates specific molecular pathways in salt-associative learning, such as MAP kinase and cAMP-mediated pathways, as well as specific neuronal classes including pharyngeal neurons, and specific sensory neurons, interneurons, and motor neurons. The authors then screen a representative group of hits from the proteome analysis. They find that mutants of candidate genes from the MAP kinase pathway, namely dlk-1 and uev-3, do not affect performance in the learning paradigm. Instead, multiple acetylcholine signaling mutants, as well as a protein-kinase-A mutant, significantly affected performance in the associative memory assay (e.g., acc-1, acc-3, lgc-46, and kin-2). Finally, the authors demonstrate that protein-kinase-A mutants, as well as acetylcholine signaling mutants, do not exhibit a phenotype in a related but distinct conditioning paradigm-aversive salt conditioning-suggesting their effect is specific to appetitive salt conditioning.

Overall, the authors addressed the concerns raised in the previous review round, including the statistics of the chemotaxis experiments and the systems-level analysis of the neuron class expression patterns of their hits. I also appreciate the further attempt to equalize the sample size of the chemotaxis experiments and the transparent reporting of the sample size and statistics in the figure captions and Table S9. The new results from the panneuronal overexpression of the kin-2 gain-of-function allele also contribute to the manuscript. Together, these make the paper more compelling. The additional tested hits provide a comprehensive analysis of the main molecular pathways that could have affected learning. However, the revised manuscript includes more information and analysis, raising additional concerns.

Major comments:

As reviewer 4 noted, and as also shown to be relevant for C30G12.6 presented in Figure 6, the backcrossing of the mutants is important, as background mutations may lead to the observed effects. Could the authors add to Table 1, sheet 1, the outcrossing status of the tested mutants? It is important to validate that the results of the positive hits (where learning was affected), such as acc-1, acc-3, and lgc-46, do not stem from background mutations.

The fold change in the number of hits for different neurons in the CENGEN-based rank analysis requires a statistical test (discussed on pages 17-19 and summarized in Table S7). Similar to the other gene enrichment analyses presented in the manuscript, the new rank analysis also requires a statistical test. Since the authors extensively elaborate on the results from this analysis, I think a statistical analysis is especially important for its interpretation. For example, if considering the IL1 neurons, which ranked highest, and assuming random groups of genes-each having the same size as those of the ranked neurons (209 genes in total for IL1 in Table S7)-how common would it be to get the calculated fold change of 1.38 or higher? Such bootstrapping analysis is common for enrichment analysis. Perhaps the authors could consult with an institutional expert (Dr. Pawel Skuza, Flinders University) for the statistical aspects of this analysis.

The learning phenotypes from Figure S8, concerning acc-1, acc-3, and lgc-46 mutants, are summarized in a scheme in Figure 4; however, the chemotaxis results are found in the supplemental Figure S8. Perhaps I missed the reasoning, but for transparency, I think the relevant Figure S8 results should be shown together with their summary scheme in Figure 4.

Reviewer #2 (Public review):

Summary:

In this study by Rahmani in colleagues, the authors sought to define the "learning proteome" for a gustatory associative learning paradigm in C. elegans. Using a cytoplasmic TurboID expressed under the control of a pan-neuronal promoter, the authors labeled proteins during the training portion of the paradigm, followed by proteomics analysis. This approach revealed hundreds of proteins potentially involved in learning, which the authors describe using gene ontology and pathway analysis. The authors performed functional characterization of over two dozen of these genes for their requirement in learning using the same paradigm. They also compared the requirement for these genes across various learning paradigms and found that most hits they characterized appear to be specifically required for the training paradigm used for generating the "learning proteome".

Strengths:

- The authors have thoughtfully and transparently designed and reported the results of their study. Controls are carefully thought-out, and hits are ranked as strong and weak. By combining their proteomics with behavioral analysis, the authors also highlight the biological significance of their proteomics findings, and support that even weak hits are meaningful.

- The authors display a high degree of statistical rigor, incorporating normality tests into their behavioral data which is beyond the field standard.

- The authors include pathway analysis that generates interesting hypotheses about processes involved learning and memory

-The authors generally provide thoughtful interpretations for all of their results, both positive and negative, as well as any unexpected outcomes.

Weaknesses:

- The authors use the Cengen single cell-transcriptomic atlas to predict where the proteins in the "learning proteome" are likely to be expressed and use this data to identify neurons that are likely significant to learning, and building hypothetical circuit. This is an excellent idea; however, the Cengen dataset only contains transcriptomic data from juvenile L4 animals, while the authors performed their proteome experiments in Day 1 Adult animals. It is well documented that the C. elegans nervous system transcriptome is significant different between these two stages (Kaletsky et al., 2016, St. Ange et al., 2024), so the authors might be missing important expression data, resulting in inaccurate or incomplete networks. The adult neuronal single-cell atlas data (https://cestaan.princeton.edu/) would be better suited to incorporate into neuronal expression analysis.

- The authors offer many interpretations for why mutants in "learning proteome" hits have no detectable phenotype, which is commendable. They are however overlooking another important interpretation, it is possible that these changes to the proteome are important for memory, which is dependent upon translation and protein level changes, and is molecularly distinct from learning. It is well established in the field mutating or knocking down memory regulators in other paradigms will often have no detectable effect on learning. Incorporating this interpretation into the discussion and highlighting it as an area for future exploration would strengthen the manuscript.

-A minor weakness - In the discussion, the authors state that the Lakhina, et al 2015 used RNA-seq to assess memory transcriptome changes. This study used microarray analysis.

Significance:

The approach used in this study is interesting and has the potential to further our knowledge about the molecular mechanisms of associative behaviors. There have been multiple transcriptomic studies in the worm looking at gene expression changes in the context of behavioral training. This study compliments and extends those studies, by examining how the proteome changes in a different training paradigm. This approach here could be employed for multiple different training paradigms, presenting a new technical advance for the field. This paper would be of interest to the broader field of behavioral and molecular neuroscience. Though it uses an invertebrate system, many findings in the worm regarding learning and memory translate to higher organisms, making this paper of interest and significant to the broader field of behavioral neuroscience.

Reviewer #4 (Public review):

Summary:

In this manuscript, authors used a learning paradigm in C. elegans; when worms were fed in a saltless plate, its chemotaxis to salt is greatly reduced. To identify learning-related proteins, authors employed nervous system-specific transcriptome analysis to compare whole proteins in neurons between high-salt-fed animals and saltless-fed animals. Authors identified "learning-specific proteins" which are observed only after saltless feeding. They categorized these proteins by GO analyses, pathway analyses and expression site analyses, and further stepped forward to test mutants in selected genes identified by the proteome analysis. They find several mutants that are defective or hyper-proficient for learning, including acc-1/3 and lgc-46 acetylcholine receptors, F46H5.3 putative arginine kinase, and kin-2, a cAMP pathway gene. These mutants were not previously reported to have abnormality in the learning paradigm.

Concerns:

Upon revision, authors addressed all concerns of this reviewer, and the results are now presented in a way that facilitates objective evaluation. Authors' conclusions are supported by the results presented, and the strength of the proteomics approach is persuasively demonstrated.

Significance:

(1) Total neural proteome analysis has not been conducted before for learning-induced changes, though transcriptome analysis has been performed for odor learning (Lakhina et al., http://dx.doi.org/10.1016/j.neuron.2014.12.029). This warrants the novelty of this manuscript, because for some genes, protein levels may change even though mRNA levels remain the same. Although in a few reports TurboID has been used in C. elegans, this is the first report of a systematic analysis of tissue-specific differential proteomics.

(2) Authors found five mutants that have abnormality in the salt learning. These genes have not been described to have the abnormality, providing novel knowledge to the readers, especially those who work on C. elegans behavioural plasticity. Especially, involvement of acetylcholine neurotransmission has not been addressed before. Although transgenic rescue experiments have not been performed except kin-2, and the site of action (neurons involved) has not been tested in this manuscript, it will open the venue to further determine the way in which acetylcholine receptors, cAMP pathway etc. influences the learning process.

Reviewer #1 (Public review):

Summary:

The authors show that corticotropin-releasing factor (CRF) neurons in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) monosynaptically target cholinergic interneurons (CINs) in the dorsal striatum of rodents. Functionally, activation of CRFR1 receptors increases CIN firing rate, and this modulation was reduced by pre-exposure to ethanol. This is an interesting finding, with potential significance for alcohol use disorders, but some conclusions could use additional support.

Strengths:

Well-conceived circuit mapping experiments identify a novel pathway by which the CeA and BNST can modulate dorsal striatal function by controlling cholinergic tone. Important insight into how CRF, a neuropeptide that is important in mediating aspects of stress, affective/motivational processes, and drug-seeking, modulates dorsal striatal function.

Weaknesses:

(1) Tracing and expression experiments were performed both in mice and rats (in a mostly non-overlapping way). While these species are similar in many ways, some conclusions are based on assumptions of similarities that the presented data do not directly show. In most cases, this should be addressed in the text (but see point number 2).

(2) Experiments in rats show that CRFR1 expression is largely confined to a subpopulation of striatal CINs. Is this true in mice, too? Since most electrophysiological experiments are done in various synaptic antagonists and/or TTX, it does not affect the interpretation of those data, but non-CIN expression of CRFR1 could potentially have a large impact on bath CRF-induced acetylcholine release.

(3) Experiments in rats show that about 30% of CINs express CRFR1 in rats. Did only a similar percentage of CINs in mice respond to bath application of CRF? The effect sizes and error bars in Figure 5 imply that the majority of recorded CINs likely responded. Were exclusion criteria used in these experiments?

(4) The conclusion that prior acute alcohol exposure reduces the ability of subsequent alcohol exposure to suppress CIN activity in the presence of CRF may be a bit overstated. In Figure 6D (no ethanol pre-exposure), ethanol does not fully suppress CIN firing rate to baseline after CRF exposure. The attenuated effect of CRF on CIN firing rate after ethanol pre-treatment (6E) may just reduce the maximum potential effect that ethanol can have on firing rate after CRF, due to a lowered starting point. It is possible that the lack of significant effect of ethanol after CRF in pre-treated mice is an issue of experimental sensitivity. Related to this point, does pre-treatment with ethanol reduce the later CIN response to acute ethanol application (in the absence of CRF)?

(5) More details about the area of the dorsal striatum being examined would be helpful (i.e., a-p axis).

Reviewer #2 (Public review):

Summary:

Essoh and colleagues present a thorough and elegant study identifying the central amygdala and BNST as key sources of CRF input to the dorsal striatum. Using monosynaptic rabies tracing and electrophysiology, they show direct connections to cholinergic interneurons. The study builds on previous findings that CRF increases CIN firing, extending them by measuring acetylcholine levels in slices and applying optogenetic stimulation of CRF+ fibers. It also uncovers a novel interaction between alcohol and CRF signaling in the striatum, likely to spark significant interest and future research.

Strengths:

A key strength is the integration of anatomical and functional approaches to demonstrate these projections and assess their impact on target cells, striatal cholinergic interneurons.

Weaknesses:

The nature of the interaction between alcohol and CRF actions on cholinergic neurons remains unclear. Also, further clarification of the ACh sensor used and others is required

Reviewer #3 (Public review):

Summary:

The authors demonstrate that CRF neurons in the extended amygdala form GABAergic synapses onto cholinergic interneurons and that CRF can excite these neurons. The evidence is strong, however, the authors fail to make a compelling connection showing CRF released from these extended amygdala neurons is mediating any of these effects. Further, they show that acute alcohol appears to modulate this action, although the effect size is not particularly robust.

Strengths:

This is an exciting connection from the extended amygdala to the striatum that provides a new direction for how these regions can modulate behavior. The work is rigorous and well done.

Weaknesses:

While the authors show that opto stim of these neurons can increase firing, this is not shown to be CRFR1 dependent. In addition, the effects of acute ethanol are not particularly robust or rigorously evaluated. Further, the opto stim experiments are conducted in an Ai32 mouse, so it is impossible to determine if that is from CEA and BNST, vs. another population of CRF-containing neurons. This is an important caveat.

Reviewer #4 (Public review):

Summary:

This manuscript presents a compelling and methodologically rigorous investigation into how corticotropin-releasing factor (CRF) modulates cholinergic interneurons (CINs) in the dorsal striatum - a brain region central to cognitive flexibility and action selection-and how this circuit is disrupted by alcohol exposure. Through an integrated series of anatomical, optogenetic, electrophysiological, and imaging experiments, the authors uncover a previously uncharacterized CRF⁺ projection from the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) to dorsal striatal CINs.

Strengths:

Key strengths of the study include the use of state-of-the-art monosynaptic rabies tracing, CRF-Cre transgenic models, CRFR1 reporter lines, and functional validation of synaptic connectivity and neurotransmitter release. The finding that CRF enhances CIN excitability and acetylcholine (ACh) release via CRFR1, and that this effect is attenuated by acute alcohol exposure and withdrawal, provides important mechanistic insight into how stress and alcohol interact to impair striatal function. These results position CRF signaling in CINs as a novel contributor to alcohol use disorder (AUD) pathophysiology, with implications for relapse vulnerability and cognitive inflexibility associated with chronic alcohol intake.

The study is well-structured, with a clear rationale, thorough methodology, and logical progression of results. The discussion effectively contextualizes the findings within broader addiction neuroscience literature and suggests meaningful future directions, including therapeutic targeting of CRFR1 signaling in the dorsal striatum.

Weaknesses:

Minor areas for improvement include occasional redundancy in phrasing, slightly overlong descriptions in the abstract and significance sections, and a need for more concise language in some places. Nevertheless, these do not detract from the manuscript's overall quality or impact.

Overall, this is a highly valuable contribution to the fields of addiction neuroscience and striatal circuit function, offering novel insights into stress-alcohol interactions at the cellular and circuit level, which requires minor editorial revisions.

Reviewer #1 (Public review):

Summary:

Fogel & Ujfalussy report an extension of a visualization tool that was originally designed to enable an understanding of detailed biophysical neuron models. Named "extended currentscape", this new iteration enables visual assessment of individual currents across a neuron's spatially extended dendritic arbor with simultaneous readout of somatic currents and voltage. The overall aim was to permit a visually intuitive understanding for how a model neuron's inputs determine its output. This goal was worthwhile and the authors achieved it. Their manuscript makes two additional contributions of note: (1) a clever algorithmic approach to model the axial propagation of ionic currents (recursively traversing acyclic graph subsections) and (2) interesting, albeit not easily testable, insights into important neurophysiological phenomena such as complex spike generation and place field dynamics. Overall, this study provides a valuable and well-characterized biophysical modeling resource to the neuroscience community.

Strengths:

The authors significantly extended a previously published open-source biophysical modeling tool. Beyond providing important new capabilities, the potential impact of "extended currentscape" is boosted by its integration with preexisting resources in the field.

The code is well-documented and freely available via GitHub.

The author's clever portioning algorithm to relate dendritic/synaptic currents to somatic yielded multiple intriguing observations regarding when and why CA1 pyramidal neurons fire complex spikes versus single action potentials. This topic carries major implications for how the hippocampus represents and stores information about an animal's environment.

Weaknesses:

While extended currentscape is clearly a valuable contribution to the neuroscience community, this reviewer would argue that it is framed in a way that oversells its capabilities. The Abstract, Introduction, Results, and Methods all contain phrases implying that extended currentscape infers dendritic/synaptic currents contributing to somatic output., i.e. backwards inference of unknown inputs from a known output. This is not the case; inputs are simulated and then propagated through the model neuron using a clever partitioning algorithm that essentially traverses a biologically undirected graph structure by treating it like a time series of tiny directed graphs. This is an impressive solution, but it does not infer a neuron's input structure.

Because a directed acyclic graph architecture is shown in Figure 2, it is unintuitive that the authors can infer bidirectional current flow, e.g. Figure 3 showing current flowing from basal dendrites and axon to soma, and further towards the apical dendrites. This is explained in Methods, but difficult to parse from Results amidst lots of rather abstract jargon (target, reference, collision, compartment). Figure 2 would have presented an opportunity to clearly illustrate the author's portioning algorithm by (1) rooting it in the exact morphology of one of their multicompartmental model neurons and (2) illustrating that "target" and "reference" have arbitrary morphological meanings; they describe the direction of current flow which is reevaluated at each time step.

Analyses in Figure 7, C and D, are insightfully devised and illuminating. However, they could use some reconciliation with Figure 5 regarding initiation of individual APs versus CSBs within place fields.

The intriguing observations generated by extended currentscape also point to its main weakness, which the authors openly acknowledge: as of now, no experimental methods exist to conclusively tests its predictions.

Reviewer #2 (Public review):

Summary


The electrical activity of neurons and neuronal circuits is dictated by the concerted activity of multiple ionic currents. Because directly investigating these currents experimentally isn't possible with current methods, researchers rely on biophysical models to develop hypotheses and intuitions about their dynamics. Models of neural activity produce large amounts of data that is hard to visualize and interpret. The currentscape technique helps visualize the contributions of currents to membrane potential activity, but it's limited to model neurons without spatial properties. The extended currentscape technique overcomes this limitation by tracking the contributions of the different currents from distant locations. This extension allows tracking not only the types of currents that contribute to the activity in a given location, but also visualizing the spatial region where the currents originate. The method is applied to study the initiation of complex spike bursts in a model hippocampal place cell. 



Strengths.


The visualization method introduced in this work represents a significant improvement over the original currentscape technique. The extended currentscape method enables investigation of the contributions of currents in spatially extended models of neurons and circuits. 



Weaknesses.


The case study is interesting and highlights the usefulness of the visualization method. A simpler case study may have been sufficient to exemplify the method, while also allowing readers to compare the visualizations against their own intuitions of how currents should flow in a simpler setting.

Reviewer #1 (Public review):

Summary:

The authors used fine-level resolution epidemiological data to describe the spatiotemporal patterns of dengue, chikungunya and Zika. They assessed which factors best captured the historic transmission dynamics in Brazil. It was used epidemiological data from 2013 to 2020. They tested the association between arbovirus incidence and environment, human connectivity and socioeconomic, and climate variables, including extreme weather conditions.

Strengths:

The authors used granular epidemiological data at the subnational level and weekly case notification time series. Furthermore, they considered more than one hundred variables. Among the variables, it is highlighted that they also considered human connectivity and extreme weather events.

The authors used appropriate statistical methods accounting for the spatiotemporal structure and used the negative binomial to handle overdispersion; They applied a systematic covariate screening, using WAIC and performed sensitivity analysis. Their results suggest an important role of climate variables such as El Niño South Oscillation Anomalies, and that extremes in wetness and drought may drive infections outside regular patterns; it also suggests that temperature variations and extremes may be more associated with the incidence than the mean temperature; in addition, human connectivity networks are also pointed out as a key driver factor at fine level scale.

Weaknesses:

The authors have not accounted for the correlation between diseases. They have not considered the co-occurrence of diseases by applying a joint modelling approach, nor have they discussed this as a possibility for future work. Still, regarding the methods, they used a simplified lag treatment. They could have included into the discussion, examples of methods like Distributed Lag Models. This can be used in contexts when analysing meteorological covariates and extreme weather events.

They also have not considered the population's immunity to the different serotypes of dengue, which can reflect in peaks of incidence when a new serotype starts to circulate in a certain region. It is important to bring this into the discussion section.

Whether the authors achieved their aims, and whether the results support their conclusions:

The authors assess variables which may be associated with different vector-borne disease incidence and the magnitude of these associations. Conducting a fine-scale resolution analysis (spatial and temporal), they emphasised the role of environmental and extreme weather conditions. Their findings are coherent with their analysis and corroborate some of the existing literature.

Discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

Their work shows how the different vector-borne diseases are influenced by environmental and climatic factors and that human connectivity may play an important role at the fine level spatial and temporal scale. This work brings a picture of the spatial and temporal distributions of dengue, chikungunya and Zika, at the municipal level in Brazil (2013-2020). The material and methods are well described, and the source is made available, allowing reproducibility by other researchers and academics.

Reviewer #2 (Public review):

Summary:

This manuscript looks at a wide variety of likely important drivers of arbovirus transmission across municipalities in Brazil. The results are intriguing due to their relevance and breadth, but the approach also brings challenges, which make the results hard to interpret.

Strengths:

Important and complex problem, excellent spatiotemporal resolution, collection of important covariates, and holistic analysis.

Weaknesses:

There are two key weaknesses. First, it is difficult to understand the actual contributions of each included covariate. The principal fit metric is WAIC, and importance is characterized by rank based on univariate fit. WAIC is a valuable comparison metric, but does not indicate how well the best model (or any other) fits the data. Figures 5B and S2-S4 show what look like good fits, but it also seems possible that most of this fit could be coming from the random effects rather than the covariates. It would be helpful to show the RE-only model as a comparator in these figures and also to consider other metrics that could help show overall fit (e.g., R^2). How much variance is actually being explained by the covariates?

Relatedly, the mean absolute errors reported are approximately 2-8 across the viruses, which sounds good on the surface. But many of the actual counts are zeros, so it's hard to tell if this is really good. Comparison to the mean and median observed case counts would be helpful.

Second, some of the results/discussion on specific variables and covariates were confusing. For example, the relationships between relative humidity and temperature vary substantially between pathogens and minimum or maximum temperature values. However, as transmission of three viruses relies on the same mosquito and minimum and maximum temperatures are highly correlated, we would expect these relationships to be very similar. One concern is clarity, and another is that some of the findings may be spurious - potentially related to how much of the variance is accounted for by the random effects alone (see above) and the wide range of covariates assessed (thus increasing the chance of something improving fit).

Underlying much of this are likely nonlinear relationships. The authors comment on this as a likely reason for some of the specific relationships, but it is not a very strong argument because the variable selection process is completely based on (generalized) linear univariate regressions.

Lastly, the mischaracterization of arboviral disease is a big challenge, as noted in the discussion. Only a subset of cases in Brazil are laboratory confirmed, but I couldn't find any statement about whether the cases used here were laboratory confirmed or not. I suspect that they are a combination of confirmed and suspect cases. A sensitivity analysis with only confirmed cases would increase confidence in the results.

Reviewer #1 (Public review):

Summary:

Many studies have investigated adaptation to altered sensorimotor mappings or to an altered mechanical environment. This paper asks a different but also important question in motor control and neurorehabilitation: how does the brain adapt to changes in the controlled plant? The authors addressed this question by performing a tendon transfer surgery in two monkeys during which the swapped tendons flexing and extending the digits. They then monitored changes in task performance, muscle activation and kinematics post-recovery over several months, to assess changes in putative neural strategies.

Strengths:

(1) The authors performed complicated tendon transfer experiments to address their question of how the nervous system adapts to changes in the organisation of the neuromusculoskeletal system, and present very interesting data characterising neural (and in one monkey, also behavioural) changes post tendon transfer over several months.

(2) The fact that the authors had to employ to two slightly different tasks -one more artificial, the other more naturalistic- in the two monkeys and yet found qualitatively similar changes across them makes the findings more compelling.

(3) The paper is quite well written, and the analyses are sound, although some analyses could be improved (suggestions below).

Weaknesses:

(1) I think this is an important paper, paper but I'm puzzled about a tension in the results. On the one hand, it looks like the behavioural gains post-TT happen rather smoothly over time (Figure 5). On the other, muscle synergy activations changes abruptly at specific days (around day ~65 for Monkey A and around day ~45 for monkey B; e.g., Figure 6). How do the authors reconcile this tension? In other words, how do they think that this drastic behavioural transition can arise from what appears to be step-by-step, continuous changes in muscle coordination? Is it "just" subtle changes in movements/posture exploiting the mechanical coupling between wrist and finger movements combined with subtle changes in synergies and they just happen to all kick in at the same time? This feels to me the core of the paper and should be addressed more directly.

(2) The muscles synergy analyses, which are an important part of the paper, could be improved. In particular:

(2a) When measuring the cross-correlation between the activation of synergies, the authors should include error bars, and should also look at the lag between the signals.

(2b) Figure 7C and related figures, the authors state that the activation of muscle synergies revert to pre-TT patterns toward the end of the experiments. However, there are noticeable differences for both monkeys (at the end of the "task range" for synergy B for monkey A, and around 50 % task range for synergy B for monkey B). The authors should measure this, e.g., by quantifying the per-sample correlation between pre-TT and post-TT activation amplitudes. Same for Figures 8I,J, etc.

(2c) In Figures 9 and 10, the authors show the cross-correlation of the activation coefficients of different synergies; the authors should also look at the correlation between activation profiles because it provides additional information.

(2d) Figure 11: the authors talk about a key difference in how Synergy B (the extensor finger) evolved between monkeys post-TT. However, to me this figure feels more like a difference in quantity -the time course- than quality, since for both monkeys the aaEMG levels pretty much go back to close to baseline levels -even if there's a statistically significant difference only for Monkey B. What am I missing?

(2e) Lines 408-09 and above: The authors claim that "The development of a compensatory strategy, primarily involving the wrist flexor synergy (Synergy C), appears crucial for enabling the final phase of adaptation", which feels true intuitively and also based on the analysis in Figure 8, but Figure 11 suggests this is only true for Monkey A . How can these statements be reconciled?

(3) Experimental design: at least for the monkey who was trained on the "artificial task" (Monkey A), it would have been good if the authors had also tested him on naturalistic grasping, like the second monkey, to see to what extent the neural changes generalise across behaviours or are task-specific. Do the authors have some data that could be used to assessed this even if less systematically?

(4) Monkey's B behaviour pre-tendon transfer seems more variable than that of Monkey A (e.g., the larger error bars in Figure 5 compared to monkey A, the fluctuating cross-correlation between FDS pre and EDC post in Figure 6Q), this should be quantified to better ground the results since it also shows more variability post-TT.

(5) Minor: Figure 12 is interesting and supports the idea that monkeys may exploit the biomechanical coupling between wrist and fingers as part of their function recovery. It would be interesting to measure whether there is a change in such coupling (tenodesis) over time, e.g., by plotting change in wrist angle vs change in MCP angle as a scatter plot (one dot per trial), and in the same plot show all the days, colour coded by day. Would the relationship remain largely constant or fluctuate slightly early on? I feel this analysis could also help address my point (1) above.

Reviewer #2 (Public review):

Summary:

This study tackles an important question for both basic science understanding and translational relevance - how does the nervous system learn to control a changing body? Of course, all bodies change slowly over time, including basic parameters like size and weight distribution, but many types of diseases and injuries also alter the body and require neural adaptation to sustain normal control. A dramatic example from the clinic is the use of tendon transfer surgery in patients with near tetraplegia that allows them to use more proximal arm muscles to control the hand. Here, the authors sought to ask what strategies may be used when an animal adapts its motor control in response to tendon transfer. They focus on whether recovered functions leverage fractionated control over each muscle separately or, alternatively, whether there is evidence for modular control in which pre-existing synergies are recruited differently after the surgery. Overall, this work is very promising and advances the use of tendon transfer in animal models as a powerful way to study motor control flexibility, but the incomplete data and difficulty comparing between the two subjects mean that evidence is lacking for some of the conclusions.

Strengths:

A major strength of this paper is its motivating idea of using tendon transfer between flexor and extensor muscles in non-human primate wrist control to ask what adaptations are possible, how they evolve over time, and what might be the underlying neural control strategies. This is a creative and ambitious approach. Moreover, these surgeries are likely very challenging to do properly, and the authors rigorously documented the effectiveness of the transfer, particularly for Monkey A.

The results are promising, and there are two very interesting findings suggested by the data. First, when a single muscle out of a related group is manipulated, there is aberrant muscle activity detected across related muscles that are coordinated with each other and impacted as a group. For example, when the main finger extensor muscle now becomes a flexor, the timing of its activation is changed, and this is accompanied by similar changes in a more minor finger extensor as well as in wrist extensor muscles. This finding was observed in both monkeys and likely reflects a modular adaptive response. Second, there is a biphasic response in the weeks following injury, with an early phase in which the magnitude of an extensor synergy was increased and the timing of flexor and extensor recruitment was altered, followed by a later phase in which the timing and overall magnitude are restored.

Weaknesses:

The most notable weakness of the study is the incompleteness of the data. Monkey A has excellent quality EMG in all relevant muscles, but no analysis of video data, while Monkey B has some video data kinematics and moderate quality EMG, but the signal in the transferred FDS muscle was lost. These issues could be overcome by aligning data between the two monkeys, but the behavior tasks performed by each monkey are different, and so are the resulting muscle synergies detected (e.g., for synergies C and D), and different timepoints were analyzed in each monkey. As a result, it is difficult to make general conclusions from the study, and it awaits further analysis or the addition of another subject.

A second weakness is the insufficient analysis of the movements themselves, particularly for Monkey A. The main metrics analyzed were the time from task engagement (touch) to action onset and the time spent in an off-target location - neither of these measures can be related directly to muscle activity or the movement. Since the authors have video data for both monkeys, it is surprising that it was not used to extract landmarks for kinematic analysis, or at least hand/endpoint trajectory, and how it is adjusted over time. Adding more behavior data and aligning it with the EMG data would be very helpful for characterizing motor recovery and is needed to support conclusions about underlying neural control strategies for functional improvement.

Considering specific conclusions, the statement that the monkeys learned to use "tenodesis" over time by increasing activation of a wrist flexor muscle synergy does not seem to be fully supported by the data. Monkey A data includes EMG for two wrist flexors and a clear wrist flexor synergy, but it seems that, when comparing baseline and the final post-surgery timepoints, the main change is decreased activity around grasp after tendon transfer (at 0% of the task range if I understand this correctly) (Figure 8E and Figure S2-H vs R and -I vs S). It is clear that Monkey B increases the flexion of the wrist joint over time from the kinematic data, but the activity pattern in the only recorded wrist flexor (PL) doesn't change much with time (Figure S2-AN) and this monkey does not have a clear wrist flexor synergy (PL is active in the flexor synergy A while synergy C mainly reflects deltoid activity). Given these issues, it is not clear how to align the EMG and kinematic data and interpret these findings.

A more minor point regarding conclusions: statements about poor task performance and high energy expenditure being the costs that drive exploration for a new strategy are speculative and should be presented as such. Although the monkeys did take longer to complete the tasks after the surgery, they were still able to perform it successfully and in less than a second and no measurements of energy expenditure were taken.

A small concern is whether the tendon transfer effect may fail over time, either due to scar tissue formation or tendon tearing, and it would be ideal if the integrity of the intervention were re-assessed at the end of the study.

Reviewer #3 (Public review):

Summary:

In this study, Philipp et al. investigate how a monkey learns to compensate for a large, chronic biomechanical perturbation - a tendon transfer surgery, swapping the actions of two muscles that flex and extend the fingers. After performing the surgery and confirming that the muscle actions are swapped, the authors follow the monkeys' performance on grasping tasks over several months. There are several main findings:

(1) There is an initial stage of learning (around 60 days), where monkeys simply swap the activation timing of their flexors and extensors during the grasp task to compensate for the two swapped muscles.

(2) This is (seemingly paradoxically) followed by a stage where muscle activation timing returns almost to what it was pre-surgery, suggesting that monkeys suddenly swap to a new strategy that is better than the simple swap.

(3) Muscle synergies seem remarkably stable through the entire learning course, indicating that monkeys do not fractionate their muscle control to swap the activations of only the two transferred muscles.

(4) Muscle synergy activation shows a similar learning course, where the flexion synergy and extension synergy activations are temporarily swapped in the first learning stage and then revert to pre-surgery timing in the second learning stage.

(5) The second phase of learning seems to arise from making new, compensatory movements (supported by other muscle synergies) that get around the problem of swapped tendons.

Strengths:

This study is quite remarkable in scope, studying two monkeys over a period of months after a difficult tendon-transfer surgery. As the authors point out, this kind of perturbation is an excellent testbed for the kind of long-term learning that one might observe in a patient after stroke or injury, and provides unique benefits over more temporary perturbations like visuomotor transformations and studying learning through development. Moreover, while the two-stage learning course makes sense, I found the details to be genuinely surprising--specifically the fact that: (1) muscle synergies continue to be stable for months after the surgery, despite now being maladaptive; and (2) muscle activation timing reverts to pre-surgery levels by the end of the learning course. These two facts together initially make it seem like the monkey simply ignores the new biomechanics by the end of the learning course, but the authors do well to explain that this is mainly because the monkeys develop a new kind of movement to circumvent the surgical manipulation.

I found these results fascinating, especially in comparison to some recent work in motor cortex, showing that a monkey may be able to break correlations between the activities of motor cortical neurons, but only after several sessions of coaching and training (Oby et al. PNAS 2019). Even then, it seemed like the monkey was not fully breaking correlations but rather pushing existing correlations harder to succeed at the virtual task (a brain-computer interface with perturbed control).

Weaknesses:

I found the analysis to be reasonably well considered and relatively thorough. However, I do have a few suggestions that I think may elevate the work, should the authors choose to pursue them.

First, I find myself wondering about the physical healing process from the tendon transfer surgery and how it might contribute to the learning. Specifically, how long does it take for the tendons to heal and bear forces? If this itself takes a few months, it would be nice to see some discussion of this.

Second, I see that there are some changes in the muscle loadings for each synergy over the days, though they are relatively small. The authors mention that the cosine distances are very small for the conserved synergies compared to distances across synergies, but it would be good to get a sense for how variable this measure is within synergy. For example, what is the cosine similarity for a conserved synergy across different pre-surgery days? This might help inform whether the changes post-surgery are within a normal variation or whether they reflect important changes in how the muscles are being used over time.

Last, and maybe most difficult (and possibly out of scope for this work): I would have ideally liked to see some theoretical modeling of the biomechanics so I could more easily understand what the tendon transfer did or how specific synergies affect hand kinematics before and after the surgery. Especially given that the synergies remained consistent, such an analysis could be highly instructive for a reader or to suggest future perturbations to further probe the effects of tendon transfer on long-term learning.

Reviewer #1 (Public review):

Summary:

The authors propose a new technique which they name "Multi-gradient Permutation Survival Analysis (MEMORY)" that they use to identify "Genes Steadily Associated with Prognosis (GEARs)" using RNA-seq data from the TCGA database. The contribution of this method is one of the key stated aims of the paper. The majority of the paper focuses on various downstream analyses that make use of the specific GEARs identified by MEMORY to derive biological insights, with a particular focus on lung adenocarcinoma (LUAD) and breast invasive carcinoma (BRCA) which are stated to be representative of other cancers and are observed to have enriched mitosis and immune signatures, respectively. Through the lens of these cancers, these signatures are the focus of significant investigation in the paper.

Strengths:

The approach for MEMORY is well-defined and clearly presented, albeit briefly. This affords statisticians and bioinformaticians the ability to effectively scrutinize the proposed methodology and may lead to further advancements in this field. The scientific aspects of the paper (e.g., the results based on the use of MEMORY and the downstream bioinformatics workflows) are conveyed effectively and in a way that is digestible to an individual that is not deeply steeped in the cancer biology field.

Weaknesses:

Comparatively little of the paper is devoted to the justification of MEMORY (i.e., the authors' method) for identification of genes that are important broadly for the understanding of cancer. The authors' approach is explained in the methods section of the paper, but no comparison or reference is made to any other methods that have been developed for similar purposes, and no results are shown to illustrate the robustness of the proposed method (e.g., is it sensitive to subtle changes in how it is implemented).

For example, in the first part of the MEMORY algorithm, gene expression values are dichotomized at the sample median, and a log-rank test is performed. This would seemingly result in an unnecessary loss of information for detecting an association between gene expression and survival. Moreover, while dichotomizing gene expressions at the median is optimal from an information theory perspective (i.e., it creates equally sized groups), there is no reason to believe that median-dichotomization is correct vis-à-vis the relationship between gene expression and survival. If a gene really matters and expression only differentiates survival more towards the tail of the empirical gene expression distribution, median-dichotomization could dramatically lower power to detect group-wise differences. Notwithstanding this point, the reviewer acknowledges that dichotomization offers a straightforward approach to model gene expression and is widely used. This approach is nonetheless an example of a limitation of the current version of MEMORY that could be addressed to improve the methodology.

If I understand correctly, for each cancer the authors propose to search for the smallest subsample size (i.e., the smallest value of k_{j}) were there is at least one gene with a survival analysis p-value <0.05 for each of the 1000 sampled datasets. Then, any gene with a p-value <0.05 in 80% of the 1000 sampled datasets would be called a GEAR for that cancer. The 80% value here is arbitrary but that is a minor point. I acknowledge that something must be chosen.

Presumably the gene with the largest effect for the cancer will define the value of K_{j} and, if the effect is large, this may result in other genes with smaller effects not being defined as a GEAR for that cancer by virtue of the 80% threshold. Thus, a gene being a GEAR is related to the strength of association for other genes in addition to its own strength of association. One could imagine that a gene that has a small-to-moderate effect consistently across many cancers may not show up as GEAR in any of them (if there are [potentially different] genes with more substantive effects for those cancers). Is this desirable?

The term "steadily associated" implies that a signal holds up across subsample gradients. Effectively this makes the subsampling a type of indirect adjustment to ensure the evidence of association is strong enough. How well this procedure performs in repeated use (i.e., as a statistical procedure) is not clear.

Assuredly subsampling sets the bar higher than requiring a nominal p-value to be beneath the 0.05 threshold based on analysis of the full data set. The author's note that the MEMORY has several methodological limitations, "chief among them is the need for rigorous, large-scale multiple-testing adjustment before any GEAR list can be considered clinically actionable." The reviewer agrees and would add that it may be difficult to address this limitation within the author's current framework. Moreover, should the author's method be used before such corrections are available given their statement? Perhaps clarification of what it means to be clinically actionable could help here. If a researcher uses MEMORY to screen for GEARs based on the current methodology, what do the authors recommend be done to select a subset of GEARs worthy of additional research/investment?

Reviewer #2 (Public review):

Summary:

The authors are trying to come up with a list of genes (GEAR genes) that are consistently associated with cancer patient survival based on TCGA database. A method named "Multi-gradient Permutation Survival Analysis" was created based on bootstrapping and gradually increasing the sample size of the analysis. Only the genes with consistent performance in this analysis process are chosen as potential candidates for further analyses.

Strengths:

The authors describe in details their proposed method and the list of the chosen genes from the analysis. Scientific meaning and potential values of their findings are discussed in the context of published results in this field.

Weaknesses:

Some steps of the proposed method (especially the definition survival analysis similarity (SAS) need further clarification or details since it would be difficult if anyone tries to reproduce the results.

If the authors can improve the clarity of the manuscript, including the proposed method and there is no major mistake there, the proposed approach can be applied to other diseases (assuming TCGA type of data is available for them) to identify potential gene lists, based on which drug screening can be performed to identify potential target for development.

Reviewer #4 (Public review):

Thank you to the authors for their detailed responses and changes in relation to my questions. They have addressed all my concerns around methodological and inference clarity. I would still recommend against the use of feature/pathway selection techniques where there is no way of applying formal error control. I am pleased to read, however, that the authors are planning to develop this in future work. My edited review reflects these changes:

The authors apply what I gather is a novel methodology titled "Multi-gradient Permutation Survival Analysis" to identify genes that are robustly associated with prognosis ("GEARs") using tumour expression data from 15 cancer types available in the TCGA. The resulting lists of GEARs are then interrogated for biological insights using a range of techniques including connectivity and gene enrichment analysis.

I reviewed this paper primarily from a statistical perspective. Evidently an impressive amount of work has been conducted, concisely summarised, and great effort has been undertaken to add layers of insight to the findings. I am no stranger to what an undertaking this would have been. My primary concern, however, is that the novel statistical procedure proposed, and applied to identify the gene lists, as far as I can tell offers no statistical error control nor quantification. Consequently we have no sense what proportion of the highlighted GEAR genes and networks are likely to just be noise.

