Reviewer #2 (Public review):

The manuscript by Jackman et al. explores the role of a candidate enhancer of dlx2b in zebrafish tooth formation.

They have mapped the dental epithelium and mesenchyme activity of a 4kb promoter proximal region previously identified as a candidate enhancer region. They identified candidate TFBS and candidate transcription factors regulating this enhancer and proposed that their findings reveal principles of enhancer function during vertebrate organogenesis (tooth development) and the power of dissecting cis regulatory architecture. The study offer valuable genetic tagging resource for studying tooth development while further experiments and analyses would be needed to support the suggestion for novel findings on in cis-regulatory principles of tooth development. In the lack of functional evidence on endogenous target gene pr tooth development, some of the claims of the paper may need rephrasing.

(1) The candidate enhancer region has previously been published, this study narrows the enhancer effect to a well-conserved region within. To what degree the element is unique in the locus for tooth development and to what degree this element is required for tooth morphogenesis, is not addressed.

(2) The knock-in approach is convenient for reporter activity based analyses, however it lacks the precision that would be necessary to conclude on enhancer- autonomous effects or enhancer effects on the endogenous target promoter. The HSP promoter inserted in within a 5kb(?) insert in the UTR region of dlx2b creates an chimeric E-P context. The expression profile of the knock-in reporter is substantially different from the endogenous gene (Figure 1B and C) suggesting E-P interaction dependent expression profile, which may confuse what in the expression comes solely from the enhancer and not as a result of the HSP promoter interaction with the enhancer. An alternative heterologous promoter would help in defining the enhancer specific effects.

(3) Function of the candidate enhancer: The MTE enhancer effect is measured by gain of function towards dlx2b regulation. The deletion assays are limited to plasmids designed to test the enhancer in isolation from the endogenous enhancer architecture, or to a deletion in the knock-in, which may be impacted by the chimeric regulatory interaction with a heterologous HSP promoter. As a result we do not learn whether the enhancer targets or needs for endogenous target gene activity. This design allows a conclusion on tissue activity of the enhancer but not the requirement for tooth development.

(4) Since the locus is scattered by candidate enhancers (see genome annotation resources) it is feasible that additional E-P interactions lead to potential enhancer redundancies with the MTE. For a conclusive functional test/requirement of the MTE enhancer, the authors would need to delete it in the endogenous locus context. The knock-in could theoretically be used for an enhancer function on dlx2b activity, if the authors show that there is interaction with the endgogenous promoter (3C type experiment); and that the MTE enhancer-driven GFP activity was identical to the endogenous tagged dlx2b activity. This does not appear to be the case, as ectopic expression in Fig 1C as compared to B is shown. Of note, RNA detection by WISH would be more precise for comparisons. The figure likely compares protein (legend is unclear, but text suggests protein) to mRNA, which is imprecise.

(5) There is an experimental design question arising with generating the MTE deletion in the knock-in (line 391): the authors describe generating the transgenic lines by screening for reduced reporter activity first. This suggests the authors pre-emptively looked for an effect as result they predicted when generating the transgenic lines, which would create a circular argument. All transgenic lines carrying the deletion (tested by sequencing first) would need to be assayed for activity change and then can conclusion could be made on effect of MTE loss by statistical analyses of reporter activities in the generated lines.

(6) Most transgenic work described are based on single transgenic lines. Enhancer promoter contexts may be affected either by position effects (in case of the reporter constructs) or by the heterologous promoter context of the knock which may be affected by unexpected recombination events. Such unintended confound effects can be excluded by replicates.

(7) GFP protein detection does not allow precise spatio-temporal resolution due to varying protein stability in tissues, which potentially impacts endogenous gene activity comparison, and accurate determination of activity dynamics towards conclusions on lineage determining/maintenance roles of the dlx2b enhancer.

(8) The expression pattern change upon MTE loss (retention of mesenchyme, loss of epithelium) is an interesting observation, which would benefit from more comprehensive analysis of the grammar (TFBS contributions) to the pattern variation by dissection of the combination of TFBS contributions. Without such, enhancer grammar remains mostly unclear, thus, principles of morphogenesis may not have been uncovered.

(9) The diagrammatic models of the conclusions are illustrating simple logic which does not add to the text.

(10) Author contributions need to be explained in more detail to be sufficiently granular for fair credit.

Reviewer #3 (Public review):

In the manuscript entitled "A Minimal tooth Enhancer Regulates dlx2b Expression During Zebrafish Tooth 1 Formation: Insights into Cis-Regulatory Logic in Organogenesis", the authors explore the cis-regulatory logic of a dlx2b minimal enhancer capable of directing dlx2b gene expression to the developing tooth germs. The study combines (1) CRISPR-mediated GFP knock-in to track endogenous gene expression; (2) a promoter-bashing approach to identify a minimal tooth enhancer (MTE); (3) site-directed mutagenesis coupoled with transgenesis to assess the individual role of conserved TF binding sites; and (4) in vivo deletgion of the MTE to examine the consequences for gene expression. Overall, this is a technically solid study that provides some novel insights into tooth development and extends previous observations by the authors (Jackman & Stock, 2006; PNAS). However, the added value of the manuscript is limited by both the narrow experimental scope and the relatively modest impact of the findings for the broader field of developmental biology.

Main concerns:

(1) My main concern is that the study restricts the search for cis-regulatory information to the 5' region 4kb upstream of the TSS of the gene, rather than encompassing the full genomic locus. This is particularly limiting given that a knock-in allele was generated, which in principle allows interrogation of regulatory elements across the entire locus, and that the authors acknowledge the availability of genome-wide regulatory datasets (e.g. DANIO-CODE) in the Discussion. Despite this, no systematic effort is made to test additional regulatory elements beyond the proximal promoter/enhancers.<br /> This has important implications for the interpretation of the current work as: (a) dlx2b, as many developmental genes, resides in a gene desert enriched in open chromatin regions that may function as distal enhancers, and (b) the deletion of the MTE unmasked a cis-regulatory activity which nature cannot be explained with the information provided, and that may seem relevant for the expression of the gene in the dental mesenchyme.

(2) A second concern is the absence of information on the functional consequences of deleting the gene or the MTE on tooth primordium development. From the description of the KI strategy, it is unclear whether the GFP insertion results in a functional fusion protein. The cytoplasmic GFP distribution and the schematic in Figure S1 instead suggest the presence of a terminal stop codon in the GFP sequence, which would result in a dlx2b loss-of-function allele. If this interpretation is correct, the manuscript does not describe the developmental consequences in homozygous embryos. Similar concerns apply to the MTE deletion: it remains unclear whether loss of this enhancer results in any detectable morphological or developmental defects.

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Reviewer #1 (Public review):

Summary:

The manuscript by Ma et al. provides robust and novel evidence that the noctuid moth Spodoptera frugiperda (Fall Armyworm) possesses a complex compass mechanism for seasonal migration that integrates visual horizon cues with Earth's magnetic field (likely its horizontal component). This is an important and timely study: apart from the Bogong moth, no other nocturnal Lepidoptera has yet been shown to rely on such a dual-compass system. The research therefore expands our understanding of magnetic orientation in insects with both theoretical (evolution and sensory biology) and applied (agricultural pest management, a new model of magnetoreception) significance.

The study uses state-of-the-art methods and presents convincing behavioural evidence for a multimodal compass. It also establishes the Fall Armyworm as a tractable new insect model for exploring the sensory mechanisms of magnetoreception, given the experimental challenges of working with migratory birds. Overall, the experiments are well designed, the analyses are appropriate, and the conclusions are generally well supported by the data.

Strengths:

• Novelty and significance: First strong demonstration of a magnetic-visual compass in a globally relevant migratory moth species, extending previous findings from the Bogong moth and opening new research avenues in comparative magnetoreception.<br /> • Methodological robustness: Use of validated and sophisticated behavioural paradigms and magnetic manipulations consistent with best practices in the field. The use of 5 min bins to study a dynamic nature of magnetic compass which is anchored to a visual cue but updated with latency of several minutes is an important finding and a new methodological aspect in insect orientation studies.<br /> • Clarity of experimental logic: The cue-conflict and visual cue manipulations are conceptually sound and capable of addressing clear mechanistic questions.<br /> • Ecological and applied relevance: Results have implications for understanding migration in an invasive agricultural pest with expanding global range.<br /> • Potential model system: Provides a new, experimentally accessible species for dissecting the sensory and neural bases of magnetic orientation.

Weaknesses:

Overall, this is a strong study, and the authors have completed an excellent major revision.

Reviewer #2 (Public review):

Summary:

The work titled "Geomagnetic and visual cues guide seasonal migratory orientation in the nocturnal fall armyworm, the world's most invasive insect" provided experimental evidence on how geomagnetic and visual cues are integrated, and visual cues are indispensable for magnetic orientation in the nocturnal fall armyworm.

Strengths:

It has been demonstrated that the Australian Bogon moth could integrate global stellar cues with the geomagnetic field for long distance navigation. However, data are lacking for other insects. This study suggested that the integration of geomagnetic and visual cues may represent a conserved navigational mechanism broadly employed across migratory insects.

Weaknesses:

The visual cues used in the indoor experimental system designed by the authors may have some limitations in ecological relevance. The author may need more explanations on this experimental system.

In the revised manuscript, the authors have added explanations in the discussion section. I am fine with the revision.

Reviewer #1 (Public review):

Summary:

The researchers aimed to identify which neurotransmitter pathways are required for animals to withstand chronic oxidative stress. This work thus has important implications for disease processes that are caused/linked to oxidative stress. This work identified specific neurotransmitters and receptors that coordinate stress resilience, both prior to and during stress exposure. Further, the authors identified specific transcriptional programs coordinated by neurotransmission that may provide stress resistance.

Strengths:

The manuscript is very clearly written with a well-formulated rationale. Standard C. elegans genetic analysis and rescue experiments were performed to identify key regulators of the chronic oxidative stress response. These findings were enhanced by transcriptional profiling that identified differentially expressed genes that likely affect survival when animals are exposed to stress.

Weaknesses:

Where the gar-3 promoter drives expression was not discussed in the context of the rescue experiments in Fig 7.

Comments on revisions:

This issue has now been appropriately addressed in the revision.

Reviewer #2 (Public review):

In this paper, Biswas et al. describe the role of acetylcholine (ACh) signaling in protection against chronic oxidative stress in C. elegans. They showed that disruption of ACh signaling in either unc-17 mutant or gar-3 mutants led to sensitivity to toxicity caused by chronic paraquat (PQ) treatment. Using RNA seq, they found that approximately 70% of the genes induced by chronic PQ exposure in wild type failed to upregulate in these mutants. The overexpression of gar-3 selectively in cholinergic neurons was sufficient to promote protection against chronic PQ exposure in an ACh-dependent manner. The study points to a previously undescribed role for ACh signaling in providing organism-wide protection from chronic oxidative stress likely through the transcriptional regulation of numerous oxidative stress-response genes. The paper is well-written, and the data are robust, though some conclusions seem preliminary and are not fully support the current data (see below). While the study identifies the muscarinic ACh receptor gar-3 as an important regulator of the response to PQ, the specific neurons in which gar-3 functions were not unambiguously identified, and the sources of ACh that regulate GAR-3 signaling and the identities of the tissues targeted by gar-3 were not addressed.

Comments on revisions:

The authors addressed my comments adequately in their revised submission. Please include representative images to accompany the quantification of the new results presented in Fig S4A.

Reviewer #1 (Public review):

This study makes a fundamental contribution to our understanding of interocular suppression, particularly continuous flash suppression (CFS). Using neuroimaging data from two macaque monkeys, the study provides compelling evidence that CFS suppresses orientation responses in neurons within V1. These findings enrich the CFS literature by demonstrating that neural activity under CFS may prevent high-level visual and cognitive processing.

Comments on revisions:

The authors have addressed all my previous comments.

Reviewer #2 (Public review):

Summary:

The goal of this study was to investigate the degree to which low-level stimulus features (i.e., grating orientation) are processed in V1 when stimuli are not consciously perceived under conditions of continuous flash suppression (CFS). The authors measured the activity of a population of V1 neurons at single neuron resolution in awake fixating monkeys while they viewed dichoptic stimuli that consisted of an oriented grating presented to one eye and a noise stimulus to the other eye. Under such conditions, the mask stimulus can prevent conscious perception of the grating stimulus. By measuring the activity of neurons (with Ca2+ imaging) that preferred one or the other eye, the authors tested the degree of orientation processing that occurs during CFS.

Strengths:

The greatest strength of this study is the spatial resolution of the measurement and the ability to quantify stimulus representations during CSF in populations of neurons preferring the eye stimulated by either the grating or the mask. There have been a number of prominent fMRI studies of CFS, but all of them have had the limitation of pooling responses across neurons preferring either eye, effectively measuring the summed response across ocular dominance columns. The ability to isolate separate populations offers an exciting opportunity to study the precise neural mechanisms that give rise to CFS, and potentially provide insights into nonconscious stimulus processing.

Weaknesses:

However, while this is an impressive experimental setup, the major weakness of this study is that the experiments don't advance any theoretical account of why CFS occurs or what CFS implies for conscious visual perception. There are two broad camps of thinking with regard to CFS. On the one hand, Watanabe et al., 2011 reported that V1 activity remained intact during CFS, implying that CFS interrupts stimulus processing downstream of V1. On the other hand, Yuval-Greenberg and Heeger (2013) showed that V1 activity is in fact reduced during CFS. By using a parametric experimental design, they measured the impact of the mask on the stimulus response as a function of contrast, and concluded that the mask reduces the gain of neural responses to the grating stimulus. They presented a theoretical model in which the mask effectively reduced the SNR of the grating, making it invisible in the same way that reducing contrast makes a stimulus invisible.

In the first submission of the manuscript, the authors incorrectly described the Yuval-Greenberg & Heeger (2013) paper and Watanabe et al. (2011) papers, suggesting that they had observed the same or similar effects of CFS on V1 activity, when in fact they had described opposite results. Reviewer 1 also observed that the authors appeared to be confused in their reading of these highly relevant papers. In the revision, the authors have reworked this paragraph, now correctly describing these sets of opposing results. However, I still do not understand what the authors are trying to argue: "...these studies were not designed to quantify the pure effect of CFS on stimulus-evoked V1 responses." I do not understand what is meant by "pure" in this case. Regardless, it is clear that the measurements in the present study strongly support the interpretation of Yuval-Greenberg & Heeger (i.e., that V1 activity is degraded by CFS, 'akin' to a loss in the contrast-to-noise ratio of neural activity). It would be appropriate for the authors to communicate this clearly.

I continue to be of the opinion that this study is lacking an adequate model of interocular interactions that might explain the Ca2+ imaging. The machine learning results are not terribly surprising - multivariate methods, such as SVMs, are more sensitive than univariate approaches. So it is plausible that an SVM can support decoding of the coarse orientation information, even when no tuning is evident in the univariate analyses. However, the link between this result and the underlying neurophysiology is opaque. The failure to model the neural data with an explicit model is a missed opportunity.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. Binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge.

Weaknesses:

The strength of CFS suppression varies across animals, but the authors attribute this to comparable heterogeneity in the human psychophysics literature.

Comments on revisions:

The authors have addressed my comments from the previous round of review, and I have no further comments

Reviewer #1 (Public review):

This study by Li and colleagues examines how defensive responses to visual threats during foraging are modulated by both reward level and social hierarchy. Using a naturalistic paradigm, the authors test how the availability of water or sucrose, with sucrose being more rewarding than water, shapes escape behavior in mice exposed to looming stimuli of different intensities, which are used to probe perceived threat level and defensive responses. In parallel, the study compares dominant and subordinate animals to assess how social rank biases the trade off between reward seeking and threat avoidance. By combining detailed behavioral analyses with computational modeling, the work addresses how reward level and social context jointly influence escape decisions in an ethologically relevant setting.

Across the different experimental conditions, perceived threat level is the main determinant of behavior. The authors show that looming stimuli associated with higher threat (contrast) consistently elicit faster and more robust escape responses than lower threat stimuli. This effect is particularly evident during early exposures, when animals are highly vigilant and have not yet habituated to the looming stimulus (learned that it is not dangerous). Later they described that as animals gain experience and habituate, behavior becomes more flexible, and reward level begins to exert a graded modulation of the escape response. Importantly, the authors show that under high threat conditions increasing reward value leads to more frequent and faster escape rather than greater reward pursuit. This finding is particularly relevant, as it suggests that highly valued rewards can heighten vigilance and thereby enhance responsiveness to threat, highlighting that reward does not simply compete with defensive behavior but can also reshape it depending on the perceived level of danger, in contrast to low threat conditions, where threat can be more easily outweighed by reward. Thus, an important conceptual contribution of the study is the introduction of vigilance as a useful framework to interpret these effects. Vigilance is treated as a behavioral state reflecting heightened attention to potential danger. In line with what is known from natural foraging, mice initially maintain high vigilance when confronted with an innate threat. This perspective helps clarify a finding that might otherwise appear counterintuitive. One might expect higher rewards to motivate animals to tolerate risk, explore more, and habituate faster in any scenario. Instead, the data suggest that highly rewarding outcomes can elevate vigilance, making animals more responsive to threat and leading to faster or more frequent escape under high threat conditions. In this sense, reward does not simply compete with threat but can also amplify sensitivity to it, depending on the internal state of the animal.

The social results are particularly interesting in this context as well. Dominant mice consistently prioritize avoidance over reward, showing stronger escape responses and slower habituation than subordinates. This behavior is well captured by the vigilance framework proposed by the authors: dominant animals appear to maintain higher vigilance, which biases decisions toward threat avoidance. The authors further suggest that stable social relationships sustain high vigilance and slow habituation, framing this as an evolutionarily conserved strategy that may enhance survival. This interpretation provides a valuable perspective on how social structure shapes defensive behavior beyond immediate physical interactions. At the same time, there are important limitations to this interpretation. All experiments were conducted in male mice, and it is possible that the relationship between social hierarchy, vigilance, and defensive behavior would differ substantially in females. In addition, the idea that stable social relationships maintain elevated vigilance does not straightforwardly align with broader views of social stability as protective for mental health and as a buffer against anxiety and stress. These points do not undermine the findings but suggest that the social effects described here should be interpreted with caution and within the specific context of the task and sex studied.

Another important limitation is that the neural mechanisms underlying these effects remain speculative. The manuscript includes an extensive discussion of candidate circuits, particularly involving the superior colliculus and downstream structures, but this section is necessarily based on prior literature rather than on data presented in the study. Given the complexity of the circuits involved in integrating internal state, reward, social context, and vigilance, the current work should be viewed as providing a strong behavioral and conceptual framework rather than direct insight into underlying neural mechanisms.

Methodologically, the behavioral paradigm is well suited for studying escape decisions in socially housed animals, and the machine learning based classification of defensive responses is a clear strength. The computational model provides a useful formalization of how threat level, reward level, and vigilance interact and may be valuable for other laboratories studying escape, approach avoidance, or conflict situations, particularly as a way to classify behavioral outcomes after pose estimation. More generally, the work will be of interest to the neuroethology community for its detailed characterization of escape behavior under naturalistic conditions.

Given the ethological nature of the study and the high inter individual variability reported by the authors, clarity and precision in the methods are especially important for reproducibility. While the revised manuscript addresses many earlier concerns, some aspects remain slightly difficult to follow. For example, the main text states that animals were not water deprived to avoid differences in internal state, whereas parts of the methods describe conditions in which animals were water deprived, suggesting that internal state manipulation may differ across experiments. Clearer separation and explanation of these conditions would further strengthen confidence in the work.

Overall, this study provides a rich and thoughtful analysis of how reward level and social hierarchy modulate defensive behavior through changes in vigilance. It offers a useful conceptual advance for thinking about escape behavior in naturalistic settings and lays a solid foundation for future work aimed at linking these behavioral states to underlying neural circuits.

Reviewer #2 (Public review):

Zhe Li and colleagues investigate how mice exposed to visual threats and rewards balance their decisions in favour of consuming rewards or engaging in defensive actions. By varying threat intensity and reward value, they first confirm previous findings showing that defensive responses increase with threat intensity and that there is habituation to the threat stimulus. They then find that water-deprived mice have a reduced probability of escaping from low contrast visual looming stimuli when water or sucrose are offered in the environment, but that when the stimulus contrast is high, the presence of sucrose or water increases the probability of escape. By analysing behaviour metrics such as the latency to flee from the threat stimulus, they suggest that this increase in threat sensitivity is due to increased vigilance. Analysis of this behaviour as a function of social hierarchy shows that dominant mice have higher threat sensitivity, which is also interpreted as being due to increased vigilance. These results are captured by a drift diffusion model variant that incorporates threat intensity and reward value.

The main contribution of this work is quantifying how the presence of water or sucrose in water-deprived mice affects escape behaviour. The differential effects of reward between the low and high contrast conditions are intriguing, but I find the interpretation that vigilance plays a major in this process not supported by the data. The idea that reward value exerts some form of graded modulation of the escape response is also not supported by the data. In addition, there is very limited methodological information, which makes assessing the quality of some of the analyses difficult, and there is no quantification on the quality of the model fits.

(1) The main measure of vigilance in this work is reaction time. While reaction time can indeed be affected by vigilance, reaction times can vary as a function of many variables, and be different for the same level of vigilance. For example, a primate performing the random dot motion task exhibits differences in reaction times that can be explained entirely by the stimulus strength. Reaction time is therefore not a sound measure of vigilance, and if a goal of this work is to investigate this parameter, then it should be measured. There is some attempt at doing this for a subset of the data in Figure 3H, by looking at differences in the action of monitoring the visual field (presumably a rearing motion, though this is not described) between the first and second trials in the presence of sucrose. I find this an extremely contrived measure. What is the rationale for analysing only the difference between the first and second trials? Also, the results are only statistically significant because the first trial in the sucrose condition happens to have zero up action bouts, in contrast to all other conditions. I am afraid that the statistics are not solid here. When analysing the effects of dominance, a vigilance metric is the time spent in the reward zone. Why is this a measure of vigilance? More generally, measuring vigilance of threats in mice requires monitoring the position of the eyes, which previous work has shown is biased to the upper visual field, consistent with the threat ecology of rodents.

(2) In both low and high contrast conditions, there are differences in escape behaviour between no reward and water or sucrose presence, but no statistically significant differences between water and sucrose (eg: Figure 3B). I therefore find that statements about reward value are not supported by the data, which only show differences between the presence or absence of reward. Furthermore, there is a confound in these experiments, because according to the methods, mice in the no-reward condition were not water-deprived. It is thus possible that the differences in behaviour arise from differences in the underlying state.

(3) There is very little methodological information on behavioural quantification. For example, what is hiding latency? Is this the same are reaction time? Time to reach the safe zone? What exactly is distance fled? I don't understand how this can vary between 20 and 100cm. Presumably, the 20cm flights don't reach the safe place, since the threat is roughly at the same location for each trial? How is the end of a flight determined? How is duration measured in reward zone measures, e.g., from when to when? How is fleeing onset determined?

(4) There is little methodological information on how the model was fit (for example, it is surprising that in the no reward condition, the r parameter is exactly 0. What this constrained in any way), and none of the fit parameters have uncertainty measures so it is not possible to assess whether there are actually any differences in parameters that are statistically significant.

Comments on the revised manuscript:

The manuscript has been revised and improved significantly by the addition of methodological details and new analysis. I remain, however, unconvinced by the argument that increased vigilance in the presence of reward leads to heightened escape behaviour.

In response to my criticism that the work does not measure vigilance directly, the authors have included measures of foraging interval and foraging speed, which they state are "two direct behavioral analyses of vigilance". I disagree - like reaction time, foraging speed and foraging interval can be modulated, for example, by changes in threat sensitivity. Increased threat sensitivity comes with diverse behavioral changes that may well include increased vigilance, but foraging interval and foraging speed can certainly change without the animal expressing increased vigilance behaviors. A bigger issue I still have though, is with the conclusion that the presence of reward increases "direct escape behaviors". Comparing the no reward, water and sucrose groups indeed shows a difference (which is now clear after the split into early and late phases), but the issue is that these are different mice. As the text is written, is sounds like introducing reward will acutely increase escape. But if we look at the raw data show in Figure 2C, what I think is happening is that the presence of reward is decreasing habituation to the stimulus. The data for trials 1 and 10 in the three conditions show this - there is habituation with no reward (reaction times are all shifting to the right), a bit less with water and very little with sucrose. This is interesting in its own right and we can speculate why it might be happening, but I think this is conceptually different from what the authors are proposing.

Reviewer #3 (Public review):

Male mice were tested in a classic behavioral "flee the looming stimulus" paradigm. This is a purely behavioral study; no neural analyses were done. Mice were housed socially, but faced the looming stimulus individually, using an elegant automated tunnel (see videos for clarity).

The additional changes made to the paper clarify the work done. While there are some limitations (male mice, weird stimulus), the general results are interesting and a valuable addition to the experimental literature. The main claim of the paper is that the different rewards (none, water, sucrose) did not change the escape properties early in learning, but did late, particularly that in the late (already experienced) conditions, reward value (assuming sucrose > water > no reward) interacted with the salience of the looming stimulus (light gray, dark gray). (Panels 3D, 3G, 3K, 3N).

For readers, I want to note that one of the most interesting results is actually in Figure S2, where they find that a looming stimulus behind the mouse still makes a mouse run to the nest. In these conditions, the mouse runs past the looming stimulus to get to safety! (I also do love the video of the mouse running around the barriers like a snake to get home.)

I have a few minor clarification questions and a few notes that I think would be useful additions for authors and readers to think about.

Dominance: What does the mouse social science literature say about the "test tube" test? What can we conclude from this test? This would be useful when trying to understand what is causing the dominance/submissive difference in responses. Figure 4 shows that the dominant mice are more risk-averse than the submissive mice. Is "dominance" in the test-tube actually a measure of risk-seeking? Is the issue that the submissive mice don't think they can get back to the food-site easily, so they are less willing to sacrifice the current (if dangerous) foraging opportunity? Is the issue that the submissive mice can't get back to the nest? As I understand it, the nest was always available to all the mice, so I suspect inability to get to the nest is an unlikely hypotheses. Is the issue that the submissive mice also don't feel safe in the nest?

Limitations of the study: There is an acknowledged limitation to male mice, and the limitations of the small data sets that are typical of such experiments. In addition, however, it is also worth noting the strangeness of the looming stimulus, which is revealed clearly in the videos. The stimulus is a repeating growing circle, growing in a single location within the environment. The stimulus repeats 10 times, once per second. This is not what an attacking hawk or owl would look like. (I now have this image of an owl diving down, and then teleporting up and diving down again.) Note - I am fine with this stimulus. It produces an interesting experiment and interesting results. I do not think the authors need to change anything in their paper, but readers need to recognize that this is not a "looming predator".

These "limitations" are better seen as "caveats" when folding these results in with the rest of the literature that has gone before and the literature to come. (Generally, I do not believe that science works by studies making discoveries that change how we think about problems - instead, science works by studies adding to the literature that we integrate in with the rest of the literature.) Thus, these caveats should not be taken as problems with the study or as fixes that need to be done. Instead, they are notes for future researchers to notice if differences are found in any future studies.

Thus, my only suggestion is that I think authors could write a more careful paper by using the past and subjunctive tense appropriately. Experimental observations should be in past tense, as in "the influence of reward was context-dependent and emerged in the late phase" instead of "the influence of reward is context-dependent and emerges in the late phase" - it emerged in the late phase this once - it might not in future experiments, not due to any fault in this experiment nor due to replicability problems, but rather due to unexpected differences between this and those future experiments. At which point, it will be up to those future experiments to determine the difference. Similarly, large conclusions should be in the subjunctive tense, as in "these data suggest that threat intensity is likely to be the primary determinant of decision making" rather than "threat intensity is the primary determinant of decision making", because those are hypotheses not facts.

Reviewer #1 (Public review):

In this manuscript, the authors report that GPR55 activation in presynaptic terminals of Purkinje cells decrease GABA release at the PC-DCN synapse. The authors use an impressive array of techniques (including highly challenging presynaptic recordings) to show that GPR55 activation reduces the readily releasable pool of vesicle without affecting presynaptic AP waveform and presynaptic Ca2+ influx. This is an interesting study, which is seemingly well-executed and proposes a novel mechanism for the control of neurotransmitter release. However, the authors' main conclusions are heavily, if not solely, based on pharmacological agents that most often than not demonstrate affinity at multiple targets. Below are points that the authors should consider in a revised version.

Major points:

(1) There is no clear evidence that GPR55 is specifically expressed in presynaptic terminals at the PC-DCN synapse. The authors cited Ryberg 2007 and Wu 2013 in the introduction, mentioning that GPR55 is potentially expressed in PCs. Ryberg (2007) offers no such evidence, and the expression in PC suggested by Wu (2013) does not necessarily correlate with presynaptic expression. The authors should perform additional experiments to demonstrate presynaptic expression of GPR55 at PC-DCN synapse.

(2) The authors' conclusions rest heavily on pharmacological experiments, with compounds that are sometimes not selective for single targets. Genetic deletion of GPR55 would be a more appropriate control. The authors should also expand their experiments with occlusion experiments, showing if the effects of LPI are absent after AM251 or O-1602 treatment. In addition, the authors may want to consider AM281 as a CB1R antagonist without reported effects at GPR55.

(3) It is not clear how long the different drugs were applied, and at what time the recording were performed during or following drug application. It appears that GPR55 agonists can have transient effects (Sylantyev, 2013; Rosenberg, 2023), possibly due to receptor internalization. The timeline of drug application should be reported, where IPSC amplitude is shown as a function of time and drug application windows are illustrated.

(4) A previous investigation on the role of GPR55 in the control of neurotransmitter release is not cited nor discussed Sylantyev et al., (2013, PNAS, Cannabinoid- and lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central synapses). Similarities and differences should be discussed.

Minor point:

(1) What is the source of LPI? What isoform was used? The multiple isoforms of LPI have different affinities for GPR55.

Comments on revisions:

In this revised version, the authors have addressed my major concerns. Notably, they used CRISPR/Cas9 genetic knockdown of GPR55 to independently validate their original findings. The main conclusions are now well supported and represent an important contribution to the field.

Reviewer #2 (Public review):

Summary:

This paper investigates the mode of action of GPR55, a relatively understudied type of cannabinoid receptors, in presynaptic terminals of Purkinje cells. The authors use demanding techniques of patch clamp recording of the terminals, sometimes coupled with another recording of the postsynaptic cell. They find a lower release probability of synaptic vesicles after activation of GPR55 receptors, while presynaptic voltage-dependent calcium currents are unaffected. They propose that the size of a specific pool of synaptic vesicles supplying release sites is decreased upon activation of GPR55 receptors.

Strengths:

The paper uses cutting edge techniques to shed light on a little studied, potentially important type of cannabinoid receptors. The results are clearly presented, and the conclusions are sound.

Weaknesses:

The nature of the vesicular pool that is modified following activation of GPR55 is not definitively characterized.

Comments on revisions:

The authors have done a good job in answering the criticisms of reviewers. Consequently, the revised version offers a substantial improvement over the first version.

Reviewer #3 (Public review):

Inoshita and Kawaguchi investigated the effects of GPR55 activation on synaptic transmission in vitro. To address this question, they performed direct patch-clamp recordings from axon terminals of cerebellar Purkinje cells and fluorescent imaging of vesicular exocytosis utilizing synapto-pHluorin. They found that exogenous activation of GPR55 suppresses GABA release at Purkinje cell to deep cerebellar nuclei (PC-DCN) synapses by reducing the readily releasable pool (RRP) of vesicles. This mechanism may also operate at other synapses.

Strengths:

The main strength of this study lies in combining patch-clamp recordings from axon terminals with imaging of presynaptic vesicular exocytosis to reveal a novel mechanism by which activation of GPR55 suppresses inhibitory synaptic strength. The results strongly suggest that GPR55 activation reduces the RRP size without altering presynaptic calcium influx.

Weaknesses:

The study relies on the exogenous application of GPR55 agonists. It remains unclear whether endogenous ligands released by physiological or pathological processes would have similar effects. There is also little evidence that GPR55 is expressed in Purkinje cell axon boutons. This study would benefit from the use of GPR55 knockout (KO) mice. The downstream mechanism by which GPR55 mediates the suppression of GABA release remains unknown.

Comments on revisions:

The authors have addressed all my concerns effectively. I have no further comments and want to commend their comprehensive study.

Reviewer #1 (Public review):

Summary:

The "number sense" refers to an imprecise and noisy representation of number. Many researchers propose that the number sense confers a fixed (exogenous) subjective representation of number that adheres to scalar variability, whereby the variance of the representation of number is linear in the number.

This manuscript investigates whether the representation of number is fixed, as usually assumed in the literature, or whether it is endogenous. The two dimensions on which the authors investigate this endogeneity are the subject's prior beliefs about stimuli values and the task objective. Using two experimental tasks, the authors collect data that are shown to violate scalar variability and are instead consistent with a model of optimal encoding and decoding, where the encoding phase depends endogenously on prior and task objectives. I believe the paper asks a critically important question. The literature in cognitive science, psychology, and increasingly in economics, has provided growing empirical evidence of decision-making consistent with efficient coding. However, the precise model mechanics can differ substantially across studies. This point was made forcefully in a paper by Ma and Woodford (2020, Behavioral & Brain Sciences), who argue that different researchers make different assumptions about the objective function and resource constraints across efficient coding models, leading to a proliferation of different models with ad-hoc assumptions. Thus, the possibility that optimal coding depends endogenously on the prior and the objective of the task, opens the door to a more parsimonious framework in which assumptions of the model can be constrained by environmental features. Along these lines, one of the authors' conclusions is that the degree of variability in subjective responses increases sublinearly in the width of the prior. And importantly, the degree of this sublinearity differs across the two tasks, in a manner that is consistent with a unified efficient coding model.

Comments on revisions:

The authors have done an excellent job addressing my main concerns from the previous round. The new analyses that address the alternative model of "no cognitive noise and only motor noise" are compelling and provide quantitative evidence that bolsters the paper's overall contribution. The authors also went above and beyond by reanalyzing the Frydman and Jin (2022) dataset to provide new and very interesting analyses that provide an additional out of sample test of the model proposed in the current paper.

Reviewer #2 (Public review):

Summary:

This paper provides an ingenious experimental test of an efficient coding objective based on optimization as a task success. The key idea is that different tasks (estimation vs discrimination) will, under the proposed model, lead to a different scaling between the encoding precision and the width of the prior distribution. Empirical evidence in two tasks involving number perception supports this idea.

Strengths:

- The paper provides an elegant test of a prediction made by a certain class of efficient coding models previously investigated theoretically by the authors.<br /> The results in experiments and modeling suggest that competing efficient coding models, optimizing mutual information alone, may be incomplete by missing the role of the task.

- The paper carefully considers how the novel predictions of the model interact with the Weber/Fechner law.

Weaknesses:

- The claims would be even more strongly validated if data were present at more than two widths in the discrimination experiment (also noted in Discussion).

Reviewer #3 (Public review):

Summary:

This work investigates whether human imprecision in numeric perception is a fixed structural constraint or an endogenous property that adapts to environmental statistics and task objectives. By measuring behavioral variability across different uniform prior distributions in both estimation and discrimination tasks, the authors show that perceptual imprecision increases sublinearly with prior width. They demonstrate that the specific exponents of this scaling (1/2 for estimation and 3/4 for discrimination) can be derived from an efficient-coding model, wherein decision-makers optimally balance task-specific expected rewards against the metabolic costs of neural coding. The revised manuscript expands this framework to accommodate logarithmic representations and validates the core model against an independent dataset of risky choices.

Strengths:

The authors have effectively addressed my previous concerns with rigorous additions:

(1) The mathematical formulation has been revised into a discrete signal accumulation framework, making the objective function and resource trade-offs much more transparent and mathematically tractable.

(2) The incorporation of the logarithmic representation resolves prior ambiguities regarding structural constraints.

(3) The new split-half analysis effectively addresses the temporal dynamics of adaptation. The stability of the sublinear scaling across the experiment provides solid evidence that human subjects utilize rapid, top-down modulation to adjust their encoding strategy when explicitly informed about the environment.

(4) Validating the derived scaling exponents on an independent risky-choice dataset robustly supports the generalizability of the theoretical framework beyond a single cognitive domain.

Weaknesses:

The methodological and theoretical issues raised in the first round have been thoroughly resolved, and the evidence supporting the claims regarding response variance is convincing.

There is one remaining theoretical point that warrants discussion to provide a complete picture of the proposed generative model. The manuscript exquisitely models and predicts response variance (imprecision), but it remains largely silent on the closed-form predictions for the mean estimation (i.e., bias). Under the assumption of optimal Bayesian decoding combined with specific encoding schemes (e.g., linear vs. logarithmic), the model implicitly generates mathematical predictions for the subjects' mean estimates. Specifically, varying the scaling exponent (α) and the prior width (w) should systematically alter the predicted bias in different conditions.

While fitting or explicitly explaining this mean bias is not strictly necessary for the core claims regarding variance scaling, acknowledging what the optimal decoder analytically predicts for the mean estimation-and how it aligns or contrasts with typical empirical observations-would strengthen the theoretical transparency of the paper.

Reviewer #1 (Public review):

Summary:

The authors aimed to assess the variability in expression of surface protein multigene families between amastigote and trypomastigote Trypanosoma cruzi, as well as between individuals within each population. The analysis presented shows higher expression of multigene family transcripts in trypomastigotes compared to amastigotes and that there is variation in which copies are expressed between individual parasites. Notably, they find no clear subpopulations expressing previously characterised trans-sialidase groups and that no patterns of coexpressed TcS genes were evident within individual cells or subpopulations. They also note that TcS encoded in the core genome are more often expressed, compared to TcS genes encoded in other genome compartments.

Strengths:

Additionally, the authors successfully process methanol fixed parasites with the 10x Genomics platform. This approach is valuable for other studies where using live parasites for these methods is logistically challenging.

In this second submission the authors show the kallisto mapping approach used is as robust as possible, and that this approach outperforms STAR mapping.

Weaknesses:

The authors describe a single experiment, which lacks repeats, controls or complementation with other approaches and the investigation is limited to the trans-sialidase transcripts.

Comments on revised version:

Thank you to the authors for taking the time to thoroughly address the peer review. The main concerns have now been addressed, and the manuscript edited to make points of confusion clearer.

Reviewer #2 (Public review):

Summary:

This manuscript presents a valuable single-cell RNA-seq study on Trypanosoma cruzi, an important human parasite. It investigates the expression heterogeneity of surface proteins, particularly those from the trans-sialidase-like (TcS) superfamily, within amastigote and trypomastigote populations. The findings suggest a previously underappreciated level of diversity in TcS expression, which could have implications for understanding parasite-host interactions and immune evasion strategies. The use of single cell approaches to delve into population heterogeneity is strong. However, the study does have some limitations that need to be addressed.

The focus on single-cell transcriptional heterogeneity in surface proteins, especially the TcS family, in T. cruzi is novel. Given the important role of these proteins in parasite biology and host interaction, the findings have potential significance.

Strengths:

The key finding of heterogeneous TcS expression in trypomastigotes is well-supported. The analysis comparing multigene families, single-copy genes, and ribosomal proteins highlights the unusual nature of the variation in surface protein coding genes.

Weaknesses:

While the manuscript identifies TcS heterogeneity, the functional implications of the different expression profiles remain speculative. The authors state it may reflect differences in infectivity, but no direct experimental evidence supports this.

The manuscript lacks any functional validation of the single-cell findings. For instance, do the trypomastigote subpopulations identified based on TcS expression exhibit differences in infectivity, host cell tropism, or immune evasion? Such experiments would greatly strengthen the study.

The authors identify a subpopulation of TcS genes that are highly expressed in many cells. However, it is unclear if these correspond to previously characterized TcS members with specific functions.

The authors hypothesize that observed heterogeneity may relate to chromatin regulation. However, the study does not directly address these mechanisms. There are interesting connections to be made with what they identify as colocalization of genes within chromatin folding domains, but the authors do not fully explore this. It would be insightful to address these mechanisms in future work. [...]

Comments on revisions:

The novel version of the manuscript has improved and satisfied this reviewer.

Reviewer #3 (Public review):

The study aimed to address a fundamental question in T. cruzi and Chagas disease biology - how much variation is there in gene expression between individual parasites? This is particularly important with respect to the surface protein-encoding genes, which are mainly from massive repetitive gene families with 100s to 1000s of variant sequences in the genome. There is very little direct evidence for how expression of these genes is controlled. The authors conducted a single cell RNAseq experiment of in vitro cultured parasites with a mixture of amastigotes and trypomastigotes. Most of the analysis focused on the heterogeneity of gene expression patterns amongst trypomastigotes. They show that heterogeneity was very high for all gene classes, but surface-protein encoding genes were the most variable. Interestingly, in the case of the trans-sialidase genes, many sequence variants were detected in fewer than 5% of parasites while a subset of 31 others was detected in >40% if parasites, hinting at compartmentalised expression control within the gene family. The biology of the parasite (e.g. extensive post-transcriptional regulation) and potential technical caveats (e.g. high dropout rates across the genome) make it difficult to infer connections to actual protein expression on the parasite surface, but the results are a significant advance for the field.

(1) Limit of detection and gene dropouts.

An average of ~1100 genes are detected per parasite which indicates a dropout rate of over 90%. It appears that RNA for the "average" single copy 'core' gene is only detected in around 3% of the parasites sampled (Figure 2c: ~100 / 3192). While comparable with some other trypanosome scRNAseq studies, this remains a caveat to the interpretation that high cell-to-cell variability in gene expression is explained by biological factors. The argument would be more convincing if the dropout rates and expression heterogeneity were minimal for highly expressed housekeeping genes. The authors are appropriately cautious in their interpretation and acknowledge the need for further validation.

(2) Heterogeneity across the board.

The authors focus on the relative heterogeneity in RNA abundance for surface proteins from the multicopy gene families vs core genes. While multicopy gene sequences do show significantly more cell-to-cell variability, there is still surprisingly high inequality of expression amongst genes in other classes including single copy housekeeping and ribosomal genes. Again the biological relevance of the comparison is uncertain and the authors acknowledge the need for further investigation.

This study provides some tantalising evidence that the expression of surface genes may vary substantially between individual parasites in a single clonal population. The study is also amongst the very first to apply scRNAseq to T. cruzi, so the broader data set will be an important resource for researchers in the field.

Comment on revised version:

The manuscript is significantly improved. The revised explanations and figures make several aspects of the data analysis and interpretation much clearer to me now. Thanks to the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rupasinghe and co-authors introduce a new statistical model for spiking neurons. Building on earlier work, they propose to model spikes as arising from a Poisson process whereby the firing rate is the product of stimulus drive and a stimulus-independent gain signal. The critical innovation of this work is that the gain signal is modeled in continuous time. Earlier explorations of this statistical construction treated the gain-signal as constant within a trial. This innovation is elegant and important. It makes the model richer, more plausible, and more broadly applicable. The authors show that the model parameters are recoverable from realistic amounts of data and then apply the framework to previously studied datasets. They show that the new model outperforms earlier models and alternative candidates in capturing spiking data across four visual areas of the macaque monkey. Analysis of the model parameters replicates some earlier findings and uncovers several new insights. The model and fitting methods can be broadly applied to partition different types of signals and noise from spiking data and are likely to be widely adopted in the systems neuroscience community.

Strengths:

(1) Through clever use of advanced statistical techniques, the authors manage to infer critical information from single-trial single-cell data.

