Reviewer #4 (Public review):

The paper by Xie et al. investigates the micro-evolutionary dynamics of sex-biased gene expression across somatic and gonadal tissues in four mouse taxa, with comparative analyses in humans. The study introduces a new metric, the Sex-Bias Index (SBI), to quantify individual-level variation in sex-biased gene expression, and explores the evolutionary turnover, variance, and adaptive evolution of these genes.

These strengths of the paper are not in dispute:

Novelty: The study is among the first to systematically analyze sex-biased gene expression at a micro-evolutionary scale in outbred animals, using closely related mouse taxa. This contrasts with most previous work, which focused on macro-evolutionary comparisons between distant species.

Controlled Sampling: The use of age-matched, outbred individuals raised under standardized conditions minimizes environmental confounders, allowing for robust within- and between-taxon comparisons.

Somatic vs. Gonadal Focus: Unlike many earlier studies that emphasized gonadal tissues, this work provides a detailed analysis of somatic organs, revealing rapid evolutionary turnover and mosaicism in sex-biased gene expression.

Sex-Bias Index (SBI): The SBI offers a cumulative, individual-level measure of sex-biased gene expression, facilitating visualization of variance and overlap between sexes within tissues. While one can argue about whether a new metric is necessary (as the authors argue), the combination of fold-change cutoffs, non-parametric Wilcoxon tests, and FDR correction reduces false positives, addressing concerns raised in the field about inflated detection of sex-biased genes.

Evolutionary implications: The study demonstrates that sex-biased gene expression in somatic tissues evolves more rapidly than in gonads, and that this turnover is often accompanied by signatures of adaptive protein evolution. The lack of correlation in SBI across tissues within individuals supports a mosaic model of sex-biased gene expression, challenging binary models of sexual differentiation.

The weaknesses are already listed by previous rounds of review but I will add one more: in an attempt to be comprehensive, the writing is quite dry and the main conclusions sort of get hidden within the less important observations.

Since the debate is mostly about what words to use to describe the importance and the strength of evidence, I thought it would be useful to directly compare this study to other studies that address the same topic:

Naqvi et al. Science 2019 (David Page lab): Conservation, acquisition, and functional impact of sex-biased gene expression in mammals

Oliva et al. Science 2020 (Stranger lab): The impact of sex on gene expression across human tissues

Rodríguez-Montes et al. Science 2023 (Kaessman, Cardoso-Moreira labs)

Let's start with the fact that all three peer studies have had a major impact. Second, although Naqvi et al. (2019) and Oliva et al. (2020) provided foundational cross-species and cross-tissue analyses of sex-biased gene expression, but did not address micro-evolutionary turnover or individual-level variance. Third, Rodríguez-Montes et al. (2023) focused on developmental and evolutionary patterns of sex-biased expression, but at a broader phylogenetic scale and without the individual-level or module-based analyses presented here. None of the peer studies addressed the possibility of mosaicism within individuals, none of them addressed the relations between expression bias and adaptive evolution. So the comparison is really a bit of an apples to oranges comparison: the peer studies are about patterns in deep phylogeny, whereas the present study is an amazing (to me) analysis of inter-individual mosaicism, which is at the heart of this kind of variation, which would totally be missed or worse misinterpreted in deep phylogenetic analyses. Having said that, in my subjective opinion, all three related papers are better written than the present one, but to me there is no question this belongs in the same pedestal as all of them.

Reviewer #1 (Public review):

Summary:

In this manuscript, the roles of the insulin receptor and the insulin growth factor receptor were investigated in podocytes. Mice in which both receptors were deleted developed glomerular dysfunction and developed proteinuria and glomerulrosclerosis over several months. Because of concerns about incomplete KO, the authors generated podocyte cell lines where both receptors were deleted. Loss of both receptors was highly deleterious with greater than 50% cell death. To elucidate the mechanism, the authors performed global proteomics and find that spliceosome proteins are down-regulated. They confirm this by using long-range sequencing. These results suggest a novel role for these pathways in podocytes.

This is primarily a descriptive study. The mechanism of how insulin and IGF1 signaling are linked to the spliceosome is not addressed and the phenotype of the mice is only superficially explored. The main issues are that the completeness of the mouse KO is never assessed nor is the completeness of the KO in cell lines. The absence of this data is a significant weakness. The mouse experiments would be improved if the serum creatinines were measured to provide some idea about the severity of the kidney injury. An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful. If this didn't rescue the phenotype, an explanation in the text would suffice. As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on. Lastly, in the cell line experiments, the authors should discuss the caveats associated with studying the 50% of the cells that survive vs the ones that died.

Significance:

With the GLP1 agonists providing renal protection, there is great interest in understanding the role of insulin and other incretins in kidney cell biology. It is already known that Insulin and IGFR signaling play important roles in other cells of the kidney, therefore, there is great interest in understanding these pathways in podocytes. The major advance is that these two pathways appear to have a role in RNA metabolism, the major limitations are the lack of information regarding the completeness of the KO's. If, for example, they can determine that in the mice, the KO is complete, that the GFR is relatively normal, then the phenotype they describe is relatively mild.

Comments on revision plan:

I agree with the suggested experiments especially, the experiments to examine whether insulin/IGF1 signaling have effects on splicing proteins. An alternative experiment would be to ask whether rescue of IR or IGF1R would ameliorate the splicing effects.

Reviewer #1 (Public review):

This study by Gangadharan and colleagues provides significant progress towards a quantitative biochemical mechanism for Stu2 polymerase activity. A key conceptual advance is the novel application of an enzyme-like model, initially developed for the actin polymerase Ena/VASP, to Stu2.

New refined affinity measurements for a Stu2 TOG domain using Bio-layer interferometry show more than an order of magnitude higher affinity of TOG domains to tubulin compared to previously published reports.

The findings reinforce the "concentrating reactants" or, more specifically, for TOG-domain proteins, the "tubulin-shuttling antenna" model, compared to the "polarized unfurling" model, a more speculative structural hypothesis.

The manuscript builds upon a series of previous manuscripts that showcase the profound intellectual engagement with microtubule polymerization mechanisms by TOG-domain proteins from the Rice lab, a thought leader in microtubule polymerization for over a decade.

Minor remarks:

(1) A major new experimental finding of this paper is the affinity of TOG domains, which is more than an order of magnitude lower (10 nM) than previous measurements from the same lab (~200 nM). The authors attribute this change to ionic strength differences between buffer conditions, citing the lab's previous work (Ayaz et al., 2014). This argument left me contemplating what the buffer conditions are in both experiments, and I wonder if other readers would feel the same. After going down the rabbit hole, I believe the difference in ionic strength is ~2.3 fold, and at least on the back of my envelope, this works out beautifully with the measured differences in affinities. A short version of this argument may strengthen the manuscript.

(2) I am wondering if there may be an alternative explanation to tubulin binding by TOG being the kinetically rate-limiting step for polymerase function:

TOG + Tubulin ⇌ TOG:Tubulin (fast binding rate, high-affinity binding)<br /> TOG:Tubulin + MT_end → TOG:MT (tubulin is incorporated into MT, fast transfer rate)<br /> The binding rate is 3/s, and the transfer rate is 5/s.

I was wondering if the following step should be considered, which involves a conformational change of tubulin (e.g., straightening) TOG:MT → TOG + MT (rate-limiting straightening and unbinding of TOG from the lattice).

Presumably, the affinity of TOGs for straight tubulin is practically zero for the purpose of this discussion, as there is no lattice binding, which means unbinding is likely very rapid; however, straightening may be the rate-limiting factor here.

In theory, straightening should also be rapid; however, we lack measurements of how fast or slow this step occurs within the context of a TOG domain, which presumably skews the process towards curved tubulin.

A hypothetical Stu2, when bound to the microtubule end and with the TOG domain not disengaged from tubulin, would not permit the processivity of that molecule or the binding of a new molecule.<br /> To emphasize the importance of unbinding, when it is not efficient, as reported for the T238 mutant that results in Stu2 lattice binding (Geyer et al., 2018), the polymerase becomes inefficient.

Reviewer #1 (Public review):

Summary:

This study on potassium ion transport by the protein complex KdpFABC from E. coli reveals a 2.1 Å cryo-EM structure of the nanodisc-embedded transporter under turnover conditions. The results confirm that K+ ions pass through a previously identified tunnel that connects the channel-like subunit with the P-type ATPase-type subunit.

Strengths:

The excellent resolution of the structure and the thorough analysis of mutants using ATPase and ion transport measurements help to strengthen new and previous interpretations. The evidence supporting the conclusions is solid, including biochemical assays and analysis of mutants. The work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis.

Weaknesses:

There is insufficient credit and citation of previous work.

Reviewer #1 (Public review):

In this study, Ma et al. aimed to determine previously uncharacterized contributions of tissue autofluorescence, detector afterpulse, and background noise on fluorescence lifetime measurement interpretations. They introduce a computational framework they named "Fluorescence Lifetime Simulation for Biological Applications (FLiSimBA)" to model experimental limitations in Fluorescence Lifetime Imaging Microscopy (FLIM) and determine parameters for achieving multiplexed imaging of dynamic biosensors using lifetime and intensity. By quantitatively defining sensor photon effects on signal to noise in either fitting or averaging methods of determining lifetime, the authors contradict any claims of FLIM sensor expression insensitivity to fluorescence lifetime and highlight how these artifacts occur differently depending on analysis method. Finally, the authors quantify how statistically meaningful experiments using multiplexed imaging could be achieved.

A major strength of the study is the effort to present results in a clear and understandable way given that most researcher do not think about these factors on a day-to-day basis. Additionally, the model code is readily available in Matlab and Python, which should allow for open access to a larger community.

Overall, the authors' achieved their aims of demonstrating how common factors (autofluorescence, background, and sensor expression) will affect lifetime measurements and they present a clear strategy for understanding how sensor expression may confound results if not properly considered. This work should bring to awareness an issue that new users of lifetime biosensors may not be aware of and that experts, while aware, have not quantitatively determine the conditions where these issues arise. This work will also point to future directions for improving experiments using fluorescence lifetime biosensors and the development of new sensors with more favorable properties.

Joint Public Review:

Summary:

In this paper the authors examined the effects of strip cropping, a relatively new agricultural technique of alternating crops in small strips of several meters wide, on ground beetle diversity. The results show an increase in species diversity (i.e. abundance and species richness) of the ground beetle communities compared to monocultures.

Strengths:

The article is well written; it has an easily readable tone of voice without too much jargon or overly complicated sentence structure. Moreover, as far as reviewing the models in depth without raw data and R scripts allows, the statistical work done by the authors looks good. They have well thought out how to handle heterogenous, unbalanced and taxonomically unspecific yet spatially and temporarily correlated field data. The models applied and the model checks performed are appropriate for the data at hand. Combining RDA and PCA axes together is a nice touch. Moreover, after the first round of reviews, the authors have done a great job at rewriting the paper to make it less overstated, more relevant to the data at hand and more solid in the findings. Many of the weaknesses noted in the first review have been dealt with. The overall structure of the paper is good, with a clear introduction, hypotheses, results section and discussion.

Reviewer #1 (Public review):

Summary:

The authors report four cryoEM structures (2.99 to 3.65 Å resolution) of the 180 kDa, full-length, glycosylated, soluble Angiotensin-I converting enzyme (sACE) dimer, with two homologous catalytic domains at the N- and C-terminal ends (ACE-N and ACE-C). ACE is a protease capable of effectively degrading Aβ. The four structures are C2 pseudo-symmetric homodimers and provide insight into sACE dimerization. These structures were obtained using discrete classification in cryoSPARC and show different combinations of open, intermediate, and closed states of the catalytic domains, resulting in varying degrees of solvent accessibility to the active sites.

To deepen the understanding of the gradient of heterogeneity (from closed to open states) observed with discrete classification, the authors performed all-atom MD simulations and continuous conformational analysis of cryo-EM data using cryoSPARC 3DVA, cryoDRGN, and RECOVAR. cryoDRGN and cryoSPARC 3DVA revealed coordinated open-closed transitions across four catalytic domains, whereas RECOVAR revealed independent motion of two ACE-N domains, also observed with cryoSPARC focused classification. The authors suggest that the discrepancy in the results of the different methods for continuous conformational analysis in cryo-EM could results from different approaches used for dimensionality reduction and trajectory generation in these methods.

Strengths:

This is an important study that shows, for the first time, the structure and the snapshots of the dynamics of the full-length sACE dimer. Moreover, the study highlights the importance of combining insights from different cryo-EM methods that address questions difficult or impossible to tackle experimentally, while lacking ground truth for validation.

Weaknesses (from the last round of review):

The open, closed, and intermediate states of ACE-N and ACE-C in the four cryo-EM structures from discrete classification were designated quantitatively (based on measured atomic distances on the models fitted into cryo-EM maps). Unfortunately, atomic models were not fitted into cryo-EM maps obtained with cryoSPARC 3DVA, cryoDRGN, and RECOVAR, and the open/closed states in these cases were designated based on a qualitative analysis.

Reviewer #1 (Public review):

Summary:

In their study, the authors investigated the F. graminearum homologue of the Drosophila Misato-Like Protein DML1 for a function in secondary metabolism and sensitivity to fungicides.

Strengths:

Generally, the topic of the study is interesting and timely, and the manuscript is well written, albeit in some cases, details on methods or controls are missing.

Weaknesses:

However, a major problem I see is with the core result of the study, the decrease in the DON content associated with the deletion of FgDML1. Although some growth data are shown in Figure 6, indicating a severe growth defect, the DON production presented in Figure 3 is not related to biomass. Also, the method and conditions for measuring DON are not described. Consequently, it could well be concluded that the decreased amount of DON detected is simply due to decreased growth, and the specific DON production of the mutant remains more or less the same.

To alleviate this concern, it is crucial to show the details on the DON measurement and growth conditions and to relate the biomass formation under the same conditions to the DON amount detected. Only then can a conclusion as to an altered production in the mutant strains be drawn.

Reviewer #1 (Public review):

Summary:

The authors make a bold claim that a combination of repetitive transcranial magnetic stimulation (intermittent theta burst-iTBS) and transcranial alternating current stimulation (gamma tACS) causes slight improvements in memory in a face/name/profession task.

Strengths:

The idea of stimulating the human brain non-invasively is very attractive because, if it worked, it could lead to a host of interesting applications. The current study aims to evaluate one such exciting application.

Weaknesses:

(1) The title refers to the "precuneus-hippocampus" network. A clear definition of what is meant by this terminology is lacking. More importantly, mechanistic evidence that the precuneus and the hippocampus are involved in the potential effects of stimulation remains unconvincing.

(2) The question of the extent to which the stimulation approach and the stimulation parameters used in these experiments causes specific and functionally relevant neural effects remains open. Invasive recordings that could address this question remain out of the scope of this non-invasive study. The authors conducted scalp EEG experiments in an attempt to address this question using non-invasive methods. However, the results shown in Fig. 3 are unclear. The results are inconsistently reported in units of microvolts squared in some panels (3A, 3B) and in units of microvolts in other panels (3C). Also, there is insufficient consideration of potential contamination by signal components reflecting eye movements, other muscle artifacts, or another volume-conducted signal reflecting aggregate activity inside the brain.

(3) Figure 3 indicates "Precuneus oscillatory activity ...", but evidence that the activity presented reflects precuneus activity is lacking. The maps shown at the bottom of Figure 3C suggest that the EEG signals recorded with scalp EEG reflect activity generated across a wide spatial range, with a peak encompassing at least tens of centimeters. Thus, evidence that effects specifically reflect precuneus activity, as the paper's title and text throughout the manuscript suggest, is lacking.

(4) The paper as currently presented (e.g., Figure 3) also lacks rigorous evidence of relevant oscillatory activity. Prior to filtering EEG signals in a particular frequency band, clear evidence of oscillations in the frequency band of interest should be shown (e.g., demonstration of a clear peak that emerges naturally in the frequency range of interest when spectral analysis is applied to "raw" signals). The authors claim that gamma oscillations change because of the stimulation, but a clear peak in the gamma range prior to stimulation is not apparent in the data as currently presented. Thus, the extent to which spectral measurements during stimulation reflect physiological gamma oscillations remains unclear.

(5) Concerns remain regarding the rigor of statistical analyses in the revised manuscript (see also point 8 below). Figure 3B shows an undefined statistical test with p<0.05. The statistical test that was used is not explained. Also, a description of how corrections for multiple comparisons were made is missing. Figures 3A and 3C are not accompanied by statistics, making the results difficult to interpret. For Figure 4C, a claim was made based on a significant p-value for one statistical test and a non-significant p-value in another test. This is a common statistical mistake (see Figure 1 and accompanying discussion in Makin and Orban de Xivry (2019) Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript. eLife 8:e48175).

(6) In the second question posed in the original review, I highlighted that it was unclear how such stimulation would produce memory enhancement. The authors replied that, in the absence of mechanisms, there are many other studies that suffer from the same problem. This raises the question of placebo effects. The paper does not sufficiently address or discuss the possibility that any potential stimulation effects may reflect placebo effects.

(7) The third major concern in the original review was the lack of evidence for a mechanism that is specific to the precuneus. Evidence for specific involvement of the precuneus remains lacking in the revised manuscript. The authors state: "the non-invasive stimulation protocol was applied to an individually identified precuneus for each participant". However, the meaning of this statement is unclear. Specifically, it is unclear how the authors know that they are specifically targeting the precuneus. Without directly recording from the precuneus and directly demonstrating effects, which is outside of the scope of the study, specific involvement of the precuneus seems speculative. Also, it does not seem as though a figure was included in the paper to show how the stimulation protocol specifically targets the precuneus. In their response to the original reviews, the authors state that posterior medial parietal areas are the only regions that show significant differences following the stimulation, but they did not cite a specific figure, or statistics reported in the text, that show this. In any event, posterior medial parietal areas encompass a wide area of the brain, so this would still not provide evidence for an effect specifically involving the precuneus.