Major comments:

The main methodology used to identify the GEAR genes, "Multi-gradient Permutation Survival Analysis" does not formally account for multiple testing and offers no formal error control. Meaning we are left without knowing what the family wise (aka type 1) error rate is among the GEAR lists, nor the false discovery rate. I appreciate the emphasis on reproducibility, but I would generally recommend against the use of any feature selection methodology which does not provide error quantification because otherwise we do not know if we are encouraging our colleagues and/or readers to put resource into lists of genes that contain more noise than not. I am glad though and appreciative that the authors intend to develop this in future work.

The authors make a good point that, despite lack of validation in an external independent dataset, it is still compelling work given the functional characterisation and literature validation. I am pleased though that the authors agree validation in an independent dataset is an important next step, and plan to do so in future work.

Reviewer #1 (Public review):

Summary:

The Neuronal microtubule cytoskeleton is essential long long-range transport in axons and dendrites. The axon-specific plus-end out microtubule organization vs the dendritic-specific plus-end in organization allows for selective transport into each neurite, setting up neuronal polarity. In addition, the dendritic microtubule organization is thought to be important for dendritic pruning in Drosophila during metamorphosis. However, the precise mechanisms that organize microtubules in neurons are still incompletely understood.

In the current manuscript, the authors describe the spectraplakin protein Shot as important in developmental dendritic pruning. They find that Shot has dendritic microtubule polarity defects, which, based on their rescues and previous work, is likely the reason for the pruning defect.

Since Shot is a known actin-microtubule crosslinker, they also investigate the putative role of actin and find that actin is also important for dendritic pruning. Finally, they find that several factors that have been shown to function as a dendritic MTOC in C. elegans also show a defect in Drosophila upon depletion.

Strengths:

Overall, this work was technically well-performed, using advanced genetics and imaging. The author reports some interesting findings identifying new players for dendritic microtubule organization and pruning.

Weaknesses:

The evidence for Shot interacting with actin for its functioning is contradictory. The Shot lacking the actin interaction domain did not rescue the mutant; however, it also has a strong toxic effect upon overexpression in wildtype (Figure S3), so a potential rescue may be masked. Moreover, the C-terminus-only construct, which carries the GAS2-like domain, was sufficient to rescue the pruning. This actually suggests that MT bundling/stabilization is the main function of Shot (and no actin binding is needed). On the other hand, actin depolymerization leads to some microtubule defects and subtle changes in shot localization in young neurons (not old ones). More importantly, it did not enhance the microtubule or pruning defects of the Shot domain, suggesting these act in the same pathway. Interesting to note is that Mical expression led to microtubule defects but not to pruning defects. This argues that MT organization effects alone are not enough to cause pruning defects. This may be be good to discuss. For the actin depolymerization, the authors used overexpression of the actin-oxidizing Mical protein. However, Mical may have another target. It would be good to validate key findings with better characterized actin targeting tools.

In analogy to C. elegans, where RAB-11 functions as a ncMTOC to set up microtubules in dendrites, the authors investigated the role of these in Drosophila. Interestingly, they find that rab-11 also colocalizes to gamma tubulin and its depletion leads to some microtubule defects. Furthermore, they find a genetic interaction between these components and Shot; however, this does not prove that these components act together (if at all, it would be the opposite). This should be made more clear. What would be needed to connect these is to address RAB-11 localization + gamma-tubulin upon shot depletion.

All components studied in this manuscript lead to a partial reversal of microtubules in the dendrite. However, it is not clear from how the data is represented if the microtubule defect is subtle in all animals or whether it is partially penetrant stronger effect (a few animals/neurons have a strong phenotype). This is relevant as this may suggest that other mechanisms are also required for this organization, and it would make it markedly different from C. elegans. This should be discussed and potentially represented differently.

Reviewer #2 (Public review):

Summary:

In their manuscript, the authors reveal that the spectraplakin Shot, which can bind both microtubules and actin, is essential for the proper pruning of dendrites in a developing Drosophila model. A molecular basis for the coordination of these two cytoskeletons during neuronal development has been elusive, and the authors' data point to the role of Shot in regulating microtubule polarity and growth through one of its actin-binding domains. The authors also propose an intriguing new activity for a spectraplakin: functioning as part of a microtubule-organizing center (MTOC).

Strengths:

(1) A strength of the manuscript is the authors' data supporting the idea that Shot regulates dendrite pruning via its actin-binding CH1 domain and that this domain is also implicated in Shot's ability to regulate microtubule polarity and growth (although see comments below); these data are consistent with the authors' model that Shot acts through both the actin and microtubule cytoskeletons to regulate neuronal development.

(2) Another strength of the manuscript is the data in support of Rab11 functioning as an MTOC in young larvae but not older larvae; this is an important finding that may resolve some debates in the literature. The finding that Rab11 and Msps coimmunoprecipitate is nice evidence in support of the idea that Rab11(+) endosomes serve as MTOCs.

Weaknesses:

(1) A significant, major concern is that most of the authors' main conclusions are not (well) supported, in particular, the model that Shot functions as part of an MTOC. The story has many interesting components, but lacks the experimental depth to support the authors' claims.

(2) One of the authors' central claims is that Shot functions as part of a non-centrosomal MTOC, presumably a MTOC anchored on Rab11(+) endosomes. For example, in the Introduction, last paragraph, the authors summarize their model: "Shot localizes to dendrite tips in an actin-dependent manner where it recruits factors cooperating with an early-acting, Rab11-dependent MTOC." This statement is not supported. The authors do not show any data that Shot localizes with Rab11 or that Rab11 localization or its MTOC activity is affected by the loss of Shot (or otherwise manipulating Shot). A genetic interaction between Shot and Rab11 is not sufficient to support this claim, which relies on the proteins functioning together at a certain place and time. On a related note, the claim that Shot localization to dendrite tips is actin-dependent is not well supported: the authors show that the CH1 domain is needed to enrich Shot at dendrite tips, but they do not directly manipulate actin (it would be helpful if the authors showed the overexpression of Mical disrupted actin, as they predict).

(3) The authors show an image that Shot colocalizes with the EB1-mScarlet3 comet initiation sites and use this representative image to generate a model that Shot functions as part of an MTOC. However, this conclusion needs additional support: the authors should quantify the frequency of EB1 comets that originate from Shot-GFP aggregates, report the orientation of EB1 comets that originate from Shot-GFP aggregates (e.g., do the Shot-GFP aggregates correlate with anterogradely or retrogradely moving EB1 comets), and characterize the developmental timing of these events. The genetic interaction tests revealing ability of shot dsRNA to enhance the loss of microtubule-interacting proteins (Msps, Patronin, EB1) and Rab11 are consistent with the idea that Shot regulates microtubules, but it does not provide any spatial information on where Shot is interacting with these proteins, which is critical to the model that Shot is acting as part of a dendritic MTOC.

(4) It is unclear whether the authors are proposing that dendrite pruning defects are due to an early function of Shot in regulating microtubule polarity in young neurons (during 1st instar larval stages) or whether Shot is acting in another way to affect dendrite pruning. It would be helpful for the authors to present and discuss a specific model regarding Shot's regulation of dendrite pruning in the Discussion.

(5) The authors argue that a change in microtubule polarity contributes to dendrite pruning defects. For example, in the Introduction, last paragraph, the authors state: "Loss of Shot causes pruning defects caused by mixed orientation of dendritic microtubules." The authors show a correlative relationship, not a causal one. In Figure 4, C and E, the authors show that overexpression of Mical disrupts microtubule polarity but not dendrite pruning, raising the question of whether disrupting microtubule polarity is sufficient to cause dendrite pruning defects. The lack of an association between a disruption in microtubule polarity and dendrite pruning in neurons overexpressing Mical is an important finding.

(6) The authors show that a truncated Shot construct with the microtubule-binding domain, but no actin-binding domain (Shot-C-term), can rescue dendrite pruning defects and Khc-lacZ localization, whereas the longer Shot construct that lacks just one actin-binding domain ("delta-CH1") cannot. Have the authors confirmed that both proteins are expressed at equivalent levels? Based on these results and their finding that over-expression of Shot-delta-CH1 disrupts dendrite pruning, it seems possible that Shot-delta-CH1 may function as a dominant-negative rather than a loss-of-function. Regardless, the authors should develop a model that takes into account their findings that Shot, without any actin-binding domains and only a microtubule-binding domain, shows robust rescue.

(7) The authors state that: "The fact that Shot variants lacking the CH1 domain cannot rescue the pruning defects of shot[3] mutants suggested that dendrite tip localization of Shot was important for its function." (pages 10-11). This statement is not accurate: the Shot C-term construct, which lacks the CH1 domain (as well as other domains), is able to rescue dendrite pruning defects.

(8) The authors state that: "In further support of non-functionality, overexpression of Shot[deltaCH1] caused strong pruning defects (Fig. S3)." (page 8). Presumably, these results indicate that Shot-delta-CH1 is functioning as a dominant-negative since a loss-of-function protein would have no effect. The authors should revise how they interpret these results. This comment is related to another comment about the ability of Shot constructs to rescue the shot[3] mutant.

Reviewer #1 (Public review):

Summary:

This study aims to address an important and timely question: how does the mesoscale architecture of cortical and subcortical circuits reorganize during sensorimotor learning? By using high-density, chronically implanted ultra-flexible electrode arrays, the authors track spiking activity across ten brain regions as mice learn a visual Go/No-Go task. The results indicate that learning leads to more sequential and temporally compressed patterns of activity during correct rejection trials, alongside changes in functional connectivity ranks that reflect shifts in the relative influence of visual, frontal, and motor areas throughout learning. The emergence of a more task-focused subnetwork is accompanied by broader and faster propagation of stimulus information across recorded regions.

Strengths:

A clear strength of this work is its recording approach. The combination of stable, high-throughput multi-region recordings over extended periods represents a significant advance for capturing learning-related network dynamics at the mesoscale. The conceptual framework is well motivated, building on prior evidence that decision-relevant signals are widely distributed across the brain. The analysis approach, combining functional connectivity rankings with information encoding metrics is well motivated but needs refinement. These results provide some valuable evidence of how learning can refine both the temporal precision and the structure of interregional communication, offering new insights into circuit reconfiguration during learning.

Weaknesses:

The technical approach is strong and the conceptual framing is compelling, but several aspects of the evidence remain incomplete. In particular, it is unclear whether the reported changes in connectivity truly capture causal influences, as the rank metrics remain correlational and show discrepancies with the manipulation results. The absolute response onset latencies also appear slow for sensory-guided behavior in mice, and it is not clear whether this reflects the method used to define onset timing or factors such as task structure or internal state. Furthermore, the small number of animals, combined with extensive repeated measures, raises questions about statistical independence and how multiple comparisons were controlled. The optogenetic experiments, while intended to test the functional relevance of rank-increasing regions, leave it unclear how effectively the targeted circuits were silenced. Without direct evidence of reliable local inhibition, the behavioral effects or lack thereof are difficult to interpret. Details on spike sorting are limited.

Reviewer #2 (Public review):

Summary:

Wang et al. measure from 10 cortical and subcortical brain as mice learn a go/no-go visual discrimination task. They found that during learning, there is a reshaping of inter-areal connections, in which a visual-frontal subnetwork emerges as mice gain expertise. Also visual stimuli decoding became more widespread post-learning. They also perform silencing experiments and find that OFC and V2M are important for the learning process. The conclusion is that learning evoked a brain-wide dynamic interplay between different brain areas that together may promote learning.

Strengths:

The manuscript is written well and the logic is rather clear. I found the study interesting and of interest to the field. The recording method is innovative and requires exceptional skills to perform. The outcomes of the study are significant, highlighting that learning evokes a widespread and dynamics modulation between different brain areas, in which specific task-related subnetworks emerge.

Weaknesses:

I had several major concerns:

(1) The number of mice was small for the ephys recordings. Although the authors start with 7 mice in Figure 1, they then reduce to 5 in panel F. And in their main analysis, they minimize their analysis to 6/7 sessions from 3 mice only. I couldn't find a rationale for this reduction, but in the methods they do mention that 2 mice were used for fruitless training, which I found no mention in the results. Moreover, in the early case, all of the analysis is from 118 CR trials taken from 3 mice. In general, this is a rather low number of mice and trial numbers. I think it is quite essential to add more mice.

(2) Movement analysis was not sufficient. Mice learning a go/no-go task establish a movement strategy that is developed throughout learning and is also biased towards Hit trials. There is an analysis of movement in Figure S4, but this is rather superficial. I was not even sure that the 3 mice in Figure S4 are the same 3 mice in the main figure. There should be also an analysis of movement as a function of time to see differences. Also for Hits and FAs. I give some more details below. In general, most of the results can be explained by the fact that as mice gain expertise, they move more (also in CR during specific times) which leads to more activation in frontal cortex and more coordination with visual areas. More needs to be done in terms of analysis, or at least a mention of this in the text.

(3) Most of the figures are over-detailed, and it is hard to understand the take-home message. Although the text is written succinctly and rather short, the figures are mostly overwhelming, especially Figures 4-7. For example, Figure 4 presents 24 brain plots! For rank input and output rank during early and late stim and response periods, for early and expert and their difference. All in the same colormap. No significance shown at all. The Δrank maps for all cases look essentially identical across conditions. The division into early and late time periods is not properly justified. But the main take home message is positive Δrank in OFC, V2M, V1 and negative Δrank in ThalMD and Str. In my opinion, one trio map is enough, and the rest could be bumped to the Supplementary section, if at all. In general, the figure in several cases do not convey the main take home messages. See more details below.

(4) The analysis is sometimes not intuitive enough. For example, the rank analysis of input and output rank seemed a bit over complex. Figure 3 was hard to follow (although a lot of effort was made by the authors to make it clearer). Was there any difference between the output and input analysis? Also, the time period seems redundant sometimes. Also, there are other network analysis that can be done which are a bit more intuitive. The use of rank within the 10 areas was not the most intuitive. Even a dimensionality reduction along with clustering can be used as an alternative. In my opinion, I don't think the authors should completely redo their analysis, but maybe mention the fact that other analyses exist.

Reviewer #3 (Public review):

Summary:

In the manuscript " Dynamics of mesoscale brain network during decision-making learning revealed by chronic, large-scale single-unit recording", Wang et al investigated mesoscale network reorganization during visual stimulus discrimination learning in mice using chronic, large-scale single-unit recordings across 10 cortical/subcortical regions. During learning, mice improved task performance mainly by suppressing licking on no-go trials. The authors found that learning induced restructuring of functional connectivity, with visual (V1, V2M) and frontal (OFC, M2) regions forming a task-relevant subnetwork during the acquisition of correct No-Go (CR) trials.

Learning also compressed sequential neural activation and broadened stimulus encoding across regions. In addition, a region's network connectivity rank correlated with its timing of peak visual stimulus encoding.

Optogenetic inhibition of orbitofrontal cortex (OFC) and high order visual cortex (V2M) impaired learning, validating its role in learning. The work highlights how mesoscale networks underwent dynamic structuring during learning.

Strengths:

The use of ultra-flexible microelectrode arrays (uFINE-M) for chronic, large-scale recordings across 10 cortical/subcortical regions in behaving mice represents a significant methodological advancement. The ability to track individual units over weeks across multiple brain areas will provide a rare opportunity to study mesoscale network plasticity.

While limited in scope, optogenetic inhibition of OFC and V2M directly ties connectivity rank changes to behavioral performance, adding causal depth to correlational observations.

Weaknesses:

The weakness is also related to the strength provided by the method. It is demonstrated in the original method that this approach in principle can track individual units for four months (Luan et al, 2017). The authors have not showed chronically tracked neurons across learning. Without demonstrating that and taking advantage of analyzing chronically tracked neurons, this approach is not different from acute recording across multiple days during learning. Many studies have achieved acute recording across learning using similar tasks. These studies have recorded units from a few brain areas or even across brain-wide areas.

Another weakness is that major results are based on analyses of functional connectivity that is calculated using the cross-correlation score of spiking activity (TSPE algorithm). Functional connection strengthen across areas is then ranked 1-10 based on relative strength. Without ground truth data, it is hard to judge the underlying caveats. I'd strongly advise the authors to use complementary methods to verify the functional connectivity and to evaluate the mesoscale change in subnetworks. Perhaps the authors can use one key information of anatomy, i.e. the cortex projects to the striatum, while the striatum does not directly affect other brain structures recorded in this manuscript.

Reviewer #1 (Public review):

Summary:

This study by Akhtar et al. aims to investigate the link between systemic metabolism and respiratory demands, and how sleep and the circadian clock regulate metabolic states and respiratory dynamics. The authors leverage genetic mutants that are defective in sleep and circadian behavior in combination with indirect respirometry and steady-state LC-MS-based metabolomics to address this question in the Drosophila model.

First, the authors performed respirometry (on groups of 25 flies) to measure oxygen consumption (VO2) and carbon dioxide production (VCO2) to calculate the respiratory quotient (RQ) across the 24-hour day (12h:12h light-dark cycle) and assess metabolic fuel utilization. They observed that among all the genotypes tested, wild type (WT) flies and per0 flies in LD and WT flies in DD exhibit RQ >1. They concluded the >1 RQ is consistent with active lipogenesis. In contrast, the short-sleep mutants fumin (fmn) and sleepless (sss) showed significantly different RQ; the fmn exhibits a slight reduction in RQ values, suggesting increased reliance on carbohydrate metabolism, while sss exhibits even lower RQ (0.94), consistent with a shift toward lipid and protein catabolism.

The authors then proceeded to bin these measurements in 12-hour partitions, ZT0-12 and ZT12-24, to assess diurnal differences in average values of VO2, VCO2, and RQ. They observed significant day-night differences in metabolic rates in WT-LD flies, with higher rates during the day. The diurnal differences remain in the short-sleep mutants, but the overall metabolic rates are higher. WT-DD flies exhibit the lowest respiratory activity, although the day-night differences remain in free-running conditions. Finally, per01 mutants exhibit no significant change in day-night respiratory rates, suggesting that a functional circadian clock is necessary for diurnal differences in metabolic rates.

They then performed finer-resolution 24-hour rhythmic analysis (RAIN and JTK) to determine if VO2, VCO2, and RQ exhibit 24-hour rhythmic and if there are genotype-specific differences. Based on their criteria, VCO2 is rhythmic in all conditions tested, while VO2 is rhythmic in all conditions except in fmn-LD. Finally, RQ is rhythmic in all 3 mutants but not in WT-LD and WT-DD. Peak phases for the rhythms were deduced using JTK lag values.

The authors proceeded to leverage a previously published steady-state metabolite dataset to investigate the potential association of RQ with metabolite profiles. Spearman correlation was performed to identify metabolites that exhibit coupling to respiratory output. Positive and negative lag analysis were subsequently performed to further characterize these associations based on the timing of the metabolite peak changes relative to RQ fluctuations. The authors suggest that a positive lag indicates that metabolite changes occur after shifts in RQ, and a negative lag signifies that metabolite changes precede RQ changes. To visualize metabolic pathways that exhibit these temporal relationships, a clustered heatmap and enrichment analysis were performed. Through these analyses, they concluded that both sleep and circadian systems are essential for aligning metabolic substrate selection with energy demands, and different metabolic pathways are misregulated in the different mutants with sleep and circadian defects.

Strength:

The research questions this study explores are significant, given that metabolism and respiratory demand are central to animal biology. The experimental methods used, including the well-characterized fly genetic mutants, the newly developed method for indirect calorimetry measurements, and LC-MS-based metabolomics, are all appropriate. This study provides insights into the impact of sleep and circadian rhythm disruption on metabolism and respiratory demand and serves as a foundation for future mechanistic investigations.

Weaknesses:

There are some conceptual flaws that the authors need to address regarding circadian biology, and some of the conclusions can be better supported by additional analysis to provide a stronger foundation for future functional investigation. At times, the methods, especially the statistical analysis, are not well articulated; they need to be better explained.

Reviewer #2 (Public review):

This is an innovative and technically strong study that integrates dual-gas respirometry with LC-MS metabolomics to examine how sleep and circadian disruption shape metabolism in Drosophila. The combination of continuous O₂/CO₂ measurements with high-temporal-resolution metabolite profiling is novel and provides fresh insight into how wild-type flies maintain anticipatory fuel alignment, while mutants shift to reactive or misaligned metabolism. The use of lag-shift correlation analysis is particularly clever, as it highlights temporal coordination rather than static associations. Together, the findings advance our understanding of how circadian clocks and sleep contribute to metabolic efficiency and redox balance.

However, there are several areas where the manuscript could be strengthened. The authors should acknowledge that their findings may be gene-specific. Because sleep deprivation was not performed, it remains uncertain whether the observed metabolic shifts generalize to sleep loss broadly or are restricted to the fmn and sss mutants. This concern also connects to the finding of metabolic misalignment under constant darkness despite an intact clock. The conclusion that external entrainment is essential for maintaining energy homeostasis in flies may not translate to mammals. It would help to reference supporting data for the finding and discuss differences across species. Ideally, complementary circadian (light-dark cycle disruption) or sleep deprivation (for several hours) experiments, or citation of comparable studies, would strengthen the generality of the findings. Figures 1-4 are straightforward and clear, but when the manuscript transitions to the metabolite-respiration correlations, there is little description of the metabolomics methods or datasets, which should be clarified. The Discussion is at times repetitive and could be tightened, with the main message (i.e., wild-type flies align metabolism in advance, while mutants do not) kept front and center. Terms such as "anticipatory" and "reactive" should be defined early and used consistently throughout.

Overall, this is a strong and novel contribution. With clarification of scope, refinement of presentation, and a more focused Discussion, the paper will make a significant impact.

Reviewer #3 (Public review):

Summary:

The authors investigate how sleep loss and circadian disruption affect whole-organism metabolism in Drosophila melanogaster. They used chamber-based flow-through respirometry to measure oxygen consumption and carbon dioxide production in wild-type flies and in mutants with impaired sleep or circadian function. These measurements were then integrated with a previously published metabolomics dataset to explore how respiratory dynamics align with metabolic pathways. The central claim is that wild-type flies display anticipatory coordination of metabolic processes with circadian time, while mutants exhibit reactive shifts in substrate use, redox imbalance, and signs of mitochondrial stress.

Strengths:

The study has several strengths. Continuous high-resolution respirometry in flies is challenging, and its application across multiple genotypes provides good comparative insight. The conceptual framework distinguishing anticipatory from reactive metabolic regulation is interesting. The translational framing helps place the work in a broader context of sleep, circadian biology, and metabolic health.

Weaknesses:

At the same time, the evidence supporting the conclusions is somewhat limited. The metabolomics data were not newly generated but repurposed from prior work, reducing novelty. The biological replication in the respirometry assays is low, with only a small number of chambers per genotype. Importantly, respiratory parameters in flies are strongly influenced by locomotor activity, yet no direct measurements of activity were included, making it difficult to separate intrinsic metabolic changes from behavioral differences in mutants. In addition, repeated claims of "mitochondrial stress" are not directly substantiated by assays of mitochondrial function. The study also excluded female flies entirely, despite well-documented sex differences in metabolism, which narrows the generality of the findings.

Reviewer #2 (Public review):

Summary:

This article presents Morphonet 2.0, a software designed to visualise and curate segmentations of 3D and 3D+t data. The authors demonstrate its capabilities on five published datasets, showcasing how even small segmentation errors can be automatically detected, easily assessed and corrected by the user. This allows for more reliable ground truths which will in turn be very much valuable for analysis and training deep learning models. Morphonet 2.0 offers intuitive 3D inspection and functionalities accessible to a non-coding audience, thereby broadening its impact.

Strengths:

The work proposed in this article is expected to be of great interest for the community, by enabling easy visualisation and correction of complex 3D(+t) datasets. Moreover, the article is clear and well written making MorphoNet more likely to be used. The goals are clearly defined, addressing an undeniable need in the bioimage analysis community. The authors use a diverse range of datasets, successfully demonstrating the versatility of the software.

We would also like to highlight the great effort that was made to clearly explain which type of computer configurations are necessary to run the different dataset and how to find the appropriate documentation according to your needs. The authors clearly carefully thought about these two important problems and came up with very satisfactory solutions.

Weaknesses:

Sometimes, it can be a bit difficult to assess the strength of the improvements made by the proposed methods, but this is not something the authors could easily address, given the great complexity of the samples

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors employ diaphragm denervation in rats and mice to study titin‑based mechanosensing and longitudinal muscle hypertrophy. By integrating bulk RNA‑seq, proteomics, and phosphoproteomics, they map the stretch‑responsive signalling landscape, uncovering robust induction of the muscle‑ankyrin‑repeat proteins (MARP1‑3) together with enhanced phosphorylation of titin's N2A element. Genetic ablation of MARPs in mice amplifies longitudinal fibre growth and is accompanied by activation of the mTOR pathway, whereas systemic rapamycin treatment suppresses the hypertrophic response, highlighting mTORC1 as a key downstream effector of titin/MARP signalling.

Strengths:

The authors address a clear biological question: "how titin‑associated factors translate mechanical stretch into longitudinal fibre growth" using a unique and clinically relevant animal model of diaphragm denervation. Using a comprehensive multiomics approach, the authors identify MARPs as potential mediators of these effects and use a genetic mouse model to provide compelling evidence supporting causality. Additionally, connecting these findings to rapamycin, a drug widely used clinically, further increases the relevance and potential impact of the study.

Weaknesses:

There are several areas where the manuscript could be substantially improved.

(1) The statistical analysis of multi-omics data needs clarification. Typically, analyses across multiple experimental groups require controlling the false discovery rate (FDR) simultaneously to avoid reporting false-positive findings. It would be very helpful if the authors could specify whether adjusted p-values were calculated using a multi-factorial statistical model (e.g., ~group) or through separate pairwise contrasts.

(2) There are three separate points regarding MARP3 that could be improved. First, the authors report that MARP3-KO mice exhibit smaller increases in muscle mass after diaphragm denervation compared to wild-type mice (a -13% difference), indicating MARP3 likely promotes rather than attenuates hypertrophy. However, the manuscript currently states the opposite (lines 215-216); this interpretation should be revisited. Second, it would be valuable if the authors could provide data showing whether MARP3 transcript or protein levels change response to denervation - if they do not, discussing mechanisms behind the observed phenotype would help clarify the findings. Finally, given that some MARP-KO mice already exhibit baseline differences, employing and reporting the full two-way ANOVA ( including genotype × treatment interaction) would allow a direct statistical assessment of whether MARP deficiency modifies the muscle's response to stretch. This analysis would help clearly resolve any existing ambiguity.

(3) The current presentation of multi-omics data is somewhat difficult to follow, making it challenging to determine whether observed changes occur at the transcript or protein level due to inconsistent gene/protein naming and capitalization (e.g., proper forms are mTOR, p70 S6K, 4E-BP1). Clearly organizing and presenting transcript and protein-level changes side-by-side, especially for key molecules discussed in later experiments, would make the data more accessible and provide clearer insights into the biology of titin-mediated mechanosensing.

(4) The current analysis relies on total protein measurements downstream of mTOR, yet mTOR's primary mode of action is to change phosphorylation status. Because the authors have already generated a phosphoproteomic dataset, it would be very helpful to report - or at least comment on - whether known mTOR target phosphosites were detected and how they respond to denervation and rapamycin. Including even a brief summary of canonical sites such as S6K1 Thr389 or 4E‑BP1 Thr37/46 would make the link between mTOR activity and hypertrophy much clearer.

(5) Finally, since rapamycin blocks only a subset of mTOR signalling, a brief discussion that distinguishes rapamycin‑sensitive from rapamycin‑insensitive pathways would be valuable. Clarifying whether diaphragm stretch relies exclusively on the sensitive branch or also engages the resistant branch would place the results in a broader mTOR context and deepen the mechanistic narrative.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

This thoughtful and thorough mechanistic and functional study reports ARHGAP36 as a direct transcriptional target of FOXC1, which regulates Hedgehog signaling (SUFU, SMO, and GLI family transcription factors) through modulation of PKAC. Clinical outcome data from patients with neuroblastoma, one of the most common extracranial solid malignancies in children, demonstrate that ARHGAP36 expression is associated with improved survival. Although this study largely represents a robust and near-comprehensive set of focused investigations on a novel target of FOXC1 activity, several significant omissions undercut the generalizability of the findings reported.

(1) It is notable that the volcano plot in Figure 1a does now show evidence of canonical Hedgehog gene regulation, even though the subsequent studies in this paper clearly demonstrate that ARHGAP36 regulates Hedgehog signal transduction. Is this because canonical Hedgehog target genes (GLI1, PTCH1, SUFU) simply weren't labeled? Or is there a technical limitation that needs to be clarified? A note about Hedgehog target genes is made in conjunction with Table S1, but the justification or basis of defining these genes as Hedgehog targets is unclear. More broadly, it would be useful to see ontology analyses from these gene expression data to understand FOXC1 target genes more broadly. Ontology analyses are included in a supplementary table, but network visualizations would be much preferred.

(2) Likewise, the ChIP-seq data in Figure 2 are under-analyzed, focusing only on the ARHGAP36 locus and not more broadly on the FOXC1 gene expression program. This is a missed opportunity that should be remedied with unbiased analyses intersecting differentially expressed FOXC1 peaks with differentially expressed genes from RNA-sequencing data displayed in Figure 1.

(3) RNA-seq and ChIP-seq data strongly suggest that FOXC1 regulates ARHGAP36 expression, and the authors convincingly identify genomic segments at the ARHGAP36 locus where FOXC1 binds, but they do not test if FOXC1 specifically activates this locus through the creation of a luciferase or similar promoter reporter. Such a reagent and associated experiments would not only strengthen the primary argument of this investigation but could serve as a valuable resource for the community of scientists investigating FOXC1, ARHGAP36, the Hedgehog pathway, and related biological processes. CRISPRi targeting of the identified regions of the ARHGAP locus is a useful step in the right direction, but these experiments are not done in a way to demonstrate FOXC1 dependency.

(4) It would be useful to see individual fluorescence channels in association with images in Figure 3b.

(5) Perhaps the most significant limitation of this study is the omission of in vivo data, a shortcoming the authors partly mitigate through the incorporation of clinical outcome data from pediatric neuroblastoma patients in the context of ARHGAP36 expression. The authors also mention that high levels of ARHGAP36 expression were also detected in "specific CNS, breast, lung, and neuroendocrine tumors," but do not provide clinical outcome data for these cohorts. Such analyses would be useful to understand the generalizability of their findings across different cancer types. More broadly, how were high, medium, and low levels of ARHGAP36 expression identified? "Terciles" are mentioned, but such an approach is not experimentally rigorous, and RPA or related approaches (nested rank statistics, etc) are recommended to find optimal cutpoints for ARHGAP36 expression in the context of neuroblastoma, "specific CNS, breast, lung, and neuroendocrine" tumor outcomes.

Reviewer #2 (Public review):

FOXC1 is a transcription factor essential for the development of neural crest-derived tissues and has been identified as a key biomarker in various cancers. However, the molecular mechanisms underlying its function remain poorly understood. In this study, the authors used RNA-seq, ChIP-seq, and FOXC1-overexpressing cell models to show that FOXC1 directly activates transcription of ARHGAP36 by binding to specific cis-regulatory elements. Elevated expression of FOXC1 or ARHGAP36 was found to enhance Hedgehog (Hh) signaling and suppress PKA activity. Notably, overexpression of either gene also conferred resistance to Smoothened (SMO) inhibitors, indicating ligand-independent activation of Hh signaling. Analysis of public gene expression datasets further revealed that ARHGAP36 expression correlates with improved 5-year overall survival in neuroblastoma patients. Together, these findings uncover a novel FOXC1-ARHGAP36 regulatory axis that modulates Hh and PKA signaling, offering new insights into both normal development and cancer progression.

The main strengths of the study are:

(1) Identification of a novel signaling pathway involving FOXC1 and ARHGAP36, which may play a critical role in both normal development and cancer biology.

(2) Mechanistic investigation using RNA-seq, ChIP-seq, and functional assays to elucidate how FOXC1 regulates ARHGAP36 and how this axis modulates Hh signaling.

(3) Clinical relevance demonstrated through analysis of neuroblastoma patient datasets, linking ARHGAP36 expression to improved 5-year overall survival.

The main weaknesses of the study are:

(1) Lack of validation in neuroblastoma models - the study does not directly test its findings in neuroblastoma cell models, limiting translational relevance.

(2) Incomplete mechanistic insight into PKA regulation - the study does not fully elucidate how FOXC1-ARHGAP36 regulates PKAC activity at the molecular level.

(3) Insufficient discussion of clinical outcome data - while ARHGAP36 expression correlates with improved survival in neuroblastoma, the manuscript lacks a clear interpretation of this unexpected finding, especially given the known oncogenic roles of FOXC1, ARHGAP36, and Hh signaling.

Reviewer #3 (Public review):

Summary:

The focus of the research is to understand how transcription factors with high expression in neural crest cell-derived cancers (e.g., neuroblastoma) and roles in neural crest cell development function to promote malignancy. The focus is on the transcription factor FOXC1 and using murine cell culture, gain- and loss-of-function approaches, and ChIP profiling, among other techniques, to place PKAC inhibitor ARHGAP36 mechanistically between FOXC1 and another pathway associated with malignancy, Sonic Hedgehog (SHH).

Strengths:

Major strengths are the mechanistic approaches to identify FOXC1 direct targets, definitively showing that FOXC1 transcriptional regulation of ARHGAP36 leads to dysregulation of SHH signaling downstream of ARHGAP36 inhibition of PKC. Starting from a screen of Foxc1 OE to get to ARHGAP36 and then using genetic and pharmacological manipulation to work through the mechanism is very well done. There is data that will be of use to others studying FOXC1 in mesenchymal cell types, in particular, the FOXC1 ChIP-seq.

Weaknesses:

Work is almost all performed in NIH3T3 or similar cells (mouse cells, not patient or mouse-derived cancer cells), so the link to neuroblastoma that forms the major motivation of the work is not clear. The authors look at ARHGAP36 levels in association with the neuroblastoma patient survival; however, the finding, though interesting and quite compelling, is misaligned with what the literature shows about FOXC1 and SHH, their high expression is associated with increased malignancy (also maybe worse outcomes?). Therefore, ARHGAP36 expression may be more complicated in a tumor cell or may be unrelated to FOXC1 or SHH, leaving one to wonder what the work in NIH3T3 cells, though well done, is telling us about the mechanisms of FOXC1 as an oncogene in neuroblastoma cells or in any type of cancer cell. Does it really function as an SHH activator to drive tumor growth? The 'oncogenic relevance' and 'contribution to malignancy' claimed in the last paragraph of the introduction are currently weakly supported by the data as presented. This could be improved by studying some of these mechanisms in patient-derived neuroblastoma cells with high FOXC1 expression. Does inhibiting FOXC1 change SHH and ARHGAP36 and have any effect on cell proliferation or migration? Alternatively, does OE of FOXC1 in NIH3T3 cells increase their migration or stimulate proliferation in some way, and is this dependent on ARHGAP36 or SHH? Application of their mechanistic approaches in cancer cells or looking for hallmarks of cancer phenotypes with FOXC1 OE (and dependent on SHH or ARHGAP36) could help to make a link with cellular phenotypes of malignant cells.

Reviewer #1 (Public review):

Summary:

This study set out to investigate potential pharmacological drug-drug interactions between the two most common antimalarial classes, the artemisinins and quinolines. There is a strong rationale for this aim, because drugs from these classes are already widely used in Artemisinin Combination Therapies (ACTs) in the clinic, and drug combinations are an important consideration in the development of new medicines. Furthermore, whilst there is ample literature proposing many diverse mechanisms of action and resistance for the artemisinins and quinolines, it is generally accepted that the mechanisms for both classes involve heme metabolism in the parasite, and that artemisinin activity is dependent on activation by reduced heme. The study was designed to measure drug-drug interactions associated with a short pulse exposure (4 h) that is reminiscent of the short duration of artemisinin exposure obtained after in vivo dosing. Clear antagonism was observed between dihydroartemisinin (DHA) and chloroquine, which became even more extensive in chloroquine-resistant parasites. Antagonism was also observed in this assay for the more clinically-relevant ACT partner drugs piperaquine and amodiaquine, but not for other ACT partners mefloquine and lumefantrine, which don't share the 4-aminoquinoline structure or mode of action. Interestingly, chloroquine induced an artemisinin resistance phenotype in the standard in vitro Ring-stage Survival Assay, whereas this effect was not apparent for piperaquine.

The authors also utilised a heme-reactive probe to demonstrate that the 4-aminoquinolines can inhibit heme-mediated activation of the probe within parasites, which suggests that the mechanism of antagonism involves the inactivation of heme, rendering it unable to activate the artemisinins. Measurement of protein ubiquitination showed reduced DHA-induced protein damage in the presence of chloroquine, which is also consistent with decreased heme-mediated activation, and/or with decreased DHA activity more generally.

Overall, the study clearly demonstrates a mechanistic antagonism between DHA and 4-aminoquinoline antimalarials in vitro. It is interesting that this combination is successfully used to treat millions of malaria cases every year, which may raise questions about the clinical relevance of this finding. However, the conclusions in this paper are supported by multiple lines of evidence, and the data are clearly and transparently presented, leaving no doubt that DHA activity is compromised by the presence of chloroquine in vitro. It is perhaps fortunate that the clinical dosing regimens of 4-aminoquinoline-based ACTs have been sufficient to maintain clinical efficacy despite the non-optimal combination. Nevertheless, optimisation of antimalarial combinations and dosing regimens is becoming more important in the current era of increasing resistance to artemisinins and 4-aminoquinolines. Therefore, these findings should be considered when proposing new treatment regimens (including Tripe-ACTs) and the assays described in this study should be performed on new drug combinations that are proposed for new or existing antimalarial medicines.

Strengths:

This manuscript is clearly written, and the data presented are clear and complete. The key conclusions are supported by multiple lines of evidence, and most findings are replicated with multiple drugs within a class, and across multiple parasite strains, thus providing more confidence in the generalisability of these findings across the 4-aminoquinoline and peroxide drug classes.

A key strength of this study was the focus on short pulse exposures to DHA (4 h in trophs and 3 h in rings), which is relevant to the in vivo exposure of artemisinins. Artemisinin resistance has had a significant impact on treatment outcomes in South-East Asia, and is now emerging in Africa, but is not detected using a 'standard' 48 or 72 h in vitro growth inhibition assay. It is only in the RSA (a short pulse of 3-6 h treatment of early ring stage parasites) that the resistance phenotype can be detected in vitro. Therefore, assays based on this short pulse exposure provide the most relevant approach to determine whether drug-drug interactions are likely to have a clinically relevant impact on DHA activity. These assays clearly showed antagonism between DHA and 4-aminoquinolines (chloroquine, piperaquine, amodiaquine, and ferroquine) in trophozoite stages. Interestingly, whilst chloroquine clearly induced an artemisinin-resistant phenotype in the RSA, piperaquine did not appear to impact the early ring stage activity of DHA, which may be fortunate considering that piperaquine is a currently recommended DHA partner drug in ACTs, whereas chloroquine is not!

The evaluation of additional drug combinations at the end of this paper is a valuable addition, which increases the potential impact of this work. The finding of antagonism between piperaquine and OZ439 in trophozoites is consistent with the general interactions observed between peroxides and 4-aminoquinolines, and it would be interesting to see whether piperaquine impacts the ring-stage activity of OZ439.

The evaluation of reactive heme in parasites using a fluorescent sensor, combined with the measurement of K48-linked ubiquitin, further supports the findings of this study, providing independent read-outs for the chloroquine-induced antagonism.