(2) The question of which aspect of a spike train is signal and which is noise is omnipresent in neuroscience. By improving our ability to characterize the distinct factors that shape spiking activity, this work makes a fundamental contribution to the literature.

Weaknesses:

Overall, I find the work impressive and important. I have a couple of questions and suggestions.

(1) The work is entirely focused on single-cell data. While this is a great starting point, expanding the approach to spiking activity in neural populations is an important future goal.

(2) Line 49-53: These statements seem incorrect to me. The modulated Poisson model, as introduced in Goris et al (2014), is a process model that can perfectly be used to generate spike trains (within a trial, spiking emerges from a Poisson process, which can be homogeneous or inhomogeneous). Moreover, the model contains a parameter that represents the duration of the counting window (delta t). The dependency of over-dispersion on the size of the time bins for real neurons is shown in Figure 1b (inset plot) of that paper (and shown to resemble the model prediction). This time-dependency was further explored by the same authors in Goris et al (2018 - Journal of Vision) and also in Hénaff et al (2020 - Nature Communications ). I suggest that the authors rephrase this argument (here and at some later points in the paper). They could just say that the Goris model makes the simplistic and implausible assumption that, within a given trial, gain does not fluctuate. This is clearly an important limitation and the key difference with the continuous model introduced here.

(3) Line 54-55: I think the first part of the claim is a bit misleading. There is nothing in the Goris model that would inherently limit it to homogeneous Poisson processes, as seems to be implied by this description. The model is built on the assumption that spike generation within a trial arises from a Poisson process. This may very well be an inhomogeneous Poisson process (i.e., a stimulus-dependent time-varying firing rate). Homogeneous and inhomogeneous Poisson processes both give rise to Poisson distributed spike counts (and thus a mixture of Poisson distributions across trials in the Goris model). I suggest the authors clarify this description a bit. Note that the two model variants illustrated in Figure 1b and c were also explored in Hénaff et al (2020 - Nature Communications).

(4) The extension to the continuous case is very elegant!

(5) I find the result shown in Appendix 3 critically important. The recoverability of the model for realistic amounts of data is foundational for the rest of the paper. I would consider including this analysis in the main results section. Not all readers may check Appendix 3, but they should know about this result.

(6) Figure 3: I am wondering whether the inferred gain is capturing some response fluctuations that originate from the cell's phase-selectivity. Could the authors compute the trial-averaged inferred gain (ideally, aligned to stimulus-phase at the start of the trial if this experimental parameter varied across repeats)? If they have successfully partitioned the response variance, the trial-averaged gain should have no systematic temporal structure. If it has a sinusoidal modulation, it may partially capture stimulus-drive. This could be an interesting test to run on all model fits to further validate that the partitioning into a signal and noise component succeeded as intended.

(7) One common observation that is currently not explored is the quenching of neuronal response variability following stimulus onset (Churchland et al 2010 - Nature Neuroscience), which was suggested to reflect a quenching of gain variability in Goris et al (2024 - Nature Reviews Neuroscience). Building on the previous suggestion, the authors could compute the temporal evolution of cross-trial gain variability from the inferred gain traces. Do they recognize a reduction in gain variability following stimulus onset? If so, it would be worthwhile to show this.

(8) Line 543-565: I want to make sure I understand the Baseline Poisson model and Poisson-GP correctly. For the baseline model, I had imagined that the authors would simply use the stimulus-conditioned PSTH as an estimate of the time-dependent firing rate, coupled with an inhomogeneous Poisson process assumption. But they additionally assume a Gamma prior on the firing rate to compensate for the sparseness of the data (sometimes only 5 repeats per condition). The Poisson-GP includes exactly the same model components, but now the time-dependent firing rate is modeled by a Gaussian process. Doing this massively improves the goodness-of-fit (Fig 4A). Do I understand this correctly?

Reviewer #2 (Public review):

Summary:

Neurons have varied responses to external stimuli that cannot be explained by naive Poisson models. Previous work has quantified and partitioned higher-than-Poisson variability in the brain into different components. The authors improve on these methods to infer how both the stimulus drive and internal gain dynamics impact neuronal variability continuously in time. The clean and well-reasoned model is rigorously developed and then applied to neural data across the visual hierarchy. This lends new insights into how variability is partitioned, agreeing with and extending previous work on how that variability changes from early visual areas (LGN, V1) through to higher, motion-sensitive areas (area MT). Another key contribution is that this partitioning can be fully addressed as a continuous-time process, which allows for the dissection of how the timescale of fluctuations in these two components changes across the brain's processing arc.

Strengths:

(1) The model is cleanly derived and thoroughly documented, including usable code shared in a GitHub repo. This makes the method immediately portable to other neural systems.

(2) This is a clear and well-presented piece of work. The figures and writing are clear and understandable, and all pieces of the derivations are included in the main text and supplementary information.

(3) Comparisons to other models, particularly the one from Goris et al., 2014 shows how this Continuous Modulated Poisson (CMP) model outperforms previous work.

(4) New insights about how variability partitioning changes across the visual stream from LGN to MT are revealed, including how the gain fluctuates on longer timescales in higher visual areas. Another key result about the anticorrelation between the variance in stimulus drive and gain fluctuations comports with theories about how neurons maintain efficient, reliable encoding.

(5) In addition to the results reported here, this work will serve as an excellent tutorial for students and postdocs first delving into the sources of variability in the brain.

Weaknesses:

The work is somewhat incremental, building on previous studies of the partitioning of variability in the brain, but it provides important new extensions, as noted above.

The only major gap I would suggest addressing in the Discussion is the observation of sub-Poisson variability in the brain. It seems clear that this model can extend to sub-Poisson variability and its partitioning and perhaps even show how that varies in real time, with an animal's attentional state. That is, of course, beyond the scope of the current work, but could be mentioned in the Discussion.

Reviewer #1 (Public review):

Summary:

Maigler et al. set out to test the hypothesis that individual differences in taste preferences are (in part) due to individual differences in central taste processing. The first tested rats' preferences for a variety of taste stimuli on multiple days. They then recorded responses of neurons in the taste cortex to the same tastes on two consecutive days.

Strengths:

The authors collected high-resolution behavioral data from the same animals across multiple days, allowing for a detailed characterization of individual variation in taste preferences. They then performed recordings from the same set of animals in response to the same stimuli, allowing them to draw parallels between behavioral and neural responses. They convincingly show that preference ranks for a variety of basic tastes change over time and that the correlation between neural responses and preferences is not stable, correlating more strongly with more recent measures of preference.

Weaknesses:

Behavioral analysis: Data presentation does not show how preferences change over the course of testing. In particular, it is unclear whether there are any systematic changes in preferences over the course of testing that could explain the observed changes in correlation with neural responses, such as changes due to learning (e.g., flavor nutrient conditioning, relief of neophobia), changes in deprivation state, or habituation to/proficiency with the BAT setup. A secondary point is whether any changes in preference are attributed to internal individual versus external contextual factors. Both types of variation (i.e., across individuals and across time within an individual) are mentioned in the introduction, but it is not clear what the authors believe about the nature or neural representation of these sources of variation.

With respect to neural data analysis, no individual animal/day data are shown, making it difficult to assess the extent to which differences in correlation match individual differences in preferences and/or changes in preference with time within individuals. The correlation analysis is also lacking control for the fact that there is a certain degree of "chance" associated with behavioral and neural measures having matching ranks.

Finally, the conclusion that correlations between final day preferences and neural responses obtained from the second recording session are the result of experience needs more justification; it is unclear to what extent changes in correlation may be attributed to overall changes in responsiveness of the neural population.

Reviewer #2 (Public review):

Summary:

The study from Maigler et al investigates how between- and within-animal differences in taste preference relate to differences in neural responsiveness. The experiments rely on an elegant combination of behavioral assays to measure preference (e.g., repeated brief access testing, BAT) and electrophysiological recordings to monitor the activity of ensembles of neurons in the gustatory cortex (GC) of rats.

BAT with distinct batteries of tastants revealed pronounced variability in preference (measured as licking bout size) across individuals. This variability across individuals persisted after repeated testing. Repeated BAT also revealed that each rat's preference for different tastants changed across time.

Electrophysiological responses of GC neurons to batteries of tastants showed that firing in the "late epoch" of taste processing (i.e., 500ms post taste delivery) correlated more strongly with the individualized rat's BAT preference rather than with a canonical preference ranking. Importantly, this correlation was stronger for the last BAT session compared to the first. Finally, the author shows that the correlation disappeared in a second, consecutive recording session, indicating that exposure to tastants reconfigures preferences.

Strengths:

(1) The experimental design allows for an unprecedented look at the relationship between individual variability in taste preferences and neural processing.

(2) The study demonstrates that taste preference variability is not mere experimental noise but reflects the dynamic nature of taste. A key strength is the clear evidence that behavioral variability is reflected in neural activity patterns, establishing a strong correlation between brain and behavior.

(3) The evidence that simple exposure to familiar tastes can reconfigure preferences and taste representations is interesting.

Weaknesses:

(1) The manuscript could use additional corollary analyses to provide a more complete picture of the phenomenon. For instance, how many neurons (per animal and in total) have significant correlations with the final BAT patterns? And with the first BAT? Can a time course of such counts be provided? Can some decoding analyses be performed at a single session level to reconstruct a rat's behavioral preference pattern from its neural activity?

(2) The manuscript could benefit from additional polishing, both in the text as well as in the figures.

Reviewer #3 (Public review):

Summary:

Maigler & Lin et al present a compelling set of behavioral and electrophysiological experiments exploring how individual differences in taste preference map onto neural responses in the gustatory cortex (GC). They go on to examine how both preferences and neural responses shift following intervening taste experience. Their experiments are strengthened by examining tastes of distinct identities and palatability (sweet, sour, salty, bitter) and corresponding each animal's individual preference to the palatability-related late phase of the neural response.

Strengths:

(1) They demonstrate a relationship between the behavioral expression of taste preference and palatability-related GC neural responses. The direct correlation of expression of taste preference with GC neural responses indicates that taste preference behavior may be less noisy than previously thought, reflecting actual neural activity.

(2) They address the stability of individual taste preference by comparing within and between session expression. This finding indicates that individual preference on any given test session can differ from canonical palatability.

(3) They provide evidence that representational drift in palatability coding may arise from sensory experience rather than from the passive passage of time. The findings are novel and potentially impactful. The results are relatively complete.

Weaknesses:

Experiments require further clarification. The interpretations would be strengthened by reorganizing the experimental design.

(1) Figures 5-6 show shifts in palatability-related GC responses from recording day 1 to recording day 2. The authors propose that this drift is due to the taste experience during recording day 1, but the study, as designed, does not directly test this idea. Without a behavioral measure collected after recording day 1 intraoral exposure, it is not possible to determine whether taste preference was altered by that experience, nor whether the neural responses collected on recording day 2 represent current or most recent palatability expression vs something else. The authors' conclusion would be strengthened by adding an intervening brief access test between recording days 1 and 2. The authors could then determine whether the behavioral preferences changed after intraoral taste exposure on recording day 1, as well as whether the new set of taste-related palatability responses correlates with the updated taste preferences.

(2) The current experimental design exposes animals to 3 distinct sets of substances. These substances differ in identity (some rats never experienced sweet, while others did not experience bitter during the recording sessions) and concentration (ranging from very aversive to slightly aversive or possibly even neutral). Because palatability is known to be comparative depending on the other substances available and concentration-dependent, this introduces challenges to interpretation.

The authors state that "no differences in effects were observed between taste batteries" (Methods), but it is not clear which analyses were performed to determine the lack of difference, especially considering that many of the analyses are within-animal. Without more clarity, it is difficult to evaluate whether the interaction of different tastes within the sets of stimuli biases the main conclusions.

(3) Responses to sweet tastes are not reported in the electrophysiology data. This is seemingly the case because rats given set 1 received no sweet stimulus while rats given set 2 received to 2 distinct sweet tastes. Finally, rats given set 3 did not receive quinine, yet quinine is reported in electrophysiology data.

(4) The choice of reporting average lick cluster size is problematic because the authors use thirsty rats with 10-second-long trials. Thirsty rats are likely to lick in relatively long clusters, especially for neutral and palatable tastes. If the rat is mid-cluster when the trial ends, the final cluster would be cut off prematurely, resulting in shorter overall average lick cluster size, disproportionately affecting neutral and palatable tastes over aversive tastes.

(5) Canonical palatability rankings may not apply to the concentrations selected in every stimulus set. This is particularly true for set 1, which included two concentrations of citric acid and quinine for the behavior. It is also not clear which concentrations are reported in Figures 3A2 and 3B2. Meanwhile, the concentrations of quinine and citric acid used for electrophysiology are quite low.

Reviewer #1 (Public review):

Summary:

The authors provide extensive immunoreactivity and expression data to map monoaminergic neurotransmitter production sites in Pristionchus pacificus. This nematode is relatively distantly related to the popular model nematode Caenorhabditis elegans, for which such information is already available. They find that dopamine, tyramine, and octopamine are present in the same neurons in both species, but differences are observed for serotonin. This forms the basis for a comparison of serotonergic neurons across 22 nematode species. In addition, they evaluate monoaminergic effects on egg-laying, head movement during reversals, and nictation behavior, to find that monoaminergic control over the latter differs between C. elegans and P. pacificus. This shows that some anatomical flexibility supports similar outcomes, whereas in other cases it is the basis of evolved regulatory differences.

Strengths:

The comparative efforts are laudable and valuable, including a thorough revisiting of old data and corrections of what is judged as a historic misannotation. The expected continued value of this work is also appreciated, because nematodes have similar anatomies and behaviors, cellular-resolution data of different species permits the study of functional evolution of neurotransmitter usage in homologous neurons.

Despite the strong experimental approach, there are some points that require addressing:

(1) Not all the concepts of the introduction ('feeding behaviors', to a lesser extent also 'evolution of neurotransmitter usage in homologous neurons') are followed up upon in the results or discussion sections.

(2) The choice of nematodes ('only' 13 species) may affect what is perceived as ancestral. Also, identifying their cells based on comparisons with Ce or Ppa identifications only is understandable but mildly risky: there are many cells in the head, and mistakes would go unnoticed until detailed analysis in each species can provide conclusive evidence.

(3) It is not reported whether the nictation-defective mutants have general locomotion defects; therefore, whether the reported problem is specific to this host-finding behavior or not.

(4) The section on RIP neurons makes sense for Ppa, but not for Ce (dauers in fact have weakened IL2-to-RIP connections), and should be revised. The nictation data also do not support the breadth of the conclusions, which should either be toned down or rephrased as hypothetical.

(5) The discussion mostly reiterates the results, leaving little room for the author's interpretations and opinions. I would suggest reworking in favor of conceptual discussion.

Reviewer #2 (Public review):

Summary:

This paper makes important contributions to our understanding of how nervous systems evolve, with a particular focus on whether changes in neurotransmitter usage within homologous neurons represent a mechanism for evolutionary adaptation without large-scale changes to circuitry. Comparing the predatory nematode P. pacificus with C. elegans, this study systematically examines monoamine-producing neurons, assesses how their neurotransmitter identities differ between homologous neural types, and determines how these differences relate to behavior.

Strengths:

The major strength of this work is its breadth, rigor, and data quality. It combines multiple, independent lines of evidence to assign neurotransmitter identity for neurons with homology grounded in lineage, morphology, and connectomics, which is essential for meaningful cross-species comparisons. Additionally, by extending the analysis beyond P. pacificus and C. elegans to other nematodes, the authors convincingly argue that features observed in P. pacificus likely reflect an ancestral state. This depth greatly enhances the significance of the conclusions.

This work is likely to have a significant impact on the fields of comparative neurobiology and nervous system evolution. It demonstrates a powerful system and approach for linking molecular identity, cell-type homology, circuit context, and behavior across species. The data generated here will be a valuable resource for the community and provide a strong foundation for future mechanistic studies.

More broadly, the study reinforces the idea that evolutionary change in nervous systems can occur through modulation of chemical signaling within conserved circuits, rather than through complete rewiring. This conceptual framework is likely to influence how researchers think about neural evolution in other systems.

Weaknesses:

Given the availability of detailed connectivity information for both species, a more explicit comparison of the local circuit context of key neurons would further strengthen the link between molecular identity and circuit function.

Reviewer #3 (Public review):

Summary:

The study by Hong, Loer, Hobert, and colleagues is a comprehensive description of monoaminergic neurons in the nematode Pristionchus pacificus. The work used multiple, complementary approaches, including immunostaining and expression of genes involved in neurotransmitter synthesis or transport, to identify neurons that express a monoamine neurotransmitter. Moreover, this study characterized the phenotypes of various mutants to study their organismal function. Extensive comparisons are made to C. elegans, the nematode model that, in a way, anchors the model studied here, and new outgroup species were examined for some features so that the polarity of their evolution could be inferred. Although there is no simple or groundbreaking punchline to distill from the manuscript (i.e., other than some things are the same as in C. elegans, and some things are different), and while the study is basically descriptive in nature, the scope of the project warrants broad attention.

Strengths:

This manuscript offers a tremendous resource for those who use this species as a model, which, based on the author list alone, includes many labs. This study sets the bar for what can be done in a "satellite" model system.

Given the complementarity of approaches used, such as the position of cell bodies, the connectivity and morphology of dendrites, and a previously published atlas of the connectome for this species, the identification of specific neurons (which, as the authors point out, can be easily mistaken) is convincing throughout. Likewise, appropriate caution is observed where neuron identities are ambiguous, e.g., unlabelled cells in Figure 5, or ambiguous identities in other species, as shown in Figure 10. There was a lot of data to unpack in this manuscript, but I could not find any obvious flaws in neuron identification.

Also, the phenotypic assays were straightforward and informative.

Weaknesses:

No serious weaknesses were noted. One minor comment is that in general, I think the Methods could use some additional text to describe what the goal of any given technique was. For example, although there is a description of the HCR protocol in the methods, nowhere does it say what genes this method would be used for. In addition to what is shown in Figure 4, this information should be given in the Methods.

Reviewer #1 (Public review):

Summary:

This manuscript examines whether retrieval practice protects memory-based inference from acute stress and proposes rapid neural reactivation of a bridging memory element as the underlying mechanism. Using a two-day associative inference paradigm combined with EEG decoding, the authors report that stress impairs inference accuracy and speed, while retrieval practice eliminates these deficits and restores neural signatures associated with bridge-element reactivation. The study addresses an important and timely question by integrating research on retrieval-based learning, stress effects on memory, and neural dynamics of inference. While the work provides promising multi-level evidence linking behavioral and neural findings, limitations in experimental design, causal interpretation, and decoding specificity weaken the strength of the mechanistic claims and suggest that further work is needed to disentangle strengthened associative memory from inference-specific protection effects

Strengths:

(1) Strong theoretical integration<br /> The study integrates three influential frameworks: memory integration through associative inference, stress-induced retrieval impairment, and the testing effect. The authors present a clear theoretical narrative linking these domains and derive testable hypotheses that retrieval practice protects inference by strengthening neural reactivation of a bridge element. The conceptual framing is well-grounded in prior literature and addresses an important gap regarding neural dynamics during inference.

(2) Multi-level evidence<br /> The study provides converging behavioral and neural evidence. The authors demonstrate that stress reduces inference accuracy and speed, while retrieval practice eliminates these deficits. EEG decoding further suggests that bridge element reactivation predicts successful inference. The combination of behavioral performance and neural decoding strengthens the overall argument.

(3) Transparent experimental implementation<br /> The procedures are described in substantial detail, including stimulus construction, stress manipulation, and decoding pipelines. Data and code availability are also strengths, facilitating reproducibility.

Weaknesses:

(1) Insufficient evidence that retrieval practice specifically protects inference rather than strengthening associative memories

A central claim of the manuscript is that retrieval practice specifically protects inference ability rather than simply strengthening underlying associative memories. However, the current data do not convincingly distinguish between these possibilities. Although the authors limited analyses to trials in which AB and BC pairs were correctly retrieved in the subsequent memory test, this procedure does not fully rule out the possibility that improved inference performance reflects stronger base associative memories rather than enhanced integrative processes.

Importantly, the direct memory retrieval test used a two-alternative forced-choice (2AFC) format, which inherently allows a substantial proportion of correct responses to arise from guessing. Consequently, trials classified as "successfully retrieved" may still include weak associative memory traces, making it difficult to conclude that failures in inference reflect deficits in integration rather than incomplete associative learning.

The authors further argue that retrieval practice does not improve inference in the absence of stress, suggesting independence between inference and associative memory strength. However, this null effect does not sufficiently rule out mediation through strengthened premise memory. A factorial design and/or mediation analysis would be necessary to determine whether inference resilience emerges independently of premise memory strength.

(2) Apparent below-chance inference performance raises interpretational concerns

One surprising aspect of the results is that inference performance across experiments and groups appears to fall below the theoretical chance level (0.33) in Figure 4A. This is particularly unexpected because analyses were restricted to trials in which participants correctly retrieved both AB and BC associations.

If performance is indeed below chance, this raises concerns regarding whether participants fully understood the task instructions or whether other methodological factors influenced performance. Additionally, below-chance performance complicates the interpretation of subsequent behavioral and neural analyses. It is possible that this reflects my misunderstanding of the figure; therefore, clarification from the authors regarding how inference accuracy is calculated and presented would be helpful.

(3) Between-experiment implementation of retrieval practice weakens causal inference

The retrieval practice manipulation was implemented as a separate experiment rather than as part of a factorial design. Experiment 2 was conducted after results from Experiment 1 were known, and the authors acknowledge this post hoc decision. This design introduces several potential confounds, including cohort differences between experiments, possible differences in participant motivation or task familiarity, and reduced ability to rigorously test interaction effects.

Although the authors combined data across experiments to test interactions between stress and retrieval practice, such post hoc aggregation cannot fully substitute for a factorial design. A within-experiment 2 × 2 design (Stress × Retrieval Practice) would provide substantially stronger causal evidence and reduce confounding influences.

(4) Lack of an appropriate comparison condition for retrieval practice limits the interpretation of the mechanism

Although acknowledged briefly in the discussion, the absence of an appropriate comparison condition for retrieval practice represents a critical limitation. Without a matched re-exposure or restudy control condition, it remains unclear whether observed benefits are attributable specifically to retrieval practice or to additional exposure to AB and BC associations.

Furthermore, it is unclear whether retrieval practice operates at the trial level or the participant level. Retrieval practice could enhance memory representations for specific practiced items, making those trials more resistant to stress, or it could induce a more global change in cognitive strategy or stress resilience across participants. One way to address this issue would be to analyze inference performance separately for trials that were successfully retrieved during the retrieval practice phase versus those that were not.

(5) Interpretation of EEG decoding as bridge-element reactivation may be overstated

The neural decoding results form the mechanistic foundation of the manuscript; however, the interpretation that decoding reflects reactivation of specific bridging memories may be overstated. The classifier distinguishes between face and building categories, and because the bridging element belongs to one of these categories, successful decoding may reflect category-level semantic activation rather than reinstatement of item-specific episodic representations.

Alternative explanations include category-level retrieval, strategic task differences, or even attentional biases. Because only two categories were used, the decoding analysis lacks the specificity necessary to distinguish between category-level and item-level reactivation. As such, conclusions regarding the reinstatement of specific bridging memories should be tempered or supported with additional analyses.

Reviewer #2 (Public review):

Summary:

Guo et al. investigate the neural and behavioral mechanisms of stress-induced impairments in memory-based inference. Across two well-powered experiments (N=136), the authors demonstrate that acute stress disrupts the rapid neural reactivation of "bridge" elements necessary for novel inferences. Crucially, they identify retrieval practice as a robust behavioral buffer that restores both inferential performance and the underlying neural signatures of memory reactivation.

Strengths:

(1) The use of two independent experiments provides high confidence in the behavioral findings.

(2) Utilizing time-resolved EEG decoding allows the authors to pinpoint the "online" moment of inferential failure, a significant advancement over the lower temporal resolution of fMRI.

Weaknesses:

(1) The authors correctly timed the inference task to begin approximately 20 minutes after the onset of the stressor. While this window aligns with the expected peak of the glucocorticoid (HPA) response, it also represents a period where the rapid adrenergic (SAM) response, confirmed by heart rate elevation, is still highly influential. As the authors acknowledge, because they did not collect saliva samples due to safety protocols, they cannot definitively separate the influence of peak cortisol from the tail-end of the adrenergic surge on the observed memory impairments.

(2) Figures 4 and 6: Without asterisks is really difficult to compare the significant group differences.

Appraisal and Impact:

This study provides high-quality evidence that acute stress impairs the rapid neural reactivation of "bridge" elements necessary for novel memory-based inferences. By leveraging the high temporal resolution of EEG decoding, the authors identify the specific neural "chokepoint" where inferential failure occurs. The research is strengthened by two independent experiments and the identification of retrieval practice as a powerful buffer that not only preserves but also enhances neural reactivation under pressure. The findings have significant implications for both cognitive neuroscience and applied learning science.

Reviewer #3 (Public review):

Summary:

In this study, Guo and colleagues investigated the effects of stress and retrieval practice on memory inference. In the first experiment, they found that memory inference was significantly worse after induced stress. Conversely, when participants received retrieval practice in the second experiment, they found no significant differences between these conditions. They monitored EEG during the inference phase and applied multivariate decoding analysis to examine evidence of neural reactivation. Complementing the behavioural findings of the first experiment, they found that they were able to decode the stimulus category of the inference item with more fidelity in the no stress condition. Surprisingly, they found the opposite direction when participants had retrieval practice, with stronger evidence of reactivation in the stress condition than in the control condition.

Strengths:

(1) The authors have carefully designed two studies investigating the effects of stress and memory retrieval on memory inference.

(2) The use of multivariate decoding on the inference phase data sheds new light on how stress and retrieval may impact the neural signatures of inference processing.

Weaknesses:

(1) There are some key gaps in the reporting of the data. In particular, data is missing on how many trials were included in the inference phase and how many were retrieved in the direct memory task. This is important to know as the main conclusions are based on inference trials proportional to the direct retrieval trials. Considering that the direct retrieval performance differs significantly between the experiments, there could be issues with floor/ceiling effects (in the behaviour) and statistical power (in the EEG results) that confound the comparisons between experiments. Without the data, it is difficult to draw conclusions.

(2) There are some relatively strong conclusions drawn without the data to support them. An important example is the title suggesting a mechanistic role of memory reactivation for these effects; however, the data instead suggest a relationship between successful inference and evidence of reactivation. Additionally, one-tailed t-tests have been used in follow-up tests, and, as I understand it, no multiple comparisons corrections have been applied to the post-hoc tests, suggesting that these findings should be interpreted with caution.

(3) In places, the structure is unclear, making the narrative difficult to follow, often making it necessary for the reader to go back and forth between the sections to understand the study and analyses. I have made some recommendations for how to improve this.

Reviewer #1 (Public review):

Summary:

The authors report a novel binding partner of the TolC channel protein that forms complexes with the two principal classes of transporter-based tripartite assemblies (both ABC- and RND-transporter based) and appears to modulate their function, while also anchoring TolC into the outer membrane, compensating for the lack of direct lipidation seen in other members of the OMF family.

The newly identified protein, YbjP, is comprehensively characterized from both phylogenetic and structural perspectives. Two independent cryo-EM structures (MacAB-TolC-YbjP and AcrABZ-TolC-YbjP) provide strong structural evidence for its role and are generated using peptidiscs, mimicking the membrane environment. These findings are further supported by pull-down experiments (including state-of-the-art in vivo photo crosslinking) and functional assays for a well-rounded characterisation of the protein, and a significant amount of modelling and phylogenetic analysis. This work sheds light on the function of the members of the DUF3828-containing protein family, which appear to anchor TolC to the outer membrane and influence the expression of the TnaB and YojI transporters.

Strengths:

The strengths of the manuscript are numerous, and it presents a well-rounded package of structural biology complemented by functional and computational studies.

The full assemblies of both MacAB-TolC-YbjP and AcrABZ-TolC-YbjP are reconstituted and resolved to near-atomic resolution using cryo-EM for unambiguous assignment of binding interfaces, which are then validated using a number of techniques, including ITC, in vitro and in vivo binding assays and cross-linking.

The evolutionary analysis is particularly notable, and provides genuine insight into the DUF3828-containing proteins, the function of which remains enigmatic till now. Similarly, the involvement of YbjP in trafficking of TolC and the analysis of the impact of YbjP deletion of the full E. coli proteome is commendable.

Overall, this is a very solid piece of work, competently executed and presented, which significantly advances the field.

Weaknesses:

None obvious, however the presentation and especially main-text illustrative material seems to focus disproportionately on MacAB-TolC-YbjP complex, and the AcrABZ-TolC-YbjP is relegated to supplementary data which is somewhat confusing. There is no high-resolution side view of the AcrABZ-TolC-YbjP side-by-side to MacAB-TolC-YbjP which may be helpful to spot parallels and differences in the organisation of the two systems.

Supplementary Figure 2 may also be better presented in the main text, as it shows specific displacements of residues upon binding of the YbjP relative to the apo-complexes, although this can be left at the authors' discretion.

Reviewer #2 (Public review):

This article focuses on the study of two E. coli tripartite efflux pumps both using TolC as partner in the outer membrane, namely MacAB-TolC and AcrABZ-TolC.

By preparing MacAB-TolC in Peptidiscs rather than in detergent for cryo-EM structure determination, they visualized an extra protein localized around TolC. The resolution was sufficient to build part of the structure, and using the AlphaFold2 database and DALI topology recognition program, they identified it as the lipoprotein YbjP. This protein has an anchorage in the outer membrane, and it was suggested that it could act as a support for TolC that is the only OMF that does not have an N-terminal extension anchored in the outer membrane, which is very puzzling for the community working in this field of research.

Authors used a large number of different approaches to evaluate the importance of YbjP (structure, genomic evolution, microbiology, photocrosslink in vivo, proteomic profile), but did not succeed in finding it a clear role so far, even if it could be important depending on environmental stress. Nevertheless, their results are of main interest for the comprehension of the complexity of such systems and deserve publication.

The different analyses are properly performed and presented, and support the conclusions.

My only concern is for the photocrosslink presented in Figures 3 and S3. My impression is that the bands do not migrate at the proper size after the crosslink.

A second point that could be discussed further is the comparison of the structure of the pump in the presence of the peptidoglycan with the images previously obtained by tomography. It is not totally clear to me if YbjP could have been positioned in these maps.

Reviewer #1 (Public review):

Summary:

The authors introduce EMUsort, an open-source algorithm for the automatic decomposition of high-resolution intramuscular EMG recordings. The method builds upon the Kilosort4 framework and incorporates modifications designed to better handle the spatial and temporal characteristics of intramuscular signals. The performance of EMUsort is evaluated on openly available datasets and compared against KS4 and MUEdit, demonstrating improved motor unit accuracy.

Strengths:

(1) The manuscript is clearly written, technically detailed, and well structured.

(2) The open-source software is thoroughly documented, both within the manuscript and in the accompanying repository README, facilitating adoption by the community.

(3) The availability of both code and datasets is a major strength, enabling reproducibility and independent validation.

(4) The authors provide quantitative comparisons with existing decomposition algorithms, which is essential for contextualizing the proposed method.

(5) The methodological details are sufficiently described to allow replication and further development by other researchers.

Weaknesses:

While the manuscript is strong overall, I have several suggestions that could further strengthen its impact and clarity.

(1) Benchmarking and community integration

A recent work has proposed standardized datasets and benchmarking pipelines for high-density surface EMG decomposition ("MUniverse: A Simulation and Benchmarking Suite for Motor Unit Decomposition", Mamidanna*, Klotz*, Halatsis* et al, NeurIPS 2025). A similar effort for intramuscular EMG would be highly valuable to the field. The authors may consider discussing how their dataset and algorithm could be integrated into broader benchmarking initiatives (e.g., platforms such as MUniverse), enabling systematic comparisons across multiple datasets and decomposition methods.

(2) Comparison with additional decomposition algorithms

Since the manuscript compares EMUsort with MUEdit, it would be appropriate to also include a comparison with Swarm-Contrastive Decomposition (SCD), which has been proposed for both surface and intramuscular EMG signals. Including this comparison, or explicitly discussing why it was not feasible, would strengthen the positioning of EMUsort relative to the current state of the art.

(3) Manual editing and post-processing

In practical EMG decomposition workflows, manual inspection and editing of motor units are often required after automatic decomposition. It would be useful for readers to know whether EMUsort provides (or is compatible with) a graphical interface or workflow for manual refinement, or how the authors envision this step being handled.

(4) Ablation analysis of algorithmic modifications

EMUsort is described as an extension of Kilosort4. An ablation analysis examining the impact of the main modifications introduced relative to KS4 would help clarify which changes contribute most to the observed performance improvements and under which conditions.

(5) Failure modes and limitations

A more explicit discussion of when EMUsort is likely to fail or degrade in performance would be valuable. For example, sensitivity to the number of channels, recording duration, signal quality, or motor unit density could be discussed to guide users.

(6) Generalisability to surface EMG

Given the shared methodological foundations between surface and intramuscular EMG decomposition, it would be helpful to know whether EMUsort has been tested on high-density surface EMG datasets or whether the authors expect limitations when applied outside the intramuscular domain.

(7) Applicability to human intramuscular recordings

The authors could clarify whether EMUsort has been tested on human intramuscular EMG, and discuss any expected differences in performance due to anatomical or physiological factors.

(8) Parameter sensitivity

Clustering-based methods can be sensitive to parameter choices. Reporting a parameter sensitivity analysis, or at least discussing the robustness of EMUsort to parameter variations, would increase confidence in the method's reliability and ease of use.

(9) Differences between template matching and BSS methods

Since the manuscript proposes a new template matching algorithm, but it compares its performance with a BSS one (MUedit), BSS algorithms should be described in the introduction. The differences between the methodologies should be highlighted, and the pros and cons of each described.

Conclusion:

The authors largely achieve their stated aims, and the results mostly support the main conclusions. EMUsort represents a meaningful contribution to the EMG decomposition literature, particularly for researchers working with high-resolution intramuscular recordings. With additional clarification regarding benchmarking, algorithmic ablations, and limitations, the manuscript would be further strengthened and likely to have a substantial impact on the field.

Reviewer #2 (Public review):

Summary:

This work presents a new spike sorter, EMUsort, to target the challenging task of spike sorting Motor Unit Action Potentials (MUAP). EMUsort is essentially a modified version of Kilosort, with some key extensions to target EMG data: correct for large delays due to propagation across channels, spike detection of highly overlapping and large units via multiple thresholds, an increased number of waveform templates for spike detection, and an extended representation of waveforms to grasp complex MUAP spike shapes. The results on simulated data show solid evidence that the applied modifications make a difference for EMG recordings. All in all, I believe that EMUsort will greatly improve spike sorting performance for high-density EMG data.

Strengths:

The manuscript is well written, and the methods and modifications to the Kilosort pipeline are well-motivated, well-explained, and clear. The simulation results provide strong evidence that the presented modifications make spike sorting of high-density EMG data more accurate.

Weaknesses:

The method is overall only validated on 15 simulated motor units. The monkey dataset, in particular, seems too "easy" and not challenging enough to highlight weaknesses of any of the spike sorters. A second weakness is in the distribution of the code, which is shipped with submodules for Kilosort and SpikeInterface, and makes it hard to maintain long-term, and pins to old versions of these key dependencies.

Reviewer #3 (Public review):

Summary

This paper introduces EMUsort, an extension of Kilosort4 designed to sort motor unit action potentials from high-density intramuscular EMG recordings. Using rat and monkey forelimb recordings, the authors generate realistic simulated datasets with known ground truth and demonstrate that EMUsort substantially outperforms Kilosort4 and MUedit, particularly during periods of high motor unit overlap.

Strengths

This is a timely study in light of recent advances in intramuscular muscle recording technologies and the growing interest in automated methods for decoding neural and neuromuscular signals. The work leverages state-of-the-art electrode arrays and combines them with advanced signal processing tools to address a challenging and relevant problem in motor unit analysis.

Weaknesses

There are several aspects of the study that substantially limit the interpretation of the main results and conclusions. The following major points should be carefully considered by the authors.

(1) Choice of experimental model and validation framework: The study aims to validate a new methodology for EMG decomposition, yet the rationale for the chosen experimental models is unclear. Specifically, it is not evident why the authors focused on intramuscular recordings from two animal models performing dynamic tasks. Given the extensive literature on the development and validation of EMG decomposition methods, the choice of an experimental design that substantially deviates from established approaches is insufficiently justified. In particular, it is unclear why the authors did not consider more standard validation paradigms based on (i) isometric contractions, (ii) human data, (iii) surface EMG recordings, or (iv) combinations of their recording technologies with previously validated motor unit identification methods. This methodological divergence makes it difficult to interpret the findings in the context of existing evidence.

(2) Lack of manual EMG decomposition as reference: Related to the previous point, it is unclear why standard manual EMG decomposition methods were not used to generate reference datasets for validation. Manual decomposition has been shown to be reliable under specific conditions (low contraction levels, slow dynamics, etc.) and would have substantially strengthened the validation of the proposed algorithm.

(3) Neglect of muscle deformation effects: While the manuscript discusses several factors that complicate EMG decomposition relative to brain recordings, it does not address the well-known effects of muscle deformation during contractions on motor unit action potential shapes. There is extensive literature demonstrating that dynamic muscle contractions lead to systematic changes in action potential morphology, representing a major challenge for EMG decomposition and a fundamental difference from brain recordings. Additionally, even small relative movements of intramuscular electrodes can produce waveform changes that are large relative to muscle fiber dimensions. These issues are particularly relevant given the highly dynamic tasks studied here (e.g., treadmill walking in rats), yet they are not discussed or incorporated into the analysis.

(4) Exclusive reliance on simulated data for validation: The primary validation of EMUsort is based on simulated data, which represents a major limitation of the study. This reliance should be clearly and explicitly stated in the abstract, introduction, and discussion. Moreover, the simulation approach itself raises concerns. The simulated EMG signals are generated using templates derived from the same sorting framework being validated, which introduces a potential methodological bias. The linear combination of components used to synthesize waveforms constitutes an unjustified modeling assumption that may favor template-based approaches such as EMUsort. Additionally, the spike time generation procedure appears unnecessarily complex and insufficiently justified. Previous validation studies typically modeled motor units as firing at relatively stable levels along their recruitment curves, producing long spike trains with pseudo-random relative timing and diverse overlap conditions. This framework would likely provide a more robust and interpretable validation. If the authors believe their simulation approach is superior, a stronger justification is required. Finally, the limited number of simulated motor units is difficult to reconcile with the expected level of motor unit recruitment during the studied behaviors, and this choice is not adequately justified.

(5) Incomplete reporting and visualization of experimental data: The manuscript would benefit from a clearer description of the number of rats and monkeys used, which should be reported explicitly in the abstract. In addition, visualizations of the raw multichannel EMG data across different task phases and activation levels would substantially improve transparency. Providing comprehensive visualizations of motor unit action potential shapes across all channels and identified units (for both rats and monkeys) would also help readers assess the spatiotemporal features that underpin unit identification and sorting reliability.

(6) Physiological limitations of conduction delay correction: The proposed method for correcting conduction delays across channels is physiologically suboptimal. First, motor unit conduction velocities differ substantially across units, implying that delay correction should be applied at the unit level rather than uniformly across channels. Second, conduction delays depend on fiber orientation and distance relative to electrode geometry; if fibers are oriented at different angles with respect to the array, a single delay correction becomes invalid. Additionally, the schematic in Figure 2A appears to contradict the proposed correction approach: if electrode threads are arranged perpendicular to muscle fibers, conduction delays across channels within a single thread should be minimal.

(7) Clarity issues in Figure 4: Figure 4 (panels A-D) is potentially misleading. It should be clearly stated whether the signals shown are artificial examples or derived from real recordings; ideally, real data should be used to illustrate the advantages of dynamic thresholds. In panels B-D, the depiction of overlapping action potentials is difficult to interpret due to the thickness of the traces, and it is unclear whether overlaps with neighboring action potentials are absent by design or expected to occur in real data. If overlaps are expected, one would also expect to observe contamination in the extracted waveforms, which is not evident in the figure.

(8) Concerns regarding method comparisons: The comparison with existing methods raises methodological concerns. It appears that EMUsort was carefully optimized, whereas the competing algorithms were not equivalently fine-tuned. The literature clearly shows that EMG decomposition performance depends strongly on adapting algorithms to the signal type (intramuscular vs. surface, species, electrode geometry). Furthermore, it is surprising that MUedit is reported to perform particularly poorly during periods of motor unit overlap, as blind source separation methods were explicitly developed to handle convolutive mixtures and overlapping sources, especially in surface EMG (which is an extreme case of motor unit overlapping). This discrepancy requires further explanation.

(9) Insufficient characterization of motor unit firing properties: The study does not provide sufficient information about the firing characteristics of the identified motor units in experimental data. Relevant metrics that should be reported include average, minimum, and maximum firing rates; coefficients of variation of discharge rate; signal-to-noise ratios of motor unit action potentials; potential evidence of motor unit rotation over time; and stability of firing behavior across recording intervals.

(10) Lack of theoretical framing: Given the scope and claims of the paper, it would be valuable to include a more theory-driven introduction explaining why different sorting approaches (e.g., template matching vs. blind source separation) may be more or less suitable depending on the nature of the recorded signals. A clearer conceptual rationale for why the proposed approach is expected to outperform existing methods would substantially strengthen the manuscript.

(11) Limitations of validation metrics: Some of the metrics used to evaluate performance are not ideal. For example, reporting 0% accuracy for a unit is misleading and should instead be described as a failure to identify that unit. Similarly, comparisons of total spike counts are of limited interpretive value and may be misleading, as correct spike counts do not necessarily imply correct spike identities.

(12) Clarification of computational performance claims: Finally, the discussion of computation times should clarify that some existing methods require substantial time for offline estimation of projection vectors but can operate in near real time once these vectors are learned and remain stable. This distinction is important for a fair comparison of practical usability.

Reviewer #1 (Public review):

Summary:

Freas and Wystrach present a computational model of steering in insects. In this model, the central complex provides an error signal indicating the animal should turn left or right; this error signal biases the function of an oscillator composed of two mutually inhibiting self-exciting units. The output of these units generates a "steering signal" that is used both to set the direction and speed of the ant. Additionally, a separate module induces pauses, and an inverse relation between forward speed and turning speed is externally imposed. Statistics of the trajectories generated by the model are compared to the measured behaviors of ants.

Strengths:

While the model is very simple compared to state-of-the-art models, that simplicity makes it a potentially useful guide to researchers studying insect navigation. Some predictions that emerge from the model appear to be experimentally testable, although a more complete description of the model and its parameters, as well as an analysis of how this model's predictions differ from previous models' predictions, would be required to design these experiments.

Weaknesses:

I found it difficult to identify evidence in the paper supporting central elements of the abstract. Hopefully, these difficulties can be resolved with a clearer presentation and the addition of supporting detail, especially in the methods.

(1) The model is not clearly described

In the Materials and Methods, there is no description of the model, just "The computational model is presented in Figure 1." (This is probably a typo and may refer to Figure 2A-C), and a link to Matlab source code. It is inappropriate to ask readers or reviewers to examine source code in lieu of providing a method, but I attempted to do so anyway. To my eye, the source code does not match the model presented in 2A-C. For instance, in 2C, "Steering signal" inhibits "Freeze", but I couldn't find this in the source. "Freeze" is shown to inhibit "steering signal," but as "steering signal" is a signed quantity, it's not clear what this means. Literally, since "ang_speed_raw = L-R," it would seem to indicate the "freeze" would bias towards right turns. In the code, "freeze" appears to be implemented through the boolean variable "speed_inhibition_time." The logic controlled by this variable doesn't appear to inhibit the "steering signal" but instead (depending on control parameters) either reduces the movement speed and amplifies the turning rate, or it turns the angular speed output into a temporal integral of the control signal.