(8) Regarding chance levels, it is unfortunate that the authors cannot quantify what chance levels are in the immediate and delayed recall conditions. This makes interpretation of the results challenging. In the immediate and delayed conditions, the authors state that the chance level is 33%. It would be useful to mark this in the figures. If I understand correctly, chance is 33% in Fig. 2A. If this is the case and if I am interpreting the figure correctly:<br /> Gray bars for the sham condition appear to be below chance (~20-25%). Why is this condition associated with an accuracy level that is lower than chance?<br /> Cyan bars and red bars do not appear to be significantly different from chance (i.e., 33%), with red slightly higher than cyan. What statistic was performed to obtain the level of significance indicated in the figure? The highest average value for the red condition appears to be around 35%. More details are needed to fully explain this figure and to support the claims associated with this figure.

(9) In the revised version of the paper, the authors did not address concerns associated with the block design (please see question 4d in the original review).

In sum, this study presents an admirable aspirational goal, the notion that a non-invasive stimulation protocol could modulate activity in specific brain regions to enhance memory. However, the evidence presented at the behavioral level and at the mechanistic level (e.g. the putative involvement of specific brain regions) remains unconvincing.

Reviewer #1 (Public review):

Summary:

The authors use longitudinal in vivo 1-photon calcium recordings in mouse prefrontal cortex throughout the learning of an odor-guided spatial memory task, with the goal of examining the development of task-related prefrontal representations over the course of learning in different task stages and during sleep sessions. They report replication of their previous results, Muysers et al. 2025, that task and representations in prefrontal cortex arise de novo after learning, comprising of goal selective cells that fire selectively for left or right goals during the spatial working memory component of the task, and generalized task phase selective cells that fire equivalently in the same place irrespective of goal, together comprising task-informative cells. The number of task-informative cells increases over learning, and covariance structure changes resulting in increased sequential activation in the learned condition, but with limited functional relevance to task representation. Finally, the authors report that similar to hippocampal trajectory replay, prefrontal sequences are replayed at reward locations.

Strengths:

The major strength of the study is the use of longitudinal recordings, allowing identification of task-related activity in the prefrontal cortex that emerges de novo after learning, and identification of sub-second sequences at reward wells.

Weaknesses:

(1) The study mainly replicates the authors' previously reported results about generalized and trajectory-specific coding of task structure by prefrontal neurons, and stable and changing representations over learning (Muysers et al., 2024, PMID: 38459033; Muysers et al., 2025, PMID: 40057953), although there are useful results about changes in goal-selective and task-phase selective cells over learning. There are basic shortcomings in the scientific premise of two new points in this manuscript, that of the contribution of pre-existing spatial representations, and the role of replay sequences in the prefrontal cortex, both of which cannot be adequately tested in this experimental design.

(2) The study denotes neurons that show precise spatial firing equivalently irrespective of goal, as generalized task representations, and uses this as a means to testing whether pre-existing spatial representations can contribute to task coding and learning. A previous study using this data has already shown that these neurons preferentially emerge during task learning (Muysers et al., 2025, PMID: 40057953). Furthermore, in order to establish generalization for abstract task rules or cognitively flexibility, as motivated in the manuscript, there is a need to show that these neurons "generalize" not just to firing in the same position during learning of a given task, but that they can generalize across similar tasks, e.g., different mazes with similar rules, different rules with similar mazes, new odor-space associations, etc. For an adequate test of pre-existing spatial structure, either a comparison task, as in the examples above, is needed, or at least a control task in which animals can run similar trajectories without the task contingencies. An unambiguous conclusion about pre-existing spatial structure is not possible without these controls.

(3) The scientific premise for the test of replay sequences is motivated using hippocampal activity in internally guided spatial working memory rule tasks (Fernandez-Ruiz et al., 2019, PMID: 31197012; Kay et al., PMID: 32004462; Tang et al., 2021, PMID: 33683201), and applied here to prefrontal activity in a sensory-cue guided spatial memory task (Muysers et al., 2024, PMID: 38459033; Symanski et al., PMID: 36480255; Taxidis et al, 2020, PMID: 32949502). There are several issues with the conclusion in the manuscript that prefrontal replay sequences are involved in evaluating behavioral outcomes rather than planning future outcomes.

(4) First, odor sampling in odor-guided memory tasks is an active sensory processing state that leads to beta and other oscillations in olfactory regions, hippocampus, prefrontal cortex, and many other downstream networks, as documented in a vast literature of studies (Martin et al., 2007, PMID: 17699692; Kay, 2014, PMID: 24767485; Martin et al., 2014; Ramirez-Gordillo, 2022, PMID: 36127136; Symanski et al., 2022, PMID: 36480255). This is an active sensory state, not conducive to internal replay sequences, unlike references used in this manuscript to motivate this analysis, which are hippocampal spatial memory studies with internally guided rather than sensory-cue guided decisions, where internal replay is seen during immobility at reward wells. These two states cannot be compared with the expectation of finding similar replay sequences, so it is trivially expected that internal replay sequences will not be seen during odor sampling.

(5) Second, sequence replay is not the only signature of reactivation. Many studies have quantified prefrontal replay using template matching and reactivation strength metrics that do not involve sequences (Peyrache et al., 2009, PMID: 19483687; Sun et al., 2024, PMID: 38872470). Third, previous studies have explicitly shown that prefrontal activity can be decoded during odor sampling to predict future spatial choices - this uses sensory-driven ensemble activity in prefrontal cortex and not replay, as odor sampling leads to sensory driven processing and recall rather than a reactivation state (Symanski et al., 2022, PMID: 36480255). It is possible that 1-photon recordings do not have the temporal resolution and information about oscillatory activity to enable these kinds of analyses. Therefore, an unambiguous conclusion about the existence and role of prefrontal reactivation is not possible in this experimental and analytical design.

Reviewer #1 (Public review):

Summary:

This manuscript provides an open-source tool including hardware and software, and a dataset to facilitate and standardize behavioral classification in laboratory mice. The hardware for behavioral phenotyping was extensively tested for safety. The software is GUI-based, facilitating the usage of this tool across the community of investigators who do not have a programming background. The behavioral classification tool is highly accurate, and the authors deposited a large dataset of annotations and pose tracking for many strains of mice. This tool has great potential for behavioral scientists who use mice across many fields; however, there are many missing details that currently limit the impact of this tool and publication.

Strengths:

(1) There is software-hardware integration for facilitating cross-lab adaptation of the tool and minimizing the need to annotate new data for behavioral classification.

(2) Data from many strains of mice were included in the classification and genetic analyses in this manuscript.

(3) A large dataset was annotated and deposited for the use of the community.

(4) The GUI-based software tool decreases barriers to usage across users with limited coding experience.

Weaknesses:

(1) The authors only report the quality of the classification considering the number of videos used for training, but not considering the number of mice represented or the mouse strain. Therefore, it is unclear if the classification model works equally well in data from all the mouse strains tested, and how many mice are represented in the classifier dataset and validation.

(2) The GUI requires pose tracking for classification, but the software provided in JABS does not do pose tracking, so users must do pose tracking using a separate tool. Currently, there is no guidance on the pose tracking recommendations and requirements for usage in JABS. The pose tracking quality directly impacts the classification quality, given that it is used for the feature calculation; therefore, this aspect of the data processing should be more carefully considered and described.

(3) Many statistical and methodological details are not described in the manuscript, limiting the interpretability of the data presented in Figures 4,7-8. There is no clear methods section describing many of the methods used and equations for the metrics used. As an example, there are no details of the CNN used to benchmark the JABS classifier in Figure 4, and no details of the methods used for the metrics reported in Figure 8.

Reviewer #1 (Public review):

The authors note that very premature infants experience the visual world early and, as a consequence, sustain lasting deficits including compromised motion processing. Here they investigate the effects of early eye opening in ferret, choosing a time point after birth when both retinal waves and light traveling through closed lids drive sensory responses. The laboratory has long experience in quantitative studies of visual response properties across development and this study reflects their expertise.

The investigators find little or no difference in mean orientation and direction selectivity, or in spatial frequency tuning, as a result of early eye opening but marked differences in temporal frequency tuning. These changes are especially interesting as they relate to deficits seen in prematurely delivered children. Temporal frequency bandwidth for responses evoked from early-opened contralateral eyes were broader than for controls; this is the case for animals in which either one or both eyes were opened prematurely. Further, when only one eye was opened early, responses to low temporal frequencies were relatively stronger.

The investigators also found changes in firing rate and sign of response to visual stimuli. Premature eye-opening increased spontaneous rates in all test configurations. When only one eye was opened early, firing rates recorded from the ipsilateral cortex were strongly suppressed, with more modest effects in other test cases.

As the authors' discussion notes, these observations are just a starting point for studies underlying mechanism. The experiments are so difficult to perform and so carefully described that the results will be foundational for future studies of how premature birth influences cortical development.

Joint Public Review:

Summary:

This work aims to improve our understanding of the factors that influence female-on-female aggressive interactions in gorilla social hierarchies, using 25 years of behavioural data from five wild groups of two gorilla species. Researchers analysed aggressive interactions between 31 adult females, using behavioural observations and dominance hierarchies inferred through Elo-rating methods. Aggression intensity (mild, moderate, severe) and direction (measured as the rank difference between aggressor and recipient) were used as key variables. A linear mixed-effects model was applied to evaluate how aggression direction varied with reproductive state (cycling, trimester-specific pregnancy, or lactation) and sex composition of the group. This study highlights the direction of aggressive interactions between females, with most interactions being directed from higher- to lower-ranking adult females close in social rank. However, the results show that 42% of these interactions are directed from lower- to higher-ranking females. Particularly, lactating and pregnant females targeted higher-ranking individuals, which the authors suggest might be due to higher energetic needs, which increase risk-taking in lactating and pregnant females. Sex composition within the group also influenced which individuals were targeted. The authors suggest that male presence buffers female-on-female aggression, allowing females to target higher-ranking females than themselves. In contrast, females targeted lower-ranking females than themselves in groups with a larger ratio of females, which supposes a lower risk for the females since the pool of competitors is larger. The findings provide an important insight into aggression heuristics in primate social systems and the social and individual factors that influence these interactions, providing a deeper understanding of the evolutionary pressures that shape risk-taking, dominance maintenance, and the flexibility of social strategies in group-living species.

The authors achieved their aim by demonstrating that aggression direction in female gorillas is influenced by factors such as reproductive condition and social context, and their results support the broader claim that aggression heuristics are flexible. However, some specific interpretations require further support. Despite this, the study makes a valuable contribution to the field of behavioural ecology by reframing how we think about intra-sexual competition and social rank maintenance in primates.

Strengths:

One of the study's major strengths is the use of an extensive dataset that compiles 25 years of behavioural data and 6871 aggressive interactions between 31 adult females in five social groups, which allows for a robust statistical analysis. This study uses a novel approach to the study of aggression in social groups by including factors such as the direction and intensity of aggressive interactions, which offers a comprehensive understanding of these complex social dynamics. In addition, this study incorporates ecological and physiological factors such as the reproductive state of the females and the sex composition of the group, which allows an integrative perspective on aggression within the broader context of body condition and social environment. The authors successfully integrate their results into broader evolutionary and ecological frameworks, enriching discussions around social hierarchies and risk sensitivity in primates and other animals.

Reviewer #1 (Public review):

Summary:

Can a plastic RNN serve as a basis function for learning to estimate value. In previous work this was shown to be the case, with a similar architecture to that proposed here. The learning rule in previous work was back-prop with an objective function that was the TD error function (delta) squared. Such a learning rule is non-local as the changes in weights within the RNN, and from inputs to the RNN depends on the weights from the RNN to the output, which estimates value. This is non-local, and in addition, these weights themselves change over learning. The main idea in this paper is to examine if replacing the values of these non-local changing weights, used for credit assignment, with random fixed weights can still produce similar results to those obtained with complete bp. This random feedback approach is motivated by a similar approach used for deep feed-forward neural networks.

This work shows that this random feedback in credit assignment performs well but is not as well as the precise gradient-based approach. When more constraints due to biological plausibility are imposed performance degrades. These results are consistent with previous results on random feedback.

Strengths:

The authors show that random feedback can approximate well a model trained with detailed credit assignment.

The authors simulate several experiments including some with probabilistic reward schedules and show results similar to those obtained with detailed credit assignments as well as in experiments.

The paper examines the impact of more biologically realistic learning rules and the results are still quite similar to the detailed back-prop model.

Reviewer #1 (Public Review):

The authors reported that mutations were identified in the ZC3H11A gene in four adolescents from 1015 high myopia subjects in their myopia cohort. They further generated Zc3h11a knockout mice utilizing the CRISPR/Cas9 technology.

The main claims are only partially supported. The reviewers still have the concerns of 1) the modes of inheritance for the families need to be shown; 2) the phenotype of heterozygous mutant mice is too weak; 3) the authors still have not addressed the biological question of whether there are fewer bipolar cells or decreased expression of the marker protein. This would involve counting cells, which they have not done. The blots they show do not appear to support their quantifications. Considering the sensitivity of quantifying nearly saturated blots, the authors should show blots that are not exposed to that level of saturation.

Reviewer #1 (Public review):

Summary:

This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

Strengths/weaknesses:

The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of the neural symptons in ciliopathy patients.

Comments on revisions:

The authors addressed most of the points I raised in my original review.

Reviewer #1 (Public review):

Summary:

The study by Zhuomin Yin and colleagues focuses on the relationship between cell-free HPV (cfHPV) DNA and metastatic or recurrent cervical cancer patients. It expands the application of cfHPV DNA in tracking disease progression and evaluating treatment response in cervical cancer patients. The study is overall well-designed, including appropriate analyses.

Strengths:

The findings provide valuable reference points for monitoring drug efficacy and guiding treatment strategies in patients with recurrent and metastatic cervical cancer. The concordance between HPV cfDNA fluctuations and changes in disease status suggests that cfDNA could play a crucial role in precision oncology, allowing for more timely interventions. As with similar studies, the authors used Droplet Digital PCR to measure cfDNA copy numbers, a technique that offers ultrasensitive nucleic acid detection and absolute quantification, lending credibility to the conclusions.

Weaknesses:

Despite including 28 clinical cases, only 7 involved recurrent cervical cancer, which may not be sufficient to support some of the authors' conclusions fully. Future studies on larger cohorts could solidify HPV cfDNA's role as a standard in the personalized treatment of recurrent cervical cancer patients.

Comments on revisions:

Thanks for your additional efforts and for addressing my concerns.

Reviewer #1 (Public review):

Summary:

Chao et al. produced an updated version of the SpliceAI package using modern deep learning frameworks. This includes data preprocessing, model training, direct prediction, and variant effect prediction scripts. They also added functionality for model fine-tuning and model calibration. They convincingly evaluate their newly trained models against those from the original SpliceAI package and investigate how to extend SpliceAI to make predictions in new species. While their comparisons to the original SpliceAI models are convincing on the grounds of model performance, their evaluation of how well the new models match the original's understanding of non-local mutation effects is incomplete. Further, their evaluation of the new calibration functionality would benefit from a more nuanced discussion of what set of splice sites their calibration is expected to hold for, and tests in a context for which calibration is needed.

Strengths:

(1) They provide convincing evidence that their new implementation of SpliceAI matches the performance of the original model on a similar dataset while benefiting from improved computational efficiencies. This will enable faster prediction and retraining of splicing models for new species as well as easier integration with other modern deep learning tools.

(2) They produce models with strong performance on non-human model species and a simple, well-documented pipeline for producing models tuned for any species of interest. This will be a boon for researchers working on splicing in these species and make it easy for researchers working on new species to generate their own models.

(3) Their documentation is clear and abundant. This will greatly aid the ability of others to work with their code base.

Weaknesses:

(1) The authors' assessment of how much their model retains SpliceAI's understanding of "non-local effects of genomic mutations on splice site location and strength" (Figure 6) is not sufficiently supported. Demonstrating this would require showing that for a large number of (non-local) mutations, their model shows the same change in predictions as SpliceAI or that attribution maps for their model and SpliceAI are concordant even at distances from the splice site. Figure 6A comes close to demonstrating this, but only provides anecdotal evidence as it is limited to 2 loci. This could be overcome by summarizing the concordance between ISM maps for the two models and then comparing across many loci. Figure 6B also comes close, but falls short because instead of comparing splicing prediction differences between the models as a function of variants, it compares the average prediction difference as a function of the distance from the splice site. This limits it to only detecting differences in the model's understanding of the local splice site motif sequences. This could be overcome by looking at comparisons between differences in predictions with mutants directly and considering non-local mutants that cause differences in splicing predictions.

(2) The utility of the calibration method described is unclear. When thinking about a calibrated model for splicing, the expectation would be that the models' predicted splicing probabilities would match the true probabilities that positions with that level of prediction confidence are splice sites. However, the actual calibration that they perform only considers positions as splice sites if they are splice sites in the longest isoform of the gene included in the MANE annotation. In other words, they calibrate the model such that the model's predicted splicing probabilities match the probability that a position with that level of confidence is a splice site in one particular isoform for each gene, not the probability that it is a splice site more broadly. Their level of calibration on this set of splice sites may very well not hold to broader sets of splice sites, such as sites from all annotated isoforms, sites that are commonly used in cryptic splicing, or poised sites that can be activated by a variant. This is a particularly important point as much of the utility of SpliceAI comes from its ability to issue variant effect predictions, and they have not demonstrated that this calibration holds in the context of variants. This section could be improved by expanding and clarifying the discussion of what set of splice sites they have demonstrated calibration on, what it means to calibrate against this set of splice sites, and how this calibration is expected to hold or not for other interesting sets of splice sites. Alternatively, or in addition, they could demonstrate how well their calibration holds on different sets of splice sites or show the effect of calibrating their models against different potentially interesting sets of splice sites and discuss how the results do or do not differ.

(3) It is difficult to assess how well their calibration method works in general because their original models are already well calibrated, so their calibration method finds temperatures very close to 1 and only produces very small and hard to assess changes in calibration metrics. This makes it very hard to distinguish if the calibration method works, as it doesn't really produce any changes. It would be helpful to demonstrate the calibration method on a model that requires calibration or on a dataset for which the current model is not well calibrated, so that the impact of the calibration method could be observed.