The in-depth discussion of the interpretation and implications of the results is an additional strength of this manuscript. Whilst the discussion section is rather lengthy, there are important caveats to the interpretation of some of these results, and clear relevance to the future management of malaria that require these detailed explanations.

Overall, this is a high-quality manuscript describing an important study that has implications for the selection of antimalarial combinations for new and existing malaria medicines.

Weaknesses:

This study is an in vitro study of parasite cultures, and therefore, caution should be taken when applying these findings to decisions about clinical combinations. The drug concentrations and exposure durations in these assays are intended to represent clinically relevant exposures, although it is recognised that the in vitro system is somewhat simplified and there may be additional factors that influence in vivo activity. I think this is reasonably well acknowledged in the manuscript.

It is also important to recognise that the majority of the key findings regarding antagonism are based on trophozoite-stage parasites, and one must show caution when generalising these findings to other stages or scenarios. For example, piperaquine showed clear antagonism in trophozoite stages, but not in ring stages under these assay conditions.

The key weakness in this manuscript is the over-interpretation of the mechanistic studies that implicate heme-mediated artemisinin activation as the mechanism underpinning antagonism by chloroquine. In particular, the manuscript title focuses on heme-mediated activation of artemisinins, but this study did not directly measure the activation of artemisinins. The data obtained from the activation of the fluorescent probe are generally supportive of chloroquine suppressing the heme-mediated activation of artemisinins, and I think this is the most likely explanation, but there are significant caveats that undermine this conclusion. Primarily, the inconsistency between the fluorescence profile in the chemical reactions and the cell-based assay raises questions about the accuracy of this readout. In the chemical reaction, mefloquine and chloroquine showed identical inhibition of fluorescence, whereas piperaquine had minimal impact. On the contrary, in the cell, chloroquine and piperaquine had similar impacts on fluorescence, but mefloquine had minimal impact. This inconsistency indicates that the cellular fluorescence based on this sensor does not give a simple direct readout of the reactivity of ferrous heme, and therefore, these results should be interpreted with caution. Indeed, the correlation between fluorescence and antagonism for the tested drugs is a correlation, not causation. There could be several reasons for the disconnect between the chemical and biological results, either via additional mechanisms that quench fluorescence, or the presence of biomolecules that alter the oxidation state or coordination chemistry of heme or other potential catalysts of this sensor. It is possible that another factor that influences the H-FluNox fluorescence in cells also influences the DHA activity in cells, leading to the correlation with activity. It should be noted that H-FluNox is not a chemical analogue of artemisinins. Its activation relies on Fenton-like chemistry, but with an N-O rather than O-O bond, and it possesses very different steric and electronic substituents around the reactive centre, which are known to alter reactivity to different iron sources. Despite these limitations, the authors have provided reasonable justification for the use of this probe to directly visualise heme reactivity in cells, and the results are still informative, but additional caution should be provided in the interpretation, and the results are not conclusive enough to justify the current title of the paper.

Another interesting finding that was not elaborated by the authors is the impact of chloroquine on the DHA dose-response curves from the ring stage assays. Detection of artemisinin resistance in the RSA generally focuses on the % survival at high DHA concentrations (700 nM) as there is minimal shift in the IC50 (see Figure 2), however, chloroquine clearly induces a shift in the IC50 (~5-fold), where the whole curve is shifted to the right, whereas the increase in % survival is relatively small. This different profile suggests that the mechanism of chloroquine-induced antagonism is different from the mechanism of artemisinin resistance. Current evidence regarding the mechanism of artemisinin resistance generally points towards decreased heme-mediated drug activation due to a decrease in hemoglobin uptake, which should be analogous to the decrease in heme-mediated drug activation caused by chloroquine. However, these different dose-response curves suggest different mechanisms are primarily responsible. Additional mechanisms have been proposed for artemisinin resistance, involving redox or heat stress responses, proteostatic responses, mitochondrial function, dormancy, and PI3K signaling, among others. Whilst the H-FluNox probe generally supports the idea that chloroquine suppresses heme-mediated DHA activation, it remains plausible that chloroquine could induce these, or other, cellular responses that suppress DHA activity.

The other potential weakness in the current manuscript is the interpretation of the OZ439 clinical data. Whilst the observed interaction with piperaquine and ferroquine may have been a contributing factor, it should also be recognised that the low pharmacokinetic exposure in these studies was the primary reason for treatment failure (Macintyre 2017).

Impact:

This study has important implications for the selection of drugs to form combinations for the treatment of malaria. The overall findings of antagonism between peroxide antimalarials and 4-aminoquinolines in the trophozoite stage are robust, and this carries across to the ring stage for chloroquine (but not piperaquine).

The manuscript also provides a plausible mechanism to explain the antagonism, although future work will be required to further explore the details of this mechanism and to rule out alternative factors that may contribute.

Overall, this is an important contribution to the field and provides a clear justification for the evaluation of potential drug combinations in relevant in vitro assays before clinical testing.

Reviewer #2 (Public review):

Summary:

This manuscript by Rosenthal and Goldberg investigates interactions between artemisinins and their quinoline partner drugs currently used for treating uncomplicated Plasmodium falciparum malaria. The authors show that chloroquine (CQ), piperaquine, and amodiaquine antagonize dihydroartemisinin (DHA) activity, and in CQ-resistant parasites, the interaction is described as "superantagonism," linked to the pfcrt genotype. Mechanistically, application of the heme-reactive probe H-FluNox indicates that quinolines render cytosolic heme chemically inert, thereby reducing peroxide activation. The work is further extended to triple ACTs and ozonide-quinoline combinations, with implications for artemisinin-based combination therapy (ACT) design, including triple ACTs.

Strengths:

The manuscript is clearly written, methodologically careful, and addresses a clinically relevant question. The pulsing assay format more accurately models in vivo artemisinin exposure than conventional 72-hour assays, and the use of H-FluNox and Ac-H-FluNox probes provides mechanistic depth by distinguishing chemically active versus inert heme. These elements represent important refinements beyond prior studies, adding nuance to our understanding of artemisinin-quinoline interactions.

Weaknesses:

Several points warrant consideration. The novelty of the work is somewhat incremental, as antagonism between artemisinins and quinolines is well established. Multiple prior studies using standard fixed-ratio isobologram assays have shown that DHA exhibits indifferent or antagonistic interactions with chloroquine, piperaquine, and amodiaquine (e.g., Davis et al., 2006; Fivelman et al., 2007; Muangnoicharoen et al., 2009), with recent work highlighting the role of parasite genetic background, including pfcrt and pfmdr1, in modulating these interactions (Eastman et al., 2016). High-throughput drug screens likewise identify quinoline-artemisinin combinations as mostly antagonistic. The present manuscript adds refinement by applying pulsed-exposure assays and heme probes rather than establishing antagonism de novo.

The dataset focuses on several parasite lines assayed in vitro, so claims about broad clinical implications should be tempered, and the discussion could more clearly address how in vitro antagonism may or may not translate to clinical outcomes. The conclusion that artemisinins are predominantly activated in the cytoplasm is intriguing but relies heavily on Ac-H-FluNox data, which may have limitations in accessing the digestive vacuole and should be acknowledged explicitly. The term "superantagonism" is striking but may appear rhetorical; clarifying its reproducibility across replicates and providing a mechanistic definition would strengthen the framing. Finally, some discussion points, such as questioning the clinical utility of DHA-PPQ, should be moderated to better align conclusions with the presented data while acknowledging the complexity of in vivo pharmacology and clinical outcomes.

Despite these mild reservations, the data are interesting and of high quality and provide important new information for the field.

Reviewer #3 (Public review):

Summary:

The authors present an in vitro evaluation of drug-drug interactions between artemisinins and quinoline antimalarials, as an important aspect for screening the current artemisinin-based combination therapies for Plasmodium falciparum. Using a revised pulsing assay, they report antagonism between dihydroartemisinin (DHA) and several quinolines, including chloroquine, piperaquine (PPQ), and amodiaquine. This antagonism is increased in CQ-resistant strains in isobologram analyses. Moreover, CQ co-treatment was found to induce artemisinin resistance even in parasites lacking K13 mutations during the ring-stage survival assay. This implies that drug-drug interactions, not just genetic mutations, can influence resistance phenotypes. By using a chemical probe for reactive heme, the authors demonstrate that quinolines inhibit artemisinin activation by rendering cytosolic heme chemically inert, thereby impairing the cytotoxic effects of DHA. The study also observed negative interactions in triple-drug regimens (e.g., DHA-PPQ-Mefloquine) and in combinations involving OZ439, a next-generation peroxide antimalarial. Taken together, these findings raise significant concerns regarding the compatibility of artemisinin and quinoline combinations, which may promote resistance or reduce efficacy.

Throughout the manuscript, no combinations were synergistic, which necessitates comparing the claims to a synergistic combination as a control. The lack of this positive control makes it difficult to contextualize the observed antagonism. Including a known synergistic pair (e.g., artemisinin + lumefantrine) throughout the study would have provided a useful benchmark to assess the relative impact of the drug interactions described.

Strengths:

This study demonstrates the following strengths:

(1) The use of a pulsed in vitro assay that is more physiologically relevant than the traditional 48h or 72h assays.

(2) Small molecule probes, H-FluNox, and Ac-H-FluNox to detect reactive cytosolic heme, demonstrating that quinolines render heme inert and thereby block DHA activation.

(3) Evaluates not only traditional combinations but also triple-drug combinations and next-generation artemisinins like OZ439. This broad scope increases the study's relevance to current treatment strategies and future drug development.

(4) By using the K13 wild-type parasites, the study suggests that resistance phenotypes can emerge from drug-drug interactions alone, without requiring genetic resistance markers.

Weaknesses:

(1) No combinations are shown as synergistic: it could be valuable to have a combination that shows synergy as a positive control (e.g, artemisinin + lumefantrine) throughout the manuscript. The absence of a synergistic control combination in the experimental design makes it more challenging to evaluate the relative impact of the described drug interactions.

(2) Evaluation of the choice of drug-drug interactions: How generalizable are the findings across a broad range of combinations, especially those with varied modes of action?

(3) The study would also benefit from a characterization of the molecular basis for the observed heme inactivation by quinolines to support this hypothesis - while the probe experiments are valuable, they do not fully elucidate how quinolines specifically alter heme chemistry at the molecular level.

(4) Suggestion of alternative combinations that show synergy could have improved the significance of the work.

(5) All data are derived from in vitro experiments, without accompanying an in vivo validation. While the pulsing assay improves physiological relevance, it still cannot fully capture the complexity of drug pharmacokinetics, host-parasite interactions, or immune responses present in living organisms.

(6) The absence of pharmacokinetic/pharmacodynamic modeling leaves questions about how the observed antagonism would manifest under real-world dosing conditions.

Reviewer #1 (Public review):

Summary:

In this manuscript, Nührenberg et al., describe vassi, a Python package for mutually exclusive behavioral classification of social behaviors. This package imports and organizes trajectory data and manual behavior labels, and then computes feature representations for use with available Python machine learning-based classification tools. These representations include all possible dyadic interactions within an animal group, enabling classification of social behaviors between pairs of animals at a distance. The authors validate this package by reproducing the behavior classification performance on a previously published dyadic mouse dataset, and demonstrate its use on a novel cichlid group dataset. The authors have created a package that is agnostic to the mechanism of tracking and will reduce the barrier of data preparation for machine learning, which can be a stumbling block for non-experts. The package also evaluates the classification performance with helpful visualizations and provides a tool for inspection of behavior classification results.

Strengths:

(1) A major contribution of this paper was creating a framework to extend social behavior classification to groups of animals such that the actor and receiver can be any member of the group, regardless of distance. To implement this framework, the authors created a Python package and an extensive documentation site, which is greatly appreciated. This package should be useful to researchers with a knowledge of Python, virtual environments, and machine learning, as it relies on scripts rather than a GUI interface and may facilitate the development of new machine learning algorithms for behavior classification.

(2) The authors include modules for correctly creating train and test sets, and evaluation of classifier performance. This is extremely useful. Beyond evaluation, they have created a tool for manual review and correction of annotations. And they demonstrate the utility of this validation tool in the case of rare behaviors where correct classification is difficult, but the number of examples to review is reasonable.

(3) The authors provide well-commented step-by-step instructions for the use of the package in the documentation.

Weaknesses:

(1) While the classification algorithm was not the subject of the paper, as the authors used off-the-shelf methods and were only able to reproduce the performance of the CALMS21 dyadic dataset, they did not improve upon previously published results. Furthermore, the results from the novel cichlid fish dataset, including a macro F1 score of 0.45, did not compellingly show that the workflow described in the paper produces useful behavioral classifications for groups of interacting animals performing rare social behaviors. I commend the authors for transparently reporting the results both with the macro F1 scores and the confusion matrices for the classifiers. The mutually exclusive, all-vs-all data annotation scheme of rare behaviors results in extremely unbalanced datasets such that categorical classification becomes a difficult problem. To try to address the performance limitation, the authors built a validation tool that allows the user to manually review the behavior predictions.

(2) The pipeline makes a few strong assumptions that should be made more explicit in the paper.

First, the behavioral classifiers are mutually exclusive and one-to-one. An individual animal can only be performing one behavior at any given time, and that behavior has only one recipient. These assumptions are implicit in how the package creates the data structure, and should be made clearer to the reader. Additionally, the authors emphasize that they have extended behavior classification to animal groups, but more accurately, they have extended behavioral classification to all possible pairs within a group.

Second, the package expects comprehensive behavior labeling of the tracking data as input. Any frames not manually labeled are assumed to be the background category. Additionally, the package will interpolate through any missing segments of tracking data and assign the background behavioral category to those trajectory segments as well. The effects of these assumptions are not explored in the paper, which may limit the utility of this workflow for naturalistic environments.

(3) Finally, the authors described the package as a tool for biologists and ethologists, but the level of Python and machine learning expertise required to use the package to develop a novel behavior classification workflow may be beyond the ability of many biologists. More accessible example notebooks would help address this problem.

Reviewer #2 (Public review):

Summary:

The authors present a novel supervised behavioral analysis pipeline (vassi), which extends beyond previously available packages with its innate support of groups of any number of organisms. Importantly, this program also allows for iterative improvement upon models through revised behavioral annotation.

Strengths:

vassi's support of groups of any number of animals is a major advancement for those studying collective social behavior. Additionally, the built-in ability to choose different base models and iteratively train them is an important advancement beyond current pipelines. vassi is also producing behavioral classifiers with similar precision/recall metrics for dyadic behavior as currently published packages using similar algorithms.

Weaknesses:

vassi's performance on group behaviors is potentially too low to proceed with (F1 roughly 0.2 to 0.6). Different sources have slightly different definitions, but an F1 score of 0.7 or 0.8 is often considered good, while anything lower than 0.5 can typically be considered bad. There has been no published consensus within behavioral neuroscience (that I know of) on a minimum F1 score for use. Collective behavioral research is extremely challenging to perform due to hand annotation times, and there needs to be a discussion in the field as to the trade-off between throughput and accuracy before these scores can be either used or thrown out the door. It would also be useful to see the authors perform a few rounds of iterative corrections on these classifiers to see if performance is improved.

While the interaction networks in Figure 2b-c look visually similar based on interaction pairs, the weights of the interactions appear to be quite different between hand and automated annotations. This could lead to incorrect social network metrics, which are increasingly popular in collective social behavior analysis. It would be very helpful to see calculated SNA metrics for hand versus machine scoring to see whether or not vassi is reliable for these datasets.

Reviewer #1 (Public review):

In this manuscript, Hoon Cho et al. presents a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.

Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.

Highlights of the Manuscript:

The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.

The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.

Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.

Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells. However, several major points need to be addressed:

Major Comments:

Figures 1 and 2

The authors conclude, based on the results from these two figures, that PexRAP promotes the homeostatic maintenance and proliferation of B cells. In this section, the authors first use a tamoxifen-inducible full Dhrs7b knockout (KO) and afterwards Dhrs7bΔ/Δ-B model to specifically characterize the role of this molecule in B cells. They characterize the B and T cell compartments using flow cytometry (FACS) and examine the establishment of the GC reaction using FACS and immunofluorescence. They conclude that B cell numbers are reduced, and the GC reaction is defective upon stimulation, showing a reduction in the total percentage of GC cells, particularly in the light zone (LZ).

The analysis of the steady-state B cell compartment should also be improved. This includes a more detailed characterization of MZ and B1 populations, given the role of lipid metabolism and lipid peroxidation in these subtypes.

Suggestions for Improvement:

- B Cell compartment characterization: A deeper characterization of the B cell compartment in non-immunized mice is needed, including analysis of Marginal Zone (MZ) maturation and a more detailed examination of the B1 compartment. This is especially important given the role of specific lipid metabolism in these cell types. The phenotyping of the B cell compartment should also include an analysis of immunoglobulin levels on the membrane, considering the impact of lipids on membrane composition.

- GC Response Analysis Upon Immunization: The GC response characterization should include additional data on the T cell compartment, specifically the presence and function of Tfh cells. In Fig. 1H, the distribution of the LZ appears strikingly different. However, the authors have not addressed this in the text. A more thorough characterization of centroblasts and centrocytes using CXCR4 and CD86 markers is needed.<br /> The gating strategy used to characterize GC cells (GL7+CD95+ in IgD− cells) is suboptimal. A more robust analysis of GC cells should be performed in total B220+CD138− cells.

- The authors claim that Dhrs7b supports the homeostatic maintenance of quiescent B cells in vivo and promotes effective proliferation. This conclusion is primarily based on experiments where CTV-labeled PexRAP-deficient B cells were adoptively transferred into μMT mice (Fig. 2D-F). However, we recommend reviewing the flow plots of CTV in Fig. 2E, as they appear out of scale. More importantly, the low recovery of PexRAP-deficient B cells post-adoptive transfer weakens the robustness of the results and is insufficient to conclusively support the role of PexRAP in B cell proliferation in vivo.

- In vitro stimulation experiments: These experiments need improvement. The authors have used anti-CD40 and BAFF for B cell stimulation; however, it would be beneficial to also include anti-IgM in the stimulation cocktail. In Fig. 2G, CTV plots do not show clear defects in proliferation, yet the authors quantify the percentage of cells with more than three divisions. These plots should clearly display the gating strategy. Additionally, details about histogram normalization and potential defects in cell numbers are missing. A more in-depth analysis of apoptosis is also required to determine whether the observed defects are due to impaired proliferation or reduced survival.

Reviewer #2 (Public review):

Summary:

In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.

Strengths:

Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.

Weaknesses:

More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In the minor part, there are issues with the interpretation of the data which might cause confusion for the readers.

Reviewer #1 (Public review):

Summary:

This paper aims to characterize the relationship between affinity and fitness in the process of affinity maturation. To this end, the authors develop a model of germinal center reaction and a tailored statistical approach, building on recent advances in simulation-based inference. The potential impact of this work is hindered by the poor organization of the manuscript. In crucial sections, the writing style and notations are unclear and difficult to follow.

Strengths:

The model provides a framework for linking affinity measurements and sequence evolution and does so while accounting for the stochasticity inherent to the germinal center reaction. The model's sophistication comes at the cost of numerous parameters and leads to intractable likelihood, which are the primary challenges addressed by the authors. The approach to inference is innovative and relies on training a neural network on extensive simulations of trajectories from the model.

Weaknesses:

The text is challenging to follow. The descriptions of the model and the inference procedure are fragmented and repetitive. In the introduction and the methods section, the same information is often provided multiple times, at different levels of detail. This organization sometimes requires the reader to move back and forth between subsections (there are multiple non-specific references to "above" and "below" in the text).

The choice of some parameter values in simulations appears arbitrary and would benefit from more extensive justification. It remains unclear how the "significant uncertainty" associated with these parameters affects the results of inference. In addition, the performance of the inference scheme on simulated data is difficult to evaluate, as the reported distributions of loss function values are not very informative.

Finally, the discussion of the similarities and differences with an alternative approach to this inference problem, presented in Dewitt et al. (2025), is incomplete.

Reviewer #2 (Public review):

Summary:

This paper presents a new approach for explicitly transforming B-cell receptor affinity into evolutionary fitness in the germinal center. It demonstrates the feasibility of using likelihood-free inference to study this problem and demonstrates how effective birth rates appear to vary with affinity in real-world data.

Strengths:

(1) The authors leverage the unique data they have generated for a separate project to provide novel insights into a fundamental question.

(2) The paper is clearly written, with accessible methods and a straightforward discussion of the limits of this model.

(3) Code and data are publicly available and well-documented.

Weaknesses (minor):

(1) Lines 444-446: I think that "affinity ceiling" and "fitness ceiling" should be considered independent concepts. The former, as the authors ably explain, is a physical limitation. This wouldn't necessarily correspond to a fitness ceiling, though, as Figure 7 shows. Conversely, the model developed here would allow for a fitness ceiling even if the physical limit doesn't exist.

(2) Lines 566-569: I would like to see this caveat fleshed out more and perhaps mentioned earlier in the paper. While relative affinity is far more important, it is not at all clear to me that absolute affinity can be totally ignored in modeling GC behavior.

(3) One other limitation that is worth mentioning, though beyond the scope of the current work to fully address: the evolution of the repertoire is also strongly shaped by competition from circulating antibodies. (Eg: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600904/, http://www.sciencedirect.com/science/article/pii/S1931312820303978). This is irrelevant for the replay experiment modeled here, but still an important factor in general repertoires.

Reviewer #1 (Public review):

Summary:

The authors develop a set of biophysical models to investigate whether a constant area hypothesis or a constant curvature hypothesis explains the mechanics of membrane vesiculation during clathrin-mediated endocytosis.

Strengths:

The models that the authors choose are fairly well-described in the field and the manuscript is well-written.

Weaknesses:

One thing that is unclear is what is new with this work. If the main finding is that the differences are in the early stages of endocytosis, then one wonders if that should be tested experimentally. Also, the role of clathrin assembly and adhesion are treated as mechanical equilibrium but perhaps the process should not be described as equilibria but rather a time-dependent process. Ultimately, there are so many models that address this question that without direct experimental comparison, it's hard to place value on the model prediction.

While an attempt is made to do so with prior published EM images, there is excessive uncertainty in both the data itself as is usually the case but also in the methods that are used to symmetrize the data. This reviewer wonders about any goodness of fit when such uncertainty is taken into account.

Comments on revisions:

I appreciate the authors edits, but I found that the major concerns I had still hold. Therefore, I did not alter my review.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors employ theoretical analysis of an elastic membrane model to explore membrane vesiculation pathways in clathrin-mediated endocytosis. A complete understanding of clathrin-mediated endocytosis requires detailed insight into the process of membrane remodeling, as the underlying mechanisms of membrane shape transformation remain controversial, particularly regarding membrane curvature generation. The authors compare constant area and constant membrane curvature as key scenarios by which clathrins induce membrane wrapping around the cargo to accomplish endocytosis. First, they characterize the geometrical aspects of the two scenarios and highlight their differences by imposing coating area and membrane spontaneous curvature. They then examine the energetics of the process to understand the driving mechanisms behind membrane shape transformations in each model. In the latter part, they introduce two energy terms: clathrin assembly or binding energy, and curvature generation energy, with two distinct approaches for the latter. Finally, they identify the energetically favorable pathway in the combined scenario and compare their results with experiments, showing that the constant-area pathway better fits the experimental data.

Strengths:

The manuscript is well-written, well-organized, and presents the details of the theoretical analysis with sufficient clarity.<br /> The calculations are valid, and the elastic membrane model is an appropriate choice for addressing the differences between the constant curvature and constant area models.<br /> The authors' approach of distinguishing two distinct free energy terms-clathrin assembly and curvature generation-and then combining them to identify the favorable pathway is both innovative and effective in addressing the problem.<br /> Notably, their identification of the energetically favorable pathways, and how these pathways either lead to full endocytosis or fail to proceed due to insufficient energetic drives, is particularly insightful.

Comments on revisions:

The authors have carefully addressed all my comments, and the revised manuscript is now clear, rigorous, and satisfactory.

Reviewer #1 (Public review):

Summary:

This manuscript by Pournejati et al investigates how BK (big potassium) channels and CaV1.3 (a subtype of voltage-gated calcium channels) become functionally coupled by exploring whether their ensembles form early-during synthesis and intracellular trafficking-rather than only after insertion into the plasma membrane. To this end, the authors use the PLA technique to assess the formation of ion channel associations in the different compartments (ER, Golgi or PM), single-molecule RNA in situ hybridization (RNAscope), and super-resolution microscopy.

Strengths:

The manuscript is well written and addresses an interesting question, combining a range of imaging techniques. The findings are generally well-presented and offer important insights into the spatial organization of ion channel complexes, both in heterologous and endogenous systems.

Weaknesses:

The authors have improved their manuscript after revisions, and some previous concerns have been addressed. Still, the main concern about this work is that the current experiments do not quantitatively or mechanistically link the ensembles observed intracellularly (in the endoplasmic reticulum (ER) or Golgi) to those found at the plasma membrane (PM). As a result, it is difficult to fully integrate the findings into a coherent model of trafficking. Specifically, the manuscript does not address what proportion of ensembles detected at the PM originated in the ER. Without data on the turnover or half-life of these ensembles at the PM, it remains unclear how many persist through trafficking versus forming de novo at the membrane. The authors report the percentage of PLA-positive ensembles localized to various compartments, but this only reflects the distribution of pre-formed ensembles. What remains unknown is the proportion of total BK and CaV1.3 channels (not just those in ensembles) that are engaged in these complexes within each compartment. Without this, it is difficult to determine whether ensembles form in the ER and are then trafficked to the PM, or if independent ensemble formation also occurs at the membrane. To support the model of intracellular assembly followed by coordinated trafficking, it would be important to quantify the fraction of the total channel population that exists as ensembles in each compartment. A comparable ensemble-to-total ratio across ER and PM would strengthen the argument for directed trafficking of pre-assembled channel complexes.

Reviewer #2 (Public review):

Summary:

The co-localization of large conductance calcium- and voltage activated potassium (BK) channels with voltage-gated calcium channels (CaV) at the plasma membrane is important for the functional role of these channels in controlling cell excitability and physiology in a variety of systems.

An important question in the field is where and how do BK and CaV channels assemble as 'ensembles' to allow this coordinated regulation - is this through preassembly early in the biosynthetic pathway, during trafficking to the cell surface or once channels are integrated into the plasma membrane. These questions also have broader implications for assembly of other ion channel complexes.

Using an imaging based approach, this paper addresses the spatial distribution of BK-CaV ensembles using both overexpression strategies in tsa201 and INS-1 cells and analysis of endogenous channels in INS-1 cells using proximity ligation and superesolution approaches. In addition, the authors analyse the spatial distribution of mRNAs encoding BK and Cav1.3.

The key conclusion of the paper that BK and CaV1.3 are co-localised as ensembles intracellularly in the ER and Golgi is well supported by the evidence. However, whether they are preferentially co-translated at the ER, requires further work. Moreover, whether intracellular pre-assembly of BK-CaV complexes is the major mechanism for functional complexes at the plasma membrane in these models requires more definitive evidence including both refinement of analysis of current data as well as potentially additional experiments.

Strengths & Weaknesses

(1) Using proximity ligation assays of overexpressed BK and CaV1.3 in tsa201 and INS-1 cells the authors provide strong evidence that BK and CaV can exist as ensembles (ie channels within 40 nm) at both the plasma membrane and intracellular membranes, including ER and Golgi. They also provide evidence for endogenous ensemble assembly at the Golgi in INS-1 cells and it would have been useful to determine if endogenous complexes are also observe in the ER of INS-1 cells. There are some useful controls but the specificity of ensemble formation would be better determined using other transmembrane proteins rather than peripheral proteins (eg Golgi 58K).

(2) Ensemble assembly was also analysed using super-resolution (dSTORM) imaging in INS-1 cells. In these cells only 7.5% of BK and CaV particles (endogenous?) co-localise that was only marginally above chance based on scrambled images. More detailed quantification and validation of potential 'ensembles' needs to be made for example by exploring nearest neighbour characteristics (but see point 4 below) to define proportion of ensembles versus clusters of BK or Cav1.3 channels alone etc. For example, it is mentioned that a distribution of distances between BK and Cav is seen but data are not shown.

(3) The evidence that the intracellular ensemble formation is in large part driven by co-translation, based on co-localisation of mRNAs using RNAscope, requires additional critical controls and analysis. The authors now include data of co-localised BK protein that is suggestive but does not show co-translation. Secondly, while they have improved the description of some controls mRNA co-localisation needs to be measured in both directions (eg BK - SCN9A as well as SCN9A to BK) especially if the mRNAs are expressed at very different levels. The relative expression levels need to be clearly defined in the paper. Authors also use a randomized image of BK mRNA to show specificity of co-localisation with Cav1.3 mRNA, however the mRNA distribution would not be expected to be random across the cell but constrained by ER morphology if co-translated so using ER labelling as a mask would be useful?

(4) The authors attempt to define if plasma membrane assemblies of BK and CaV occur soon after synthesis. However, because the expression of BK and CaV occur at different times after transient transfection of plasmids more definitive experiments are required. For example, using inducible constructs to allow precise and synchronised timing of transcription. This would also provide critical evidence that co-assembly occurs very early in synthesis pathways - ie detecting complexes at ER before any complexes at Golgi or plasma membrane.

(5) While the authors have improved the definition of hetero-clusters etc it is still not clear in superesolution analysis, how they separate a BK tetramer from a cluster of BK tetramers with the monoclonal antibody employed ie each BK channel will have 4 binding sites (4 subunits in tetramer) whereas Cav1.3 has one binding site per channel. Thus, how do authors discriminate between a single BK tetramer (molecular cluster) with potential 4 antibodies bound compared to a cluster of 4 independent BK channels.

(6) The post-hoc tests used for one way ANOVA and ANOVA statistics need to be defined throughout

Reviewer #3 (Public review):

Summary:

The authors present a clearly written and beautifully presented piece of work demonstrating clear evidence to support the idea that BK channels and Cav1.3 channels can co-assemble prior to their assertion in the plasma membrane.

Strengths:

The experimental records shown back up their hypotheses and the authors are to be congratulated for the large number of control experiments shown in the ms.

Reviewer #2 (Public review):

In the manuscript by Fu et al., the authors developed a chemo-immunological method for the reliable detection of Kacac, a novel post-translational modification, and demonstrated that acetoacetate and AACS serve as key regulators of cellular Kacac levels. Furthermore, the authors identified the enzymatic addition of the Kacac mark by acyltransferases GCN5, p300, and PCAF, as well as its removal by deacetylase HDAC3. These findings indicate that AACS utilizes acetoacetate to generate acetoacetyl-CoA in the cytosol, which is subsequently transferred into the nucleus for histone Kacac modification. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveal the broad impacts of Kacac on diverse cellular processes and various pathophysiological conditions. This study provides valuable additional insights into the investigation of Kacac and would serve as a helpful resource for future physiological or pathological research.

Comments on revised version:

The authors have made efforts to revise this manuscript and address my concerns. The revisions are appropriate and have improved the quality of the manuscript.

Reviewer #3 (Public review):

Summary:

This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemo-immunological method using the reducing reagent NaBH4 to transform Kacac into lysine β-hydroxybutyrylation (Kbhb).

Strengths:

This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing its potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.

Weaknesses:

The experimental evidence presented in the article is insufficient to fully support the authors' conclusions. In the in vitro assays, the proteins used appear to be highly inconsistent with their expected molecular weights, as shown by Coomassie Brilliant Blue staining (Figure S3A). For example, p300, which has a theoretical molecular weight of approximately 270 kDa, appeared at around 37 kDa; GCN5/PCAF, expected to be ~70 kDa, appeared below 20 kDa. Other proteins used in the in vitro experiments also exhibited similarly large discrepancies from their predicted sizes. These inconsistencies severely compromise the reliability of the in vitro findings. Furthermore, the study lacks supporting in vivo data, such as gene knockdown experiments, to validate the proposed conclusions at the cellular level.

Reviewer #1 (Public review):

Kong et al.'s work describes a new approach that does exactly what the title states: "Correction of local beam-induced sample motion in cryo-EM images using a 3D spline model." I find the method appropriate, logical, and well-explained. Additionally, the work suggests using 2DTM-related measurements to quantify the improvement of the new method compared to the old one in cisTEM, Unblur. I find this part engaging; it is straightforward, accurate, and, of course, the group has a strong command of 2DTM, presenting a thorough study.

However, everything in the paper (except some correct general references) refers to comparisons with the full-frame approach, Unblur. Still, we have known for more than a decade that local correction approaches perform better than global ones, so I do not find anything truly novel in their proposal of using local methods (the method itself- Unbend- is new, but many others have been described previously). In fact, the use of 2DTM is perhaps a more interesting novelty of the work, and here, a more systematic study comparing different methods with these proposed well-defined metrics would be very valuable. As currently presented, there is no doubt that it is better than an older, well-established approach, and the way to measure "better" is very interesting, but there is no indication of how the situation stands regarding newer methods.

Regarding practical aspects, it seems that the current implementation of the method is significantly slower than other patch-based approaches. If its results are shown to exceed those of existing local methods, then exploring the use of Unbend, possibly optimizing its code first, could be a valuable task. However, without more recent comparisons, the impact of Unbend remains unclear.

Reviewer #2 (Public review):

Summary:

The authors present a new method, Unbend, for measuring motion in cryo-EM images, with a particular emphasis on more challenging in situ samples such as lamella and whole cells<br /> (that can be more prone to overall motion and/or variability in motion across a field of view). Building on their previous approach of full-frame alignment (Unblur), they now perform full-frame alignment followed by patch alignment, and then use these outputs to generate a 3D cubic spline model of the motion. This model allows them to estimate a continuous, per-pixel shift field for each movie frame that aims to better describe complex motions and so ultimately generate improved motion-corrected micrographs. Performance of Unbend is evaluated using the 2D template matching (2DTM) method developed previously by the lab, and results are compared to using full-frame correction alone. Several different in situ samples are used for evaluation, covering a broad range that will be of interest to the rapidly growing in situ cryo-EM community.

Strengths:

The method appears to be an elegant way of describing complex motions in cryo-EM samples, and the authors present convincing data that Unbend generally improves SNR of aligned micrographs as well as increases detection of particles matching the 60S ribosome template when compared to using full-frame correction alone. The authors also give interesting insights into how different areas of a lamella behave with respect to motion by using Unbend on a montage dataset collected previously by the group. There is growing interest in imaging larger areas of in situ samples at high resolution, and these insights contribute valuable knowledge. Additionally, the availability of data collected in this study through the EMPIAR repository will be much appreciated by the field.

Weaknesses:

While the improvements with Unbend vs. Unblur appear clear, it is less obvious whether Unbend provides substantial gains over patch motion correction alone (the current norm in the field). It might be helpful for readers if this comparison were investigated for the in situ datasets. Additionally, the authors are open that in cases where full motion correction already does a good job, the extra degrees of freedom in Unbend can perhaps overfit the motions, making the corrections ultimately worse. I wonder if an adaptive approach could be explored, for example, using the readout from full-frame or patch correction to decide whether a movie should proceed to the full Unbend pipeline, or whether correction should stop at the patch estimation stage.

Reviewer #3 (Public review):

Summary

Kong and coauthors describe and implement a method to correct local deformations due to beam-induced motion in cryo-EM movie frames. This is done by fitting a 3D spline model to a stack of micrograph frames using cross-correlation-based local patch alignment to describe the deformations across the micrograph in each frame, and then computing the value of the deformed micrograph at each pixel by interpolating the undeformed micrograph at the displacement positions given by the spline model. A graphical interface in cisTEM allows the user to visualise the deformations in the sample, and the method has been proven to be successful by showing improvements in 2D template matching (2DTM) results on the corrected micrographs using five in situ samples.

Impact

This method has great potential to further streamline the cryo-EM single particle analysis pipeline by shortening the required processing time as a result of obtaining higher quality particles early in the pipeline, and is applicable to both old and new datasets, therefore being relevant to all cryo-EM users.

Strengths

(1) One key idea of the paper is that local beam induced motion affects frames continuously in space (in the image plane) as well as in time (along the frame stack), so one can obtain improvements in the image quality by correcting such deformations in a continuous way (deformations vary continuously from pixel to pixel and from frame to frame) rather than based on local discrete patches only. 3D splines are used to model the deformations: they are initialised using local patch alignments and further refined using cross-correlation between individual patch frames and the average of the other frames in the same patch stack.

(2) Another strength of the paper is using 2DTM to show that correcting such deformations continuously using the proposed method does indeed lead to improvements. This is shown using five in situ datasets, where local motion is quantified using statistics based on the estimated motions of ribosomes.

Weaknesses

(1) While very interesting, it is not clear how the proposed method using 3D splines for estimating local deformations compares with other existing methods that also aim to correct local beam-induced motion by approximating the deformations throughout the frames using other types of approximation, such as polynomials, as done, for example MotionCor2.

(2) The use of 2DTM is appropriate, and the results of the analysis are enlightening, but one shortcoming is that some relevant technical details are missing. For example, the 2DTM SNR is not defined in the article, and it is not clear how the authors ensured that no false positives were included in the particles counted before and after deformation correction. The Jupyter notebooks where this analysis was performed have not been made publicly available.

(3) It is also not clear how the proposed deformation correction method is affected by CTF defocus in the different samples (are the defocus values used in the different datasets similar or significantly different?) or if there is any effect at all.

Reviewer #1 (Public review):

Summary:

In this manuscript, Chengjian Zhao et al. focused on the interactions between vascular, biliary, and neural networks in the liver microenvironment, addressing the critical bottleneck that the lack of high-resolution 3D visualization has hindered understanding of these interactions in liver disease.

Strengths:

This study developed a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized CUBIC tissue clearing. This method enables the simultaneous 3D visualization of spatial networks of the portal vein, hepatic artery, bile ducts, and central vein in the mouse liver. The authors reported a perivascular structure termed the Periportal Lamellar Complex (PLC), which is identified along the portal vein axis. This study clarifies that the PLC comprises CD34⁺Sca-1⁺ dual-positive endothelial cells with a distinct gene expression profile, and reveals its colocalization with terminal bile duct branches and sympathetic nerve fibers under physiological conditions.

Weaknesses:

This manuscript is well-written, organized, and informative. However, there are some points that need to be clarified.

(1) After MCNP-dye injection, does it remain in the blood vessels, adsorb onto the cell surface, or permeate into the cells? Does the MCNP-dye have cell selectivity?

(2) All MCNP-dyes were injected after the mice were sacrificed, and the mice's livers were fixed with PFA. After the blood flow had ceased, how did the authors ensure that the MCNP-dyes were fully and uniformly perfused into the microcirculation of the liver?

(3) It is advisable to present additional 3D perspective views in the article, as the current images exhibit very weak 3D effects. Furthermore, it would be better to supplement with some videos to demonstrate the 3D effects of the stained blood vessels.

(4) In Figure 1-I, the authors used MCNP-Black to stain the central veins; however, in addition to black, there are also yellow and red stains in the image. The authors need to explain what these stains are in the legend.

(5) There is a typo in the title of Figure 4F; it should be "stem cell".

(6) Nuclear staining is necessary in immunofluorescence staining, especially for Figure 5e. This will help readers distinguish whether the green color in the image corresponds to cells or dye deposits.