There are a number of parameters in the source code that aren't described at all in the paper, including the internal oscillator parameters.

Together, these limitations make it difficult to understand what is being simulated, what parts of the model are tied to biology, and where the model improves on or departs from previous work.

It is absolutely essential that authors fully describe the computational model, that they explain the meaning of all parameters of the model, and that they explain how the particular values of these parameters were chosen.

(2) The biological inspiration is unclear

A central claim of the paper is that the model is "biologically grounded." But some elements, for instance, using a signed quantity to represent left-right steering drive, are not biologically possible; at best, these are shorthand for biologically possible implementations, e.g., opposing groups of left-right driving neurons.

The mechanism that produces fixations and saccades - the "freeze" module - is not tied to any particular anatomy of the insect brain. Initiation of a freeze occurs at a specific time coded into the model by the authors; it is not generated by an internal model signal. Release of a freeze is by drawing a random variable; there is no neural mechanism proposed to generate this signal.

In some versions of the model, instead of directly controlling the signal, during fixations, the angular drive signal is integrated into a variable "cumul_drive." No neural substrate is proposed for this integrator. In the code, if cumul_drive passes a threshold, the angular heading of the ant changes (saccades), but only if this threshold is passed before the Poisson process ends the fixation. No neural substrate is proposed for any of this logic.

The model steps forward in time by a fixed increment - the actual duration (in seconds) of this time step is not specified. From Figure 4F, G, it appears a simulation time step is meant to be about 10ms. This would imply an oscillator frequency of about 2 Hz (Fig 2B), that the heading oscillates at a similar frequency (2G), and that a forward crawling ant stops moving every 500 ms (2I). Are these plausible? Can they be compared to an experiment?

Model parameters, including the ones that control the frequency of the oscillator, are non-dimensionalized. It is not possible to evaluate whether these parameters are biologically plausible or match experimental results.

(3) Claims that behaviors emerge from the model may be overstated

The abstract claims that steering correction and fixations/saccades emerge naturally from the same model. But it appears to me that fixations/saccades are externally imposed by the specification of specific times for a "freeze." Faster angular rotation during saccades than during course correction is imposed and does not emerge naturally from neural simulations.

(4) Citations to previous literature are difficult to follow, and modeling results are presented as though they are experimental data

I would ask the authors to be much clearer in their description and citation of previous work. It should be clear whether the cited work was experimental or computational. To the extent possible, the actual measurement should be described succinctly. Instead of grouping references together to support a sentence with multiple claims, references should be cited for each claim. Studies of computational models should not be presented as proving a biological result.

For example:

a) Lines 141-146:<br /> "Previous studies have established many key components of insect navigation, including .... the intrinsic oscillatory dynamics in the lateral accessory lobes (LALs) that support continuous zigzagging locomotion (Clément et al., 2023; Kanzaki, 2005; Namiki and Kanzaki, 2016; Steinbeck et al., 2020)."

The first reference is to one author's previous modeling work - it hypothesizes that oscillations in the LAL support zigzagging but includes no data that would "establish" the fact. Kanzaki et al. 2005 describes numerical modeling and simulation with a physical robot. Namiki and Kanzaki, 2016 is a review article that links the LAL to zigzagging behavior. It describes the LAL as a winner-take-all bistable network but does not describe or hypothesize that the LAL has intrinsic oscillatory dynamics. Steinbeck et al. 2020 is a more comprehensive review; it reinforces that the LAL is a winner-take-all bistable network that drives left-right steering, including during zig-zagging behavior. But in my reading, I could not find a statement that the LAL has intrinsic oscillatory dynamics (the closest is Steinbeck et al. saying the activity pattern switches regularly, as does the behavior; this doesn't imply that the LAL is intrinsically oscillatory.)

b) Lines 701-703:<br /> "In plume-tracking moths, CX output has been shown to modulate LAL flip-flop neurons driving zigzagging (Adden et al., 2022)."

This reads as though an experimental measurement was made, but in fact, this is modeling work.

c) Lines 703-706:<br /> "In ants, strong goal signals in the CX - whether elicited by the path integrator or visual familiarity (Wehner et al., 2016; Wystrach et al., 2020b, 2015) do not only sharpen directional accuracy but also increase oscillation frequency (Clément et al., 2023)."

Here again, modeling results are presented as though they were experimental data.

Reviewer #2 (Public review):

Summary:

The paper by Freas and Wystrach is an interesting computational study, exploring the detailed mechanisms of how simple neural circuits could explain complex behavioral patterns observed in navigating ants. The authors compare detailed, high-speed video recordings of Australian desert ants (Melophorus bagoti) with predictions made by their new computational model and find convincing similarities between the model and the behavioral data, at a level of detail not previously studied. Particularly interesting are emerging properties of the model, yielding behavioral motifs it was not designed to reproduce, but which occur in natural ant behavior.

Strengths:

A strength of the study is that the model is based on previous models, without making major novel explicit assumptions. It combines existing models of the insect central complex with a model of the lateral accessory lobe and adds a stochastic inhibition of forward velocity to the interaction of central complex and lateral accessory lobes. The central complex provides corrective steering signals when the goal direction and the current heading of an insect are not aligned, while the lateral accessory lobes provide an intrinsic oscillator underlying the behavioral oscillations shown by walking ants at all times. These background oscillations are modulated by the steering signals from the central complex. Depending on which phase of the intrinsic oscillations coincides with the corrective signals, and how fast the ant is moving forward during this time, a complex set of behaviors emerges. Most prominently, scanning behaviors, which are regularly carried out by the ants, are recapitulated in great detail by the model. Additionally, other behaviors, such as full loops, emerge naturally from the model. While computational models are not to be seen as definite evidence for any biological reality, they can provide strong support for particular neural implementations. The current study is an excellent example in that it provides evidence for a serial arrangement of central complex circuits upstream of the lateral accessory lobe circuits, modulated by speed-regulating input. While the latter is hypothetical, it yields a clear hypothesis that can be validated by connectomics studies and functional work in the future.

The study shows that even complex behavioral motifs do not require dedicated neural modules, but can rather emerge from the interplay of already known circuits - highlighting the efficiency of insect brains and possibly providing the path towards embodied hardware solutions of such circuits in autonomous agents.

Weaknesses:

There are several weaknesses in the paper as it is.

Firstly, the model is not described in the methods, but only found when following the link to the authors' GitHub repository. This is clearly not sufficient and prevents readers from evaluating the model's assumptions directly. Most importantly, how natural do the emerging properties indeed emerge from the model? What parameters need to be tuned to generate a match between data and model?

Second, it is often not entirely clear what is biological data and what is a computational model. This relates to figures, text, and references. As a reader, this makes it difficult to clearly judge what is new in the current paper, how it adds to previous models, and what the predictions and assumptions are for biology.

Third, while neural data from bees and flies are taken to motivate and design the computational model, the discussion and interpretation revolve almost exclusively around ants. For the most part, this is justified, as the behavioral data used to benchmark the model are taken from ants. Nevertheless, more broadly discussing the newly defined circuit in the context of flying insects would give a better idea of the broad relevance of the neural circuits predicted by the model.

Reviewer #1 (Public review):

Summary:

D. Fuller et al. set out to study the molecular partners that cooperate with ATG2A, a lipid transfer protein essential for phagophore elongation, during the process of autophagy. Through a series of experiments combining microscopy and biochemistry, the authors identify ARFGAP1 and Rab1A as components of early autophagic membranes, which accumulate at the periphery of aberrant pre-autophagosomal structures induced by loss of ATG2. While ARFGAP1 has no apparent function in autophagy, the authors show that RAB1A is implicated in autophagy, although the precise mechanisms are not explored in the manuscript.

Strengths:

The work presented by Fuller et al. provides new insights into the composition of early autophagic membranes. The authors provide a series of MS experiments identifying proteins in close proximity to ATG2A, which is a valuable dataset for the field. Furthermore, they show for the first time the interaction between ATG2A and RAB1A both in fed and starved conditions, which extends the characterisation of the pre-autophagosomal structures observed in ATG2 DKO cells.

Weaknesses / Specific comments:

(1) The authors claim that Rab1A/B knockdown phenocopies the LC3-II accumulation observed in ATG2 DKO cells. While LC3-II accumulation is consistent with this interpretation, depletion of many autophagy-related proteins can give rise to a similar phenotype, even when they function at distinct stages of the autophagic cascade. Therefore, LC3-II accumulation alone is insufficient to support phenocopying in my vew. Immunofluorescence analyses demonstrating comparable cellular phenotypes-such as membrane accumulation of pre-autophagosomal structures-following Rab1 knockdown should be provided. Moreover, p62 does not accumulate upon Rab1 depletion, suggesting that loss of Rab1 does not fully phenocopy ATG2 deficiency. Consequently, it remains unclear whether Rab1A depletion truly phenocopies ATG2A depletion with respect to autophagy progression or the accumulation of pre-autophagosomal structures.

(2) Interpretation of the significance of the data

(2.1) The significance statement asserts that "this study elucidates the role of early secretory membranes in autophagosome biogenesis." While the data convincingly demonstrate an association between the RAB1A GTPase and ATG2A, the study does not provide mechanistic insight into how this interaction functionally contributes to autophagy. As presented, the findings support a correlative relationship rather than a defined role in autophagosome biogenesis.

(2.2) The title states that ATG2A "engages" Rab1A- and ARFGAP1-positive membranes during autophagosome formation. However, both Rab1A and ARFGAP1 are shown to localize to pre-autophagosomal structures independently of ATG2A. In the absence of evidence demonstrating a functional or causal dependency, the term "engages" appears overstated. A more descriptive term, such as "associates," would more accurately reflect the data.

(2.3) In the Discussion, the authors state that previous studies have reported increased LC3-II levels following knockdown of Rab1 proteins (refs. 38 and 49). However, it is unclear where this observation is documented in the cited references.

(3) Some concerns remain in specific figures, as outlined below:<br /> • Quantification is missing in Fig S2D.<br /> • The authors claim: "siRNA against ARFGAP1 had very little effect" but the quantification and blots show actually no effect.<br /> • Conclusions drawn from KD experiments in Fig. S2 should be interpreted with caution, as knockdown efficiency is very low, particularly for ARFGAP1/3 in the triple knockdown.<br /> • In New Fig. 4, the representative blot is not representative of the results showed in the quantification as previously noted.

Reviewer #2 (Public review):

The mechanisms governing autophagic membrane expansion remain incompletely understood. ATG2 is known to function as a lipid transfer protein critical for this process; however, how ATG2 is coordinated with the broader autophagic machinery and endomembrane systems has remained elusive. In this study, the authors employ an elegant proximity labeling approach and identify two ER-Golgi intermediate compartment (ERGIC)-localized proteins-Rab1 and ARFGAP1-as novel regulators of ATG2 during autophagic membrane expansion.

Their findings support a model in which autophagosome formation occurs within a specialized subdomain of the ER that is enriched in both ER exit sites (ERES) and ERGIC, providing valuable mechanistic insight. The overall study is well executed and offers an important contribution to our understanding of autophagy. I support its publication in eLife and offer the following minor comments for clarification and improvement.

Specific Comments

(1) Integration with Prior Literature<br /> The data convincingly implicate the ERES-ERGIC interface in autophagosome biogenesis. It would strengthen the manuscript to discuss previous studies reporting ERES and ERGIC remodeling and formation of ERERS-ERGIC contact sites (PMID: 34561617; PMID: 28754694) in the context of the current findings.

(2) Figure Labeling<br /> The font size in Figure 1A and Supplementary Figure S1G is too small for comfortable reading. Please consider enlarging the labels to improve clarity.

(3) Experimental Conditions<br /> In Figures 2A-C and Figure 4, it is unclear how the cells were treated. Were they starved in EBSS? Please include this information in the corresponding figure legends.

(4) LC3 Lipidation vs. Cleavage<br /> In Figure 2A, ARFGAP1 knockdown appears to reduce LC3 lipidation without affecting Halo-LC3 cleavage. Clarifying this observation would help readers better understand the functional specificity of ARFGAP1 in the pathway.

(5) Use of HT-mGFP in Figure 2C<br /> Please clarify why the assay in Figure 2C was performed in the presence of HT-mGFP. Explaining the rationale would aid interpretation of the results.

(6) FIB-SEM Imaging<br /> For the FIB-SEM images in Figures 3 and S3, directly labeling the cellular structures in the images would greatly facilitate interpretation for the reader.

(7) Supplementary Figures<br /> Many of the supplemental figures are high quality and contain key data. If space permits, I suggest moving these into the main figures. In particular, the FLASH-PAINT experiment could be presented as part of Figure 1.

(8) Text Revision for Clarity<br /> In line 242, the phrase "but protein-protein interactions appear to be limited to RAB1" would benefit from clarification. A more precise formulation could be: "but stable protein-protein interactions appear to be limited to RAB1."

(9) COPII Inhibition Strategy<br /> The authors used the dominant-active SAR1(H79G) mutant to inhibit COPII function. While this is effective in in vitro budding assays, the GDP-locked mutant SAR1(T39N) has been shown to be more effective in blocking COPII-mediated trafficking in cells. Including SAR1(T39N) in the analysis would provide stronger support for the conclusions.

Reviewer #3 (Public review):

The manuscript by Fuller et al describes a crosstalk between ARTG2A with components of the early secretory pathway, namely RAB1A and ARFGAP1. They show that ATG2A is recruited to membranes positive for RAB1A, which they also show to interact with ATG2A. In agreement with earlier findings by other groups, silencing RAB1A negatively affects autophagy. While ARFGAP1 was also found on ATG2A positive membranes, silencing ARFGAP1 had no impact autophagy. Notably, these ARFGAP1 positive membranes are not Golgi membranes.

The findings are interesting and the data are in general of good quality. I think the story is good enough to be published in eLife and I have the following questions, which the authors may attend to:

(1) Are the membranes to which ATG2A is recruited a form of ERGIC?

(2) Figure 3A/B: Is it possible to show a better example? The difference is barely detectable by eye. Since Immunoblotting is not really a quantitative method, I think that such a weak effect is prone to be wrong. Is there another tool/assay to validate this result?

(3) Is the curvature-sensitive region of ARFGAP1 required for its co-localization with ATG2A?

(4) What does Rab1A do? What is its effector? Or does the GTPase itself remodel the membrane?

(5) What about Arf1? It appears that this role of ARFGAP1 is unrelated to Arf1 and COPI? Thus, one would predict that Arf1 does not localize to these structures and does not affect ATG2A function

(6) Does ARFGAP1 promote fission of the membrane from its donor compartment?

(7) What are ARFGAP1 and Rab1A recruited to? What is the lipid composition, or protein that recruits these two players to regulate autophagy?

Comments on the latest version:

The revisions carried out by the authors are fine. The new data on ArfGAP1 and about the indirectness of the ATG2A and Rab1A interaction improve both clarity and strength of the manuscript. I have no further comments.

Reviewer #1 (Public review):

Summary:

Participants learned a graph-based representation, but, contrary to the hypotheses, failed to show neural replay shortly after. This prompted a critical inquiry into temporally delayed linear modeling (TDLM)--the algorithm used to find replay. First, it was found that TDLM detects replay only at implausible numbers of replay events per second. Second, it detects replay-to-cognition correlations only at implausible densities. Third, there are concerning baseline shifts in sequenceness across participants. Fourth, spurious sequences arise in control conditions without a ground truth signal. Fifth, the revised manuscript adapts a previously published synthetic simulation to show that previous validations/support of TDLM may have overestimated TDLM sensitivity because synthetic assumptions can produce unrealistically high pattern separability and reduced baseline confounds.

Strengths:

- This work is meticulous and meets a high standard of transparency and open science, with preregistration, code and data sharing, external resources such as a GUI with the task and material for the public.

- The writing is clear, balanced, and matter-of-fact.

- By injecting visually evoked empirical data into the simulation, many surface-level problems are avoided, such as biological plausibility and questions of signal-to-noise ratio.

- The investigation of sequenceness-to-cognition correlations is an especially useful add-on because much of the previous work uses this to make key claims about replay as a mechanism.

- In the revised version, the authors foreshadow ways to improve sequenceness detection by introducing a sign-flipping analysis.

Weaknesses:

Many of the weaknesses are not so much flaws in the analyses, but shortcomings when it comes to interpretation and a lack of making these findings as useful as they could be. Furthermore, as I will explain below, some weaknesses have been partially improved in the last round of revisions.

- I found the bigger picture analysis to be lacking, though improved in the latest version. Let us take stock: in other work during active cognition, including at least one study from the Authors, TDLM shows significant sequenceness. But the evidence provided here suggests that even very strong localizer patterns injected into the data cannot be detected as replay except at implausible speeds. How can both of these things be true? Assuming these analyses are cogent, do these findings not imply something more destructive about all studies that found positive results with TDLM? In the revisions, the manuscript concentrates a bit more on criteria that influence detection of sequences, though it is still not entirely clear what consequences there are for previous work.

- All things considered, TDLM seems like a fairly vanilla and low assumption algorithm for finding event sequences. Although the authors have improved their discussion of "boundary conditions" or factors for why TDLM might fail, it remains not fully clear to what extent the core problem is TDLM on an algorithmic/mathematical level (intrinsic factor), vs data quality, power, window size (extrinsic factors).

- The new sign-flip analysis underscores the authors' goal of being solution-oriented, though it is important to emphasize that a comprehensive way forward is not yet provided. This is fine, but the manuscript could be improved further through a concrete alternative or a revised version of the original approach.

Reviewer #2 (Public review):

Summary:

Kern et al. investigated whether temporally delayed linear modeling (TDLM) can uncover sequential memory replay from a graph-learning task in human MEG during an 8 minute post-learning rest period. After failing to detect replay events, they conduct a simulation study in which they insert synthetic replay events, derived from each participants' localizer data, into a control rest period prior to learning. The simulations suggest that TDLM only reveals sequences when replay occurs at very high densities (> 80 per minute) and that individual differences in baseline sequenceness may lead to spurious and/or lacklustre correlations between replay strength and behavior.

Strengths:

The approach is extremely well documented and rigorous. The authors have done an excellent job re-creating the TDLM methodology that is most commonly used, reporting the different approaches and parameters that they used, and reporting their preregistrations. The hybrid simulation study is creative and provides a new way to assess the efficacy of replay decoding methods, and its comparison to earlier published TDLM simulations is particularly useful. The authors remain measured in the scope/applicability of their conclusions, constructive in their discussion, and end with a useful set of recommendations for how to best apply TDLM in future studies. I also want to commend this work for not only presenting a null result, but thoroughly exploring the conditions under which such a null result is expected. I think this paper is interesting and will be generally quite useful for the field.

In the revised version, the authors have adequately addressed each of the weaknesses I raised previously. In brief, they:

(i) Added new power analyses of sequenceness for bootstrapped sample sizes, along with a new permutation test (Supplemental Fig 11),

(ii) Qualified their conclusions with added limitations and clarified several points that I found previously unclear,

(iii) Added several new analyses to the Appendices

(iv) Demonstrated that previous simulations validating TDLM overestimated TDLM sensitivity relative to the hybrid simulation.

(v) Added a new and extensive appendix on the relationship between TDLM and replay characteristics.

Weaknesses:

The remaining weaknesses of the work relate primarily to explaining the cause of measured non-random fluctuations in the simulated correlations between replay detection and performance at different time lags, as well as a lack of general recommendations of parameter choices for applying TDLM in future work. But these are minor weaknesses that can be left to future work.

Reviewer #3 (Public review):

Summary:

Kern et al. critically assess the sensitivity of temporally delayed linear modelling (TDLM), a relatively new method used to detect memory replay in humans via MEG. While TDLM has recently gained traction and been used to report many exciting links between replay and behavior in humans, Kern et al. were unable to detect replay during a post-learning rest period. To determine whether this null result reflected an actual absence of replay or sensitivity of the method, the authors ran a simulation: synthetic replay events were inserted into a control dataset, and TDLM was used to decode them, varying both replay density and its correlation with behavior. The results revealed that TDLM could only reliably detect replay at unrealistically (not-physiological) high replay densities, and the authors were unable to induce strong behavior correlations. These findings highlight important limitations of TDLM, particularly for detecting replay over extended, minutes long time periods.

Strengths:

Overall, I think this is an extremely important paper, given the growing use of TDLM to report exciting relationships between replay and behavior in humans. I found the text clear, the results compelling, and the critique of TDLM quite fair: it is not that this method can never be applied, but just that it has limits in its sensitivity to detect replay during minutes long periods. Further, I greatly appreciated the authors efforts to describe ways to improve TDLM: developing better decoders and applying them to smaller time windows.

The power of this paper comes from the simulation whereby the authors inserted replay events and attempted to detect them using TDLM. Regarding their first study, there are many alternative explanations or possible analysis strategies that the authors do not discuss; however, none of these are relevant if replayed, under conditions where it is synthetically inserted, cannot be detected.

Further, the authors provide a simulation and series of analyses aimed at replicating previous TDLM-based replay studies. They demonstrate methodological flaws, and show that previous simulations greatly overestimated the sensitivity of TDLM. This work emphasizes the need to cast a critical eye over both past and future studies applying TDLM to detect replay.

Finally, the authors are relatively clear about which parameters they chose, why they chose them, and how well they match previous literature (they seem well matched); and provide suggestions for how others can determine the best parameters for TDLM within their own experimental contexts.

Comments on revisions:

The authors thoroughly addressed my previous comments; the added analyses and discussion significantly strengthen the paper's clarity, utility, and impact.

Reviewer #1 (Public Review):

Summary:

Zeng et al. have investigated the impact of inhibiting lactate dehydrogenase (LDH) on glycolysis and the tricarboxylic acid cycle. LDH is the terminal enzyme of aerobic glycolysis or fermentation that converts pyruvate and NADH to lactate and NAD+ and is essential for the fermentation pathway as it recycles NAD+ needed by upstream glyceraldehyde-3-phosphate dehydrogenase. As the authors point out in the introduction, multiple published reports have shown that inhibition of LDH in cancer cells typically leads to a switch from fermentative ATP production to respiratory ATP production (i.e., glucose uptake and lactate secretion are decreased, and oxygen consumption is increased). The presumed logic of this metabolic rearrangement is that when glycolytic ATP production is inhibited due to LDH inhibition, the cell switches to producing more ATP using respiration. This observation is similar to the well-established Crabtree and Pasteur effects, where cells switch between fermentation and respiration due to the availability of glucose and oxygen. Unexpectedly, the authors observed that inhibition of LDH led to inhibition of respiration and not activation as previously observed. The authors perform rigorous measurements of glycolysis and TCA cycle activity, demonstrating that under their experimental conditions, respiration is indeed inhibited. Given the large body of work reporting the opposite result, it is difficult to reconcile the reasons for the discrepancy. In this reviewer's opinion, a reason for the discrepancy may be that the authors performed their measurements 6 hours after inhibiting LDH. Six hours is a very long time for assessing the direct impact of a perturbation on metabolic pathway activity, which is regulated on a timescale of seconds to minutes. The observed effects are likely the result of a combination of many downstream responses that happen within 6 hours of inhibiting LDH that causes a large decrease in ATP production, inhibition of cell proliferation, and likely a range of stress responses, including gene expression changes.

Strengths:

The regulation of metabolic pathways is incompletely understood, and more research is needed, such as the one conducted here. The authors performed an impressive set of measurements of metabolite levels in response to inhibition of LDH using a combination of rigorous approaches.

Weaknesses:

Glycolysis, TCA cycle, and respiration are regulated on a timescale of seconds to minutes. The main weakness of this study is the long drug treatment time of 6 hours, which was chosen for all the experiments. In this reviewer's opinion, if the goal was to investigate the direct impact of LDH inhibition on glycolysis and the TCA cycle, most of the experiments should have been performed immediately after or within minutes of LDH inhibition. After 6 hours of inhibiting LDH and ATP production, cells undergo a whole range of responses, and most of the observed effects are likely indirect due to the many downstream effects of LDH and ATP production inhibition, such as decreased cell proliferation, decreased energy demand, activation of stress response pathways, etc.

Comments on revisions:

Based on the response to comments that the authors have submitted, I do not think I need to make any changes to my review, as the time course experiment that could have explained the difference between reported results and extensive prior literature has not been performed.

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.

Comments on revisions:

The authors very nicely addressed all concerns from this reviewer. There are no further concerns and comments.

Reviewer #3 (Public review):

Xu, Cao and colleagues 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 adult liver portal veins.<br /> Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting mRNA presence of genes linked to neurodevelopment, bile acid transport, 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. 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. The importance of the CD34+/Sca1+ endothelial cell population and claims based on transcriptomic re-analysis require future assessment by functional experimental approaches to decipher the functional molecules involved in PLC formation, maintenance, and the involvement in injury response before establishing their role in biliary, arterial, and neural liver systems.

Strengths:

The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.<br /> This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - PLCs.

Weaknesses:

The importance of CD34+Sca1+ endothelial cell sub-population for PLC formation and function was not tested and warrants further validation.

Comments on revisions:

I appreciate the author's effort to revise the text so it more rigorously adheres to the presented evidence. Following a thorough read of the revised text, a few remaining minor issues were identified in the Discussion.

(1) From where comes the hard evidence for PLC being the stem cell niche in the following sentence?<br /> for the two following statements:

This suggests that the PLC may not only provide structural support but also serve as a perivascular stem cell niche specific to the portal region, potentially involved in hematopoiesis and tissue regeneration.

The PLC serves as a directional scaffold for ductal growth, a specialized stem cell niche, and a potential site of neurovascular coupling.

(2) In the following paragraph, I lack references to the previously published evidence of liver innervation guidance mechanisms, such as the mesenchyme-mediated guidance (CD31- population) Gannoun et al., 2023 https://doi.org/10.1242/dev.201642, an important context for your finding.

Further analysis showed significant upregulation of genes involved in neurodevelopment and axonal guidance in the CD34⁺Sca-1⁺ cluster, along with activation of neuronal signaling pathways. Immunostaining confirmed the presence of TH⁺ sympathetic nerve fibers wrapping around the PLC in a "beads-on-a-string" pattern (Fig. 6), consistent with a classic neurovascular unit(Adori et al., 2021). Previous studies have shown that sympathetic nerves enter the liver along collagen fibers of Glisson's capsule and interact with hepatic arteries, portal veins, and bile duct epithelium, supporting the PLC as a scaffold for intrahepatic neurovascular integration.

(3) Several sentences have issues with a lack of space between words.

Reviewer #1 (Public review):

Summary:

This study by Xu et al. investigates how clathrin-independent endocytosis in cancer cells influences T cell activation. Using a combination of biochemical approaches and imaging, the authors identify ICAM1, the ligand for the T cell integrin LFA-1, as a novel cargo of EndoA3-mediated endocytosis.

The authors then explore the functional consequences of EndoA3 depletion in cancer cells on T cell function using cytokine measurements, surface marker analyses, cytotoxicity assays and imaging. Loss of EndoA3 results in reduced T cell cytokine production, while expression of activation and exhaustion markers such as TIM-3, PD-1, and CD137 remains largely unchanged. EndoA3 knockout is associated with reduced ICAM1 surface levels and increased ALCAM levels in cancer cells. Imaging experiments further reveal directional transport of ICAM1 toward the immunological synapse, seemingly slightly reduced ICAM1 levels at the synapse upon EndoA3 depletion and an enlarged contact area between T cells and cancer cells.

Based on these observations, the authors propose a model in which EndoA3-mediated endocytosis and retrograde trafficking of ICAM1 (and ALCAM) supplies the immunological synapse with ligands for adhesion molecules. In the absence of EndoA3, T cells are suggested to compensate for suboptimal ICAM1 availability by enlarging the synaptic contact area, altering synapse architecture, leading to reduced cytokine secretion but modestly enhanced cytotoxicity.

Overall, the study provides convincing evidence for a modulatory role of EndoA3-mediated endocytosis in regulating T cell-cancer cell interactions. However, the choice of cellular model systems, the limited number of biological replicates and insufficiently supported mechanistic interpretations weaken the manuscript and weaken the strength of its conclusions.

Strengths:

The authors employ a rigorous and innovative experimental strategy that convincingly identifies ICAM1 as a novel cargo of EndoA3-mediated endocytosis with convincing visualization of directional ICAM1 transport toward the immunological synapse. In addition, the study provides a comprehensive characterization of how EndoA3 depletion in cancer cells affects T cell cytokine production, activation, proliferation and cytotoxic function, representing a valuable contribution to our understanding of how membrane trafficking pathways in target cells can modulate immune responses.

Comments on revised version:

Thank you very much for submitting your revised manuscript. I appreciated your efforts to answer all of the reviewers questions. While in my opinion the manuscript truly improved I think there are still lingering questions, in particular regarding the following points:

(1) Limited biological replication:

The LB33-MEL system remains problematic, as also noted by other reviewers. While it clearly represents an improvement over highly derived model systems such as Jurkat or Raji cells, it nevertheless effectively restricts the study to a single biological replicate. In this context, it may be more appropriate to compare the chosen approach to more state-of-the-art systems, such as expression of HLA-A*02:01, peptide loading (e.g. NY-ESO), and introduction of the matching TCR into donor-derived primary T cells. Such an approach would allow the use of multiple T cell donors and would substantially strengthen the generalizability of the conclusions.

(2) Expression levels of ICAM1:

Based on available database information (e.g. UniProt) and published literature (PMID: 9371813), ICAM1 appears to be expressed at relatively low levels in both HeLa and LB33-MEL cells. While the effects on T cells are initially discussed in terms of broader changes in EndoA3-mediated recycling of multiple surface proteins, including ICAM1 and ALCAM (and potentially others), the focus of the manuscript increasingly shifts toward ICAM1 as the primary driver of the observed phenotypes. Given the comparatively low endogenous expression of ICAM1 in the chosen model systems, it is unclear whether this emphasis is fully justified. In addition, if ICAM1 polarization toward the immunological synapse was assessed using ICAM1 overexpression, whereas other phenotypes (such as enlarged contact area) were analyzed under endogenous expression conditions, this further complicates the interpretation. As a first step toward clarifying these issues, it would be helpful to include representative flow cytometry histograms showing surface expression levels of ICAM1 and ALCAM, rather than only normalized quantifications.

(3) Cell-cell contact dynamics:

The manuscript suggests that altered contact dynamics may underlie the observed increase in cytotoxicity upon EndoA3 depletion. However, these claims are not directly tested. Such effects could be addressed with relatively straightforward experiments, for example by directly measuring T cell-cancer contact duration in co-culture assays.

Reviewer #2 (Public review):

The manuscript by Xu et al. studies the relevance of endophilin A3-dependent endocytosis and retrograde transport of immune synapse components and in the activation of cytotoxic CD8 T cells. First, the authors show that ICAM1 and ALCAM, known component of immune synapses, are endocytosed via endoA3-dependent endocytosis and retrogradely transported to the Golgi. The authors then show that blocking internalization or retrograde trafficking reduces the activation of CD8 T cells. Moreover, this diminished CD8 T cells activation resulted the formation of an enlarged immune synapse with reduced ICAM1 recruitment.

Comments on revisions:

The authors have addressed all my comments adequately.

Reviewer #3 (Public review):

Shiqiang Xu and colleagues have examined the importance of ICAM-1 and ALCAM internalization and retrograde transport in cancer cells on formation of a polarized immunological synapse with cytotoxic CD8+ T cells. They find that internalization is mediated by Endophilin A3 (EndoA3) while retrograde transport to the Golgi apparatus is mediated by the retromer complex. Perturbing these trafficking pathways reduces cytokine release, but increases cytolytic killing. The paper is building on previous findings from corresponding author Henri-François Renard showing that ALCAM is an EndoA3 dependent cargo in clathrin-independent endocytosis.

The work is interesting as it describes a novel mechanism by which cancer cells might influence CD8+ T cell activation and immunological synapse formation, and the authors have used a variety of cell biology and immunology methods to study this. The authors have also made substantial efforts to address the reviewers comments to the first version of the paper. However, there are still some points which could be further improved to underpin their conclusions:

The movies and the related micrographs of EndoA3-mediated ICAM-1 endocytosis could be more convincing. Is the invagination of large membrane patches visible by volumetric imaging (e.g. confocal z-stacks) or brightfield microscopy?

There is still a lack of quantitative evidence for polarized transport of ICAM-1 positive vesicles towards the immunological synapse. Only one example is shown and the authors state that the data is from a single movie representative of two independent experiments. If there are multiple cells per experiment, the number of cells should be stated and more examples should be included.

Reviewer #1 (Public review):

Summary:

The authors investigate how infestation of rice plants by the small brown planthopper (Laodelphax striatellus), an important pest in rice cultivation, alters host plant carbohydrate metabolism and how these changes affect insect physiology and fitness. They show that planthopper infestation leads to a density-dependent increase in glucose levels in rice plants, which the authors suggest results from a redistribution of carbohydrates from roots to shoots. Elevated glucose levels in plants are reflected by increased glucose contents in the insects themselves, an effect that is particularly pronounced in gravid females and associated with enhanced fecundity.

In addition, the authors demonstrate that increased glucose availability enhances tolerance of the small brown planthopper to the neonicotinoid insecticide imidacloprid. These findings suggest that insect-mediated changes in plant carbohydrate allocation may benefit insect fitness in multiple ways, including increased reproductive output and enhanced tolerance to insecticides, both of which are relevant for understanding insect population dynamics in agroecosystems.

Beyond these physiological observations, the authors aim to elucidate the underlying molecular mechanisms. They propose that glucose functions not only as a nutritional resource but also as a signaling molecule. Specifically, they show that increased glucose availability is associated with activation of the Target Of Rapamycin (TOR) pathway, a conserved nutrient-sensing signaling pathway regulating growth and metabolism across eukaryotes. Activation of TOR signaling is linked to increased juvenile hormone levels, which in turn stimulate vitellogenesis and likely contribute to increased fecundity. Furthermore, elevated juvenile hormone levels are associated with increased expression of glutathione S-transferases, suggesting a mechanism contributing to enhanced detoxification capacity. Independent of this pathway, increased glucose availability also leads to higher expression of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis. Together, these mechanisms provide a non-exclusive explanation for the observed increase in imidacloprid tolerance and form the basis of the authors' proposed mechanistic framework linking glucose availability to reproduction and detoxification.

Strengths:

A major strength of the manuscript is its substantial mechanistic depth and the extensive use of complementary experimental approaches that converge on a coherent mechanistic interpretation. The authors combine plant manipulations, dietary supplementation, injection assays, RNAi-mediated gene silencing, pharmacological inhibition, and rescue experiments to systematically test the role of glucose as a signaling molecule linking plant-derived nutrition to insect reproduction and insecticide tolerance. Results obtained from independent experimental strategies are highly consistent, and the different datasets collectively support the central conclusions of the study.

The role of glucose is supported by multiple lines of evidence demonstrating that increased glucose availability, whether induced by prior planthopper feeding, dietary supplementation, or direct injection, consistently results in elevated glucose levels in insects, increased oviposition, and enhanced expression of vitellogenesis-related genes (LsVg and LsVgR). The specificity of this effect is further strengthened by experiments using alternative carbohydrates that release glucose upon enzymatic cleavage, as well as inhibitor and rescue experiments, supporting the interpretation that glucose acts beyond a purely nutritional role.

The authors further establish a mechanistic link between glucose availability, TOR signaling, juvenile hormone regulation, and vitellogenesis. Activation of TOR signaling by glucose, demonstrated at the level of protein phosphorylation, together with RNAi knockdown and pharmacological inhibition, allows causal placement of TOR upstream of juvenile hormone signaling. Consistent reductions in juvenile hormone titers, vitellogenesis-related gene expression, and oviposition following TOR inhibition, as well as rescue of reproductive output by juvenile hormone analog treatment, provide strong functional support for a glucose-TOR-juvenile hormone axis regulating fecundity. The absence of additive effects following combined knockdown of TOR and juvenile hormone synthesis components further supports the interpretation that these factors act within the same signaling cascade.

Similarly, the authors provide a detailed mechanistic analysis of glucose-mediated effects on imidacloprid tolerance. Functional assays demonstrate that glutathione S-transferases contribute to detoxification in this species and that increased glucose availability enhances GST activity, glutathione synthesis, and overall glutathione levels. Transcriptomic analyses and targeted RNAi experiments further identify specific GSTs contributing to insecticide tolerance and indicate that glucose enhances detoxification through both TOR-dependent and TOR-independent mechanisms. The combined knockdown experiments, which produce additive effects on mortality, provide particularly strong support for the involvement of multiple interacting glucose-dependent pathways.

Weaknesses:

While I am impressed by the mechanistic depth of the study and the clarity with which the authors dissect the underlying physiological pathways, I am less convinced by the current conceptual framing of the phenomenon as a sophisticated adaptive strategy "co-opted" by the small brown planthopper. The data convincingly demonstrate that glucose availability activates conserved nutrient-sensing and endocrine pathways, including TOR signaling and juvenile hormone regulation, which in turn affect reproduction and detoxification capacity. However, these pathways are deeply conserved and likely operate in many insects in response to nutritional status. As such, the results may reflect a general physiological response to elevated carbohydrate availability rather than a species-specific, evolved strategy. Relatedly, herbivory-induced changes in plant carbohydrate allocation appear to be relatively common across plant-insect systems, and it would be helpful to discuss how specific (or general) the observed phenomenon is likely to be.

In particular, I encourage the authors to more clearly distinguish between (i) a conserved nutrient-responsive signaling cascade and (ii) an adaptive mechanism that evolved specifically under selection imposed by insecticide exposure. The presented data strongly support the former interpretation, whereas evidence for the latter is less clear. The increased tolerance to imidacloprid appears to arise as a consequence of enhanced metabolic and detoxification capacity under elevated glucose conditions, rather than as a trait shaped directly by insecticide-driven selection. Framing this phenomenon as an adaptation to insecticide stress may therefore overextend the conclusions that can be drawn from the data. A more cautious discussion acknowledging that glucose-mediated activation of conserved metabolic and endocrine pathways may incidentally enhance insecticide tolerance, without necessarily having evolved under insecticide selection, would strengthen the conceptual clarity of the manuscript.

Reviewer #2 (Public review):

Summary:

Zhang and colleagues investigate the molecular mechanisms by which the small brown planthopper (SBPH, Laodelphax striatellus) manipulates host rice carbohydrate metabolism to enhance its own fitness. Using a combination of molecular, pharmacological, and biochemical approaches, they demonstrate that SBPH infestation induces systemic glucose reallocation in rice, as evidenced by the upregulation of glucose levels in aerial tissues and a simultaneous reduction in root glucose levels. Notably, host-derived glucose acts as a central signaling molecule, driving two key adaptive traits: enhanced fecundity via the glucose-TOR-JH-Vg signaling cascade, and increased imidacloprid tolerance through synergistic metabolic (GCL-GSH) and regulatory (TOR-JH-GST) pathways targeting GST activity. These findings uncover a sophisticated resource-manipulation strategy in SBPH and identify nutrient-sensing and detoxification pathways as potential targets for pest control.

Strengths:

(1) The study addresses a gap in plant-insect coevolution research by identifying glucose as a dual-function signaling molecule that coordinates SBPH reproduction and insecticide tolerance, providing valuable insights into how herbivores exploit host nutritional signals.

(2) The experimental design is well structured and multifaceted, integrating RNAi, RT-qPCR, Western blotting, pharmacological inhibition, and biochemical assays. The use of appropriate controls (e.g., osmotic controls with mannitol and hydrolase-inhibitor rescue experiments) strengthens the causal interpretation of the results.

(3) The mechanistic framework is clear and well-supported. The authors delineate two interconnected molecular cascades (glucose-TOR-JH-Vg for fecundity and GCL-GSH/TOR-JH-GST for tolerance) with hierarchical validation (e.g., rescue experiments with JHA), ensuring the reliability of conclusions.

Weaknesses:

(1) The study focuses exclusively on SBPH without validating whether the observed phenomena and mechanisms are conserved in closely related planthopper species (e.g., brown planthopper Nilaparvata lugens). This limitation restricts the generalizability of the findings to other economically important rice pests.

(2) The specific upstream signals that trigger glucose reallocation in rice (e.g., SBPH salivary effectors or oviposition-associated factors) are not identified. Although this represents a complex and independent research direction, the absence of such information limits the depth and completeness of the mechanistic framework and leaves open questions regarding the initiation of host metabolic manipulation.

(3) Insecticide tolerance assays are limited to imidacloprid. Extending these analyses to one or two additional commonly used insecticides (e.g., thiamethoxam) would help determine whether the glucose-mediated detoxification pathway is specific to imidacloprid or reflects a broader resistance mechanism, thereby strengthening conclusions regarding the generality of the GST activation cascade.

(4) Given the study's potential implications for pest management, the manuscript would benefit from a brief discussion of possible practical applications, such as manipulating rice glucose metabolism through breeding strategies or developing small-molecule inhibitors targeting the TOR-JH axis. Including such perspectives would enhance the translational relevance of the work by linking mechanistic insights to real-world pest control strategies.

Reviewer #1 (Public review):

Summary:

In their manuscript, Richter and colleagues comprehensively investigate the cell wall recycling pathway in the model alphaproteobacterium Caulobacter crescentus using biochemical, imaging, and genetic approaches. They clearly demonstrate that this organism encodes a functional peptidoglycan recycling pathway and demonstrate the activities of many enzymes and transporters within this pathway. They leverage imaging and growth assays to demonstrate that mutants in peptidoglycan recycling have varying degrees of beta-lactam sensitivity as well as morphological and cell division defects. They propose that, rather than impacting the levels or activity of the major beta-lactamase, BlaA, defects in PG recycling lead to beta-lactam sensitivity by limiting the availability of new cell wall precursors. The findings will be of interest to those in the field of bacterial cell wall biochemistry, antibiotics and antibiotic resistance, and bacterial morphogenesis.

Strengths:

Overall the manuscript is laid out logically, and the data are comprehensive, quantitative, and rigorous. The mutants and their phenotypes will be a valuable resource for Caulobacter researchers, and the findings may be relevant to cell wall recycling in other organisms.

Weaknesses:

No major weaknesses are noted.

Comments on revisions:

The authors addressed all of our concerns with the initial submission.

Reviewer #3 (Public review):

Summary:

The authors propose a new version of idTracker.ai for animal tracking. Specifically, they apply contrastive learning to embed cropped images of animals into a feature space where clusters correspond to individual animal identities. By doing this, they address the requirement for so-called global fragments - segments of the video, in which all entities are visible/detected at the same time. In general, the new method reduces the long tracking times from the previous versions, while also increasing the average accuracy of assigning the identity labels.

Comments on revisions:

I have no additional comments, the authors have responded to all the points I raised previously.

Reviewer #1 (Public review):

Summary:

This paper by Karimian et al proposes an oscillator model tuned implementing binding by (gamma) synchrony principles in a visual task. The authors set out to show how well these principles explain human behavior in a figure-ground segregation tasks. The model is inspired by electrophysiological findings in non-human primates suggesting that gamma oscillations in early visual cortex implement feature-binding through a synchronization of feature-selective neurons. The psychophysics experiment involves the identification of a figure consisting of gabor annuli, presented on a background of gabor annuli. The participants' task is to identify the orientation of the figure. The task difficulty is varied based on the contrast and density of the gabor annuli that make up the figure. The same figures are used as inputs to the oscillator model. The authors report that both the discrimination accuracy in the psychophysics experiment and the synchrony of the oscillators in the proposed model follow a similar "Arnold Tongue" relationship when depicted as a function of the texture-defining features of the figure. This finding is interpreted as evidence for gamma synchrony being the underlying mechanism of the figure-ground segregation.