Reviewer #1 (Public review):

Summary:

This fundamental work employed multidisciplinary approaches and conducted rigorous experiments to study how a specific subset of neurons in the dorsal striatum (i.e., "patchy" striatal neurons) modulates locomotion speed depending on the valence of naturalistic contexts.

Strengths:

The scientific findings are novel and original and significantly advance our understanding of how the striatal circuit regulates spontaneous movement in various contexts.

Weaknesses:

This is extensive research involving various circuit manipulation approaches. Some of these circuit manipulations are not physiological. A balanced discussion of the technical strengths and limitations of the present work would be helpful and beneficial to the field.

Reviewer #1 (Public review):

Summary:

The paper describes the initial characterization of Eml3 knockout mice. Eml3 global inactivation leads to delayed embryonic development, perinatal lethality apparently due to failure to inflate lungs, and a cobblestone brain-like phenotype represented by focal neuronal ectopias in the marginal zone or subarachnoid space of dorsal telencephalon. The neural ectopias are associated with interruptions in the pial basal membrane (PBM), which appear around E11.5. The authors also confirmed previously described protein interactions, using coIP-MS experiments of placenta and embryonic tissues (TUBB3, several 14-3-3 proteins, and DYNLL). The authors generated mice carrying a TQT86AAA homozygous mutation in EML3 (a motif required for EML3-DYNLL interactions) that were normal and showed no focal neuronal ectopias, indicating that this particular protein interaction is dispensable. The authors propose Eml3 knockout mice as a model of cobblestone brain malformation.

Strengths:

The brain phenotype described in this work is relevant for the neural development field and with potential clinical relevance. The initial phenotyping is appropriate but will require additional experiments to establish the cause of the failure to inflate the lungs. The study shows convincing data regarding the main characteristics of the brain phenotype and data supporting the timing when these abnormalities arise during development.

Weaknesses:

The study would benefit from clearer evidence and additional experiments that would help to establish the molecular and cellular mechanisms underlying the brain phenotype, the central topic of the work.

Reviewer #1 (Public review):

During early Drosophila pupal development, a subset of larval abdominal muscles (DIOMs) is remodelled using an autophagy dependent mechanism.

To better understand this not very well studied process, the authors have generated a systematic transcriptomics time course using dissected larval abdominal muscles of various stages from wild type and autophagy deficient mutants. The authors have further identified a function for BNIP3 for executing mitophagy during DIOM remodelling.

Strengths:

The paper does provide a detailed mRNA time course resource for the DIOM remodelling.

The paper does find an interesting BNIP3 loss of function phenotype, a block of mitophagy during muscle remodelling and hence identifies a specific linker between mitochondria and the core autophagy

machinery. This adds to the mechanism how mitochondria are degraded.

Sophisticated fly genetics demonstrates that the larval muscle mitochondria are, to a large extend, degraded by autophagy during DIOM remodelling.

Quantitative electron microscopy data show that BNIP3 is required for initiating mito-phagosomes. It needs either its LIR and MER domain for function.

Weakness:

Mitophagy during DIOM remodelling is not novel (earlier papers from Fujita et al.).

Other weaknesses have been eliminated during the revision.

Reviewer #1 (Public review):

Summary:

This work uses a novel, ethologically relevant behavioral task to explore decision-making paradigms in C. elegans foraging behavior. By rigorously quantifying multiple features of animal behavior as they navigate in a patch food environment, the authors provide strong evidence that worms exhibit one of three qualitatively distinct behavioral responses upon encountering a patch: (1) "search", in which the encountered patch is below the detection threshold; (2) "sample", in which animals detect a patch encounter and reduce their motor speed, but do not stay to exploit the resource and are therefore considered to have "rejected" it; and (3) "exploit", in which animals "accept" the patch and exploit the resource for tens of minutes. Interestingly, the probability of these outcomes varies with the density of the patch as well as the prior experience of the animal. Together, these experiments provide an interesting new framework for understanding the ability of the C. elegans nervous system to use sensory information and internal state to implement behavioral state decisions.

Strengths:

The work uses a novel, neuroethologically-inspired approach to studying foraging behavior

The studies are carried out with an exceptional level of quantitative rigor and attention to detail

Powerful quantitative modeling approaches including GLMs are used to study the behavioral states that worms enter upon encountering food, and the parameters that govern the decision about which state to enter

The work provides strong evidence that C. elegans can make 'accept-reject' decisions upon encountering a food resource

Accept-reject decisions depend on the quality of the food resource encountered as well as on internally represented features that provide measurements of multiple dimensions of internal state, including feeding status and time.

Joint Public Review:

In this manuscript, Wafer and Tandon et al. present a thoughtful and well-designed genetic screen for regulators of adipose remodeling using zebrafish as a model system. The authors cross-referenced several human adipocyte-related transcriptomic and genetic association datasets to identify candidate genes, which they then tested in zebrafish. Importantly, the authors devised an unbiased microscopy-based screening platform to document quantitative adipose phenotypes with whole animal imaging, while also employing rigorous statistical methods. From their screen, the authors identified 6 genes that resulted in robust adipose phenotypes out of a total of 25 that were tested. Overall, this work will be a useful resource for the field because of both the genes identified and the quantitative, rigorous screening pipeline. However, there are limitations that preclude a definitive distinction between developmental and remodeling effects that should be acknowledged and discussed, or addressed with new experiments.

Strengths:

(1) This work combines multiple omic datasets to identify candidate genes that informed a CRISPR-based screen to identify genes underlying adipose tissue development and adaptation. This approach offers a new avenue to improve our understanding and testing of new genetic mechanisms underlying the development of obesity.

(2) Using a clever screening approach, this study identifies new genes that are associated with adipose tissue lipid droplet size change. Importantly, the study provides further validation using a stable CRISPR line to show the phenotype in basal and high-fat diet conditions.

(3) The experiments are well-designed and rigorous. Sample sizes are large. Statistical analyses are highly rigorous, contributing to a high-quality study.

Weaknesses:

(1) The image quantification established in Figures 3 and 4 and used in CRISPR screening showed the relationship among zebrafish development, adipose tissue size, and lipid droplet size. Although adipose tissue development patterning is linked with adipose tissue adaptation, as shown by the evidence provided in this paper, it will be more powerful if the imaging method and pipeline were established to directly access the adipose tissue plasticity rather than just the developmental patterning. Furthermore, the authors should perform additional analysis of their existing data to more accurately determine lipid droplet size along the AP axis in response to HFD.

(2) In the absence of tissue-specific manipulations, definitively establishing the mechanisms underlying the genetic regulation of adipose tissue physiology presents limitations.

Reviewer #1 (Public review):

Summary:

Two important factors in visual performance are the resolving power of the lens and the signal-to-noise ratio of the photoreceptors. These both compete for space: a larger lens has improved resolving power over a smaller one, and longer photoreceptors capture more photons and hence generate responses with lower noise. The current paper explores the tradeoff of these two factors, asking how space should be allocated to maximize eye performance (measured as encoded information).

The revisions, to my read, have greatly improved the paper. Most of this was due to setting clear expectations from the start of the paper. Nice work!

Reviewer #1 (Public review):

The objectives of this research are to understand how key selector transcription factors, Tal1, Gata2, Gata3, determine GABAergic vs glutamatergic neuron fate from the rhombencephalic V2 precursor domain and how their spatiotemporal expression is controlled by upstream regulators. Toward these goals, the authors have generated an impressive array of scRNA, scATAC-seq, and CUT&Tag datasets obtained from dissociated E12.5 ventral R1 dissections. The rV2 was subsetted with well-known markers. The authors use an extensive set of computational approaches to identify temporal patterns of chromatin accessibility, TF motif binding activities (footprints), gene expression and regulatory motifs at the different selector gene loci. These analyses are used to predict upstream regulators, candidate accessible CREs, and DNA binding motifs through which the selectors may be controlled in rV2 by upstream regulators. Further analyses predict auto- and cross-regulatory interactions for maintenance of selector expression and the downstream effectors of alternative transmitter identities controlled by the selectors. The authors have achieved their aim of making predictions about upstream and downstream selector TF regulatory networks; their conclusions and predictions are largely well supported. The work clearly illustrates the daunting gene regulatory complexity likely at play in controlling rV2 transmitter fate.

This is data-rich study and a valuable resource for future hypothesis testing, through perturbation approaches, of the many putative regulators and motifs identified in the study. The strengths of this work are the overall high quality of the datasets and in depth analyses. Through its comprehensive data and predictions, it is likely to have impact in advancing the understanding of GABAergic vs glutamatergic neuron fate decisions. The authors present a "simplified" gene regulatory model. However, the model does not illustrate the complexity of potential stage-specific upstream TF interactions with Tal1 and Vsx2 selector genes uncovered in TF footprinting analyses. While this seems nearly impossible to achieve given the plethora of potential functional TF inputs, the authors should consider assembling a focussed model by selectively illustrating the most robust, evidence-backed upstream TF input predictions, which are considered the strongest candidates for future hypothesis-driven perturbation experiments. It seems Insm1, Sox4, E2f1, Ebf1 and Tead2 TFs might be the strongest upstream candidates for future testing of Tal1 activation given the extensive analyses of their spatiotemporal expression patterns relative to Tal1, presented in Fig 4.

Reviewer #1 (Public review):

Summary:

In the present manuscript, Mashiko and colleagues describe a novel phenotype associated with deficient SLC35G3, a testis-specific sugar transporter that is important in glycosylation of key proteins in sperm function. The study characterizes a knockout mouse for this gene and the multifaceted male infertility that ensues. The manuscript is well-written and describes novel physiology through a broad set of appropriate assays.

Strengths:

Robust analysis with detailed functional and molecular assays

Weaknesses:

(1) The abstract references reported mutations in human SLC35G3, but this is not discussed or correlated to the murine findings to a sufficient degree in the manuscript. The HEK293T experiments are reasonable and add value, but a more detailed discussion of the clinical phenotype of the known mutations in this gene and whether they are recapitulated in this study (or not) would be beneficial.

(2) Can the authors expand on how this mutation causes such a wide array of phenotypic defects? I am surprised there is a morphological defect, a fertilization defect, and a transit defect. Do the authors believe all of these are present in humans as well?

Reviewer #1 (Public review):

Summary:

GPR3 is an orphan receptor that plays a crucial role in central nervous system development and cold-induced thermogenesis, with potential implications for treating neurodegenerative and metabolic diseases. Although previous structural studies of GPR3 have been reported, Qiu et al. presented both active and inactive structures of GPR3 in its dimeric form. Notably, they identified AF64394 as a negative allosteric modulator that binds at the dimerization interface. This interface, primarily formed by transmembrane helices TM5 and TM6, is significantly larger than the dimerization interfaces previously reported for class A GPCRs. The authors further elucidate GPR3's activation mechanism and propose that dimerization may serve as a regulatory feature of GPR3 function. Overall, the study is well-executed, and the conclusions are sound.

Strengths:

Reported a unique dimerization interface of GPR3 and identified AF64394 as a negative allosteric modulator that binds at the dimerization interface.

Weaknesses:

There are some minor issues in the figure presentation.

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 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:

The authors claim that this study elucidates the role of early secretory membranes in phagophore formation. However, this work is largely observational, which presents compelling evidence on the association between RAB1A GTPase and ATG2A without providing mechanistic insights into the functional relevance of this interaction. It remains unclear whether Rab1A depletion phenocopies ATG2A depletion in terms of autophagy progression or accumulation of pre-autophagosomal structures.

Furthermore, this research is conducted exclusively in HEK293 cells. Including at least one additional cell line would significantly strengthen the main findings (i.e., effects on LC3-II accumulation observed for RAB1A/B knockdown, given the previously published data on this topic).

A notable weakness of this manuscript, in this reviewer's opinion, lies in the discussion of the data in the context of existing literature. The discussion is rather short, mostly focused on the phenotype observed in ATG2 DKO cells. While this phenotype is certainly intriguing, it feels the discussion overlooks some important aspects, as outlined in the comments to the authors.

Reviewer #1 (Public review):

Summary:

In this lovely paper, McDermott and colleagues tackle an enduring puzzle in the cognitive neuroscience of perceptual prediction. Though many scientists agree that top-down predictions shape perception, previous studies have yielded incompatible results - with studies showing 'sharpened' representations of expected signals, and others showing a 'dampening' of predictable signals to relatively enhance surprising prediction errors. To deepen the paradox further, it seems like there are good reasons that we would want to see both influences on perception in different contexts.

Here, the authors aim to test one possible resolution to this 'paradox' - the opposing process theory (OPT). This theory makes distinct predictions about how the timecourse of 'sharpening' and 'dampening' effects should unfold. The researchers present a clever twist on a leading-trailing perceptual prediction paradigm, using AI to generate a large dataset of test and training stimuli, so that it is possible to form expectations about certain categories without repeating any particular stimuli. This provides a powerful way of distinguishing expectation effects from repetition effects - a perennial problem in this line of work.

Using EEG decoding, the researchers find evidence to support the OPT. Namely, they find that neural encoding of expected events is superior in earlier time ranges (sharpening-like) followed by a relative advantage for unexpected events in later time ranges (dampening-like). On top of this, the authors also show that these two separate influences may emerge differently in different phases of learning - with superior decoding of surprising prediction errors being found more in early phases of the task, and enhanced decoding of predicted events being found in the later phases of the experiment.

Strengths:

As noted above, a major strength of this work lies in important experimental design choices. Alongside removing any possible influence of repetition suppression mechanisms in this task, the experiment also allows us to see how effects emerge in 'real time' as agents learn to make predictions. This contrasts with many other studies in this area - where researchers 'over-train' expectations into observers to create the strongest possible effects, or rely on prior knowledge that was likely to be crystallised outside the lab.

Weaknesses:

This study reveals a great deal about how certain neural representations are altered by expectation and learning on shorter and longer timescales, so I am loath to describe certain limitations as 'weaknesses'. But one limitation inherent in this experimental design is that, by focusing on implicit, task-irrelevant predictions, there is not much opportunity to connect the predictive influences seen at the neural level to perceptual performance itself (e.g., how participants make perceptual decisions about expected or unexpected events, or how these events are detected or appear).

Reviewer #1 (Public review):

Summary:

The issue of how the brain can maintain the serial order of presented items in working memory is a major unsolved question in cognitive neuroscience. It has been proposed that this serial order maintenance could be achieved thanks to periodic reactivations of different presented items at different phases of an oscillation, but the mechanisms by which this could be achieved by brain networks, as well as the mechanisms of read-out, are still unclear. In an influential 2008 paper, the authors have proposed a mechanism by which a recurrent network of neurons could maintain multiple items in working memory, thanks to `population spikes' of populations of neurons encoding for the different items, occurring at alternating times. These population spikes occur in a specific regime of the network and are a result of synaptic facilitation, an experimentally observed type of synaptic short-term dynamics with time scales of order hundreds of ms.

In the present manuscript, the authors extend their model to include another type of experimentally observed short-term synaptic plasticity termed synaptic augmentation, which operates on longer time scales on the order of 10s. They show that while a network without augmentation loses information about serial order, augmentation provides a mechanism by which this order can be maintained in memory thanks to a temporal gradient of synaptic efficacies. The order can then be read out using a read-out network whose synapses are also endowed with synaptic augmentation. Interestingly, the read-out speed can be regulated using background inputs.

Strengths:

This is an elegant solution to the problem of serial order maintenance that only relies on experimentally observed features of synapses. The model is consistent with a number of experimental observations in humans and monkeys. The paper will be of interest to a broad readership, and I believe it will have a strong impact on the field.

Weaknesses:

(1) The network they propose is extremely simple. This simplicity has pros and cons: on the one hand, it is nice to see the basic phenomenon exposed in the simplest possible setting. On the other hand, it would also be reassuring to check that the mechanism is robust when implemented in a more realistic setting, using, for instance, a network of spiking neurons similar to the one they used in the 2008 paper. The more noisy and heterogeneous the setting, the better.

(2) One major issue with the population spike scenario is that (to my knowledge) there is no evidence that these highly synchronized events occur in delay periods of working memory experiments. It seems that highly synchronized population spikes would imply (a) a strong regularity of spike trains of neurons, at odds with what is typically observed in vivo (b) high synchronization of neurons encoding for the same item (and also of different items in situations where multiple items have to be held in working memory), also at odds with in vivo recordings that typically indicate weak synchronization at best. It would be nice if the authors at least mention this issue, and speculate on what could possibly bridge the gap between their highly regular and synchronized network, and brain networks that seem to lie at the opposite extreme (highly irregular and weakly synchronized). Of course, if they can demonstrate using a spiking network simulation that they can bridge the gap, even better.

Reviewer #1 (Public review):

Summary:

The authors introduce a novel algorithm for the automatic identification of long-range axonal projections. This is an important problem as modern high-throughput imaging techniques can produce large amounts of raw data, but identifying neuronal morphologies and connectivities requires large amounts of manual work. The algorithm works by first identifying points in three-dimensional space corresponding to parts of labelled neural projections, these are then used to identify short sections of axon using an optimisation algorithm and the prior knowledge that axonal diameters are relatively constant. Finally, a statistical model that assumes axons tend to be smooth is used to connect the sections together into complete and distinct neural trees. The authors demonstrate that their algorithm is far superior to existing techniques, especially when a dense labelling of the tissue means that neighbouring neurites interfere with the reconstruction. Despite this improvement, however, the accuracy of reconstruction remains below 90%, so manual proof-reading is still necessary to produce accurate reconstructions of axons.

Strengths:

The new algorithm combines local and global information to make a significant improvement on the state-of -the-art for automatic axonal reconstruction. The method could be applied more broadly and might have applications to reconstructions of electron microscopy data, where similar issues of high-throughput imaging and relatively slow or inaccurate reconstruction remain.

Weaknesses:

There are three weaknesses with the algorithm and manuscript.

(1) The best reconstruction accuracy is below 90%, which does not fully solve the problem of needing manual proof-reading.

(2) The 'minimum information flow tree' model the authors use to construct connected axonal trees has the potential to bias data collection. In particular, the assumption that axons should always be as smooth as possible is not always correct. This is a good rule-of-thumb for reconstructions, but real axons in many systems can take quite sharp turns and this is also seen in the data presented in the paper (Fig 1C). I would like to see explicit acknowledgement of this bias in the current manuscript and ideally a relaxation of this rule in any later versions of the algorithm.