Reviewer #2 (Public review):

Summary:

The present manuscript of Xu et al. reports a novel clearing and imaging method focusing on the liver. The authors simultaneously visualized the portal vein, hepatic artery, central vein, and bile duct systems by injecting metal compound nanoparticles (MCNPs) with different colors into the portal vein, heart left ventricle, inferior vena cava, and the extrahepatic bile duct, respectively. The method involves: trans-cardiac perfusion with 4% PFA, the injection of MCNPs with different colors, clearing with the modified CUBIC method, cutting 200 micrometer thick slices by vibratome, and then microscopic imaging. The authors also perform various immunostaining (DAB or TSA signal amplification methods) on the tissue slices from MCNP-perfused tissue blocks. With the application of this methodical approach, the authors report dense and very fine vascular branches along the portal vein. The authors name them as 'periportal lamellar complex (PLC)' and report that PLC fine branches are directly connected to the sinusoids. The authors also claim that these structures co-localize with terminal bile duct branches and sympathetic nerve fibers, and contain endothelial cells with a distinct gene expression profile. Finally, the authors claim that PLC-s proliferate in liver fibrosis (CCl4 model) and act as a scaffold for proliferating bile ducts in ductular reaction and for ectopic parenchymal sympathetic nerve sprouting.

Strengths:

The simultaneous visualization of different hepatic vascular compartments and their combination with immunostaining is a potentially interesting novel methodological approach.

Weaknesses:

This reviewer has several concerns about the validity of the microscopic/morphological findings as well as the transcriptomics results. In this reviewer's opinion, the introduction contains overstatements regarding the potential of the method, there are severe caveats in the method descriptions, and several parts of the Results are not fully supported by the documentation. Thus, the conclusions of the paper may be critically viewed in their present form and may need reconsideration by the authors.

Reviewer #3 (Public review):

Summary:

In the reviewed manuscript, researchers aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the portal vein axis. The PLC originates from the portal vein and is characterized by a unique population of CD34⁺Sca-1⁺ dual-positive endothelial cells. Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting the mRNA presence of genes linked to neurodevelopment, biliary function, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver at the same time. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists; however, some claims need more thorough assessment by functional experimental approaches to decipher the functional molecules and the sequence of events before establishing the PLC as the key hub governing the activity of biliary, arterial, and neuronal liver systems. Similarly, the level of detail of the methods section does not appear to be sufficient to exactly recapitulate the performed experiments, which is of concern, given that the new technique is a cornerstone of the manuscript.

Nevertheless, the work does bring a clear new insight into the liver structure and functional units and greatly improves the methodological toolbox to study it even further, and thus fully deserves the attention of readers.

Strengths:

The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.

This work proposes a new biological framework between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - the Periportal Lamellar Complexes (PLCs).

Weaknesses:

Possible overinterpretation of the CD34+Sca1+ findings was built on re-analysis of one scRNAseq dataset.

Lack of detail in the materials and methods section greatly limits the usefulness of the new technique to other researchers.

Reviewer #1 (Public review):

Summary:

The authors used an in vitro microfluidic system where HUVECs are exposed to high, low, or physiologic (normal) shear stress to demonstrate that both high and low shear stress for 24 hours resulted in decreased KLF6 expression, decreased lipid peroxidation, and increased cell death, which was reversible upon treatment with Fer-1, the ferroptosis inhibitor. RNA sequencing (LSS vs normal SS) revealed decreased steroid synthesis and UPR signaling in low shear stress conditions, which they confirmed by showing reduced expression of proteins that mitigate ER stress under both LSS and HSS. Decreased KLF6 expression after exposure to HSS/LSS was associated with decreased expression of regulators of ER stress (PERK, BiP, MVD), which was restored with KLF6 overexpression. Overexpression of KLF6 also restored SLC7A11 expression, Coq10, and reduced c11 bodipy oxidation state- all markers of lipid peroxidation and ferroptosis. The authors then used vascular smooth muscle cells (atherosclerotic model) with HUVECs and monocytes to show that KLF6 overexpression reduces the adhesion of monocytes and lipid accumulation in conditions of low shear stress.

Strengths:

(1) The use of a microfluidic device to simulate shear stress while keeping the pressure constant when varying the shear stress applied is improved and more physiologic compared to traditional cone and shearing devices. Similarly, the utilization of both low and high shear stress in most experiments is a strength.

(2) This study provides a link between disturbed shear stress and ferroptosis, which is novel, and fits nicely with existing knowledge that endothelial cell ferroptosis promotes atherosclerosis. This concept was also recently reported in September 2025, when a publication also demonstrated that LSS triggers ferroptosis in vascular endothelial cells (PMID: 40939914), which partly validates these findings.

Weaknesses:

(1) While HUVECs are commonly used in endothelial in vitro studies, it would be preferable to confirm the findings using an arterial cell line, such as human coronary artery cells, when studying mechanisms of early atherosclerosis. Furthermore, physiologic arterial shear stress is higher than venous shear stress, and different vascular beds have varying responses to altered shear stress; as such, the up- and downregulated pathways in HUVECs should be confirmed in an arterial system.

(2) The authors provide convincing evidence of disturbances in shear stress inducing endothelial ferroptosis with assays for impaired lipid peroxidation and increased cell death that was reversed with a ferroptosis inhibitor. However, more detailed characterization of ferroptosis with iron accumulation assays, as well as evaluating GPX4 activity as a consequence of the impaired mevalonate pathway, and testing for concomitant apoptosis in addition to ferroptosis, would add to the data.

(3) The authors state that KLF2 and KLF4 are not amongst the differentially expressed genes downregulated by reduced shear stress, which is contrary to previous data, where both KLF2 and KLF4 are well studied to be upregulated by physiologic laminar shear stress. While this might be due to the added pressure in their microfluidic system, it also might be due to changes in gene expression over time. In this case, a time course experiment would be needed. It is possible that KLF2, KLF4 and KLF6 are all reduced in low (and high) shear stress and cooperatively regulate the endothelial cell phenotype. Both KLF2 and KLF4 have been shown to be protective against atherosclerosis.

Reviewer #2 (Public review):

Summary:

The manuscript by Cui et al. titled "abnormal shear stress induces ferroptosis in endothelial cells via KLF6 downregulation" investigated in a microfluidic device the effect of 24-hour low, medium, and high shear stress levels upon human vein endothelial cells. The authors found that KLF6 is an important regulator of endothelial cell ferroptosis through the BiP-PERK-Slc7a11 and MVD-ID11-CoQ10 axis under both low and high shear stress, postulating this may explain the spatial preference of atherosclerosis at bifurcations of the arteries.

Strengths:

The main strength of the study is the use of a microfluidic device within which the authors could vary the shear stress (low, medium, high), whilst keeping fluid pressure near the physiological range of 70 mmHg. Deciding to focus on transcription factors that respond to shear stress, the authors found KLF6 in their dataset, for which they provide compelling evidence that endothelial cell ferroptosis is triggered by both excessive and insufficient shear stress, inversely correlating with KLF6 expression. Importantly, it was demonstrated that cell death in endothelial cells during HSS and LSS was prevented through the addition of Fer-1, supporting the role of ferroptosis. Moreso, the importance of KLF6 as an essential regulator was demonstrated through KLF6 overexpression.

Weaknesses:

There are some major concerns with the results:

(1) Inappropriate statistical tests were used (i.e., an unpaired t-test cannot be used to compare more than two groups).<br /> (2) Inconsistencies in western blot normalization as different proteins seem to have been used (GAPDH and B-actin) without specifying which is used when and why this differs.<br /> (3) Absence of transcriptomic analysis on HSS-exposed endothelial cells (which is not explained).

Moreso, the conclusions are predominantly based on an in vitro microfluidic chip model seeded with HUVECs. Although providing mechanistic insight into the effects of shear stress on (venous) endothelial cells, it does not recapitulate the in vivo complexity. The absence of validation (a.o. levels of KLF6) in clinical samples and/or animal models limits the translatability of the reported findings towards atherosclerosis. Among others, assessing the spatial heterogeneity of KLF6 abundance in atherosclerotic plaques depending on its proximity to arterial bifurcations may be interesting.

Points to be addressed:

(1) As a statistical test, the authors report having used unpaired t-tests; however, often three groups are compared for which t-tests are inadequate. This is faulty as, amongst other things, it does not take multiple comparison testing into account.

(2) Both B-Actin and GAPDH seem to have been used for protein-level normalization. Why? The Figure 2HL first panel reports B-actin, whereas the other three report GAPDH. The same applies to Figures 3E-F, where both are shown, and it is not mentioned which of the two has been used. Moreso, uncropped blots seem to be unavailable as supplementary data for proper review. These should be provided as supplementary data.

(3) LSS and MSS were compared based on transcriptomic analysis. Conversely, RNA sequencing was not reported for the HSS. Why is this data missing? It would be valuable to assess transcriptomics following HSS, and also to allow transcriptomic comparison of LSS and HSS.

(4) Actual sample sizes should be reported rather than "three or more". Moreso, it would be beneficial to show individual datapoints in bar graphs rather than only mean with SD if sample sizes are below 10 (e.g., Figures 1B-H, Figure 2G, etc.).

(5) The authors claim that by modifying the thickness of the middle layer, shear stress could be modified, whilst claiming to keep on-site pressure within physiological ranges (approx. 70 mmHg) as a hallmark of their microfluidic devices. Has it been experimentally verified that pressures indeed remain around 70 mmHg?

(6) A coculture model (VSMC, EC, monocytes) is mentioned in the last part of the results section without any further information. Information on this model should be provided in the methods section (seeding, cell numbers, etc.). Moreover, comparison of LSS vs LSS+KLF6 OE and HSS vs HSS+KLF6 OE is shown. It would benefit the interpretation of the outcomes if MSS were also shown. I twould also be beneficial to demonstrate differences between LSS, MSS, and HSS in this coculture model (without KLF6 OE).

(7) The experiments were solely performed with a venous endothelial cell line (HUVECs). Was the use of an arterial endothelial cell line considered? It may translate better towards atherosclerosis, which occurs within arteries. HUVECs are not accustomed to the claimed near-physiological pressures.

Reviewer #2 (Public review):

Summary

The authors completed a statistically rigorous analysis of the synchronization of sharp-wave ripples in the hippocampal CA1 across and within hemispheres. They used a publicly available dataset (collected in the Buzsaki lab) from 4 rats (8 sessions) recorded with silicon probes in both hemispheres. Each session contained approximately 8 hours of activity recorded during rest. The authors found that the characteristics of ripples did not differ between hemispheres, and that most ripples occurred almost simultaneously on all probe shanks within a hemisphere as well as across hemispheres. The differences in amplitude and exact timing of ripples between recording sites increased slightly with distance between recording sites. However, the phase coupling of ripples (in the 100-250 Hz range), changed dramatically with distance between recording sites. Ripples in opposite hemispheres were about 90% less coupled than ripples on nearby tetrodes in the same hemisphere. Phase coupling also decreased with distance within the hemisphere. Finally, pyramidal cell and interneuron spikes were coupled to the local ripple phase and less so to ripples at distant sites or the opposite hemisphere.

The authors also analyzed the changes in ripple coupling in relation to a couple of behavioral variables. Interestingly, while exposure to a novel track increased ripple abundance by ~5%, it did not change any form of ripple coupling within or between hemispheres.

Strengths

The analysis was well-designed and rigorous. The authors used statistical tests well suited to the hypotheses being tested, and clearly explained these tests. The paper is very clearly written, making it easy to understand and reproduce the analysis. The authors included an excellent review of the literature to explain the motivation for their study.

Weaknesses

The authors have addressed all of my concerns and recommendations.

This paper presents an important and unique analysis of ripple coupling. The same method could be used in the future to analyze the effects of other behavioral variables, such as satiety versus hunger, sleep deprivation, or enrichment, to address potential functions and causes of ripple coupling.

Reviewer #1 (Public review):

Lu & Golomb combined EEG, artificial neural networks, and multivariate pattern analyses to examine how different visual variables are processed in the brain. The conclusions of the paper are mostly well supported.

The authors find that not only real-world size is represented in the brain (which was known), but both retinal size and real-world depth is represented, at different time points or latencies, which may reflect different stages of processing. Prior work has not been able to answer the question of real-world depth due to stimuli used. The authors made this possible by assess real-world depth and testing it with appropriate methodology, accounting for retinal and real-world size. The methodological approach combining behavior, RSA, and ANNs is creative and well thought out to appropriately assess the research questions, and the findings may be very compelling if backed up with some clarifications and further analyses.

The work will be of interest to experimental and computational vision scientists, as well as the broader computational cognitive neuroscience community as the methodology is of interest and the code is or will be made available. The work is important as it is currently not clear what the correspondence between many deep neural network models are and the brain are, and this work pushes our knowledge forward on this front. Furthermore, the availability of methods and data will be useful for the scientific community.

Reviewer #3 (Public review):

The authors used an open EEG dataset of observers viewing real-world objects. Each object had a real-world size value (from human rankings), a retinal size value (measured from each image), and a scene depth value (inferred from the above). The authors combined the EEG and object measurements with extant, pre-trained models (a deep convolutional neural network, a multimodal ANN, and Word2vec) to assess the time course of processing object size (retinal and real-world) and depth. They found that depth was processed first, followed by retinal size, and then real-world size. The depth time course roughly corresponded to the visual ANNs, while the real-world size time course roughly corresponded to the more semantic models.

The time course result for the three object attributes is very clear and a novel contribution to the literature. The authors have revised the ANN motivations to increase clarity. Additionally, the authors have appropriately toned down some of the language about novelty, and the addition of a noise ceiling has helped the robustness of the work.

While I appreciate the addition of Cornet in the Supplement, I am less compelled by the authors' argument for Word2Vec over LLMs for "pure" semantic embeddings. While I'm not digging in on this point, this choice may prematurely age this work.

Reviewer #1 (Public review):

This study presents cryoEM-derived structures of the Trypanosome aquaporin AQP2, in complex with its natural ligand, glycerol, as well as two trypanocidal drugs, pentamidine and melarsoprol, which use AQP2 as an uptake route. The structures are high quality and the density for the drug molecules is convincing, showing a binding site in the centre of the AQP2 pore.

The authors then continue to study this system using molecular dynamics simulations. Their simulations indicate that the drugs can pass through the pore and identify a weak binding site in the centre of the pore, which corresponds with that identified through cryoEM analysis. They also simulate the effect of drug resistance mutations which suggests that the mutations reduce the affinity for drugs and therefore might reduce the likelihood that the drugs enter into the centre of the pore, reducing the likelihood that they progress through into the cell.

While the cryoEM and MD studies are well conducted, it is a shame that the drug transport hypothesis was not tested experimentally. For example, did they do cryoEM with AQP2 with drug resistance mutations and see if they could see the drugs in these maps? They might not bind, but another possibility is that the binding site shifts, as seen in Chen et al? Do they have an assay for measuring drug binding? I think that some experimental validation of the drug binding hypothesis would strengthen this paper. The authors describe in their response why these experiments are challenging.

Reviewer #2 (Public review):

Summary:

The authors present 3.2-3.7 Å cryo-EM structures of Trypanosoma brucei aquaglyceroporin-2 (TbAQP2) bound to glycerol, pentamidine or melarsoprol and combine them with extensive all-atom MD simulations to explain drug recognition and resistance mutations. The work provides a persuasive structural rationale for (i) why positively selected pore substitutions enable diamidine uptake, and (ii) how clinical resistance mutations weaken the high-affinity energy minimum that drives permeation. These insights are valuable for chemotherapeutic re-engineering of diamidines and aquaglyceroporin-mediated drug delivery.

My comments are on the MD part

Strengths:

The study

Integrates complementary cryo-EM, equilibrium and applied voltage MD simulations, and umbrella-sampling PMFs, yielding a coherent molecular-level picture of drug permeation.

Offers direct structural rationalisation of long-standing resistance mutations in trypanosomes, addressing an important medical problem.

Comments on revisions:

Most of the weaknesses have been resolved during the revision process.

Reviewer #3 (Public review):

Summary:

Recent studies have established that trypanocidal drugs, including pentamidine and melarsoprol, enter the trypanosomes via the glyceroaquaporin AQP2 (TbAQP2). Interestingly, drug resistance in trypanosomes is, at least in part, caused by recombination with the neighbouring gene, AQP3, which is unable to permeate pentamidine or melarsoprol. The effect of the drugs on cells expressing chimeric proteins is significantly reduced. In addition, controversy exists regarding whether TbAQP2 permeates the drugs like an ion channel, or whether it serves as a receptor that triggers downstream processes upon drug binding. In this study the authors set out to achieve these objectives: 1) to understand the molecular interactions between TbAQP2 and glycerol, pentamidine, and melarsoprol, and 2) to determine the mechanism by which mutations that arise from recombination with TbAQP3 result in reduced drug permeation.

The cryo-EM structures provide details of glycerol and drug binding, and show that glycerol and the drugs occupy the same space within the pore. Finally, MD simulations and lysis assays are employed to determine how mutations in TbAQP2 result in reduced permeation of drugs by making entry and exit of the drug relatively more energy-expensive. Overall, the strength of evidence used to support the author's claims is solid.

Strengths:

The cryo-EM portion of the study is strong, and while the overall resolution of the structures is in the 3.5Å range, the local resolution within the core of the protein and the drug binding sites is considerably higher (~2.5Å).<br /> I also appreciated the MD simulations on the TbAQP2 mutants and the mechanistic insights that resulted from this data.

Weaknesses:

(1) The authors do not provide any experimental validation the drug binding sites in TbAQP2 due to lacking resources. However, the claims have been softened in the revised paper.

Reviewer #1 (Public review):

Summary:

Roseby and colleagues report on a body region-specific sensory control of the fly larval righting response, a body contortion performed by fly larvae to correct their posture when they find themselves in an inverted (dorsal side down) position. This is an important topic because of the general need for animals to move about in the correct orientation and the clever methodologies used in this paper to uncover the sensory triggers for the behavior. Several innovative methodologies are developed, including a body region-specific optogenetic approach along different axial positions of the larva, region-specific manipulation of surface contacts with the substrate, and a 'water unlocking' technique to initiate righting behaviors, a strength of the manuscript. The authors found that multidendritic neurons, particularly the daIV neurons, are necessary for righting behavior. The contribution of daIV neurons had been shown by the authors in a prior paper (Klann et al, 2021), but that study had used constitutive neuronal silencing. Here, the authors used acute inactivation to confirm this finding. Additionally, the authors describe an important role for anterior sensory neurons and a need for dorsal substrate contact. Conversely, ventral sensory elements inhibit the righting behavior, presumably to ensure that the ventral-side-down position dominates. They move on to test the genetic basis for righting behavior and, consistent with the regional specificity they observe, implicate sensory neuron expression of Hox genes Antennapedia and Abdominal-b in self-righting.

Strengths:

Strengths of this paper include the important question addressed and the elegant and innovative combination of methods, which led to clear insights into the sensory biology of self-righting, and that will be useful for others in the field. This is a substantial contribution to understanding how animals correct their body position. The manuscript is very clearly written and couched in interesting biology.

Limitations:

(1) The interpretation of functional experiments is complicated by the proposed excitatory and inhibitory roles of dorsal and ventral sensory neuron activity, respectively. So, while silencing of an excitatory (dorsal) element might slow righting, silencing of inputs that inhibit righting could speed the behavior. Silencing them together, as is done here, could nullify or mask important D-V-specific roles. Selective manipulation of cells along the D-V axis could help address this caveat.

(2) Prior studies from the authors implicated daIV neurons in the righting response. One of the main advances of the current manuscript is the clever demonstration of region-specific roles of sensory input. However, this is only confirmed with a general md driver, 190(2)80, and not with the subset-specific Gal4, so it is not clear if daIV sensory neurons are also acting in a regionally specific manner along the A-P axis.

(3) The manuscript is narrowly focused on sensory neurons that initiate righting, which limits the advance given the known roles for daIV neurons in righting. With the suite of innovative new tools, there is a missed opportunity to gain a more general understanding of how sensory neurons contribute to the righting response, including promoting and inhibiting righting in different regions of the larva, as well as aspects of proprioceptive sensing that could be necessary for righting and account for some of the observed effects of 109(2)80.

(4) Although the authors observe an influence of Hox genes in righting, the possible mechanisms are not pursued, resulting in an unsatisfying conclusion that these genes are somehow involved in a certain region-specific behavior by their region-specific expression. Are the cells properly maintained upon knockdown? Are axon or dendrite morphologies of the cells disrupted upon knockdown?

(5) There could be many reasons for delays in righting behavior in the various manipulations, including ineffective sensory 'triggering', incoherent muscle contraction patterns, initiation of inappropriate behaviors that interfere with righting sequencing, and deficits in sensing body position. The authors show that delays in righting upon silencing of 109(2)80 are caused by a switch to head casting behavior. Is this also the case for silencing of daIV neurons, Hox RNAi experiments, and silencing of CO neurons? Does daIII silencing reduce head casting to lead to faster righting responses?

(6) 109(2)80 is expressed in a number of central neurons, so at least some of the righting phenotype with this line could be due to silenced neurons in the CNS. This should at least be acknowledged in the manuscript and controlled for, if possible, with other Gal4 lines.

Other points

(7) Interpretation of roles of Hox gene expression and function in righting response should consider previous data on Hox expression and function in multidendritic neurons reported by Parrish et al. Genes and Development, 2007.

(8) The daIII silencing phenotype could conceivably be explained if these neurons act as the ventral inhibitors. Do the authors have evidence for or against such roles?

Reviewer #2 (Public review):

Summary

This work explores the relationship between body structure and behavior by studying self-righting in Drosophila larvae, a conserved behavior that restores proper orientation when turned upside-down. The authors first introduce a novel "water unlocking" approach to induce self-righting behavior in a controlled manner. Then, they develop a method for region-specific inhibition of sensory neurons, revealing that anterior, but not posterior, sensory neurons are essential for proper self-righting. Deep-learning-based behavioral analysis shows that anterior inhibition prolongs self-righting by shifting head movement patterns, indicating a behavioral switch rather than a mere delay. Additional genetic and molecular experiments demonstrate that specific Hox genes are necessary in sensory neurons, underscoring how developmental patterning genes shape region-specific sensory mechanisms that enable adaptive motor behaviors.

Strengths

The work of Roseby et al. does what it says on the tin. The experimental design is elegant, introducing innovative methods that will likely benefit the fly behavior community, and the results are robustly supported, without overstatement.

Weaknesses:

The manuscript is clearly written, flows smoothly, and features well-designed experiments. Nevertheless, there are areas that could be improved. Below is a list of suggestions and questions that, if addressed, would strengthen this work:

(1) Figure 1A illustrates the sequence of self-righting behavior in a first instar larva, while the experiments in the same figure are performed on third instar larvae. It would be helpful to clarify whether the sequence of self-righting movements differs between larval stages. Later on in the manuscript, experiments are conducted on first instar larvae without explanation for the choice of stage. Providing the rationale for using different larval stages would improve clarity.

(2) What was the genotype of the larvae used for the initial behavioral characterization (Figure 1)? It is assumed they were wild type or w1118, but this should be stated explicitly. This also raises the question of whether different wild-type strains exhibit this behavior consistently or if there is variability among them. Has this been tested?

(3) Could the observed slight leftward bias in movement angles of the tail (Figure 1I and S1) be related to the experimental setup, for example, the way water is added during the unlocking procedure? It would be helpful to include some speculation on whether the authors believe this preference to be endogenous or potentially a technical artifact.

(4) The genotype of the larvae used for Figure 2 experiments is missing.

(5) The experiment shown in Figure 2E-G reports the proportion of larvae exhibiting self-righting behavior. Is the self-righting speed comparable to that measured using the setup in Figure 1?

(6) Line 496 states: "However, the effect size was smaller than that for the entire multidendritic population, suggesting neurons other than the daIVs are important for self-righting". Although I agree that this is the more parsimonious hypothesis, an alternative interpretation of the observed phenomenon could be that the effect is not due to the involvement of other neuronal populations, but rather to stronger Gal4 expression in daIVs with the general driver compared to the specific one. Have the authors (or someone else) measured or compared the relative strengths of these two drivers?

(7) Is there a way to quantify or semi-quantify the expression of the Hox genes shown in Figure 6A? Also, was this experiment performed more than once (are there any technical replicates?), or was the amount of RNA material insufficient to allow replication?

(8) Since RNAi constructs can sometimes produce off-target effects, it is generally advisable to use more than one RNAi line per gene, targeting different regions. Given that Hox genes have been extensively studied, the RNAis used in Figure 6B are likely already characterized. If this were the case, it would strengthen the data to mention it explicitly and provide references documenting the specificity and knockdown efficiency of the Hox gene RNAis employed. For example, does Antp RNAi expression in the 109(2)80 domain decrease Antp protein levels in multidendritic anterior neurons in immunofluorescence assays?

(9) In addition to increasing self-righting time, does Antp downregulation also affect head casting behavior or head movement speed? A more detailed behavioral characterization of this genetic manipulation could help clarify how closely it relates to the behavioral phenotypes described in the previous experiments.

(10) Does down-regulation of Antp in the daIV domain also increase self-righting time?

Reviewer #1 (Public review):

The importance of RNA editing in producing protein diversity is a widespread process that can regulate how genes function in various cellular contexts. Despite the importance of the process, we still lack a thorough knowledge of the profile of RNA editing targets in known cells. Crane and colleagues take advantage of a recently acquired scRNAseq database for Drosophila type Ib and Is larval motoneurons and identify the RNA editing landscape that differs in those cells. They find both canonical (A --> I) and non-canonical sites and characterize the targets, their frequencies, and determine some of the "rules" that influence RNA editing. They compare their database with existing databases to determine a reliance on the most well-known deaminase enzyme ADAR, determine the activity-dependence of editing profiles, and identify editing sites that are specific to larval Drosophila, differing from adults. The authors also identify non-canonical editing sites, especially in the newly appreciated and identified regulator of synaptic plasticity, Arc1.

The paper represents a strong analysis of recently made RNAseq databases from their lab and takes a notable approach to integrate this with other databases that have been recently produced from other sources. One of the places where this manuscript succeeds is in a thorough approach to analyzing the considerable amount of data that is out there regarding RNAseq in these differing motoneurons, but also in comparing larvae to adults. This is a strong advance. It also enables the authors to begin to determine rules for RNA editing. From an analytical standpoint, this paper is a notable advance in seeking to provide a biological context for massive amounts of data in the field. Further, it addresses some biological aspects in comparing WT and adar mutants to assess one potential deaminase, addresses activity-dependence, and begins to reveal profiles of canonical and non-canonical editing.

Reviewer #2 (Public review):

Summary:

The study uses single-neuron Patch-seq RNA sequencing in two subgroups of Drosophila larval motoneurons (1s and 1b) and identifies 316 high-confidence canonical mRNA edit sites, which primarily (55%) occur in the coding regions of the mRNAs (CDS). Most of the canonical mRNA edits in the CDS regions include neuronal and synaptic proteins such as Complexin, Cac, Para, Shab, Sh, Slo, EndoA, Syx1A, Rim, RBP, Vap33, and Lap, which are involved in neuronal excitability and synaptic transmission. Of the 316 identified canonical edit sites, 60 lead to missense RNAs in a range of proteins (nAChRalpha5, nAChRalpha6, nAChRbeta1, ATPalpha, Cacophony, Para, Bsk, Beag, RNase Z) that are likely to have an impact on the larval motoneurons' development and function. Only 27 sites show editing levels higher than 90% and a similar editing profile is observed between the 1s and 1b motoneurons when looking at the number of edit sites and the fraction of reads edited per cell, with only 26 RNA editing sites showing a significant difference in the editing level. The variability of edited and unedited mRNAs suggests stochastic editing. The two subsets of motoneurons show many noncanonical editing sites, which, however, are not enriched for neuron-specific genes, therefore causing more silent changes compared to canonical editing sites. Comparison of the mRNA editing sites and editing rate of the single neuron Patch-seq RNA sequencing dataset to three other RNAseq datasets, one from same stage larval motoneurons and two from adult heads nuclei, show positive correlations in editing frequencies of CDS edits between the patch-sec larval 1b + 1s MNs and all other three datasets, with stronger correlations for previously annotated edits and weaker correlations for unannotated edits. Several of the identified editing targets are only present in the single neuron Patch-seq RNA sequencing dataset, suggesting cell-type-specific or developmental-specific editing. Editing appears to be resistant to changes in neuronal activity as only a few sites show evidence of being activity-regulated.

Strengths:

The study employs GAL4 driver lines available in the Drosophila model to identify two subtypes of motoneurons with distinct biophysical and morphological features. In combination with single-neuron Patch-seq RNA sequencing, it provides a unique opportunity to identify RNA editing sites and rates specific to specific motoneuron subtypes. The RNA seq data is robustly analysed, and high-confidence mRNA edit sites of both canonical and noncanonical RNA editing are identified.

The mRNA editing sites identified from the single neuron Patch-seq RNA sequencing data are compared to editing sites identified across other RNAseq datasets collected from animals at similar or different developmental stages, allowing for the identification of editing sites that are common to all or specific to a single dataset.

Weaknesses:

Although the analysed motoneurons come from two distinct subtypes, it is unclear from how many Drosophila larvae the motoneurons were collected and from which specific regions along the ventral nerve cord (VNC). Therefore, the study does not consider possible differences in editing rate between samples from different larvae that could be in different active states or neurons located at different regions of the VNC, which would receive inputs from slightly different neuronal networks.

The RNA samples include RNAs located both in the nucleus and the cytoplasm, introducing a potential compartmental mismatch between the RNA and the enzymes mediating the editing, which could influence editing rate. Similarly, the age of the RNAs undergoing editing is unknown, which may influence the measured editing rates.

Reviewer #3 (Public review):

Summary:

The study consists of extensive computational analyses of their previously released Patch-seq data on single MN1-Ib and MNISN-Is neurons. The authors demonstrate the diversity of A>I editing events at single-cell resolution in two different neuronal cell types, identifying numerous A>I editing events that vary in their proportion, including those that cause missense mutations in conserved amino acids. They also consider "noncanonical" edits, such as C>T and G>A, and integrate publicly available data to support these analyses.

In general, the study contains a valuable resource to assess RNA editing in single neurons and opens several questions regarding the diversity and functional implications of RNA editing at single-cell resolution. The conclusions from the study are generally supported by their data; however, the study is currently based on computational predictions and would therefore benefit from experimentation to support their hypotheses and demonstrate the effects of the editing events identified on neuronal function and phenotype.

Strengths:

The study uses samples that are technically difficult to prepare to assess cell-type-specific RNA editing events in a natural model. The study also uses public data from different developmental stages that demonstrate the importance of considering cell type and developmental stage-specific RNA regulation. These critical factors, particularly that of developmental timing, are often overlooked in mechanistic studies.

Extensive computational analysis, using public pipelines, suitable filtering criteria, and accessible custom code, identifies a number of RNA editing events that have the potential to impact conserved amino acids and have subsequent effects on protein function. These observations are supported through the integration of several public data sets to investigate the occurrence of the edits in other data sets, with many identified across multiple data sets. This approach allowed the identification of a number of novel A>I edits, some of which appear to be specific to this study, suggesting cell/developmental specificity, whilst others are present in the public data sets but went unannotated.

The study also considers the role of Adar in the generation of A>I edits, as would be expected, by assessing the effect of Adar expression on editing rates using public data from adar mutant tissue to demonstrate that the edits conserved between experiments are mainly Adar-sensitive. This would be stronger if the authors also performed Patch-seq experiments in adar mutants to increase confidence in the identified edit sites.

Weaknesses:

Whilst the study makes interesting observations using advanced computational approaches, it does not demonstrate the functional implications of the observed editing events. The functional impact of the edits is inferred from either the nature of the change to the coding sequence and the amino acid conservation, or through integration of other data sets. Although these could indeed imply function, further experimentation would be required to confirm such as using their Alphafold models to predict any changes in structure. This limitation is acknowledged by the authors, but the overall strength of the interpretation of the analysis could be softened to represent this.

The study uses public data from more diverse cellular populations to confirm the role of Adar in introducing the A>I edits. Whilst this is convincing, the ideal comparison to support the mechanism behind the identified edits would be to perform patch-seq experiments on 1b or 1s neurons from adar mutants. However, although this should be considered when interpreting the data, these experiments would be a large amount of work and beyond the scope of the paper.

By focusing on the potential impact of editing events that cause missense mutations in the CDS, the study may overlook the importance of edits in noncoding regions, which may impact miRNA or RNA-binding protein target sites. Further, the statement that noncanonical edits and those that induce silent mutations are likely to be less impactful is very broad and should be reconsidered. This is particularly the case when suggesting that silent mutations may not impact the biology. Given the importance of codon usage in translational fidelity, it is possible that silent mutations induced by either A>I or noncanonical editing in the CDS impact translation efficiency. Indeed, this could have a greater impact on protein production and transcript levels than a single amino acid change alone.

Reviewer #1 (Public review):

In this manuscript, Hoon Cho et al. present a novel investigation into the role of PexRAP, an intermediary in ether lipid biosynthesis, in B cell function, particularly during the Germinal Center (GC) reaction. The authors profile lipid composition in activated B cells both in vitro and in vivo, revealing the significance of PexRAP. Using a combination of animal models and imaging mass spectrometry, they demonstrate that PexRAP is specifically required in B cells. They further establish that its activity is critical upon antigen encounter, shaping B cell survival during the GC reaction.

Mechanistically, they show that ether lipid synthesis is necessary to modulate reactive oxygen species (ROS) levels and prevent membrane peroxidation.

Highlights of the Manuscript:

The authors perform exhaustive imaging mass spectrometry (IMS) analyses of B cells, including GC B cells, to explore ether lipid metabolism during the humoral response. This approach is particularly noteworthy given the challenge of limited cell availability in GC reactions, which often hampers metabolomic studies. IMS proves to be a valuable tool in overcoming this limitation, allowing detailed exploration of GC metabolism.

The data presented is highly relevant, especially in light of recent studies suggesting a pivotal role for lipid metabolism in GC B cells. While these studies primarily focus on mitochondrial function, this manuscript uniquely investigates peroxisomes, which are linked to mitochondria and contribute to fatty acid oxidation (FAO). By extending the study of lipid metabolism beyond mitochondria to include peroxisomes, the authors add a critical dimension to our understanding of B cell biology.

Additionally, the metabolic plasticity of B cells poses challenges for studying metabolism, as genetic deletions from the beginning of B cell development often result in compensatory adaptations. To address this, the authors employ an acute loss-of-function approach using two conditional, cell-type-specific gene inactivation mouse models: one targeting B cells after the establishment of a pre-immune B cell population (Dhrs7b^f/f, huCD20-CreERT2) and the other during the GC reaction (Dhrs7b^f/f; S1pr2-CreERT2). This strategy is elegant and well-suited to studying the role of metabolism in B cell activation.

Overall, this manuscript is a significant contribution to the field, providing robust evidence for the fundamental role of lipid metabolism during the GC reaction and unveiling a novel function for peroxisomes in B cells.

Comments on revisions:

There are still some discrepancies in gating strategies. In Fig. 7B legend (lines 1082-1083), they show representative flow plots of GL7+ CD95+ GC B cells among viable B cells, so it is not clear if they are IgDneg, as the rest of the GC B cells aforementioned in the text.

Western blot confirmation: We understand the limitations the authors enumerate. Perhaps an RT-qPCR analysis of the Dhrs7b gene in sorted GC B cells from the S1PR2-CreERT2 model could be feasible, as it requires a smaller number of cells. In any case, we agree with the authors that the results obtained using the huCD20-CreERT2 model are consistent with those from the S1PR2-CreERT2 model, which adds credibility to the findings and supports the conclusion that GC B cells in the S1PR2-CreERT2 model are indeed deficient in PexRAP

Lines 222-226: We believe the correct figure is 4B, whereas the text refers to 4C.

Supplementary Figure 1 (line 1147): The figure title suggests that the data on T-cell numbers are from mice in a steady state. However, the legend indicates that the mice were immunized, which means the data are not from steady-state conditions.

Reviewer #2 (Public review):

Summary:

In this study, Cho et al. investigate the role of ether lipid biosynthesis in B cell biology, particularly focusing on GC B cell, by inducible deletion of PexRAP, an enzyme responsible for the synthesis of ether lipids.

Strengths:

Overall, the data are well-presented, the paper is well-written and provides valuable mechanistic insights into the importance of PexRAP enzyme in GC B cell proliferation.

Weaknesses:

More detailed mechanisms of the impaired GC B cell proliferation by PexRAP deficiency remain to be further investigated. In minor part, there are issues for the interpretation of the data which might cause confusions by readers.

Comments on revisions:

The authors improved the manuscript appropriately according to my comments.

Reviewer #1 (Public review):

Summary:

The authors use the theory of planned behavior to understand whether or not intentions to use sex as a biological variable (SABV), as well as attitude (value), subjective norm (social pressure), and behavioral control (ability to conduct behavior), across scientists at a pharmacological conference. They also used an intervention (workshop) to determine the value of this workshop in changing perceptions and misconceptions. Attempts to understand the knowledge gaps were made.

Strengths:

The use of SABV is limited in terms of researchers using sex in the analysis as a variable of interest in the models (and not a variable to control). To understand how we can improve on the number of researchers examining the data with sex in the analyses, it is vital we understand the pressure points that researchers consider in their work. The authors identify likely culprits in their analyses. The authors also test an intervention (workshop) to address the main bias or impediments for researchers' use of sex in their analyses.

Reviewer #2 (Public review):

Summary:

The investigators tested a workshop intervention to improve knowledge and decrease misconceptions about sex inclusive research.

Strengths:

The investigators included control groups and replicated the study in a second population of scientists. The results appear to be well substantiated. Figures are easy to understand.

Weaknesses: None noted

Comments on revised version:

The authors have responded appropriately to all of my concerns.

Reviewer #3 (Public review):

Summary:

This manuscript aims to determine cultural biases and misconceptions in inclusive sex research and evaluate the efficacy of interventions to improve knowledge and shift perceptions to decrease perceived barriers for including both sexes in basic research.

Overall, this study demonstrates that despite the intention to include both sexes and a general belief in the importance of doing so, relatively few people routinely include both sexes. Further, the perceptions of barriers to doing so are high, including misconceptions surrounding sample size, disaggregation, and variability of females. There was also a substantial number of individuals without the statistical knowledge to appropriately analyze data in studies inclusive of sex. Interventions increased knowledge and decreased perception of barriers.

Strengths:

(1) This manuscript provides evidence for the efficacy of interventions for changing attitudes and perceptions of research.

(2) This manuscript also provides a training manual for expanding this intervention to broader groups of researchers.

Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal significant structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are well-designed and executed.

Comments on revised version:

My concerns have been addressed.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of ANKRD5 (ANKEF1) as a component of the N-DRC complex in sperm motility and male fertility. Using Ankrd5 knockout mice, the study demonstrates that ANKRD5 is essential for sperm motility and identifies its interaction with N-DRC components through IP-mass spectrometry and cryo-ET. The results provide insights into ANKRD5's function, highlighting its potential involvement in axoneme stability and sperm energy metabolism.

Strengths:

The authors employ a wide range of techniques, including gene knockout models, proteomics, cryo-ET, and immunoprecipitation, to explore ANKRD5's role in sperm biology.

Comments on revised version:

The authors have already addressed the issues I am concerned about.

Reviewer #3 (Public review):

Summary:

By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wildtype protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway.

Strengths:

The high resolution (2.1 Å) of the current structure is impressive, and allows many new densities in the potassium transport pathway to be resolved. The authors are judicious about assigning these as potassium ions or water molecules, and explain their structural interpretations clearly. In addition to the nice structural work, the mechanistic work is thorough. A series of thoughtful experiments involving ATP hydrolysis/transport coupling under various pH and potassium concentrations bolsters the structural interpretations and lends convincing support to the mechanistic proposal. The SSME experiments are rigorous.

Reviewer #1 (Public review):

The study presents significant findings on the role of mitochondrial depletion in axons and its impact on neuronal proteostasis. It effectively demonstrates how the loss of axonal mitochondria and elevated levels of eIF2β contribute to autophagy collapse and neuronal dysfunction. The use of Drosophila as a model organism and comprehensive proteome analysis adds robustness to the findings.