Strengths:

The design of the proposed model is well-informed by electrophysiological findings, and the idea of using computational modeling to bridge between intracranial recordings in non-human primates and behavioral results in human participants is interesting. Previous work has criticized the gamma synchrony theories based on the observation that synchronization in the gamma-band is highly localized and the frequency of the oscillation depends on the visual features of the stimulus. I appreciate how the authors demonstrate that frequency-dependence and local synchronization can be features of gamma synchrony, and not contradictory to the theory. As such, I feel that this work has the potential to contribute meaningfully to the debate on whether binding by gamma synchrony is a biophysically realistic model of feature-binding in visual cortex.

I also acknowledge the additional simulations the authors present in this version of the manuscript, showing that the model is able to segregate figure from ground.

Weaknesses:

The authors have addressed my previous concerns regarding the quantification of effect sizes. I also appreciate the authors argument that the results support the idea of feature-binding through synchronization in the gamma-band, as the model's parameters were informed by electrophysiological recordings from non-human primates. Personally, I would have been curious to see if the intrinsic frequencies of the model are indeed in the gamma-band, I don't believe the authors include a figure on that. Weaknesses are still the absence of electrophysiological recordings to support the frequency-specificity of the claims, e.g. in the form of EEG/MEG recordings, but I understand that these may be difficult to obtain, as gamma oscillations are relatively weak in response to static gratings. As the authors emphasize in this updated version, they present one possible mechanism of feature binding that is not contrasted to alternative mechanisms such as binding by increased firing rates. Understandably, implementing a second model would be out of scope.

The presented simulations and behavioural results support the authors aim of presenting an oscillator model informed by gamma synchronization in V1 that supports figure-ground segregation.

Likely impact:

This work makes several predictions about the degree of synchronization for different visual properties of the figure, that could be tested with electrophysiological methods. I therefore believe that the paper has the potential to motivate interesting follow-up studies to understand how visual cortex solves the binding problem.

Comment on revised version:

In this reviewed version of the manuscript, the authors present several follow-up simulations and clarifications that address previously outlined weaknesses.

Reviewer #2 (Public review):

The authors aimed to investigate whether gamma synchrony serves a functional role in figure-ground perception. They specifically sought to test whether the stimulus-dependence of gamma synchrony, often considered a limitation, actually facilitates perceptual grouping. Using the theory of weakly coupled oscillators (TWCO), they developed a framework wherein synchronization depends on both frequency detuning (related to contrast heterogeneity) and coupling strength (related to proximity between visual elements). Through psychophysical experiments with texture discrimination tasks and computational modeling, they tested whether human performance follows patterns predicted by TWCO and whether perceptual learning enhances synchrony-based grouping.

Strengths:

(1) The theoretical framework connecting TWCO to visual perception is innovative and well-articulated, providing a potential mechanistic explanation for how gamma synchrony might contribute to both feature binding and separation.

(2) The methodology combines psychophysical measurements with computational modeling, with a solid quantitative agreement between model predictions and human performance.

(3) In particular, the demonstration that coupling strengths can be modified through experience is remarkable and suggests gamma synchrony could be an adaptable mechanism that improves with visual learning.

(4) The cross-validation approach, wherein model parameters derived from macaque neurophysiology successfully predict human performance, strengthens the biological plausibility of the framework.

Likely Impact and Utility:

This work offers a fresh perspective on the functional role of gamma oscillations in visual perception. The integration of TWCO with perceptual learning provides a novel theoretical framework that could influence future research on neural synchrony.

The computational model, with parameters derived from neurophysiological data, offers a useful tool for predicting perceptual performance based on synchronization principles. This approach might be extended to study other perceptual phenomena and could inspire designs for artificial vision systems.

The learning component of the study may have a particular impact, as it suggests a mechanism by which perceptual expertise develops through modified coupling between neural assemblies. This could influence thinking about perceptual learning more broadly, but also raises questions about the underlying mechanism.

Additional Context:

Historically, the functional significance of gamma oscillations has been debated, with early theories of temporal binding giving way to skepticism based on gamma's stimulus-dependence. This study reframes this debate by suggesting that stimulus-dependence is exactly what makes gamma useful for perceptual grouping.

The successful combination of computational neuroscience and psychophysics is a significant strength of this study.

The field would benefit from future work extending (if possible) these findings to more naturalistic stimuli and directly measuring neural activity during perceptual tasks. Additionally, studies comparing predictions from synchrony-based models against alternative mechanisms would help establish the specificity of the proposed framework.

Comments on revised version:

The authors now soften their claim. However, the paper demonstrates that TWCO-derived predictions quantitatively match human figure-ground perception in texture stimuli, and that a synchrony-based readout provides a viable mapping from stimulus to behavior. Given that they cite (and do not show in this paper) the link to synchrony, what they actually establish is that this particular transformation of stimulus features maps better onto behavior. That's meaningful, but it is not a demonstration of mechanism.

Reviewer #2 (Public review):

Summary:

The manuscript by Hathaway et al. describes a set of elegant behavioral experiments designed to understand which aspects of cue-reward contingencies drive risky choice behavior. The authors developed several clever variants of the well established rodent gambling task (also developed by this group) to understand how audiovisual cues alter learning, choice behavior, and risk. Computational and sophisticated statistical approaches were used to provide evidence that: 1) audiovisual cues drive risky choice if they are paired with rewards and decrease risk if only paired with loss, 2) pairing cues with rewards reduces learning from punishment, and 3) differences in risk taking seem to be present early on in training.

Strengths:

The paper is well written, the experiments well designed, and the results are highly interesting particularly for understanding how cues can motivate and invigorate normal and abnormal behavior.

Comments on revisions:

The authors have done an exceptional job at addressing my initial concerns and questions regarding the evidence to support their claims. I have no additional suggestions or concerns.

Reviewer #3 (Public review):

Summary:

In this work, Hathaway and colleagues aim to understand how audiovisual cues at time of outcome promote selection of risky choices. A real life illustration of this effect is used in electronic gambling machines which signal a win with flashing lights and jingles, encouraging the player to keep betting. More specifically, the author asks whether the cue has to be paired exclusively to wins, or whether it can be paired to both outcomes, or exclusively loss outcomes, or occur randomly. To tackle this question, they employ a version of the Iowa Gambling Task adapted to rats, and test the effect of different rules of cue-outcome associations on the probability of selecting the riskier options; they then test the effect of prior reward devaluation on the task; finally, the optimise computational models on the early phases of the experiment to investigate potential mechanisms underlying the behavioural differences.

Strengths:

The experimental approach is very thorough, in particular the choice of the different task variants cover a wide range of different potential hypotheses. Using this approach, they find that, although rats prefer the optimal choices, there is a shift towards selecting riskier options in the variants of the task where the cue is paired to win outcomes. They analyse this population average shift by showing that there is a concurrent increase in the number of risk-taking individuals in these tasks. They also make the novel discovery that pairing cues with loss outcomes instead reduces the tendency for risky decisions.

The computational strategy is appropriate and in keeping with the accepted state of the art: defining a set of candidate models, optimising them, comparing them, simulating the best ones to ensure they replicate the main experimental results, then analysing parameter estimates in the different tasks to speculate abut potential mechanisms.

Weaknesses:

While the overall computational approach is excellent, there is a missed opportunity in the computational modelling section due to the choice of models which is dependent on a preceding study by Langdon et al. (2019). Loss trials come at a double cost: firstly the lost opportunity of not having selected a winning option which is reflected straightforwardly in Q-learning by the fact that r=0, secondly a waiting period which will affect the overall reward rate. The authors combine these costs by converting the time penalty into "reward currency" using three different functions which make up the three different tested models. This means the question when comparing models is not something along the lines of "are individuals in the paired win-cue tasks more sensitive to risk? or less sensitive to time? etc." but rather "what is the best way of converting time into Q-value currency to fit the data?". Instead, the authors could have contrasted other models which explicitly track time as a separate variable (see for example "Impulsivity and risk-seeking as Bayesian inference under dopaminergic control" (Mikhael & Gershman 2021)) or give actions an extra risk bonus (as in "Nicotinic receptors in the VTA promote uncertainty seeking" (Naude et al 2016)) to better disentangle the mechanisms at play.

Reviewer #1 (Public review):

Summary:

During vertebrate gastrulation, mesendoderm cells are initially specified by morphogens (e.g. Nodal) and segregate into endoderm and mesoderm in part based on Nodal concentrations. Using zebrafish genetics, live imaging, and single-cell multi-omics, the manuscript by Cheng et al presents evidence to support a claim that anterior endoderm progenitors derive primarily from prechordal plate progenitors, with transcriptional regulators goosecoid (gsc) and ripply1 playing key roles in this cell fate determination. Such a finding would represent a significant advance in our understanding of how anterior endoderm is specified in vertebrate embryos.

Strengths:

Live imaging based tracking of PP and endo reporters (Fig 2) are well executed and convincing, though a larger number of individual cell tracks will be needed. In the first round of review, only a single cell track (n=1) was quantified. Now, more tracks have been collected but these data are still not clearly reported in a way that warrants their evaluation.

Weaknesses:

(1) While the authors have made an effort to include a gsc:CRE lineage tracing component to their study, the experimental data now presented (Figure S4E and reviewer figures) could be much stronger and more thorough. In the new panel, authors show a single microscopy image containing both red and green fluorescent cells. The green signal, which seems to mark the PP, is presumably derived from Tg(gsc:EGFP). The red mCherry signal is presumably derived from the combined effects of a Tg(gsc:CRE) and Tg(sox17-lox-STOP-lox-mCherry), i.e., labeling the progeny of gsc+ progenitors which expressed CRE and underwent recombination to create a productive endoderm-specific Tg(sox17:mCherry) reporter. The result appears to be promising and in line with the authors' predictions. However, this result should be strengthened by performing the experiment in stable transgenic lines (not just freshly injected F0 embryos) and should be properly quantified. The authors state in the legend that "the experiment was performed on at least 3 independent replicates", but offer no further detail, explanations, or quantifications. This issue is reminiscent of concerns from the previous round of review, where live tracking data derived from examining just a single (n=1) cell were presented. These standards might be adequate for generating preliminary insights, but fall far below what we would have previously expected from an Elife publication.

(2) I found the authors' rebuttal to my concerns about URD-trajectory derived insights and gsc/sox17 expression timing confusing. The authors claim that they get different results regarding gsc expression prevalence in the hypothetical PP/endoderm progenitor cluster when comparing scRNAseq data from embryos vs explants. Then they seem to use this difference to justify the use of the explants over the embryos - presumably because the explants enriched for the behavior that they wanted to see? They conclude that "directly using embryonic data to dissect the mechanism of fate separation between PP and anterior endoderm might not yield highly accurate results." I strongly disagree with this. I would argue that the whole-embryo dataset is likely doing a better job of cleanly separating these trajectories from each other.

(3) My concern about the use of n=1 cell for live tracking has been partially but not fully addressed. The authors should plot data point from each individual cell in the revised Figure 2D, instead of just saying "multiple cells" they should report the total number of cells that are actually included now (n=?), and should provide representative movies for a few additional examples.

At present the authors' data, as presented, still only partially support their aims and conclusions.

Reviewer #2 (Public review):

Summary:

During vertebrate gastrulation, the mesoderm and endoderm arise from a common population of precursor cells and are specified by similar signaling events, raising questions as to how these two germ layers are distinguished. Here, Cheng and colleagues use zebrafish gastrulation as a model for mesoderm and endoderm segregation. By reanalyzing published single cell sequencing data, they identify a common progenitor population for anterior endoderm and the mesodermal prechordal plate (PP). They find that expression levels of PP genes gsc and ripply are among the earliest differences between these populations, and that their increased expression suppresses the expression of endoderm markers. Further analysis of chromatin accessibility and Ripply CUT-and-TAG is consistent with direct repression of endoderm by this PP marker. This study demonstrates roles for Gsc and Ripply in suppressing anterior endoderm fate, but this role for Gsc was already known and the effect of Ripply is limited to a small population of anterior endoderm.

Strengths:

Integrated single cell ATAC- and RNA-seq convincingly demonstrate changes in chromatin accessibility that may underlie segregation of mesoderm and endoderm lineages, including gsc and ripply. Identification of Ripply-occupied genomic regions augments this analysis. The genetic mutants for both genes provide strong evidence for their function anterior mesendoderm development, although these phenotypes are subtle.

Weaknesses:

The use of zebrafish embryonic explants for cell fate trajectory analysis (rather than intact embryos) is not justified. Much of the work is focused on the role of Nodal in the mesoderm/endoderm fate decision, but the results largely confirm previous studies and again provide few new insights. The authors similarly confirm previous findings that FGF signaling likely plays a larger role in this fate decision, but these results are largely overlooked by the authors.

Reviewer #3 (Public review):

Summary of work:

Cheng, Liu, Dong, et al. demonstrate that anterior endoderm cells can arise from prechordal plate progenitors, which is suggested by pseudotime reanalysis of published scRNAseq data, pseudotime analysis of new scRNAseq data generated from Nodal-stimulated explants, live imaging from sox17:DsRed and gsc:eGFP transgenics, fluorescent in situ hybridization, and a Cre/Lox system. Early fate mapping studies already suggested that progenitors at the dorsal margin give rise to both of these cell types (Warga), and live imaging from the Heisenberg lab (Sako 2016, Barone 2017) convincingly showed this previously. However, the data presented for this point are very nice and further cement this result. Though better demonstrated by previous work (Alexander 1999, Gritsman 1999, Gritsman 2000, Sako 2016, Rogers 2017, others), the manuscript presents confirmatory data that high Nodal signaling is required for both cell types. The manuscript generates new single-cell RNAseq data from Nodal-stimulated explants with increased (lft1 KO) or decreased (ndr1 KD) Nodal signaling and multi-omic ATAC+scRNAseq data from wild-type 6 hpf embryos, which can be used as a resource, though few new conclusions are drawn from it in this manuscript. Lastly, the manuscript presents suggests that SWI/SNF remodelers and Ripply1 may be involved in the anterior endoderm - prechordal plate decision, but these data are less convincing. The SWI/SNF remodeler experiments are unconvincing because the demonstration that these factors are differentially expressed or active between the two cell types are weak. The Ripply1 gain-of function experiments are unconvincing because they are based on incredibly high overexpression of ripply1 (500 pg or 1000 pg) that generates a phenotype that is not in line with previously demonstrated overexpression studies (with phenotypes from 10-20x lower expression). Similarly, the cut-and-tag data seems low quality and is based on high overexpression, so may not support direct binding of ripply1 to these loci.

During revision, the authors addressed some comments, including eliminating references to "lineage" when referring to pseudotime trajectories, eliminating conclusions drawn from locations of cells on UMAP plots, and reducing use of the term "cooperative" which may have been confusing in this context, as well as increasing the number of embryos analyzed for some experiments. The authors also point out that whole-embryo transcriptional trajectories typically do not associate endodermal cells with prechordal plate cells, despite classical evidence that they are related. This is most likely because endodermal cells arise from several different previous transcriptional states in different regions of the embryonic margin and are, as the authors point out, difficult to computationally sort into dorsal, lateral, and ventral populations. Thus, there is value in generating data to more specifically look at the relationship between dorsal mesodermal and endodermal populations. However, the decision to use an artificial Nodal-treated explant system, rather than isolating the relevant population from whole embryos (such as by dissection prior to dissociation) remains a weakness of the manuscript, since it is unclear whether endodermal specification has been altered in this system (there seem to be few endodermal cells produced and the system involves manipulating one of the signals under study in this work). Concerns about the rigor of experiments concerning ripply1 and SWI/SNF experiments remains. While the authors improved peak calling in their ripply1 cut-and-tag, it is still based on massive overexpression of ripply1 that may drive binding outside of its endogenous loci.

In the end, this study provides some additional details in the cell fate decision between the prechordal plate and anterior endoderm and generates new data that may be useful for reanalysis by other experts in the field. However, this work does not make clear how Nodal signaling, FGF signaling, and elements of the gene regulatory network (including gsc, possibly ripply1, and other factors) interact to make the decision. I suggest that this manuscript is of interest to Nodal signaling or zebrafish germ layer patterning afficionados, but may not be of interest to a broad audience. While it provides new datasets and observations, it does not weave these into a convincing story that advances our understanding of the specification of these cell types.

Reviewer #1 (Public review):

Summary:

The authors aimed to elucidate the recruitment order and assembly of the Cdv proteins during Sulfolobus acidocaldarius archaeal cell division using a bottom-up reconstitution approach. They employed liposome-binding assays, EM, and fluorescence microscopy with in vitro reconstitution in dumbbell-shaped liposomes to explore how CdvA, CdvB, and the homologues of ESCRT-III proteins (CdvB, CdvB1, and CdvB2) interact to form membrane remodeling complexes.<br /> The study sought to reconstitute the Cdv machinery by first analyzing their assembly as two sub-complexes: CdvA:CdvB and CdvB1:CdvB2ΔC. The authors report that CdvA binds lipid membranes only in the presence of CdvB and localizes preferentially to membrane necks. Similarly, the findings on CdvB1:CdvB2ΔC indicate that truncation of CdvB2 facilitates filament formation and enhances curvature sensitivity in interaction with CdvB1. Finally, the authors reconstitute a quaternary CdvA:CdvB:CdvB1:CdvB2 complex and demonstrate its enrichment at membrane necks. The mechanistic details of how these complexes drive membrane remodeling, particularly through subcomplex removal by the proteasome and/or CdvC, remain insufficiently addressed, and the study therefore mainly provides an experimental framework for future mechanistic investigation.

Strengths:

The study of machinery assembly and its involvement in membrane remodeling, particularly using bottom-up reconstituted in vitro systems, presents significant challenges. This is particularly true for systems like the ESCRT-III complex, which localizes uniquely at the lumen of membrane necks prior to scission. The use of dumbbell-shaped liposomes in this study provides a promising experimental model to investigate ESCRT-III and ESCRT-III-like protein activity at membrane necks.<br /> The authors present intriguing evidence regarding the sequential recruitment of ESCRT-III proteins in crenarchaea-a close relative of eukaryotes.

Weaknesses:

The findings of this study suggest that the hierarchical recruitment characteristic of eukaryotic systems may predate eukaryogenesis, which represents a significant and exciting contribution. However, the broader implications of these findings for membrane remodeling mechanisms remain largely unexplored. Nevertheless, this study provides a valuable experimental framework to address these questions in the future.

Reviewer #2 (Public review):

Summary:

The Crenarchaeal Cdv division system represents a reduced form of the universal and ubiquitous ESCRT membrane reverse-topology scission machinery, and therefore a prime candidate for synthetic and reconstitution studies. The work here represents a convincing extension of previous work in the field, clarifying the order of recruitment of Cdv proteins to curved membranes.

Strengths:

The use of a recently developed approach to produce dumbbell-shaped liposomes (De Franceschi et al. 2022), which allowed the authors to assess recruitment of various Cdv assemblies to curved membranes or membrane necks; reconstitution of a quaternary Cdv complex at a membrane neck.

Weaknesses:

The initial manuscript was a bit light on quantitative detail, across the various figures - addressing this would make the paper much stronger. The authors could also include in the discussion a short paragraph on implications for our understanding of ESCRT function in other contexts and/or in archaeal evolution - for the interests of a broad audience. These issues have been addressed in the authors' revision.

Reviewer #3 (Public review):

In this revised report, De Franceschi et al. purify components of the Cdv machinery in archaeon M. sedula and probe their interactions with membrane and with one-another in vitro using two main assays - liposome flotation and fluorescent imaging of encapsulated proteins. This has the potential to add to the field by showing how the order of protein recruitment seen in cells is related to the differential capacity of individual proteins to bind membranes when alone or when combined.

Using the floatation assay, they demonstrate that CdvA, CdvB, and CdvB1 bind liposomes. CdvB2 lacking its C-terminus is not efficiently recruited to membranes unless CdvAB or CdvB1 are present. The authors then employ a clever liposome assay that generates chained spherical liposomes connected by thin membrane necks, which allows them to accurately control the buffer composition inside and outside of the liposome. With this, they show that all four proteins accumulate in necks of dumbbell-shaped liposomes that mimic the shape of constricting necks in cell division, possibly indicating a sensing of catenoid membrane geometry. Taken altogether, these data lead them to propose that Cdv proteins are sequentially recruited to the membrane as has also been suggested by in vivo studies of ESCRT-III dependent cell division in crenarchaea.

In their revision, the authors have addressed the vast majority of our previous concerns. The paper is much improved as a result. The Figures are improved and the authors have added appropriate controls and additional experiments, strengthening their conclusions.

There are still some discrepancies between these results and what is know about Sulfolobus division. Since the initial submission, other work has shown that in S. acidocaldarius, CdvA is the first component to assemble a ring (in absence of CdvB , doi.org/10.1073/pnas.2513939122) and that CdvB2 is able to bind membranes in vitro (doi.org/10.1073/pnas.2525941123). This might reflect differences between Sulfolobus and Metallosphaera, but probably should be discussed.

Reviewer #1 (Public review):

In this manuscript, the authors aim to define how rapid eye movement sleep supports memory consolidation by identifying the brain circuits that are selectively engaged during this sleep state. They focus on a pathway linking a hypothalamic region involved in sleep regulation to the medial septum and onward to a hippocampal subregion that is critical for social memory. By combining recordings of neural activity with sleep-state-specific circuit manipulations, the study seeks to explain how information is routed during sleep to support distinct types of memory.

A major strength of the work is the use of state-of-the-art circuit-based approaches to link sleep dynamics to defined long-range connections and behavioral outcomes. The authors show that neurons in the lateral supramammillary region projecting to the medial septum are selectively active during rapid eye movement sleep, and that silencing this pathway during this sleep state disrupts consolidation of both social and contextual fear memories. Further dissection of downstream circuitry reveals that inhibition of the medial septum-to-hippocampal CA2 pathway during rapid eye movement sleep selectively impairs social memory. These results provide support for functional specialization within parallel pathways and suggest that this circuit acts as a hub for routing memory-related information during sleep.

While the evidence supporting a role for this circuit in sleep-dependent memory consolidation is compelling, several important mechanistic details remain unresolved. The chemical signaling used by the neurons connecting the hypothalamus to the medial septum is not clearly defined, leaving open whether these cells release excitatory signals, inhibitory signals, or a combination of both. In addition, the medial septum contains multiple neuronal populations with distinct downstream targets, and the specific cell types receiving input from this pathway are not clearly identified. Similarly, the nature of the signals delivered from the medial septum to the hippocampus remains unclear, making it difficult to link circuit anatomy to the observed behavioral specificity. Finally, because different circuit segments are manipulated independently, the causal relationship between upstream and downstream pathways remains suggestive rather than definitive and should be discussed explicitly as a limitation or addressed experimentally.

Overall, the authors largely achieve their aims by identifying a rapid eye movement sleep-specialized circuit that contributes to memory consolidation in a modality-specific manner. The findings are likely to have a meaningful impact on the field by advancing understanding of how sleep organizes memory through parallel neural pathways and by providing a useful framework for future studies of sleep-dependent brain state regulation. With additional clarification of circuit mechanisms or a clearer discussion of current limitations, the study would offer even greater value to the neuroscience community.

Reviewer #2 (Public review):

Summary:

This study systematically characterizes the activity patterns of a lateral supramammillary nucleus (SuM)-medial septum (MS)-hippocampus circuit across sleep-wake cycles and its role in memory consolidation. The authors demonstrate that the lateral SuM-MS projection is specifically active during REM sleep, and that REM-selective inhibition of this circuit, and of its downstream MS-CA2 pathway, impairs the consolidation of social memory. The work is well-designed, and the data are robust in supporting clear and significant conclusions. It provides important new insights into how distinct memory modalities could be processed by parallel, sleep-active subcortical-hippocampal circuits. The manuscript is of high quality overall, with some points to address as detailed below.

Strengths:

(1) Novel finding:<br /> The identification of a REM-specialized subpopulation within the lateral SuM-MS pathway and its specific role in social memory consolidation via the lateral SuM-MS-CA2 projection is a significant advance. It effectively complements the previously described direct SuM-CA2 pathway and supports a model of the SuM as a "REM-hub" routing information through dedicated downstream targets.

(2) Technical rigor:<br /> The combination of retrograde tracing, in vivo calcium imaging, single-unit identification via optrode recording, and temporally precise (REM-sleep-specific) optogenetic manipulation provides strong correlative and causal evidence.

(3) Appropriate controls:<br /> Behavioral experiments include crucial controls for optogenetic inhibition (GtACR1 group, NREM/Wake inhibition control, mCherry control), effectively ruling out nonspecific effects of light or timing.

Weaknesses:

(1) Figure titles/descriptions:<br /> For clarity, the authors should consider specifying the recording method in the figure titles or legends. For instance, Figure 2: "Bulk Ca2+ activity (fiber photometry) of lateral SuM-MS projecting neurons..." and Figure 3: "Single-unit activity patterns (optrode recordings) of lateral SuM-MS projecting neurons...".

(2) Statistical details:<br /> The use of "LSD post-hoc comparison" following ANOVA is noted. LSD is sensitive but can increase Type I error risk with multiple comparisons. Please justify its use or consider employing a more conservative post-hoc test (e.g., Tukey's or Bonferroni) for key comparisons like the social preference index in Figure 4h to bolster robustness.

(3) Data presentation:<br /> When reporting statistical results in figure legends (e.g., Figures 2d, 3i-k), please provide the specific test statistic values (e.g., F, χ²) and exact P values where possible, rather than only significance asterisks.

(4) Deepening mechanistic insight:<br /> The study excellently demonstrates "what" the circuit does. The discussion could be strengthened by further exploring "how" it might work. The finding that SuM-MS inhibition does not affect CA1 theta power is interesting and distinguishes it from other MS/hippocampal pathways. The suggestion of a theta-independent mechanism is plausible. Could the authors hypothesize more specifically? For example, might this circuit modulate reactivation events in the local CA2 network, neurochemical milieu (e.g., acetylcholine), or synapse-specific plasticity during REM sleep to facilitate social memory consolidation?

(5) Implications of regional heterogeneity:<br /> The functional divergence between lateral (90% REM-active) and medial SuM-MS neurons is intriguing. A brief discussion on the potential anatomical basis (differential inputs/outputs) and functional significance (e.g., integration of specific affective or arousal signals) of this subdivision would be valuable.

Reviewer #3 (Public review):

Summary:

Kroeg et al. introduced a novel method for generating 3D cortical layer-like organization in hiPSC-derived models, achieving remarkably consistent topography within compact dimensions. Their approach involves seeding frontal cortex-patterned iPSC-derived neural progenitor cells into 384-well plates, which triggers the spontaneous assembly of adherent cortical organoids comprising diverse neuronal subtypes, astrocytes, and oligodendrocyte-lineage cells.

Strengths:

Compared with existing brain organoid models, these adherent cortical organoids exhibit enhanced reproducibility and improved cell viability during prolonged culture, thereby providing versatile opportunities for high-throughput drug discovery, neurotoxicological screening, and investigation of brain disorder pathophysiology. Overall, this study addresses an important and timely need for advancing current brain organoid systems.

Weaknesses:

Highlighting the consistency of differentiation across different cell lines and standardizing functional outputs are crucial to emphasize the broad future potential of this new organoid system for large-scale pharmacological screening. The authors provided a substantial amount of new data during the revision process to support the reproducibility of neuronal activity. The next step would be to leverage this platform for functional screening of chemical and genetic perturbations to identify new drug candidates.

Comments on revisions:

Most of my previous concerns were adequately addressed through the revision.

Reviewer #2 (Public review):

Summary:

Xu et al. used fMRI to examine the neural correlates associated with retrieving temporal information from an external compared to internal perspective ('mental time watching' vs. 'mental time travel'). Participants first learned a fictional religious ritual composed of 15 sequential events of varying durations. They were then scanned while they either (1) judged whether a target event happened in the same part of the day as a reference event (external condition); or (2) imagined themselves carrying out the reference event and judged whether the target event occurred in the past or will occur in the future (internal condition). Behavioural data suggested that the perspective manipulation was successful: RT was positively correlated with sequential distance in the external perspective task, while a negative correlation was observed between RT and sequential distance for the internal perspective task. Neurally, the two tasks activated different regions, with the external task associated with greater activity in the supplementary motor area and supramarginal gyrus, and the internal condition with greater activity in default mode network regions. Of particular interest, only a cluster in the posterior parietal cortex demonstrated a significant interaction between perspective and sequential distance, with increased activity in this region for longer sequential distances in the external task but increased activity for shorter sequential distances in the internal task. Only a main effect of sequential distance was observed in the hippocampus head, with activity being positively correlated with sequential distance in both tasks. No regions exhibited a significant interaction between perspective and duration, although there was a main effect of duration in the hippocampus body with greater activity for longer durations, which appeared to be driven by the internal perspective condition. On the basis of these findings, the authors suggest that the hippocampus may represent event sequences allocentrically, whereas the posterior parietal cortex may process event sequences egocentrically.

Strengths:

The topic of egocentric vs. allocentric processing has been relatively under-investigated with respect to time, having traditionally been studied in the domain of space. As such, the current study is timely and has the potential to be important for our understanding of how time is represented in the brain in the service of memory. The study is well thought out and the behavioural paradigm is, in my opinion, a creative approach to tackling the authors' research question. A particular strength is the implementation of an imagination phase for the participants while learning the fictional religious ritual. This moves the paradigm beyond semantic/schema learning and is probably the best approach besides asking the participants to arduously enact and learn the different events with their exact timings in person. Importantly, the behavioural data point towards successful manipulation of internal vs. external perspective in participants, which is critical for the interpretation of the fMRI data. The use of syllable length as a sanity check for RT analyses as well as neuroimaging analyses is also much appreciated.

Suggestions:

The authors have satisfactorily addressed my last remaining suggestion.

Reviewer #1 (Public review):

The authors use Flow cytometry and scRNA seq to identify and characterize the defect in gdT17 cell development from HEB f/f, Vav-icre (HEB cKO) and Id3 germline-deficient mice. HEB cKO mice showed defects in the gdT17 program at an early stage, and failed to properly upregulate expression of Id3 along with other genes downstream of TCR signaling. Id3KO mice showed a later defect in maturation. The results together indicate HEB and Id3 act sequentially during gdT17 development. The authors further showed that HEB and TCR signaling synergize to upregulate Id3 expression in the Scid-adh DN3-like T cell line. Analysis of previously published Chip-seq data revealed binding of HEB (and Egr2) at overlapping regulatory regions near Id3 in DN3 cells.The study provides insight into mechanisms by which HEB and Id3 act to mediate gdT17 specification and maturation. The work is well performed and clearly presented.

Comments on revisions:

The authors have answered all of my questions. I am strongly supportive of the revised work.

Reviewer #2 (Public review):

Summary:

The manuscript by Selvaratnam et al. defines how the transcription factor HEB integrates with TCR signaling to regulate Id3 expression in the context of gdT17 maturation in the fetal thymus. Using conditional HEB ablation driven by Vav Cre, flow cytometry, scRNA-seq, and reanalysis of ChIP-seq data the authors, provide evidence for a sequential model in which HEB and TCR-induced Egr2 cooperatively upregulate Id3, enabling gdT17 maturation and limiting diversion to the ab lineages. The work provides an important mechanistic insight into how the E/ID-protein axis coordinates gd T cell specification and effector maturation.

Strengths include:

(1) The proposed model that HEB primes, TCR induces, and Id3 stabilizes gdT17 cells in embryonal development is elegant and consistent with the findings.

(2) The choice of animal models and the study of a precise developmental window.

(3) The cross-validation of flow, scRNA-seq, and ChIP-seq reanalyses strengthens the conclusions.

(4) The study clarifies the dual role of Id3, first as an HEB-dependent maturation factor for gdT17 cells, and as a suppressor of diversion to the ab lineages.

Comments on revisions:

In this revised version of their manuscript the authors have effectively addressed all of my previous concerns. In its current form the study represents a significant advancement in our understanding of how the transcription factor HEB integrates with TCR signaling to regulate Id3 expression in the context of gdT17 maturation in the fetal thymus. In this revised version of their manuscript the authors have effectively addressed all of my previous concerns. In its current form the study represents a significant advancement in our understanding of how the transcription factor HEB integrates with TCR signaling to regulate Id3 expression in the context of gdT17 maturation in the fetal thymus.

Reviewer #3 (Public review):

Summary:

The authors of this manuscript have addressed a key concept in T cell development: how early thymus gd T cells subsets are specified and the elements that govern gd T17 versus other gd T cell subset or ab T cell subsets are specified. They show that the transcriptional regulator HEB/Tcf12 plays a critical role in specifying the gd T17 lineage and, intriguingly that it up regulates the inhibitor Id3 which is later required for further gd T17 maturation.

Strengths:

The conclusions drawn by the authors are amply supported by a detailed analysis of various stages of T cell maturation in WT and KO mouse strains at the single cell level both phenotypically, by flow cytometry for various diagnostic surface markers, and transcriptionally, by single cell sequencing. Their conclusions are balanced and well supported by the data and citations of previous literature.

Weaknesses:

I actually found this work to be quite comprehensive.

Comments on revisions:

Nothing to add here. The authors were very thorough in their original submission, and all minor issues identified have been addressed to my satisfaction.

Reviewer #2 (Public review):

Summary:

The premise of the manuscript by Matteucci et al. is interesting and elaborates a mechanism via which TNFa regulates monocyte activation and metabolism to promote murine survival during Plasmodium infection. The authors show that TNF signaling (via an unknown mechanism) induces nitrite synthesis, which (via yet an unknown mechanism), and stabilizes the transcription factor HIF1a. Furthermore, that HIF1a (via an unknown mechanism) increases GLUT1 expression and increases glycolysis in monocytes. The authors demonstrate that this metabolic rewiring towards increased glycolysis in a subset of monocytes is necessary for monocyte activation including cytokine secretion, and parasite control.

Strengths:

The authors provide elegant in vivo experiments to characterize metabolic consequences of Plasmodium infection, and isolate cell populations whose metabolic state is regulated downstream of TNFa. Furthermore, the authors tie together several interesting observations to propose an interesting model regarding

Weaknesses:

The main conclusion of this work - that "Reprogramming of host energy metabolism mediated by the TNF-iNOS-HIF1a axis plays a key role in host resistance to Plasmodium infection" is unsubstantiated. The authors show that TNFa induces GLUT1 in monocytes, but never show a direct role for GLUT1 or glucose uptake in monocytes in host resistance to infection (nor the hypoglycemia phenotype they describe).

Comments on revisions:

The demonstration that the established TNF-iNOS-HIF-1α-glycolysis axis operates in vivo during P. chabaudi infection is valuable and relevant. However, it constitutes contextual validation and must be carefully described as such. This distinction, i.e., "what has already been shown vs. what is new" is not consistently reflected in the framing of the manuscript raising overstatement concerns. This is particularly evident in the abstract and other conclusive statements, where mechanistic novelty is implied, even when the underlying pathways/mechanisms are already known. To improve the manuscript, all sentences that refer to already established findings should be accurately described as such.

For example, the abstract states: "Here, we show that TNF signaling hampers physical activity, food intake, and energy expenditure while enhancing glucose uptake by the liver and spleen as well as controlling parasitemia in P. chabaudi-infected mice." In this sentence, the effects of TNF signaling on physical activity, food intake, energy expenditure, glucose metabolism and control of parasitemia are unequivocally established and therefore do not, in themselves, constitute new findings. Feeding behavior, not cell-intrinsic metabolism, may drive glycemic differences

The authors propose that TNF signaling leads to GLUT1 upregulation (in inflammatory monocytes, MO-DCs, and within the liver and spleen) during Plasmodium infection, and that this results in increased glucose uptake contributing to systemic hypoglycemia. While this is an intriguing hypothesis, we urge the authors to consider an alternative explanation that, at present, is not adequately ruled out. Given that glycemia serves as a central functional readout in the manuscript, this distinction is essential to clarify.

The observed regulation of glycemia is likely not a direct consequence of increased glucose uptake by immune cells or by tissues but may instead reflect broader differences in disease severity across genotypes. The iNOS KO, TNFR KO, and HIF-19775ΔαLyz2 mice likely experience a dampened inflammatory response, which would blunt infection-induced anorexia and help preserve overall metabolic homeostasis. This alternate interpretation is supported by the authors' metabolic cage data showing increased physical activity in TNFR KO mice and the elevated food intake shown in Figure 2B.

Since anorexia and energy expenditure are tightly coupled to the inflammatory milieu, it is plausible that these behavioral and systemic differences-not monocyte nor tissue GLUT1 expression per se-are the primary contributors to the observed glycemic patterns. To support their current interpretation, the authors should perform a pair-feeding experiment in which (at least) TNFR KO mice are restricted to the same food intake as infected WT controls. This would help disentangle whether differences in glycemia truly reflect immune-driven metabolic rewiring or are secondary to differences in caloric intake.

The contribution of monocyte-specific glucose metabolism to host resistance remains unresolved.

We appreciate the authors' effort to address the mechanistic role of glycolysis in host resistance using in vivo 2-deoxyglucose (2DG) treatment. However, I would like to point out that while this experiment is informative, it does not fully resolve the specific concern raised regarding the cell-intrinsic role of TNF-induced glycolysis in monocytes. 2DG acts systemically, inhibiting glycolysis across a wide range of cell types-including hepatocytes, endothelial cells, lymphocytes, and myeloid populations. Therefore, the observed increase in parasitemia following 2DG treatment may reflect the broad importance of glycolysis for host defense, or alternatively, may result from elevated circulating glucose levels induced by 2DG (PMID: 35841892), which could enhance parasite growth by increasing nutrient availability. Therefore, this experiment does not allow for a specific conclusion about the requirement for TNF-driven metabolic reprogramming in monocytes.

Reviewer #1 (Public review):

Summary:

The manuscript of Heydasch et al. addresses the spatiotemporal regulation of Rho GTPase signaling in living cells and its coupling to the mechanical state of the cell. They focus on a GAP of RhoA, the Rho specific GAP Deleted in Liver Cancer 1 (DLC1). They first show that removing DLC1 either by a CRISPR KO or by downregulation using siRNA leads to an increased contractility and globally elevated RhoA activity, as revealed by a FRET biosensor. This result was expected, since DLC1 is deactivating RhoA its absence should lead to increasing amounts of active RhoA. To go beyond global and steady levels of RhoA activity, the authors developed an acute optogenetic system to study transient RhoA activity dynamics in different genetic and subcellular contexts. In WT cells, they found that pulses of activation lead to an increased RhoA activity at focal adhesions (FA) compared to plasma membrane (PM), which suggests that FAs contain less RhoA GAPs, more RhoA, or that FAs involve positive feedbacks implying others GEFs for example. In DLC1 KO cells, they found that the RhoA response upon pulses of optogenetic activation was increased (higher peak) both at FA and PM, which could be expected since less GAP should increase the amount of active RhoA. But surprisingly, they observed also a higher rate of RhoA deactivation in DLC1 KO cells, which is counterintuitive: less GAP should result in a slower rate of deactivation. Less GAP should also lead to a lower rate of observed RhoA activation (smaller koff) and delayed peak. Using a modeling approach and control experiments (to monitor the optogenetic intrinsic dynamics), the authors propose that there is a negative feedback in WT cell between activated RhoA and the activity of its GAPs (other than DLC1). More active RhoA decreases GAP activity such that active RhoA relaxation to its basal state is relatively slow. This negative feedback would be absent in DCL1-deficient cells, explaining the relatively faster relaxation. This hypothesis is convincing given the data and the model, and it shows that there are compensatory mechanisms at play when DLC1 is knocked down. Further on, the authors study the dynamics of DLC1 on FAs depending on the mechanical state and nicely show a causal decrease of DLC1 enrichment at FA upon FA reinforcement, hereby probing a positive feedback where RhoA activation is further amplified as the force exerted at FA is increasing. Altogether, this work highlight the extremely fine regulation in space and time of RhoGTPases that is only revealed through acute perturbations, while at the cell scale and long time scale, complex compensatory mechanisms are at play rendering knock-down or overexpression experiments not always straightforward to interpret (in the present case, knock-down of a deactivator lead to an increase of deactivation rate through the induced absence of other activity dependent-deactivators).

Strengths:

- Experiments are precise and well done.

- Technically, the work brings original and interesting data. The use of transient optogenetic activation within focal adhesions together with a biosensor of activity is new and elegant.

- The link between DLC1 and global contractility/RhoA activity is clear and convincing.

- The surprising higher rate of RhoA deactivation in DLC1 KO cells is convincing, as well as the differences in the dynamics of RhoA between focal adhesions and plasma membrane.

- The model is very helpful to support the hypothesis of the negative feedback loop.

- The correlation between DLC1 enrichment and focal adhesion dynamics is very clear.

Weaknesses:

- The negative and positive feedback loops could have been dug more deeply molecularly (in particular discover what are the compensatory mechanisms at play), but this could be the purpose of future work.

Comments on revised version:

I thank the authors for the great improvement of their work and their detailed answers to my comments. The modeling work is great and really brings novelty to the story. It also helps a lot to have the data for the optoLARG recruitment. I suggest authors move to the Version of Record.

Reviewer #1 (Public review):

Summary:

The authors were seeking to identify a molecular mechanism whereby the small molecule RY785 selectively inhibits Kv2.1 channels. Specifically, the authors sought to explain some of the functional differences that RY785 exhibits in experimental electrophysiology experiments as compared to other Kv inhibitors, namely the charged and non-specific inhibitor tetraethylammonium (TEA). The authors used a recently published cryo-EM Kv2.1 channel structure in the open activated state and performed a series of multi-microsecond-long all-atom molecular dynamics simulations to study Kv2.1 channel conduction under the applied membrane voltage with and without RY785 or TEA present. They observed that while TEA directly blocks K+ permeation by occluding ion permeation pathway, RY785 binds to multiple non-polar residues near the hydrophobic gate of the channel driving it to a semi-closed non-conductive state. They confirmed this mechanism using an additional set of simulations and used it to explain experimental electrophysiology data,

Strengths:

The total length of simulation time is impressive, totaling many tens of microseconds. The authors develop their own forcefield parameters for the RY785 molecule based on extensive QM based parameterization. The computed permeation rate of K+ ions through the channel observed under applied voltage conditions is in reasonable agreement with experimental estimates of the single channel conductance. The authors have performed extensive simulations with the apo channel as well as both TEA and RY785. The simulations with TEA reasonably demonstrate that TEA directly blocks K+ permeation by binding in the center of the Kv2.1 channel cavity, preventing K+ ions from reaching the SCav site. The authors conclude that RY785 likely stabilizes a partially closed conformation of the Kv2.1 channel and thereby inhibits K+ current. This conclusion is plausible given that RY785 makes stable contacts with multiple hydrophobic residues in the S6 helix, which they can also validate using a recently published closed-state Kv2.1 channel cryo-EM structure. This further provides a possible mechanism for the experimental observations that RY785 speeds up the deactivation kinetics of Kv2 channels from a previous experimental electrophysiology study.