(3) The writing of the manuscript is not always as clear as it could be. The manuscript would benefit from careful copy editing for language, and the Methods section in particular should be expanded to more clearly explain what each algorithm is doing. The pseudo code of the Supplemental Information could be brought into the Methods if possible as these algorithms are so fundamental to the manuscript.

Comments on revisions: I have no further comments or recommendations.

Reviewer #1 (Public review):

Summary:

The authors tried to identify the relationships between gut microbiota, lipid metabolites and the host in type 2 diabetes (T2DM) by using spontaneously developed T2DM in macaques, considered among the best human models.

Strengths:

The authors compared comprehensively the gut microbiota, plasma fatty acids between spontaneous T2DM and the control macaques, and tried verified the results with macaques in high-fat diet-fed mice model.

Weaknesses:

The observed multi-omics on macaques can be done on humans, which weakens the conclusion of the manuscript, unless the observation/data on macaques could cover during the onset of T2DM that would be difficult to obtain from humans.<br /> Regarding the metabolomic analysis on fatty acids, the authors did not include the results obtained form the macaque fecal samples which should be important considering the authors claimed the importance of gut microbiota in the pathogenesis of T2DM. Instead, the authors measured palmitic acid in the mouse model and tried to validate their conclusions with that.

In murine experiments, palmitic acid-containing diet were fed to mice to induce diabetic condition, but this does not mimic spontaneous T2DM in macaques, since the authors did not measure in macaque feces (or at least did not show the data from macaque feces of) palmitic acid or other fatty acids; instead, they assumed from blood metabolome data that palmitic acid would be absorbed from the intestine to affect the host metabolism, and added palmitic acid in the diet in mouse experiments. Here involves the probable leap of logic to support their conclusions and title of the study.

In addition, the authors measured omics data after, but not before, the onset of spontaneous T2DM of macaques. This can reveal microbiota dysbiosis driven purely by disease progression, but does not support the causative effect of gut microbiota on T2DM development that the authors claims.

Reviewer #1 (Public review):

Summary:

Cheong et al. use a synapse-resolution wiring map of the fruit fly nerve cord to comprehensively investigate circuitry between descending neurons (DNs) from the brain and motor neurons (MNs) that enact different behaviours. These neurons were painstakingly identified, categorised, and linked to existing genetic driver lines; this allows the investigation of circuitry to be informed by the extensive literature on how flights walk, fly, and escape from looming stimuli. New motifs and hypotheses of circuit function were presented. This work will be a lasting resource for those studying nerve cord function.

Strengths:

The authors present an impressive amount of work in reconstructing and categorising the neurons in the DN to MN pathways. There is always a strong link between the circuitry identified and what is known in the literature, making this an excellent resource for those interested in connectomics analysis or experimental circuits neuroscience. Because of this, there are many testable hypotheses presented with clear predictions, which I expect will result in many follow-up publications. Most MNs were mapped to the individual muscles that they innervate by linking this connectome to pre-existing light microscopy datasets. When combined with past fly brain connectome datasets (Hemibrain, FAFB) or future ones, there is now a tantalising possibility of following neural pathways from sensory inputs to motor neurons and muscle.

Weaknesses:

As with all connectome datasets, the sample size is low, limiting statistical analyses. Readers should keep this in mind, but note that this is the current state-of-the-art. Some figures are weakened by relying too much on depictions of wiring diagrams without additional quantification of connectivity. Readers may find the length of this work challenging, particularly the initial anatomical descriptions of the dataset, which span many figures and may not be of interest to those outside of the subfield.

Reviewer #1 (Public review):

Summary:

Advances in machine vision and computer learning have meant that there are now state-of-the-art and open-source toolboxes that allow for animal pose estimation and action recognition. These technologies have the potential to revolutionize behavioral observations of wild primates but are often held back by labor intensive model training and the need for some programming knowledge to effectively leverage such tools. The study presented here by Fuchs et al unveils a new framework (ASBAR) that aims to automate behavioral recognition in wild apes from video data. This framework combines robustly trained and well tested pose estimate and behavioral action recognition models. The framework performs admirably at the task of automatically identifying simple behaviors of wild apes from camera trap videos of variable quality and contexts. These results indicate that skeletal-based action recognition offers a reliable and lightweight methodology for studying ape behavior in the wild and the presented framework and GUI offer an accessible route for other researchers to utilize such tools.

Given that automated behavior recognition in wild primates will likely be a major future direction within many subfields of primatology, open-source frameworks, like the one presented here, will present a significant impact on the field and will provide a strong foundation for others to build future research upon.

Strengths:

Clearly articulated the argument as to why the framework was needed and what advantages it could convey to the wider field.

For a very technical paper it was very well written. Every aspect of the framework the authors clearly explained why it was chosen and how it was trained and tested. This information was broken down in a clear and easily digestible way that will be appreciated by technical and non-technical audiences alike.

The study demonstrates which pose estimation architectures produce the most robust models for both within context and out of context pose estimates. This is invaluable knowledge for those wanting to produce their own robust models.

The comparison of skeletal-based action recognition with other methodologies for action recognition are helpful in contextualizing the results.

Weaknesses:

While I note that this is a paper most likely aimed at the more technical reader, it will also be of interest to a wider primatological readership, including those who work extensively in the field. When outlining the need for future work I felt the paper offered almost exclusively very technical directions. This may have been a missed opportunity to engage the wider readership and suggest some practical ways those in the field could collect more ASBAR friendly video data to further improve accuracy.

Comments on latest version:

I think the new version is an improvement and applaud the authors on a well-written article that conveys some very technical details excellently. The authors have addressed my initial comments about reaching out to a wider, sometimes less technical, primatological audience by encouraging researchers to create large annotated datasets and make these publicly accessible. I also agree that fostering interdisciplinary collaboration is the best way to progress this field of research. These additions have certainly strengthened the paper but I still think some more practical advice for the actual collection of high-quality training data used to improve the pose estimates and behavioral classification in tough out-of-context environments could have been added. This doesn't detract from the quality of the paper though.

Reviewer #1 (Public review):

Summary:

This work by Ding et al uses agent-based simulations to explore the role of the structure of molecular motor myosin filaments in force generation in cytoskeletal structures. The focus of the study is on disordered actin bundles which can occur in the cell cytoskeleton and can be investigated with in vitro purified protein experiments. A key finding is that the force generation depends on the number of myosin motor heads and the spatial distribution of the myosin thick filaments in relation to passive crosslinkers.

Strengths:

The work develops a model where the detailed structure of the myosin motor filaments with multiple heads is represented. This allows the authors to test the dependence of myosin-generated forces on the number of myosin heads and their spatial distribution.

The work highlights that forces from multiple myosin motors within a disordered actin bundle may not simply add up, but depend on their spatial distribution in relation to passive crosslinkers.

This may explain prior experimental observations in in vitro reconstituted actomyosin bundles that the tension developed in the bundle was proportional to the number of myosin motor heads per filament rather than the number of myosin filaments. More generally, this type of modeling can guide fundamental understanding of the relationship between structure and mechanical force production.

Weaknesses:

The work focuses on the structure of myosin filaments but ignores other processes that may determine contractility of actomyosin structures such as the dynamics of crosslinker binding/unbinding and actin polymerization/depolymerization.

The authors did not vary the relative concentration of myosin motors and passive crosslinkers. This would have revealed interesting competing effects between motor and crosslink density and distribution, that their model and other studies suggest are important.

Given the above factors and the lack of direct quantitative comparisons with the experiment, the physiological significance of the work remains hard to ascertain.

Reviewer #1 (Public review):

Summary:

This study uses optogenetics to activate CA3, while recording from CA1 neurons and characterizing the excitation/inhibition (E/I) balance. They observe use-dependent alterations in the E/I balance as a result of STP, and they develop a model to describe these observations. This is a very ambitious paper that deals with many issues using both experimental and modeling approaches.

Strengths:

This paper examines important principles regarding the manner in which synaptic circuitry and use-dependent synaptic plasticity can transform inputs and perform computations.

Weaknesses:

The use of selective ChR2 expression in CA3 cells is a good approach, but there are numerous issues that cause concern regarding the applicability of their slice recordings to physiological conditions and that make some aspects of their results difficult to interpret. Experiments are not performed under physiological conditions (high external calcium and low temperature), which makes the interpretation of their findings difficult. In addition, the reliability of stimulating action potentials in CA3 pyramidal cells needs to be determined, particularly during high-frequency trains. If it is unreliable, there are alternative approaches that might prove to be superior, such as the use of somatically targeted ChR2. In addition, a clearer, more detailed discussion of their model that distinguishes it from previous modeling studies would be helpful (and would make it seem less incremental).

Reviewer #1 (Public review):

Summary:

Zare‑Eelanjegh et al. investigate how the endoplasmic reticulum, the nucleus, and the cell periphery are mechanically linked by indenting intact cells with specially shaped atomic‑force probes that double as drug injection devices. Fluorescence‑lifetime imaging of the membrane tension reporter Flipper‑TR reveals that these three compartments are mechanically linked and that the actin cytoskeleton, microtubules, and lamins modulate this coupling in complex ways.

Strengths:

(1) The study makes an important advance by applying FluidFM to probe organelle mechanics in living cells, a technically demanding but powerful approach.

(2) Experimental design is quantitative, the data are clearly presented, and the conclusions are broadly consistent with the measurements.

Weaknesses:

(1) Calcium‑dependent effects: Indentation can evoke cytoplasmic Ca²⁺ elevations that drive myosin contraction and reshape the internal membrane network (e.g., vesiculation: PMID : 9200614, 32179693) possibly confounding the Flipper-TR responses; without simultaneous/matching Ca²⁺ imaging, cell viability assays (e.g., Sytox), and intracellular Ca²⁺ sequestration or myosin inhibition experiments, a more complex mechanochemical coupling cannot be excluded, weakening conclusions.

(2) Baseline measurements: Flipper‑TR lifetime images acquired without indentation do not exclude potential light‑induced or time‑dependent changes, which weaken the conclusions.

(3) Indentation depth versus nuclear stiffness/tension: Because lamin‑A/C depletion softens nuclei, a given force may produce a deeper pit and thus greater membrane stretch. It is unclear how the cytoskeletal perturbations affect indentation depth, which weakens the conclusions.

Reviewer #1 (Public review):

In this meta-analysis, Ng and colleagues review the association between slow-oscillation spindle coupling during sleep and overnight memory consolidation. The coupling of these oscillations (and also hippocampal sharp-wave ripples) have been central to theories and mechanistic models of active systems consolidation, that posit that the coupling between ripples, spindles, and slow oscillations (SOs) coordinate and drive the coordinated reactivation of memories in hippocampus and cortex, facilitating cross-regional information and ultimately memory strengthening and stabilisation.

Given the importance that these coupling mechanisms have been given in theory, this is a timely and important contribution to the literature in terms of determining whether these theoretical assumptions hold true in human data. The results show that the timing of sleep spindles relative to the SO phase, and the consistency of that timing, predicted overnight memory consolidation in meta-analytic models. The overall amount of coupling events did not show as strong a relationship. Coupling phase in particular was moderated by a number of variables including spindle type (fast, slow), channel location (frontal, central, posterior), age, and memory type. The main takeaway is that fast spindles that consistently couple close to the peak of the SO in frontal channel locations are optimal for memory consolidation, in line with theoretical predictions. These findings will be very useful for future researchers in terms of determining necessary sample sizes to observe coupling - memory relationships, and in the selection and reporting of relevant coupling metrics.

Although the meta-analysis covers the three main coupling metrics that are typically assessed (occurrence, timing, and consistency), the meta-analysis also includes spindle amplitude. This may be confusing to readers, as this is not a measurement of SO-spindle coupling but instead a measurement of spindles in general (which may or may not be coupled).

Review #1 (Public Review):

Summary:

The authors employ a combination of repetitive transcranial magnetic stimulation (intermittent theta burst-iTBS) and transcranial alternating current stimulation (gamma tACS) as an approach aimed to improve memory in a face/name/profession task.

Strengths:

The paper has many strengths. The approach of stimulating the human brain non-invasively is potentially impactful because it could lead to a host of interesting applications. The current study aims to evaluate one such exciting application. The paper contains an unusual combination of noninvasive stimulation and brain imaging data, and includes independent replication samples.

Weaknesses:

(1) It remains unclear how this stimulation protocol is proposed to enhance memory. Memories are believed to be stored by precise inputs to specific neurons and highly tuned changes in synaptic strengths. It remains unclear whether proposed neural activity generated by the stimulation reflects the activation of specific memories or generally increased activity across all classes of neurons.

(2) The claim that effects directly involve the precuneus lacks strong support. The measurements shown in Figure 3 appear to be weak (i.e., Figure 3A top and bottom look similar, and Figure 3C left and right look similar). The figure appears to show a more global brain pattern rather than effects that are limited to the precuneus. Related to this, it would perhaps be useful to show the different positions of the stimulation apparatus. This could perhaps show that the position of the stimulation matters and could perhaps illustrate a range of distances over which position of the stimulation matters.

(3) Behavioral results showing an effect on memory would substantiate claims that the stimulation approach produces significant changes in brain activity. However, placebo effects can be extremely powerful and useful, and this should probably be mentioned. Also, in the behavioral results that are currently presented, there are several concerns:

a) There does not appear to be a significant effect on the STMB task.

b) The FNAT task is minimally described in the supplementary material. Experimental details that would help the reader understand what was done are not described. Experimental details are missing for: the size of the images, the duration of the image presentation, the degree of image repetition, how long the participants studied the images, whether the names and occupations were different, genders of the faces, and whether the same participant saw different faces across the different stimulation conditions. Regarding the latter point, if the same participant saw the same faces across the different stimulation conditions, then there could be memory effects across different conditions that would need to be included in the statistical analyses. If participants saw different faces across the different stimulus conditions, then it would be useful to show that the difficulty was the same across the different stimuli.

c) Also, if I understand FNAT correctly, the task is based on just 12 presentations, and each point in Figure 2A represents a different participant. How the performance of individual participants changed across the conditions is unclear with the information provided. Lines joining performance measurements across conditions for each participant would be useful in this regard. Because there are only 12 faces, the results are quantized in multiples of 100/12 % in Figure 3A. While I do not doubt that the authors did their homework in terms of the statistical analyses, it seems as though these 12 measurements do not correspond to a large effect size. For example, in Figure 3A for the immediate condition (total), it seems that, on average, the participants may remember one more face/name/occupation.

d) Block effects. If I understand correctly, the experiments were conducted in blocks. This is potentially problematic. An example study that articulates potential problems associated with block designs is described in Li et al (TPAMI 2021, https://ieeexplore.ieee.org/document/9264220). It is unclear if potential problems associated with block designs were taken into consideration.

e) In the FNAT portion of the paper, some results are statistically significant, while others are not. The interpretation of this is unclear. In Figure 3A, it seems as though the authors claim that iTBS+gtACS > iTBS+sham-tACS, but iTBS+gtACS ~ sham+sham. The interpretation of such a result is unclear. Results are also unclear when separated by name and occupation. There is only one condition that is statistically significant in Figure 3A in the name condition, and no significant results in the occupation condition. In short, the statistical analyses, and accompanying results that support the authors’ claims, should be explained more clearly.

Reviewer #1 (Public review):

Summary:

In this study, Liu et al use optogenetics and genetically encoded neuromodulator sensors to test the extent to which dopamine neuron stimulation produces striatal serotonin release, and vice versa. The study is timely given growing interest in dopamine/serotonin interactions and in the context of recent work showing bidirectional and dynamic regulation of striatal dopamine by another neuromodulator, acetylcholine. The authors find that striatal dopamine and serotonin afferents function largely independently, with dopamine neuron stimulation producing no striatal serotonin release and serotonin neuron stimulation producing minimal striatal dopamine release. This work will inform future work seeking to dissect the contributions of striatal dopamine, serotonin, and their interactions to various motivated behaviors. While the paper's main conclusions are adequately supported (see Strengths), additional controls and experiments would significantly broaden the paper's impact (see Weaknesses). Finally, this draft of the work is poorly presented with numerous errors, omissions, and inconsistencies evident throughout the text and the figures that should be addressed.

Strengths:

The study employs optogenetic stimulation simultaneously with fiber photometry recording of dopamine or serotonin release measured with genetically encoded sensors. These methods are state-of-the-art, offering tighter temporal control compared to pharmacological methods for manipulating dopamine and serotonin and improved selectivity over techniques like electrochemistry and microdialysis used to record neuromodulator release in previous studies on the subject. As a result, the paper's main conclusions are well supported.

Weaknesses:

(1) The electrophysiology experiments in Figure 3 are only tangentially related to the focus of the study, and their findings are almost entirely irrelevant to the paper's main conclusions. The results of these experiments are also not novel. Glutamate corelease from 5HT neurons has been previously shown, including in the OFC and VTA (Ren et al, 2018, Cell, McDevitt et al, 2014, Cell Rep, Liu et al 2014, Neuron; and others). The authors should explain more clearly what they think these data add to the manuscript and/or consider removing them altogether.

(2) Related to the point above, as far as I can tell, the only value the electrophysiology data add is to suggest that perhaps activation of serotonin neurons may drive minimal striatal dopamine release via glutamate corelease in the VTA. The evidence provided in this version of the manuscript is insufficient to support that claim, but the manuscript would be significantly strengthened if the authors tested this hypothesis more directly. One way to do that could be to stimulate serotonin axons in the striatum (as opposed to the serotonin cell bodies) and record striatal dopamine release. A complementary anatomical approach would be to use retrograde tracing to test whether the DR 5HT neurons projecting to the striatum are the same or different from the VTA projecting population.

(3) The findings would be strengthened by the addition of a fluorophore-only control group lacking opsin expression in all experiments in Figures 1 and 2.

(4) The experiment of stimulating serotonin neurons and recording serotonin release in the NAc was not performed. It would be useful to be able to compare the magnitudes of evoked serotonin release in these two striatal regions, though it is not central to the main claims of the paper.