In this revision, the authors have responded thoughtfully to previous concerns. In particular, they have addressed the need for a quantitative analysis of age-dependent changes in eIF2β and eIF2α. By adding western blot data from multiple time points (7 to 63 days), they show that eIF2β levels gradually increase until middle age, then decline. In milton knockdown flies, this pattern appears shifted, supporting the idea that mitochondrial defects may accelerate aging-related molecular changes. These additions clarify the temporal dynamics of eIF2β and improve the overall interpretation.

Other updates include appropriate corrections to figures and quantification methods. The authors have also revised some of their earlier mechanistic claims, presenting a more cautious interpretation of their findings.

Overall, this work provides new insights into how mitochondrial transport defects may influence aging-related proteostasis through eIF2β. The manuscript is now more convincing, and the revisions address the main points raised earlier. I find the updated version much improved.

Reviewer #2 (Public review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria, which they suggest accelerates age-dependent changes rather than increasing their magnitude.

Strong caution is necessary regarding the interpretation of translational regulation resulting from the milton KD. The effect of milton KD on translation appears subtle, if present at all, in the puromycin incorporation experiments in both the initial and revised versions. Additionally, the polysome profiling data in the revised manuscript lack the clear resolution for ribosomal subunits, monosomes, and polysomes that is typically expected in publications.

Reviewer #1 (Public review):

The aim of this study was a better understanding of the reproductive life history of acoels. The acoel Hofstenia miamia, an emerging model organism, is investigated; the authors nevertheless acknowledge and address the high variability in reproductive morphology and strategies within Acoela.

The morphology of male and female reproductive organs in these hermaphroditic worms is characterised through stereo microscopy, immunohistochemistry, histology, and fluorescent in situ hybridization. The findings confirm and better detail historical descriptions. A novelty in the field is the in situ hybridization experiments, which link already published single-cell sequencing data to the worms' morphology. An interesting finding, though not further discussed by the authors, is that the known germline markers cgnl1-2 and Piwi-1 are only localized in the ovaries and not in the testes.

The work also clarifies the timing and order of appearance of reproductive organs during development and regeneration, as well as the changes upon de-growth. It shows an association of reproductive organ growth to whole body size, which will be surely taken into account and further explored in future acoel studies. This is also the first instance of non-anecdotal degrowth upon starvation in H. miamia (and to my knowledge in acoels, except recorded weight upon starvation in Convolutriloba retrogemma [1]).

Egg laying through the mouth is described in H. miamia for the first time as well as the worms' behavior in egg laying, i.e. choosing the tanks' walls rather than its floor, laying eggs in clutches, and delaying egg-laying during food deprivation. Self-fertilization is also reported for the first time.

The main strength of this study is that it expands previous knowledge on the reproductive life history traits in H. miamia and it lays the foundation for future studies on how these traits are affected by various factors, as well as for comparative studies within acoels. As highlighted above, many phenomena are addressed in a rigorous and/or quantitative way for the first time. This can be considered the start of a novel approach to reproductive studies in acoels, as the authors suggest in the conclusion. It can be also interpreted as a testimony of how an established model system can benefit the study of an understudied animal group.

The main weakness of the work is the lack of convincing explanations on the dynamics of self-fertilization, sperm storage, and movement of oocytes from the ovaries to the central cavity and subsequently to the pharynx. These questions are also raised by the authors themselves in the discussion. Another weakness (or rather missing potential strength) is the limited focus on genes. Given the presence of the single-cell sequencing atlas and established methods for in situ hybridization and even transgenesis in H. miamia, this model provides a unique opportunity to investigate germline genes in acoels and their role in development, regeneration, and degrowth. It should also be noted that employing Transmission Electron Microscopy would have enabled a more detailed comparison with other acoels, since ultrastructural studies of reproductive organs have been published for other species (cfr e.g. [2],[3],[4]). This is especially true for a better understanding of the relation between sperm axoneme and flagellum (mentioned in the Results section), as well as of sexual conflict (mentioned in the Discussion).

(1) Shannon, Thomas. 2007. 'Photosmoregulation: Evidence of Host Behavioral Photoregulation of an Algal Endosymbiont by the Acoel Convolutriloba Retrogemma as a Means of Non-Metabolic Osmoregulation'. Athens, Georgia: University of Georgia [Dissertation].

(2) Zabotin, Ya. I., and A. I. Golubev. 2014. 'Ultrastructure of Oocytes and Female Copulatory Organs of Acoela'. Biology Bulletin 41 (9): 722-35.

(3) Achatz, Johannes Georg, Matthew Hooge, Andreas Wallberg, Ulf Jondelius, and Seth Tyler. 2010. 'Systematic Revision of Acoels with 9+0 Sperm Ultrastructure (Convolutida) and the Influence of Sexual Conflict on Morphology'.

(4) Petrov, Anatoly, Matthew Hooge, and Seth Tyler. 2006. 'Comparative Morphology of the Bursal Nozzles in Acoels (Acoela, Acoelomorpha)'. Journal of Morphology 267 (5): 634-48.

Reviewer #2 (Public review):

Summary:

While the phylogenetic position of Acoels (and Xenacoelomorpha) remains still debated, investigations of various representative species are critical to understanding their overall biology.

Hofstenia is an Acoels species that can be maintained in laboratory conditions and for which several critical techniques are available. The current manuscript provides a comprehensive and widely descriptive investigation of the productive system of Hofstenia miamia.

Strengths:

(1) Xenacoelomorpha is a wide group of animals comprising three major clades and several hundred species, yet they are widely understudied. A comprehensive state-of-the-art analysis on the reprodutive system of Hofstenia as representative is thus highly relevant.

(2) The investigations are overall very thorough, well documented, and nicely visualised in an array of figures. In some way, I particularly enjoyed seeing data displayed in a visually appealing quantitative or semi-quantitative fashion.

(3) The data provided is diverse and rich. For instance, the behavioral investigations open up new avenues for further in-depth projects.

Weaknesses:

While the analyses are extensive, they appear in some way a little uni-dimensional. For instance the two markers used were characterized in a recent scRNAseq data-set of the Srivastava lab. One might have expected slightly deeper molecular analyses. Along the same line, particularly the modes of spermatogenesis or oogenesis have not been further analysed, nor the proposed mode of sperm-storage.

[Editors' note: In their response, the authors have suitably addressed these concerns or have satisfactorily explained the challenges in addressing them.]

Reviewer #1 (Public review):

This study investigates the contribution of renal dysfunction to systemic and neuronal decline in Drosophila models of Gaucher disease (Gba1b mutants) and Parkinson's disease (Parkin mutants). While lysosomal and mitochondrial pathways are known drivers in these disorders, the role of kidney-like tissues in disease progression has not been well explored.

The authors use Drosophila melanogaster to model renal dysfunction, focusing on Malpighian tubules (analogous to renal tubules) and nephrocytes (analogous to podocytes). They employ genetic mutants, tissue-specific rescues, imaging of renal architecture, redox probes, functional assays, nephrocyte dextran uptake, and lifespan analyses. They also test genetic antioxidant interventions and pharmacological treatment.

The main findings show that renal pathology is progressive in Gba1b mutants, marked by Malpighian tubule disorganization, stellate cell loss, lipid accumulation, impaired water and ion regulation, and reduced nephrocyte filtration. A central theme is redox dyshomeostasis, reflected in whole-fly GSH reduction, paradoxical mitochondrial versus cytosolic redox shifts, reduced ROS signals, increased lipid peroxidation, and peroxisomal impairment. Antioxidant manipulations (Nrf2, Sod1/2, CatA, and ascorbic acid) consistently worsen outcomes, suggesting a fragile redox balance rather than classical oxidative stress. Parkin mutants also develop renal degeneration, with impaired mitophagy and complete nephrocyte dysfunction by 28 days, but their mechanism diverges from that of Gba1b. Rapamycin treatment rescues several renal phenotypes in Gba1b but not in Parkin, highlighting distinct disease pathways.

The authors propose that renal dysfunction is a central disease-modifying feature of Gaucher and Parkinson's disease models, driven by redox imbalance and differential engagement of lysosomal (Gba1b) vs. mitochondrial (Parkin) mechanisms. They suggest that maintaining renal health and redox balance may represent therapeutic opportunities and biomarkers in neurodegenerative disease. This is a significant manuscript that reframes GD/PD pathology through the lens of renal health. The data are extensive. However, several claims are ahead of the evidence and should be supported with additional experiments.

Major Comments:

(1) The abstract frames progressive renal dysfunction as a "central, disease-modifying feature" in both Gba1b and Parkin models, with systemic consequences including water retention, ionic hypersensitivity, and worsened neuro phenotypes. While the data demonstrates renal degeneration and associated physiological stress, the causal contribution of renal defects versus broader organismal frailty is not fully disentangled. Please consider adding causal experiments (e.g., temporally restricted renal rescue/knockdown) to directly establish kidney-specific contributions.

(2) The manuscript shows multiple redox abnormalities in Gba1b mutants (reduced whole fly GSH, paradoxical mitochondrial reduction with cytosolic oxidation, decreased DHE, increased lipid peroxidation, and reduced peroxisome density/Sod1 mislocalization). These findings support a state of redox imbalance, but the driving mechanism remains broad in the current form. It is unclear if the dominant driver is impaired glutathione handling or peroxisomal antioxidant/β-oxidation deficits or lipid peroxidation-driven toxicity, or reduced metabolic flux/ETC activity. I suggest adding targeted readouts to narrow the mechanism.

(3) The observation that broad antioxidant manipulations (Nrf2 overexpression in tubules, Sod1/Sod2/CatA overexpression, and ascorbic acid supplementation) consistently shorten lifespan or exacerbate phenotypes in Gba1b mutants is striking and supports the idea of redox fragility. However, these interventions are broad. Nrf2 influences proteostasis and metabolism beyond redox regulation, and Sod1/Sod2/CatA may affect multiple cellular compartments. In the absence of dose-response testing or controls for potential off-target effects, the interpretation that these outcomes specifically reflect redox dyshomeostasis feels ahead of the data. I suggest incorporating narrower interpretations (e.g., targeting lipid peroxidation directly) to clarify which redox axis is driving the vulnerability.

(4) This manuscript concludes that nephrocyte dysfunction does not exacerbate brain pathology. This inference currently rests on a limited set of readouts: dextran uptake and hemolymph protein as renal markers, lifespan as a systemic measure, and two brain endpoints (LysoTracker staining and FK2 polyubiquitin accumulation). While these data suggest that nephrocyte loss alone does not amplify lysosomal or ubiquitin stress, they may not fully capture neuronal function and vulnerability. To strengthen this conclusion, the authors could consider adding functional or behavioral assays (e.g., locomotor performance)

(5) The manuscript does a strong job of contrasting Parkin and Gba1b mutants, showing impaired mitophagy in Malpighian tubules, complete nephrocyte dysfunction by day 28, FRUMS clearance defects, and partial rescue with tubule-specific Parkin re-expression. These findings clearly separate mitochondrial quality control defects from the lysosomal axis of Gba1b. However, the mechanistic contrast remains incomplete. Many of the redox and peroxisomal assays are only presented for Gba1b. Including matched readouts across both models (e.g., lipid peroxidation, peroxisome density/function, Grx1-roGFP2 compartmental redox status) would make the comparison more balanced and strengthen the conclusion that these represent distinct pathogenic routes.

(6) Rapamycin treatment is shown to rescue several renal phenotypes in Gba1b mutants (water retention, RSC proliferation, FRUMS clearance, lipid peroxidation) but not in Parkin, and mitophagy is not restored in Gba1b. This provides strong evidence that the two models engage distinct pathogenic pathways. However, the therapeutic interpretation feels somewhat overstated. Human relevance should be framed more cautiously, and the conclusions would be stronger with mechanistic markers of autophagy (e.g., Atg8a, Ref(2)p flux in Malpighian tubules) or with experiments varying dose, timing, and duration (short-course vs chronic rapamycin).

(7) Several systemic readouts used to support renal dysfunction (FRUMS clearance, salt stress survival) could also be influenced by general organismal frailty. To ensure these phenotypes are kidney-intrinsic, it would be helpful to include controls such as tissue-specific genetic rescue in Malpighian tubules or nephrocytes, or timing rescue interventions before overt systemic decline. This would strengthen the causal link between renal impairment and the observed systemic phenotypes.

Reviewer #2 (Public review):

Summary:

In the present study, the authors tested renal function in Gba1b-/- flies and its possible effect on neurodegeneration. They showed that these flies exhibit progressive degeneration of the renal system, loss of water homeostasis, and ionic hypersensitivity. They documented reduced glomerular filtration capacity in their pericardial nephrocytes, together with cellular degeneration in microtubules, redox imbalance, and lipid accumulation. They also compared the Gba1b mutant flies to Parkin mutants and evaluated the effect of treatment with the mTOR inhibitor rapamycin. Restoration of renal structure and function was observed only in the Gba1b mutant flies, leading the authors to conclude that the mutants present different phenotypes due to lysosomal stress in Gba1b mutants versus mitochondrial stress in Parkin mutant flies.

Comments:

(1) The authors claim that: "renal system dysfunction negatively impacts both organismal and neuronal health in Gba1b-/- flies, including autophagic-lysosomal status in the brain." This statement implies that renal impairments drive neurodegeneration. However, there is no direct evidence provided linking renal defects to neurodegeneration in this model. It is worth noting that Gba1b-/- flies are a model for neuronopathic Gaucher disease (GD): they accumulate lipids in their brains and present with neurodegeneration and decreased survival, as shown by Kinghorn et al. (The Journal of Neuroscience, 2016, 36, 11654-11670) and by others, which the authors failed to mention (Davis et al., PLoS Genet. 2016, 12: e1005944; Cabasso et al., J Clin Med. 2019, 8:1420; Kawasaki et al., Gene, 2017, 614:49-55).

(2) The authors tested brain pathology in two experiments:

(a) To determine the consequences of abnormal nephrocyte function on brain health, they measured lysosomal area in the brain of Gba1b-/-, Klf15LOF, or stained for polyubiquitin. Klf15 is expressed in nephrocytes and is required for their differentiation. There was no additive effect on the increased lysosomal volume (Figure 3D) or polyubiquitin accumulation (Figure 3E) seen in Gba1b-/- fly brains, implying that loss of nephrocyte viability itself does not exacerbate brain pathology.

(b) The authors tested the consequences of overexpression of the antioxidant regulator Nrf2 in principal cells of the kidney on neuronal health in Gba1b-/- flies, using the c42-GAL4 driver. They claim that "This intervention led to a significant increase in lysosomal puncta number, as assessed by LysoTrackerTM staining (Figure 5D), and exacerbated protein dyshomeostasis, as indicated by polyubiquitin accumulation and increased levels of the ubiquitin-autophagosome trafficker Ref(2)p/p62 in Gba1b-/- fly brains (Figure 5E). Interestingly, Nrf2 overexpression had no significant effect on lysosomal area or ubiquitin puncta in control brains, demonstrating that the antioxidant response specifically in Gba1b-/- flies negatively impacts disease states in the brain and renal system."<br /> Notably, c42-GAL4 is a leaky driver, expressed in salivary glands, Malpighian tubules, and pericardial cells (Beyenbach et al., Am. J. Cell Physiol. 318: C1107-C1122, 2020). Expression in pericardial cells may affect heart function, which could explain deterioration in brain function.

Taken together, the contribution of renal dysfunction to brain health remains debatable.

Based on the above, I believe the title should be changed to: Redox Dyshomeostasis Links Renal and Neuronal Dysfunction in Drosophila Models of Gaucher disease. Such a title will reflect the results presented in the manuscript.

(3) The authors mention that Gba1b is not expressed in the renal system, which means that no renal phenotype can be attributed directly to any known GD pathology. They suggest that systemic factors such as circulating glycosphingolipids or loss of extracellular vesicle-mediated delivery of GCase may mediate renal toxicity. This raises a question about the validity of this model to test pathology in the fly kidney. According to Flybase, there is expression of Gba1b in renal structures of the fly.

(4) It is worth mentioning that renal defects are not commonly observed in patients with Gaucher disease. Relevant literature: Becker-Cohen et al., A Comprehensive Assessment of Renal Function in Patients With Gaucher Disease, J. Kidney Diseases, 2005, 46:837-844.

(5) In the discussion, the authors state: "Together, these findings establish renal degeneration as a driver of systemic decline in Drosophila models of GD and PD..." and go on to discuss a brain-kidney axis in PD. However, since this study investigates a GD model rather than a PD model, I recommend omitting this paragraph, as the connection to PD is speculative and not supported by the presented data.

(6) The claim: "If confirmed, our findings could inform new biomarker strategies and therapeutic targets for GBA1 mutation carriers and other at-risk groups. Maintaining renal health may represent a modifiable axis of intervention in neurodegenerative disease," extends beyond the scope of the experimental evidence. The authors should consider tempering this statement or providing supporting data.

(7) The conclusion, "we uncover a critical and previously overlooked role for the renal system in GD and PD pathogenesis," is too strong given the data presented. As no mechanistic link between renal dysfunction and neurodegeneration has been established, this claim should be moderated.

(8) The relevance of Parkin mutant flies is questionable, and this section could be removed from the manuscript.

Reviewer #3 (Public review):

Summary:

Hull et al examine Drosophila mutants for the Gaucher's disease locus GBA1/Gba1b, a locus that, when heterozygous, is a risk factor for Parkinson's. Focusing on the Malpighian tubules and their function, they identify a breakdown of cell junctions, loss of haemolymph filtration, sensitivity to ionic imbalance, water retention, and loss of endocytic function in nephrocytes. There is also an imbalance in ROS levels between the cytoplasm and mitochondria, with reduced glutathione levels, rescue of which could not improve longevity. They observe some of the same phenotypes in mutants of Parkin, but treatment by upregulation of autophagy via rapamycin feeding could only rescue the Gba1b mutant and not the Parkin mutant.

Strengths:

The paper uses a range of cellular, genetic, and physiological analyses and manipulations to fully describe the renal dysfunction in the GBa1b animals. The picture developed has depth and detail; the data appears sound and thorough.

Weaknesses:

The paper relies mostly on the biallelic Gba1b mutant, which may reflect dysfunction in Gaucher's patients, though this has yet to be fully explored. The claims for the heterozygous allele and a role in Parkinson's is a little more tenuous, making assumptions that heterozygosity is a similar but milder phenotype than the full loss-of-function.

Reviewer #1 (Public review):

Summary:

The authors used weighted ensemble enhanced sampling molecular dynamics (MD) to test the hypothesis that a double mutant of Abl favors the DFG-in state relative to the WT and therefore causes the drug resistance to imatinib.

Strengths:

The authors employed three novel progress coordinates to sample the DFG flip of ABl. The hypothesis regarding the double mutant's drug resistance is novel.

Weaknesses:

The study contains many uncertain aspects. As such, major conclusions do not appear to be supported.

Comments on revisions:

The authors have addressed some of my concerns, but these concerns remain to be addressed:

(1) Definition of the DFG conformation (in vs out). The authors specified their definition in the revised manuscript, but it has not been validated for a large number of kinases to distinguish between the two states. Thus, I recommend that the authors calculate the FES using another definition (see Tsai et al, JACS 2019, 141, 15092−15101) to confirm their findings. This FES can be included in the SI.

(2) There is no comparison to previous computational work. I would like to see a comparison between the authors' finding of the DFG-in to DFG-out transition and that described in Tsai et al, JACS 2019, 141, 15092−15101.

(3) My previous comment: "The study is not very rigorous. The major conclusions do not appear to be supported. The claim that it is the first unbiased simulation to observe DFG flip is not true. For example, Hanson, Chodera et al (Cell Chem Biol 2019), Paul, Roux et al (JCTC 2020), and Tsai, Shen et al (JACS 2019) have also observed the DFG flip." has not been adequately addressed.

The newly added paragraph clearly does not address my original comment.

"Through our work, we have simulated an ensemble of DFG flip pathways in a wild-type kinase and its variants with atomistic resolution and without the use of biasing forces, also reporting the effects of inhibitor-resistant mutations in the broader context of kinase inactivation likelihood with such level of detail. "

(4) My previous comment, "Setting the DFG-Asp to the protonated state is not justified, because in the DFG-in state, the DFG-Asp is clearly deprotonated." has not been addressed.

In the authors's response stated:

According to previous publications, DFG-Asp is frequently protonated in the DFG-in state of Abl1 kinase. For instance, as quoted from Hanson, Chodera, et al., Cell Chem Bio (2019), "Consistent with previous simulations on the DFG-Asp-out/in interconversion of Abl kinase we only observe the DFG flip with protonated Asp747 ( Shan et al., 2009 ). We showed previously that the pKa for the DFG-Asp in Abl is elevated at 6.5."

Since the pKa of DFG-Asp is 6.5, it should be deprotonated at the physiological pH 7.5. Thus, the fact that the authors used protonated DFG-Asp contradicts this. I am not requesting the authors to redo the entire simulations, but they need to acknowledge this discrepancy and add a brief discussion. See a constant pH study that demonstrates the protonation state population shift for DFG-Asp as the DFG transitions from in to out state (see Tsai et al, JACS 2019, 141, 15092−15101).

Reviewer #2 (Public review):

Summary:

This is a well-written manuscript on the mechanism of the DFG flip in kinases. This conformational change is important for the toggling of kinases between active (DFG-in) and inactive (DFG-out) states. The relative probabilities of these two states are also an important determinant of the affinity of inhibitors for a kinase. However, it is an extremely slow/rare conformational change, making it difficult to capture in simulations. The authors show that weighted ensemble simulations can capture the DFG flip and then delve into the mechanism of this conformational change and the effects of mutations.

Strengths:

The DFG flip is very hard to capture in simulations. Showing that this can be done with relatively little simulation by using enhanced sampling is a valuable contribution. The manuscript gives a nice description of the background for non-experts.

Weaknesses:

The anecdotal approach to presenting the results is disappointing. Molecular processes are stochastic and the authors have expertise in describing such processes. However, they chose to put most statistical analysis in the SI. The main text instead describes the order of events in single "representative" trajectories. The main text makes it sound like these were most selected as they were continuous trajectories from the weighted ensemble simulations. It is preferable to have a description of the highest probability pathway(s) with some quantification of how probable they are. That would give the reader a clear sense of how representative the events described are.

Reviewer #1 (Public review):

Summary:

The authors used weighted ensemble enhanced sampling molecular dynamics (MD) to test the hypothesis that a double mutant of Abl favors the DFG-in state relative to the WT and therefore causes the drug resistance to imatinib.

Strengths:

The authors employed the state-of-the-art weighted ensemble MD simulations with three novel progress coordinates to explore the conformational changes the DFG motif of Abl kinase. The hypothesis regarding the double mutant's drug resistance is novel.

Weaknesses:

The study contains many uncertain aspects. A major revision is needed to strengthen the support for the conclusions.

(1) Specifically, the authors need to define the DFG conformation using criteria accepted in the field, for example, see https://klifs.net/index.php.

(2) Convergence needs to be demonstrated for estimating the population difference between different conformational states.

(3) The DFG flip needs to be sampled several times to establish free energy difference.

(4) The free energy plots do not appear to show an intermediate state as claimed.

(5) The trajectory length of 7 ns in both Figure 2 and Figure 4 needs to be verified, as it is extremely short for a DFG flip that has a high free energy barrier.

(6) The free energy scale (100 kT) appears to be one order of magnitude too large.

(7) Setting the DFG-Asp to the protonated state is not justified, because in the DFG-in state, the DFG-Asp is clearly deprotonated.

(8) Finally, the authors should discuss their work in the context of the enormous progress made in theoretical studies and mechanistic understanding of the conformational landscape of protein kinases in the last two decades, particularly with regard to the DFG flip.

Reviewer #2 (Public review):

Summary:

This is a well-written manuscript on the mechanism of the DFG flip in kinases. This conformational change is important for the toggling of kinases between active (DFG-in) and inactive (DFG-out) states. The relative probabilities of these two states are also an important determinant of the affinity of inhibitors for a kinase. However, it is an extremely slow/rare conformational change, making it difficult to capture in simulations. The authors show that weighted ensemble simulations can capture the DFG flip and then delve into the mechanism of this conformational change and the effects of mutations.

Strengths:

The DFG flip is very hard to capture in simulations. Showing that this can be done with relatively little simulation by using enhanced sampling is a valuable contribution. The manuscript gives a nice description of the background for non-experts.

Weaknesses:

I was disappointed by the anecdotal approach to presenting the results. Molecular processes are stochastic and the authors have expertise in describing such processes. However, they chose to put most statistical analysis in the SI. The main text instead describes the order of events in single "representative" trajectories. The main text makes it sound like these were most selected as they were continuous trajectories from the weighted ensemble simulations. I would much rather hear a description of the highest probability pathway(s) with some quantification of how probable they are. That would give the reader a clear sense of how representative the events described are.

I appreciated the discussion of the strengths/weaknesses of weighted ensemble simulations. Am I correct that this method doesn't do anything to explicitly enhance sampling along orthogonal degrees of freedom? Maybe a point worth mentioning if so.

I don't understand Figure 3C. Could the authors instead show structures corresponding to each of the states in 3B, and maybe also a representative structure for pathways 1 and 2?

Why introduce S1 and DFG-inter? And why suppose that DFG-inter is what corresponds to the excited state seen by NMR?

It would be nice to have error bars on the populations reported in Figure 3.

I'm confused by the attempt to relate the relative probabilities of states to the 32 kca/mol barrier previously reported between the states. The barrier height should be related to the probability of a transition. The DFG-out state could be equiprobable with the DFG-in state and still have a 32 kcal/mol barrier separating them.

How do the relative probabilities of the DFG-in/out states compare to experiments, like NMR?

Do the staggered and concerted DFG flip pathways mentioned correspond to pathways 1 and 2 in Figure 3B, or is that a concept from previous literature?

Reviewer #1 (Public review):

Domínguez-Rodrigo and colleagues make a moderately convincing case for habitual elephant butchery by Early Pleistocene hominins at Olduvai Gorge (Tanzania), ca. 1.8-1.7 million years ago. They present this at the site scale (the EAK locality, which they excavated), as well as across the penecontemporaneous landscape, analyzing a series of findspots that contain stone tools and large-mammal bones. The latter are primarily elephants, but giraffids and bovids were also butchered in a few localities. The authors claim that this is the earliest well-documented evidence for elephant butchery; doing so requires debunking other purported cases of elephant butchery in the literature, or in one case, reinterpreting elephant bone manipulation as being nutritional (fracturing to obtain marrow) rather than technological (to make bone tools). The authors' critical discussion of these cases may not be consensual, but it surely advances the scientific discourse. The authors conclude by suggesting that an evolutionary threshold was achieved at ca. 1.8 ma, whereby regular elephant consumption rich in fats and perhaps food surplus, more advanced extractive technology (the Acheulian toolkit), and larger human group size had coincided.

The fieldwork and spatial statistics methods are presented in detail and are solid and helpful, especially the excellent description (all too rare in zooarchaeology papers) of bone conservation and preservation procedures. However, the methods of the zooarchaeological and taphonomic analysis - the core of the study - are peculiarly missing. Some of these are explained along the manuscript, but not in a standard Methods paragraph with suitable references and an explicit account of how the authors recorded bone-surface modifications and the mode of bone fragmentation. This seems more of a technical omission that can be easily fixed than a true shortcoming of the study. The results are detailed and clearly presented.

By and large, the authors achieved their aims, showcasing recurring elephant butchery in 1.8-1.7 million-year-old archaeological contexts. Nevertheless, some ambiguity surrounds the evolutionary significance part. The authors emphasize the temporal and spatial correlation of (1) elephant butchery, (2) Acheulian toolkits, and (3) larger sites, but do not actually discuss how these elements may be causally related. Is it not possible that larger group size or the adoption of Acheulian technology have nothing to do with megafaunal exploitation? Alternative hypotheses exist, and at least, the authors should try to defend the causation, not just put forward the correlation. The only exception is briefly mentioning food surplus as a "significant advantage", but how exactly, in the absence of food-preservation technologies? Moreover, in a landscape full of aggressive scavengers, such excess carcass parts may become a death trap for hominins, not an advantage. I do think that demonstrating habitual butchery bears very significant implications for human evolution, but more effort should be invested in explaining how this might have worked.

Overall, this is an interesting manuscript of broad interest that presents original data and interpretations from the Early Pleistocene archaeology of Olduvai Gorge. These observations and the authors' critical review of previously published evidence are an important contribution that will form the basis for building models of Early Pleistocene hominin adaptation.

Reviewer #2 (Public review):

The authors argue that the Emiliano Aguirre Korongo (EAK) assemblage from the base of Bed II at Olduvai Gorge shows systematic exploitation of elephants by hominins about 1.78 million years ago. They describe it as the earliest clear case of proboscidean butchery at Olduvai and link it to a larger behavioral shift from the Oldowan to the Acheulean.

The paper includes detailed faunal and spatial data. The excavation and mapping methods appear to be careful, and the figures and tables effectively document the assemblage. The data presentation is strong, but the behavioral interpretation is not supported by the evidence.

The claim for butchery is based mainly on the presence of green-bone fractures and the proximity of bones and stone artifacts. These observations do not prove human activity. Fractures of this kind can form naturally when bones break while still fresh, and spatial overlap can result from post-depositional processes. The studies cited to support these points, including work by Haynes and colleagues, explain that such traces alone are not diagnostic of butchery, but this paper presents them as if they were.

The spatial analyses are technically correct, but their interpretation extends beyond what they can demonstrate. Clustering indicates proximity, not behavior. The claim that statistical results demonstrate a functional link between bones and artifacts is not justified. Other studies that use these methods combine them with direct modification evidence, which is lacking in this case.

The discussion treats different bodies of evidence unevenly. Well-documented cut-marked specimens from Nyayanga and other sites are described as uncertain, while less direct evidence at EAK is treated as decisive. This selective approach weakens the argument and creates inconsistency in how evidence is judged.

The broader evolutionary conclusions are not supported by the data. The paper presents EAK as marking the start of systematic megafaunal exploitation, but the evidence does not show this. The assemblage is described well, but the behavioral and evolutionary interpretations extend far beyond what can be demonstrated.

Reviewer #1 (Public review):

Summary:

This manuscript investigates mutations and expression patterns of zinc finger proteins in Kenyan breast cancer patients.

Strengths:

Whole-exome sequencing and RNA-seq were performed on 23 breast cancer samples alongside matched normal tissues in Kenyan breast cancer patients. The authors identified mutations in ZNF217, ZNF703, and ZNF750.

Weaknesses:

(1) Research scope:

The results primarily focus on mutations in ZNF217, ZNF703, and ZNF750, with limited correlation analyses between mutations and gene expression. The rationale for focusing only on these genes is unclear. Given the availability of large breast cancer cohorts such as TCGA and METABRIC, the authors should compare their mutation profiles with these datasets. Beyond European and U.S. cohorts, sequencing data from multiple countries, including a recent Nigerian breast cancer study (doi: 10.1038/s41467-021-27079-w), should also be considered. Since whole-exome sequencing was performed, it is unclear why only four genes were highlighted and why comparisons to previous literature were not included.

(2) Language and Style Issues:

Several statements read somewhat 'unnaturally', and I strongly recommend proofreading.

(3) Methods and Data Analysis Details:

The methods section is vague, with general descriptions rather than specific details of data processing and analysis. The authors should provide:

(a) Parameters used for trimming, mapping, and variant calling (rather than referencing another paper such as Tang et al. 2023).

(b) Statistical methods for somatic mutation/SNP detection.

(c) Details of RNA purification and RNA-seq library preparation.

Without these details, the reproducibility of the study is limited.

(4) Data Reporting:

This study has the potential to provide a valuable resource for the field. However, data-sharing plans are unclear. The authors should:

(a) deposit sequencing data in a public repository.

(b) provide supplementary tables listing all detected mutations and all differentially expressed genes (DEGs).

(c) clarify whether raw or adjusted p-values were used for DEG analysis.

(d) perform DEG analyses stratified by breast cancer subtypes, since differential expression was observed by HER2 status, and some zinc finger proteins are known to be enriched in luminal subtypes.

(5) Mutation Analysis:

Visualizations of mutation distribution across protein domains would greatly strengthen interpretation. Comparing mutation distribution and frequency with published datasets would also contextualize the findings.

Reviewer #2 (Public review):

Summary:

This work integrated the mutational landscape and expression profile of ZNF molecules in 23 Kenyan women with breast cancer.

Strengths:

The mutation landscape of ZNF217, ZNF703, and ZNF750 was comprehensively studied and correlated with tumor stage and HER2 status to highlight the clinical significance.

Weaknesses:

The current study design is relatively simple, and there is a limited cohort size, which is relatively small to reach significant findings. Thus, sample size enrichment, along with more analytic work, is needed.

Targeted exploration of the ZNF family without emphasizing the reason or clinical significance hinders the overall significance of the entire work.

Reviewer #3 (Public review):

Summary:

The authors aimed to define the somatic mutational landscape and transcriptomic expression of the ZNF217, ZNF703, and ZNF750 genes in breast cancers from Kenyan women and to investigate associations with clinicopathological features like HER2 status and cancer stage. They employed whole-exome and RNA-sequencing on 23 paired tumor-normal samples to achieve this.

Strengths:

(1) A major strength is the focus on a Kenyan cohort, addressing a critical gap in genomic studies of breast cancer, which are predominantly based on European or Asian populations.

(2) The integration of DNA- and RNA-level data from the same patients provides a comprehensive view, linking genetic alterations to expression changes.

Weaknesses:

(1) The small cohort size (n=23) significantly limits the statistical power to detect associations between genetic features and clinical subgroups (e.g., HER2 status, stage), rendering the negative findings inconclusive.

(2) The study is primarily descriptive. While it effectively catalogs mutations and expression changes, it does not include functional experiments to validate the biological impact of the identified alterations.

Reviewer #1 (Public review):

Summary:

Using single-cell RNA sequencing and bioinformatics approaches, the authors aimed to discover if and how cells carrying mutations common to clonal haematopoiesis were more adherent to endothelial cells.

Strengths:

(1) The authors used matched blood and adipose tissue samples from the same patients (with the exception of the control people) to conduct their analysis.

(2) The use of bioinformatics and in-silico approaches helped to fast-track their aims to test specific inhibitors in their model cell adhesion system.

Weaknesses:

(1) The analysis was done on pooled cells; it would have been interesting to know if the same adhesion gene signatures were observed across the donors.

(2) The adhesion assays were conducted under static conditions; shear flow adhesion experiments would have been better. Mixed cultures using cell trackers would have been even better.

(3) In the intervention studies, the authors should have directly targeted the monocytes (not the endothelial cells) and should have also included DNMT3A mutant/KO cells to show specificity to TET2 CHIP.

Reviewer #2 (Public review):

Summary:

The authors describe potential mechanisms underlying the changes in endothelial-monocyte interactions in patients with clonal hematopoiesis of indeterminate potential (CHIP), including reduced velocity and increased ligand interactions of CHIP-mutated monocytes. They use a combination of transcriptomics (some for the first time in these tissues in patients with CHIP), in silico analyses, and ex vivo approaches to outline the changes that occur in blood monocytes derived from patients with CHIP. These findings advance the current field, which has previously mostly used mice and/or has been focused on cancer outcomes. The authors identify distinct alterations in signaling downstream of DNTM3A or TET2 mutations, which further distinguish two major mutations that contribute to CHIP.

Strengths:

(1) Combinatorial transcriptomics was used to identify potential therapeutic targets, which is an important proof-of-concept for multiple fields.

(2) The authors identify distinct ligand interactions downstream of TET2 and DNMT3A mutations.

Weaknesses:

(1) The authors extrapolate findings in adipose tissue in diabetic patients to vascular disease (ostensibly in the carotid or cardiac arteries), citing the difficulty of using tissue-matched samples. Broad-reaching conclusions need to be backed up in the relevant systems, considering how different endothelial cells in various vascular beds react. Considering these data were obtained with n=3 patients being sufficient to identify these changes, it seems that this can be performed (perhaps in silico) in the correct tissue.

(2) The selection/exclusion criteria for the diabetes samples are not noted, and therefore, the relevant conclusions cannot be fully evaluated, nor is the source of adipose tissue stated.

Appraisal:

While authors describe how to as well as the technical feasibility of integrating a number of transcriptomic techniques, they do not seem to do so to produce highly compelling data or targets within this manuscript. The potential is there to drill down to mechanisms; however, the data gathered herein do not highlight novel targets. For example, CXCL2 and 3 are already shown to be differentially expressed in TET2 loss combined with LDL treatment in the macrophages of mice. Furthermore, these authors then show that in humans, the prototypical CXC chemokine, IL8 (which mice lack), is significantly higher in TET2-mutated patients (DOI: 10.1056/NEJMoa1701719). The authors should demonstrate the utility of their transcriptomics by identifying and testing novel targets and focusing on the proper disease states. This could easily be a deep dive into CHIP in adipose tissue in diabetic patients.

Reviewer #1 (Public review):

Summary:

The study by Castro et al. presents an interesting blueprint for designing influenza immunogens that can induce cross-group influenza-specific antibodies. The authors used a structure-based design to transplant receptor binding site (RBS) residues from H5 and H3 into an H1 scaffold. In addition, they assembled the transplanted structures as heterotrimers. They characterized the constructs structurally and used them to immunize mice to define ELISA binding and neutralizing antibodies (Abs) to different influenza strains.

Strengths and Weaknesses:

The authors succeeded in generating the different, correctly folded immunogens. The heterotrimers would benefit from more characterization: it remains unclear whether they are even formed or whether the sample is a mix of homotrimers and whether some combinations are more likely than others. While some of these questions are complex to answer, authors should at least confirm the presence of heterotrimers.

While all constructs were able to elicit H1-specific Abs, different immunogens displayed differential ability to induce a response to the transplanted epitope. While H3-transplant resulted in H3-specific Abs, this was not the case for H5 or the heterotrimers. The importance of the finding is that authors are able to elicit polyclonal Abs neutralizing group 1 and group 2 influenza viruses with a single immunogen. A more in-depth discussion on why the H3-transplant but not the H5-transplant resulted in those specific Abs could be beneficial.

Overall, the work is a proof of concept that H1-H3 chimeric proteins can be produced and an important first step towards computational vaccines, inducing Abs to multiple groups.

Reviewer #2 (Public review):

Summary:

The manuscript from Castro et al describes the engineering of influenza hemagglutinin H1-based head domains that display receptor-binding-site residues from H5 and H3 HAs. The initial head-only chimeras were able to bind to FluA20, which recognizes the trimer interface, but did not bind well to H5 or H3-specific antibodies. Furthermore, these constructs were not particularly stable in solution as assessed by low melting temperatures. Crystal structures of each chimeric head in complex with FluA20 were obtained, demonstrating that the constructs could adopt the intended conformation upon stabilization with FluA20. The authors next placed the chimeric heads onto an H1 stalk to create homotrimeric HA ectodomains, as well as a heterotrimeric HA ectodomain. The homotrimeric chimeric HAs were better behaved in solution, and H3- and H5-specific antibodies bound to these trimers with affinities that were only about 10-fold weaker compared to their respective wildtype HAs. The heterotrimeric chimeric HA showed transient stability in solution and could bind more weakly to the H3- and H5-specific antibodies. Mice immunized with these trimers elicited cross-reactive binding antibodies, although the cross-neutralizing titers were less robust. The most positive result was that the H1H3 trimer was able to elicit sera that neutralized both H1 and H3 viruses.

Strengths:

The manuscript is very well-written with clear figures. The biophysical and structural characterizations of the antigen were performed to a high standard. The engineering approach is novel, and the results should provide a basis for further iteration and improvement of RBS transplantation.