Weaknesses:

The authors, however, did not directly observe this semi-closed channel conformation and in fact acknowledge that more direct simulation evidence would require extensive enhanced-sampling simulations beyond the scope of this study. They have not estimated the effect of RY785 binding on the protein-based hydrophobic pore constriction, which may further substantiate their proposed mechanism. And while the authors quantified K+ permeation, they have not made any estimates of the ligand binding affinities or rates, which could have been potentially compared to experiment and used to validate their models.

However, despite those relatively minor weaknesses, the conclusions of the study are convincing, and overall this is a solid study helping us to understand two distinct molecular mechanisms of the voltage-gated potassium channel Kv2.1 inhibition by TEA and RY785, respectively.

Reviewer #2 (Public review):

Summary

In this manuscript, Zhang et al. investigate the conduction and inhibition mechanisms of the Kv2.1 channel, with a particular focus on the distinct effects of TEA and RY785 on Kv2 potassium channels. Using microsecond-scale molecular dynamics simulations, the authors characterize K⁺ ion permeation and RY785-mediated inhibition within the central pore. Their results reveal an inhibition mechanism that differs from those described for other Kv channel inhibitors.

Strengths

The study identifies a distinctive inhibitory mode for RY785, which binds along the channel walls in the open-state structure while still permitting a reduced level of K⁺ conduction. In addition, the authors propose a long-range allosteric coupling between RY785 binding in the central pore and changes in the structural dynamics of Kv2.1. Overall, this is a well-organized and carefully executed study, employing robust simulation and analysis methodologies. The work provides novel mechanistic insights into voltage-gated potassium channel inhibition and may offer useful guidance for future structure-based drug design efforts.

Weaknesses:

The study needs to consider the possibility of multiple binding sites for PY785, particularly given its impact on voltage sensors and gating currents. Specifically, the potential for allosteric binding sites in the voltage-sensing domain (VSD) should be assessed, as some allosteric modulators with thiazole moieties are known to bind VSD domains in multiple voltage-gated sodium channels (Ahuja et al., 2015; Li et al., 2022; McCormack et al., 2013; Mulcahy et al., 2019). Increasing structural and functional evidence supports the existence of multiple ligand-binding modes in voltage-gated ion channels. For example, polyunsaturated fatty acids have been shown to bind to KCNQ1 at both the voltage sensor domain and the pore domain (https://doi.org/10.1085/jgp.202012850). Similarly, cannabidiol has been structurally resolved in Nav1.7 at two distinct sites, one in a fenestration and another near the IFM-binding pocket (https://doi.org/10.1038/s41467-023-39307-6). These advances illustrate that ligand effects cannot always be interpreted based solely on a single binding site identified previously.

Reviewer #2 (Public review):

Summary:

The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.

Strengths:

The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.

Overall impact:

Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These observations help solidify the field. The work raises an important unanswered question: why are rifampicin and many protein synthesis inhibitors bacteriostatic with E. coli but bactericidal with pathogenic mycobacteria?

Comments on revisions:

I call attention to word choice, because it can indicate how familiar the authors are with the field. An issue that caught my attention was the use of the words persistence and tolerance, because they are not uniformly used in the generally accepted way (see Balaban 2019 Nat Rev Micro). In this consensus statement persistence refers specifically to a subpopulation and as such has survival kinetics that are distinct from those seen with tolerance, a phenomenon that refers to the entire population. I notice that the Balaban paper is not in the reference list. My suggestion is to take a look at the Balaban paper and then examine every use of the words tolerance and persistence in the manuscript to be sure that they fit the Balaban definition.

Reviewer #1 (Public review):

Summary:

The authors created a metric to score the toxicity of specific amino acid homorepeats that accounts for differences in physicochemical properties. This "neutrality" score reflects how often a particular homorepeat appears in nature across the proteomes of different species. This can be used to understand known proteins and their characteristics, as well as inform on the upcoming field of protein design.

Strengths:

This study represents a very careful and thorough study of the amino acid homorepeats and does a remarkable job of accounting for the effects of the fluorescent protein tags.

Weaknesses:

The initial characterization of the neutrality score is missing a control of a known toxic homorepeat to help validate this method of characterizing amino acid homorepeats.

The authors did achieve their aim of developing a metric by which to score the toxicity and properties of amino acid homorepeats. This can be used in the future with other common amino acid motifs that are not homorepeats and can help scientists refine computer models for rational protein design.

Reviewer #2 (Public review):

Summary

The aim of this study was to assess which amino acid stretches are tolerated/favoured in the course of evolution, considering their physico-chemical properties, metabolic costs and proteotoxicity. To address this question, the authors expressed PolyX variants in yeast, E. coli and also referred to COS cells. The PolyX constructs were tagged with GFP or a different fluorescence reporter to assess expression levels and localization at the C-terminus with or without a cleavable linker or to study topological effects. The PolyX stretch was also embedded between two different fluorescent proteins. The authors used growth rate and expression levels as judged by fluorescence intensities to calculate the relative neutrality in comparison to GFP alone.

They could show that harmful/beneficial effects depend on the specific amino acid (aa) and polar aa are tolerated well, whereas hydrophobic and positively charged aa are harmful to the cell. This is not surprising as hydrophobic and positively charged aa are known to be aggregation-prone. They could further show that the topology matters for some, but not all, PolyX variants. The PolyX stretch can affect the subcellular localization and aggregation propensity of the GFP it is fused to. Interestingly, overexpression of PolyG, PolyQ or PolyS was not harmful, and overexpression of PolyE was potentially even beneficial for the cell. The authors concluded their study with a theoretical analysis of the presence of aa stretches in various species and identified a high correlation between their expression in yeast and other species, suggesting that the selection of aa stretches is conserved and follows biochemical rules (trade-off between tolerance of expression levels, solubility, sub-cellular localization, and maybe metabolic costs).

Strengths:

The authors performed a high number of experiments and systematically assessed the expression and tolerance of 10mer stretches of 20 aa fused to GFP or other fluorophores in yeast and E. coli. This is an impressive effort.

Weaknesses:

(1) The analysis of expression levels of the various PolyX variants should not rely only on fluorescence intensities. The fusion of the PolyX stretch may affect the fluorescence properties (brightness, photostability) of the fluorescent partner and may or may not affect abundance. A quantitative analysis of PolyX-GFP (same applies to the other fusion constructs shown in Figure 3) is needed. Preferably by an MS-based proteomic analysis via peptide count. Western blot is less ideal as it would rely on epitope recognition of the respective antibody, and the epitope accessibility might be altered upon fusion with different PolyX stretches. In addition, the authors should analyse the PolyX stretch without an attached fluorophore as a control.

(2) The images shown in Figure 4 are not very informative. The constructs should be subjected to FRAP to assess the solubility of the PolyX variants and Ssa1 (Hsp70). FCS could be an alternative as well.

(3) The observation of the lack of mCherry fluorescence for PolyK and PolyP (Figure 4) can also be interpreted as an instability of the fusion protein (partial truncation and degradation) or quenching. The authors should test different fluorophores and different linker lengths between the PolyX stretch and the fluorophores. Fluorophore swapping (N/C-terminally) would also be a good control.

(4) The study would benefit from a consideration of a large body of literature on protein aggregation and the contribution of amino acid composition. The here identified amino acids that as 10mer stretch are harmful to the cell and are known to be aggregation-prone and are also recognised by molecular chaperones to prevent their aggregation.

(5) The study could further benefit from ex vivo and in vitro analyses of the PolyX constructs. They could isolate the PolyX variants and study their solubility by, e.g. light scattering outside of the cellular context.

Reviewer #3 (Public review):

Summary:

The constraints limiting the usage of especially repetitive amino acid sequences in proteins remain enigmatic. In their manuscript, Murase et al. analyse the impact of polyamino acid homorepeats (PolyX) on the expression of EGFP-variants with PolyX modifications. Introducing a new measure, relative neutrality, allows us to rate beneficial versus harmful sequences. The authors find that especially hydrophobic repeats (I, V, W, F, Y) show harmful effects on the respective proteins, enhancing their aggregation. Hydrophilic repeats (E, S, N, Q), on the other hand, show beneficial properties but suppress proteotoxic stress. Interestingly, these observations correlate with the occurrence of such PolyX in natural proteins across the proteomes of different organisms.

Strengths:

The manuscript seems especially valuable in the context of rational or de novo protein design. The observations on the one hand should allow for enhancing the solubility of proteins by using beneficial PolyX. On the other hand, they explain very well why some PolyX do not occur in natural proteins. The authors present a sound, broad and well-analysed dataset. The study is well designed, the manuscript is very well written, and the conclusions drawn are overall valid.

Weaknesses:

The whole data set relies on the definition of the newly introduced "relative neutrality" score. Besides being a well-chosen tool, this score is limited and biased as it does not directly include a measure for "solubility" but relies on "fluorescence emission" derived from the respective EGFP-fusion-proteins.

A second major weakness is that the influence of PolyX-modifications on secondary structure is neither analysed nor discussed.

Reviewer #1 (Public review):

Summary:

This manuscript describes a putative clinical association between ARID5B genetic variants and a novel neurodevelopmental syndrome characterized by global developmental delay, intellectual disability, and occasional neuroinflammatory episodes. While the identification of 29 individuals with overlapping phenotypes and the use of a CRISPR-Cas9 mouse model suggest a potential gene-disease link, the study suffers from significant methodological gaps in variant prioritization and a lack of robust mechanistic evidence to support its primary claims. Specifically, the "neuroinflammation" component is over-emphasized despite appearing in only a minor subset of the cohort, and the molecular pathogenesis remains insufficiently explored beyond initial protein localization assays.

Strengths:

(1) The study proposes a new clinical syndrome associated with the ARID5B gene, distinguishing it from established Coffin-Siris syndromes related to other ARID family members.

(2) The recruitment of a relatively large cohort of 29 individuals from diverse geographical and ethnic backgrounds strengthens the initial phenotypic description.

(3) The combination of human clinical data, in vitro localization assays, and an in vivo mouse model provides a multi-level framework for investigating the gene's function.

(4) The identification of variants in the exceptionally long final exon of ARID5B that escape nonsense-mediated mRNA decay (NMD) offers an interesting perspective on the molecular pathology of this gene.

Weaknesses:

(1) The description of the genomic methodology appears limited. A more detailed explanation of the filtration and selection process for variant prioritization is essential. The authors should provide a comprehensive summary of evidence (e.g., CADD scores, allele frequencies in gnomAD, and segregation analysis) to justify the selection of the reported variants, even if they do not strictly meet all ACMG/AMP criteria.

(2) The cohort includes several inherited variants and missense mutations that require more robust evidence of pathogenicity. For example, the presence of the variant in population databases (gnomAD) suggests the need for careful re-evaluation of its causality. A more rigorous assessment using diverse computational metrics, such as PhyloP scores and conservation analysis, is necessary to confirm the pathogenicity of the missense variants.

It is recommended that the authors re-evaluate the cohort to ensure that only variants with strong evidence of causality are included to maintain a clear genotype-phenotype correlation.

(3) The proposed molecular mechanism would benefit from further empirical support. The claim of NMD escape is currently supported by only a small number of cases, and a much more detailed explanation is also required for the experimental data provided.

Although the mouse model exhibits developmental abnormalities, it does not recapitulate the other systemic features reported in humans. In addition, given that "brain development" is a central theme, the manuscript lacks detailed neuroanatomical data, histopathology, or other molecular biological (e.g., RNA-seq) evidence from brain specimens to substantiate these claims at a molecular level.

(4) The emphasis on "neuroinflammation" in the title may be disproportionate to its observed frequency. Central nervous system inflammation was identified in only a small subset of the cohort (2 of 29 individuals).

Without additional experimental validation, such as immunological challenges in the Arid5b mouse model, it is premature to characterize this as a hallmark feature. Additionally, the inconsistent response to immunotherapy suggests that the autoimmune component requires further investigation.

(5) Supplementary tables require reorganization to improve clarity. The current structures make it difficult for readers to effectively analyze the data, and a more standardized format is recommended.

(6) As the manuscript proposes a novel disease entity, a more comprehensive clinical discussion is warranted. The authors should provide a more systematic description of the core clinical features and, crucially, address the genotype-phenotype correlation. Specifically, a more detailed analysis is required to determine whether the clinical severity or the presence of specific features varies according to the location of the variant or the type of mutation. Such insights are essential for clinicians to differentiate this syndrome from other ARID-related disorders.

Reviewer #2 (Public review):

Summary:

The authors compiled 29 patients with various neurodevelopmental symptoms due to the ARID5B mutations. Although not directly, the mouse model demonstrated that the heterozygous mutant mouse showed mild behavioral problems. It would be interesting to see if the mice carry craniofacial features.

Strengths:

The HEK293T model showed that the mutant protein mis-localized, but did not show whether the mutation caused any changes in epigenetic status. Nevertheless, this paper delivers clear support for genotype-phenotype correlation.

Weaknesses:

(1) The paper would be improved by providing pedigrees of some of the patients with inherited variants.

(2) Figure 3d could provide more species for an accurate conservation assessment.

Reviewer #3 (Public review):

Summary:

In the present study, through international gene-matching efforts, the authors present 29 individuals with rare, heterozygous ARID5B variants and find that these individuals have a newly recognizable neurodevelopmental syndrome. A recurring clinical syndrome of developmental delay/intellectual disability, behavioral difficulties, renal malformation, and recurrent infections is described. 19 of these variants were confirmed to be de novo, and only one was inherited from an unaffected parent. 24/29 of these variants introduce premature termination codons in the final exon and are predicted to escape nonsense-mediated decay. The ARID5B p.Q522Ter variant was studied in a mouse heterozygous knock-in model, found to be associated with behavioral abnormalities. The well-described genetic and phenotypic data for this cohort provide convincing clinical evidence for a novel neurodevelopmental syndrome. The functional evidence provided is preliminary, and further studies are needed to understand disease mechanisms.

Strengths:

(1) The authors give a good description of a novel clinical syndrome manifesting as developmental delay/intellectual disability, facial dysmorphism, and behavioral challenges.

(2) The authors create a mouse model harboring an Arid5b(Q522*/+) variant and identify subtle behavioral changes.

(3) Attempts are made to functionally characterize a subset of ARID5B variants in human cell lines.

Weaknesses:

(1) The title - "ARID5B mutations cause a neurodevelopmental syndrome with neuroinflammation episodes" - should be revised. 2/29 individuals (7%) had CNS inflammation; this does not appear to be a core feature of the disease and should not be highlighted as such. If this is going to be a feature that is highlighted, then more details are needed. MRI images of cerebellitis and/or ADEM would be helpful, as well as lumbar puncture results and supplemental information detailing the treatment course.

(2) The abstract states that "Remarkably, 19 of 29 variants (66%) cluster within the first quarter of exon 10, are de novo, and escape nonsense-mediated mRNA decay (NMD), which we confirmed for two variants affecting seven individuals." The authors state in the Results that they "indeed found no signs of NMD". In Figure 3f, when assessing for transcript amount, there appears to be a great deal of variability. Three ARID5B variant lines are tested. Transcript amounts in two lines appear to be near control levels, but one LCL ARID5B Ile497AsnfsTer31 line appears to demonstrate significantly lower levels of transcript. The control lines also show a great deal of variability. No explanation is given for this large difference between LCL ARID5B Ile497AsnfsTer31 lines and for the variability in control lines, making these data uninterpretable. A major theme of the paper is that early truncating variants in exon 10 escape NMD and lead to the described phenotypes, so this is an important point that needs to be resolved, either by testing more patient-derived lines or knocking in these variants into cell lines.

(3) The Arid5b(Q522*/+) mice are not sufficiently molecularly characterized. Does the variant transcript escape NMD? What happens at the protein level? Is there mislocalization of the protein?

(4) For the HEK293T cell experiments, variants are overexpressed and compared to a control. These experiments appear to leave endogenous ARID5B intact. What might the authors expect to see if these variants were knocked in?

(5) The functional consequences of the missense variants are not tested. The authors suggest that missense variants may be more associated with macrocephaly and possibly ASD. Are these missense variants causing loss-of-function or gain-of-function? Is there preserved protein function?

(6) There are a number of functional assays performed, but it remains unclear if the tested variants are operating through a loss- or gain-of-function. Are truncating variants early in exon 10 leading to a partial loss-of-function? Or do they prevent the functioning of the other allele through a dominant negative mechanism? These possibilities are not directly tested.

Reviewer #1 (Public review):

Summary:

This is an excellent and strong paper. The authors not only show the mechanisms of action of destabilizing mutations in VHL, but notably, they also go on to computationally design and experimentally test an inhibitor that restores wild-type pVHL function, offering starting points for a new class of kidney cancer drugs. The approach that the authors take here can be used to target destabilizing mutations in repressor proteins, common in diseases, including cancer.

Strengths:

This paper is the culmination of an extraordinary amount of work, over years, including method development and testing by a broad range of tools and experiments. It is thorough and comprehensive. It is also well-written and easy to follow.

Reviewer #2 (Public review):

Summary:

Inactivating VHL mutations are common in clear cell renal cell carcinoma, and about half of those mutations unfold/destabilize the protein rather than directly interfering with critical protein-protein interactions. The authors identify a compound that can stabilize/refold mutant VHL and seemingly restore its ability to downregulate its major downstream targets.

Strengths:

The authors use a clever combination of virtual and cell-based screens, followed by suitable biophysical and cell-based validation assays, to arrive at a VHL refolder. This compound is suboptimal from an ADME point of view, but could be a starting point for further medicinal chemistry optimization. Success would have implications for other diseases linked to similar loss-of-function mutations.

Weaknesses:

The authors need to tighten up the evidence that the VHL refolder is downregulating HIF2 in a strictly "on-target" manner.

(1) In Figure 3C, the increase in VHL stability looks very modest. Taking into account the increased abundance of the VHL protein at time 0 in the presence of CP4 compared to control, it is not so clear that VHL is decaying much more slowly in the presence of CP4. I understand that the fact that the signal is low in the absence of CP4 at time 1 hour makes it hard to quantify the half-life of p30 in the absence of the drug (is a longer exposure needed?). However, perhaps the authors could try to quantify the p19 half-life.

(2) In going from CP4 to CP4.29 the authors screened based on downregulation of HIF. This is logical but also introduces the danger of identifying chemicals that can downregulate HIF in an "off-target" manner, i.e. non-specifically. It is therefore essential to clearly show that CP4.29 downregulates steady-state levels of HIF and HIF target genes in cells with suitable (hydrophobic core) VHL mutants but not in isogenic cells lacking VHL. Another prediction is that these chemicals should be inert in cells with VHL mutations that directly abrogate HIF binding. So Figure 4E (HIF2 target genes) needs the use of the isogenic VHL-/- cells described later in the paper. And the steady-state levels of HIF2 should be measured in the isogenic cells (mutant VHL vs -/-) with or without CP4.29. Figure 4G, as it is done now, is too indirect and not very compelling. I don't understand why the "time 0" HIF2 levels aren't lower in the presence of CP4.29, and I think the half-life differences with treatment are very subtle to my eyeball densitometer (although I applaud the authors' attempt to quantify), with the exception of I180N. I agree that Figure 4F is encouraging, but hypoxia has many effects, and this experiment is not as "clean" as the VHL-/- experiments. The same applies to some of the pharmacologic agents in Figure 5.

Reviewer #1 (Public review):

The current study is a follow-up to a previously published article in eLife in 2021, demonstrating that the transcription factor MYRF-1 interacts with the transmembrane protein PAN-1, which is required for the stability and targeting of MYRF-1 to the plasma membrane. There, MYRF-1 undergoes self-catalytic cleavage of its intracellular domain and translocates to the nucleus. Here, the authors analyze the activation of MYRF-1 during the larval development of C. elegans. They nicely show that MYRF-1 cleavage and nuclear translocation oscillate with larval stage transitions. They further identify two regions in MYRF-1 and PAN-1 that negatively regulate MYRF-1 cleavage and activation, and show that relief of this negative regulation causes premature lin-4 activation and overrides nutrient-responsive developmental checkpoints. The experiments are elegant and accurately support the conclusions raised. There are only minor comments and suggestions to improve the manuscript.

Reviewer #2 (Public review):

In this study, Xu et al. investigated the regulatory mechanisms controlling intramolecular cleavage of the transmembrane transcription factor MYRF-1, an important event that controls developmental progression in C. elegans.

The authors made important advances in several aspects:

(1) Through endogenous gene editing/tagging, further supported by western blots, the authors convincingly demonstrate the novel finding that the intramolecular cleavage and nuclear translocation of MYRF-1 is not static, but temporally controlled within each developmental stage: with nuclear translocation peaking at the late stage and then declining into lethargus/molts between developmental stages (Figure 1).

(2) They demonstrate that this cleavage and nuclear translocation is controlled by external stimuli, namely starvation.

(3) They reveal modes of regulation of the intramolecular cleavage that is mildly regulated by MYRF-1's own JM domain as well as the CCT tail of interacting partner PAN-1.

The conclusions of this paper are mostly well supported by data, but some aspects of the manuscript and conclusions should be clarified and extended to strengthen its findings.

(1) The authors concluded that the intramolecular cleavage and nuclear localization of MYRF-1 were similarly temporally-regulated in all tissue types. However, the data/image presented was limited to specific regions/cell types that were inconsistently chosen across developmental windows. For example, for the cleavage/nuclear translocation across L1 into lethargus (Figures 1B, E, F, G), the heads of the worm were shown to comprise mostly neurons and muscles. While across the rest of the larval stages, only mid-body pictures were shown, comprising mostly hypodermal and some intestinal cells. A complete coverage of all tissues across all time points would better support the author's conclusion that this temporal regulation occurs similarly in all tissue types. Additionally, the authors should clearly indicate which tissue/cell-types were used in the quantifications, as these were not done for several figure panels (including but not limited to Figure 1I and J).

(2) Related to point 1 above, this inconsistency in tissue assessment was also true for downstream experiments (Figures 2-6; e.g., starvation, JM, and CCT regulation, etc.). Broad tissue specific assessment for all downstream experiments would greatly enhance the strength and relevance of the findings. Judging by the current data presented (Figures 3, 5, 6), it seems to suggest that there are tissue/cell-type differences in the regulation of MYRF-1 nuclear translocation.

(3) Developmental progression was superficially and inconsistently assessed across the study. Developmental progression was mainly assessed by hypodermal (V-lineage) division patterns and worm length in this study. Several glaring omissions that should have been examined were the lengths of larval stages/lethargus and molting defects, as well as gonad development, to help identify which developmental landmarks were affected vs. not.

(4) The phosphorylation within MYRF-1's JM domain was insufficiently investigated. There were two serine phosphorylation sites that were discovered through mass spectrometry experiments, however the authors only investigated one of the serine (S623) residues without any justifications for the choice. Additional investigation of the other residues, as well as both together, would strengthen the relevance of these phosphorylation events to cleavage and nuclear translocation, especially considering the minimal effect observed with only mutating the one residue.

Reviewer #3 (Public review):

Summary:

In this paper, the authors identified dual inhibitory mechanisms, an intrinsic juxtamembrane (JM) region and an extrinsic cytoplasmic tail (CCT) domain in the binding protein PAN-1, that suppress MYRF-1 cleavage in C. elegans. The authors showed that MYRF-1 cleavage oscillates across larval stages, peaking in mid-to-late phases and being suppressed during molts. This oscillatory pattern is consistent with MYRF-1's role in promoting transitions of larval stages, particularly in late-L1 involving lin-4 activation and DD neuron remodeling.

Strengths:

This work generated several knock-in strains of fluorescent tags and mutations in the endogenous myrf-1 and pan-1gene loci, which will provide resources for future identification and characterization of the underlying molecular mechanisms regulating MYRF-1 cleavage inhibition.

The results presented in the paper are solid enough to support the paper's main conclusions.

This study is valuable for establishing MYRF-1 cleavage as a key gatekeeper of the C. elegans developmental timing. Findings from C. elegans MYRF-1 may provide insight into the regulation and function of mammalian MYRF.

Weaknesses:

The following points should be discussed to further support the authors' model that MYRF-1 cleavage is a key gatekeeper of developmental timing.

(1) Recent findings by Helge Großhans and Jordan Ward groups showed that KIN-20 (CK1δ) and LIN-42 (PERIOD) are required for proper molt timing in C. elegans, and that loss of LIN-42 binding or of the phosphorylated LIN-42 tail impairs nuclear accumulation of KIN-20, resulting in arrhythmic molts (EMBO J. 44, 6368-6396, 2025). In this paper, the authors concluded that PAN-1 promotes MYRF trafficking to the cell membrane, where MYRF-1 cleavage and nuclear translocation occur, and that oscillates with developmental molting cycles in C. elegans. It is unclear whether MYRF-1 and KIN-20 interact in the nucleus and, if so, how this interaction controls developmental timing.

(2) Separately, it was previously shown that the let-7 primary transcript (pri-let-7) exhibits oscillating, pulse-like expression that peaks during each larval stage, rather than a steady increase, and directly correlates with developmental molting cycles. It is unclear whether the nuclear-localized MYRF-1 fragment regulates the oscillatory primary let-7 expression during larval transition (McCulloch and Rougvie, 2014; Van Wynsberghe et al., 2011).

Reviewer #1 (Public review):

Summary:

This manuscript by Feng et al. uses mouse models to study the embryonic origins of HSPCs. Using multiple types of genetic lineage tracing, the authors aimed to identify whether BM-resident endothelial cells retain hematopoietic capacity in adult organisms. Through an important mix of various labeling methodologies (and various controls), they reach the conclusion that BM endothelial cells contribute up to 3-4% of hematopoietic cells in young mice.

Strengths:

The major strength of the paper lies in the combination of various labeling strategies, including multiple Cdh5-CreER transgenic lines, different CreER lines (col1a2), and different reporters (ZsGreen, mTmG), including a barcoding-type reporter (PolyLox). This makes it highly unlikely that the results are driven by a rare artifact due to one random Cre line, or one leaky reporter. The transplantation control (where the authors show no labeling of transplanted LSKs from the Cdh5 model) is also very supportive of their conclusions.

Weaknesses:

While the updated manuscript now provides strong evidence for Cdh5-CreER+ cells as a source of myeloid-biased hematopoiesis, the true identity of these "adult EHT stem cells", their differentiation hierarchy, the kinetics, the EHT mechanism, and the physiological relevance of this process remain unaddressed.

Reviewer #2 (Public review):

Summary:

Feng, Jing-Xin et al. studied the hemogenic capacity of the endothelial cells in the adult mouse bone marrow. Using Cdh5-CreERT2 in vivo inducible system, though rare, they characterized a subset of endothelial cells expressing hematopoietic markers which was transplantable. They suggested that the endothelial cells need the support of stromal cells to acquire blood forming capacity ex vivo. This endothelial cells were transplantable and contribute to hematopoiesis with ca. 1% chimerism in a stress hematopoiesis condition (5-FU) and recruited to peritoneal cavity upon Thioglycolate treatment. Ultimately, the authors detailed the blood lineage generation of the adult endothelial cells in a single cell fashion suggesting a predominant HSPCs-independent blood formation by adult bone marrow endothelial cells, in addition to the discovery of Col1a2+ endothelial cells with blood forming potential corresponds to its high Runx1 expressing property.

Comments on revised version:

Overall, the authors have addressed our main concerns, and the revised manuscript is improved. The new data now more strongly supports long-term multilineage reconstitution of the adult hemogenic ECs. However, critical data, especially regarding the ECs' hematopoietic identity and functional capacity remains insufficient, which limits the strength of hemogenic claim, especially to assert that these adult hemogenic ECs generate bona fide HSCs.

Points that are sufficiently addressed:

(1) Exclusion of the potential contamination during cell sorting for the ex vivo CD45- ZsGreen+ fraction culture has been explicitly shown to be of a high purity in Fig. 2B.

(2). The pre-cultured ZsG+ fraction is shown to having a long-term multilineage reconstituting capacity (10 months chimerism, Fig. 2J), which increases confidence that the fraction is not limited to short-lived progenitors.

Points that are insufficiently addressed:

(1) As noted in the "Limitation of Study", the absence of LT-HSC phenotyping and/or secondary transplantation data of ZsG+ donor limits confidence in concluding that the adult hemogenic BM-ECs generate HSPCs.

(2) The lack of early to the end of reconstitution kinetics in Fig 2E-2J restricts interpretation of whether the donor fraction contains rapid reconstituting transient progenitor versus sustained repopulating HSCs.

Reviewer #1 (Public review):

Summary:

The goal of this paper was to determine whether the T cell receptor (TCR) repertoire differs between a male or female human. To address this, this group sequenced TCRs from double-positive and single-positive thymocytes in male and female humans of various ages. Such an analysis on sorted thymocyte subsets has not been performed in the past. The only comparable dataset is a pediatric thymocyte dataset where total thymocytes were sorted.

They report on participant ages and sexes, but not on ethnicity, race, nor provide information about HLA typing of individuals. The experiments are heroic, yet do represent a relatively small sampling of diverse humans. They observed no differences in TCRbeta or TCRalpha usage, combinational diversity, or differences in the length of the CDR3 region, or amino acid usage in the CD3aa region between males or females. Though they observed some TCRbeta CD3aa sequence motifs that differed between males and females, these findings could not be replicated using an external dataset and therefore were not generalizable to the human population.

They also compared TCRbeta sequences against those identified in the past databases using computational approaches to recognize cancer-, bacterial-, viral-, or autoimmune-antigens. They found little overlap of their sequences with these annotated sequences (depending on the individual, ranged from 0.82-3.58% of sequences). Within the sequences that were in overlap, they found that certain sequences against autoimmune or bacterial antigens were significantly over-represented in female versus male CD8 SP cells. Since no other comparable dataset is available, they could not conclude whether this is a generalizable finding in the human population.

Strengths:

It is a novel dataset that attempts to understand sex differences in the T cell repertoire in humans. Overall, the methodologies are sound and are the current state-of-the-art. There was an attempt to replicate their findings in cases where an appropriate dataset was available. I agree that there are no gross differences in TCR diversity between males and females. This is an important negative result.

Weaknesses:

Overall, the sample size is small given that it is an outbred population. This reviewer recognizes the difficulty in obtaining samples for this experiment (which were from deceased donors), and this limitation was appropriately discussed. Their analysis was limited by the current availability of other TCR sequences. These weaknesses were appropriately discussed and considered.

Reviewer #2 (Public review):

Summary:

This study addresses the hypothesis that the strikingly higher prevalence of autoimmune diseases in women could be the result of biased thymic generation or selection of TCR repertoires. The biological question is important and the hypothesis is valuable. Although the topic is conceptually interesting and the dataset is rich, the study has a number of major issues. In particular, the majority of "autoimmunity-related TCRs" considered in this study are in fact specific to type 1 diabetes (T1D). Notably, T1D incidence is higher in males, which directly contradicts the stated objective of the study - to explain the higher prevalence of autoimmune diseases in women. Given this conceptual inconsistency, the evidence presented does not support the authors' conclusions.

Strengths:

The key strength of this work is the newly generated dataset of TCR repertoires from sorted thymocyte subsets (DP and SP populations). This approach enables the authors to distinguish between biases in TCR generation (DP) and thymic selection (SP). Bulk TCR sequencing allows deeper repertoire coverage than single-cell approaches, which is valuable here, although the absence of TRA-TRB pairing and HLA context limits the interpretability of antigen specificity analyses. Importantly, this dataset represents a valuable community resource and should be openly deposited rather than being "available upon request."

Weaknesses:

I thank the authors for their detailed responses to my previous comments. Several concerns were addressed satisfactorily; however, important issues remain unresolved, and a new major concern has emerged from the revised manuscript.

Major concerns:

(1) Autoimmune specificity is dominated by T1D, contradicting the study's premise. Newly added supplementary Table 3 shows that the authors considered only 14 autoimmune-related epitopes, of which 12 are associated with type 1 diabetes (T1D) and 2 with celiac disease (CeD). (I guess this is because identification of particular peptide autoantigens is an extremely difficult task and was only successful in T1D and CeD.) Thus conclusions of this work mostly relate to T1D. However, the incidence of T1D is higher in males than in females (e.g. doi:10.1111/j.1365-2796.2007.01896.x; doi:10.25646/11439.2). This directly contradicts the stated objective of the study - to explain the higher prevalence of autoimmune diseases in women. As a result, the authors' conclusions (a) cannot be generalized to autoimmune disease as a whole as the authors only considered T1D and CeD antigens and (b) are internally inconsistent with the stated objective of the study.

(2) By contrast, CeD does show a female bias (~60/40 female/male; doi:10.1016/j.cgh.2018.11.013). However, the manuscript does not allow evaluation of how much the reported "autoimmune TCR enrichment" derives from T1D versus CeD. Despite my previous request, the authors did not provide per-donor and per-epitope distributions of autoimmune-specific TCR matches. I therefore explicitly request a table in which: each row corresponds to a specific autoimmune antigen; each column corresponds to a donor (with metadata available including sex); each cell reports the number of unique TCRs specific to that antigen in that donor. Without such data, the conclusions cannot be evaluated.

(3) It is scientifically inappropriate to generalize findings to "autoimmune diseases" when only T1D and CeD were analyzed. Moreover, given that T1D and CeD show opposite directions of sex bias, combining them into a single "AID" category is misleading. All analyses presented in Figure 8 and Supplementary Figure 16 should be repeated and shown separately for T1D and CeD, rather than combined.

(4) The McPAS database contains TCRs associated with other autoimmune diseases (e.g., multiple sclerosis, rheumatoid arthritis), although the exact autoantigens in these contexts are unknown. Why didn't the authors perform the search for such TCRs? I believe disease association even without particular known antigen could still be insightful.

(5) Misuse of the concept of polyspecificity. I appreciate the authors' reference to Don Mason's work; however, the concept of polyspecificity discussed there is fundamentally different from the authors' usage. Mason, Sewell (doi:10.1074/jbc.M111.289488), Garcia (doi:10.1016/j.cell.2014.03.047), and others demonstrated that individual TCRs can recognize multiple peptides, possibly around 1 million. But importantly these peptides are not random but share some sequence motif. This is a general feature of TCRs, i.e. 100% of TCRs are polyspecific in this sense.<br /> In contrast, the authors define polyspecificity as TRB sequences annotated as specific to unrelated epitopes in TCR databases such as VDJdb. These databases are well known to contain substantial numbers of false-positive annotations (see, e.g., Ton Schumacher's preprint https://www.biorxiv.org/content/10.1101/2025.04.28.651095.abstract). The authors acknowledge that, under their definition, polyspecificity has been experimentally validated for only one (!) TCR (Quiniou et al.). In the absence of robust experimental validation, use of the term "polyspecificity" in this context is misleading. I strongly recommend removing all analyses and conclusions related to polyspecificity from the manuscript unless supported by independent functional validation.

(6) I agree that comparing specificity enrichment between sexes is meaningful. However, enrichment relative to the database composition itself is not biologically interpretable, as acknowledged by the authors in their response. I therefore recommend removing Supplementary Figure 15, which is potentially misleading.

(7) In contrast, Supplementary Figure 16 represents the most convincing result of the study (keeping in mind that the AID group should be splitted to T1D and CeD with T1D and that T1D and CeD have opposing directions of sex biases) and should be shown as a main figure, replacing Figure 8A-B which is less convincing as it doesn't show per-donor distribution.

(8) The authors argue that applying mixed-effects modeling to Rényi entropy would require assuming a common sex effect across subsets. I do not find this assumption unreasonable. For example, if sex effects are mediated through AIRE-dependent negative selection, one would indeed expect a consistent direction of effect across subsets. The lack of statistical significance in Figure 3 may reflect limited sample size rather than true absence of the difference. Moreover, the title's phrasing "comparable TCR repertoire diversity" is vague: what is the statistical definition of "comparable"?

Reviewer #1 (Public review):

Summary:

This paper tackles an important question: What drives the predictability of pre-stimulus brain activity? The authors challenge the claim that "pre-onset" encoding effects in naturalistic language data have to reflect the brain predicting the upcoming word. They lay out an alternative explanation: because language has statistical structure and dependencies, the "pre-onset" effect might arise from these dependencies, instead of active prediction. The authors analyze two MEG datasets with naturalistic data.

Strengths:

The paper proposes a very interesting alternative hypothesis for claims in prior work (e.g., Goldstein et al., 2022). In contrast to claims in prior work, the current paper convincingly demonstrates that prior results can be explained by inherent stimulus dependencies in natural language, as opposed to the brain actively predicting future linguistic content.

Two independent datasets are analyzed. The analyses with the most and least predictive words is clever, and is nicely complementing the more naturalistic analyses. The work emphasizes how claims about linguistic prediction cannot be trivially drawn using encoding models in naturalistic designs.

Reviewer #2 (Public review):

Summary:

At a high-level, the reviewers demonstrate that there is a explanation for pre-word-onset predictivity in neural responses that does not invoke a theory of predictive coding or processing. The paper does this by demonstrating that this predictivity can be explained solely as a property of the local mutual information statistics of natural language. That is, the reason that pre-word onset predictivity exist could simply boil down to the common prevalence of redundant bigram or skip-gram information in natural language.

Strengths:

The paper addresses a problem of significance and uses methods from modern NeuroAI encoding model literature to do so. The arguments, both around stimulus dependencies and the problems of residualization, are compellingly motivated and point out major holes in the reasoning behind several influential papers in the field, most notably Goldstein et al. This result, together with other papers that have pointed out other serious problems in this body of work, should provoke a reconsideration of papers from encoding model literature that have promoted predictive coding. The paper also brings to the forefront issues in extremely common methods like residualization that are good to raise for those who might be tempted to use or interpret these methods incorrectly.

Weaknesses:

After author revision, I see no major weaknesses in the underlying arguments or data processing steps.

Reviewer #3 (Public review):

Summary:

The study by Schönmann et al. presents compelling analyses based on two MEG datasets, offering strong evidence that the pre-onset response observed in a highly influential study (Goldstein et al., 2022) can be attributed to stimulus dependencies-specifically, the auto-correlation in the stimuli-rather than to predictive processing in the brain. Given that both the pre-onset response and the encoding model are central to the landmark study, and that similar approaches have been adopted in several influential works, this manuscript is likely to be of high interest to the field. Overall, this study encourages more cautious interpretation of pre-onset responses in neural data, and the paper is well written and clearly structured.

Strengths:

• The authors provide clear and convincing evidence that inherent dependencies in word embeddings can lead to pre-activation of upcoming words, previously interpreted as neural predictive processing in many influential studies.

• They demonstrate that dependencies across representational domains (word embeddings and acoustic features) can explain the pre-onset response, and that these effects are not eliminated by regressing out neighboring word embeddings-an approach used in prior work.

• The study is based on two large MEG datasets and one ECoG dataset, showing that results previously observed in ECoG data can be replicated in MEG. Moreover, the stimulus dependencies appear to be consistent across the three datasets.

Weaknesses:

• While this study shows that stimulus dependency can account for pre-onset responses, it remains unclear whether this fully explains them, or whether predictive processing still plays a role. The more important question is whether pre-activation remains after accounting for these confounds.

Comments on revisions:

I appreciate the added analyses. This study raises an important methodological concern regarding an influential paper and will certainly have a high impact on our field.

Reviewer #1 (Public review):

Summary:

The authors investigated the population structure of the invasive weed Lantana camara from 36 localities in India using 19,008 genome-wide SNPs obtained through ddRAD sequencing.

Strengths:

The manuscript is well-written, the analyses are sound, and the figures are of great quality.

Weaknesses:

The narrative almost completely ignores the fact that this plant is popular in horticultural trade and the different color morphs that form genetic populations are most likely the result of artificial selection by humans for certain colors for trade, and not the result of natural selfing. Although it may be possible that the genetic clustering of color morphs is maintained in the wild through selfing, there is no evidence in this study to support that. The high levels of homozygosity are more likely explained as a result of artificial selection in horticulture and relatively recent introductions in India. Therefore, the claim of the title that "the population structure.. is shaped by its mating system" is in part moot, because any population structure is in large part shaped by the mating system of the organism, but further misleading because it is much more likely artificial selection that caused the patterns observed.

Update after manuscript was revised by authors:

The authors added a selfing experiment, showing that the wild plants are selfing and not outcrossing, which limits the genetic exchange. This supports their claims, but a link with the horticultural industry is still lacking in the study, and conclusions should still be viewed in the regional context of India rather than globally.

Reviewer #2 (Public review):

Summary:

The authors performed a series of population genetic analyses in Lantana camara using 19,008 genome-wide SNPs data from 359 individuals in India. They found clear population structure that did not show a geographical pattern, and flower color was rather associated with population structure. Excess of homozygosity indicate high selfing rate, which may lead to fixation of alleles in local populations and explain the presence of population structure without a clear geographic pattern. Authors also performed a forward simulation analysis, theoretically confirming that selfing promotes fixation of alleles (higher Fst) and reduction in genetic diversity (lower heterozygosity).

Strengths:

Biological invasion is a critical driver of biodiversity loss, and it is important to understand how invasive species adapt to novel environments despite limited genetic diversity (genetic paradox of biological invasion). Lantana camara is one of the hundred most invasive species in the world (IUCN 2000), and the authors collected 359 plants from a wide geographical range in India, where L. camara has invaded. The scale of the dataset and the importance of the target species are the strength of the present study. Coalescent-based analysis nicely supports the authors' claim that multiple introductions may have contributed the population structure of this species.

Weaknesses:

The main findings of the SLiM-based simulation were that inbreeding promotes fixation of alleles and reduction in genetic diversity. These are theoretically well known, and such findings themselves are not novel, although it may have become interesting if these findings are quantitatively integrated with their empirical findings in the studied species.

Reviewer #1 (Public review):

Summary:

In this paper, Chen et al. identified a role for the circadian photoreceptor CRYPTOCHROME (CRY) in promoting wakefulness under short photoperiods. This research is potentially important as hypersomnolence is often seen in patients suffering from SAD during winter times. The mechanisms underlying these sleep effects are poorly known.

Strengths:

The authors clearly demonstrated that mutations in cry lead to elevated sleep under 4:20 Light-Dark (LD) cycles. Furthermore, using RNAi, they identified GABAergic neurons as a primary site of CRY action to promote wakefulness under short photoperiods. They then provide genetic and pharmacological evidence demonstrating that CRY acts on GABAergic transmission to modulate sleep under such conditions.

Weaknesses:

The authors then went on to identify the neuronal location of this CRY action on sleep. This is where this reviewer is much more circumspect about the data provided. The authors hypothesize that the l-LNvs which are known to be arousal promoting may be involved in the phenotypes they are observing. To investigate this, they undertook several imaging and genetic experiments.

While the authors have made improvements in this resubmitted manuscript, there are still multiple concerns about the paper. I think the authors provide enough evidence suggesting that CRY plays a role in sleep under short photoperiod. The data also supports that CRY acts in GABAergic neurons. However, there are still major issues with the quality of the confocal images presented throughout the paper. In many cases it appears that the images are oversaturated with poor resolution, making it hard to understand what is going on. In addition, none of the drivers used in this study are specific to the neurons the authors aim to manipulate. Therefore, the identity of the GABAergic neurons involved in this CRY dependent sleep mechanism remains unclear. Similarly, whether l-LNvs are the target of this GABA mediated sleep regulation under short photoperiod is not fully demonstrated. The data presented suggests that but does not prove it.

Major concerns:

(1) While the authors provided sleep parameters like consolidation or waking activity for some experiments. These measurements are still not shown for several experiments (for example Figures 2E, 3, 4, 5, and 6). These data are essential, these metrics must be reported for all sleep experiments.

(2) Line 144 "We fed flies with agonists of GABA-A (THIP) and GABA-B receptor (SKF-97541) (Ki and Lim, 2019; Matsuda et al., 1996; Mezler et al., 2001). Both drugs enhance sleep in WT," The proper citation is needed here, Dissel et al., 2015 PMID:25913403. Both THIP and SKF-97541 were used in that paper.