(5) The interpretation of the results from Figure 2 is described inconsistently throughout the manuscript. The title implies there is significant crosstalk between the dopamine and serotonin systems. The abstract calls the crosstalk "transient", which is a description of its temporal dynamics, not its magnitude. Then the introduction figures and discussion all suggest the crosstalk is minimal. I suggest the authors describe the main findings - minimal crosstalk between the dopamine and serotonin systems - clearly and consistently in the title, abstract, and main text.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors aim to address significant limitations of existing experimental paradigms used to study dyadic social interactions by introducing a novel experimental setup - the Dyadic Interaction Platform (DIP). The DIP uniquely allows participants to interact dynamically, face-to-face, with simultaneous access to both social cues and task-related stimuli. The authors demonstrate the versatility and utility of this platform across several exemplary scenarios, notably highlighting cases of significant behavioral differences in conditions involving direct visibility of a partner.

Major strengths include comprehensive descriptions of previous paradigms, detailed explanations of the DIP's technical features, and clear illustrations of multimodal data integration. These elements greatly enhance the reproducibility of the methods and clarify the potential applications across various research domains and species. Particularly compelling is the authors' demonstration of behavioral impacts related to transparency in interactions, as evidenced by the macaque-human experiments using the Bach-or-Stravinsky game scenario.

Strengths:

The DIP represents a methodological advance in the study of social cognition. Its transparent, touch-sensitive display elegantly solves the problem of enabling participants to attend to both their social partner and task stimuli simultaneously without requiring attention switching. This paper marks a notable step forward toward more options for naturalistic yet still lab-based studies of social decision-making, an area where the field is actively moving, especially given recent research highlighting significant differences in neural activity depending upon the context in which an action is performed. The DIP offers researchers a valuable tool to bridge the gap between tightly controlled laboratory paradigms and the dynamic, bidirectional nature of real-world social interactions.

The authors do well to provide comprehensive documentation of the technical specifications for the four different implementations of the platform, allowing other researchers to adapt and build upon their work. The detailed information about hardware configurations demonstrates careful attention to practical implementation details. They also highlight numerous options for integration with other tools and software, further demonstrating the versatility of this apparatus and the variety of research questions to which it could be applied.

The historical review of dyadic experimental paradigms is thorough and effectively positions the DIP as addressing a critical gap in existing methodologies. The authors convincingly argue that studying continuous, dynamic social interactions is essential for understanding real-world social cognition, and that existing paradigms often force unnatural attention-splitting or turn-taking behaviors that don't reflect naturalistic interaction patterns.

The four example applications showcase the DIP's versatility across diverse research questions. The Bach-or-Stravinsky economic game example is particularly compelling, demonstrating how continuous access to partners' actions substantially changes coordination strategies in non-human primates. This highlights a key strength of the DIP, which is that it removes a level of abstraction that can make tasks more difficult for non-human primates to learn. By being able to see their partner and actions directly, rather than having to understand that a cursor on a screen represents a partner, the platform makes the task more accessible to non-human primates and possibly children as well. This opens up important avenues for enhanced cross-species investigations of cognition, allowing researchers to study social dynamics in a setting that remains naturalistic yet controlled across different populations.

Weaknesses:

Some of the experimental applications would benefit from stronger evidence demonstrating the unique advantages of the transparent setup. For instance, in the dyadic foraging example, it's not entirely clear how participants' behavior differs from what might be observed when simply tracking each other's cursor movements in a non-transparent setup. More evidence showing how direct visibility of the partner, beyond simply being able to track the position of the partner's cursor, influences behavior would strengthen this example. Similarly, in the continuous perceptual report (CPR) task, the subjects could perform this task and see feedback from their partners' actions without having to see their partner through the transparent screen. Evidence showing that 1) subjects do indeed look at their partner during the task and 2) viewing their partner influences their performance on the task would significantly strengthen the claim that the ability to view the partner brings in a new dimension to this task. These additions would better demonstrate the specific value added by the transparent nature of the DIP beyond what could be achieved with standard cursor-tracking paradigms.

A significant limitation that is inadequately addressed relates to neural investigations. While the authors position the platform's ability to merge attention to social stimuli and task stimuli as a key advantage, they don't sufficiently acknowledge the challenges this creates for dissociating neural signals attributed to social cues versus task-based stimuli. More traditional lab-based experiments intentionally separate components like task-stimulus perception, social perception, and decision-making periods so that researchers can isolate the neural signals associated with each process. This deliberate separation, which the authors frame as a weakness, actually serves an important functional purpose in neural investigations. The paper would be strengthened by explicitly discussing this limitation and offering potential approaches to address it in experimental design or data analysis. For instance, the authors could suggest methodological innovations or analytical techniques that might help disentangle the overlapping neural signals that would inevitably arise from the integrated presentation of social and task stimuli in the DIP setup.

Furthermore, the authors' suggestion to arrange task stimuli around the periphery of the screen to maintain a clear middle area for viewing the partner appears to contradict their own critique of traditional paradigms. This recommended arrangement would seemingly reintroduce the very problem of attentional switching between task stimuli and social partners that the authors identified as a limitation of previous approaches. The paper would be strengthened by discussing the potential trade-offs associated with their suggested stimulus arrangement. Additionally, offering potential approaches to address these limitations in experimental design or data analysis would enhance the paper's contribution to the field.

Reviewer #1 (Public review):

Summary:

In this study titled 'The Lipocone Superfamily: A Unifying Theme In Metabolism Of Lipids, Peptidoglycan, And Exopolysaccharides, Inter-Organismal Conflicts And Immunity' from L. Aravind's group, the authors report the identification of a novel domain superfamily termed "Lipocone" superfamily. This superfamily unifies Wnt protein with a spectrum of domains from about 30 families, including those from phosphatidylserine synthases (PTDSS1/2), TelC toxin, VanZ proteins, and the animal Serum Amyloid A (SAA). The authors provide evidence that this superfamily originated as membrane proteins, with few (including Wnt and SAA) evolving into soluble domains. The authors also provide contextual evidence for the Lipocone members recruited as effectors in biological conflicts in both prokaryotes and eukaryotes. Importantly, to my knowledge, this study is the first to decipher the origins of Wnt signaling (emerging from a membrane protein context) and provide novel insights into immunity.

- The study is well-executed and provides many interesting leads for further experimental studies, which makes it very important. One of the significant hypotheses in this context is metazoan Wnt Lipocone domain interactions with lipids, which remain to be explored.

- The manuscript is generally navigable for interesting reading despite being content-rich.

- Overall, the figures are easy to follow.

Significance:

This study not only provides a plausible solution to the origins of metazoan Wnt signaling but also hypothesizes, based on retained ancestral substrate binding pocket, potential lipid interactions for lipocone wnt domains. The study also predicts novel enzymatic roles for many poorly characterized proteins that are involved in immunity across lineages/superkingdoms. This work is likely to inspire numerous experimental studies attempting to verify the hypotheses described in the study.

Reviewer #1 (Public review):

This paper concerns mechanisms of foraging behavior in C. elegans. Upon removal from food, C. elegans first executes a stereotypical local search behavior in which it explores a small area by executing many random, undirected reversals and turns called "reorientations." If the worm fails to find food, it transitions to a global search in which it explores larger areas by suppressing reorientations and executing long forward runs (Hills et al., 2004). At the population level, reorientation rate declines gradually. Nevertheless, about 50% of individual worms appear to exhibit an abrupt transition between local and global search, which is evident as a discrete transition from high to low reorientation rate (Lopez-Cruz et al., 2019). This observation has given rise to the hypothesis that local and global search correspond to separate internal states with the possibility of sudden transitions between them (Calhoun et al., 2014). The objective of the paper is to demonstrate that is not necessary to posit distinct internal states to account for discrete transitions from high to low reorientation rate. On the contrary, discrete transitions can occur simply because of the stochastic nature of the reorientation behavior itself.

Major strengths and weaknesses of the methods and results

The model was not explicitly designed to match the sudden, stable changes in reorientation rates observed in the experimental data from individual worms. Kinetic parameters were simply chosen to match the average population behavior. Nevertheless, many sudden stable changes in reorientation rates occurred. This is a strong argument that apparent state changes can arise as an epiphenomenon of stochastic processes.

The new stochastic model is more parsimonious than reorientation-state change model because it posits one state rather than two.

A prominent feature of the empirical data is that 50% of the worms exhibit a single (apparent) state change and the rest show either no state changes or multiple state changes. Does the model reproduce these proportions? This obvious question was not addressed.

There is no obvious candidate for the neuronal basis of the decaying factor M. The authors speculate that decreasing sensory neuron activity might be the correlate of M but then provide contradictory evidence that seems to undermine that hypothesis. The absence of a plausible neuronal correlate of M weakens the case for the model.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions

The authors have made a convincing case that is not necessary to posit distinct internal states to account for discrete transitions from high to low reorientation rate. On the contrary, discrete transitions can occur simply because of the stochastic nature of the reorientation behavior itself.

Impact of the work on the field, and the utility of the methods and data to the community

Posting hidden internal states to explain behavioral sequences is gaining acceptance in behavioral neuroscience. The likely impact of the paper is to establish a compelling example of how statistical reasoning can reduce the number of hidden states to achieve models that are more parsimonious.

Reviewer #1 (Public review):

Dovek and colleagues aimed at investigating the cellular and circuitry mechanisms underlying the recruitment of dentate gyrus neurons (including two morpho-physiologically-distinct subpopulations of excitatory cells called granular cells or GCs, and semilunar cells or SGCs) into memory representations, also known as engrams. To this end, the authors used TRAP2 mice to investigate the dentate gyrus "engram" neurons that were activated or not (i.e., labeled or not) in a non-fear-based context (mostly enriched environment or EE, but also Barnes Maze or BM).

A significant proportion of dentate gyrus neurons are labeled after EE exposure (35%) or after BM acquisition (15%). SGCs, distinguished from GCs using morphology-based classification, showed disproportionately context-dependent recruitment. Consistent with previous observations (Erwin et al., 2022), SGCs account for a third of behaviorally recruited "engram" neurons, although they represent less than 5% of excitatory neurons in the dentate gyrus.

Then, the authors compared the intrinsic physiological properties of GCs and SGCs that are recruited or not during EE. Consistent with previous observations (Williams et al., 2007, Afrasiabi et al., 2022), SGCs and GCs exhibited numerous differences (e.g., Rin, firing frequency) regardless of whether they were behaviorally activated or not. Differences in physiology between excitatory neuron subtypes might explain the preferential recruitment of SGCs. Interestingly, "engram" SGCs displayed lower values of adaptation in firing rate than non-recruited SGCs.

To examine how GCs and SGCs activated during EE are integrated into the local dentate gyrus microcircuits, the authors next performed a dual patch-clamp recording combined with wide-field optogenetics. Despite the presence of spontaneous EPSCs, no direct functional glutamatergic interconnection was observed between pairs of "engram" GCs and SGCs. In addition, although optogenetic stimulation of a large, random, population of neurons evokes IPSCs (indicating efficient lateral inhibition as in Stefanelli et al., 2016), the specific stimulation of behaviorally recruited GCs or SGCs rarely elicits IPSCs onto surrounding non-engram excitatory neurons.

To assess whether neurons recruited or not during EE receive differential glutamatergic drive, the authors recorded spontaneous excitatory inputs received by labeled and unlabeled GCs and SGCs. They observed that sEPSCs in labeled GCs and SGCs are more frequent and larger than in unlabeled GCs and SGCs, respectively.

Last, the authors investigated whether neurons (without discriminating GCs and SGCs) recruited in the same context were characterized by a higher propensity to receive temporally correlated inputs. To this end, they performed dual patch-clamp and analyzed the temporal correlation of spontaneous EPSCs received by pairs of neurons (either two dentate gyrus "engram" neurons, or one "engram" neuron and one "non-engram" neuron in an EE context). They observed that the temporal correlation of excitatory events received by pairs of engram neurons was greater than that of pairs of neurons that do not belong to the same ensemble, and that expected by chance.

Altogether, the data suggest that the context-dependent recruitment of dentate gyrus excitatory neurons, particularly SGCs is correlated to distinctive intrinsic properties and (correlated) excitatory afferent. Contrary to a leading hypothesis, the authors found no evidence that recruited neurons drive robust feedforward excitation of other engram neurons or feedback inhibition of non-engram neurons.

Strengths:

This article provides some information about the mechanisms that may be involved in the recruitment of neural ensembles that form non-fear-based memory engrams in the dentate gyrus. I find it interesting that the authors considered not only granular cells, the main population of excitatory neurons in the dentate gyrus, but also a sparse subpopulation of semilunar cells, a relatively understudied type of dentate excitatory neuron.

Weakness:

Most of the data presented are descriptive and based on correlation rather than causation.

Reviewer #1 (Public review):

Summary:

Frelih et al. investigated both periodic and aperiodic activity in EEG during working memory tasks. In terms of periodic activity, they found post-stimulus decreases in alpha and beta activity, while in terms of aperiodic activity, they found a bi-phasic post-stimulus steepening of the power spectrum, which was weakly predictive of performance. They conclude that it is crucial to properly distinguish between aperiodic and periodic activity in event-related designs as the former could confound the latter. They also add to the growing body of research highlighting the functional relevance of aperiodic activity in the brain.

Strengths:

This is a well-written, timely paper that could be of interest to the field of cognitive neuroscience, especially to researchers investigating the functional role of aperiodic activity. The authors describe a well-designed study that looked at both the oscillatory and non-oscillatory aspects of brain activity during a working memory task. The analytic approach is appropriate, as a state-of-the-art toolbox is used to separate these two types of activity. The results support the basic claim of the paper that it is crucial to properly distinguish between aperiodic and periodic activity in event-related designs as the former could confound the latter. They also add to the growing body of research highlighting the functional relevance of aperiodic activity in the brain. Commendably, the authors include replications of their key findings on multiple independent data sets.

Weaknesses:

The authors also claim that their results speak to the interplay between oscillatory and non-oscillatory activity, and crucially, that task-related changes in the theta frequency band - often attributed to neural oscillations in the field - are in fact only a by-product of non-oscillatory changes. I believe these claims are too bold and are not supported by compelling evidence in the paper. Some control analyses - e.g., contrasting the scalp topographies of purportedly theta and non-oscillatory effects - could help strengthen the latter argument, but it may be safest to simply soften these two claims.

In terms of the methodology used, I suggest the authors make it clearer to readers that the primary results were obtained on a sample of middle-aged-to-older-adults, some with subjective cognitive complaints, and note that while stimulus-locked event-related potentials (ERPs) were removed from the data prior to analyses, response-locked ERPs were not. This could potentially confound aperiodic findings. Contrasting the scalp topographies of response-related ERPs and the identified aperiodic components, especially the later one, could bring some clarity here too.

I have also found certain parts of the introduction to be somewhat confusing.

Comments on the latest version:

The authors have addressed several of the weaknesses I noted in my original review, specifically, they softened their claims regarding the theta findings, while simultaneously strengthening these findings with additional analyses (using simulations as well as a new measure of rhythmicity, the phase autocorrelation function, pACF). Most of the other suggested control analyses were also implemented. While I believe the fact that the participants in the main sample were not young adults could be made even more explicit, and the potential interaction between age and aperiodic changes could be unpacked a little in the discussion, the age of the sample is definitely addressed upfront.

Reviewer #1 (Public review):

Summary:

Most studies in sensory neuroscience investigate how individual sensory stimuli are represented in the brain (e.g., the motion or color of a single object). This study starts tackling the more difficult question of how the brain represents multiple stimuli simultaneously and how these representations help to segregate objects from cluttered scenes with overlapping objects.

Strengths

The authors first document the ability of humans to segregate two motion patterns based on differences in speed. Then they show that a monkey's performance is largely similar; thus establishing the monkey as a good model to study the underlying neural representations.

Careful quantification of the neural responses in the middle temporal area during the simultaneous presentation of fast and slow speeds leads to the surprising finding that, at low average speeds, many neurons respond as if the slowest speed is not present, while they show averaged responses at high speeds. This unexpected complexity of the integration of multiple stimuli is key to the model developed in this paper.

One experiment in which attention is drawn away from the receptive field supports the claim that this is not due to the involuntary capture of attention by fast speeds.

A classifier using the neuronal response and trained to distinguish single speed from bi-speed stimuli shows a similar overall performance and dependence on the mean speed as the monkey. This supports the claim that these neurons may indeed underlie the animal's decision process.

The authors expand the well-established divisive normalization model to capture the responses to bi-speed stimuli. The incremental modeling (eq 9 and 10) clarifies which aspects of the tuning curves are captured by the parameters.

Reviewer #1 (Public review):

Summary:

This study used explicit-solvent simulations and coarse-grained models to identify the mechanistic features that allow for the unidirectional motion of SMC on DNA. Shorter explicit-solvent models describe relevant hydrogen bond energetics, which were then encoded in a coarse-grained structure-based model. In the structure-based model, the authors mimic chemical reactions as signaling changes in the energy landscape of the assembly. By cycling through the chemical cycle repeatedly, the authors show how these time-dependent energetic shifts naturally lead SMC to undergo translocation steps along DNA that are on a length scale that has been identified.

Strengths:

Simulating large-scale conformational changes in complex assemblies is extremely challenging. This study utilizes highly-detailed models to parameterize a coarse-grained model, thereby allowing the simulations to connect the dynamics of precise atomistic-level interactions with a large-scale conformational rearrangement. This study serves as an excellent example for this overall methodology, where future studies may further extend this approach to investigated any number of complex molecular assemblies.

Weaknesses:

The only relative weakness is that the text does not always clearly communicate which aspects of the dynamics are expected to be robust. That is, which aspects of the dynamics/energetics are less precisely described by this model? Where are the limits of the models, and why should the results be considered within the range of applicability of the models?

Reviewer #1 (Public review):

Summary:

This paper addresses an important and topical issue: how temporal context, at various time scales, affects various psychophysical measures, including reaction times, accuracy, and localization. It offers interesting insights, with separate mechanisms for different phenomena, which are well discussed.

Strengths:

The paradigm used is original and effective. The analyses are rigorous.