Weaknesses:

The main limitation of the study is that there are no statistical tests performed for the immunogenicity results shown in Figures 4 and 5. It is therefore unknown whether the differences observed are statistically significant. Additionally, fits of the BLI data in Figure 3 to the binding model used to determine the binding constants should be shown.

Reviewer #1 (Public review):

Summary:

This is a careful and comprehensive study demonstrating that effector-dependent conformational switching of the MT lattice from compacted to expanded deploys the alpha tubulin C-terminal tails so as to enhance their ability to bind interactors.

Strengths:

The authors use 3 different sensors for the exposure of the alpha CTTs. They show that all 3 sensors report exposure of the alpha CTTs when the lattice is expanded by GMPCPP, or KIF1C, or a hydrolysis-deficient tubulin. They demonstrate that expansion-dependent exposure of the alpha CTTs works in tissue culture cells as well as in vitro.

Weaknesses:

There is no information on the status of the beta tubulin CTTs. The study is done with mixed isotype microtubules, both in cells and in vitro. It remains unclear whether all the alpha tubulins in a mixed isotype microtubule lattice behave equivalently, or whether the effect is tubulin isotype-dependent. It remains unclear whether local binding of effectors can locally expand the lattice and locally expose the alpha CTTs.

Appraisal:

The authors have gone to considerable lengths to test their hypothesis that microtubule expansion favours deployment of the alpha tubulin C-terminal tail, allowing its interactors, including detyrosinase enzymes, to bind. There is a real prospect that this will change thinking in the field. One very interesting possibility, touched on by the authors, is that the requirement for MAP7 to engage kinesin with the MT might include a direct effect of MAP7 on lattice expansion.

Impact:

The possibility that the interactions of MAPS and motors with a particular MT or region feed forward to determine its future interaction patterns is made much more real. Genuinely exciting.

Reviewer #2 (Public review):

The unstructured α- and β-tubulin C-terminal tails (CTTs), which differ between tubulin isoforms, extend from the surface of the microtubule, are post-translationally modified, and help regulate the function of MAPs and motors. Their dynamics and extent of interactions with the microtubule lattice are not well understood. Hotta et al. explore this using a set of three distinct probes that bind to the CTTs of tyrosinated (native) α-tubulin. Under normal cellular conditions, these probes associate with microtubules only to a limited extent, but this binding can be enhanced by various manipulations thought to alter the tubulin lattice conformation (expanded or compact). These include small-molecule treatment (Taxol), changes in nucleotide state, and the binding of microtubule-associated proteins and motors. Overall, the authors conclude that microtubule lattice "expanders" promote probe binding, suggesting that the CTT is generally more accessible under these conditions. Consistent with this, detyrosination is enhanced. Mechanistically, molecular dynamics simulations indicate that the CTT may interact with the microtubule lattice at several sites, and that these interactions are affected by the tubulin nucleotide state.

Strengths:

Key strengths of the work include the use of three distinct probes that yield broadly consistent findings, and a wide variety of experimental manipulations (drugs, motors, MAPs) that collectively support the authors' conclusions, alongside a careful quantitative approach.

Weaknesses:

The challenges of studying the dynamics of a short, intrinsically disordered protein region within the complex environment of the cellular microtubule lattice, amid numerous other binders and regulators, should not be understated. While it is very plausible that the probes report on CTT accessibility as proposed, the possibility of confounding factors (e.g., effects on MAP or motor binding) cannot be ruled out. Sensitivity to the expression level clearly introduces additional complications. Likewise, for each individual "expander" or "compactor" manipulation, one must consider indirect consequences (e.g., masking of binding sites) in addition to direct effects on the lattice; however, this risk is mitigated by the collective observations all pointing in the same direction.

The discussion does a good job of placing the findings in context and acknowledging relevant caveats and limitations. Overall, this study introduces an interesting and provocative concept, well supported by experimental data, and provides a strong foundation for future work. This will be a valuable contribution to the field.

Reviewer #3 (Public review):

Summary:

In this study, the authors investigate how the structural state of the microtubule lattice influences the accessibility of the α-tubulin C-terminal tail (CTT). By developing and applying new biosensors, they reveal that the tyrosinated CTT is largely inaccessible under normal conditions but becomes more accessible upon changes to the tubulin conformational state induced by taxol treatment, MAP expression, or GTP-hydrolysis-deficient tubulin. The combination of live imaging, biochemical assays, and simulations suggests that the lattice conformation regulates the exposure of the CTT, providing a potential mechanism for modulating interactions with microtubule-associated proteins. The work addresses a highly topical question in the microtubule field and proposes a new conceptual link between lattice spacing and tail accessibility for tubulin post-translational modification.

Strengths:

(1) The study targets a highly relevant and emerging topic-the structural plasticity of the microtubule lattice and its regulatory implications.

(2) The biosensor design represents a methodological advance, enabling direct visualization of CTT accessibility in living cells.

(3) Integration of imaging, biochemical assays, and simulations provides a multi-scale perspective on lattice regulation.

(4) The conceptual framework proposed lattice conformation as a determinant of post-translational modification accessibility is novel and potentially impactful for understanding microtubule regulation.

Weaknesses:

There are a number of weaknesses in the paper, many of which can be addressed textually. Some of the supporting evidence is preliminary and would benefit from additional experimental validation and clearer presentation before the conclusions can be considered fully supported.

In particular, the authors should directly test in vitro whether Taxol addition can induce lattice exchange (see comments below).

Reviewer #1 (Public review):

The remodeling of macromolecular substrates by AAA+ proteins is an essential aspect of life at the molecular scale, and understanding conserved and divergent features of substrate recognition across the AAA+ protein family remains an ongoing area of research. AAA+ proteins are highly modular and typically combine N-terminal recognition domain(s) with ATPase domain(s) to recognize and unfold some macromolecular target, such as dsDNA or protein substrates. This can be coupled to activity by additional C-terminal domains that further modify the substrate, such as a protease domain that hydrolyzes the extended, unstructured protein chain that emerges from the ATPase domain during substrate processing.

This work focuses on one such AAA+ protease, LONP1. LONP1 is an essential AAA+ protein involved in mitochondrial proteostasis, and disruption of its function in vivo has serious developmental consequences. This work explores the processing of two new mitochondrial protein substrates (StAR, TFAM) by LONP1 and presents new conformational states of LONP1 with closed configurations and no substrate threaded through the ATPase pores. The quality of the reconstructions and models is very good. Critically, one of these states (LONP1C3) has a completely occluded ATPase pore from the N-terminal side of the ATPase ring, where three of the six NTDs/CCDs interact tightly to form a C3-symmetric substructure preventing substrate ingress. The authors note several key interactions between amino acids forming these substructures, and perform ATPase assays on mutant LONP1 proteins to determine hydrolysis rates in the absence or presence of substrate. These patterns are recapitulated in casein disassembly assays as well. Based on these results, the authors note that the mutants have differential effects depending on the "foldedness" of the substrate, and surmise that disruption of the C3-symmetric substructure from the EM experiments is responsible for these effects - an intriguing idea. In addition to the C3 state, the authors observe additional intermediates which they place on the same conformational coordinate. One such structure is the LONP1C2 state with two splits, hinting at a conformational transition from LONP1C3 to the closed/active state.

Taken together, these results form the basis of an interesting story. However, I feel that more experimentation and analysis are needed to address several key points, or that the conclusions should be toned down. First and foremost, I note that while the hypothesis that the LONP1C3 state is a critical step in recognizing substrate "foldedness" is an interesting one, the claim is made solely on the basis of biochemical experiments with mutant LONP1, and that there is no substrate density associated with LONP1C3. In the absence of substrate density and/or structural data for the mutants, this seems like a very strong claim. More generally, the manuscript invokes the conformational landscape of LONP1C3 in multiple instances, but no such landscape is presented to show how LONP1C3 and the other states are quantitatively linked. Finally, I note the prevalence of ADP-only active sites in these intermediates, and am concerned that this might be related to the depletion of ATP under the on-grid reaction conditions. The inclusion of an ATP regeneration system may be a useful way to ensure that ATP/ADP concentrations are more physiological and that excessive ADP will not bias the conformations of the ring systems.

In summary, I believe this manuscript is exciting but would benefit from a paring back of claims, or the inclusion of some additional data to fill in some of the conceptual gaps outlined above.

Reviewer #2 (Public review):

This paper by Mindrebo et al. reveals multiple novel conformations of the human LONP1 protease. AAA+ proteases, like LONP1, are needed for maintaining proteostasis in cells and organelles. While structures of fully active (closed) and fully inactive (open) conformations of LONP1 are now established, the dynamics between these states and how changes in conformations may contribute to or be triggered by substrates and nucleotides are unclear. In this work, the authors characterize a novel C3-symmetric state of LONP1 bound to TFAM (a native substrate), suggesting that this C3-state is an intermediate in the open to closed cycle, and make mutations to test this model biochemically. Deeper inspection of their TFAM-bound LONP1 dataset reveals additional conformations, including a C2-symmetric and two asymmetric intermediates. All these conformations are synthesized by the authors to propose a model for how LONP1 transitions from an inactive OFF state to an active ENZ state. There are clear, interesting structural aspects to this work, revealing alternate conformations to shed light on the dynamics of LONP1. However, some of the conclusions interpret well beyond the scope of the experiments shown, and this is discussed below.

Overall, there are two major comments with the work as written that, if addressed, would make the results more compelling. First, the order of events and existence of intermediate states is primarily from static structural snapshots and fitting these structures to a possible mechanism. It would be ideal to have some biochemical or kinetic data supporting these steps and the existence of these intermediates. For example, the model is that the C3-state is an ADP-bound intermediate that blocks access and acts as a checkpoint for progression to the ENZ state of LONP1. The major evidence for this comes from a mutation (D449A) that fails to degrade TFAM as well as StAR or casein, which is taken as evidence that failure to form the C3 state reduces the ability to degrade more 'folded' substrates. A prediction of this model would be that destabilizing TFAM through mutation should improve D449A degradation. Ideally, other measures of conformational changes, such as FRET or HDX-MS, could be used to visualize this C3-state in unmutated LONP1 during the process of substrate engagement and degradation. At a minimum, using ATP hydrolysis as a proxy for forming the ENZ state and the assumption that different substrates will differentially promote formation of the C3-state means that measuring ATP hydrolysis of wt LONP1 with different substrates will be informative.

The second major comment is that the primary evidence for the importance of the C3 state is a mutation (D449A) that, based on the cryoEM structure, is incompatible with this conformation but should not affect any other state. A concern that arises is whether this mutation is doing more than simply destabilizing the C3 state and affecting substrate recognition/enzymatic activity in some other manner. To address this point, the authors could perform cryoEM characterization of the D449A mutant, which should show reduced or no presence of the C3-state, but still an intact ability to form the closed ENZ state.

Reviewer #3 (Public review):

Summary:

The AAA+ protease LON1P is a central component of mitochondrial protein quality control and has crucial functions in diverse processes. Cryo-EM structures of LON1P defined inactive and substrate-processing active states. Here, the authors determined multiple new LON1P structural states by cryo-EM in the presence of diverse substrates. The structures are defined as on-pathway intermediates to LON1P activation. A C3-symmetry state is suggested to function as a checkpoint to scan for LON1P substrates and link correct substrate selection to LON1P activation.

Strengths:

The determination of multiple structures provides relevant information on substrate-triggered activation of LON1P. The authors support structural data by biochemical analysis of structure-based mutants.

Weaknesses:

How substrate selection is achieved remains elusive, also because substrates are not detectable in the diverse structures. It also remains in parts unclear whether mutant phenotypes can be specifically linked to a single structural state (C3). Some mutant phenotypes appear complex and do not seem to be in line with the model proposed.

Reviewer #1 (Public review):

Summary:

This work provides evidence that slender T. brucei can initiate and complete cyclical development in Glossina morsitans without GlcNAc supplementation, in both sexes, and importantly in non-teneral flies, including salivary-gland infections.

Comparative transcriptomics show early divergence between slender- and stumpy-initiated differentiation (distinct GO enrichments), with convergence by ~72 h, supporting an alternative pathway into the procyclic differentiation program.

The work addresses key methodological criticisms of earlier studies and supports the hypothesis that slender forms may contribute to transmission at low parasitaemia.

Strengths:

(1) Directly tackles prior concerns (no GlcNAc, both sexes, non-teneral flies) with positive infections through to the salivary glands.

(2) Transcriptomic time course adds some mechanistic depth.

(3) Clear relevance to the "transmission paradox"; advances an important debate in the field.

Weaknesses:

(1) Discrepancy with Ngoune et al. (2025) remains unresolved; no head-to-head control for colony/blood source or microbiome differences that could influence vector competence.

(2) Lacks in vivo feeding validation (e.g., infecting flies directly on parasitaemic mice) to strengthen ecological relevance.

(3) Mechanistic inferences are largely correlative (although not requested, there is no functional validation of genes or pathways emerging from the transcriptomics).

(4) Reliance on a single parasite clone (AnTat 1.1) and one vector species limits external validity.

Reviewer #2 (Public review):

Summary:

This paper is an exciting follow-up to two recent publications in eLife: one from the same lab, reporting that slender forms can successfully infect tsetse flies (Schuster, S et al., 2021), and another independent study claiming the opposite (Ngoune, TMJ et al., 2025). Here, the authors address four criticisms raised against their original work: the influence of N-acetyl-glucosamine (NAG), the use of teneral and male flies, and whether slender forms bypass the stumpy stage before becoming procyclic forms.

Strengths:

We applaud the authors' efforts in undertaking these experiments and contributing to a better understanding of the T. brucei life cycle. The paper is well-written and the figures are clear.

Weaknesses:

We identified several major points that deserve attention.

(1) What is a slender form? Slender-to-stumpy differentiation is a multi-step process, and most of these steps unfortunately lack molecular markers (Larcombe et al, 2023). In this paper, it is essential that the authors explicitly define slender forms. Which parameters were used? It is implicit that slender forms are replicative and GFP::PAD1-negative. Isn't it possible that some GFP::PAD1-negative cells were already transitioning toward stumpy forms, but not yet expressing the reporter? Transcriptomically, these would be early transitional cells that, upon exposure to "tsetse conditions" (in vitro or in vivo), could differentiate into PCF through an alternative pathway, potentially bypassing the stumpy stage (as suggested in Figure 4). Given the limited knowledge of early molecular signatures of differentiation, we cannot exclude the possibility that the slender forms used here included early differentiating cells. We suggest:

1.1 Testing the commitment of slender forms (e.g., using the plating assay in Larcombe et al., 2023), assessing cell-cycle profile, and other parameters that define slender forms.

1.2 In the Discussion, acknowledging the uncertainty of "what is a slender?" and being explicit about the parameters and assumptions.

1.3 Clarifying in the Materials and Methods how cultures were maintained in the 3-4 days prior to tsetse infections, including daily cell densities. Ideally, provide information on GFP expression, cell cycle, and morphology. While this will not fully resolve the concern, it will allow future reinterpretation of the data when early molecular events are better understood.

(2) Figure 1: This analysis lacks a positive control to confirm that NAG is working as expected. It would strengthen the paper if the authors showed that NAG improves stumpy infection. Once confirmed, the authors could discuss possible differences in the tsetse immune response to slender vs. stumpy forms to explain the absence of an effect on slender infections.

(3) Figure 2. To conclude that teneral flies are less infected than non-teneral flies, data from Figures 1 and 2 must be directly comparable. Were these experiments performed simultaneously? Please clarify in the figure legends. Moreover, the non-teneral flies here are still relatively young (6-7 days old), limiting comparisons with Ngoune, TMJ et al. 2025, where flies were 2-3 weeks old.

(4) Figure 3. The PCA plot (A) appears to suggest the opposite of the authors' interpretation: slender differentiation seems to proceed through a transcriptome closer to stumpy profiles. Plotting DEG numbers (panel C) is informative, but how were paired conditions selected? Besides, plotting of the number of DEGs between consecutive time points within and between parasite types is also necessary. There may also be better computational tools to assess temporal relationships. Finally, how does PAD1 transcript abundance change over time in both populations? It would also be important to depict the upregulation of procyclic-specific genes.

(5) Could methylcellulose in the medium sensitize parasites to QS-signal, leading to more frequent and/or earlier differentiation, despite low densities? If so, cultures with vs. without methylcellulose might yield different proportions of early-differentiating (yet GFP-negative) parasites. This could explain discrepancies between the Engstler and Rotureau labs despite using the same strain. The field would benefit from reciprocal testing of culture conditions. Alternatively, the authors could compare infectivity and transcriptomes of their slender forms under three conditions: (i) in vitro with methylcellulose, (ii) in vitro without methylcellulose, and (iii) directly from mouse blood.

Reviewer #1 (Public review):

This is a re-review following an author revision. I will go point-by-point in response to my original critiques and the authors' responses. I appreciate the authors taking the time to thoughtfully respond to the reviewer critiques.

Query 1. Based on the authors' description of their contribution to the algorithm design, it sounds like a hyperparameter search wrapped around existing software tools. I think that the use of their own language to describe these modules is confusing to potential users as well as unintentionally hides the contributions of the original LigBuilder developers. The authors should just explain the protocol plainly using language that refers specifically to the established software tools. Whether they use LigBuilder or something else, at the end of the day the description is a protocol for a specific use of an existing software rather than the creation of a new toolkit.

Query 2. I see. Correct me if I am mistaken, but it seems as though the authors are proposing using the Authenticator to identify the best distributions of compounds based on an in silico oracle (in this case, Vina score), and train to discriminate them. This is similar to training QSAR models to predict docking scores, such as in the manuscript I shared during the first round of review. In principle, one could perform this in successive rounds to create molecules that are increasingly composed of features that yield higher docking scores. This is an established idea that the authors demonstrate in a narrow context, but it also raises concern that one is just enriching for compounds with e.g., an abundance of hydrogen bond donors and acceptors. Regarding points (4) and (5), it is unclear to me how the authors perform train/test splits on unlabeled data with supervised machine learning approaches in this setting. This seems akin to a Y-scramble sanity check. Finally, regarding the discussion on the use of experimental data or FEP calculations for the determination of HABs and LABs, I appreciate the authors' point; however, the concern here is that in the absence of any true oracle the models will just learn to identify and/or generate compounds that exploit limitations of docking scores. Again, please correct me if I am mistaken. It is unclear to me how this advances previous literature in CADD outside of the specific context of incorporating some ideas into a GPCR-Gprotein framework.

Query 3. The authors mention that the hyperparameters for the ML models are just the package defaults in the absence of specification by the user. I would be helpful to know specifically what the the hyperparameters were for the benchmarks in this study; however, I think a deeper concern is still that these models are almost certainly far overparameterized given the limited training data used for the models. It is unclear why the authors did not just build a random forest classifier to discriminate their HABs and LABs using ligand- or protein-ligand interaction fingerprints or related ideas.

Query 4. It is good, and expected, that increasing the fraction of the training set size in a random split validation all the way to 100% would allow the model to perfectly discriminate HABs and LABs. This does not demonstrate that the model has significant enrichment in prospective screening, particularly compared to simpler methods. The concern remains that these models are overparameterized and insufficiently validated. The authors did not perform any scaffold splits or other out-of-distribution analysis.

Query 5. The authors contend that Gcoupler uniquely enables training models when data is scarce and ultra-large screening libraries are unavailable. Today, it is rather straightforward to dock a minimum of thousands of compounds. Using tools such as QuickVina2-GPU (https://pubs.acs.org/doi/10.1021/acs.jcim.2c01504), it is possible to quite readily dock millions in a day with a single GPU and obtain the AutoDock Vina score. GPU-acclerated Vina has been combined with cavity detection tools likely multiple times, including here (https://arxiv.org/abs/2506.20043). There are multiple cavity detection tools, including the ones the authors use in their protocol.

Query 6. The authors contend that the simulations are converged, but they elected not to demonstrate stability in the predicting MM/GBSA binding energies with block averaging across the trajectory. This could have been done through the existing trajectories without additional simulation.

Reviewer #1 (Public review):

This is a re-review following an author revision. I will go point-by-point in response to my original critiques and the authors' responses. I appreciate the authors taking the time to thoughtfully respond to the reviewer critiques.

Query 1. Based on the authors' description of their contribution to the algorithm design, it sounds like a hyperparameter search wrapped around existing software tools. I think that the use of their own language to describe these modules is confusing to potential users as well as unintentionally hides the contributions of the original LigBuilder developers. The authors should just explain the protocol plainly using language that refers specifically to the established software tools. Whether they use LigBuilder or something else, at the end of the day the description is a protocol for a specific use of an existing software rather than the creation of a new toolkit.

Query 2. I see. Correct me if I am mistaken, but it seems as though the authors are proposing using the Authenticator to identify the best distributions of compounds based on an in silico oracle (in this case, Vina score), and train to discriminate them. This is similar to training QSAR models to predict docking scores, such as in the manuscript I shared during the first round of review. In principle, one could perform this in successive rounds to create molecules that are increasingly composed of features that yield higher docking scores. This is an established idea that the authors demonstrate in a narrow context, but it also raises concern that one is just enriching for compounds with e.g., an abundance of hydrogen bond donors and acceptors. Regarding points (4) and (5), it is unclear to me how the authors perform train/test splits on unlabeled data with supervised machine learning approaches in this setting. This seems akin to a Y-scramble sanity check. Finally, regarding the discussion on the use of experimental data or FEP calculations for the determination of HABs and LABs, I appreciate the authors' point; however, the concern here is that in the absence of any true oracle the models will just learn to identify and/or generate compounds that exploit limitations of docking scores. Again, please correct me if I am mistaken. It is unclear to me how this advances previous literature in CADD outside of the specific context of incorporating some ideas into a GPCR-Gprotein framework.

Query 3. The authors mention that the hyperparameters for the ML models are just the package defaults in the absence of specification by the user. I would be helpful to know specifically what the the hyperparameters were for the benchmarks in this study; however, I think a deeper concern is still that these models are almost certainly far overparameterized given the limited training data used for the models. It is unclear why the authors did not just build a random forest classifier to discriminate their HABs and LABs using ligand- or protein-ligand interaction fingerprints or related ideas.

Query 4. It is good, and expected, that increasing the fraction of the training set size in a random split validation all the way to 100% would allow the model to perfectly discriminate HABs and LABs. This does not demonstrate that the model has significant enrichment in prospective screening, particularly compared to simpler methods. The concern remains that these models are overparameterized and insufficiently validated. The authors did not perform any scaffold splits or other out-of-distribution analysis.

Query 5. The authors contend that Gcoupler uniquely enables training models when data is scarce and ultra-large screening libraries are unavailable. Today, it is rather straightforward to dock a minimum of thousands of compounds. Using tools such as QuickVina2-GPU (https://pubs.acs.org/doi/10.1021/acs.jcim.2c01504), it is possible to quite readily dock millions in a day with a single GPU and obtain the AutoDock Vina score. GPU-acclerated Vina has been combined with cavity detection tools likely multiple times, including here (https://arxiv.org/abs/2506.20043). There are multiple cavity detection tools, including the ones the authors use in their protocol.

Query 6. The authors contend that the simulations are converged, but they elected not to demonstrate stability in the predicting MM/GBSA binding energies with block averaging across the trajectory. This could have been done through the existing trajectories without additional simulation.

Reviewer #1 (Public review):

The authors have implemented several clarifications in the text and improved the connection between their findings and previous work. As stated in my initial review, I had no major criticisms of the previous version of the manuscript, and I continue to consider this a solid and well-written study. However, the revised manuscript still largely reiterates existing findings and does not offer novel conceptual or experimental advances. It supports previous conclusions suggesting a likely conserved sex determination locus in aculeate hymenopterans, but does so without functional validation (i.e., via experimental manipulation) of the candidate locus in O. biroi. I also wish to clarify that I did not intend to imply that functional assessments in the Pan et al. study were conducted in more than one focal species; my previous review explicitly states that the locus's functional role was validated in the Argentine ant.

Reviewer #3 (Public review):

The authors have made considerable efforts to conduct functional analyses to the fullest extent possible in this study; however, it is understandable that meaningful results have not yet been obtained. In the revised version, they have appropriately framed their claims within the limits of the current data and have adjusted their statements as needed in response to the reviewers' comments.

Reviewer #1 (Public review):

Summary:

In this manuscript, Subhramanian et al. carefully examined how microglia adapt their surveillance strategies during chronic neurodegeneration, specifically in prion-infected mice. The authors used ex vivo time-lapse imaging and in vitro strategies, finding that reactive microglia exhibit a highly mobile, "kiss-and-ride" behavior, which contrasts with the more static surveillance typically observed in homeostatic microglia. The manuscript provides fundamental mechanistic insights into the dynamics of microglia-neuron interactions, implicates P2Y6 signaling in regulating mobility, and suggests that intrinsic reprogramming of microglia might underlie this behavior. The conclusions are therefore compelling.

Strengths:

(1) The novelty of the study is high, in particular, the demonstration that microglia lose territorial confinement and dynamically migrate from neuron to neuron under chronic neurodegeneration.

(2) The possible implications of a stimulus-independent high mobility in reactive microglia are particularly striking. Although this is not fully explored (see comments below).

(3) The use of time-lapse imaging in organotypic slices rather than overexpression models provided a more physiological approach.

(4) Microglia-neuron interactions in neurodegeneration have broad implications for understanding the progression of other diseases that are associated with chronic inflammation, such as Alzheimer's and Parkinson's.

Weaknesses:

(1) The Cx3cr1/EGFP line labels all myeloid cells, which makes it difficult to conclude that all observed behaviors are attributable to microglia rather than infiltrating macrophages. The authors refer to this and include it as a limitation. Nonetheless, complementary confirmation by additional microglia markers would strengthen their claims.

(2) Although the authors elegantly describe dynamic surveillance and envelopment hypothesis, it is unclear what the role of this phenotype is for disease progression, i.e., functional consequences. For example, are the neurons that undergo sustained envelopment more likely to degenerate?

(3) Moreover, although the increase in mobility is a relevant finding, it would be interesting for the authors to further comment on what the molecular trigger(s) is/are that might promote this increase. These adaptations, which are at least long-lasting, confer apparent mobility in the absence of external stimuli.

(4) The authors performed, as far as I could understand, the experiments in cortical brain regions. There is no clear rationale for this in the manuscript, nor is it clear whether the mobility is specific to a particular brain region. This is particularly important, as microglia reactivity varies greatly depending on the brain region.

(5) It would be relevant information to have an analysis of the percentage of cells in normal, sub-clinical, early clinical, and advanced stages that became mobile. Without this information, the speed/distance alone can have different interpretations.

Reviewer #2 (Public review):

This is a nice paper focused on the response of microglia to different clinical stages of prion infection in acute brain slices. The key here is the use of time-lapse imaging, which captures the dynamics of microglial surveillance, including morphology, migration, and intracellular neuron-microglial contacts. The authors use a myeloid GFP-labeled transgenic mouse to track microglia in SSLOW-infected brain slices, quantifying differences in motility and microglial-neuron interactions via live fluorescence imaging. Interesting findings include the elaborate patterns of motility among microglia, the distinct types and duration of intracellular contacts, the potential role of calcium signaling in facilitating hypermobility, and the fact that this motion-promoting status is intrinsic to microglia, persisting even after the cells have been isolated from infected brains. Although largely a descriptive paper, there are mechanistic insights, including the role of calcium in supporting movement of microglia, where bursts of signaling are identified even within the time-lapse format, and inhibition studies that implicate the purinergic receptor and calcium transient regulator P2Y6 in migratory capacity.

Strengths:

(1) The focus on microglia activation and activity in the context of prion disease is interesting.

(2) Two different prions produce largely the same response.

(3) Use of time-lapse provides insight into the dynamics of microglia, distinguishing between types of contact - mobility vs motility - and providing insight into the duration/transience and reversibility of extensive somatic contacts that include brief and focused connections in addition to soma envelopment.

(4) Imaging window selection (3 hours) guided by prior publications documenting preserved morphology, activity, and gene expression regulation up to 4 hours.

(5) The distinction between high mobility and low mobility microglia is interesting, especially given that hyper mobility seems to be an innate property of the cells.

(6) The live-imaging approach is validated by fixed tissue confocal imaging.

(7) The variance in duration of neuron/microglia contacts is interesting, although there is no insight into what might dictate which status of interaction predominates.

(8) The reversibility of the enveloping action, that is not apparently a commitment to engulfment, is interesting, as is the fact that only neurons are selected for this activity.

(9) The calcium studies use the fluorescent dye calbryte-590 to pick up neuronal and microglial bursts - prolonged bursts are detected in enveloped neurons and in the hyper-mobile microglia - the microglial lead is followed up using MRS-2578 P2Y6 inhibitor that blunts the mobility of the microglia.

Weaknesses:

(1) The number of individual cells tracked has been provided, but not the number of individual mice. The sex of the mice is not provided.

(2) The statistical approach is not clear; was each cell treated as a single observation?

(3) The potential for heterogeneity among animals has not been addressed.

(4) Validation of prion accumulation at each clinical stage of the disease is not provided.

(5) How were the numerous captures of cells handled to derive morphological quantitative values? Based on the videos, there is a lot of movement and shape-shifting.

(6) While it is recognized that there are limits to what can be measured simultaneously with live imaging, the authors appear to have fixed tissues from each time point too - it would be very interesting to know if the extent or prion accumulation influences the microglial surveillance - i.e., do the enveloped ones have greater pathology>

Reviewer #3 (Public review):

Summary:

The authors developed a new phenological lag metric and applied this analytical framework to a global dataset to synthesize shifts in spring phenology and assess how abiotic constraints influence spring phenology.

Strengths:

The dataset developed in this study is extensive, and the phenological lag metric is valuable.

Weaknesses:

The stability of the method used to calculate forcing requirements needs improvement, for example by including different base temperature thresholds. In addition, the visualization of the results should be improved.

Reviewer #1 (Public review):

In the current article, Octavia Soegyono and colleagues study "The influence of nucleus accumbens shell D1 and D2 neurons on outcome-specific Pavlovian instrumental transfer", building on extensive findings from the same lab. While there is a consensus about the specific involvement of the Shell part of the Nucleus Accumbens (NAc) in specific stimulus-based actions in choice settings (and not in General Pavlovian instrumental transfer - gPIT, as opposed to the Core part of the NAc), mechanisms at the cellular and circuitry levels remain to be explored. In the present work, using sophisticated methods (rat Cre-transgenic lines from both sexes, optogenetics and the well-established behavioral paradigm outcome-specific PIT - sPIT), Octavia Soegyono and colleagues decipher the differential contribution of dopamine receptors D1 and D2 expressing-spiny projection neurons (SPNs).

After validating the viral strategy and the specificity of the targeting (immunochemistry and electrophysiology), the authors demonstrate that while both NAc Shell D1- and D2-SPNs participate in mediating sPIT, NAc Shell D1-SPNs projections to the Ventral Pallidum (VP, previously demonstrated as crucial for sPIT), but not D2-SPNs, mediates sPIT. They also show that these effects were specific to stimulus-based actions, as value-based choices were left intact in all manipulations.

This is a well-designed study and the results are well supported by the experimental evidence. The paper is extremely pleasant to read and add to the current literature.

Reviewer #2 (Public review):

Summary:

This manuscript by Soegyono et a. describes a series of experiments designed to probe the involvement of dopamine D1 and D2 neurons within the nucleus accumbens shell in outcome-specific Pavlovian-instrumental transfer (osPIT), a well-controlled assay of cue-guided action selection based on congruent outcome associations. They used an optogenetic approach to phasically silence NAc shell D1 (D1-Cre mice) or D2 (A2a-Cre mice) neurons during a subset of osPIT trials. Both manipulations disrupted cue-guided action selection but had no effects on negative control measures/tasks (concomitant approach behavior, separate valued guided choice task), nor were any osPIT impairments found in reporter only control groups. Separate experiments revealed that selective inhibition of NAc shell D1 but not D2 inputs to ventral pallidum were required for osPIT expression, thereby advancing understanding of the basal ganglia circuitry underpinning this important aspect of decision making.

Strengths:

The combinatorial viral and optogenetic approaches used here were convincingly validated through anatomical tract-tracing and ex vivo electrophysiology. The behavioral assays are sophisticated and well-controlled to parse cue and value guided action selection. The inclusion of reporter only control groups is rigorous and rules out nonspecific effects of the light manipulation. The findings are novel and address a critical question in the literature. Prior work using less decisive methods had implicated NAc shell D1 neurons in osPIT but suggested that D2 neurons may not be involved. The optogenetic manipulations used in the current study provides a more direct test of their involvement and convincingly demonstrate that both populations play an important role. Prior work had also implicated NAc shell connections to ventral pallidum in osPIT, but the current study reveals the selective involvement of D1 but not D2 neurons in this circuit. The authors do a good job of discussing their findings, including their nuanced interpretation that NAc shell D2 neurons may contribute to osPIT through their local regulation of NAc shell microcircuitry.

Weaknesses:

The current study exclusively used an optogenetic approach to probe the function of D1 and D2 NAc shell neurons. Providing a complementary assessment with chemogenetics or other appropriate methods would strengthen conclusions, particularly the novel demonstration for D2 NAc shell involvement. Likewise, the null result of optically inhibiting D2 inputs to ventral pallidum leaves open the possibility that a more complete or sustained disruption of this pathway may have impaired osPIT.

Conclusions:

The research described here was successful in providing critical new insights into the contributions of NAc D1 and D2 neurons in cue-guided action selection. The authors' data interpretation and conclusions are well reasoned and appropriate. They also provide a thoughtful discussion of study limitations and implications for future research. This research is therefore likely to have a significant impact on the field.

Comments on the previous version:

I have reviewed the rebuttal and revised manuscript and have no remaining concerns.

Reviewer #1 (Public review):

The paper reports some interesting patterns in epistasis in a recently published large fitness landscape dataset. The results may have implications for our understanding of fitness landscapes and protein evolution. However, this version of the paper remains fairly descriptive and has significant deficiencies in clarity and rigor.

The authors have addressed some of my criticisms (e.g., I appreciate the additional analysis of synonymous mutations, and a more rigorous approach to calling fitness peaks), but many of the issues raised in my first round of review remain in the current version. Frankly, I am quite disappointed that the authors did not address my comments point by point, which is the norm. The remaining (and some new) issues are below.

(1a) (Modified from first round) I previously suggested to dissect what appears to be three different patterns of epistasis: "strong" and "weak" global epistasis and what one can could "purely idiosyncratic", i.e., not dependent on background fitness. The authors attempted to address this, but I don't think what they have done is sufficient. They make a statement "The lethal mutations have a slope smaller than -0.7 and average slope of -0.98. The remaining mutations all have a slope greater than -0.56" (LL 274-276)", but there is no evidence provided to support this claim. This is a strong and I think interesting statement (btw, how is "lethal" defined?) and warrants a dedicated figure. This statement suggests that the mixed patterns shown in Figure 5 can actually be meaningfully separated. Why don't the authors show this? Instead, they still claim "overall, global epistasis is not very strong on the folA landscape" (LL. 273-274). I maintain that this claim does not quite capture the observations.

Later in the text there is a whole section called "Only a small fraction of mutations exhibit strong global epistasis", which also seems related to this issue. First, I don't follow the logic here. Why is this section separate from this initial discussion? Second, here the authors claim "only a small subset of mutations exhibits strong global epistasis (R^2 > 0.5)" and then "This sharp contrast suggests a binary behavior of mutations: they either exhibit strong global epistasis (R2 > 0.5), or not (R2 < 0.5)." But this R^2 threshold seems arbitrary, and I don't see any statistical support for this binary nature.

(1b) (Verbatim from first round) Another rather remarkable feature of this plot is that the slopes of the strong global epistasis patterns sem to be very similar across mutations. Is this the case? Is there anything special about this slope? For example, does this slope simply reflect the fact that a given mutation becomes essentially lethal (i.e., produces the same minimal fitness) in a certain set of background genotypes?

(1c) (Verbatim from first round) Finally, how consistent are these patterns with some null expectations? Specifically, would one expect the same distribution of global epistasis slopes on an uncorrelated landscape? Are the pivot points unusually clustered relative to an expectation on an uncorrelated landscape?

(1d) (Verbatim from first round) The shapes of the DFE shown in Figure 7 are also quite interesting, particularly the bimodal nature of the DFE in high-fitness (HF) backgrounds. I think this bimodalilty must be a reflection of clustering of mutation-background combinations mentioned above. I think the authors ought to draw this connection explicitly. Do all HF backgrounds have a bimodal DFE? What mutations occupy the "moving" peak?

(1e) (Modified from first round). I still don't understand why there are qualitative differences in the shape of the DFE between functional and non-functional backgrounds (Figure 8B,C). Why is the transition between bimodal DFE in Figure 8B and unimodal DFE in Figure 8C is so abrupt? Perhaps the authors can plot the DFEs for all backgrounds on the same plot and just draw a line that separates functional and non-functional backgrounds so that the reader can better see whether DFE shape changes gradually or abruptly.

(1f) (Modified from first round) I am now more convinced that synonymous mutations alter epistasis and behave differently than non-synonymous mutations, but I still have some questions. (i) I would have liked a side-by-side comparison of synonymous and non-synonymous mutations, both in terms of their effects on fitness and on epistasis.<br /> (ii) The authors claim (LL 278-286) that "synonymous substitutions tend to follow two recurring behaviors" but this is not shown. To demonstrate this, the authors ought to plot (for example) the distribution of slopes of regression lines. Is this distribution actually bimodal? (iii) Later in the same paragraph the authors say "synonymous changes do not exhibit very strong background fitness-dependence". I don't see how this follows from the previous discussion.

(2) The authors claim to have improved statistical rigor of their analysis, but the Methods section is really thin and inadequate for understanding how the statistical analyses were done.

(3) In general, I notice a regrettable lack of attention to detail in the text, which makes me worried about a similar problem in the actual data analysis. Here are a few examples. (i) Throughout the text, the authors now refer to functional and non-functional genotypes, but several figures and captions retained the old HF and LF designations. (ii) Figure 7 is called Figure 8. (iii) Figure 3B is not discussed, though it logically precedes Figure 3A and 3C. (iv) Many of my comments, especially minor, were not addressed at all.

Reviewer #3 (Public review):

Summary:

The authors have studied a previously published large dataset on the fitness landscape of a 9 base-pair region of the folA gene. The objective of the paper is to understand various aspects of epistasis in this system, which the authors have achieved through detailed and computationally expensive exploration of the landscape. The authors describe epistasis in this system as "fluid", meaning that it depends sensitively on the genetic background, thereby reducing the predictability of evolution at the genetic level. However, the study also finds some robust patterns. The first is the existence of a "pivot point" for a majority of mutations, which is a fixed growth rate at which the effect of mutations switches from beneficial to deleterious (consistent with a previous study on the topic). The second is the observation that the distribution of fitness effects (DFE) of mutations is predicted quite well by the fitness of the genotype, especially for high-fitness genotypes. While the work does not offer a synthesis of the multitude of reported results, the information provided here raises interesting questions for future studies in this field.

Strengths:

A major strength of the study is its multifaceted approach, which has helped the authors tease out a number of interesting epistatic properties. The study makes a timely contribution by focusing on topical issues like global epistasis, the existence of pivot points, and the dependence of DFE on the background genotype and its fitness.

The authors have classified pairwise epistasis into six types, and found that the type of epistasis changes depending on background mutations. Switches happen more frequently for mutations at functionally important sites. Interestingly, the authors find that even synonymous mutations can alter the epistatic interaction between mutations in other codons, and this effect is uncorrelated with the direct fitness effects of the synonymous mutations. Alongside the observations of "fluidity", the study reports limited instances of global epistasis (which predicts a simple linear relationship between the size of a mutational effect and the fitness of the genetic background in which it occurs). Overall, the work presents strong evidence for the genetic context-dependent nature of epistasis in this system.