(3) Figure 2C and 2F: it appears that the control data is the same in both panels. That is not acceptable.

(4) Figure 4A: With the quality of the images, it is impossible to assess whether GABA levels are increased at the l-LNvs soma.

(5) Fig 4 S1A shows colabeling of l-LNvs and Gad1-Gal4 expressing neurons. They are almost 100% overlapping signals. This would indicate that the l-LNvs are GABAergic themselves, or that there is a problem with this experiment.

(6) Fig 4 S1B: Again, I can see colabelling of the GFP and PDF staining, suggesting that Gad1-Gal4 expresses in l-LNvs.

(7) Line 184: "Consistently, knocking down Rdl in the l-LNvs rescues the long sleep phenotype of cry mutants (Figure 4-figure supplement 1D)." This statement is incorrect as the driver used for this experiment, 78G01-GAL4 is not specific to the l-LNvs, so it is possible that the phenotypes observed are not coming from these neurons.

(8) Figure 4G-K: None of these manipulations are specific to the l-LNvs. The authors describe 10H10-GAL4 and 78G01-GAL4 as l-LNvs specific tools, but this is not the case. Why not use the SS00681 Split-GAL4 line described in Liang et al., 2017 PMID: 28552314? It is possible that some of the effects reported in this manuscript are not caused by manipulating the l-LNvs.

(9) Similarly for the manipulation of s-LNvs, the authors cannot rule out effect that are coming from other cells as R6-GAL4 is not specific to s-LNvs.

(10) The staining presented in Fig 5 S1 is not very convincing. Difficult to see whether Gad1-GAL4 only expresses in the s-LNvs.

Reviewer #3 (Public review):

Summary:

In humans, short photoperiods are associated with hypersomnolence. The mechanisms underlying these effects is however, unknown. Chen et al. use the fly Drosophila to determine the mechanisms regulating sleep under short photoperiods. They find that mutations in the circadian photoreceptor cryptochrome (cry) increase sleep specifically under short photoperiods (e.g. 4h light : 20 h dark). They go on to show that cry is required in GABAergic neurons and that the effects of the cry mutation on sleep are mediated by alterations in GABA signalling. Further, they suggest that the relevant subset of GABAergic neurons are the well-studied small ventral lateral neurons that they suggest inhibit the arousal promoting large ventral neurons via GABA signalling

Strengths:

Genetic analysis to show that cryptochrome (but not other core clock genes) mediates the increase in sleep in short photoperiods, and circuit analysis to localise cry function to GABAergic neurons.

Weaknesses:

The authors' have substantially revised their manuscript, and the manuscript is better for the revisions. However, the conclusion that the sLNvs are GABAergic is unfortunately still not well supported by the data. A key sticking point remains the anti GABA immunostaining, and specific driver lines for sLNvs and lLNvs.

The authors should tone down their conclusions to reflect the fact that their data, as presented, does not support the model that cry acts in sLNvs to modulate GABA signalling onto lLNvs and thus modulate sleep.

Reviewer #4 (Public review):

Summary:

Short photoperiod is an important experimental manipulation in neurobiology, endocrinology, and metabolism studies. However, the molecular mechanisms by which short photoperiod gives rise to behavioral phenotypes that are seen in seasonal affective disorders remain unknown. Using the classic circadian model organism Drosophila, this study examines short photoperiod-induced hypersomnolence and identifies the circadian photoreceptor cryptochrome as a regulator of GABAergic tone within the clock neural circuit to promote wakefulness under short photoperiod conditions. The discovery has broad implications for understanding how short photoperiod modulates neural inhibition in circadian circuits in regulating sleep.

Strengths:

The Drosophila model provided a powerful platform to dissect the molecular mechanisms underlying short photoperiod-induced hypersomnolence. A battery of behavioral, imaging, circuit-manipulation approaches was employed to test the novel hypothesis that the circadian photoreceptor cryptochrome modulates GABAergic tone within the clock neural circuit to promote wakefulness under short photoperiod conditions.

Weaknesses:

The current model proposed by the authors suggests that the small ventral lateral neurons of the Drosophila clock circuit are GABAergic; however, this remains unclear. At present, the field lacks sufficient data and validated reagents to definitively establish the GABAergic identity of these neuropeptidergic neurons.

Reviewer #1 (Public review):

Summary:

The authors developed a new autofocusing method, LUNA (Locking Under Nanoscale Accuracy), to address severe focus drift-a major challenge in time-lapse microscopy. Using this method, they tackle a fundamental question in bacterial cold shock response: whether cells halt growth and division following an abrupt temperature downshift. Overall, the experimental design, modeling, and data analysis are solid and well executed. However, several points require clarification or further support to fully substantiate the authors' conclusions.

Strengths:

(1) The LUNA method outperforms existing autofocusing systems with nanoscale precision over a large focusing range. The focusing time is reasonable for the presented experiments, and the authors note potential improvements by using faster motors and optimized control algorithms, suggesting broad applicability. The theoretical simulations and experimental validation provide solid support for the robustness of the method.

(2) Using LUNA, the authors address a long-standing question in bacterial physiology: whether cells arrest growth and division after an abrupt cold shock. Single-cell analyses monitoring the entire course of cold adaptation and steady-state growth reveal features that are obscured in bulk-culture studies: cells continue to grow at reduced rates with smaller cell sizes, resulting in an apparently unchanged population-level OD. The experiments are well designed and analyses are generally solid and largely support the authors' conclusions.

(3) The authors also propose a model describing how population-level OD measurements depend on cell dry mass density, volume, and concentration. This provides a valuable conceptual contribution to the interpretation of OD-based growth measurements, which remain a gold-standard method in microbiology.

Weaknesses:

(1) It is unclear whether the author's model explaining the population-level OD during acclimation is broadly applicable. Most analyses focus on a shift from 37˚C to 14˚C, where the model agrees well with experimental data. However, in the 37˚C to 12˚C experiment, OD600 decreases after cold shock (Fig. 5e), and the computed OD does not match the experimental measurements (Fig. S16a). Although the authors attribute this discrepancy to a "complicated interplay," no further explanation is provided, which limits confidence in the model's general applicability.

(2) The manuscript proposes that cell-cycle progression becomes synchronized across the population after cold shock, but the supporting evidence is not fully convincing. If synchronization refers primarily to the uniform reduction in growth rate following cold shock, this could plausibly arise from global translation inhibition affecting all cells. However, the additional claim that "cells encountering a relatively late CSR will accelerate division to maintain synchronization" is not strongly supported by the presented data.

(3) Several technical terms used in the method development section are not clearly defined and may be unfamiliar to a broad readership, which makes it difficult to fully understand the methodology and evaluate its performance. Examples include depth of focus, focusing precision, focusing time, focusing frequency, and drift threshold value. In addition, the reported average focusing time per location (~0.6 s) lacks sufficient context, limiting the reader's ability to assess its significance relative to existing autofocusing methods.

Reviewer #2 (Public review):

Summary:

This study presents LUNA, an autofocus method that compensates for focus drift during rapid temperature changes. Using this approach, the authors show that E. coli cells continue to grow and divide during cold shock, revealing a coordinated, multi-phase adaptation process that could not be deduced from traditional population measurements. They propose a scattering-theory-based model that reconciles the paradox between growth differences of the bacteria at the single-cell level vs population level.

Strengths:

(1) The LUNA approach is pretty creative, turning coma aberration from what is normally a nuisance into an exploit. LUNA enabled long-term single-cell imaging during rapid temperature downshifts.

(2) The authors show that the long-assumed growth arrest during cold shock from population-level measurements is misleading. At the single-cell level, bacteria do not stop growing or dividing but undergo a continuous, three-phase adaptation process. Importantly, this behavior is highly synchronized across the population and not based on bet-hedging.

(3) Finally, the authors propose a model to resolve a long-standing paradox between single-cell vs population behavior: if cells keep growing, why does optical density (OD) of the culture stop increasing? Using light-scattering theory, they show that OD depends not only on cell number but also on cell volume, which decreases after cold shock. As a result, OD can remain flat, or even decrease, despite continued biomass accumulation. This demonstrates that OD is not a reliable proxy for growth under non-steady conditions.

Weaknesses:

(1) While the authors theoretically explain the advantages of LUNA over existing autofocus methods, it is unclear whether practical head-to-head comparisons have been performed, apart from the comparison to Nikon PFS shown in Video S1. As written, the manuscript gives the impression that only LUNA can solve this problem, but such a claim would require more systematic and rigorous benchmarking against alternative approaches.

(2) No mutants/inhibitors used to test and challenge the proposed model.

(3) Cells display a high degree of synchronization, but they are grown in confined microfluidic channels under highly uniform conditions. It is unclear to what extent this synchrony reflects intrinsic biology versus effects imposed by the microfluidic environment.

(4) To further test and generalize the model, it would be informative to also examine bacterial responses at intermediate temperatures rather than focusing primarily on a single cold-shock condition.

Reviewer #1 (Public review):

Summary

The manuscript by Peden-Asarch et al. introduces MPS, a new open-source software package for processing miniscope data. The authors aim to provide a fast, end-to-end analysis pipeline tailored to miniscope users with minimal experience in coding or version control. The work addresses an important practical barrier in the field by focusing on usability and accessibility.

Strengths

The authors identify a clear and well-motivated need within the miniscope community. Existing pipelines for miniscope data analysis are often complex, difficult to install, and challenging to maintain. In addition, users frequently encounter technical limitations such as out-of-memory errors, reflecting the substantial computational demands of these workflows-resources that are not always available in many laboratories. MPS is presented as an attempt to alleviate these issues by offering a more streamlined, accessible, and robust processing framework.

Weaknesses

The authors state that "MPS is the first implementation of Constrained Non-negative Matrix Factorization (CNMF) with Nonnegative Double Singular Value Decomposition (NNDSVD) initialization." However, NNDSVD initialization is the default method in scikit-learn's NMF implementation and is also used in CaIMAN. I recommend rephrasing this claim in the abstract to more accurately reflect MPS's novelty, which appears to lie in the specific combination of constrained NMF with NNDSVD initialization, rather than being the first use of NNDSVD initialization itself.

At present, there are practical issues that limit the usability of the software. The link to the macOS installer on the documentation website is not functional. Furthermore, installation on a MacBook Pro was unsuccessful, producing the following error:<br /> "rsync(95755): error: ... Permission denied ... unexpected end of file."

For the purposes of this review, resolving this issue would significantly improve the evaluation of the software and its accessibility to users.

More broadly, the authors propose self-contained installers as a solution to the "package-management burden" commonly associated with scientific software. While this approach is appealing and potentially useful for novice users, current best practices in software development increasingly rely on continuous integration and continuous deployment (CI/CD) pipelines to ensure reproducibility, testing, and long-term maintenance. In this context, it has become standard for Python packages to be distributed via PyPI or Conda. Without dismissing the value of standalone installers, the overall quality and sustainability of MPS would be greatly enhanced by also supporting conventional environment-based installations.

Reviewer #2 (Public review):

Summary:

This manuscript introduces Miniscope Processing Suite (MPS), a novel no-code GUI-based pipeline built to easily process long-duration one-photon calcium imaging data from head-mounted Miniscopes. MPS aims to address two large problems that persist despite the rapid proliferation of Miniscope use across the field. The first issue is concerned with the high technical barrier to using existing pipelines (e.g., CaImAn, MIN1PIPE, Minian, CaliAli) that require users to have coding skills to analyze data. The second problem addressed is the intense memory limitations of these pipelines, which can prevent analysis of long-duration (multi-hour) recordings without state-of-the-art hardware. The MPS toolbox takes inspiration from what existing pipelines do well, innovates new modules like Window Cropping, NNDSVD initialization, Watershed-based segmentation, and improves the user experience to improve access to calcium imaging analysis without the need for new training in new coding languages. In many ways, MPS achieves this aim, and thus will be of interest to a growing, broad audience of new calcium imagers.

There are, however, some concerns with the current manuscript and pipeline that, if addressed, would greatly improve the impact of this work. Currently, the manuscript provides insufficient evidence that MPS can generate good results efficiently on various data sets, and it is not properly benchmarked against other established packages. Additionally, considering the goal of MPS is to attract novices to attempt Miniscope analysis, better tutorials, documentation, and walkthroughs of expected vs inaccurate results should be provided so that it is clear when the user can trust the output. Otherwise, this simplified approach may end up leading new users to erroneous results.

Strengths:

The manuscript itself is well-organized, clear, and easy to follow. MPS is clearly designed to remove the computational barrier for entry for a broad neuroscience community to record and analyze calcium data. The development of several well-detailed algorithmic innovations merits recognition. Firstly, MPS is extremely easy to install, keep updated, and step through. Having each step save every output automatically is a well-thought-out feature that will allow users to enter back into the pipeline at any step and compare results.

The implementation of an erroneous frame identifier and remover during preprocessing is an important new feature that is typically done offline with custom-built code. Interactive ROI cropping early in the pipeline is an efficient way to lower pixel load, and NNDSVD initialization is a new way to provide nonnegative, biologically interpretable starting spatial and temporal factors for later CNMF iterations. Parallel temporal-first update ordering cuts down dramatically on later computational load. Together, all these features, neatly packaged into a no-code GUI like the Data Explorer for manual curation, are practical additions that will benefit end users.

Weaknesses:

A major limitation of this manuscript is that the authors don't validate the accuracy of their source extraction using ground-truth data or any benchmark against existing pipelines. The paper uses their own analysis of processing speeds, component counts, signal-to-noise ratio improvements, and morphological characteristics of detected cells, but it needs to be reworked to include some combination of validation against manually annotated ground truth data sets, simulated data with known cell locations and activity patterns, or cross-validation with established pipelines on identical datasets. Without this kind of validation, it is impossible to truly determine whether MPS produces biologically acceptable results that help distinguish it from what is currently already available. For example, line 57 refers to the CaImAn pipeline having near-human efficiency (Figures 3-5 and Tables 1 and 2 of the CaImAn paper), but no specific examples for MPS performance benchmarks are made. Figure 15 of the Minian paper provides other examples of how to show this.

Considering one of the main benefits of MPS is its low memory demand and ability to run on unsophisticated hardware, the authors should include a figure that shows how processing times and memory usage scale with dataset sizes (FOV, number of frames and/or neurons, sparsity of cells) and differing pipelines. Figure 8 of the CaImAn paper and Figure 18 of the Minian paper show this quite nicely. Table 1 currently references how "traditional approaches" differ methodologically from MPS innovations, but runtime comparisons on identical datasets processed through MPS, CaImAn, Minian, or CaliAli would be necessary to substantiate performance claims of MPS being "10-20X faster". Additionally, while the paper does mention the type of hardware used by the experimenters, a table with a full breakdown of components may be useful for reproducibility. As well as the minimum requirements for smooth processing.

The current datasets used for validating MPS are not described in the manuscript. The manuscript appears to have 28 sessions of calcium imaging, but it is unclear if this is a single cohort or even animal, or whether these data are all from the same brain region. Importantly, the generalizability of parameter choices and performance could vary for others based on brain region differences, use of alternative calcium indicators (anything other than GCaMP8f used in the paper), etc. This leads to another limitation of the paper in its current form. While MPS is aimed at eliminating the need to code, users should not be expected to blindly trust default or suggested parameter selections. Instead, users need guidance on what each modifiable parameter does to their data and how each step analysis output should be interpreted. Perhaps including a tutorial with sample test data for parameter investigation and exploration, like many other existing pipelines do, is warranted. This would also increase the transparency and reproducibility of this work.

Currently, the documentation and FAQ website linked to MPS installation does not do an adequate job of describing parameters or their optimization. The main GitHub repository does contain better stepwise explanations, but there needs to be a centralized location for all this information. Additionally, a lack of documentation on the graphs created by each analysis step makes it hard for a true novice to interpret whether their own data is appropriately optimized for the pipeline. Greater detail on this would greatly improve the quality and impact of MPS.

Reviewer #2 (Public review):

Summary:

In this work, the authors investigate the role of fluid flow in shaping the colony size of a freshwater cyanobacterium Microcystis. To do so, they have created a novel assay by combining a rheometer with a bright field microscope. This allows them to exert precise shear forces on cyanobacterial cultures and field samples, and then quantify the effect of these shear forces on the colony size distribution. Shear force can affect the colony size in two ways: reducing size by fragmentation and increasing size by aggregation. They find limited aggregation at low shear rates, but high shear forces can create erosion-type fragmentation: colonies do not break in large pieces, but many small colonies are sheared off the large colonies. Overall, bacterial colonies from field samples seem to be more inert to shear than laboratory cultures, which the authors explain in terms of enhanced intercellular adhesion mediated by secreted polysaccharides.

Strengths:

-This study is timely, as cyanobacterial blooms are an increasing problem in freshwater lakes. They are expected to increase in frequency and severeness because of rising temperatures, and it is worthwhile learning how these blooms are formed. More generally, how physical aspects such as flow and shear influence colony formation is often overlooked, at least in part because of experimental challenges. Therefore, the method developed by the authors is useful and innovative, and I expect applications beyond the presented system here.

-A strong feature of this paper is the highly quantitative approach, combining theory with experiments, and the combination of laboratory experiments and field samples.

Weaknesses:

This study has no major weaknesses. Although the initial part of the introduction seems to imply that fluid flow is the predominant factor in shaping cyanobacterial colony (de)formation, the ensuing discussion is sufficiently nuanced for the reader to understand that the multicellular lifestyle of cyanobacterium Microcystis is shaped by multiple effects, that include bacterial behavior (e.g. which and how much EPS is produced), environmental variables that control cellular aggregation or adhesion and, indeed, fluid flow.

Joint Public Review:

Summary:

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Comments from the editors on the latest version:

In the latest communication, the authors were asked to (i) justify their selection of metrics (i.e. why these specific five behavioural metrics were chosen from the many recorded), (ii) discuss the variation in ICCs, and (iii) in light of this variation and the reliance on a few selected behavioural parameters, tone down the general claim so as not to overstate that individuality persists across all behaviours.

We note that the justification for choosing the five metrics and the discussion of ICC variation are purely qualitative, and, despite the edits, the manuscript continues to frame individual behaviours as broadly stable.

Reviewer #1 (Public review):

Summary:

The authors present a compelling case for the necessity of age-specific templates in functional hyperalignment. Given that the brain undergoes substantial developmental, structural, and functional changes across the lifespan, a 'one-size-fits-all' canonical template is often insufficient. This study effectively demonstrates that incorporating age-congruent features significantly enhances the performance and sensitivity of hyperalignment models. By validating these findings across two independent datasets (Cam-CAN and DLBS), the paper provides robust evidence that accounting for age-related functional organization is a critical prerequisite for accurate functional alignment in lifespan research.

Strengths:

(1) The authors used three metrics to evaluate performance. Across all metrics, they found that age-congruent templates outperformed age-incongruent templates, suggesting that age-specific templates can improve alignment.

(2) These findings highlight the superiority of age-congruent templates for hyperalignment. This work underscores the importance of age-matching in cross-subject functional mapping and represents a vital step forward for the methodology.

Weaknesses:

(1) Participant Demographics and Group Separation:

The study defines the 'older' cohort as 65-90 years and the 'younger' cohort as 18-45 years. While this 20-year gap (ages 46-64) effectively maximizes the contrast between groups, the results in Figure 4a suggest that the predicted individualized connectomes follow a continuous distribution. Given this continuity, could the authors provide the average median trends for Figures 2a and 2b to illustrate how the model behaves across the missing age range?

(2) Request for Implementation:

I have been unable to locate the source code associated with this publication. Could the authors please provide a link to the repository or clarify if the implementation is available for reproduction?

(3) Analysis of Prediction Performance and Distribution:

While Figures 3b and 5b clearly demonstrate that the congruent template improves correlation, Figure 4a shows a distinct shift in the scatter distribution. Could the authors provide a detailed explanation of the prediction performance metrics used? Specifically, I would like to understand how the underlying method accounts for the distribution differences observed when applying the congruent template.

Reviewer #2 (Public review):

Summary:

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

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

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

Strengths:

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

Weaknesses:

While the authors appropriately highlight the potential applications of this result (e.g., to different clinical statuses), it is not apparent how to appropriately extend this methodology to many common experimental paradigms. For example, in case-control studies (where researchers are interested in comparing clinical and non-clinical participants) the use of two different functional templates may complicate rather than ease analyses. Providing this as a potential limitation of the current template construction method, or providing recommendations to researchers interested in comparing across groups, would help to increase the impact of this work.

Reviewer #1 (Public review):

Summary:

The manuscript examines the factors that restrict the induction of IL-17-producing T cells during Mycobacterium tuberculosis (Mtb) infection. The authors show that neither the infectious route nor the duration of infection is responsible. But they do show that mice that lack the Th1-defining transcription factor, a finding consistent with prior reports in the field of immunology. They also show that 2 highly attenuated Mtb mutants in ESX-1 and PDIM, two well-known Mtb virulence factors, do induce IL-17-producing T cells. In contrast, Mtb mutants in mmpl4 are also similarly attenuated, but do not induce IL-17-producing T cells, suggesting that this property is not simply a result of attenuation but due to specific properties of ESX-1 and PDIM-deficient mutants.

Strengths:

(1) It is interesting that mice infected with ESX-1 and PDIM mutants have increased induction of Th17 cells.

(2) The data are solid and convincing throughout.

Weaknesses:

There are two main criticisms:

(1) It is not clear how much the factors uncovered here are true beyond B6 mice. B6 mice, compared to humans, are known to be very Th1-skewed, and Tbet is a strong inhibitor of Th17-specific T cells. Many people make IL-17-producing T cells in response to Mtb infection.

(2) Very few novel insights are mechanistically revealed about how Th17 induction is restricted by Mtb. Tbet induction is known to restrict Th17 development, and this is a T-cell intrinsic mechanism. In contrast, the IL-23 association revealed seems to be extrinsic to T cells and to act on T cells. How, if at all, are these factors related to each other in restricting Th17 induction? Also, the conclusion that it is not a result of attenuation is not completely convincing.

Other points:

(1) The authors show that mice infected with a deficiency in ESX-1 have more IL-17-producing CD4 T cells in response to stimulation with an ESAT-6 peptide pool (Figure 3B). Because ESAT-6 is encoded by ESX-1, why do mice infected with this Mtb mutant have any ESAT-6-specific T cells? Is it an incomplete knockdown?

(2) The manuscript states, "Under the conditions where Th17s are highly induced, mice infected with either ΔESX-1 or PDIM lacking Mtb, the Il17a-/- mice had ~3-5 fold higher CFU than WT mice (Figures 3F-G). These results indicate that the induction of Th17s is not dependent on the attenuation of Mtb in general, but instead Mtb utilizes ESX-1 and PDIM to suppress the induction of a Th17 response that enhances protection against Mtb infection." I don't think the last sentence is necessarily true. I can imagine a scenario in which the induction of the Th17s is, in fact, due to the attenuation, and the Th17 induction still contributes to protection.

(3) ESX-1, PDIM, and mmpl4 mutants all have similarly reduced CFUs in the lung, but what about the LN? The bacterial burden in the LN may be more important for regulating T-bet, IL-23, and Th17 differentiation, since the LN is where T cell priming occurs, than the CFU in the lung. Perhaps ESX-1 and PDIM mutants have reduced CFU in the LN, but mmpl4 does not. This difference in LN burdens may be the primary driver of Th17 priming, as high avidity interactions are thought to be an important driver of T-bet induction.

(4) Do LN cDC1 and high levels of IL-12 p35 in mice infected with the mmpl4 mutant? Likewise, LN cDC2's express low levels of IL-12 p19 (akin to those infected with WT Mtb)? If these observations for ESX-1 and PDIM mutants are mechanistically linked to the increased numbers of Th17 cells, then you would expect mice infected with mmpl4 mutants to be more like those infected with WT Mtb than those infected with ESX-1 and PDIM mutants.

(5) ESX-1 and PDIM are very different virulence factors - a protein secretory pathway and cell wall lipid, respectively? Mechanistically, how would mutants in these pathways give very similar outcomes regarding Th17 cells unless it was simply as an aspect of their attenuation? Perhaps, mmpl4 mutants simply differ in some aspects of their attenuation, such as bacterial burdens in LNs, or their interaction with cDCs?

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors tackle an important question of why IL-17 production and TH17 responses are lower than expected during Mtb infection. The authors identify an axis of cross-regulation between TH1 and TH17 cells and provide data to support roles for Mtb virulence factors ESX1 and PDIM in promoting TH1 responses and/or suppressing TH17 responses.

Strengths:

The strengths include the significance of the work, the combination of host and Mtb genetic models to dissect the mechanistic basis for regulation of IL-17 production from T cells during infection, and the rigor of the experiments. There are a number of exciting findings from the work, including the cross-talk between T cell responses and the impact of ESX1 and PDIM on these responses.

Weaknesses:

The following conclusions and interpretations should be revisited, rephrased, and re-evaluated:

(1) The manuscript neglects to analyze T cell responses in the dLN, which is the critical site where these responses are initiated (only DC cytokine production is measured in the dLN). The differences in the lungs could reflect trafficking of T cells to the lungs, local lung T cell responses, or durability of the T cell responses in the lungs. The authors state in the last results section that "These results indicate that the ESX-1 and PDIM virulence factors impact naïve T cell differentiation at the draining mediastinal lymph node..." but T cell responses are never measured in the dLN.

(2) Figure 2: The authors state that "Importantly, IFN-γ deficient mice did not exhibit elevated levels of IL-17A producing CD4 T cells demonstrating that IFN-γ production is not the mechanism by which Th1 T cells limit a Th17 response during Mtb infection", but the difference is significantly different and even more obvious in Panel B. In fact, if the Panel D y-axis was on a log scale, the Ifng-/- would likely look more like Tbet-/- than WT. Based on this data, it seems like IFNg is having an effect and should not be completely discounted. Does the deletion of Ifng affect the number of Tbet+ T cells?

In addition, the deletion of Tbet results in an increased number of IFNg+IL-17+ double positive T cells (Figure 2B), in addition to a sizable IFNg single positive T cell population maintained in the Tbet-/- mice (10x the negative control of Ifng-/-). Is this why Tbet deletion is not as severe as Ifng deletion, because T cells are still making IFNg?

Along these lines, the statement in the text that, "Tbet-/-Il17a-/- mice completely lacked both IFN-γ producing...." T cells is not supported by the data in Figure 2C. Tbet-/-Il17a-/- mice look to have more gamma-producing T cells than Tbet-/- mice (which is already 10x the negative control of Ifng-/- in panel 2B if one includes the gamma single positive and IFNg/IL-17 double positive).

(3) In the Results sections describing Figures 3, 4, and 5, the authors equate IL-17 production by T cells with TH17 responses and IFNg expression with TH1, but Tbet and RORgt expression in the T cells should be measured to make conclusions about TH1 and TH17. Or the authors can rephrase their findings to specifically state the observations as IFNg or IL-17 expressing CD4+ T cells.

(4) Conceptually, do the authors think that ESX1/PDIM promotes TH1 responses and this blocks TH17 or are ESX1/PDIM blocking TH17 responses directly, allowing for increased TH1 responses? It would be helpful to clarify the model in this regard, describe how the data supports one model or the other, and then make sure the language is consistent throughout. Can these effects on T cell responses be tested and recapitulated in vitro using infected APC and T cell co-cultures?

Reviewer #3 (Public review):

Summary:

The manuscript by Zilinskas et al seeks to understand the mechanisms underlying the ability of Mtb to suppress Th17 differentiation. As Th17 responses are needed for protective immunity against TB, this is an important topic of investigation. They use Mtb mutants that lack eccC1 (from the ESX-1 locus) and fadD28 (encoding PDIM) and implicate a Tbet-dependent pathway by which Mtb modulates Th17 differentiation. The mechanism by which ESX-1/PDIM function to impact Th17 differentiation is, however, unclear, which limits the novelty of the results.

Strengths:

Understanding how Mtb limits Th17 differentiation has implications for vaccine development. Comparative study of KO mice and Mtb mutants is a strength.

Weaknesses:

(1) The authors should acknowledge and reference key findings from the literature that have identified suppression of Th17 differentiation as an Mtb virulence mechanism, e.g., the role of the Hip1 protease and CD40 signaling (Madan-Lala JI 2014, Sia Plos Path 2017, Enriquez iScience 2022) and Khader JI 2005, showing the requirement of IL-23 for Th17 responses in vivo in a TB mouse model.

(2) Addressing several questions related to the Tbet KO mouse experiments would strengthen the study. Do the Tbet KO mice have elevated IL-4/5/13 (which has been previously reported in non-TB studies) in addition to IL-17? The lack of Th17 cells in the IFNg KO compared to the Tbet KO may be due to a difference in timing, since only 3-week data are shown; earlier and later time points would provide better interpretation. The authors do not present any data on neutrophil infiltration in WT vs Tbet KO vs IFNg KO mice. Since IL-17 is known to be important for recruiting neutrophils to the lung, data on neutrophils are important for clarifying the mechanism for the CFU outcomes.

(3) While IL-23 is important for sustaining IL-17 production, IL-6, TGF-b and/or IL-1β are necessary for Th17 polarization. What were the levels of these cytokines in DCs in the lung? (Figure 5). Additionally, Tbet-deficient DCs exhibit impaired activation of antigen-specific Th1 cells and have reduced IL-12 production. Given the data showing higher IL-17 levels in Tbet KO mice, the authors should provide information on the DC phenotype (IL-23, IL-6, etc.) in the Tbet KO experiments.

(4) The mechanism by which ESX-1/PDIM function to impact Th17 differentiation is not clear. While data showing a role for ESX-1 and PDIMs in inhibiting Th17 responses is interesting, there is no insight into the potential mechanism of action. Figure 3 showing reduction in IFNg+ CD4 T cells after infection with eccC1 and fadD28 mutants suggests that this outcome is due to a lower bacterial load relative to WT Mtb at the 3-week time point. Since IFNg is known to suppress IL-17, the higher levels of Th17 cells could be due to the reduction in IFNg due to the attenuated growth of the mutants. Additionally, what was the level of Type I IFNs elicited by these mutants?

(5) Since macrophages have been implicated in the reduced cytokines seen in the ESX-1 mutant, IL-23 and other cytokine data on lung macrophages would complement the DC data.

(6) Figure 5. There are many fewer DCs overall in the eccC1 and fadD28 mutant groups, which could account for the increased % IL-23p19 in DCs (5D). What were the levels of IL-23 in DC1s?

Reviewer #1 (Public review):

Summary:

In this study, the authors examine the effect of Chlorpyrifos (CPF) exposure on zebrafish social development. They expose larval zebrafish to CPF (0 - 3 dpf), and report social deficits at juvenile stages. They show that the gut microbial metabolite butyrate can rescue these social deficits, proposing that butyrate acts as a histone deacetylase (HDAC) inhibitor, given that inhibition of some HDACs can also rescue social deficits. They also show that CPF changes neuronal gene expression, and butyrate partially rescues these changes. Finally, they demonstrate changes in gut microbiome and metabolome composition, pointing to potential modulation of nitrogen metabolism pathways. They then hypothesise that NO can modulate HDAC activity and attempt to link the NO pathway to social behavior.

Strengths:

The authors demonstrate an interesting link between early Chlorpyrifos (CPF) exposure and later-life social deficits, such as changes in neuronal gene expression, including some autism-related genes, and provide solid evidence that butyrate and epigenetic modulation (histone deacetylase inhibition) may be involved.

They also comprehensively characterise the microbiome and metabolome of CPF-exposed zebrafish, providing a useful resource for further investigation into its gut-brain mechanisms.

They are cautious in framing some of their conclusions as a hypothesis and provide some suggestions for future analyses.

Weaknesses:

The claim that butyrate's effects on CPF-induced social deficits and neuron activity changes are mediated by histone deacetylase inhibition is lacking some additional controls and, hence, is not completely supported.

Details on the social behavior assay performed and other potential morphological or behavioral changes were not provided.

Claims on the mechanism of action of CPF are inconclusive. The causal role of the gut microbiome is not established, especially since gut microbial dysbiosis may also be a downstream consequence of direct effects of CPF on the host, such as changes in host gut gene expression. Evidence for the role of nitrogen metabolism is also incomplete, and the authors have not discussed or ruled out the potential alternative mechanism of reduced butyrate production due to gut microbiome changes.

Reviewer #2 (Public review):

Summary:

This paper by Diaz et al. uses the zebrafish model to examine how early embryonic exposure to Chlorpyrifos (CPF), a widely used organophosphate pesticide, induces social behavior deficits later in life. This paper combined behavioral testing, pharmaceutical treatment, genetic manipulation, and multi-omics to test the hypothesis that early CPF increases the abundance of denitrifying bacteria, Pseudomonas, which, in turn, enhances nitric oxide production and induces selective inhibition of HDAC8 and abnormal gene expression in the brain.

Strengths:

(1) The observation that early embryonic CPF exposure causes behavior deficits in juvenile zebrafish is very intriguing. It is especially exciting to see that CPF-induced behavior deficits can be reversed by overnight treatment with butyrate or HDAC1 inhibitors in juvenile zebrafish. In humans, CPF exposure during pregnancy causes brain abnormalities and neurological disorders such as Autism. Though it is far away from the zebrafish experimental study to human application, the experimental effects reported in the paper are still quite thought-provoking.

(2) The authors performed RNA sequencing experiments on control zebrafish, CPF-exposed zebrafish, and CPF-exposed zebrafish that were treated with Butyrate. The data not only showed large-scale transcriptomic changes in the juvenile zebrafish brain in response to embryonic CPF exposure but also showed that many CPF-induced genetic alterations can be alleviated by butyrate exposure later in life.

(3) The authors also performed untargeted metabolomics on zebrafish gut and metagenomic analysis in zebrafish feces samples. The results are interesting and support the conclusion that increased Intestinal Nitric oxide metabolism and the abundance of denitrifying bacteria, such as Pseudomonas, are associated with CPF exposure.

(4) The large datasets presented in the paper will be useful to other researchers interested in understanding how CPF or butyrate alters brain and gut function. It might be useful to generate new hypotheses to power other research lines.

(5) The social preferences, behavior testing, and experimental paradigm used by the paper may also be used by other researchers to investigate the interaction among gene, environmental factors, and brain function.

Weaknesses:

(1) The presented link between gut microbiome and CPF-induced behavior and genetic alteration is an association, but not causation. Although the research data align with the hypothesis, the hypothesis is not fully supported or tested by the data presented in the paper in the current state.

(2) The authors performed several large omic studies. However, some of the presented analyses are relatively simple and incomplete. For example, the authors performed shotgun metagenomic analysis on zebrafish feces. However, the paper only displayed the bacterial taxa differences. Are there any differences in bacterial genetic pathways, especially the pathways associated with microbial nitrogen metabolism? What is the alpha and beta diversity looking like when comparing different experimental groups?

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate how the anterior claustrum may integrate temporally separated task-relevant signals to guide behavior in a delayed escape paradigm. Because in vivo neural recordings from claustrum during this task are extremely limited - comprising single-trial data with small neuronal samples - the authors adopt a modeling-driven approach. They train recurrent neural networks (RNNs) using only behavioral data (escape latency) to reproduce task performance and then analyze the internal dynamics of the trained networks. Within these networks, they identify a subset of units whose activity exhibits persistent responses and strong correlations with behavior, which the authors label as "claustrum-like." Using dimensionality reduction, decoding, and information-theoretic analyses, they argue that these units dynamically integrate conditioned stimulus (CS) and door-opening signals via nonlinear, trajectory-based population dynamics rather than fixed-point attractor states.

To bridge model predictions and biology, the authors complement the modeling with in vitro slice experiments demonstrating recurrent excitatory connectivity and prolonged activity in the anterior claustrum that depends on glutamatergic transmission. They further compare latent neural trajectories derived from previously published in vivo claustrum recordings to those observed in the RNN, reporting qualitative similarities. Based on these results, the authors propose that the claustrum implements temporal signal integration through recurrent excitatory circuitry and dynamic population trajectories, potentially supporting broader theories of integrative brain function.

Strengths:

This study addresses an important and challenging problem: how to infer population-level computation in a brain structure for which in vivo data are sparse and experimentally constrained. The authors are commendably transparent about these limitations and seek to overcome them through a principled modeling framework. The integration of behavioral modeling, RNN analysis, and slice electrophysiology is ambitious and technically sophisticated.

Several aspects stand out as strengths. First, the behavioral RNN is carefully trained and interrogated using a rich set of modern analytical tools, including cross-temporal decoding, trajectory analysis, and partial information decomposition, providing multiple complementary views of network dynamics. Second, the slice experiments convincingly demonstrate recurrent excitatory connectivity in the anterior claustrum, lending biological plausibility to the model's reliance on recurrent dynamics. Third, the manuscript is clearly written, logically organized, and conceptually engaging, and it offers a coherent mechanistic hypothesis that could guide future large-scale recording experiments.

Importantly, the work has significant heuristic value: rather than merely fitting data, it attempts to generate testable computational ideas about claustral function in a regime where direct empirical access is currently limited.

Weaknesses:

Despite these strengths, the manuscript suffers from a recurring and substantial conceptual issue: systematic over-interpretation of model-data correspondence. While the modeling results are potentially insightful, the extent to which they are presented as recapitulating real claustral neural mechanisms goes beyond what the available data can support.

A fundamental limitation is that the RNN is trained solely on behavioral output, without being constrained by neural data at either single-unit or population levels. As a result, the internal network dynamics are underdetermined and non-unique. Many distinct internal solutions could plausibly generate identical behavior. However, the manuscript frequently treats the specific internal solution discovered in the RNN as if it were a close approximation of the actual claustrum circuit.

This issue is compounded by the sparse nature of the in vivo data used for comparison. The GPFA-based trajectory analyses rely on pseudo-populations and single-trial recordings, yet are interpreted as evidence for robust population-level dynamics. Because neurons were not recorded simultaneously, the inferred trajectories necessarily lack true population covariance and shared trial-to-trial variability, limiting their interpretability as genuine population dynamics. Similarly, conclusions about trajectory-based versus attractor-based computation are drawn almost exclusively from model analyses and then generalized to the biological system.

Overall, while the modeling framework is appropriate as a hypothesis-generating tool, the manuscript repeatedly crosses the line from proposing plausible mechanisms to asserting explanatory or even causal equivalence between the model and the brain. This undermines the otherwise strong contributions of the work.

Below are several specific points that warrant further clarification or revision:

(1) Tone of model-data correspondence

Numerous statements describe the RNN as "closely mimicking," "recapitulating," or being "nearly identical" to claustral neural dynamics, sometimes extending to claims about causal relationships between neural activity and behavior. Given that neural data were not used to train the model, and that only a small subset of trained networks showed the reported dynamics, these statements should be substantially softened throughout the manuscript. The RNN should be framed as providing one possible computational realization consistent with existing data, not as a close instantiation of the biological circuit

(2) Non-uniqueness of RNN solutions

The fact that only a small fraction of trained networks exhibited "claustrum-like" clusters deserves deeper discussion. This observation raises the possibility that the identified solution is fragile or highly specific rather than canonical. The authors should explicitly discuss the non-uniqueness of internal solutions in behavior-trained RNNs, including the range of alternative network dynamics that can reproduce the same behavior. In particular, it should be clarified why the specific network exhibiting "claustrum-like" clusters is informative about claustral computation, rather than representing one arbitrary solution among many.

(3) GPFA trajectory comparisons

The qualitative similarity between RNN trajectories and GPFA-derived trajectories from sparse in vivo data is interesting but insufficient to support claims of robustness or population-level structure. Statements suggesting that these patterns are unlikely to arise from noise or random fluctuations are not justified, given the single-trial, pseudo-population nature of the data. Either additional quantitative controls should be added, or the interpretation should be substantially tempered.

(4) Scope of functional claims

The discussion connecting the findings to broad theories of claustral function, global workspace, or consciousness extends well beyond the data presented. These speculative links should be clearly labeled as such and significantly reduced in strength and prominence.

(5) Comment on Conceptual Interpretation of the Behavioral Paradigm:

The manuscript repeatedly describes the delayed escape task as an "inference-based behavioral paradigm" and states that animals "infer that a value-neutral alternative space is likely to be safer" when the CS is presented in a novel environment. While I appreciate that the US-CS association was established in a different context and that the CS is then presented in a new environment, I am not convinced that the current behavioral evidence uniquely supports an inference interpretation.

First, it is not clear that this task is widely recognized in the literature as a canonical inference task, in the sense of, for example, sensory preconditioning, transitive inference, or model-based inference paradigms. Rather, the observed effect-that CS animals escape faster to a neutral compartment than neutral-CS controls-can be parsimoniously interpreted in terms of generalized threat value, heightened fear/anxiety, or a bias toward avoidance/escape under elevated threat, without requiring an explicit inferential step about the specific safety of the alternative compartment. The fact that no prior training is needed is compatible with flexible generalization, but does not by itself demonstrate inference in a more formal computational sense.

Second, the inference claim becomes central to the manuscript's conceptual framing (e.g., the idea that rsCla supports "inference-based escape"), yet the behavioral analyses presented here and in the cited prior work do not clearly rule out simpler accounts. Clarifying this distinction would help avoid overstating both the inferential nature of the behavior and the specific role of rsCla and the RNN's "claustrum-like" cluster in supporting inference per se, as opposed to more general integration of threat-related signals with an opportunity for escape.

Overall Assessment:

This manuscript presents an interesting and potentially valuable modeling-based framework for thinking about temporal integration in the claustrum, supported by solid slice physiology. However, in its current form, it overstates the degree to which the proposed RNN dynamics reflect actual claustral neural mechanisms. With substantial revision - especially a more cautious interpretation of model-data similarity and a clearer articulation of modeling limitations - the study could make a meaningful contribution as a hypothesis-generating work rather than a definitive mechanistic account.

Reviewer #2 (Public review):

This manuscript reports the behavior of a computational model of rat claustral neurons during the performance of a behavioral task known as the delayed escape task (in this reviewer's understanding, this behavioral task was created and implemented by this group only). These authors have argued in a prior manuscript (Han et al.) that a group of neurons located "rostral to striatum" is part of the claustrum. The group names the region the "rostral to striatum claustrum." Additionally, in the Han et al. paper, the authors argue that these cells are responsible for maintaining a signal that lasts through the delay period.

The main findings of the current paper are:

(1) The authors have built a model network that was trained to show firing similar to what was reported for rats in their prior paper.

(2) The authors' analysis of model behavior is used to suggest that the model network recapitulates biological activity, including the existence of a cluster of cells mainly responsible for the delay period firing.

(3) The authors offer evidence from patch clamp recordings for excitatory interconnections among claustral neurons that are an essential feature of the model network.

A major value of the computational network is that "trials" of the network can be performed. In experiments on animals, only single trials can be used.

Concerns:

(1) This paper is based on behavioral results and neural recordings from their prior paper (Han et al.), but data, e.g., in Figure 1, are not clearly identified as new or as coming from that source. Figure 1A, for example, appears to be taken directly from Han et al. No methods are given in this manuscript for the behavioral testing or the in vivo electrophysiology.

(2) Many other details are unclear. Examples include model training, the weight matrices and how these changed with training (p. 13), equations 2 and 3 (p. 13), the sources for the constants in the equations (p. 14), the methods (anesthesia, stereotaxic coordinates, injection specifics and details for "sparse expression") for the ChrimsonR injections.