Weaknesses:

Here I make some suggestions for the authors to consider. Most are stylistic, but the issue of precision may be important.

(1) The manuscript is quite dense, with some concepts that may prove difficult for the non-specialist. I recommend spending a few more words (and maybe some pictures) describing the difference between task-relevant and task-irrelevant planes. Nice technique, but not instantly obvious. Then we are hit with "stimulus-related", which definitely needs some words (also because it is orthogonal to neither of the above).

(2) While I understand that the authors want the three classical separations, I actually found it misleading. Firstly, for a perceptual scientist to call intervals in the order of seconds (rather than milliseconds), "micro" is technically coming from the raw prawn. Secondly, the divisions are not actually time, but events: micro means one-back paradigm, one event previously, rather than defined by duration. Thirdly, meso isn't really a category, just a few micros stacked up (and there's not much data on this). And macro is basically patterns, or statistical regularities, rather than being a fixed time. I think it would be better either to talk about short-term and long-term, which do not have the connotations I mentioned. Or simply talk about "serial dependence" and "statistical regularities". Or both.

(3) More serious is the issue of precision. Again, this is partially a language problem. When people use the engineering terms "precision" and "accuracy" together, they usually use the same units, such as degrees. Accuracy refers to the distance from the real position (so average accuracy gives bias), and precision is the clustering around the average bias, usually measured as standard deviation. Yet here accuracy is percent correct: also a convention in psychology, but not when contrasting accuracy with precision, in the engineering sense. I suggest you change "accuracy" to "percent correct". On the other hand, I have no idea how precision was defined. All I could find was: "mixture modelling was used to estimate the precision and guess rate of reproduction responses, based on the concentration (k) and height of von Mises and uniform distributions, respectively". I do not know what that means.

(4) Previous studies show serial dependence can increase bias but decrease scatter (inverse precision) around the biased estimate. The current study claims to be at odds with that. But are the two measures of precision relatable? Was the real (random) position of the target subtracted from each response, leaving residuals from which the inverse precision was calculated? (If so, the authors should say so..) But if serial dependence biases responses in essentially random directions (depending on the previous position), it will increase the average scatter, decreasing the apparent precision.

(5) I suspect they are not actually measuring precision, but location accuracy. So the authors could use "percent correct" and "localization accuracy". Or be very clear what they are actually doing.

Reviewer #1 (Public review):

Summary:

Liu et al. investigated the mechanisms by which apolipoprotein L1 (APOL1) G1 and G2 variants cause inflammation and lipid accumulation in macrophages by bone-marrow-derived macrophages from transgenic mice and human iPS cells. Although these findings are not novel, this work provides solid evidence to prove enhanced inflammation and lipid accumulation in macrophages by APOL1 G1 and G2 variants by a variety of in vitro assays and metabolomics measurements. Further, metabolomics measurements identified that the spermidine synthesis pathway was altered by APOL1 G1 and G2 variants, and the polyamine inhibitor reversed the variants-induced phenotypes.

Strengths:

Their hypothesis and choice of experiments in each section were clear and mostly solid. Mitochondrial morphological quantification by transmission electron microscopy images was convincing. The authors confirmed APOL1 localization inside macrophages and built stories based on their findings. Showing relevant positive and negative findings in line with current knowledge of APOL1-variants-driven pathologies, such as cation flux, cGAS-STING pathways, indicates a good rigor.

Weaknesses:

Although most methods in this work were solid, the choice of α-difluoromethylornithine (DFMO) as an inhibitor of spermidine synthesis was not direct. It was still unclear if DFMO was reversing the phenotypes by lowering spermidine levels. Seahorse assay results would have avoided potential variabilities in cell densities by normalization. Heatmaps showing RNA-seq results would be appreciated better with a clear description of how the color is defined and calculated.

Reviewer #1 (Public review):

This study is focused on identifying unique, innovative surface markers for mature Achilles tendons by combining the latest multi-omics approaches and in vitro evaluation, which would address the knowledge gap of controversial identity of TPSCs with unspecific surface markers. The use of multi-omics technologies, in vivo characterization, in vitro standard assays of stem cells, and in vitro tissue formation is a strength of this work and could be applied for other stem cell quantification in the musculoskeletal research. The evaluation and identification of Cd55 and Cd248 in TPSCs have not been conducted in tendon, which is considered as innovative. Additionally, the study provided solid sequencing data to confirm co-expressions of Cd55 and Cd248 with other well-described surface markers such as Ly6a, Tpp3, Pdgfra, and Cd34. Generally, the data shown in the manuscript support the claims that the identified surface antigens mark TPSCs in juvenile tendons.

Reviewer #1 (Public review):

Summary:

This manuscript describes a series of lab and field experiments to understand the role of tadpole transport in shaping the microbiome of poison frogs in early life. The authors conducted a cross-foster experiment in which R. variabilis tadpoles were carried by adults of their own species, carried by adults of another frog species, or not carried at all. After being carried for 6 hours, tadpole microbiomes resembled those of their caregiving species. Next, the authors reported higher microbiome diversity in tadpoles of two species that engage in transport-based parental care compared to one species that does not. Finally, they collected tadpoles either from the backs of an adult (i.e., they had recently been transported) or from eggs (i.e., not transported) but did not find significant overlap in microbiome composition between transported tadpoles and their parents.

Strengths:

The cross-foster experiment and the field experiment that reared transported and non-transported tadpoles are creative ways to address an important question in animal microbiome research. Together, they imply a small role for parental care in the development of the tadpole microbiome. The manuscript is generally well-written and easy to understand. The authors make an effort (improved since the first version of the manuscript) to acknowledge the limitations of their experimental design.

Weaknesses:

This manuscript has improved since the initial version and now more clearly discusses the limitations of its experimental design. I have no further revisions to request.

Reviewer #1 (Public review):

Summary

This manuscript from Azeroglu et al. presents the application of END-Seq to examine the sequence composition of chromosome termini, i.e., telomeres. END-seq is a powerful genome sequencing strategy developed in Andre Nussesweig's lab to examine the sequences at DNA break sites. Here, END-Seq is applied to explore the nucleotide sequences at telomeres and to ascertain (i) whether the terminal end sequence is conserved in cells that activate ALT telomere elongation mechanism and (ii) whether the processes responsible for telomere end sequence regulation are conserved. With these aims clearly articulated, the authors convincingly show the power of this technique to examine telomere end-processing.

Strengths

(1) The authors effectively demonstrate the application of END-seq for these purposes. They verify prior data that 5'terminal sequences of telomeres in Hela and RPE cells end in a canonical ATC sequence motif. They verify that the same sequence is present at the 5' ends of telomeres by performing END-seq across a panel of ALT cancer cells. As in non-ALT cells, the established role of POT1, a ssDNA telomere binding protein, in coordinating the mechanism that maintains the canonical ATC motif is likewise verified. However, by performing END-Seq in mouse cells lacking POT1 isoforms, POT1a and POT1b, the authors uncover that POT1b is dispensable for this process. This reveals a novel, important insight relating to the evolution of POT1 as a telomere regulatory factor.

(2) The authors then demonstrate the utility of S1-END-seq, a variation of END-Seq, to explore the purported abundance of single-stranded DNA at telomeres within telomeres of ALT cancer cells. Here, they demonstrate that ssDNA abundance is an intrinsic aspect of ALT telomeres and is dependent on the activity of BLM, a crucial mediator of ALT.

Overall, the authors have effectively shown that END-seq can be applied to examine processes maintaining telomeres in normal and cancerous cells across multiple species. Using END-Seq, the authors confirm prior cell biological and sequencing data and the role of POT1 and BLM in regulating telomere termini sequences and ssDNA abundance. The study is nice and well-written, with the experimental rationale and outcomes clearly explained.

Weaknesses

This reviewer finds little to argue with in this study. It is timely and highly valuable for the telomere field. One minor question would be whether the authors could expand more on the application of END-Seq to examine the processive steps of the ALT mechanism? Can they speculate if the ssDNA detected in ALT cells might be an intermediate generated during BIR (i.e., is the ssDNA displaced strand during BIR) or a lesion? Furthermore, have the authors assessed whether ssDNA lesions are due to the loss of ATRX or DAXX, either of which can be mutated in the ALT setting?

Comments on revisions:

The authors addressed the comments. Thank you.

Reviewer #1 (Public review):

Summary:

Genome-wide association studies have been an important approach to identifying the genetic basis of human traits and diseases. Despite their successes, for many traits, a substantial amount of variation cannot be explained by genetic factors, indicating that environmental variation and individual 'noise' (stochastic differences as well as unaccounted for environmental variation) also play important roles. The authors' goal was to address how gene expression variation in genetically identical individuals, driven by historical environmental differences and 'noise', could be used to predict reproductive trait differences.

Strengths:

To address this question, the authors took advantage of genetically identical C. elegans individuals to transcriptionally profile 180 adult hermaphrodite individuals that were also measured for two reproductive traits. A major strength of the paper is in its experimental design. While experimenters aim to control the environment that each worm experiences, it is known that there are small differences even when worms are grown together on the same agar plate - e.g., the age of their mother, their temperature, the amount of food they eat, and the oxygen and carbon dioxide levels depending on where they roam on the plate. Instead of neglecting this unknown variation, the authors design the experiment up front to create two differences in the historical environment experienced by each worm: 1) the age of its mother and 2) 8 8-hour temperature difference, either 20 or 25 C. This helped the authors interpret the gene expression differences and trait expression differences that they observed.

Using two statistical models, the authors measured the association of gene expression for 8824 genes with the two reproductive traits, considering both the level of expression and the historical environment experienced by each worm. Their data supports several conclusions. They convincingly show that gene expression differences are useful for predicting reproductive trait differences, predicting ~25-50% of the trait differences depending on the trait. Using RNAi, they also show that the genes they identify play a causal role in trait differences. Finally, they demonstrate an association with trait variation and the H3K27 trimethylation mark, suggesting that chromatin structure can be an important causal determinant of gene expression and trait variation.

Overall, this work supports the use of gene expression data as an important intermediate for understanding complex traits. This approach is also useful as a starting point for other labs in studying their trait of interest.

Weaknesses:

There are no major weaknesses that I have noted. Some important limitations of their work are worth highlighting, though (and I believe the authors would agree with these points):

(1) A large remaining question in the field of complex traits remains in splitting the role of non-genetic factors between environmental variation and stochastic noise. It is still an open question which role each of these factors plays in controlling the gene expression differences they measured between the individual worms.

(2) The ability of the authors to use gene expression to predict trait variation was strikingly different between the two traits they measured. For the early brood trait, 448 genes were statistically linked to the trait difference, while for egg-laying onset, only 11 genes were found. Similarly, the total R2 in the test set was ~50% vs. 25%. It is unclear why the differences occur, but this somewhat limits the generalizability of this approach to other traits.

(3) For technical reasons, this approach was limited to whole worm transcription. The role of tissue and cell-type expression differences is important to the field, so this limitation is relevant.

Comments on revisions: The authors have addressed my previous comments to my satisfaction.

Reviewer #1 (Public review):

Summary:

The authors address a fundamental question for cell and tissue biology using the skin epidermis as a paradigm and ask how stratifying self-renewing epithelia induce differentiation and upwards migration in basal dividing progenitor cells to generate suprabasal barrier-forming cells that are essential for a functional barrier formed by such an epithelium. The authors show for the first time that an increase in intracellular actomyosin contractility, a hallmark of barrier-forming keratinocytes, is sufficient to trigger terminal differentiation. Hence the data provide in vivo evidence of the more general interdependency of cell mechanics and differentiation. The data appear to be of high quality and the evidences are strengthened through a combination of different genetic mouse models, RNA sequencing and immunofluorescence analysis.

To generate and maintain the multilayered, barrier-forming epidermis, keratinocytes of the basal stem cell layer differentiate and move suprabasally accompanied by stepwise changes not only in gene expression but also in cell morphology, mechanics and cell position. If any of these changes are instructive for differentiation itself, and whether consecutive changes in differentiation are required, remains unclear. Also, there are few comprehensive data sets on the exact changes in gene expression between different states of keratinocyte differentiation. In this study, through genetic fluorescence labeling of cell states at different developmental timepoints the authors were able to analyze gene expression of basal stem cells and suprabasal differentiated cells at two different stages of maturation: E14 (embryonic day 14) when the epidermis comprises mostly two functional compartments (basal stem cells and suprabasal so called intermediate cells) and E16 when the epidermis comprise three (living) compartments where the spinous layer separates basal stem cells from the barrier forming granular layer, as is the case in adult epidermis. Using RNA bulk sequencing, the authors developed useful new markers for suprabasal stages of differentiation like MafB and Cox1. The transcription factor MafB was then shown to inhibit suprabasal proliferation in a MafB transgenic model.

The data indicate that early in development at E14 the suprabasal intermediate cells resemble in terms of RNA expression, the barrier-forming granular layer at E16, suggesting that keratinocyte can undergo either stepwise (E16) or more direct (E14) terminal differentiation.

Previous studies by several groups found an increased actomyosin contractility in the barrier forming granular layer and showed that this increase in tension is important for epidermal barrier formation and function. However, it was not clear whether contractility itself serves as an instructive signal for differentiation. To address this question, the authors use a previously published model to induce premature hypercontractility in the spinous layer by using spastin overexpression (K10-Spastin) to disrupt microtubules (MT) thereby indirectly inducing actomyosin contractility. A second model activates myosin contractility more directly through overexpression of a constitutively active RhoA GEF (K10-Arhgef11CA). Both models induce late differentiation of suprabasal keratinocytes regardless of the suprabasal position in either spinous or granular layer indicating that increased contractility is key to induce late differentiation of granular cells. A potential weakness is the use of the K10-spastin model that disrupts MT and likely has additional roles in altering differentiation next to the induction of hypercontractility. Their previous publications provided some evidence that the effect on differentiation is driven by the increase in contractility (Ning et al. cell stem cell 2021). Moreover, their data are now further supported by a second model activating myosin through RhoA. This manuscript extends their previous findings that indicated a role for contractility in early differentiation, now focussing on the regulation of late differentiation in barrier forming cells. This data set thus help to unravel the interdependencies of cell position, mechanical state and differentiation in the epidermis, and suggest that an increase in cellular contractility within the epidermis can induce terminal differentiation. Importantly the authors show that despite contractility induced nuclear localization of the mechanoresponsive transcription factor YAP in the barrier forming granular layer, YAP nuclear localization is not sufficient to drive premature differentiation when forced to the nucleus in the spinous layer.

Overall, this is a well written manuscript and comprehensive dataset.

Reviewer #1 (Public Review):

Summary:

The study "Impact of Maximal Overexpression of a Non-toxic Protein on Yeast Cell Physiology" by Fujita et al. aims to elucidate the physiological impacts of overexpressing non-toxic proteins in yeast cells. By identifying model proteins with minimal cytotoxicity, the authors claim to provide insights into cellular stress responses and metabolic shifts induced by protein overexpression.

Strengths:

The study introduces a neutrality index to quantify cytotoxicity and investigates the effects of protein burden on yeast cell physiology. The study identifies mox-YG (a non-fluorescent fluorescent protein) and Gpm1-CCmut (an inactive glycolytic enzyme) as proteins with the lowest cytotoxicity, capable of being overexpressed to more than 40% of total cellular protein while maintaining yeast growth. Overexpression of mox-YG leads to a state resembling nitrogen starvation probably due to TORC1 inactivation, increased mitochondrial function, and decreased ribosomal abundance, indicating a metabolic shift towards more energy-efficient respiration and defects in nucleolar formation.

Weaknesses:

While the introduction of the neutrality index seems useful to differentiate between cytotoxicity and protein burden, the biological relevance of the effects of overexpression of the model proteins is unclear.

Reviewer #1 (Public review):

Summary:

The study aimed to develop a liquid biopsy EV miRNA signature associated with radiomics features for early diagnosis of pancreatic cancer. Flawed study design and inadequate description of clinical characteristics of the enrolled samples makes the findings unconvincing.

Strengths:

The concept of developing EV miRNA signature associated with disease relevant radiomics features is a strength.

Weaknesses:

There are many weaknesses in this manuscript, which include drawing association of data derived from unmatched sample sets, selection of low abundance miRNAs for developing the signature with inadequate rationale, incomplete description of experimental methods and confusing statements in the text.

Reviewer #1 (Public review):

Summary:

Zhu et al., investigate the cellular defects in glia as a result of loss in DEGS1/ifc encoding the dihydroceramide desaturase. Using the strength of Drosophila and its vast genetic toolkit, they find that DEGS1/ifc is mainly expressed in glia and it's loss leads to profound neurodegeneration. This supports a role for DEGS1 in the developing larval brain as it safeguards proper CNS development. Loss of DEGS1/ifc leads to dihydroceramide accumulation in the CNS and induces alteration in the morphology of glial subtypes and a reduction in glial number. Cortex and ensheathing glia appeared swollen and accumulated internal membranes. Astrocyte-glia on the other hand displayed small cell bodies, reduced membrane extension and disrupted organization in the dorsal ventral nerve cord. They also found that DEGS1/ifc localizes primarily to the ER. Interestingly, the authors observed that loss of DEGS1/ifc drives ER expansion and reduced TGs and lipid droplet numbers. No effect on PC and PE and a slight increase in PS.

The conclusions of this paper are well supported by the data.

Strengths:

This is an interesting study that provides new insight into the role of ceramide metabolism in neurodegeneration.

The strength of the paper is the generation of LOF lines, the insertion of transgenes and the use of the UAS-GAL4/GAL80 system to assess the cell-autonomous effect of DEGS1/ifc loss in neurons and different glial subtypes during CNS development.

The imaging, immunofluorescence staining and EM of the larval brain and the use of the optical lobe and the nerve cord as a readout are very robust and nicely done.

Drosophila is a difficult model to perform core biochemistry and lipidomics, but the authors used the whole larvae and CNS to uncover global changes in mRNA levels related to lipogenesis and the unfolded protein responses, as well as specific lipid alterations upon DEGS1/ifc loss.

Weaknesses:

No major weaknesses identified.