Weaknesses:

Despite the wealth of information provided by the study, there are a few points of concern.

The authors find that in non-functional genotypic backgrounds, most pairs of mutations display no epistasis. However, we do not know if this simply because a significant epistatic signal is hard to detect since all the fitness values involved in calculating epistasis are small (and therefore noise-prone). A control can be done by determining whether statistically significant differences exist among the fitness values themselves. In the absence of such information, it is hard to understand whether the classification of epistasis for non-functional backgrounds into discrete categories, such as in Fig 1C, is meaningful.

The authors have looked for global epistasis (i.e. a negative dependence of mutational fitness effect on background fitness) in all 108 (9x12) mutations in the landscape. They report that the majority of the mutations (77/108 or about 71 per cent) display weak correlation between fitness effect and background fitness (R^2<0.2), and a relatively small proportion show particularly strong correlation (R^2>0.5). They therefore conclude that global epistasis in this system is 'binary'-meaning that strong global epistasis is restricted to a few sites, whereas weak global epistasis occurs in the rest (Figure 5). Precise definitions of 'strong' and 'weak' are not given in the text, but the authors do mention that they are interested here primarily in detecting whether a correlation with background fitness exists or not. This again raises the question of the extent to which the low (and possibly noisy) fitness values of non-functional backgrounds can confound the results. For example, would the results be much the same if the analysis was repeated with only high-fitness backgrounds or only those sets of genotypes where the fitness differences between backgrounds and mutants were significant?<br /> Apart from this, I am also a bit conceptually perplexed by the term 'binary behavior', which suggests that the R^2 values should belong to two distinct classes; but, even assuming that the reported results are robust, Figure S12 shows that most values are 0.2 or less whereas higher values are more or less evenly distributed in the range 0.2-1.0, rather than showing an overall bimodal pattern. An especially confusing remark by the authors in this regard is the following; "This sharp contrast suggests a binary behavior of mutations: they either exhibit strong global epistasis (R^2 > 0.5), or not (R^2 < 0.5)'.

Conclusions: As large datasets on empirical fitness landscapes become increasingly available, more computational studies are needed to extract as much information from them as possible. The authors have made a timely effort in this direction. It is particularly instructive to learn from the work that higher-order epistasis is pervasive in the studied intragenic landscape, at least in functional genotypic backgrounds. Some of the analysis and interpretations in the paper require careful scrutiny, and the lack of a synthesis of the multitude of reported results leaves something to be desired. But the paper contains intriguing observations that can fuel further research into the factors shaping the topography of complex landscapes.

Reviewer #1 (Public review):

Summary:

Dendrotweaks provides to its users a solid tool to implement, visualize, tune, validate, understand, and reduce single-neuron models that incorporate complex dendritic arbors with differential distribution of biophysical mechanisms. The visualization of dendritic segments and biophysical mechanisms therein provide users an intuitive way to understand and appreciate dendritic physiology.

Reviewer #2 (Public review):

The paper by Makarov et al. describes the software tool called DendroTweaks, intended for examination of multi-compartmental biophysically detailed neuron models. It offers extensive capabilities for working with very complex distributed biophysical neuronal models and should be a useful addition to the growing ecosystem of tools for neuronal modeling.

Strengths

• This Python-based tool allows for visualization of a neuronal model's compartments.

• The tool works with morphology reconstructions in the widely used .swc and .asc formats.

• It can support many neuronal models using the NMODL language, which is widely used for neuronal modeling.

• It permits one to plot the properties of linear and non-linear conductances in every compartment of a neuronal model, facilitating examination of model's details.

• DendroTweaks supports manipulation of the model parameters and morphological details, which is important for exploration of the relations of the model composition and parameters with its electrophysiological activity.

• The paper is very well written - everything is clear, and the capabilities of the tool are described and illustrated with great attention to details.

Weaknesses

• Not a really big weakness, but it would be really helpful if the authors showed how the performance of their tool scales. This can be done for an increasing number of compartments - how long does it take to carry out typical procedures in DendroTweaks, on a given hardware, for a cell model with 100 compartments, 200, 300, and so on? This information will be quite useful to understand the applicability of the software.

Let me also add here a few suggestions (not weaknesses, but something that can be useful, and if the authors can easily add some of these for publication, that would strongly increase the value of the paper).

• It would be very helpful to add functionality to read major formats in the field, such as NeuroML and SONATA.

• Visualization is available as a static 2D projection of the cell's morphology. It would be nice to implement 3D interactive visualization.

• It is nice that DendroTweaks can modify the models, such as revising the radii of the morphological segments or ionic conductances. It would be really useful then to have the functionality for writing the resulting models into files for subsequent reuse.

• If I didn't miss something, it seems that DendroTweaks supports allocation of groups of synapses, where all synapses in a group receive the same type of Poisson spike train. It would be very useful to provide more flexibility. One option is to leverage the SONATA format, which has ample functionality for specifying such diverse inputs.

• "Each session can be saved as a .json file and reuploaded when needed" - do these files contain the whole history of the session or the exact snapshot of what is visualized when the file is saved? If the latter, which variables are saved, and which are not? Please clarify.

Comments on revisions:

In this revised version of the paper, the authors addressed all my comments. While many of the suggestions were addressed by textual changes in the manuscript or an explanation in the response to the reviewers (rather than adding substantial new functionality to the tool), DendroTweaks in its current updated state does represent an advanced and useful tool. Further extensions can be added as the development of the tool continues, in interaction with the community.

Reviewer #1 (Public review):

Summary:

The authors conducted a human neuroimaging study investigating the role of context in the representation of fear associations when the contingencies between a conditioned stimulus and shock unconditioned stimulus switches between contexts. The novelty of the analysis centered on neural pattern similarity to derive a measure of context and cue stability and generalization across different regions of the brain. Given the complexity and nuance of the results, it is kind of difficult to provide a concise summary. But during fear and reversal, there was cue generalization (between current CS+ cues) in the canonical fear network, and "item stability" for cues that changed their association with the shock in the IFG and precuneus. Reinstatement was quantified as pattern similarity for items or sets of cues from the earlier phases to the test phases, and they found different patterns in the IFG and dmPFC. A similar analytical strategy was applied to contexts.

Strengths:

Overall, I found this to be a novel use of MVPA to study the role of context in reversal/extinction of human fear conditioning that yielded interesting results. The paper was overall well-written, with a strong introduction and fairly detailed methods and results. The lack of any univariate contrast results from the test phases was used as motivation for the neural pattern similarity approach, which I appreciated as a reader.

I have no additional or new comments. The authors adequately addressed my major comments and concerns.

Reviewer #2 (Public review):

Summary:

This is a timely and original study on the geometry of macroscopic (2.5 mm) brain representations of multiple cues and contexts in Pavlovian fear conditioning. The authors report that these representations differ between initial learning, and reversal learning, and remain stable during extinction.

Strengths:

The authors address an important question and use a rigorous experimental methodology.

Weaknesses:

The findings are limited by the chosen spatial resolution (2.5 mm) which is far away from what modern fMRI can achieve. Also, region-of-interesting findings should be considered exploratory due to the chosen FDR method for correction for multiple comparison (which is transparently reported).

Reviewer #1 (Public review):

Summary:

In this manuscript, Henning et al. examine the impact of GABAergic feedback inhibition on the motion-sensitive pathway of flies. Based on a previous behavioral screen, the authors determined that C2 and C3, two GABAergic inhibitory feedback neurons in the optic lobes of the fly, are required for the optomotor response. Through a series of calcium imaging and disruption experiments, connectomics analysis, and follow-up behavioral assays, the authors concluded that C2 and C3 play a role in temporally sharpening visual motion responses. While this study employs a comprehensive array of experimental approaches, I have some reservations about the interpretation of the results in their current form. I strongly encourage the authors to provide additional data to solidify their conclusions. This is particularly relevant in determining whether this is a general phenomenon affecting vision or a specific effect on motion vision. Knowing this is also important for any speculation on the mechanisms of the observed temporal deficiencies.

Strengths:

This study uses a variety of experiments to provide a functional, anatomical, and behavioral description of the role of GABAergic inhibition in the visual system. This comprehensive data is relevant for anyone interested in understanding the intricacies of visual processing in the fly.

Weaknesses:

(1) The most fundamental criticism of this study is that the authors present a skewed view of the motion vision pathway in their results. While this issue is discussed, it is important to demonstrate that there are no temporal deficiencies in the lamina, which could be the case since C2 and C3, as noted in the connectomics analysis, project strongly to laminar interneurons. If the input dynamics are indeed disrupted, then the disruption seen in the motion vision pathway would reflect disruptions in temporal processing in general and suggest that these deficiencies are inherited downstream. A simple experiment could test this. Block C2, C3, and both together using Kir2.1 and Shibire independently, then record the ERG. Alternatively, one could image any other downstream neuron from the lamina that does not receive C2 or C3 input.

(2) Figure 6c. More analysis is required here, since the authors claim to have found a loss in inhibition (ND). However, the difference in excitation appears similar, at least in absolute magnitude (see panel 6c), for PD direction for the T4 C2 and C3 blocks. Also, I predict that C2 & C3 block statistically different from C3 only, why? In any case, it would be good to discuss the clear trend in the PD direction by showing the distribution of responses as violin plots to better understand the data. It would also be good to have some raw traces to be able to see the differences more clearly, not only polar plots and averages.

(3) The behavioral experiments are done with a different disruptor than the physiological ones. One blocks chemical synapses, the other shunts the cells. While one would expect similar results in both, this is not a given. It would be great if the authors could test the behavioral experiments with Kir2.1, too.

Reviewer #2 (Public review):

Summary:

The work by Henning et al. explores the role of feedback inhibition in motion vision circuits, providing the first identification of inhibitory inheritance in motion-selective T4 and T5 cells of Drosophila. This work advances our current knowledge in Drosophila motion vision and sets the way for further exploring the intricate details of direction-selective computations.

Strengths:

Among the strengths of this work is the verification of the GABAergic nature of C2 and C3 with genetic and immunohistochemical approaches. In addition, double-silencing C2&C3 experiments help to establish a functional role for these cells. The authors holistically use the Drosophila toolbox to identify neural morphologies, synaptic locations, network connectivity, neuronal functions, and the behavioral output.

Weaknesses:

The authors claim that C2 and C3 neurons are required for direction selectivity, as per the publication's title; however, even with their double silencing, the directional T4 & T5 responses are not completely abolished. Therefore, the contribution of this inherited feedback in direction-selective computations is not a prerequisite for its emergence, and the title could be re-adjusted.

Connectivity is assessed in one out of the two available connectome datasets; therefore, it would make the study stronger if the same connectivity patterns were identified in both datasets.

The mediating neural correlates from C2 & C3 to T4 & T5 are not clarified; rather, Mi1 is found to be one of them. The study could be improved if the same set of silencing experiments performed for C2-Mi1 were extended to C2 &C3-Tm1 or Tm4 to find the T5 neural mediators of this feedback inhibition loop. Stating more clearly from the connectomic analysis, the potential T5 mediators would be equally beneficial. Future experiments might also disentangle the parallel or separate functions of C2 and C3 neurons.

Finally, the authors' conclusions derive from the set of experiments they performed in a logical manner. Nonetheless, the Discussion could benefited from a more extensive explanation on the following matters: why do the ON-selective C2 and C3 neurons control OFF-generated behaviors, why the T4&T5 responses after C2&C3 silencing differ between stationary and moving stimuli and finally why C2 and not C3 had an effect in T5 DS responses, as the connectivity suggests C3 outputting to two out of the four major T5 cholinergic inputs.

Reviewer #3 (Public review):

Summary:

This article is about the neural circuitry underlying motion vision in the fruit fly. Specifically, it regards the roles of two identified neurons, called C2 and C3, that form columnar connections between neurons in the lamina and medulla, including neurons that are presynaptic to the elementary motion detectors T4 and T5. The approach takes advantage of specific fly lines in which one can disable the synaptic outputs of either or both of the C2/3 cell types. This is combined with optical recording from various neurons in the circuit, and with behavioral measurements of the turning reaction to moving stimuli.

The experiments are planned logically. The effects of silencing the C2/C3 neurons are substantial in size. The dominant effect is to make the responses of downstream neurons more sustained, consistent with a circuit role in feedback or feedforward inhibition. Silencing C2/C3 also makes the motion-sensitive neurons T4/T5 less direction-selective. However, the turning response of the fly is affected only in subtle ways. Detection of motion appears unaffected. But the response fails to discriminate between two motion pulses that happen in close succession. One can conclude that C2/C3 are involved in the motion vision circuit, by sharpening responses in time, though they are not essential for its basic function of motion detection.

Strengths:

The combination of cutting-edge methods available in fruit fly neuroscience. Well-planned experiments carried out to a high standard. Convincing effects documenting the role of these neurons in neural processing and behavior.

Weaknesses:

The report could benefit from a mechanistic argument linking the effects at the level of single neurons, the resulting neural computations in elementary motion detectors, and the altered behavioral response to visual motion.

Reviewer #1 (Public review):

This work by Reitz, Z. L. et al. developed an automated tool for high-throughput identification of microbial metallophore biosynthetic gene clusters (BGCs) by integrating knowledge of chelating moiety diversity and transporter gene families. The study aimed to create a comprehensive detection system combining chelator-based and transporter-based identification strategies, validate the tool through large-scale genomic mining, and investigate the evolutionary history of metallophore biosynthesis across bacteria.

Major strengths include providing the first automated, high-throughput tool for metallophore BGC identification, representing a significant advancement over manual curation approaches. The ensemble strategy effectively combines complementary detection methods, and experimental validation using HPLC-HRMS strengthens confidence in computational predictions. The work pioneers a global analysis of metallophore diversity across the bacterial kingdom and provides a valuable dataset for future computational modeling.

Some limitations merit consideration. First, ground truth datasets derived from manual curation may introduce selection bias toward well-characterized systems, potentially affecting performance assessment accuracy. Second, the model's dependence on known chelating moieties and transporter families constrains its ability to detect novel metallophore architectures, limiting discovery potential in metagenomic datasets. Third, while the proposed evolutionary hypothesis is internally consistent, it lacks direct validation and remains speculative without additional phylogenetic studies.

The authors successfully achieved their stated objectives. The tool demonstrates robust performance metrics and practical utility through large-scale application to representative genomes. Results strongly support their conclusions through rigorous validation, including experimental confirmation of predicted metallophores via HPLC-HRMS analysis.

The work provides a significant and immediate impact by enabling the transition from labor-intensive manual approaches to automated screening. The comprehensive phylogenetic framework advances understanding of bacterial metal acquisition evolution, informing future studies on microbial metal homeostasis. Community utility is substantial, since the tool and accompanying dataset create essential resources for comparative genomics, algorithm development, and targeted experimental validation of novel metallophores.

Reviewer #2 (Public review):

Summary:

This study presents a systematic and well-executed effort to identify and classify bacterial NRP metallophores. The authors curate key chelator biosynthetic genes from previously characterized NRP-metallophore biosynthetic gene clusters (BGCs) and translate these features into an HMM-based detection module integrated within the antiSMASH platform.

The new algorithm is compared with a transporter-based siderophore prediction approach, demonstrating improved precision and recall. The authors further apply the algorithm to large-scale bacterial genome mining and, through reconciliation of chelator biosynthetic gene trees with the GTDB species tree using eMPRess, infer that several chelating groups may have originated prior to the Great Oxidation Event.

Overall, this work provides a valuable computational framework that will greatly assist future in silico screening and preliminary identification of metallophore-related BGCs across bacterial taxa.

Strengths:

(1) The study provides a comprehensive curation of chelator biosynthetic genes involved in NRP-metallophore biosynthesis and translates this knowledge into an HMM-based detection algorithm, which will be highly useful for the initial screening and annotation of metallophore-related BGCs within antiSMASH.

(2) The genome-wide survey across a large bacterial dataset offers an informative and quantitative overview of the taxonomic distribution of NRP-metallophore biosynthetic chelator groups, thereby expanding our understanding of their phylogenetic prevalence.

(3) The comparative evolutionary analysis, linking chelator biosynthetic genes to bacterial phylogeny, provides an interesting and valuable perspective on the potential origin and diversification of NRP-metallophore chelating groups.

Weaknesses:

(1) Although the rule-based HMM detection performs well in identifying major categories of NRP-metallophore biosynthetic modules, it currently lacks the resolution to discriminate between fine-scale structural or biochemical variations among different metallophore types.

(2) While the comparison with the transporter-based siderophore prediction approach is convincing overall, more information about the dataset balance and composition would be appreciated. In particular, specifying the BGC identities, source organisms, and Gram-positive versus Gram-negative classification would improve transparency. In the supplementary tables, the "Just TonB" section seems to include only BGCs from Gram-negative bacteria - if so, this should be clearly stated, as Gram type strongly influences siderophore transport systems.

Reviewer #1 (Public review):

Summary:

The study explores the use of Transport-based morphometry (TBM) to predict hematoma expansion and growth 24 hours post-event, leveraging Non-Contrast Computed Tomography (NCCT) scans combined with clinical and location-based information. The research holds significant clinical potential, as it could enable early intervention for patients at high risk of hematoma expansion, thereby improving outcomes. The study is well-structured, with detailed methodological descriptions and a clear presentation of results. However, the practical utility of the predictive tool requires further validation, as the current findings are based on retrospective data. Additionally, the impact of this tool on clinical decision-making and patient outcomes needs to be further investigated.

Strengths

(1) Clinical Relevance: The study addresses a critical need in clinical practice by providing a tool that could enhance diagnostic accuracy and guide early interventions, potentially improving patient outcomes.

(2) Feature Visualization: The visualization and interpretation of features associated with hematoma expansion risk are highly valuable for clinicians, aiding in the understanding of model-derived insights and facilitating clinical application.

(3) Methodological Rigor: The study provides a thorough description of methods, results, and discussions, ensuring transparency and reproducibility.

Comments on revisions:

The authors have addressed my concerns.

Reviewer #1 (Public review):

Summary:

In this study, Donofrio et al. investigated cerebellar Purkinje cell (PC) degeneration during normal aging using both mouse and human samples. They found that PC loss followed a stripe pattern rather than occurring randomly. Although this pattern resembled the pattern of zebrin II expression in the anterior cerebellum, the overall pattern was different from zebrin II expression. Surviving PCs exhibited severe degeneration, including thickened axons, axonal torpedoes and shrunken dendrites. These structural changes were accompanied by functional deficits in motor coordination and tremor. Understanding why certain PC subpopulations are more vulnerable than others may provide insight into regional susceptibility (or resilience) to aging and inform potential therapeutic strategies for age-related neurological disorders. Overall, the findings are novel and significant, supported by compelling evidence from structural and functional analyses. The authors have fully addressed my previous concerns and improved the clarity of their presentation. I believe this work will have a significant impact in the field.

Reviewer #2 (Public review):

Summary:

The cerebellum is known to be vulnerable to aging, yet specific cell type vulnerability remains understudied. This important study convincingly demonstrate that the normal aged mouse cerebellum exhibits Purkinje cell loss, and that the vulnerable PCs to age are arranged on the basis of known Zebrin stripe pattern that represents a particular subtype of the PCs. As the authors wrote, future studies should investigate why this PC loss phenotype occurs stochastically across the population, and whether these findings parallel human cerebellar aging.

Strength:

• Banding pattern of PC loss is very clearly demonstrated by combining immunostaining for Zebrin.

• A critical methodological concern that a standard PC marker, Calbindin, could be compromised in aging has been addressed by performing control experiments with appropriate counterstaining and a transgenic mouse.

• Parallels with neurodegenerative phenotype would be helpful to understand the mechanisms of age-related PC loss in future.

Weakness:

• Limited strain diversity: The study exclusively uses C57BL/6J mice despite known genetic and motor differences among even closely related strains like C57BL/6N, weakening the generalizability of the findings. However, on the other hand, the presence of age-related PC loss makes C57BL/6J an interesting mouse model for studying aging of the cerebellum.

• Linkages with normal human aging and cerebellar function is not supported well. It remains unclear whether this PC loss phenomenon is universal or specific to a particular individual, and whether specific to human PC subtype.

Reviewer #3 (Public review):

Donofrio et al. report a new observation that in normal aging mice, anti-calbindin whole-mount staining and coronal immunohistochemistry in the cerebellum often show a sagittally patterned loss of Purkinje cells with age. The authors address a central concern that calbindin antibody staining alone is not sufficient to definitively assess Purkinje cell loss, and corroborate their antibody staining data with transgenic Pcp2-CRE x flox-GFP reporter mice and Neutral Red staining. The authors then investigate whether this patterned Purkinje loss correlates with the known parasagittal expression of zebrin-II, finding a strong but imperfect correlation with zebrin-II antibody staining. They next draw a connection between this age-related Purkinje loss to the age-related decline in motor function in mice, with trending but non-significant statistical association between the severity/patterning of Purkinje loss and motor phenotypes within cohorts of aged mice. Finally, the authors look at post-mortem human cerebellar tissues from deceased healthy donors between 21 and 74 years of age, finding a positive correlation between Purkinje degeneration and age, but with unknown spatial patterning.

The conclusions drawn from this study are well supported by the data provided, with image quantification corroborating visual observations. The authors highlight several examples of parasagittal patterning of Purkinje cell degeneration in disease, and they show that proper methodologies must be used to account for these patterns to avoid highly variable data in the sagittal plane. The authors aptly point out that additional work is needed to investigate the spatial patterns of Purkinje cell loss in the human cerebellum.

Reviewer #1 (Public review):

In their paper entitled "Combined transcriptomic, connectivity, and activity profiling of the medial amygdala using highly amplified multiplexed in situ hybridization (hamFISH)" Edwards et al. present a new method designated as hamFISH (highly amplified multiplexed in situ hybridization) that enables sequential detection of {less than or equal to}32 genes using multiplexed branched DNA amplification. As proof-of-principle, the authors apply the new technique - in conjunction with connectivity, and activity profiling - to the medial amygdala (MeA) of the mouse, which is a critical nucleus for innate social and defensive behaviors.

As mentioned by Edwards et al., hamFISH could prove beneficial as an affordable alternative to other in situ transcriptomic methods, including commercial platforms, that are resource-intensive and require complex analysis pipelines. Thus, the authors envision that the method they present could democratize in situ cell-type identification in individual laboratories.

The data presented by Edwards et al. is convincing. The authors use the appropriate and validated methodology in line with the current state-of-the-art. The paper makes a strong case for the benefits of hamFISH when combining transcriptomics studies with connectivity tracing and immediate early gene-based activity profiling. Notably, the authors also discuss the caveats and limitations of their study/approach in an open and transparent manner.

Comments on revisions:

In their revised paper, Edwards et al. have made an effort to improve manuscript clarity. Revisions made address the non-public "recommendations for the authors." The main criticism that prevents a more enthusiastic overall assessment, i.e., absence of some more in-depth hypothesis-based analysis (though, as originally mentioned, maybe beyond the study's scope), is still valid.

Reviewer #2 (Public review):

The authors describe the development and implementation of hamFISH, a sensitive multiplexed ISH method. They leverage a pre-existing scRNA-seq dataset for the MeA to design 32 probes that combinatorically represent MeA neuronal populations - ~80% of MeA neurons express at least three of these 32 markers. Using these markers to assess the spatial organization of the MeA, the authors identify a novel population of Ndnf+ projection neurons and characterize their connectivity with anterograde and retrograde labeling. They additionally combine hamFISH with CTB labeling of three principal MeA projections sites to show that 75% of MeA neurons have only a single projection target. Finally, they engage adult male mice in encounters with other adult males (aggression), females (mating), and pups (infanticide), followed with hamFISH and c-fos labeling to relate cell identity to behavior. Their overall conclusion is that hamFISH-defined cell types are broadly active to multiple sensory stimuli. However, the data presented are not sufficient to conclude that no selectivity exists.

A strength of the manuscript is the novel hamFISH approach, which is technically innovative and could potentially be adopted by many labs. However, a weakness is that the 32 selected hamFISH marker genes employed here are predominantly neuropeptides. These genes, such as Tac1, Cartpt, Adcyap1, Calb1, and Gal, are expressed throughout the MeA, and many other brain regions and are not selective for transcriptomic cell types or developmental lineages. The use of hamFISH probes that provide a more stringent classification of cell type or cell identity could potentially provide a different picture of sensory response selectivity within the MeA. Thus, although the data in the manuscript are exemplary, the biological insight into MeA function is more limited.

Reviewer #3 (Public review):

Summary:

In this manuscript, Edwards et al. describe hamFISH, a customizable and cost-efficient method for performing targeted spatial transcriptomics. hamFISH utilizes highly amplified multiplexed branched DNA amplification, and the authors extensively describe hamFISH development and its advantages over prior variants of this approach.

The authors then used hamFISH to investigate an important circuit in the mouse brain for social behavior, the medial amygdala (MeA). To develop a hamFISH probe set capable of distinguishing MeA neurons, the authors mined published single cell RNA-sequencing datasets of the MeA, ultimately creating a panel of 32 hamFISH probes that mostly cover the identified MeA cell types. They evaluated over 600,000 MeA cells and classified neurons into 16 inhibitory and 10 excitatory types, many of which are spatially clustered.

The authors combined hamFISH with viral and other circuit tracer injections to determine whether the identified MeA cell populations sent and/or received unique inputs from connected brain regions, finding evidence that several cell types had unique patterns of input and output. Finally, the authors performed hamFISH on the brains of male mice that were placed in behavioral conditions that elicit aggressive, infanticidal, or mating behaviors, finding that some cell populations are selectively activated (as assessed by c-fos mRNA expression) in specific social contexts.

Strengths:

(1) The authors developed an optimized tissue preparation protocol for hamFISH and implemented oligopools instead of individually synthesized oligonucleotides to reduce costs. The branched DNA amplification scheme improved smFISH signal compared to previous methods, and multiple variants provide additional improvements in signal intensity and specificity. Compared to other spatial transcriptomics methods, the pipeline for imaging and analysis is streamlined, and is compatible with other techniques like fluorescence-based circuit tracing. This approach is cost-effective and has several advantages that make it a valuable addition to the list of spatial transcriptomics toolkits.

(2) Using 31 probes, hamFISH was able to detect 16 inhibitory and 10 excitatory neuron types in the MeA subregions, including the vast majority of cell types identified by other transcriptomics approaches. The authors quantified the distributions of these cell types along the anterior-posterior, dorsal-ventral, and medial-lateral axes, finding spatial segregation among some, but not all, MeA excitatory and inhibitory cell types. The authors additionally identified a class of inhibitory neurons expressing Ndnf (and a subset of these that express Chrna7) that project to multiple social chemosensory circuits.

(3) The authors combined hamFISH with MeA input and output mapping, finding cell-type biases in the projections to the MPOA, BNST, and VMHvl, and inputs from multiple regions.

(4) The authors identified excitatory and inhibitory cell types, and patterns of activity across cell types, that were selectively activated during various social behaviors, including aggression, mating, and infanticide, providing new insights and avenues for future research into MeA circuit function.

Weaknesses:

(1) Gene selection for hamFISH is likely to still be a limiting factor, even with the expanded (32-probe) capacity. This may have contributed to the lack of ability to identify sexually dimorphic cell types (Fig. S2B). This is an expected tradeoff for a method that has major advantages in terms of cost and adaptability.

(2) Adaptation of hamFISH, for example, to adapt it to other brain regions or tissues, may require extensive optimization. This does not preclude it from being highly useful for other brain regions with extra effort.

(3) Pairing this method with behavioral experiments is likely to require further optimization, as c-fos mRNA expression is an indirect and incomplete survey of neuronal activity (e.g. not all cell types upregulate c-fos when electrically active). As such, there is a risk of false negative results that limit its utility for understanding circuit function.

(4) The incompatibility of hamFISH with thicker tissue samples and minimal optical sectioning introduce additional technical limitations. For example, it would be difficult to densely sample larger neural circuits using serial 20 micron sections.

Reviewer #1 (Public review):

In the current article, Octavia Soegyono and colleagues study "The influence of nucleus accumbens shell D1 and D2 neurons on outcome-specific Pavlovian instrumental transfer", building on extensive findings from the same lab. While there is a consensus about the specific involvement of the Shell part of the Nucleus Accumbens (NAc) in specific stimulus-based actions in choice settings (and not in General Pavlovian instrumental transfer - gPIT, as opposed to the Core part of the NAc), mechanisms at the cellular and circuitry levels remain to be explored. In the present work, using sophisticated methods (rat Cre-transgenic lines from both sexes, optogenetics and the well-established behavioral paradigm outcome-specific PIT - sPIT), Octavia Soegyono and colleagues decipher the differential contribution of dopamine receptors D1 and D2 expressing-spiny projection neurons (SPNs).

After validating the viral strategy and the specificity of the targeting (immunochemistry and electrophysiology), the authors demonstrate that while both NAc Shell D1- and D2-SPNs participate in mediating sPIT, NAc Shell D1-SPNs projections to the Ventral Pallidum (VP, previously demonstrated as crucial for sPIT), but not D2-SPNs, mediates sPIT. They also show that these effects were specific to stimulus-based actions, as value-based choices were left intact in all manipulations.

This is a well-designed study and the results are well supported by the experimental evidence. The paper is extremely pleasant to read and add to the current literature.

Comments on revisions:

We thank the authors for their detailed responses and for addressing our comments and concerns.

To further improve consistency and transparency, we kindly request that the authors provide, for Supplemental Figures S1-S4, panels E (raw data for lever presses during the PIT test), the individual data points together with the corresponding statistical analyses in the figure legends.

In addition, regarding Supplemental Figure S3, panel E, we note the absence of a PIT effect in the eYFP group under the ON condition, which appears to differ from the net response reported in the main Figure 5, panel B. Could the authors clarify this apparent discrepancy?

We also note a discrepancy between the authors' statement in their response ("40 rats excluded based on post-mortem analyses") and the number of excluded animals reported in the Materials and Methods section, which adds up to 47. We kindly ask the authors to clarify this point for consistency.

Finally, as a minor point, we suggest indicating the total number of animals used in the study in the Materials and Methods section.

Reviewer #2 (Public review):

Summary:

This manuscript by Soegyono et a. describes a series of experiments designed to probe the involvement of dopamine D1 and D2 neurons within the nucleus accumbens shell in outcome-specific Pavlovian-instrumental transfer (osPIT), a well-controlled assay of cue-guided action selection based on congruent outcome associations. They used an optogenetic approach to phasically silence NAc shell D1 (D1-Cre mice) or D2 (A2a-Cre mice) neurons during a subset of osPIT trials. Both manipulations disrupted cue-guided action selection but had no effects on negative control measures/tasks (concomitant approach behavior, separate valued guided choice task), nor were any osPIT impairments found in reporter only control groups. Separate experiments revealed that selective inhibition of NAc shell D1 but not D2 inputs to ventral pallidum were required for osPIT expression, thereby advancing understanding of the basal ganglia circuitry underpinning this important aspect of decision making.

Strengths:

The combinatorial viral and optogenetic approaches used here were convincingly validated through anatomical tract-tracing and ex vivo electrophysiology. The behavioral assays are sophisticated and well-controlled to parse cue and value guided action selection. The inclusion of reporter only control groups is rigorous and rules out nonspecific effects of the light manipulation. The findings are novel and address a critical question in the literature. Prior work using less decisive methods had implicated NAc shell D1 neurons in osPIT but suggested that D2 neurons may not be involved. The optogenetic manipulations used in the current study provides a more direct test of their involvement and convincingly demonstrate that both populations play an important role. Prior work had also implicated NAc shell connections to ventral pallidum in osPIT, but the current study reveals the selective involvement of D1 but not D2 neurons in this circuit. The authors do a good job of discussing their findings, including their nuanced interpretation that NAc shell D2 neurons may contribute to osPIT through their local regulation of NAc shell microcircuitry.

Weaknesses:

The current study exclusively used an optogenetic approach to probe the function of D1 and D2 NAc shell neurons. Providing a complementary assessment with chemogenetics or other appropriate methods would strengthen conclusions, particularly the novel demonstration for D2 NAc shell involvement. Likewise, the null result of optically inhibiting D2 inputs to ventral pallidum leaves open the possibility that a more complete or sustained disruption of this pathway may have impaired osPIT.

Conclusions:

The research described here was successful in providing critical new insights into the contributions of NAc D1 and D2 neurons in cue-guided action selection. The authors' data interpretation and conclusions are well reasoned and appropriate. They also provide a thoughtful discussion of study limitations and implications for future research. This research is therefore likely to have a significant impact on the field.

Comments on revisions:

I have reviewed the rebuttal and revised manuscript and have no remaining concerns.

Reviewer #1 (Public review):

In the current article, Octavia Soegyono and colleagues study "The influence of nucleus accumbens shell D1 and D2 neurons on outcome-specific Pavlovian instrumental transfer", building on extensive findings from the same lab. While there is a consensus about the specific involvement of the Shell part of the Nucleus Accumbens (NAc) in specific stimulus-based actions in choice settings (and not in General Pavlovian instrumental transfer - gPIT, as opposed to the Core part of the NAc), mechanisms at the cellular and circuitry levels remain to be explored. In the present work, using sophisticated methods (rat Cre-transgenic lines from both sexes, optogenetics, and the well-established behavioral paradigm outcome-specific PIT-sPIT), Octavia Soegyono and colleagues decipher the differential contribution of dopamine receptors D1 and D2 expressing spiny projection neurons (SPNs).

After validating the viral strategy and the specificity of the targeting (immunochemistry and electrophysiology), the authors demonstrate that while both NAc Shell D1- and D2-SPNs participate in mediating sPIT, NAc Shell D1-SPNs projections to the Ventral Pallidum (VP, previously demonstrated as crucial for sPIT), but not D2-SPNs, mediates sPIT. They also show that these effects were specific to stimulus-based actions, as value-based choices were left intact in all manipulations.

This is a well-designed study, and the results are well supported by the experimental evidence. The paper is extremely pleasant to read and adds to the current literature.

Reviewer #2 (Public review):

Summary:

This manuscript by Soegyono et al. describes a series of experiments designed to probe the involvement of dopamine D1 and D2 neurons within the nucleus accumbens shell in outcome-specific Pavlovian-instrumental transfer (osPIT), a well-controlled assay of cue-guided action selection based on congruent outcome associations. They used an optogenetic approach to phasically silence NAc shell D1 (D1-Cre mice) or D2 (A2a-Cre mice) neurons during a subset of osPIT trials. Both manipulations disrupted cue-guided action selection but had no effects on negative control measures/tasks (concomitant approach behavior, separate valued guided choice task), nor were any osPIT impairments found in reporter-only control groups. Separate experiments revealed that selective inhibition of NAc shell D1 but not D2 inputs to ventral pallidum was required for osPIT expression, thereby advancing understanding of the basal ganglia circuitry underpinning this important aspect of decision making.

Strengths:

The combinatorial viral and optogenetic approaches used here were convincingly validated through anatomical tract-tracing and ex vivo electrophysiology. The behavioral assays are sophisticated and well-controlled to parse cue and value-guided action selection. The inclusion of reporter-only control groups is rigorous and rules out nonspecific effects of the light manipulation. The findings are novel and address a critical question in the literature. Prior work using less decisive methods had implicated NAc shell D1 neurons in osPIT but suggested that D2 neurons may not be involved. The optogenetic manipulations used in the current study provide a more direct test of their involvement and convincingly demonstrate that both populations play an important role. Prior work had also implicated NAc shell connections to ventral pallidum in osPIT, but the current study reveals the selective involvement of D1 but not D2 neurons in this circuit. The authors do a good job of discussing their findings, including their nuanced interpretation that NAc shell D2 neurons may contribute to osPIT through their local regulation of NAc shell microcircuitry.

Weaknesses:

The current study exclusively used an optogenetic approach to probe the function of D1 and D2 NAc shell neurons. Providing a complementary assessment with chemogenetics or other appropriate methods would strengthen conclusions, particularly the novel demonstration of D2 NAc shell involvement. Likewise, the null result of optically inhibiting D2 inputs to the ventral pallidum leaves open the possibility that a more complete or sustained disruption of this pathway may have impaired osPIT.

Reviewer #3 (Public review):

Summary:

The authors present data demonstrating that optogenetic inhibition of either D1- or D2-MSNs in the NAc Shell attenuates expression of sensory-specific PIT while largely sparing value-based decision on an instrumental task. They also provide evidence that SS-PIT depends on D1-MSN projections from the NAc-Shell to the VP, whereas projections from D2-MSNs to the VP do not contribute to SS-PIT.

Strengths:

This is clearly written. The evidence largely supports the authors' interpretations, and these effects are somewhat novel, so they help advance our understanding of PIT and NAc-Shell function.

Weaknesses:

I think the interpretation of some of the effects (specifically the claim that D1-MSNs do not contribute to value-based decision making) is not fully supported by the data presented.

Reviewer #1 (Public review):

Summary:

The authors used high-density probe recordings in the medial prefrontal cortex (PFC) and hippocampus during a rodent spatial memory task to examine functional sub-populations of PFC neurons that are modulated vs. unmodulated by hippocampal sharp-wave ripples (SWRs), an important physiological biomarker that is thought to have role in mediating information transfer across hippocampal-cortical networks for memory processes. SWRs are associated with reactivation of representations of previous experiences, and associated reactivation in hippocampal and cortical regions have been proposed to have a role in memory formation, retrieval, planning, and memory-guided behavior. This study focuses of awake SWRs that are prevalent during immobility periods during pauses in behavior. Previous studies have reported strong modulation of a subset of prefrontal neurons during hippocampal SWRs, with some studies reporting prefrontal reactivation during SWRs that have a role in spatial memory processes. The study seeks to extend these findings by examining activity of SWR-modulated vs. unmodulated neurons across PFC sub-regions, and whether there is a functional distinction between these two kinds of neuronal populations with respect to representing spatial information and supporting memory-guided decision making.

Strengths:

The major strength of the study is the use of Neuropixels 1.0 probes to monitor activity throughput the dorsal-ventral extent of the rodent medial prefrontal cortex, permitting an investigation of functional distinction in neuronal populations across PFC sub-regions. They are able to show that SWR-unmodulated neurons, in addition to having stronger spatial tuning than SWR-modulated neurons as previously reported, also show stronger directional selectivity, and theta-cycle skipping properties.

Weaknesses:

(1) The title and abstract have been updated to reflect the updated interpretation that prefrontal neurons are involved in spatial tuning and signaling upcoming choice independently from hippocampal SWRs, implying the negative that these functions do not happen during SWRs. The evidence presented, however, is lacking and the analyses has key limitations that preclude such a conclusion. First, the fact that prefrontal neurons decode past and future choices independently of the hippocampus, not just hippocampal SWRs, is well-established (for e.g., Baeg et al., 2003, 10.1016/s0896-6273(03)00597-x). Second, the statement that prefrontal neurons are involved in spatial tuning independently from SWRs is inconsistent, since spatial tuning is assessed during exploratory behaviors that are not associated with SWRs. Apart from showing that non-local decoding occurs in prefrontal cortex outside SWR time periods, which is already established, the conclusion needs evidence this does not occur during SWR time periods, which is not presented.

(2) The results show that SWR-modulated prefrontal neurons are more linked to hippocampal non-local representations, whereas SWR-unmodulated neurons encode upcoming choice independently of SWRs. This is logical, and implies that SWR-modulated prefrontal neurons are involved in non-local decoding during hippocampal non-local representations. This hints at potentially multiple mechanisms, one involving independent prefrontal non-local decoding, and another involving prefrontal and hippocampal non-local decoding.