(3) The explorations of model behavior are a catalog of everything tried rather than an organized demonstration of what the model can and cannot do. The figures could be reduced in number to emphasize the key comparisons of the different clusters and the model's behavior under different conditions, intended to "test" the model.

(4) On page 6, the E-E connectivity is argued from Shelton et al. (2025) and against Kim et al. (2016), but ignores Orman (2015), which, to this reviewer's knowledge, was the first to demonstrate such connectivity, including the long-duration events and impact of planes of section.

(5) Whereas the authors are entitled to their own opinion of prior work (references 3-8), it is inappropriate to misrepresent prior work as only demonstrating a "limited function" of claustum. Additional papers by Mathur's group and Citri's group are ignored.

In summary, the authors have made a computational model that recapitulates the firing of a subset of potentially claustral neurons during a particular behavioral task (delayed escape is certainly not the only behavior that involves claustrum - see e.g., attention, salience, sleep). If the conclusion is that excitatory claustral cells must be connected to other excitatory claustral cells, such a conclusion is not new, and the electrophysiological E-E metrics are not well quantified (e.g., connectivity frequency, strength of connection). If the model is intended to predict how the claustrum might accomplish any other task, there is insufficient detail to evaluate the model beyond the evidence that the model creates a subset of cells that can sustain firing during the delay period in the delayed escape task.

All relevant work must be appropriately cited throughout the manuscript.

Reviewer #2 (Public review):

Summary:

Missed diagnosis of myocardial ischemia (MI) is more common in women, and treatment is typically less aggressive. This diagnosis stems from the fact that women's ECGs commonly exhibit 12 lead ECG biomarkers that are less likely to fall within the traditional diagnostic criteria. Namely, women have shorter QRS durations and lower ST junction and T wave amplitudes, but longer QT intervals, than men. To study the impact, this study aims to quantify sex differences in heart-torso anatomy and ECG biomarkers, as well as their relative associations, in both pre- and post-MI populations. A novel computational pipeline was constructed to generate torso-ventricular geometries from cardiac magnetic resonance imaging. The pipeline was used to build models for 425 post-myocardial infarction subjects and 1051 healthy controls from UK Biobank clinical images to generate the population.

This study has a strength in that it utilizes a large patient population from the UK Biobank (425 post-MI and 1051 healthy controls) to analyze sex-based differences. The computational pipeline is state-of-the-art for constructing torso-ventricular geometries from cardiac MR and is clinically viable. It draws on novel machine learning techniques for segmentation, contour extraction, and shape modeling. This pipeline is publicly available and can help in the large-scale generation of anatomies for other studies. The study then deploys a linear regression model to relate the level of influence of various factors to ECG-based changes. This allows computation of various anatomical factors (torso volume, cavity volume, etc), and subsequent linear regression analysis on how these factors are altered before and after MI from the 12-lead ECG.

A major weakness is that a linear additive model may not adequately capture how anatomy and electrophysiology interact. Myocardial infarction dramatically alters both anatomy and electrophysiology in ways that are not easily separable and could be considered non-linear. As such, the electrophysiological factors in the model may still include factors that have an anatomical basis (i.e. the formation of scar) that were not accounted for during model generation. However, the technique remains useful for dissecting large factors beyond anatomy, as demonstrated in this study.

Reviewer #1 (Public review):

Summary:

The electrocardiogram (ECG) is routinely used to diagnose and assess cardiovascular risk. However, its interpretation can be complicated by sex-based and anatomical variations in heart and torso structure. To quantify these relationships, Dr. Smith and colleagues developed computational tools to automatically reconstruct 3D heart and torso anatomies from UK Biobank data. Their regression analysis identified key sex differences in anatomical parameters and their associations with ECG features, particularly post-myocardial infarction (MI). This work provides valuable quantitative insights into how sex and anatomy influence ECG metrics, potentially improving future ECG interpretation protocols by accounting for these factors.

Strengths:

• The study introduces an automated pipeline to reconstruct heart and torso anatomies from a large cohort (1,476 subjects, including healthy and post-MI individuals). • The 3-stage reconstruction achieved high accuracy (validated via Dice coefficient and error distances). • Extracted anatomical features enabled novel analyses of disease-dependent relationships between sex, anatomy, and ECG metrics. • Open-source code for the pipeline and analyses enhances reproducibility.

Weaknesses:

• The study attributes residual ECG differences to sex/MI status after controlling for anatomical variables. However, regression model errors could distort these estimates. A rigorous evaluation of potential deviations (e.g., variance inflation factors or alternative methods like ridge regression) would strengthen the conclusions.

Reviewer #1 (Public review):

Summary:

The electrocardiogram (ECG) is routinely used to diagnose and assess cardiovascular risk. However, its interpretation can be complicated by sex-based and anatomical variations in heart and torso structure. To quantify these relationships, Dr. Smith and colleagues developed computational tools to automatically reconstruct 3D heart and torso anatomies from UK Biobank data. Their regression analysis identified key sex differences in anatomical parameters and their associations with ECG features, particularly post-myocardial infarction (MI). This work provides valuable quantitative insights into how sex and anatomy influence ECG metrics, potentially improving future ECG interpretation protocols by accounting for these factors.

Strengths:

(1) The study introduces an automated pipeline to reconstruct heart and torso anatomies from a large cohort (1,476 subjects, including healthy and post-MI individuals).

(2) The 3-stage reconstruction achieved high accuracy (validated via Dice coefficient and error distances).

(3) Extracted anatomical features enabled novel analyses of disease-dependent relationships between sex, anatomy, and ECG metrics.

(4) Open-source code for the pipeline and analyses enhances reproducibility.

Weaknesses:

(1) The linear regression approach, while useful, may not fully address collinearity among parameters (e.g., cardiac size, torso volume, heart position). Although left ventricular mass or cavity volume was selected to mitigate collinearity, other parameters (e.g., heart center coordinates) could still introduce bias.

(2) The study attributes residual ECG differences to sex/MI status after controlling for anatomical variables. However, regression model errors could distort these estimates. A rigorous evaluation of potential deviations (e.g., variance inflation factors or alternative methods like ridge regression) would strengthen the conclusions.

(3) The manuscript's highly quantitative presentation may hinder readability. Simplifying technical descriptions and improving figure clarity (e.g., separating superimposed bar plots in Figures 2-4) would aid comprehension.

(4) Given established sex differences in QTc intervals, applying the same analytical framework to explore QTc's dependence on sex and anatomy could have provided additional clinically relevant insights.

Reviewer #2 (Public review):

Summary:

Missed diagnosis of myocardial ischemia (MI) is more common in women, and treatment is typically less aggressive. This diagnosis stems from the fact that women's ECGs commonly exhibit 12 lead ECG biomarkers that are less likely to fall within the traditional diagnostic criteria. Namely, women have shorter QRS durations and lower ST junction and T wave amplitudes, but longer QT intervals, than men. To study the impact, this study aims to quantify sex differences in heart-torso anatomy and ECG biomarkers, as well as their relative associations, in both pre- and post-MI populations. A novel computational pipeline was constructed to generate torso-ventricular geometries from cardiac magnetic resonance imaging. The pipeline was used to build models for 425 post-myocardial infarction subjects and 1051 healthy controls from UK Biobank clinical images to generate the population.

Strengths:

This study has a strength in that it utilizes a large patient population from the UK Biobank (425 post-MI and 1051 healthy controls) to analyze sex-based differences. The computational pipeline is state-of-the-art for constructing torso-ventricular geometries from cardiac MR and is clinically viable. It draws on novel machine learning techniques for segmentation, contour extraction, and shape modeling. This pipeline is publicly available and can help in the large-scale generation of anatomies for other studies. This allows computation of various anatomical factors (torso volume, cavity volume, etc), and subsequent regression analysis on how these factors are altered before and after MI from the 12-lead ECG.

Weaknesses:

Major weaknesses stem from the fact that, while electrophysiological factors appear to play a role across many leads, both post-MI and healthy, the electrophysiological factors are not stated or discussed. The computational modeling pipeline is validated for reconstructing torso contours; however, potential registration errors stemming from ventricular-torso construction are not addressed within the context of anatomical factors, such as the tilt and rotation of the heart. This should be discussed as the paper's claims are based on these results. Further analysis and explanation are needed to understand how these sex-specific results impact the ECG-based diagnosis of MI in men and women, as stated as the primary reason for the study at the beginning of the paper. This would provide a broader impact within the clinical community. Claims about demographics do not appear to be supported within the main manuscript but are provided in the supplements. Reformatting the paper's structure is required to efficiently and effectively present and support the findings and outcomes of this work.

Reviewer #1 (Public review):

Summary:

In this study, the authors trained rats on a "figure 8" go/no-go odor discrimination task. Six odor cues (3 rewarded and 3 non-rewarded) were presented in a fixed temporal order and arranged into two alternating sequences that partially overlap (Sequence #1: 5⁺-0^--1^--2⁺; Sequence #2: 3⁺-0^--1^--4⁺) --forming an abstract figure-8 structure of looping odor cues.

This task is particularly well-suited for probing representations of hidden states, defined here as the animal's position within the task structure beyond superficial sensory features. Although the task can be solved without explicit sequence tracking, it affords the opportunity to generalize across functionally equivalent trials (or "positions") in different sequences, allowing the authors to examine how OFC representations collapse across latent task structure.

Rats were first trained to criterion on the task and then underwent 15 days of self-administration of either intravenous cocaine (3 h/day) or sucrose. Following self-administration, electrodes were implanted in lateral OFC, and single-unit activity was recorded while rats performed the figure-8 task.

Across a series of complementary analyses, the authors report several notable findings. In control animals, lOFC neurons exhibit representational compression across corresponding positions in the two sequences. This compression is observed not only in trial/positions involving overlapping odor (e.g., Position 3 = odor 1 in sequence 1 vs sequence 2), but also in trials/positions involving distinct, sequence-specific odors (e.g., Position 4: odor 2 vs odor 4) --indicating generalization across functionally equivalent task states. Ensemble decoding confirms that sequence identity is weakly decodable at these positions, consistent with the idea that OFC representations collapse incidental differences in sensory information into a common latent or hidden state representation. In contrast, cocaine-experienced rats show persistently stronger differentiation between sequences, including at overlapping odor positions.

Strengths:

- Elegant behavioral design that affords the detection of hidden-state representations.<br /> - Sophisticated and complementary analytical approaches (single-unit activity, population decoding, and tensor component analysis).

Weaknesses:

-The number of subjects is small --can't fully rule out idiosyncratic, animal-specific effects.

Comments on revisions:

The authors have thoroughly addressed all of my previous comments. Congratulations on an excellent paper!

Reviewer #2 (Public review):

In the current study, the authors use an odor-guided sequence learning task described as a "figure 8" task to probe neuronal differences in latent state encoding within the orbitofrontal cortex after cocaine (n = 3) vs sucrose (n = 3) self-administration. The task uses six unique odors which are divided into two sequences that run in series. For both sequences, the 2nd and 3rd odors are the same and predict reward is not available at the reward port. The 1st and 4th odors are unique, and are followed by reward. Animals are well-trained before undergoing electrode implant and catheterization, and then retrained for two weeks prior to recording. The hypothesis under test is that cocaine-experienced animals will be less able to use the latent task structure to perform the task, and instead encode information about each unique sequence that is largely irrelevant. Behaviorally, both cocaine and sucrose-experienced rats show high levels of accuracy on task, with some group differences noted. When comparing reaction times and poke latencies between sequences, more variability was observed in the cocaine-treated group, implying animals treated these sequences somewhat differently. Analyses done at the single unit and ensemble level suggests that cocaine self-administration had increased the encoding of sequence-specific information, but decreased generalization across sequences. For example, the ability to decode odor position and sequence from neuronal firing in cocaine-treated animals was greater than controls. This pattern resembles that observed within the OFC of animals that had fewer training sessions. The authors then conducted tensor component analysis (TCA) to enable a more "hypothesis agnostic" evaluation of their data.

Overall, the paper is well written and the authors do a good job of explaining quite complicated analyses so that the reader can follow their reasoning. The findings are important, and the results are compelling. The introduction and discussion contextualize the experiments in the context of the literature, and explain the novelty and significance of the current findings. Specifically, the observation that cocaine self-administration impairs generalization across task sequences at the single unit level builds on previous observations of aberrant neuronal activity within the OFC in animals with a history of cocaine self-administration. These new data point to a neurophysiological mechanism that could explain why drug-seeking is so context dependent, and hard to ameliorate with therapeutic strategies that take place within a clinical setting.

The authors clearly acknowledge the major limitations of this work, namely that the sample size is restricted due to the technical challenges of performing in vivo electrophysiology recordings combined with self-administration, and that animals of only one sex were used. Importantly, the data from all rats within each group was remarkably homogeneous, increasing confidence in the conclusions drawn.

Reviewer #1 (Public review):

The manuscript "Heterozygote advantage cannot explain MHC diversity, but MHC diversity can explain heterozygote advantage" explores two topics. First, it is claimed that the recently published by Mattias Siljestam and Claus Rueffler conclusion (in the following referred to as [SR] for brevity) that heterozygote advantage explains MHC diversity does not withstand an even very slight change in ecological parameters. Second, a modified model that allows an expansion of MHC gene family shows that homozygotes outperform heterozygotes. This is an important topic and could be of potential interest to the readership of eLife if the conclusions are valid and non-trivial.

The resubmitted manuscript addresses several questions from my previous review. In particular, there is a more detailed description of how the code of Siljestam and Rueffler ([SR]) was used for the simulations and the calculation of the factor 2.7 x 10^43 that is the key to the alleged breakdown of the numerical reasoning presented by in [SR].

Yet I think that important aspects of my critique of the first statement of the manuscript about the flaws of [SR] model remain unanswered. I guess the discussion becomes rather general about the universality and robustness of various types of models to parameter changes. My point is that none of the models is totally universal. The model in [SR] is not phenomenological as none of the parameters or functional forms were derived empirically. Instead, it is a proof of principle demonstration that inevitably grossly simplifies the actual immune response. The choice of constants and functions used in Eqs. (1-5) is dictated by the mathematical convenience and works in a limited range of parameter values. It is shown in [SR] that for 3 pathogens and reasonable "virulence " \nu, the alleles branch. These conclusions are supported by the analytically derived Adaptive Dynamics branching criteria (7), which, contrary to the statement is the cover letter (" It is clear from Fig. 4 of Siljestam and Rueffler that the branching condition is far from sufficient for high MHC diversity.") is perfectly confirmed by the simulation data shown in Fig. 4.

The mathematical simplicity of the [SR] model generates various artifacts, such as the mentioned by the Author reduction of the "condition" by an enormous factor 2.7 x 10^43 and the resulting decrease in the "survival" induced by the addition of a new pathogen. This occurs at the very large value of \nu=20, whose effect is enormous due to the Gaussian form of (1), which, once again, was chosen for the mathematical convenience. In reality, a new pathogen cannot reduce the "survival" by such a factor as it would wipe out any resident population. So to compensate for such an artifact, the additional factor c_max was introduced to buffer such an excess. There is no reason to fix c_max once for an arbitrary number of pathogens, because varying c_max basically reflects the observation that a well-adapted individual must have a reasonable survival probability. At the same time, there are many ways in which the numerical simulation may break down when the survival rates become of the order of 10^(-43) instead of one, so it comes to no surprise that the diversification, predicted by the adaptive dynamics, does not readily occur in the scenario with an addition or removal of the 8th pathogen with a very high virulence \nu=20.

I have doubts that the reported breakdown of the [SR] model with fixed c_max remains observable with less extreme values of m and \nu (say, for \nu=7 and m=3 plus or minus 1 used in Fig. 3 in the manuscript).

So I still find the claim that " the phenomenon that leads to high diversity in the simulations of Siljestam and Rueffler depends on finely tuned parameter values" is not well substantiated.

Reviewer #2 (Public review):

Summary:

This study addresses the population genetic underpinnings of the extraordinary diversity of genes in the MHC, which is widespread among jawed vertebrates. This topic has been widely discussed and studied, and several hypotheses have been suggested to explain this diversity. One of them is based on the idea that heterozygote genotypes have an advantage over homozygotes. While this hypothesis lost early on support, a reason study claimed that there is good support for this idea. The current study highlights an important aspect that allows us to see results presented in the earlier published paper in a different light, changing strongly the conclusions of the earlier study, i.e., there is no support for a heterozygote advantage. This is a very important contribution to the field. Furthermore, this new study presents an alternative hypothesis to explain the maintenance of MHC diversity, which is based on the idea that gene duplications can create diversity without heterozygosity being important. This is an interesting idea, but not entirely new.

Strength:

(1) A careful re-evaluation of a published model, questioning a major assumption made by a previous study.

(2) A convincing reanalysis of a model that, in the light of the re-analysis-loses all support.

(3) A convincing suggestion for an alternative hypothesis.

Weakness:

(1) The title of the study is catchy, but it is explained only in the very end of the paper.

Reviewer #1 (Public review):

Summary:

This study examined whether infraslow fluctuations in noradrenaline and in heart rate are coupled and how they are affected by sleep transitions. The authors used the fluorescent NA biosensor GRAB-NE2m in the medial prefrontal cortex of mice to record extracellular NA while also recording EEG and EMG during sleep-wake episodes. They also analyzed previously published human data to reproduce relationships they found between sigma power and RR intervals in mice.

Strengths:

This is an impressive study with significant strengths, as it involves a rich set of data that includes not only observations of associations between heart rate and noradrenergic dynamics but also optogenetic manipulation of the locus coeruleus. Human data is presented to show parallels in the association between sigma power during sleep and phasic heart-rate bursts.

Weaknesses:

(1) Language could be clearer and more precise. As detailed below, in both the introduction and the discussion, the way the hypotheses and study objectives are described could use some revision to be more precise and accurate.

1A) In the introduction on p. 4: The overarching question is framed as "could the peripheral autonomous systems be a read-out of the central LC-NE system and thus be a biomarker of memory consolidation and LC dysfunction?" This gives the impression that the LC function would be the main influence on peripheral autonomous systems. There are, of course, many influences on peripheral autonomous systems, so it would be advisable for the authors to be more specific here about what signal(s) in particular would be predicted to be sensitive markers of LC function.

1B) In the discussion on p. 12: "In this study, we leveraged real-time measurements of mPFC NE levels and HR measurements from EMG recordings in mice to investigate the causal link between the two variables with high temporal resolution in freely moving sleeping mice, with similar inspection in humans." To test the causal link between mPFC NA levels and HR measures, the study would manipulate NA levels just in the mPFC and not elsewhere in the brain. However, in this study, the manipulation occurred in the LC, and so there would be broad cortical changes in NA levels. Thus, it could be that LC activity causes HR changes via a non-PFC pathway.

(2) Comparisons with the control condition need further development.

2A) While the authors did include a key YFP control condition, in the main text no direct statistical comparison between the closed-loop optogenetic stimulation (ChR2) condition and the YFP control condition was reported. (It was reported in Supplementary Figure 2c-d.) Instead, in the main text, the authors only reported that the effects of stimulation were significant in the closed-loop condition and not in the control. However, that is not the same as demonstrating that the two conditions significantly differed from each other, and it is the direct test that is important for the conclusions, so it seems important to include this result in the main presentation.

2B) In addition, the authors should address the issue that the pre-stimulation NE was consistently significantly lower in the YFP condition than in the ChR2 condition (see Supplementary Figure 2c), which is a potential confound.

2C) Direct comparison of the strengths of correlations shown in Figure 2h vs. Supplementary Figure 2f should be included. Currently, we see relatively weak correlations in both ChR2 and YFP conditions, and it is not clear if the relationships differ in the control. It seems they are still present in the control condition but weaker, which would contradict the apparently broad claim on p. 7 that "No such effects were present in the control condition" (it is not entirely clear whether this claim refers to all effects discussed in the figure or just a subset - this language should be clarified).

2D) Did the YFP controls vs. ChR2 animals show any differences in the number of NA states that triggered stimulation in the closed-loop system? With ChR2 animals, stimulation changes NA, which could change future triggering. In YFP animals, nothing changes NA (other than natural fluctuations), so the dynamics of stimulation timing could diverge between groups in a way that complicates interpretation. Specifically, if ChR2 stimulation raises NA and prevents future threshold crossings, ChR2 animals may end up receiving fewer subsequent stimulations than YFP animals (or a different temporal clustering). If the number or pattern of stimulation differed in two groups, it would be important to have a yoked control where matched animals get the same stimulation pattern but not triggered by their own NA.

(3) Some more discussion/explanation of the rationale for the closed-loop approach and how it influences how we should interpret the results could be useful. For instance, currently, it is not clear whether LC stimulation needs to be timed after an NA dip to yield the effects seen.

(4) The section on heart rate decelerations is hard to follow. In particular, I was not sure how to interpret Figure 3f-j. For Figure 3f, what does the middle line represent? The laser onset or the max RR value after laser onset? What is the baseline that is used to correct the values to obtain amplitudes? If it is the whole period before the maximal RR value or the laser onset, wouldn't baseline values differ significantly across conditions and so potentially account for differences seen between conditions in the reported HR decelerations? Larger HR decelerations may be seen in conditions with higher HR simply as a regression to the mean phenomenon.

(5) The findings regarding LC suppression could be further clarified.

5A) Page 8: "observed a response in NE decline" - please be more precise. Did NE decline more or less?

5B) It would be helpful to also show the correlation between NE and RR in the control (YFP) condition and whether there were any differences between YFP and Arch conditions (Figure 4e).

5C) This sentence took me multiple readings to understand - it would be helpful to rewrite to make it clearer: "indicating that, while HR generally did not respond strongly to LC suppression, the variability in RR responses was dependent on NE changes to the suppression (Figure 4e)."

5D) The two colors in Figure 4 are similar and hard to distinguish.

5E) The correlations shown in Figure 4j seem to be driven by just two of the cases. Are the effects significant when outliers are removed?

5D) Page 10: Were there any differences in memory performance between the Arch and YFP conditions?

5E) Page 10: "We found a correlation between RR responses to LC suppression and sigma power, suggesting that a stronger HR reduction response is linked to higher spindle power." It should be noted in the text that the correlation was not specific to sigma (it was also seen for theta and beta, Figure 4i).

(6) It is not clear which of the sigma power and RR interval findings do/do not exactly line up between the mice and humans. It could be helpful to have a table comparing them. For instance, was the finding in humans that pre-HRB sigma power was positively associated with slowing in heart rate after the HRB also seen in mice? Was there evidence in mice (as seen in the human sample) that sleep-dependent memory improvement was associated with pre-HRB sigma power?

(7) Page 18: It is not clear if the sex of mice was balanced across controls and optogenetics groups.

Reviewer #2 (Public review):

Summary:

The major part of this study reproduces previously published findings in both mice and humans and provides incremental analyses on these findings. In essence, the work reaffirms the presence of coordinated infraslow fluctuations in sigma power and heart rate during NREM sleep. It further confirms previous findings that coordination depends on noradrenaline-releasing neurons in the locus coeruleus. Also supporting previously published work in mice and humans, the authors describe a link between the strength of these infraslow fluctuations and memory consolidation in mice and humans.

Strengths:

The authors successfully replicate key previously reported phenomena across both mice and humans. Confirmatory studies and demonstrations of reproducibility are essential for progress in neuroscience. To maximize their value, such studies should clearly acknowledge their confirmatory nature and carefully situate what, in their view, are novel results, going beyond existing literature.

Weaknesses:

The authors' interpretation of their data needs to be revised. Many of their claims regarding the mechanistic basis of their findings and the predictive value of their correlative datasets are not supported by the available evidence.

In the present manuscript, several citations of literature on the work they reproduce lack precision or completeness, which reduces transparency and obscures how the reported findings relate to previously established results.

Reviewer #1 (Public review):

Summary:

Liao et al. performed a large-scale integrative analysis to explore the function of two cancer genes (BRCA1 and BRCA2) in lung cancer, which is one of the cancers with an extremely high mortality rate. The detailed genetic analysis demonstrated new roles of BRCA1/2 in causing the tumor microenvironment in lung cancer. In particular, the discovery of different mechanisms of BRCA1 and BRCA2 provides an essential foundation for developing drugs that target BRCA1 or BRCA2 in lung cancer therapy.

Strengths:

(1) This study leveraged large-scale genomic and transcriptomic datasets to investigate the prognostic implications of BRCA1/2 mutations in LUAD patients (~2,000 samples). The datasets range from genomics to single-cell RNA-seq to scTCR-seq.

(2) In particular, the scTCR-seq offers a powerful approach for understanding T cell diversity, clonal expansion, and antigen-specific immune responses. Leveraging these data, this study found that BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations were linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity.

(3) This study also performed a comprehensive analysis of genomic variation, gene expression, and clinical data from the TCGA program, which provides an independent validation of the findings from LUAD patients newly collected in this study.

(4) This study provides an exemplary integration analysis using both computational biology and wet bench experiments. The experimental testing in the A549 cell line further supports the robustness of the computational analysis.

(5) The findings of this study offer a comprehensive view of the molecular mechanisms underlying BRCA1 and BRCA2 mutations in LUAD. BRCA1 and BRCA2 are two well-known cancer-related genes in multiple cancers. However, their role in shaping the tumor microenvironment, particularly in lung cancer, is largely unknown.

(6) By focusing on PD-L1-negative LUAD patients, this study demonstrated the molecular mechanisms underlying resistance to immune therapy. These new insights highlight new opportunities for personalized therapeutic strategies to BRCA-driven tumors. For example, they found histone deacetylase (HDAC) inhibitors consistently downregulated 4-R genes in A549 cells.

(7) The deposition of raw single-cell sequencing (including scRNA-seq and scTCR-seq) data will provide an essential data resource for further discovery in this field.

Weaknesses:

(1) The finding of histone deacetylase (HDAC) inhibitors suggests the potential roles of epigenetic regulation in lung cancer. It would be interesting to explore epigenetic changes in LUAD patients in the future.

(2) For some methods, more detailed information is needed.

(3) There are grammar issues in the text that need to be fixed.

(2) Some text in the figures is not labeled well.

Reviewer #2 (Public review):

Summary:

This study investigates the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma using multi-omics approaches. The work highlights distinct roles of BRCA1 and BRCA2 mutations in shaping immune-related processes, and is logically structured with clearly presented analyses. However, the conclusions rely primarily on descriptive computational analyses and would benefit from additional immunological validation.

Strengths:

By integrating public datasets with in-house data, this study examines the impact of BRCA1/2 mutations on immunotherapy in lung adenocarcinoma from multiple perspectives using multi-omics approaches. The analyses are diverse in scope, with a clear overall logic and a well-organized structure.

Weaknesses:

The study is largely descriptive and would benefit from additional immunological experiments or validation using in vivo models. The fact that the BRCA1 and BRCA2 samples were each derived from a single patient also limits the robustness of the conclusions.

Reviewer #1 (Public review):

Summary:

Small molecule therapeutics for snakebite have received a lot of attention for their potential to close the gap between bite and treatment, where antivenom is not immediately available.

Strengths:

There has been a lot of focus on Africa, Asia, and India, but very little work related to neotropical regions. The authors seek to begin filling this gap in the preclinical literature. The authors use well-developed methods for preclinical assessment.

Weaknesses:

A clearer and more focused discussion of the limitations of the overall present work would be desirable (e.g. protection vs. rescue, why marimastat over prinomastat for in vivo assays when both have been through clinical trials for other indications; real-world feasibility of nafamostat, which has a half-life of 1-2 minutes compared to camostat, which has a half-life of hours). All of this could be be improved in a revision.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

Several aspects of the study design and framing reduce the confidence with which readers can translate the findings beyond the specific experimental context presented. The evidence base is strongest in controlled in-vitro settings, while the bridge to real-world effectiveness remains limited, particularly for understanding performance under conditions that better reflect delayed treatment and systemic exposure. As a result, the manuscript is best interpreted as a well-organized comparative screening study with promising signals, rather than a definitive demonstration of a broadly effective, deployable intervention.

Reviewer #3 (Public review):

In this work, the authors wanted to evaluate repurposed small molecule inhibitors for the treatment of envenomation by snakes of the Bothrops genus; one of the most medically relevant in the Americas. I believe the objectives of the research were clearly achieved, and compelling evidence for the ability of these molecules to neutralize enzymatic and toxic activities of metalloproteinases and phospholipases in all the tested venoms is provided. Furthermore, the work highlights the limited efficacy of the tested serineprotease inhibitor, suggesting a need for drug discovery campaigns to address toxicity caused by this protein family. The methods are well designed and performed, and the use of both in vitro and in vivo methodologies makes this a thorough and robust work.

These results are extremely relevant, since they take us one step further to a potential orally administered snakebite treatment. The existence of such a treatment could improve the outcomes for thousands of snakebite victims worldwide. I have a few comments and questions that I hope will be useful to the authors:

During the introduction, the authors mention that small-molecule inhibitors can neutralize the localized tissue damage via cytotoxicity of some venoms, and cite PLA2s, SVMPs and/or cytotoxic 3FTxs as the main causing agents of this pathology. I am not aware of any direct effect described by small molecule inhibitors on cytotoxic 3FTxs alone. Has this been observed at all? Or is it more likely that the small molecule inhibitors act on the enzymatic toxins only, preventing synergistic effects with 3FTxs?

I think it would be relevant to address the effects of non-enzymatic PLA2s, such as myotoxin II, which have been described in detail within Bothrops venoms. I believe there is some evidence of Varespladib also having a neutralizing effect on the myotoxicity caused by these non-enzymatic PLA2s. I suggest adding a comment about the contribution of these toxins in the discussion or in the section where PLA2 activity of the venoms is compared. In my opinion, right now it seems like these were overlooked.

Regarding Marimastat and the other MP inhibitors, are there any studies showing that they don't have an effect on endogenous MPs? I understand they have been approved for human use before, but is there any indication that they would not have an effect at the doses that would be required to treat envenomation?

Regarding the quenched fluorescence substrate used for enzymatic activity. Is there a possibility that some of the SVMPs would not act on this substrate, and therefore their activity or neutralization is not observed? Would it be relevant to test other substrates, such as gelatin, collagen, or even specific clotting factors?

Finally, could the authors comment or provide some bibliography regarding the translatability of the chicken embryo model in the context of envenomation?

Reviewer #1 (Public review):

Summary:

This study addresses a fundamental question in cognitive neuroscience regarding how the brain transitions from a reactive state of following external instructions to a proactive state of self-directed agency. The authors utilize an ambitious multimodal design by combining the spatial precision of fMRI with the temporal resolution of EEG across two independent datasets from the University of Florida and UC Davis. By applying multivariate pattern analysis, the work demonstrates that while both instructed and willed attention engage the Dorsal Attention Network, willed choices uniquely recruit a frontoparietal decision network including the dACC and anterior insula. Furthermore, the study shows that pre cue alpha oscillations can predict subsequent spontaneous choices. This provides a neural link between pre-existing brain states and intentional action, representing a significant technical effort to characterize the neural scaffolding of internal goal generation.

Strengths:

The primary strengths of this work include the integration of fMRI and EEG which allows the authors to bridge the gap between slow metabolic signals and fast oscillatory brain states. The use of two independent cohorts is a commendable effort to ensure the reproducibility of the willed attention effect, which is often a concern in small sample neuroimaging studies. Additionally, the move beyond univariate activation toward information based mapping demonstrates that the identified networks actually contain specific information about the direction of attention.

Weaknesses:

However, several critical weaknesses must be addressed to support the fundamental claims made in the manuscript. There are significant behavioral differences in performance between the two sites, specifically regarding the UC Davis cohort exhibiting slower reaction times and lower accuracy compared to the UF group. These discrepancies suggest potential differences in subject populations or experimental environments that are not currently accounted for in the neural models. The fMRI analysis lacks temporal precision because the use of beta series regression collapses the complex BOLD response into a single estimate per trial. This loss of temporal information obscures the evolution of the decision process and makes it difficult to distinguish whether the identified patterns represent a truly spontaneous choice or a slow building pre planned strategy.

Furthermore, the EEG decoding approach utilized the entire topography of electrodes rather than a biologically motivated posterior region of interest. Given that alpha mediated spatial attention is traditionally localized to parieto occipital sensors, using the full electrode set risks the inclusion of non neural artifacts such as micro saccades or muscle activity which can contaminate multivariate classifiers. The introduction of the neural efficiency metric also requires further validation as the current ratio is mathematically sensitive to small denominators in the BOLD contrast.

Crucially, the manuscript does not address the physiological implications of recruiting additional frontoparietal networks when behavioral performance remains identical across conditions. The activation of the anterior insula and dACC is frequently associated with increased autonomic arousal and effort. If the willed condition requires more extensive neural scaffolding to reach the same behavioral output as the instructed condition, it raises the question of whether this internal decision process is accompanied by changes in arousal levels. The authors should consider whether the lack of a behavioral tax is due to a compensatory increase in arousal, which could be reflected in the EEG data or pupil diameter if recorded, and potentially also in the amplitude of BOLD activity, which is being masked by the neural efficiency metric. Without an account of how the brain balances this increased computational demand without impacting behavioral performance, the functional significance of the willed attention network remains partially obscured.

Reviewer #2 (Public review):

Summary:

This manuscript combines fMRI and EEG investigations performed at two research sites to examine 'willed' or volitional visuospatial attention, as contrasted with more standard cued (or 'instructed') visuospatial attention. The primary findings are: 1) willed attention (vs. instructed attention) drives additional cortical circuitry across a broad fronto-parietal network; 2) the direction of willed attention, but not instructed attention can be decoded from the pre-cue EEG data and from MVPA analysis of the trial-level fMRI data; and 3) the subjects with high EEG decoding also exhibited high neural efficiency (i.e., high decoding with low BOLD signal change) in the fMRI data. The methods and data analysis are generally sound, and these results appear solid. On the negative side, it is not made clear how the present findings extend our understanding beyond prior published work from one of the senior authors. There are also three significant concerns regarding interpretation of the findings. One has to do with the causal interpretation of the pre-cue alpha EEG signal determining the direction of willed attention. The second concern is the degree to which the present research paradigm adequately examines 'willed attention.' The third is that the MVPA analysis is not sufficiently described, and Permutation testing needs to be done to validate these findings. Otherwise, this manuscript appears methodologically sound, but questions about interpretation may mute the potential impact.

Strengths:

The focus on willed attention attempts to move beyond some of the many limitations of standard laboratory investigations of attention.

The shared paradigm across two modalities and two research sites demonstrates solid reproducibility, even though a few minor differences are observed across sites.

Weaknesses:

(1) There are concerns about this experimental paradigm carrying the banner of Willed Attention, because the application of 'Will' appears quite modest. Yes, extra brain activity is exhibited for this condition vs. its control, but do the cognitive processes isolated adequately stand in for 'Willed Attention?" Willed attention, as operationally defined here, appears to involve a simple decision process prior to the shifting of spatial attention. The cue is internally generated, but after that the rest of the attentional processes appear identical to standard externally cued visuospatial attention experiments. This self-generated cue process likely involves some sort of memory/history of the recently selected cues and then some random-ish selection between A and B. This appears very similar to asking the subject to guess whether a fair coin flip will be heads or tails on each trial. A mental 'coin flip' feels like a very weak version of 'will.' As a potential remedy, it would be helpful to discuss what other phenomena might fall within 'willed attention' and what some future studies might choose to focus on, along with some potential pitfalls (e.g., the reasons why the current study avoided more robust exemplars of will).

(2) The manuscript is lacking a description of the decision processes used during the willed attention paradigm and is lacking evidence as to WHEN subjects made their willed decision. Both of these points are of major concern:

(a) The authors state: "For willed attention, participants were explicitly told to avoid relying on any stereotypical strategies of generating decisions, such as always attending the same/opposite side they attended during the previous trial, as well as to avoid randomizing or equalizing their decisions to choose left or right across trials; prior studies found that decisions to explicitly randomize decisions might invoke additional working memory related processes (Spence & Frith, 1999)." Subjects were instructed NOT to apply a simple heuristic and NOT to randomize or try to equalize their decisions, but exactly HOW the subjects made their decisions is not at all clear. What options does that leave? How does this strategy avoid the working memory-related processes mentioned in the Spence & Frith, 1999 citation? The brain regions that comprise the network of interest (aka Frontoparietal Decision Network) are activated by a very broad range of visual cognitive tasks, including many working memory paradigms. The Anterior Insula and dACC nodes Salience Network often simply reflect task difficulty. Obviously, making a choice is more cognitively demanding than not making a choice. The present experiments do not distinguish functional roles between different regions of the Frontoparietal Decision Network. On the whole, the study does very little to isolate the cognitive processes or neural bases of willed attention beyond calling out the set of 'Usual Suspects' for visual cognition.

(b) The finding that pre-cue EEG signals predicted the postcue decision is intriguing. It could mean that the seemingly irrelevant and transient state of the brain causally and unconsciously biased the subject to one direction or the other. Alternatively, it could mean that the subjects utilized the pre-cue period to make their decision and hold it in case it was needed (i.e., that it was a choice trial). While 2-8 seconds ITI variability makes sense for fMRI decoding, it is a long time for a subject to idly wait, so they might fill that time preparing for the next trial. There appears to have been a substantial amount of individual difference in the pre-cue alpha decoding, which could reflect individual differences in cognitive strategy, specifically in the use of the pre-cue period to make their decision. More efficient decision makers might have pre-decided, which might account for the neural efficiency. The experiments lack any measurement of WHEN participants made their decision. For that reason, I would ask that the authors temper their claims about the significance of the alpha decoding and its possible causality.

(3) Did individual subjects exhibit a choice bias of location for the willed trials? If not, doesn't that raise concerns that subjects were trying to equalize their trials? If they do exhibit location biases, how does that impact the decoding? A simple decoder could learn to always just guess the biased direction for a subject and would perform > 50%. Consider the example in which an individual subject chooses 'Left' 55% of the time. A classifier that simply learns to choose 'Left' on every trial will be correct on 55% of trials. The training data would likely be sufficient to learn the direction of choice bias in each individual subject. So the classifiers could perform significantly above 50% without learning anything beyond the tendency of each subject. That is to say, 50% is not truly chance in this data set. It doesn't appear that Permutation testing has been performed to empirically determine chance for an individual's data. Permutation methods, scrambling the labels 1000 or 10000 times to establish a true baseline would be preferred over simply comparing to 50% and would address concerns about individual subject biases.

(4) The novel contributions of this work beyond the two prior Bengson et al papers from Dr. Mangun's lab appear quite modest. The discussion would be enhanced by specifically stating how the present work advances understanding beyond the prior Bengson studies.

Reviewer #3 (Public review):

Summary:

This manuscript analyzes two independent datasets collected at different sites. Using the same willed-attention paradigm (instructional vs. choice cues) and combining fMRI and EEG analyses, the authors investigate how attentional direction is selected when no external instruction is provided. Their main claims are that the dorsal attention network is engaged by both cue types, whereas the choice cue additionally involves a frontoparietal decision network. Moreover, left-versus-right attentional decisions can be decoded in this decision network only on choice trials, and multichannel pre-stimulus alpha patterns predict the subsequent attentional choice. Finally, individuals with more predictive alpha patterns show greater neural efficiency in the decision network, i.e., higher decoding with lower BOLD activation.

The question is worthwhile and the two-site design is a genuine strength. At the same time, several central inferences rely on decoding analyses for which the statistical testing and cross-validation structure are not described in enough detail to assess robustness. In addition, using a ratio-based neural-efficiency measure make the interpretation more fragile than it needs to be. With a focused revision that tightens inference around MVPA and clarifies a few methodological points, I think the paper could become substantially more convincing.

Strengths:

The work extends previous willed attention studies by attempting to link pre-stimulus alpha pattern predictability to post-cue frontoparietal representations, and by testing reproducibility across two datasets. The conceptual advance beyond previous studies, e.g., Bengson et al. (2015), however, depends on how solid the decoding-based evidence is and whether alternative explanations are convincingly excluded. At present, the strength of support is limited mainly by incomplete reporting and/or controls for MVPA significance testing, as well as potential inflation of decoding estimates if folds are not independent of run structure. Concerns about statistical assessment of decoding accuracy are well documented in the literature (Combrisson & Jerbi, 2015).

Weaknesses:

(1) The manuscript describes the decoding pipeline for both fMRI and EEG MVPA. However, it does not clearly specify how "significantly above chance" is determined for the fMRI ROI decoding, nor how multiple comparisons across ROIs are handled, even though p-values are reported. The same issue applies to the time-resolved EEG analysis across many time points. For each decoding analysis, please specify the inferential test (e.g., permutation test within participant, group-level test on subject accuracies, binomial test, etc.) and report effect sizes with confidence intervals (e.g., Combrisson & Jerbi, 2015). Further, for EEG decoding over time, it would be preferable to control family-wise error, e.g., cluster-based permutation, rather than thresholding pointwise p-values. A standard approach here is the nonparametric cluster framework (e.g., Maris & Oostenveld, 2007).

(2) The cross-validation approach used here is appreciated and appropriate in principle. However, random 10-fold splits across trials can inflate accuracy if training and test folds share run-specific noise, scanner drift, or autocorrelated structure. The manuscript should indicate whether folds were blocked by run or randomized across the entire session. In addition, please report the number of trials per condition after artifact rejection and after removing short ITIs for the long prestimulus epochs (−2500 ms to 0 ms) for each dataset in the section of EEG preprocessing. Similarly, please report how often participants chose left vs. right on choice trials, and whether balanced folds (or an equivalent balancing procedure) were used if needed.

(3) Moreover, ROI definition is not sufficiently specified and independence should be clarified. The ROIs are defined based on peaks from the choice-instructed univariate contrast (Table 2) and then used for MVPA. First, are these ROIs defined as spheres around peaks or using anatomical masks? What radius or voxel count was used? This needs to be explicit. Second, I am concerned about circularity risk. Although choice-vs-instructed selection is not identical to left-vs-right decoding, ROI selection from the same dataset can still bias descriptive estimates and encourages overinterpretation if not carefully justified (Kriegeskorte et al., 2009). At minimum, the authors should explain why their selection criterion is independent of the decoded contrast under the null, and ideally provide a robustness check using either anatomical ROIs or independently defined ROIs, e.g., from prior literature or an atlas.

(4) Using an index of neural efficiency is conceptually interesting. However, if the denominator, computed as the activation difference between choice and instructional conditions, is near zero or noisy, the ratio can become unstable. I would rather see a multivariate model that treats activation and decoding as separate dependent measures, or a latent-variable approach, than a single ratio.

Reviewer #1 (Public review):

Summary:

The authors investigated the relationship between physical activity (PA) and both structural (MRI) and cognitive brain health in the LIFE-Adult Study, with total baseline recruitment of 2576. Hippocampal volume, an MRI-derived BrainAGE marker, and scores from the Trail Making Test were used as outcomes, with the majority of participants measured at baseline and subsets also measured in a follow-up session. The key findings were a lack of direct association between PA and outcomes, but longitudinal evidence for a higher BrainAge at baseline leading to lower physical capacity at follow-up. This supports a reverse-causation hypothesis in contrast to the prevailing understanding of the positive effects of physical activity on brain health.

Strengths:

The Life-Adult study is a rich and carefully acquired dataset, with multiple follow-up time points. The statistical analyses were conducted carefully with appropriate control for confounds and multiple testing. The study design enables an important assessment for reverse causality. The authors are scrupulous in their consideration of a number of factors that could potentially bias their results, performing an age-stratified analysis, and emphasising discrepancies in PA measurements (specifically, age-reporting bias) across the dataset and other limitations.

Weaknesses:

This is an observational study with inconsistent measures of physical activity. Previous studies have used physical activity interventions, and might be more strongly weighted when considering evidence for these effects (specific confounders involved in interventions notwithstanding).