Minor point: The authors performed lipidomics and RTqPCR on whole larvae and larval CNS which does not inform of any cell type-specific effects. Performing single-cell RNAseq on larval brains to tease apart the cell-type specific effect of DEGS1/ifc loss would be interesting to explore the future, but beyond the scope of the current study.

Reviewer #1 (Public review):

The study by Lotonin et al. investigates correlates of protection against African swine fever virus (ASFV) infection. The study is based on a comprehensive work, including the measurement of immune parameters using complementary methodologies. An important aspect of the work is the temporal analysis of the immune events, allowing for the capture of the dynamics of the immune responses induced after infection. Also, the work compares responses induced in farm and SPF pigs, showing the latter an enhanced capacity to induce a protective immunity. Overall, the results obtained are interesting and relevant for the field. The findings described in the study further validate work from previous studies (critical role of virus-specific T cell responses) and provide new evidence on the importance of a balanced innate immune response during the immunization process. This information increases our knowledge on basic ASF immunology, one of the important gaps in ASF research that needs to be addressed for a more rational design of effective vaccines. Further studies will be required to corroborate that the results obtained based on the immunization of pigs by a not completely attenuated virus strain are also valid in other models, such as immunization using live attenuated vaccines.

While overall the conclusions of the work are well supported by the results, I consider that the following issues should be addressed to improve the interpretation of the results:

(1) An important issue in the study is the characterization of the infection outcome observed upon Estonia 2014 inoculation. Infected pigs show a long period of viremia, which is not linked to clinical signs. Indeed, animals are recovered by 20 days post-infection (dpi), but virus levels in blood remain high until 141 dpi. This is uncommon for ASF acute infections and rather indicates a potential induction of a chronic infection. Have the authors analysed this possibility deeply? Are there lesions indicative of chronic ASF in infected pigs at 17 dpi (when they have sacrificed some animals) or, more importantly, at later time points? Does the virus persist in some tissues at late time points, once clinical signs are not observed? Has all this been tested in previous studies?

(2) Virus loads post-Estonia infection significantly differ from whole blood and serum (Figure 1C), while they are very similar in the same samples post-challenge. Have the authors validated these results using methods to quantify infectious particles, such as Hemadsorption or Immunoperoxidase assays? This is important, since it would determine the duration of virus replication post-Estonia inoculation, which is a very relevant parameter of the model.

(3) Related to the previous points, do the authors consider it expected that the induction of immunosuppressive mechanisms during such a prolonged virus persistence, as described in humans and mouse models? Have the authors analysed the presence of immunosuppressive mechanisms during the virus persistence phase (IL10, myeloid-derived suppressor cells)? Have the authors used T cell exhausting markers to immunophenotype ASFV Estonia-induced T cells?

(4) A broader analysis of inflammatory mediators during the persistence phase would also be very informative. Is the presence of high VLs at late time points linked to a systemic inflammatory response? For instance, levels of IFN are still higher at 11 dpi than at baseline, but they are not analysed at later time points.

(5) The authors observed a correlation between IL1b in serum before challenge and protection. The authors also nicely discuss the potential role of this cytokine in promoting memory CD4 T cell functionality, as demonstrated in mice previously. However, the cells producing IL1b before ASFV challenge are not identified. Might it be linked to virus persistence in some organs? This important issue should be discussed in the manuscript.

(6) The lack of non-immunized controls during the challenge makes the interpretation of the results difficult. Has this challenge dose been previously tested in pigs of the age to demonstrate its 100% lethality? Can the low percentage of protected farm pigs be due to a modulation of memory T and B cell development by the persistence of the virus, or might it be related to the duration of the immunity, which in this model is tested at a very late time point? Related to this, how has the challenge day been selected? Have the authors analysed ASFV Estonia-induced immune responses over time to select it?

(7) Also, non-immunized controls at 0 dpc would help in the interpretation of the results from Figure 2C. Do the authors consider that the pig's age might influence the immune status (cytokine levels) at the time of challenge and thus the infection outcome?

(8) Besides anti-CD2v antibodies, anti-C-type lectin antibodies can also inhibit hemadsorption (DOI: 10.1099/jgv.0.000024). Please correct the corresponding text in the results and discussion sections related to humoral responses as correlates of protection. Also, a more extended discussion on the controversial role of neutralizing antibodies (which have not been analysed in this study), or other functional mechanisms such as ADCC against ASFV would improve the discussion.

Reviewer #1 (Public review):

Summary:

Li et al describe a novel form of melanosome based iridescence in the crest of an Early Cretaceous enantiornithine avialan bird from the Jehol Group.

This is an interesting manuscript that describes never before seen melanosome structures and explores fossilised feathers through new methods. This paper creates an opening for new work to explore coloration in extinct birds.

Strengths:

A novel set of methods applied to the study of fossil melanosomes.

Comments on revised version:

The authors provided a response to the previous 9 issues, for which additional response is provided here:

(1) I respectfully disagree with the authors justification regarding the crest. They show one specimen of Confuciusornis with short feathers (which appears to be a unique feature of this species, possibly related to the fact it is beaked) but what about the more primitive Eoconfuciusornis, a referred specimen of which superficially has an enormous "crest" (Zheng et al 2017), as does Changchengornis (Ji et al 1999). Regardless, it would make more sense compare this new specimen to other enantiornithines. Although limited by the preservation of body feathers, which is not all that common, the following published enantiornithines also exhibit a "crest": bohaiornithid indet. (Peteya et al 2017); Brevirostruavis (Li et al 2021); Dapingfangornis (Li et al 2006); Eoenantiornis (Zhou et al 2005); Grabauornis (Dalsatt etal 2014); Junornis (Liu et al 2017); Longirostravis (Hou etal 2004); Monoenantiornis (Hu & O'Connor 2016); Neobohaiornis (Shen etal 2024); Orienantiornis (Liu etal 2019); Parabohaironis (Wang 2023); Parapengornis (Hu etal 2015); Paraprotopteryx (Zheng et al 2007); and every specimen of Protopteryx. In fact, every single published enantiornithine that preserves any feathering on the head has the feathers preserved perpendicular to the bone (in fact, the body feathers on all parts of the bed are splayed at a right angle to the bone due to compression), as shown in the confuciuornis specimen image provided by the authors. Since it is highly improbable they all had crests, the authors have no justification for the interpretation that this new specimen was crested. This does not mean that the feathers were not iridescent or take away from the novel methods these authors have used to explore preserved feathers.

(2) Yes, this is possible, but see above for the very strong argument against interpretation of these feathers as forming a crest.

(3) This just further makes the point that the isolated feather is not likely from the head. Since the neck feathers are missing, it is more likely that it is these feathers that have been disarticulated (and sampled) from the neck region rather than from the very complete looking head feathers; this has significant implications with regards to the birds colour pattern.

(4) Thank you for acknowledging taphonomy.

(5) An interesting hypothesis and one I look forward to seeing explored in the future.

(6) Since the compression is in a single direction, in fact it is not reasonable to assume that distortion would be random. One might predict similar distortion, as with the feathers (spread out from the bone at a 90˚ angle) and bone (crushed), which are all compressed in a single direction. However, I agree that such a consistent discovery suggests it is not an artifact of preservation, and only further studies will elucidate this

(7) I still fail to detect this hexagonal pattern - could machine learning be used to quantify this pattern? The random arrangement of white arrows does little to clarify the authors interpretations.

(8) Great to see additional sampling

(9) Thank you for the explanation.

Reviewer #1 (Public review):

Summary:

Palardy and colleagues examine how transcription factors of the SoxB1 family alter patterning within the zebrafish posterior lateral line primordium and subsequent formation of neuromast organs along the body of the developing fish. They describe how expression of soxb genes changes when Wnt and Fgf signaling pathways are altered, and in addition, how outputs of these signalling pathways change when soxb gene expression is disrupted. Together, experiments suggest a model where the expression of SoxB genes counteracts Wnt signaling. Support comes from the combined inhibition of both pathways, partially restoring the pattern of neuromast deposition. Together, the work reveals an additional layer of control over Wnt and Fgf signals that together ensure proper posterior lateral line development.

Strengths:

The authors provide a clear analysis of changes in RNA expression after systematic manipulation of gene expression and signaling pathways to construct a plausible model of how Sox factors regulate primordium patterning.

Weaknesses:

There is little attempt to capture the variation of expression patterns with each manipulation. Photomicrographs are examples, with little quantification.

While the combined loss of soxb functions shows more severe phenotypes, it is not exactly clear what underlies the apparent redundancy. It would be helpful if the soxb gene family member expression was reported after loss of each. Expression of sox1a is shown in sox2 mutants in Figure 4, but other combinations are not reported. This additional analysis would clarify whether there are alterations in expression that influence apparent redundancy.

Reviewer #1 (Public review):

Summary:

This paper introduces a new class of machine learning models for capturing how likely a specific nucleotide in a rearranged IG gene is to undergo somatic hypermutation. These models modestly outperform existing state-of-the-art efforts, despite having fewer free parameters. A surprising finding is that models trained on all mutations from non-functional rearrangements give divergent results from those trained on only silent mutations from functional rearrangements.

Strengths:

* The new model structure is quite clever and will provide a powerful way to explore larger models.<br /> * Careful attention is paid to curating and processing large existing data sets.<br /> * The authors are to be commended for their efforts to communicate with the developers of previous models and use the strongest possible versions of those in their current evaluation.

Weaknesses:

* No significant weaknesses noted

Reviewer #1 (Public review):

Summary:

The authors revisit the specific domains/signals required for redirection of an inner nuclear membrane protein, emerin, to the secretory pathway. They find that epitope tagging influences protein fate, serving as a cautionary tale for how different visualisation methods are used. Multiple tags and lines of evidence are used, providing solid evidence for the altered fate of different constructs.

Strengths:

This is a thorough dissection of domains and properties that confer INM retention vs secretion to the PM/lysosome, and will serve the community well as a caution regarding placement of tags and how this influences protein fate.

Weaknesses:

The specific biogenesis pathway for C-terminally tagged emerin might confound some interpretations. Appending the large GFP to the C-terminus may direct the fusion protein to a different ER insertion pathway than that used by the endogenous protein. How this might influence the fate of the tagged protein remains to be determined. In some ways this is beyond the scope of the current study, but should serve as a warning to epitope-tagging approaches.

Reviewer #1 (Public review):

Summary:

The authors report four cryoEM structures (2.99 to 3.65 Å resolution) of the 180 kDa, full-length, glycosylated, soluble Angiotensin-I converting enzyme (sACE) dimer, with two homologous catalytic domains at the N- and C-terminal ends (ACE-N and ACE-C). ACE is a protease capable of effectively degrading Aβ. The four structures are C2 pseudo-symmetric homodimers and provide insight into sACE dimerization. These structures were obtained using discrete classification in cryoSPARC and show different combinations of open, intermediate, and closed states of the catalytic domains, resulting in varying degrees of solvent accessibility to the active sites.

To deepen the understanding of the gradient of heterogeneity (from closed to open states) observed with discrete classification, the authors performed all-atom MD simulations and continuous conformational analysis of cryo-EM data using cryoSPARC 3DVA, cryoDRGN, and RECOVAR. cryoDRGN and cryoSPARC 3DVA revealed coordinated open-closed transitions across four catalytic domains, whereas RECOVAR revealed independent motion of two ACE-N domains, also observed with cryoSPARC focused classification. The authors suggest that the discrepancy in the results of the different methods for continuous conformational analysis in cryo-EM could results from different approaches used for dimensionality reduction and trajectory generation in these methods.

Strengths:

This is an important study that shows, for the first time, the structure and the snapshots of the dynamics of the full-length sACE dimer. Moreover, the study highlights the importance of combining insights from different cryo-EM methods that address questions difficult or impossible to tackle experimentally, while lacking ground truth for validation.

Weaknesses:

The open, closed, and intermediate states of ACE-N and ACE-C in the four cryo-EM structures from discrete classification were designated quantitatively (based on measured atomic distances on the models fitted into cryo-EM maps). Unfortunately, atomic models were not fitted into cryo-EM maps obtained with cryoSPARC 3DVA, cryoDRGN, and RECOVAR, and the open/closed states in these cases were designated based on a qualitative analysis.

Reviewer #1 (Public review):

Summary:

This study utilizes polarized second-harmonic generation (pSHG) microscopy to investigate myosin conformation in the relaxed state, distinguishing between the disordered, actin-accessible ON state and the ordered, energy-conserving OFF state. By pharmacologically modulating the ON/OFF equilibrium with a myosin activator (2-deoxyATP) and inhibitor (Mavacamten), the authors demonstrate that pSHG can sensitively quantify the ON/OFF ratio in both skeletal and cardiac muscle. Validation with X-ray diffraction supports the accuracy of the method. Applying this approach to a hypertrophic cardiomyopathy model, the study shows that R403Q/MYH7-mutated minipigs exhibit an increased ON state fraction relative to controls. This difference is eliminated under saturating concentrations of myosin modulators, indicating that the ON/OFF balance can be pharmacologically shifted to its extremes. Additionally, ATPase assays reveal elevated resting ATPase activity in R403Q samples, which persists even when the ON state is saturated, suggesting that increased energy consumption in this mutation is driven by both a shift toward the ON state and inherently higher myosin ATPase activity.

Strengths:

This is a well-written and well-conducted study that clearly reveals the power of SHG microscopy. The study clearly establishes the great utility of SHG to study thick filament regulation.

Weaknesses:

(1) Several studies have shown that the ON state of the thick filament is sensitive to both temperature and filament lattice spacing, with a common recommendation to conduct skinned fiber experiments at temperatures above 27{degree sign}C and in the presence of dextran to better preserve physiological conditions. The authors should clarify the experimental temperature used in their skinned fiber studies, indicate whether dextran was included, and discuss whether adherence to these recommended conditions would have impacted their results.

(2) On page 13, the authors report the proportion of disordered heads as approximately 30% in wild-type and 65% in R403Q fibers. They should clarify whether these values represent the percentage of total myosin heads, or rather the percentage of heads that are responsive to Mavacamten and dATP.

(3) In Figure 5, regarding ATPase measurements, the content of contractile material per unit volume of muscle preparation will influence the results. Did the authors account for this variable, and if not, how might it have affected the conclusions?

(4) For readers primarily interested in assessing the ON/OFF state of thick filaments, could the authors list the specific advantages of polarized second harmonic generation (pSHG) microscopy compared to X-ray diffraction?

(5) Given that many data points were derived from the same fiber or myocyte, how did the authors address the risk of type I errors due to non-independence of measurements? Was a nested or hierarchical statistical approach used?

Reviewer #1 (Public review):

Summary:

This manuscript presents findings on the adaptation mechanisms of Saccharomyces cerevisiae under extreme stress conditions. The authors try to generalize this to adaptation to stress tolerance. A major finding is that S. cerevisiae evolves a quiescence-like state with high trehalose to adapt to freeze-thaw tolerance independent of their genetic background. The manuscript is comprehensive, and each of the conclusions is well supported by careful experiments.

Strengths:

This is excellent interdisciplinary work.

Weaknesses: .

I have questions regarding the overall novelty of the proposal, which I would like the authors to explain.

(1) Earlier papers have shown that loss of ribosomal proteins, that slow growth, leads to better stress tolerance in S. cerevisiae. Given this, isn't it expected that any adaptation that slows down growth would, overall, increase stress tolerance? Even for other systems, it has been shown that slowing down growth (by spore formation in yeast or bacteria/or dauer formation in C. elegans) is an effective strategy to combat stress and hence is a likely route to adaptation. The authors stress this as one of the primary findings. I would like the authors to explain their position, detailing how their findings are unexpected in the context of the literature.

(2) Convergent evolution of traits: I find the results unsurprising. When selecting for a trait, if there is a major mode to adapt to that stress, most of the strains would adapt to that mode, independent of the route. According to me, finding out this major route was the objective of many of the previous reports on adaptive evolution. The surprising part in the previous papers (on adaptive evolution of bacteria or yeast) was the resampling of genes that acquired mutations in multiple replicates of an evolution experiments, providing a handle to understand the major genetic route or the molecular mechanism that guides the adaptation (for example in this case it would be - what guides the over-accumulation of trehalose). I fail to understand why the authors find the results surprising, and I would be happy to understand that from the authors. I may have missed something important.

(3) Adaptive evolution would work on phenotype, as all of selective evolution is supposed to. So, given that one of the phenotypes well-known in literature to allow free-tolerance is trehalose accumulation, I think it is not surprising that this trait is selected. For me, this is not a case of "non-genetic" adaptation as the authors point out: it is likely because perturbation of many genes can individually result in the same outcome - upregulation of trehalose accumulation. Thereby, although the adaptation is genetic, it is not homogeneous across the evolving lines - the end result is. Do the authors check that the trait is actually a non-genetic adaptation, i.e., if they regrow the cells for a few generations without the stress, the cells fall back to being similarly only partially fit to freeze-thaw cycles? Additionally, the inability to identify a network that is conserved in the sequencing does not mean that there is no regulatory pathway. A large number of cryptic pathways may exist to alter cellular metabolic states.<br /> This is a point in continuation of point #2, and I would like to understand what I have missed.

(4) To propose the convergent nature, it would be important to check for independently evolved lines and most probably more than 2 lines. It is not clear from their results section if they have multiple lines that have evolved independently.

(5) For the genomic studies, it is not clear if the authors sequenced a pool or a single colony from the evolved strains. This is an important point, since an average sequence will miss out on many mutations and only focus on the mutations inherited from a common ancestral cell. It is also not clear from the section.

Reviewer #1 (Public review):

Summary:

This study builds off prior work that focused on the molecule AA147 and its role as an activator of the ATF6 arm of the unfolded protein response. In prior manuscripts, AA147 was shown to enter the ER, covalently modify a subset of protein disulfide isomerases (PDIs), and improve ER quality control for the disease-associated mutants of AAT and GABAA. Unsuccessful attempts to improve the potency of AA147 have led the authors to characterize a second hit from the screen in this study: the phenylhydrazone compound AA263. The focus of this study on enhancing the biological activity of the AA147 molecule is compelling, and overcomes a hurdle of the prior AA147 drug that proved difficult to modify. The study successfully identifies PDIs as a shared cellular target of AA263 and its analogs. The authors infer, based on the similar target hits previously characterized for AA147, that PDI modification accounts for a mechanism of action for AA263.