(3) The analyses have key limitations. The Methods section notes that decoding was performed in 50ms bins, periods with running speed less than 15cm/s were excluded, then decoded probabilities summed for each maze segment, followed by grouping probabilities together for local and non-local decoding. This implies that decoding segments can span entire maze segments or long time periods - this needs to be clarified and quantified. When examining time-locking of decoding segments to hippocampal SWRs, only non-local segments that occurred within 2 secs of SWRs were used. This raises several concerns. First, prefrontal modulation by hippocampal SWRs lasts primarily <500ms, so a 2sec temporal proximity will lead to non-SWR modulation periods being included in the analyses. In addition, even for decoding segments that may be in close temporal proximity, these can be very long, based on the analyses description. This can lead to spurious results. Second, if only running speeds >15cm/s were included, immobility periods are necessarily being excluded, which is when SWRs occur. So, this analysis cannot be used to investigate decoding during SWRs; rather, a direct approach of extracting prefrontal activity during SWRs and then decoding this activity is required.

Reviewer #2 (Public review):

Summary:

This work by den Bakker and Kloosterman contributes to the vast body of research exploring the dynamics governing the communication between the hippocampus (HPC) and the medial prefrontal cortex (mPFC) during spatial learning and navigation. Previous research showed that population activity of mPFC neurons is replayed during HPC sharp-wave ripple events (SWRs), which may therefore correspond to privileged windows for the transfer of learned navigation information from the HPC, where initial learning occurs, to the mPFC, which is thought to store this information long term. Indeed, it was also previously shown that the activity of mPFC neurons contains task-related information that can inform about the location of an animal in a maze, which can predict the animals' navigational choices. Here, the authors aim to show that the mPFC neurons that are modulated by HPC activity (SWRs and theta rhythms) are distinct from those "encoding" spatial information. This result could suggest that the integration of spatial information originating from the HPC within the mPFC may require the cooperation of separate sets of neurons.

This observation may be useful to further extend our understanding of the dynamics regulating the exchange of information between the HPC and mPFC during learning. However, my understanding is that this finding is mainly based upon a negative result, which cannot be statistically proven by the failure to reject the null hypothesis. Moreover, in my reading, the rest of the paper mainly replicates phenomena that have already been described, with the original reports not correctly cited. My opinion is that the novel elements should be precisely identified and discussed, while the current phrasing in the manuscript, in most cases, leads readers to think that these results are new. Detailed comments are provided below.

Major concerns:

ORIGINAL COMMENT: (1) The main claim of the manuscript is that the neurons involved in predicting upcoming choices are not the neurons modulated by the HPC. This is based upon the evidence provided in Figure 5, which is a negative result that the authors employ to claim that predictive non-local representations in the mPFC are not linked to hippocampal SWRs and theta phase. However, it is important to remember that in a statistical test, the failure to reject the null hypothesis does not prove that the null hypothesis is true. Since this claim is so central in this work, the authors should use appropriate statistics to demonstrate that the null hypothesis is true. This can be accomplished by showing that there is no effect above some size that is so small that it would make the effect meaningless (see https://doi.org/10.1177/070674370304801108).

AUTHOR RESPONSE: We would like to highlight a few important points here. (1) We indeed do not intend to claim that the SWR-modulated neurons are not at all involved in predicting upcoming choice, just that the SWR-unmodulated neurons may play a larger role. We have rephrased the title and abstract to make this clearer.

REVIEWER COMMENT: The title has been rephrased but still conveys the same substantive claim. The abstract sentence also does not clearly state what was found. Using "independently" in the new title continues to imply that SWR modulation and prediction of upcoming choices are separate phenomena. By contrast, in your response here in the rebuttall you state only that "SWR-unmodulated neurons may play a larger role," which is a much more tempered claim than what the manuscript currently argues. Why is this clarification not adopted in the article? Moreover, the main text continues to use the same arguments as before; beyond the cosmetic changes of title and abstract, the claim itself has not materially changed.

AUTHOR RESPONSE: (2) The hypothesis that we put forward is based not only on a negative effect, but on the findings that: the SWR-unmodulated neurons show higher spatial tuning (Fig 3b), more directional selectivity (Fig 3d), more frequent encoding of the upcoming choice at the choice point (new analysis, added in Fig 4d), and higher spike rates during the representations of the upcoming choice (Fig 5b). This is further highlighted by the fact that the representations of upcoming choice in the PFC are not time locked to SWRs (whereas the hippocampal representations of upcoming choice are; see Fig 5a and Fig 6a), and not time-locked to hippocampal theta phase (whereas the hippocampal representations are; see Fig 5c and Fig 6c). Finally, the representations of upcoming and alternative choices in the PFC do not show a large overlap in time with the representations in the hippocampus (see updated Fig 4e were we added a statistical test to show the likelihood of the overlap of decoded timepoints). All these results together lead us to hypothesize that SWR-modulation is not the driving factor behind non-local decoding in the PFC.

REVIEWER COMMENT: I do not see how these precisions address my remark. The main claim in the title used to be "Neurons in the medial prefrontal cortex that are not modulated by hippocampal sharp-wave ripples are involved in spatial tuning and signaling upcoming choice." It is now "Neurons in the medial prefrontal cortex are involved in spatial tuning and signaling upcoming choice independently from hippocampal sharp-wave ripples." The substance has not changed. This specific claim is supported solely by Figure 5.

The other analyses cited describe functional characteristics of SWR-unmodulated neurons but, unless linked by explicit new analyses, do not substantiate independence/orthogonality between SWR modulation and non-local decoding in PFC. If there is an analysis that makes this link explicit, it should be clearly presented; as it stands, I cannot find an explanation in the manuscript for why "all these results together" justify the conclusion that "All these results together lead us to hypothesize that SWR-modulation is not the driving factor behind non-local decoding in the PFC". Also: is the main result of this work a "hypothesis"? If so, this should be clearly differentiated from a conclusion supported by results and analyses.

AUTHOR RESPONSE: (3) Based on the reviewers suggestion, we have added a statistical test to compare the phase-locking based of the non-local decoding to hippocampal SWRs and theta phase to shuffled posterior probabilities. Instead of looking at all SWRs in a -2 to 2 second window, we have now only selected the closest SWR in time within that window, and did the statistical comparison in the bin of 0-20 ms from SWR onset. With this new analysis we are looking more directly at the time-locking of the decoded segments to SWR onset (see updated Fig 5a and 6a).

REVIEWER COMMENT: I appreciate the added analysis focusing on the closest SWR and a 0-20 ms bin. My understanding is that you consider the revised analyses in Figures 5a and 6a sufficient to show that predictive non-local representations in mPFC are not linked to hippocampal SWRs and theta phase.

First, the manuscript should explicitly explain the rationale for this analysis and why it is sufficient to support the claim. From the main text it is not possible to understand what was done; the Methods are hard to follow, and the figure legends are not clearly described (e.g. the shuffle is not even defined there).

Specific points I could not reconcile:

i) The gray histograms in the revised Figures 5a and 6a now show a peak at zero lag, whereas in the previous version they were flat, although they are said to plot the same data. What changed?

ii) Why choose a 20 ms bin? A single narrow bin invites false negatives. Please justify this choice.

iii) Comparing to a shuffle is a useful control, but when the p-value is non-significant we only learn that no difference was detected under that shuffle-not that there is no difference or that the processes are independent.

ORIGINAL COMMENT: (2) The main claim of the work is also based on Figure 3, where the authors show that SWRs-unmodulated mPFC neurons have higher spatial tuning, and higher directional selectivity scores, and a higher percentage of these neurons show theta skipping. This is used to support the claim that SWRs-unmodulated cells encode spatial information. However, it must be noted that in this kind of task, it is not possible to disentangle space and specific task variables involving separate cognitive processes from processing spatial information such as decision-making, attention, motor control, etc., which always happen at specific locations of the maze. Therefore, the results shown in Figure 3 may relate to other specific processes rather than encoding of space and it cannot be unequivocally claimed that mPFC neurons "encode spatial information". This limitation is presented by Mashoori et al (2018), an article that appears to be a major inspiration for this work. Can the authors provide a control analysis/experiment that supports their claim? Otherwise, this claim should be tempered. Also, the authors say that Jadhav et al. (2016) showed that mPFC neurons unmodulated by SWRs are less tuned to space. How do they reconcile it with their results?

AUTHOR RESPONSE: The reviewer is right to assert caution when talking about claims such as spatial tuning where other factors may also be involved. Although we agree that there may be some other factors influencing what we are seeing as spatial tuning, it is very important to note that the behavioral task is executed on a symmetrical 4-armed maze, where two of the arms are always used for the start of the trajectory, and the other two arms (North and South) function as the goal (reward) arms. Therefore, if the PFC is encoding cognitive processes such as task phases related to decision-making and reward, we would not be able to differentiate between the two start arms and the two goal arms, as these represent the same task phases. Note also that the North and South arm are illuminated in a pseudo-random order between trials and during cue-based rule learning this is a direct indication of where the reward will be found. Even in this phase of the task, the PFC encodes where the animal will turn on a trial-to-trial basis (meaning the North and South arm are still differentiated correctly on each trial even though the illumination and associated reward are changing).

REVIEWER COMMENT: I appreciate that the departure location was pseudorandomized. However, this control does not rule out that PFC activity reflects motor preparation (left vs right turns) and associated perceptual decision-making/attentional processes that are inherently tied to a specific action. As such, it cannot by itself support the claim that PFC neurons "encode spatial information." Moreover, the authors acknowledge here that "other factors may also be involved," yet this caveat is not reflected in the manuscript. Why?

AUTHOR RESPONSE: Secondly, importantly, the reviewer mentions that we claimed that Jadhav et al. (2016) showed that mPFC neurons unmodulated by SWRs are less tuned to space, but this is incorrect. Jadhav et al. (2016) showed that SWR-unmodulated neurons had lower spatial coverage, meaning that they are more spatially selective (congruent with our results). We have rephrased this in the text to be clearer.

REVIEWER COMMENT: Thanks for clarifying this.

ORIGINAL COMMENT: (3) My reading is that the rest of the paper mainly consists of replications or incremental observations of already known phenomena with some not necessarily surprising new observations:<br /> a) Figure 2 shows that a subset of mPFC neurons is modulated by HPC SWRs and theta (already known), that vmPFC neurons are more strongly modulated by SWRs (not surprising given anatomy), and that theta phase preference is different between vmPFC and dmPFC (not surprising given the fact that theta is a travelling wave).

AUTHOR RESPONSE: The finding that vmPFC neurons are more strongly modulated by SWRs than dmPFC indeed matches what we know from anatomy, but that does not make it a trivial finding. A lot remains unknown about the mPFC subregions and their interactions with the hippocampus, and not every finding will be directly linked to the anatomy. Therefore, in our view this is a significant finding which has not been studied before due to the technical complexity of large-scale recordings along the dorsal-ventral axis of the mPFC.

REVIEWER COMMENT: This finding is indeed non-trivial; however, it seems completely irrelevant to the paper's main claim unless the Authors can argue otherwise.

AUTHOR RESPONSE: Similarly, theta being a traveling wave (which in itself is still under debate), does not mean we should assume that the dorsal and ventral mPFC should follow this signature and be modulated by different phases of the theta cycle. Again, in our view this is not at all trivial, but an important finding which brings us closer to understanding the intricate interactions between the hippocampus and PFC in spatial learning and decision-making.

REVIEWER COMMENT: Yes, but in what way does this support the manuscript's primary claim? This is unclear to me.

ORIGINAL COMMENT: b) Figure 4 shows that non-local representations in mPFC are predictive of the animal's choice. This is mostly an increment to the work of Mashoori et al (2018). My understanding is that in addition to what had already been shown by Mashoori et al here it is shown how the upcoming choice can be predicted. The author may want to emphasize this novel aspect.

AUTHOR RESPONSE: In our view our manuscript focuses on a completely different aspect of learning and memory than the paper the reviewer is referring to (Mashoori et al. 2018). Importantly, the Mashoori et al. paper looked at choice evaluation at reward sites and shows that disappointing reinforcements are associated with reactivations in the ACC of the unselected target. This points to the role of the ACC in error detection and evaluation. Although this is an interesting result, it is in essence unrelated to what we are focusing on here, which is decision making and prediction of upcoming choices. The fact that the turning direction of the animal can be predicted on a trial-to-trial basis, and even precedes the behavioral change over the course of learning, sheds light on the role of the PFC in these important predictive cognitive processes (as opposed to post-choice reflective processes).

REVIEWER COMMENT: Indeed, as I said, the new element here is that the upcoming choice can be predicted. This appears only incremental and could belong to another story; as the manuscript is currently written, it does not support the article's main claim. I would like to specify that, regarding this and the other points above, my inability to see how these minor results support the Authors' claim may reflect my misunderstanding; nevertheless, this suggests that the manuscript should be extensively rewritten and reorganized to make the Authors' meaning clear.

ORIGINAL COMMENT: c) Figure 6 shows that prospective activity in the HPC is linked to SWRs and theta oscillations. This has been described in various forms since at least the works of Johnson and Redish in 2007, Pastalkova et al 2008, and Dragoi and Tonegawa (2011 and 2013), as well as in earlier literature on splitter cells. These foundational papers on this topic are not even cited in the current manuscript.

AUTHOR RESPONSE: We have added these citations to the introduction (line 37).

REVIEWER COMMENT: This is an example of how the Authors fail to acknowledge the underlying problem with how the manuscript is written; the issue has not been addressed except with a cosmetic change like the one described above. The Results section contains a series of findings that are well-known phenomena described previously (see below). Prior results should be acknowledged at the beginning of each relevant paragraph, followed by an explicit statement of what is new, so that readers can distinguish replication from novelty. Here, I pointed specifically to the results of Figure 6, and the Authors deemed it sufficient simply to add the citations I indicated to an existing sentence in the Introduction, while keeping the Results description unchanged. As written, this reads as if these phenomena are being described for the first time. This is incorrect. It is hard to avoid the impression that the Authors did not take this concern seriously; the same issue appears elsewhere in the manuscript, and I fail to see how the Authors "have improved clarity of the text throughout to highlight the novelty of our results better."

Reviewer #2 (Public review):

This study presents a thorough investigation of remote memory deficits in the APP/PS1 mouse model of Alzheimer's disease, highlighting the progressive emergence of these deficits alongside selective hyperexcitability of PV interneurons in the mPFC. By combining viral-TRAP labeling and patch-clamp electrophysiology, the authors demonstrate increased inhibitory input onto engram cells in APP/PS1 mice, despite preserved engram size and reactivation. The revised manuscript successfully addresses earlier concerns by clarifying the relationship between amyloid pathology and circuit dysfunction, acknowledging the correlative nature of the findings, and integrating possible contributions of excitatory remodeling and broader network changes, including oscillatory disruptions. Although the precise mechanistic link between PV hyperexcitability, increased inhibition, and impaired remote memory remains to be empirically established, the study convincingly argues for inhibitory microcircuit alterations as an early contributor to cognitive decline in AD.

Reviewer #2 (Public review):

In this paper Chang et al follow up on their lab's previous findings about the secreted protein Shv and its role in activity-induced synaptic remodeling at the fly NMJ. Previously they reported that shv mutants have impaired synaptic plasticity. Normally a high stimulation paradigm should increase bouton size and GluR expression at synapses but this does not happen in shv mutants. The phenotypes relating to activity-dependent plasticity were completely recapitulated when Shv was knocked down only in neurons and could be completely rescued by incubation in exogenously applied Shv protein. The authors also showed that Shv activation of integrin signaling on both the pre- and post-synapse was the molecular mechanism underlying its function in plasticity. Here they extend their study to consider a role of Shv derived from glia in modulating synaptic features at baseline and remodeling conditions. The authors show evidence that Shv is expressed in both neurons and glia. Despite the fact that neuron-specific RNAi knockdown of Shv recapitulated the plasticity phenotypes seen in whole animal mutants, the authors asked whether glial-specific knockdown would have any effects. Surprisingly, knockdown of Shv only in glia also blocked plasticity, just like neuron-specific knockdown, and supporting an important role for glial-derived Shv in plasticity. Unlike neuronal knockdown, though, glial knockdown also caused abnormally high baseline GluR expression. Restoring Shv in ONLY glia in mutant animals is sufficient to completely rescue the plasticity phenotypes and baseline GluR expression, but glial-Shv does not appear to activate integrin signaling which was shown to be the mechanism for neuronally derived Shv to control plasticity. This suggests a different or indirect mechanism of action for glial-derived Shv. This led the authors to hypothesize that glial Shv might work via controlling the levels of neuronal Shv and/or extracellular glutamate. To test these hypotheses, they provide evidence that in the absence of glial Shv, synaptic levels of Shv go up overall, suggesting that glial Shv could somehow have a suppressive effect on release of neuronal Shv. This would indirectly modulate integrin signaling to control plasticity. Using an extracelluar glutamate sensor in presynaptic boutons, they also observe decreased signal (extracellular glutamate) from the sensor in glial Shv KD animals, and increased signal in glial Shv overexpression animals, supporting the hypothesis that glial Shv can regulate glutamate levels somehow. These results establish glia as an important source of Shv in these processes and identify some mechanisms for how this might be accomplished. Several outstanding questions remain-most importantly: how/why do glial-derived and neuronal-derived Shv have different effects when in the same space? No obvious isoform or size differences were found, and the same rescue construct expressed either in neurons or glia could have different effects on integrin activation or glutamate levels. Answering these questions using modified rescue constructs will be an important future direction to understand Shv function specifically and how neurons and glia work together in this context--and potentially many other contexts.

Comments on revisions:

The authors addressed my and the other reviewers' concerns from the original review adequately and this has strengthened the paper substantially.

One small omission to correct: In Figures 4 and 6, the graphs in the figures do not have a legend for the colored bars.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides compelling evidence that glia-derived Shriveled (Shv) modulates activity-dependent synaptic plasticity at the Drosophila neuromuscular junction (NMJ). This mechanism differs from the previously reported function of neuronally released Shv, which activates integrin signaling. They further show that this requirement of Shv is acute and that glial Shv supports synaptic plasticity by modulating neuronal Shv release and the ambient glutamate levels. However, there are a number of conceptual and technical issues that need to be addressed.

Major comments

(1) From the images provided for Fig 2B +RU486, the bouton size appears to be bigger in shv RNAi + stimulation, especially judging from the outline of GluR clusters.

(2) The shv result needs to be replicated with a separate RNAi.

(3) The phenotype of shv mutant resembles that of neuronal shv RNAi - no increased GluR baseline. Any insights why that is the case?

(4) In Fig 3B, SPG shv RNAi has elevated GluR baseline, while PG shv RNAi has a lower baseline. In both cases, there is no activity induced GluR increase. What could explain the different phenotypes?

(5) In Fig 4C, the rescue of PTP is only partial. Does that suggest neuronal shv is also needed to fully rescue the deficit of PTP in shv mutants?

(6) The observation in Fig 5D is interesting. While there is a reduction in Shv release from glia after stimulation, it is unclear what the mechanism could be. Is there a change in glial shv transcription, translation or the releasing machinery? It will be helpful to look at the full shv pool vs the released ones.

(7) In Fig 5E, what will happen after stimulation? Will the elevated glial Shv after neuronal shv RNAi be retained in the glia?

(8) It would be interesting to see if the localization of shv differs based on if it is released by neuron or glia, which might be able to explain the difference in GluR baseline. For example, by using glia-Gal4>UAS-shv-HA and neuronal-QF>QUAS-shv-FLAG. It seems important to determine if they mix together after release? It is unclear if the two shv pools are processed differently.

(9) Alternatively, do neurons and glia express and release different Shv isoforms, which would bind different receptors?

(10) It is claimed that Sup Fig 2 shows no observable change in gross glial morphology, further bolstering support that glial Shv does not activate integrin. This seems quite an overinterpretation. There is only one image for each condition without quantification. It is hard to judge if glia, which is labeled by GFP (presumably by UAS-eGFP?), is altered or not.

(11) The hypothesis that glutamate regulates GluR level as a homeostatic mechanism makes sense. What is the explanation of the increased bouton size in the control after glutamate application in Fig 6?

(12) What could be a mechanism that prevents elevated glial released Shv to activate integrin signaling after neuronal shv RNAi, as seen in Fig 5E?

(13) Any speculation on how the released Shv pool is sensed?

Comments on revisions:

The authors have addressed most of my previous comments and questions in their revision.

Reviewer #1 (Public review):

In this manuscript, Rishiq et al. investigate whether natural killer (NK) cells can interact with Fusobacterium nucleatum and identify the molecular mediators involved in this interaction. The authors propose that the bacterial adhesin RadD may bind to the activating NK cell receptor NKp46 (NCR1 in mice), leading to NK cell activation and tumor control. While the topic is of significant interest and the hypothesis intriguing, the manuscript lacks critical experimental evidence, contains several technical concerns, and requires substantial revisions.

Major Concerns:

(1) Lack of Direct Evidence for RadD-NKp46 Interaction

The central claim that RadD interacts with NKp46 is not formally demonstrated. A direct binding assay (e.g., Biacore, ELISA, or pull-down with purified proteins) is essential to support this assertion. The absence of this fundamental experiment weakens the mechanistic conclusions of the study.

(2) Figure 2: Binding Specificity and Bacterial Strains

A CEACAM1-Ig control should be included in all binding experiments to distinguish between specific and non-specific Ig interactions. There is differential Ig binding between strains ATCC 23726 and 10953. The authors should quantify RadD expression in each strain to determine if the difference in binding is due to variation in RadD levels.

(3) Figure 3: Flow Cytometry Inconsistencies and Missing Controls

What do the FITC-negative, Ig-negative events represent? The authors should clarify whether these are background signals, bacterial aggregates, or debris.

Panel B, CEACAM1-Ig binding appears markedly increased compared to WT bacteria. The reason for this enhancement should be discussed-does it reflect upregulation of the bacterial ligand or an artifact of overexpression? Fluorescence compensation should be carefully reviewed for the NKp46/NCR1-Ig binding assays to ensure that the signals are not due to spectral overlap or nonspecific binding. Importantly, binding experiments using the FadI/RadD double knockout strain are missing and should be included. This control is essential.

In Panel E, the basis for calculating fold-change in MFI is unclear. Please indicate the reference condition to which the change is normalized.

(4) Figure 4: Binding Inhibition and Receptor Sensitivity

Panel A lacks representative FACS plots and is currently difficult to interpret. Differences in the sensitivity of human vs. mouse NKp46 to arginine inhibition should be discussed, given species differences in receptor-ligand interactions. What are the inhibition results using F. nucleatum strains deficient in FadI?

In Panel B, CEACAM1-Ig and RadD-deficient bacteria must be included as negative controls for binding specificity upon anti-NKp46 blocking.

(5) Figure 5: Functional NK Activation and Tumor Killing

In Panels B and C, the key control condition (NK cells + anti-NKp46, without bacteria) is missing. This is needed to evaluate if NKp46 recognition is involved in tumor killing. The authors should explicitly test whether pre-incubation of NK cells with bacteria enhances their anti-tumor activity. Alternatively, could bacteria induce stress signals in tumor cells that sensitize them to NK killing? This distinction is critical.

(6) Figure 5D: Mechanism of Peripheral Activation

It is suggested that contact between bacteria and NK cells in the periphery leads to their activation. Can the authors confirm whether this pre-activation leads to enhanced killing of tumor targets, or if bacteria-tumor co-localization is required? The literature indicates that F. nucleatum localizes intracellularly within tumor cells. If so, how is RadD accessible to NKp46 on infiltrating NK cells?

(8) Figure 5E and In Vivo Relevance

Surprisingly, F. nucleatum infection is associated with increased tumor burden. Does this reflect an immunosuppressive effect? Are NK cells inhibited or exhausted in infected mice (TGIT, SIGLEC7...)? If NK cell activation leads to reduced tumor control in the infected context, the role of RadD-induced activation needs further explanation. RadD-deficient bacteria, which do not activate NK cells, result in even poorer tumor control. This paradox needs to be addressed: how can NK activation impair tumor control while its absence also reduces tumor control?

(9) NKp46-Deficient Mice: Inconsistencies

In Ncr1⁻/⁻ mice, infection with WT or RadD-deficient F. nucleatum has no impact on tumor burden. This suggests that NKp46 is dispensable in this context and casts doubt on the physiological relevance of the proposed mechanism. This contradiction should be discussed more thoroughly.

Reviewer #2 (Public review):

Summary:

In the present study, Rishiq et al. investigated whether the RadD protein expressed by Fusobacterium nucleatum subsp. Nucleatum serves as a natural ligand for the NK-activating receptor NKp46, and whether RadD-NKp46 interaction enhances NK cell cytotoxicity against tumor cells. To address this, the authors first performed an association analysis of F. nucleatum abundance and NKp46 expression in head and neck squamous cell carcinoma (HNSC) and colorectal cancer (CRC) using the TCMA and TCGA databases, respectively. While a positive association between NKp46⁺ and F. nucleatum⁺ status with improved overall survival was observed in HNSC patients, no such correlation was found in CRC.

Next, they examined the binding of NKp46-Ig to various F. nucleatum strains. To confirm that this interaction was mediated specifically by RadD, they employed a RadD-deficient mutant strain. Finally, to establish the functional relevance of the RadD-NKp46 interaction in promoting NK cell cytotoxicity and anti-tumor responses, they utilized a syngeneic mouse breast cancer model. In this setup, AT3 cells were orthotopically implanted into the mammary fat pad of C57BL/6 wild-type (WT) or Ncr1-deficient (NCR1⁻/⁻; murine orthologue of human NKp46) mice, followed by intravenous inoculation with either WT F. nucleatum or the ∆RadD mutant strain.

Strengths:

A notable strength of the work is that it identifies a previously unrecognized activating interaction between F. nucleatum RadD and the NK cell receptor NKp46, demonstrating that the same bacterial protein can engage distinct NK cell receptors (activating or inhibitory) to exert context-dependent effects on anti-tumor immunity. This dual-receptor insight adds depth to our understanding of F. nucleatum-immune interactions and highlights the complexity of microbial modulation of the tumor microenvironment.

Weaknesses:

(1) A previous study by this group (PMID: 38952680) demonstrated that RadD of F. nucleatum binds to NK cells via Siglec-7, thereby diminishing their cytotoxic potential. They further proposed that the RadD-Siglec-7 interaction could act as an immune evasion mechanism exploited by tumor cells. In contrast, the present study reports that RadD of F. nucleatum can also bind to the activating receptor NKp46 on NK cells, thereby enhancing their cytotoxic function.

While F. nucleatum-mediated tumor progression has been documented in breast and colon cancers, the current study proposes an NK-activating role for F. nucleatum in HNSC. However, it remains unclear whether tumor-infiltrating NK cells in HNSC exhibit differential expression of NKp46 compared to Siglec-7. Furthermore, heterogeneity within the NK cell compartment, particularly in the relative abundance of NKp46⁺ versus Siglec-7⁺ subsets, may differ substantially among breast, colon, and HNSC tumors. Such differences could have been readily investigated using publicly available single-cell datasets. A deeper understanding of this subset heterogeneity in NK cells would better explain why F. nucleatum is passively associated with a favorable prognosis in HNSC but correlates with poor outcomes in breast and colon cancers.

(2) The in vivo tumor data (Figure 5D-F) appear to contradict the authors' claims. Specifically, Figure 5E suggests that WT mice engrafted with AT3 breast tumors and inoculated with WT F. nucleatum exhibited an even greater tumor burden compared to mice not inoculated with F. nucleatum, indicating a tumor-promoting effect. This finding conflicts with the interpretation presented in both the results and discussion sections.

(3) Although the authors acknowledge that F. nucleatum may have tumor context-specific roles in regulating NK cell responses, it is unclear why they chose a breast cancer model in which F. nucleatum has been reported to promote tumor growth. A more appropriate choice would have been the well-established preclinical oral cancer model, such as the 4-nitroquinoline 1-oxide (4NQO)-induced oral cancer model in C57BL/6 mice, which would more directly relate to HNSC biology.

(4) Since RadD of F. nucleatum can bind to both Siglec-7 and NKp46 on NK cells, exerting opposing functional effects, the expression profiles of both receptors on intratumoral NK cells should be evaluated. This would clarify the balance between activating and inhibitory signals in the tumor microenvironment and provide a more mechanistic explanation for the observed tumor context-dependent outcomes.

Reviewer #1 (Public review):

Summary:

This is an interesting study on the role of FGF signaling in the induction of primitive streak like-cells (PS-LC) in human 2D-gastruloids. The authors use a previously characterized standard culture that generates a ring of PS-LCs (TBXT+) and correlate this with pERK staining. A requirement for FGF signaling in TBXT induction is demonstrated via pharmacological inhibition of MEK and FGFR activity. A second set of culture conditions (with no exogenous FGFs) suggests that endogenous FGFs are required for pERK and TBXT induction. The authors then characterize, via scRNA-seq, various components of the FGF pathway (genes for ligand, receptors, ERK regulators, HSPG regulation). They go on to characterize the pFGFR1, receptor isoforms and polarized localization of this receptor. Finally, they perform FGF4 inhibition and use a cell line with a limited FGF17 inactivation (heterozygous null) and show that loss of these FGFs reduce PS-LC and derivative cell types.

Strengths:

(1) As the authors point out, the role of FGF signaling in gastrulation is less well understood than other signaling pathways. Hence this is a valuable contribution to that field.

(2) The FGF4 and FGF17 loss-of-function experiments in Figure 5 are very intriguing. This is especially so given the intriguing observation that these FGFs appear to be dominating in this model of human gastrulation, in contrast to what FGFs dominate in mice, chick and frogs.

(3) In general this paper is valuable as a further development of the Human gastruloid system and the role of FGF signaling in the induction of PS-CLs. The wide net that the authors cast in characterizing FGF ligand gene, receptor isoforms, and downstream components provides a foundation for future work. As the authors write near the beginning of the Discussion "Many questions remain."

Weaknesses:

(1) FGFs are cell survival factors in various aspects of development. The authors fail to address cell death due to loss of FGF signaling in any of their experiments. For example, in Figure 1E (which requires statistical analysis) and 1G (the bottom FGFRi row), there appears to be a significant amount of cell loss. Is this due to cell death? The authors should address the question of whether the role of FGF/ERK signaling is to keep the cells alive.

(2) Regarding the sparse cells in 1G, is there a reduction in cell number only with FGFRi and not MEKi? Is this reproducible? Gattiglio et al (Development, 2023, PMID: 37530863) present data supporting a "community effect" in the FGF-induced mesoderm differentiation of mouse embryonic stem cells. Could a community effect be at play in this human system (especially given the images in the bottom row of 1G). If the authors don't address this experimentally they should at least address the ideas in Gattoglio et al.

(3) Do the FGF4 and FGF17 LOF experiments in Figure 5 affect cell number like FGFRi in Figure 1? Why examine PS-LC induction only in FGF17 heterozygous cells and not homozygous FGF17 nulls?

(4) The idea that FGF8 plays a dominant role during gastrulation of other species but not humans is so intriguing it warrants deeper testing. The authors dismiss FGF8 because its mRNA "...levels always remained low." (line 363) as well as the data published in Zhai et al (PMID: 36517595) and Tyser et al (PMID: 34789876). But there are cases in mouse development where a gene was expressed at levels so low, it might be dismissed, and yet LOF experiments revealed it played a role or even was required in a developmental process. The authors should consider FGF8 inhibition or inactivation to explore its potential role, despite its low levels of expression.

(5) Redundancy is a common feature in FGF genetics. What is the effect of inhibiting FGF4 in FGF17 LOF cells?

(6) I suggest stating that the authors take more caution describing FGF gradients. For example, in one Results heading they write "Endogenous FGF4 and FGF17 gradients underly the ERK activity pattern.", implying an FGF protein gradient. However, they only present data for FGF mRNA , not protein. This issue would be clarified if they used proper nomenclature for gene, mRNA (italics) and protein (no italics) throughout the paper.

Comments on revisions:

The authors have addressed my concerns.

Reviewer #2 (Public review):

Summary:

The role of FGFs in embryonic development and stem cell differentiation has remained unclear due to its complexity. In this study, the authors utilized a 2D human stem cell-based gastrulation model to investigate the functions of FGFs. They discovered that FGF-dependent ERK activity is closely linked to the emergence of primitive streak cells. Importantly, this 2D model effectively illustrates the spatial distribution of key signaling effectors and receptors by correlating these markers with cell fate markers, such as T and ISL1. Through inhibition and loss-of-function studies, they further corroborated the needs of FGF ligands. Their data shows that FGFR1 is the primary receptor, and FGF2/4/17 are the key ligands for primitive streak development, which aligns with observations in primate embryos. Additional experiments revealed that the reduction of FGF4 and FGF17 decreases ERK activity.

Strengths:

This study provides comprehensive data and improves our understanding of the role of FGF signaling in primate primitive streak formation. The authors provide new insights related to the spatial localization of the key components of FGF signaling and attempt to reveal the temporal dynamics of the signal propagation and cell fate decision, which has been challenging.

Reviewer #3 (Public review):

Jo and colleagues set out to investigate the origins and functions of localized FGF/ERK signaling for the differentiation and spatial patterning of primitive streak fates of human embryonic stem cells in a well-established micropattern system. They demonstrate that endogenous FGF signaling is required for ERK activation in a ring-domain in the micropatterns, and that this localized signaling is directly required for differentiation and spatial patterning of specific cell types. Through high-resolution microscopy and transwell assays, they show that cells receive FGF signals through basally localized receptors. Finally, the authors find that there is a requirement for exogenous FGF2 to initiate primitive streak-like differentiation, but endogenous FGFs, especially FGF4 and FGF17, fully take over at later stages.

Even though some of the authors' findings - such as the localized expression of FGF ligands during gastrulation and the importance of FGF/ERK signaling for cell differentiation in the primitive streak - have been reported in model organisms before, this is one of the first studies to investigate the role of FGF signaling during primitive streak-like differentiation of human cells. In doing so, the paper reports a number of interesting and valuable observations, namely the basal localization of FGF receptors which mirrors that of BMP and Nodal receptors, as well as the existence of a positive feedback loop centered on FGF signaling that drives primitive-streak differentiation. In the revised version of their work, the authors have furthermore dissected the role of different FGFs through knockdown approaches. These experiments reveal discrete functions for different FGF genes in their system, as well as interesting differences between the role of specific FGFs in human compared to model systems.

Comments on revisions:

The authors have appropriately addressed all comments and suggestions from the previous round of review. The only textual change that I would still like to suggest is to write explicitly in the main text corresponding to Fig. 1 that the mTESR1 medium used for these initial experiments already contains FGF. This is something that is probably known to experts in the field, but not necessarily to a broader readership.

Reviewer #1 (Public review):

This manuscript puts forward the concept that there is a specific time window during which YAP/TAZ driven transcription provides feedback for optimal endothelial cell adhesion, cytoskeletal organization and migration. The study follows up on previous elegant findings from this group and others which established the importance of YAP/TAZ-mediated transcription for persistent endothelial cell migration. The data presented here extends the concept at two levels: first, the data may explain why there are differences between experimental setups where YAP/TAZ activity are inhibited for prolonged times (e.g. cultures of YAP knockdown cells), versus experiments in which the transient inhibition of YAP/TAZ and (global) transcription affects endothelial cell dynamics prior to their equilibrium.

All experiments are convincing, clearly visualized and quantified.

The strength of the paper is that it clearly indicates that there are temporal controlled feedback systems, which is important knowledge for understanding the mechanisms that drive endothelial collective cell behavior.

A potential limitation of the in vivo experiments is that the inhibitors may include off-target effects as well. To solve this caveat in future research endeavours, which is beyond the scope of the current study, it would be interesting to study this process in knockout models, combined with optogenetics and transgenic zebrafish lines that visualize endothelial cell functional properties such as proliferation and migration.

Reviewer #2 (Public review):

Summary:

Here the effect of overall transcription blockade, and then specifically depletion of YAP/TAZ transcription factors was tested on cytoskeletal responses, starting from a previous paper showing YAP/TAZ-mediated effects on the cytoskeleton and cell behaviors. Here, primary endothelial cells were assessed on substrates of different stiffness and parameters such as migration, cell spreading, and focal adhesion number/length were tested upon transcriptional manipulation. Zebrafish subjected to similar manipulations were also assessed during the phase of intersegmental vessel elongation. The conclusion was that there is a feedback loop of 4 hours that is important for the effects of mechanical changes to be translated into transcriptional changes that then permanently affect the cytoskeleton.

The idea is intriguing and a previous paper contains data supporting the overall model. The fish washout data is quite interesting and supports the kinetics conclusions. New transcriptional profiling in this version supports that cytoskeletal genes are differentially regulated with YAP/TAZ manipulations.

Major strengths:

The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting. New RNA profiling supports the model.

Weaknesses:

Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability.

Comments on latest version:

I read the author response to previous reviews, and it seems they agree with the weaknesses stated in the reviews but did not provide any text or data revisions.

Reviewer #4 (Public review):

Summary:

Mason DE et al. have extended their previous study on continuous migration of cells regulated by a feedback loop that controls gene expression by YAP and TAZ. Time scale of the negative feedback loop is derived from the authors' adhesion-spreading-polarization-migration (ASPM) assay. Involvement of transcription-translation in the negative feedback loop is evidenced by the experiments using Actinomycin D. The time scale of mechanotransduction-dependent feedback demonstrated by cytoskeletal alteration in the actinomycin D-treated endothelial colony forming cells (ECFCs) and that shown in the ECFCs depleted of YAP/TAZ by siRNA. The authors examine the time scale when ECFCs are attached to MeHA matrics (soft, moderate, and stiff substrate) and show the conserved time scale among the conditions they use, although instantaneous migration, cell area, and circularity vary. Finally, they tried to confirm that the time scale of the feedback loop-dependent endothelial migration by the effect of washout of Actinomycin D (inhibition of gene transcription), Puromycin (translational inhibition), and Verteporfin (YAP/TAZ inhibitor) on ISV extension during sprouting angiogenesis. They conclude that endothelial motility required for vascular morphogenesis is regulated by a mechanotransduction-mediated feedback loop that is dependent on YAP/TAZ-dependent transcriptional regulation.

Strengths:

The authors conduct ASPM assay to find the time scale of feedback when ECFCs attach to three different matrics. They observe the common time scale of feedback. Thus, under very specific conditions they use, the reproducibility is validated by their ASPM assay. The feedback loop mediated by inhibition of gene expression by Actinomycin D is similar to that obtained from YAP/TAZ-depleted cells, suggesting the mechanotranduction might be involved in the feedback loop. The time scale representing infection point might be interesting when considering the continuous motility in cultured endothelial cells, although it might not account for the migration of endothelial cells that is controlled by a wide variety of extracellular cues. In vivo, stiffness of extracellular matrix is merely one of the cues.

Weaknesses:

ASPM assay is based on attachment-dependent phenomenon. The time scale, including the inflection point determined by ASPM experiments using cultured cells and the mechanotransduction-based theory, do not seem to fit in vivo ISV elongation. Although it is challenging to find the conserved theory of continuous cell motility of endothelial cells, the data is preliminary and does not support the authors' claim. There is no evidence that mechanotransduction solely determines the feedback loop during elongation of ISVs.

Comments on revisions:

The authors' methods using ASPM assay might suggest the feedback loop by their in vitro culture assay. They still need to confirm the loop in vivo using zebrafish intersegmental vessels. The time course of the feedback loop is supported by the ASPM assay. However, the feedback loop is not confirmed in vivo, although it might be suggested by the phenotypes of the ISV treated with drugs. Thus, in the abstract and in the results section, they had better rewrite the interpretation. They have not yet confirmed the feedback loop in vivo.