The model identifying potential reverse causality is relatively limited - it seems possible/likely that brainAge could reflect more general health status, which would expand the potential range of factors underlying this observation.

The important quantitative actigraphy subset is small (n=227), as are the longitudinal subsets. Along with the discrepancy of physical activity/capacity at baseline and follow-up, and other complexities of the dataset, it is difficult to make firm conclusions. The authors point out that the actigraphy subset was quite inactive.

Reviewer #2 (Public review):

Summary:

This population-based cohort study found no evidence that physical activity, whether self-reported or objectively measured, positively influenced brain structure (hippocampal volume or BrainAGE) or cognitive function (Trail Making Test scores). Notably, longitudinal analyses suggested the opposite temporal relationship: a higher BrainAGE at baseline predicted higher physical capacity at follow-up, more in line with reverse causation rather than a neuroprotective effect of physical activity.

Strengths:

The study's statistical approach is thorough and well-documented, and the inclusion of two measurements of physical activity (self-report questionnaire and objective accelerometer data) is a strength. The longitudinal aspect also represents a strength.

Weaknesses:

Several aspects of the measurement timing warrant consideration. Physical activity was assessed over 7-day periods, creating a potential mismatch with (commonly less dynamic) brain outcomes examined (hippocampal volume, BrainAGE), which may reflect cumulative exposures over longer timescales. Additionally, the asynchronous measurement protocol (cognitive testing preceding accelerometry, and the MRI occurring weeks after baseline visits) may introduce time lags that attenuate associations. The observed null associations may be influenced by timing misalignment rather than reflecting the absence of consistent effects of physical activity on brain health and cognition.

Other measurement characteristics also warrant consideration when interpreting the null findings. Physical activity was assessed using short-form self-report questionnaires and averaged accelerometer MET/day values, both of which have limited reliability. Additionally, the modest accelerometer subsample size and low/insufficient variation in activity levels observed in this cohort increase the likelihood of missing effects. These factors collectively raise the possibility that true physical activity-brain health associations may have been obscured.

The study's conclusions regarding brain health, structure, and cognitive functioning are broad despite the scope of the selection of outcomes examined. The analyses focus on hippocampal volume, BrainAGE (a global aging metric), and Trail Making Test performance (processing speed and executive function), while omitting other important neuroimaging markers such as cortical thickness, functional connectivity, or white matter microstructure. The null findings presented here cannot exclude positive effects of physical activity on broader constructs of brain health or cognitive functioning.

While the authors appropriately note the use of different physical activity instruments across time points (IPAQ at baseline, VSAQ at follow-up) in the limitations section, the discussion should more explicitly address the interpretive challenges this creates. The observed association between higher baseline brain age gap and lower follow-up physical activity may reflect: (1) a true temporal relationship, (2) an artifact of switching from behavior-focused (IPAQ) to capacity-focused (VSAQ) measurement, or (3) some combination of both. This ambiguity substantially limits causal inference.

Reviewer #1 (Public review):

Summary:

This manuscript by Zhao et. al investigates the canonical hedgehog pathway in testis development of Nile tilapia. They used complementary approaches with genetically modified tilapia and transfected TSL cells (a clonal stem Leydig cell line) previously derived from 3-mo old tilapia. The approach is innovative and provides a means to investigate DHH and each downstream component from the ptch receptors to the gli and sf1 transcription factors. They concluded that Dhh binds Ptch2 to stimulate Gli1 to promote an increase in Sf1 expression leading to the onset of 11-ketotesterone synthesis heralding the differentiation of Leydig cells in the developing male tilapia.'

Strengths of the methods and results:

- The use of Nile tilapia is important as it is an important aquaculture species, it shares the genetic pathway for sex determination of mammalian species, and molecular differentiation pathways are highly conserved<br /> - The approach is rigorous and incorporates a novel TSL, clonal stem Leydig cell model that they developed that is relatively faithful in following endogenous developmental steps and can produce the appropriate steroid.<br /> - Tilapia are relatively amenable to CRISPR/Cas9 targeting and, with their accelerated developmental time frame, provide an excellent model system to interrogate specific signaling pathways.<br /> - The stepwise analysis from dhh-gli-sf1 is thoughtful and well done.

Weaknesses of the methods and results:

- Line 162: need to establish and verify the PKH26-labeled TSL cells were unaffected by the dhh-/- environment. No data to support the claim that they were unaffected.<br /> - The rescued phenotype caused by the addition of ptch2-/- to the dhh-/- model is a compelling. To further define potential ptch1 contributions, it would be helpful to examine the expression level of ptch1 in the context of the ptch2-/- and ptch2-/-;dhh-/- mutant animals. Any compensatory increase in ptch1 in either case, without obvious phenotype changes, would support the dominant role for ptch2.<br /> - Activity of individual gli factors need additional reconciliation. The expression profiles for both alternative gli factors should be quantified in each knockout cell line to establish redundancy and/or compensation.<br /> - Figure 5E: An important control is missing that includes evaluation of HEK293 cells transfected with pcDNA3.1-OnGli1 without the addition of pGL3-sf1.

Achieved Aims:

The authors set out to test the hypothesis that the canonical Dhh signaling pathway for Leydig cell differentiation and steroidogenic activity is mediated via ptch2 and gli1 regulation of sf1. The results are strong, there are additional steps needed to verify that redundancy/compensation is not contributing to the outcomes.

This work is important in better understanding of nuanced commonalities and differences in developmental pathways across species. Specific to Leydig cell differentiation and steroidogenesis, their work with tilapia supports conservation of the canonical Dhh pathway; however, there appear to be some differences in downstream mediators compared to mouse. Specifically, they conclude that ptch2/gli1 stimulates sf1 and steroidogenesis in tilapia where gli1 is dispensable in mouse. Instead, Gli3 has recently been shown to play an important role to stimulate Sf1 and support the hedgehog pathway.

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:

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 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.

The revised methods section is easier to follow and better explains the approach. Inference results on simulated data are compelling and the real-data findings are compared with alternative approaches, clarifying the relationship to previous work.

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:

• The authors leverage the unique data they have generated for a separate project to provide novel insights to a fundamental question.
• The paper is clearly written, with accessible methods and straightforward discussion of the limits of this model.
• Code and data are publicly available and well-documented.

Weaknesses:

• No substantial weaknesses noted.

Reviewer #1 (Public review):

In this study, Brickwedde et al. leveraged a cross-modal task where visual cues indicated whether upcoming targets required visual or auditory discrimination. Visual and auditory targets were paired with auditory and visual distractors, respectively. The authors found that during the cue-to-target interval, posterior alpha activity increased along with auditory and visual frequency-tagged activity when subjects were anticipating auditory targets. The authors conclude that their results imply that alpha modulation does not solely regulate 'gain control' in early visual areas (also referred to as alpha inhibition hypothesis), but rather orchestrates signal transmission to later stages of the processing stream.

Comments on the first revision:

I thank the authors for their clarifications. The manuscript is much improved now, in my opinion. The new power spectral density plots and revised Figure 1 are much appreciated. However, there is one remaining point that I am unclear about. In the rebuttal, the authors state the following: "To directly address the question of whether the auditory signal was distracting, we conducted a follow-up MEG experiment. In this study, we observed a significant reduction in visual accuracy during the second block when the distractor was present (see Fig. 7B and Suppl. Fig. 1B), providing clear evidence of a distractor cost under conditions where performance was not saturated."

I am very confused by this statement, because both Fig. 7B and Suppl. Fig. 1B show that the visual- (i.e., visual target presented alone) has a lower accuracy and longer reaction time than visual+ (i.e., visual target presented with distractor). In fact, Suppl. Fig. 1B legend states the following: "accuracy: auditory- - auditory+: M = 7.2 %; SD = 7.5; p = .001; t(25) = 4.9; visual- - visual+: M = -7.6%; SD = 10.80; p < .01; t(25) = -3.59; Reaction time: auditory- - auditory +: M = -20.64 ms; SD = 57.6; n.s.: p = .08; t(25) = -1.83; visual- - visual+: M = 60.1 ms ; SD = 58.52; p < .001; t(25) = 5.23)."

These statements appear to directly contradict each other. I appreciate that the difficulty of auditory and visual trials in block 2 of MEG experiments are matched, but this does not address the question of whether the distractor was actually distracting (and thus needed to be inhibited by occipital alpha). Please clarify.

Comments on the latest version:

I am satisfied with the author's response and do not have any additional comments.

Reviewer #1 (Public Review):

Summary:

In their paper, Zhan et al. have used Pf genetic data from simulated data and Ghanaian field samples to elucidate a relationship between multiplicity of infection (MOI) (the number of distinct parasite clones in a single host infection) and force of infection (FOI). Specifically, they use sequencing data from the var genes of Pf along with Bayesian modeling to estimate MOI individual infections and use these values along with methods from queueing theory that rely on various assumptions to estimate FOI. They compare these estimates to known FOIs in a simulated scenario and describe the relationship between these estimated FOI values and another commonly used metric of transmission EIR (entomological inoculation rate).

This approach does fill an important gap in malaria epidemiology, namely estimating the force of infection, which is currently complicated by several factors including superinfection, unknown duration of infection, and highly genetically diverse parasite populations. The authors use a new approach borrowing from other fields of statistics and modeling and make extensive efforts to evaluate their approach under a range of realistic sampling scenarios. However, the write-up would greatly benefit from added clarity both in the description of methods and in the presentation of the results. Without these clarifications, rigorously evaluating whether the author's proposed method of estimating FOI is sound remains difficult. Additionally, there are several limitations that call into question the stated generalizability of this method that should at minimum be further discussed by authors and in some cases require a more thorough evaluation.

Major comments:

(1) Description and evaluation of FOI estimation procedure.

a. The methods section describing the two-moment approximation and accompanying appendix is lacking several important details. Equations on lines 891 and 892 are only a small part of the equations in Choi et al. and do not adequately describe the procedure notably several quantities in those equations are never defined some of them are important to understand the method (e.g. A, S as the main random variables for inter-arrival times and service times, aR and bR which are the known time average quantities, and these also rely on the squared coefficient of variation of the random variable which is also never introduced in the paper). Without going back to the Choi paper to understand these quantities, and to understand the assumptions of this method it was not possible to follow how this works in the paper. At a minimum, all variables used in the equations should be clearly defined.

b. Additionally, the description in the main text of how the queueing procedure can be used to describe malaria infections would benefit from a diagram currently as written it's very difficult to follow.

c. Just observing the box plots of mean and 95% CI on a plot with the FOI estimate (Figures 1, 2, and 10-14) is not sufficient to adequately assess the performance of this estimator. First, it is not clear whether the authors are displaying the bootstrapped 95%CIs or whether they are just showing the distribution of the mean FOI taken over multiple simulations, and then it seems that they are also estimating mean FOI per host on an annual basis. Showing a distribution of those per-host estimates would also be helpful. Second, a more quantitative assessment of the ability of the estimator to recover the truth across simulations (e.g. proportion of simulations where the truth is captured in the 95% CI or something like this) is important in many cases it seems that the estimator is always underestimating the true FOI and may not even contain the true value in the FOI distribution (e.g. Figure 10, Figure 1 under the mid-IRS panel). But it's not possible to conclude one way or the other based on this visualization. This is a major issue since it calls into question whether there is in fact data to support that these methods give good and consistent FOI estimates.

d. Furthermore the authors state in the methods that the choice of mean and variance (and thus second moment) parameters for inter-arrival times are varied widely, however, it's not clear what those ranges are there needs to be a clear table or figure caption showing what combinations of values were tested and which results are produced from them, this is an essential component of the method and it's impossible to fully evaluate its performance without this information. This relates to the issue of selecting the mean and variance values that maximize the likelihood of observing a given distribution of MOI estimates, this is very unclear since no likelihoods have been written down in the methods section of the main text, which likelihood are the authors referring to, is this the probability distribution of the steady state queue length distribution? At other places the authors refer to these quantities as Maximum Likelihood estimators, how do they know they have found the MLE? There are no derivations in the manuscript to support this. The authors should specify the likelihood and include in an appendix an explanation of why their estimation procedure is in fact maximizing this likelihood, preferably with evidence of the shape of the likelihood, and how fine the grid of values they tested is for their mean and variance since this could influence the overall quality of the estimation procedure.

(2) Limitation of FOI estimation procedure.

a. The authors discuss the importance of the duration of infection to this problem. While I agree that empirically estimating this is not possible, there are other options besides assuming that all 1-5-year-olds have the same duration of infection distribution as naïve adults co-infected with syphilis. E.g. it would be useful to test a wide range of assumed infection duration and assess their impact on the estimation procedure. Furthermore, if the authors are going to stick to the described method for duration of infection, the potentially limited generalizability of this method needs to be further highlighted in both the introduction, and the discussion. In particular, for an estimated mean FOI of about 5 per host per year in the pre-IRS season as estimated in Ghana (Figure 3) it seems that this would not translate to 4-year-old being immune naïve, and certainly this would not necessarily generalize well to a school-aged child population or an adult population.

b. The evaluation of the capacity parameter c seems to be quite important and is set at 30, however, the authors only describe trying values of 25 and 30, and claim that this does not impact FOI inference, however it is not clear that this is the case. What happens if the carrying capacity is increased substantially? Alternatively, this would be more convincing if the authors provided a mathematical explanation of why the carrying capacity increase will not influence the FOI inference, but absent that, this should be mentioned and discussed as a limitation.

Reviewer #2 (Public Review):

Summary:

The authors combine a clever use of historical clinical data on infection duration in immunologically naive individuals and queuing theory to infer the force of infection (FOI) from measured multiplicity of infection (MOI) in a sparsely sampled setting. They conduct extensive simulations using agent-based modeling to recapitulate realistic population dynamics and successfully apply their method to recover FOI from measured MOI. They then go on to apply their method to real-world data from Ghana before and after an indoor residual spraying campaign.

Strengths:

(1) The use of historical clinical data is very clever in this context.

(2) The simulations are very sophisticated with respect to trying to capture realistic population dynamics.

(3) The mathematical approach is simple and elegant, and thus easy to understand.

Weaknesses:

(1) The assumptions of the approach are quite strong and should be made more clear. While the historical clinical data is a unique resource, it would be useful to see how misspecification of the duration of infection distribution would impact the estimates.

(2 )Seeing as how the assumption of the duration of infection distribution is drawn from historical data and not informed by the data on hand, it does not substantially expand beyond MOI. The authors could address this by suggesting avenues for more refined estimates of infection duration.

(3) It is unclear in the example how their bootstrap imputation approach is accounting for measurement error due to antimalarial treatment. They supply two approaches. First, there is no effect on measurement, so the measured MOI is unaffected, which is likely false and I think the authors are in agreement. The second approach instead discards the measurement for malaria-treated individuals and imputes their MOI by drawing from the remaining distribution. This is an extremely strong assumption that the distribution of MOI of the treated is the same as the untreated, which seems unlikely simply out of treatment-seeking behavior. By imputing in this way, the authors will also deflate the variability of their estimates.

- For similar reasons, their imputation of microscopy-negative individuals is also questionable, as it also assumes the same distributions of MOI for microscopy-positive and negative individuals.

Reviewer #3 (Public Review):

Summary:

It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying it to simulated results from a previously published agent-based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI.

Strengths:

It would be great to be able to infer FOI from cross-sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross-sectional studies. They attempt to validate this process using a previously published agent-based model which helps us understand the complexity of parasite population genetics.

Weaknesses:

(1) I fear that the work could be easily over-interpreted as no true validation was done, as no field estimates of FOI (I think considered true validation) were measured. The authors have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion.

(2) Another aspect of the paper is adding greater realism to the previous agent-based model, by including assumptions on missing data and under-sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone).

(3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the latter before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying.

(4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method.

(5) The text is a little difficult to follow at times and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology.

Reviewer #2 (Public review):

Summary:

The authors combine a clever use of historical clinical data on infection duration in immunologically naive individuals and queuing theory to infer the force of infection (FOI) from measured multiplicity of infection (MOI) in a sparsely sampled setting. They conduct extensive simulations using agent based modeling to recapitulate realistic population dynamics and successfully apply their method to recover FOI from measured MOI. They then go on to apply their method to real world data from Ghana before and after an indoor residual spraying campaign.

Strengths:

- The use of historical clinical data is very clever in this context

- The simulations are very sophisticated with respect to trying to capture realistic population dynamics

- The mathematical approach is simple and elegant, and thus easy to understand

Weaknesses:

- The assumptions of the approach are quite strong, and the authors have made clear that applicability is constrained to individuals with immune profiles that are similar to malaria naive patients with neurosyphilis. While the historical clinical data is a unique resource and likely directionally correct, it remains somewhat dubious to use the exact estimated values as inputs to other models without extensive sensitivity analysis.

Comments on revisions:

The authors have adequately responded to all comments.

Reviewer #1 (Public review):

This manuscript adds to the recent, exciting developments in our understanding of the MmpL/S transporters from mycobacteria. This work provides solid support for the trimeric/hexameric arrangement of subunits in the complex, and reveals a possible pathway for substrate translocation.

Overall, I think this manuscript is a solid body of work that adds to several recent studies from this team and others on the structure and mechanism of the MmpL/S transporter family, particularly MmpL4/S4. The combination of AF, disulfide engineering, and experimental structure is good, though it is a bit puzzling that the experimental structure based on disulfide stabilization of the AF prediction does not recapitulate key elements (MmpS periplasmic domain docking to MmpL, and altered CCD configuration).

I have no major concerns about this manuscript.

Reviewer #2 (Public review):

Summary:

The manuscript describes the structure of the Mycobacterium tuberculosis (MmpS4)3-(MmpL4)3 hetero-heximeric transporter complex. The structure was obtained by cryogenic electron microscopy using an engineered construct that cross-links MmpS4 to MmpL4 via a disulfide bond. The position of the disulfide bond was determined using an Alphafold2 model of the hetero-heximer. Although Alphafold2 predicts a symmetric hetero-heximer, the author found that the structure of the coiled-coil domain (CCD) is asymmetric, tilted at about 60° relative to the membrane domains, and only contains two of the three alpha helical hairpins, with the third being disordered.

Strengths:

The strategy of using Alphafold2 models to guide construct design for experimental structure determination is state-of-the-art, and this work provides a great example of its applications and limitations. I.e., the experimental structure does not fully recapitulate the prediction but provides unexpected results.

The comparisons between the authors' structures and the previously published structures of the MmpL4 monomer and MmpL5 trimers strengthen the authors' findings.

Weaknesses:

A more detailed description of the current mechanistic hypothesis would strengthen the manuscript. The authors state that the two periplasmic domains "are expected to undergo rigid body movements that allow substrate transport through these periplasmic domains similar to the conformational changes observed in the E. coli multidrug efflux pump AcrB". A schematic of the proposed transport cycle, as a supplemental figure that shows the current hypothesis regarding transport, would be beneficial for understanding the previous structures and putting the current structure in context. Outside of "the mechanistic basis of how these conformational changes are coupled to protonation of the DY-pairs", what are the major controversies/open questions regarding the mechanism?

The authors provide evidence that the cysteine-depleted S4L4 construct is functional, but do not show that the construct with the introduced disulfide bond #5 (D39C MmpS4 and S434C MmpL4) is also functional. Demonstrating this would allow the authors to better interpret their resulting structures.

The analysis presented in Figure 5 and Supplementary Figure 7 seems to suggest that the authors are proposing that the CCD central cavity acts as a transport pathway for the transported substrate, but I am not sure that this hypothesis is explicitly stated. This makes the reasoning behind the analysis presented unclear. Clarity could be improved by stating that the hypothesis of direct transport of substrate through the CCD central channel is being examined using the structure prediction, and what the implications are for the structure solved with the incompletely formed CCD.

Given that the results emphasize the flexibility of the CCD, the manuscript would be strengthened by 3D variability analysis either in cryoSPARC or using cryoDRGN (or both). This would allow the authors to better quantify the degree of motion in the CCD and how it may correlate to flexibility in other regions. Further 3D flex reconstruction in cryoSPARC may improve the map quality of the CCD.

Reviewer #3 (Public review):

Summary:

This manuscript by Earp et al reports cryoEM structures of the hexameric (MmpS4)₃-(MmpL4)₃ complex from Mycobacterium tuberculosis, which belongs to the RND family of transporters and is known to have a role in the export of siderophores and contribute to drug resistance. The experimental workflow showcased involves the design of disulfide pairs using distance constraints obtained from the AlphaFold predicted structure of the hexameric complex. One such disulfide pair was used to determine the ~3.0 Å structures. The structure reveals density for the previously unresolved coiled-coil domain (CCD), a tilted CCD arrangement, and a cavity within the periplasmic domain, which the authors assert is occupied by detergent. Comparison of this complex with the monomer structure of MmpL4 shows conformational variations interpreted to implicate different domains and conserved residues involved in proton coupling, which might be related to the transport mechanism. While the methodological aspects of the manuscript are solid, enthusiasm for the overall advance/significance is less so, with doubts about the relevance of the tilted CCD structure, considering disulfide trapping and an incomplete validation of the claim that the titled CCD represents a stable intermediate conformation. A clear, updated transport mechanism is largely missing from the manuscript.

Strengths:

Beautiful structures, AF prediction-experimental validation nexus that could be fine-tuned for different systems/difficult to target complexes.

Weaknesses:

Physiological relevance of the tilted CCD conformation. No clear mechanistic model for the transport. While the CCD may indeed be a stable intermediate, the fact that the rest of the trimeric arrangement is unaffected does not fully rule out disulfide trapping as a factor in promoting this. The findings would be strengthened if the same tilted conformation is seen using a different set of disulfides. The significance of the detergent molecule and the new cavity observed could also be better discussed in terms of an updated transport model.

Reviewer #1 (Public review):

Summary:

In "Drift in Individual Behavioral Phenotype as a Strategy for Unpredictable Worlds," Maloney et al. (2026) investigate changes in individual responses over time, referred to as behavioral drift within the lifespan of an animal. Drift, as defined in the paper, complements stable behavioral variation (animal individuality/personality within a lifetime) over shorter timeframes, which the authors associate with an underlying bet-hedging strategy. The third timeframe of behavioral variability that the authors discuss occurs within seasons (across several generations of some insects), termed "adaptive tracking." This division of "adaptive" behavioral variability over different timeframes is intuitively logical and adds valuable depth to the theoretical framework concerning the ecological role of individual behavioral differences in animals.

Strengths:

While the theoretical foundations of the study are compelling, the connection between the experimental data (Fig. 1) and the modeling work (Fig. 2-4) is convincing.

Weaknesses:

In the experimental data (Fig. 1), the authors describe the changes in behavioral preferences over time. While generally plausible, I had identified three significant issues with the experiments that were addressed in the revision:

(1) All of the subsequent theoretical/simulation data is based on changing environments, yet all the experiments are conducted in unchanging environments. While this may suffice to demonstrate the phenomenon of behavioral instability (drift) over time, it does not fully link to the theory-driven work in changing environments. A full experimental investigation of this would be beyond the scope of the current work.

(2) The temporal aspect of behavioral instability has been addressed in Figure 1F.

(3) The temporal dimension leads directly into the third issue: distinguishing between drift and learning (e.g., line 56). This issue has been further discussed in the revised manuscript.

Reviewer #2 (Public review):

Summary:

This is an inspired study that merges the concept of individuality with evolutionary processes to uncover a new strategy that diversifies individual behavior that is also potentially evolutionarily adaptive.

The authors use time-resolved measurement of spontaneous, innate behavior, namely handedness or turn bias in individual, isogenic flies, across several genetic backgrounds.

They find that an individual's behavior changes over time, or drifts. This has been observed before, but what is interesting here is that by looking at multiple genotypes, the authors find the amount of drift is consistent within genotype i.e., genetically regulated, and thus not entirely stochastic. This is not in line with what is known about innate, spontaneous behaviors. Normally, fluctuations in behavior would be ascribed to a response to environmental noise. However, here, the authors go on to find what is the pattern or rule that determines the rate of change of the behavior over time within individuals. Using modeling of behavior and environment in the context of evolutionarily important timeframes such as lifespan or reproductive age, they could show when drift is favored over bet-hedging and that there is an evolutionary purpose to behavioral drift. Namely, drift diversifies behaviors across individuals of the same genotype within the timescale of lifespan, so that the genotype's chance for expressing beneficial behavior is optimally matched with potential variation of environment experienced prior to reproduction. This ultimately increases fitness of the genotype. Because they find that behavioral drift is genetically variable, they argue it can also evolve.

Strengths:

Unlike most studies of individuality, in this study, authors consider the impact of individuality on evolution. This is enabled by the use of multiple natural genetic backgrounds and an appropriately large number of individuals to come to the conclusions presented in the study. I thought it was really creative to study how individual behavior evolves over multiple timescales. And indeed this approach yielded interesting and important insight into individuality. Unlike most studies so far, this one highlights that behavioral individuality is not a static property of an individual, but it dynamically changes. Also, placing these findings in the evolutionary context was beneficial. The conclusion that individual drift and bet-hedging are differently favored over different timescales is, I think, a significant and exciting finding.

Overall, I think this study highlights how little we know about the fundamental, general concepts behind individuality and why behavioral individuality is an important trait. They also show that with simple but elegant behavioral experiments and appropriate modeling, we could uncover fundamental rules underlying the emergence of individual behavior. These rules may not at all be apparent using classical approaches to studying individuality, using individual variation within a single genotype or within a single timeframe.

Weaknesses:

I am unconvinced by the claim that serotonin neuron circuits are regulating behavioral drift, especially because of its bidirectional effect and lack of relative results for other neuromodulators. Without testing other neuromodulators, it will remain unclear if serotonin intervention increases behavioral noise within individuals, or if any other pharmacological or genetic intervention would do the same. Another issue is that the amount of drugs that the individuals ingested was not tracked. Variable amounts can result in variable changes in behavior that are more consistent with the interpretation of environmental plasticity, rather than behavioral drift. With the current evidence presented, individual behavior may change upon serotonin perturbation, but this does not necessarily mean that it changes or regulates drift.

However, I think for the scope of this study, finding out whether serotonin regulates drift or not is less important. I understand that today there is a strong push to find molecular and circuit mechanisms of any behavior, and other peers may have asked for such experiments, perhaps even simply out of habit. Fortunately, the main conclusions derived from behavioral data across multiple genetic backgrounds and the modeling are anyway novel, interesting and in fact more fundamental than showing if it is serotonin that does it or not.

To this point, one thing that was unclear from the methods section is whether genotypes that were tested were raised in replicate vials and how was replication accounted for in the analyses. This is a crucial point - the conclusion that genotypes have different amounts of behavioral drift cannot be drawn without showing that the difference in behavioral drift does not stem from differences in developmental environment.

Comments on the latest version:

The changes to the manuscript sufficiently addressed my few comments. I do not have anything else substantial to add to my review and I am comfortable with my initial assessment.

Reviewer #3 (Public review):

The paper begins by analyzing the drift in individual behavior over time. Specifically, it quantifies the circling direction of freely walking flies in an arena. The main takeaway from this dataset is that while flies exhibit an individual turning bias (when averaged over time), yet their preferences fluctuate over slow timescales.

To understand whether genetic or neuromodulatory mechanisms influence the drift in individual preference, the authors test different fly strains in a Y maze concluding that both genetic background and the neuromodulator serotonin contribute to the degree of drift (although with some contrasting results). The use of a different assay for this different dataset (Y maze istead of wide arena) is justified by previous observation of similar behavioral biases in these assay. Yet the conceptual link between the spectral power analysis used for the first dataset and the autoregressive model used for the second remains unclear.

Finally, the authors use theoretical approaches to show the potential advantage of individual drift for survival in unpredictable, fluctuating environments. They demonstrate that while bet-hedging provides an advantage over timescales matching the generation time (since reproduction is required), it offers less benefit on shorter timescales, where an increased individual drift could be advantageous.

Reviewer #1 (Public review):

This manuscript characterizes the effects of isoflurane on visual processing in layer 2/3 of the mouse primary visual cortex (V1). General anesthesia, including isoflurane, has been reported to modulate various neural processes, such as size tuning, direction selectivity, and spatial selectivity in V1. Using two-photon calcium imaging, the authors monitored neural responses to visual stimuli under isoflurane anaesthesia and found that spatial frequency preferences are also affected across cell types, with the magnitude and direction of these effects varying between cell types.

The authors performed careful and rigorous comparisons of neuronal responses between the two conditions using well-chosen nonparametric statistics. At the same time, because two-photon calcium imaging can be combined with cell-type-specific labeling, the authors labelled inhibitory neurons with tdTomato, allowing them to distinguish GCaMP activities in excitatory and specific inhibitory cell classes. We also appreciated that the manuscript provides not only summary statistics but also example GCaMP traces (Figure 1), which makes it easy for readers to understand the quality of the raw data.

We believe that the manuscript could be improved by emphasizing the following three points.

(1) The analyses are limited to the neurons that responded to visual stimuli in both the anesthetized and awake states. According to Table S1, the proportion of visually responsive neurons that met such criteria is only 27.4% for the excitatory neurons. This raises the potential concerns that the reported effects of isoflurane may not fully reflect population-level changes in visual coding. We suggest that the authors repeat the same analyses, including average tuning curves and decoding analyses, for all recorded neurons in each condition.

(2) The manuscript would benefit from tuning curves of spatial frequency preference for individual neurons, as this would help readers assess whether the reported statistics are appropriate (Figures 2A-D). In addition, more in-depth single-neuron analyses would help distinguish between the two proposed hypotheses in Figure 5 that may not be evident from average responses alone. This is because, with the current analysis, it is not clear how the shape of the tuning curves will affect the estimation of spatial frequency preference. To address this potential concern and strengthen the interpretation of the results, we suggest:<br /> a) repeating the analysis at the level of individual neuronal responses, instead of average responses, and<br /> b) using simulated data to examine how changes in tuning-curve width could affect estimated spatial frequency preference.

For example, using the neuronal responses in the awake condition, one could broaden the tuning curves and recompute the preferred spatial frequency, then compare the resulting distribution with that observed under anesthesia.

(3) We believe the manuscript's overall framing is a little broader than what is directly supported by the data. In particular:

(a) the statement "reduced sensory perception during anesthesia is linked to a degradation in spatial resolution at the cellular level" in the Abstract is an unclear and unsupported claim. We suggest removing this sentence and more directly summarizing the findings.

(b) given the discrepancy between the effects of urethane and isoflurane as laid out in the discussion, the current title "Anesthesia Lowers Spatial Frequency Preference in the Primary Visual Cortex" appears overstated and should be revised to explicitly reflect the specific anesthetic tested: "Isoflurane Anesthesia Lowers Spatial Frequency Preference in the Primary Visual Cortex".

Reviewer #2 (Public review):

Summary:

The main objective of the study was to link the changes in brain state due to anesthesia to consequences on visual neural processing, particularly effects on spatial frequency tuning. This is accomplished by 2-photon imaging of excitatory and inhibitory neurons (separating PV- and SST-positive subtypes) in mouse visual cortex during full-field visual stimulation with gratings, and tracking neuronal tuning for spatial frequency before, during, and after isoflurane anesthesia. The main finding is that anesthesia induces lower spatial frequency preferences in excitatory neurons, and this leads to poorer population representations (decoding) of higher spatial frequency responses during anesthesia. A second main finding is that anesthesia impacts inhibitory neuron subtypes in distinct ways, with the most pronounced effects of anesthesia on somatostatin inhibitory neurons.

Strengths:

(1) A main strength is that the study is that it is straightforward, and reassuringly, the results confirm multiple previous studies showing anesthesia's effects on the amplitude of cortical responses: larger and less selective responses in excitatory neurons (versus awake responses); strongly reduced responses in somatostatin inhibitory neurons (versus awake responses) (Fig. 5I-L), with less differences across anesthetized and awake states on response amplitude of PV neurons.

(2) These confirmations of prior observations (on the amplitude of responses) establish good ground for the new results on spatial frequency tuning. For excitatory neurons, spatial frequency selectivity shifts to higher values in awake versus anesthetized conditions; this is because anesthesia induces larger responses to lower spatial frequencies. In somatostatin neurons, instead, wakefulness reduces the lower spatial frequency responses present in anesthesia, and dramatically increases the overall amplitude of responses and medium and higher spatial frequencies. This is consistent with prior work showing that in awake states, somatostatin neurons exert broad inhibition in V1; this study extends that finding to the tuning of spatial frequencies.

Weaknesses:

(1) A first weakness of the study is the lack of examination of changes to single neuron receptive field sizes and/or surround suppression across conditions, and how these may relate to the effects on spatial frequency tuning with full field gratings. There is a well-known relationship between the size of the receptive field and the resulting selectivity for spatial frequencies (i.e., large receptive fields prefer lower spatial frequency stimuli). Likewise, there are many studies showing how surround suppression / spatial integration is impacted by anesthesia (and arousal). A more detailed examination of all these related quantities on an individual neuron basis would provide a greater understanding of the factors underlying the effects on spatial frequency tuning. One could imagine that receptive field changes, and/or changes in surround suppression, influence the selectivity to full-field gratings.

(2) A second weakness is the lack of examination/insight into the temporal dynamics of the effects. The experimental paradigm records activity across control, anesthesia, and recovery epochs in a single duration (~40 mins) session. The epochs are simply binned together ("Awake", "Anes.", "Recover"). It is not clear how the start of the anesthesia bin is defined, nor is it clear how the recovery period is defined. It is also not clear what the changes are to motor tone, brain state, etc., that are also strong influences on visual responses in mouse V1. Presumably, these onset/offset effects are similar enough across mice and sessions that they affect all the bins in the same way, but greater examination of the temporal effects in excitatory, PV, and SOM neurons could shed light on interactions driving the changes. Is there some temporal dependence of anesthesia on selectivity changes across the cell types? For example, at the onset of anesthesia, are SOM neurons losing broadband frequency responses before the excitatory neurons gain low frequency responses? Do PV neurons also show effects after the changes in SOM neurons (suggesting strong SOM -> PV inhibition)? Such analysis might shed light on the timing/causality of the effects among these 3 neuron types.

(3) A third weakness concerns the interpretation of the low and high arousal conditions during awake states (Figure 6). It is not clear how movement (or lack of movement) impacts the high arousal epochs, nor is it clear how the low arousal condition compares to the brain state during anesthesia. For example, deep versus light anesthesia can lead to synchronized or asynchronous states, respectively, and low arousal in wakefulness can show strong low-frequency oscillations of activity, which could promote a lower excitability state than light anesthesia. Without some more detail about commonly measured brain state or body/face motion metrics, it is difficult to know what brain states are represented by the bins and how to interpret the comparisons.

Overall, the study uses adequate methods and experimental design to demonstrate solid support for the (somewhat narrow) central finding that anesthesia lowers the spatial resolution of mouse V1 responses.

Since this is a very well-examined topic, the findings here are not totally surprising, but confirmatory and slightly extend prior findings (a good thing). As such, the study will likely have most relevance to specialists in the mouse visual system, but if the study could address some of the remaining questions discussed above, this would potentially broaden the implications of the study to general insights about the operation of cortical circuitry.

Reviewer #3 (Public review):

Summary:

This manuscript is focused on studying the spatial frequency selectivity of individual neurons in the mouse primary visual cortex (V1) in the anesthetized and awake brain states using 2-photon calcium imaging. Although previous studies have demonstrated that anesthesia decreases both size tuning and spatial selectivity in V1 neurons, the strength of this study is its focus on characterization of the same neurons in awake and anesthetized states in combination with transgenic mouse lines selectively labeling pan-inhibitory neurons and also more specific neuronal subtypes, including parvalbumin-positive (PV+) or somatostatin-positive (SOM+) interneurons. A combination of these methodologies allows for a more in-depth mechanistic study of the properties of different types of neurons. The main findings suggest that in excitatory neurons, anesthesia leads to a shift in preferred SF and broadening of SF tuning, with no changes in orientation and direction selectivity. Downward shift in preferred SF was more pronounced in both SOM+ and PV+ interneurons.

Strengths:

(1) 2-photon calcium imaging with single-cell resolution.

(2) Characterization of excitatory and two types of inhibitory neurons.

Weaknesses:

(1) VIP interneurons are critical to the neural circuit, and their characterization would be critical to the mechanistic understanding of this process, but is missing.

(2) Unfortunately, the manuscript does not lead to an additional insight into the nature of this anesthesia-induced shift in SF preference.

(3) Furthermore, it also doesn't help understand how SF preference is encoded in V1.

(4) Finally, some critical histological controls are missing.

Reviewer #1 (Public review):

Summary:

The manuscript entitled "Evaluation of Antibiotic and Peptide Vaccine Strategies for Mirror Bacterial Infections" addresses a topic that is well established in the literature. The authors investigate the activity of enantiomeric (D-form) antibiotics against bacteria and the immunogenicity of D-form peptides, proposing that D-enantiomers are ineffective both as antibacterial agents and as vaccine candidates. While the subject matter is relevant, the concepts explored are already well known, and the manuscript offers limited novelty.

The authors demonstrate that D-enantiomeric antibiotics lack antibacterial activity compared to their naturally occurring L-forms and that D-form peptides fail to elicit detectable immune responses. These observations are consistent with existing knowledge regarding molecular chirality in biological systems. However, the manuscript relies on a limited experimental dataset while extrapolating the findings broadly, which weakens the strength of the conclusions.

Strengths:

The manuscript introduces the topic of Mirror Bacterial Infections, likely to occur if no regulations or restrictions are placed immediately.

The manuscript addresses a relevant topic and has potential value, particularly in framing discussions around chirality and pathogen interactions. With a more cautious interpretation of the results, the manuscript could better justify its conceptual framework and strengthen its contribution to the field.

Weaknesses:

(1) Several sections of the manuscript are overly descriptive and would benefit from deeper comparative analysis and critical synthesis. In multiple instances, the discussion relies on hypothetical scenarios supported primarily by selective citations rather than robust experimental evidence. The introduction of the term "mirror microbiology" or "mirror bacteria" appears largely conceptual and is used to unify what are essentially two separate lines of investigation, enantioselective antibiotic activity and peptide chirality in immune recognition, without sufficient mechanistic integration.

(2) To the best of this reviewer's understanding, the manuscript does not present substantial novelty. The pronounced differences in biological activity between L- and D-forms of small molecules and peptides are well documented, including their implications for antimicrobial efficacy and immune recognition. While the manuscript is written in clear and accessible language suitable for both specialists and interdisciplinary readers, novelty remains limited.

The manuscript reiterates well-established principles of stereochemistry and biological recognition. Given the extensive existing literature demonstrating that enantiomeric antibiotics are typically inactive due to stereospecific target interactions, the failure of D-form antibiotics is expected and does not constitute a novel finding.

(3) Critical experimental details are lacking, particularly regarding the peptide design. It is unclear whether the peptides were synthesized entirely in the D-configuration or whether only select amino acids were substituted. This distinction is essential for interpreting immunogenicity results and for comparison with prior studies.

(4) The authors conclude that D-form peptides are poorly recognized by the immune system. However, the data presented indicate that neither the L- nor the D-form peptides tested elicited a measurable immune response. Without demonstrating immunogenicity of the corresponding L-form peptides, the conclusion that immune non-recognition is specific to the D-form is not sufficiently supported.

Reviewer #2 (Public review):

This paper by Kleinman et al. tackles an increasingly discussed biosecurity scenario, namely the possibility that "mirror bacteria" could evade key elements of host immunity and therefore demand bespoke medical countermeasures. The authors experimentally probe two such countermeasure concepts: (1) whether existing chiral antibiotics might still work against mirror bacteria (this is tested indirectly by measuring the activity of antibiotic enantiomers against natural-chirality bacteria), and (2) whether D-peptide antigens can be made immunogenic. Briefly, the authors show that enantiomers of four approved antibiotics have little to no activity in MIC assays, argue this implies the parent drugs would likely fail against mirror bacteria, report limited single-dose tolerability data for the enantiomers in mice, and show that selected bacterially derived D-peptides can elicit strong binding antibody titers when conjugated to a carrier protein and given with adjuvant.

Overall, the study is quite interesting but constrained by the fact that D-peptide immunogens and related ideas have been explored for decades, by prior literature showing that D-enantiomeric peptides can themselves be strongly antimicrobial vs conventional bacteria, and by a number of conceptual and experimental limitations outlined below.

(1) A blanket statement indicating that flipping chirality makes antibiotics ineffective cannot be true across all classes. Indeed, there is extensive precedent for "mirror" (D-amino-acid) peptides that retain, or even improve, antimicrobial activity against natural bacteria.

(2) The paper's key claim ("parent antibiotics won't work on mirror bacteria") is based on the observation that the enantiomers of chloramphenicol/linezolid/tedizolid/aztreonam largely lose activity against natural bacteria. This is a reasonable proxy experiment given the absence of mirror organisms, but it remains an inference and should be described as such.

(3) The chiral purity needs to be documented more rigorously. The methods mention structural confirmation by NMR and >95% purity by LC-MS/HPLC for enantiomeric compounds, but this is not the same as demonstrating high enantiomeric excess or excluding low-level contamination by the active parent enantiomer.

(4) The residual activity of ent-aztreonam is quite interesting. The authors report slight activity for ent-aztreonam (MIC of 32-128 µg/mL in a subset), still far weaker than aztreonam but nonzero.

(5) For antibiotics, MIC is a starting point, but further experiments are needed. To justify countermeasure relevance, it would help to include at least one additional pharmacodynamic readout (time-kill kinetics, post-antibiotic effect, inoculum effect, or activity in the presence of human serum).

(6) The acute toxicity study is limited (single-dose, short follow-up, small n, one sex/strain, and no histopathology).

(7) The Discussion leans on human equivalent dosing logic to reassure feasibility. Given the lack of PK, bioavailability, metabolism, and repeat-dose data, these comparisons risk overreach.

(8) The readout is ELISA endpoint binding (IgG; and IgA in BALF for one antigen), which is fine for an initial immunogenicity screen. But the manuscript then drifts toward "vaccine strategy" claims without showing any antibody functionality (opsonophagocytosis, complement deposition, neutralization, blocking adhesion, and so on) or even binding to a more native-like antigen format (e.g., D-peptide displayed on particles; D-protein fragments; or any surrogate that goes beyond plate-bound peptide).

(9) The methods report peptide conjugates containing ~10-200 EU/mL endotoxin. That is not trivial and could materially amplify immunogenicity, and should be discussed.

(10) The authors should report how many technical/biological replicates were performed for MIC determinations and for ELISAs.

Reviewer #3 (Public review):

Summary:

There is a threat of mirror life bacteria, which could possibly evade immunity and cause problems for human/animal hosts. This paper evaluates enantiomeric antibiotics and vaccines as a means to understand how this could be combatted in the future.

Strengths:

It is valuable to collect such information, as it is not always clear how an antibiotic in its enantiomeric form would interact with a bacterium in terms of its MIC or towards toxicity. The paper is scientifically sound with regard to assays and statistical methods.

Weaknesses:

The beginning of the paper could be described as hyperbolic. For a paper that demonstrates that mirror-image molecules have (expected) lower MICs and toxicity, some of the claims in the beginning that they are going to cause a pandemic of evading the immune system seem to be a bit overstated. If they are mirror images, how are these bacteria going to generate virulence factors or mediate pathogenesis mechanisms? It seems like the lack of adaptation would go both ways - supported by the empirical data gathered in this manuscript. There is also the issue of only relatively simple and accessible mirror-image antibiotics being available. This is a limitation that - to their credit - the authors do discuss in the discussion section.