Strengths:

The authors are able to establish that, like AA147, AA263 covalently targets ER PDIs. The work establishes the ability to modify the AA263 molecule to create analogs with more potency and efficacy for ATF6 activation. The "next generation" analogs are able to enhance the levels of functional AAT and GABAA receptors in cellular models expressing the Z-variant of AAT or an epilepsy-associated variant of the GABAA receptor, outlining the therapeutic potential for this molecule and laying the foundation for future organism-based studies.

Weaknesses:

Arguably, the work does not fully support the statement provided in the abstract that the study "reveals a molecular mechanism for the activation of ATF6". The identification of targets of AA263 and its analogs is clear. However, it is a presumption that the overlap in PDIs as targets of both AA263 and AA147 means that AA263 works through the PDIs. While a likely mechanism, this conclusion would be bolstered by establishing that knockdown of the PDIs lessens drug impact with respect to ATF6 activation. Alternatively, it has previously been suggested that the cell-type dependent activity of AA263 may be traced to the presence of cell-type specific P450s that allow for the metabolic activation of AA263 or cell-type specific PDIs (Plate et al 2016; Paxman et al 2018). If the PDI target profile is distinct in different cell types, and these target difference correlates with ATF6-induced activity by AA263, that would also bolster the authors' conclusion.

DOI: 10.1038/s44319-025-00511-8

Resource: None

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-ALT-151022-1

What is this?

Reviewer #1 (Public review):

Summary:

This study provided key experimental evidence for the "Solstice-as-Phenology-Switch Hypothesis" through two temperature manipulation experiments.

Strengths:

The research is data-rich, particularly in exploring the effects of pre- and post-solstice cooling, as well as daytime versus nighttime cooling, on bud set timing, showcasing significant innovation. The article is well-written, logically clear, and is likely to attract a wide readership.

Weaknesses:

However, there are several issues that need to be addressed.

(1) In Experiment 1, significant differences were observed in the impact of cooling in July versus August. July cooling induced a delay in bud set dates that was 3.5 times greater in late-leafing trees compared to early-leafing ones, while August cooling induced comparable advances in bud set timing in both early- and late-leafing trees. The study did not explain why the timing (July vs. August) resulted in different mechanisms. Can a link be established between phenology and photosynthetic product accumulation? Additionally, can the study differentiate between the direct warming effect and the developmental effect, and quantify their relative contributions?

(2) The two experimental setups differed in photoperiod: one used a 13-hour photoperiod at approximately 4,300 lux, while the other used an ambient day length of 16 hours with a light intensity of around 6,900 lux. What criteria were used to select these conditions, and do they accurately represent real-world scenarios? Furthermore, as shown in Figure S1, significant differences in soil moisture content existed between treatments - could this have influenced the conclusions?

(3) The authors investigated how changes in air temperature around the summer solstice affected primary growth cessation, but the summer solstice also marks an important transition in photoperiod. How can the influence of photoperiod be distinguished from the temperature effect in this context?

(4) The study utilized potted trees in a controlled environment, which limits the generalization of the results to natural forests. Wild trees are subject to additional variables, such as competition and precipitation. Moreover, climate differences between years (2022 vs. 2023) were not controlled. As such, the conclusions may be overgeneralized to "all temperate tree species", as the experiment only involved potted European beech seedlings. The discussion would benefit from addressing species-specific differences.

Reviewer #1 (Public review):

Summary:

The paper describes the cryoEM structure of RAD51 filament on the recombination intermediate. In the RAD51 filament, the insertion of a DNA-binding loop called the L2 loop stabilizes the separation of the complementary strand for the base-pairing with an incoming ssDNA and the non-complementary strand, which is captured by the second DNA-binding channel called the site II. The molecular structure of the RAD51 filament with a recombination intermediate provides a new insight into the mechanism of homology search and strand exchange between ssDNA and dsDNA.

Strong points:

This is the first human RAD51 filament structure with a recombination intermediate called the D-loop. The work has been done with great care, and the results shown in the paper are compelling based on cryo-EM and biochemical analyses. The paper is really nice and important for researchers in the field of homologous recombination, which gives a new view on the molecular mechanism of RAD51-mediated homology search and strand exchange.

Comments on revisions:

The authors nicely address most of the previous points.

Reviewer #1 (Public review):

Summary:

The authors extended a previous study of selective response to herbivory in Arabidopsis, in order to look specifically for selection on induced epigenetic variation ("Lamarckian evolution"). They found no evidence. In addition, the re-examined result from a previously published study arguing that environmentally induced epigenetic variation was common, and found that these findings were almost certainly artifactual.

Strengths:

The paper is very clearly written, there is no hype, and the methods used are state-of-the-art.

Weaknesses:

The result is negative, so the best you can do is put an upper bound on any effects.

Significance:

Claims about epigenetic inheritance and Lamarckian evolution continue to be made based on very shaky evidence. Convincing negative results are therefore important. In addition, the study presents results that, to this reviewer, suggest that the 2024 paper by Lin et al. [26] should probably be retracted.

Reviewer #1 (Public review):

Summary:

The authors quantified information in gesture and speech, and investigated the neural processing of speech and gestures in pMTG and LIFG, depending on their informational content, in 8 different time-windows, and using three different methods (EEG, HD-tDCS and TMS). They found that there is a time-sensitive and staged progression of neural engagement that is correlated with the informational content of the signal (speech/gesture).

Strengths:

A strength of the paper is that the authors attempted to combine three different methods to investigate speech-gesture processing.

Comments on revisions:

I thank the authors for their careful responses to my comments. However, I remain not convinced by their argumentation regarding the specificity of their spatial targeting and the time-windows that they used.

I do not believe the authors have adequately demonstrated the spatial and temporal specificity required to disentangle the contributions of the IFG and pMTG during the gesture-speech integration process. While the authors have made a sincere effort to address the concerns raised by the reviewers, and have done so with a lot of new analyses, I remain doubtful that the current methodological approach is sufficient to draw conclusions about the causal roles of the IFG and pMTG in gesture-speech integration.

Reviewer #1 (Public review):

Summary:

Kang et al. provide the first experimental insights from holographic stimulation of auditory cortex. Using stimulation of functionally-defined ensembles, they test whether overactivation of a specific subpopulation biases simultaneous and subsequent sensory-evoked network activations.

Strengths:

The investigators use a novel technique to investigate the sensory response properties in functionally defined cell assemblies in auditory cortex. These data provide the first evidence of how acutely perturbing specific frequency-tuned neurons impacts the tuning across a broader population. Their revised manuscript appropriately tempers any claims about specific plasticity mechanisms involved.

Weaknesses:

Although the single cell analyses in this manuscript are comprehensive, questions about how holographic stimulation impacts population coding are left to future manuscripts, or perhaps re-analyses of this unique dataset.

Reviewer #1 (Public review):

Summary:

The authors have developed self-amplifying RNAs (saRNAs) encoding additional genes to suppress dsRNA-related inflammatory responses and cytokine release. Their results demonstrate that saRNA constructs encoding anti-inflammatory genes effectively reduce cytotoxicity and cytokine production, enhancing the potential of saRNAs. This work is significant for advancing saRNA therapeutics by mitigating unintended immune activation.

Strengths:

This study successfully demonstrates the concept of enhancing saRNA applications by encoding immune-suppressive genes. A key challenge for saRNA-based therapeutics, particularly for non-vaccine applications, is the innate immune response triggered by dsRNA recognition. By leveraging viral protein properties to suppress immunity, the authors provide a novel strategy to overcome this limitation. The study presents a well-designed approach with potential implications for improving saRNA stability and minimizing inflammatory side effects.

Comments on revisions:

All comments have been thoroughly addressed, and the manuscript has been significantly improved.

Reviewer #1 (Public review):

Summary:

The study by Wu et al. uses endogenous bruchpilot expression in a cell-type-specific manner to assess synaptic heterogeneity in adult Drosophila melanogaster mushroom body output neurons. The authors performed genomic on locus tagging of the presynaptic scaffold protein bruchpilot (BRP) with one part of splitGFP (GFP11) using the CRISPR/Cas9 methodology and co-expressed the other part of splitGFP (GFP1-10) using the GAL4/UAS system. Upon expression of both parts of splitGFP, fluorescent GFP is assembled at the N-terminus of BRP, exactly where BRP is endogenously expressed in active zones. For manageable analysis, a high-throughput pipeline was developed. This analysis evaluated parameters like location of BRP clusters, volume of clusters, and cluster intensity as a direct measure of the relative amount of BRP expression levels on site, using publicly available 3D analysis tools that are integrated in Fiji. Analysis was conducted for different mushroom body cell types in different mushroom body lobes using various specific GAL4 drivers. To test this new method of synapse assessment, Wu et al. performed an associative learning experiment in which an odor was paired with an aversive stimulus and found that, in a specific time frame after conditioning, the new analysis solidly revealed changes in BRP levels at specific synapses that are associated with aversive learning.

Strengths:

Expression of splitGFP bound to BRP enables intensity analysis of BRP expression levels as exactly one GFP molecule is expressed per BRP. This is a great tool for synapse assessment. This tool can be widely used for any synapse as long as driver lines are available to co-express the other part of splitGFP in a cell-type-specific manner. As neuropils and thus the BRP label can be extremely dense, the analysis pipeline developed here is very useful and important. The authors have chosen an exceptionally dense neuropil - the mushroom bodies - for their analysis and convincingly show that BRP assessment can be achieved with such densely packed active zones. The result that BRP levels change upon associative learning in an experiment with odor presentation paired with punishment is likewise convincing, and strongly suggests that the tool and pipeline developed here can be used in an in vivo context.

Weaknesses:

Although BRP is an important scaffold protein and its expression levels were associated with function and plasticity, I am still somewhat reluctant to accept that synapse structure profiling can be inferred from only assessing BRP expression levels and BRP cluster volume. Also, is it guaranteed that synaptic plasticity is not impaired by the large GFP fluorophore? Could the GFP10 construct that is tagged to BRP in all BRP-expressing cells, independent of GAL4, possibly hamper neuronal function? Is it certain that only active zones are labeled? I do see that plastic changes are made visible in this study after an associative learning experiment with BRP intensity and cluster volume as read-out, but I would be reassured by direct measurement of synaptic plasticity with splitGFP directly connected to BRP, maybe at a different synapse that is more accessible.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to uncover how the Parkinson's disease-linked LRRK2 G2019S mutation affects synaptic integrity through astrocyte-intrinsic mechanisms. Specifically, they investigate whether LRRK2-driven ERM hyperphosphorylation disrupts astrocyte morphology and excitatory synapse maintenance, with a focus on regional specificity within the cortex.

Strengths:

(1) Novelty and significance: The work provides important insights into non-neuronal contributions to Parkinson's disease (PD) pathology by highlighting a previously underappreciated role of astrocytic ERM signaling in synapse maintenance. This astrocyte-specific mechanism might help explain early cognitive dysfunctions in PD.

(2) Mechanistic depth: The authors present a detailed molecular pathway where the LRRK2 G2019S mutation increases ERM phosphorylation, disrupting Ezrin-Atg7 interactions critical for astrocyte morphology.

(3) Robust methodology: The study uses a powerful combination of tools, including AAV-mediated gene delivery, BioID-based interactome mapping, PALE labeling, and patch-clamp electrophysiology to link molecular, morphological, and functional changes.

(4) Physiological relevance: Parallel findings in both mouse models and human post-mortem brains suggest conservation of the observed phenotypes and strengthen the relevance to PD pathogenesis.

Weaknesses:

(1) Causal directionality: While ERM hyperphosphorylation is clearly shown to correlate with morphological and synaptic changes, the specific causal hierarchy-especially between Ezrin-Atg7 interaction loss and synapse alteration, is inferred but not definitively proven. For example, a rescue experiment directly restoring Atg7 function alongside Ezrin manipulation could strengthen this point.

(2) Brain region specificity: Although regional differences between ACC and MOp are well documented, the underlying cause of this differential vulnerability remains speculative. Examining astrocyte heterogeneity within cortical layers or via transcriptomic/proteomic profiling could clarify these regional effects.

(3) Autophagy function: While Atg7 knockdown leads to clear morphological changes, autophagic flux (e.g., LC3-II turnover or p62 accumulation) is not directly assessed. This would strengthen the mechanistic link to autophagy disruption.

(4) GFAP-based astrogliosis interpretation: The conclusion that no astrogliosis occurs in LRRK2 G2019S mice is based solely on GFAP staining. However, GFAP-negative reactive states have been reported. Including additional markers would help validate this interpretation.

(5) Impact on neuronal populations: The authors conclude that changes in inhibitory synapse density in the MOp are not rescued by astrocytic Ezrin manipulation and suggest developmental effects on interneurons. However, this is speculative without neuronal cell-type-specific data. Including interneuron density or synaptic connectivity analysis would make this claim more robust.

(6) Despite these limitations, the authors substantially achieve their stated aims. Their results provide strong support for a model in which astrocytic ERM signaling downstream of LRRK2 contributes to region-specific synaptic changes, particularly in the anterior cingulate cortex. While certain mechanistic links-such as the role of Ezrin-Atg7 interaction in synaptic maintenance-would benefit from further functional validation, the study offers a well-supported framework for understanding astrocyte-intrinsic contributions to synaptic dysfunction in Parkinson's disease.

This work is likely to contribute meaningfully to ongoing research in neurodegeneration, glial biology, and synaptic regulation. The methodological approaches - especially the combination of in vivo models with proteomics and electrophysiology - will be of interest to others studying astrocyte function and neuron-glia interactions. More broadly, the study highlights the importance of astrocyte heterogeneity and regional specialization in shaping neural circuit vulnerability, providing a valuable foundation for future investigations.

Reviewer #1 (Public review):

Summary:

The authors have investigated the role of GAT3 in the visual system. First, they have developed a CRISPR/Cas9-based approach to locally knock out this transporter in the visual cortex. They then demonstrated electrophysiologically that this manipulation increases inhibitory synaptic input into layer 2/3 pyramidal cells. They further examined the functional consequences by imaging neuronal activity in the visual cortex in vivo. They found that the absence of GAT3 leads to reduced spontaneous neuronal activity and attenuated neuronal responses and reliability to visual stimuli, but without an effect on orientation selectivity. Further analysis of this data suggests that Gat3 removal leads to less coordinated activity between individual neurons and in population activity patterns, thereby impairing information encoding. Overall, this is an elegant and technically advanced study that demonstrates a new and important role of GAT3 in controlling the processing of visual information.

Strengths:

(1) Development of a new approach for a local knockout (GAT3).

(2) Important and novel insights into visual system function and its dependence on GAT3.

(3) Plausible cellular mechanism.

Weaknesses:

No major weaknesses were identified by this reviewer.

Reviewer #1 (Public review):

Summary:

This study aims to investigate the development of infants' responses to music by examining neural activity via EEG and spontaneous body kinematics using video-based analysis. The authors also explore the role of musical pitch in eliciting neural and motor responses, comparing infants at 3, 6, and 12 months of age.

Strengths:

A key strength of the study lies in its analysis of body kinematics and modeling of stimulus-motor coupling, demonstrating how the amplitude envelope of music predicts infant movement, and how higher musical pitch may enhance auditory-motor synchronization.

Weaknesses:

The neural data analysis is currently limited to auditory evoked potentials aligned with beat timing. A more comprehensive approach is needed to robustly support the proposed developmental trajectory of neural responses to music.

Reviewer #1 (Public review):

Summary:

This article investigates the origin of movement slowdown in weightlessness by testing two possible hypotheses: the first is based on a strategic and conservative slowdown, presented as a scaling of the motion kinematics without altering its profile, while the second is based on the hypothesis of a misestimation of effective mass by the brain due to an alteration of gravity-dependent sensory inputs, which alters the kinematics following a controller parameterization error.

Strengths:

The article convincingly demonstrates that trajectories are affected in 0g conditions, as in previous work. It is interesting, and the results appear robust. However, I have two major reservations about the current version of the manuscript that prevent me from endorsing the conclusion in its current form.

Weaknesses:

(1) First, the hypothesis of a strategic and conservative slowdown implicitly assumes a similar cost function, which cannot be guaranteed, tested, or verified. For example, previous work has suggested that changing the ratio between the state and control weight matrices produced an alteration in movement kinematics similar to that presented here, without changing the estimated mass parameter (Crevecoeur et al., 2010, J Neurophysiol, 104 (3), 1301-1313). Thus, the hypothesis of conservative slowing cannot be rejected. Such a strategy could vary with effective mass (thus showing a statistical effect), but the possibility that the data reflect a combination of both mechanisms (strategic slowing and mass misestimation) remains open.

(2) The main strength of the article is the presence of directional effects expected under the hypothesis of mass estimation error. However, the article lacks a clear demonstration of such an effect: indeed, although there appears to be a significant effect of direction, I was not sure that this effect matched the model's predictions. A directional effect is not sufficient because the model makes clear quantitative predictions about how this effect should vary across directions. In the absence of a quantitative match between the model and the data, the authors' claims regarding the role of misestimating the effective mass remain unsupported.

In general, both the hypotheses of slowing motion (out of caution) and misestimating mass have been put forward in the past, and the added value of this article lies in demonstrating that the effect depended on direction. However, (1) a conservative strategy with a different cost function can also explain the data, and (2) the quantitative match between the directional effect and the model's predictions has not been established.

Specific points:

(1) I noted a lack of presentation of raw kinematic traces, which would be necessary to convince me that the directional effect was related to effective mass as stated.

(2) The presentation and justification of the model require substantial improvement; the reason for their presence in the supplementary material is unclear, as there is space to present the modelling work in detail in the main text. Regarding the model, some choices require justification: for example, why did the authors ignore the nonlinear Coriolis and centripetal terms?

(3) The increase in the proportion of trials with subcomponents is interesting, but the explanatory power of this observation is limited, as the initial percentage was already quite high (from 60-70% during the initial study to 70-85% in flight). This suggests that the potential effect of effective mass only explains a small increase in a trend already present in the initial study. A more critical assessment of this result is warranted.

Reviewer #1 (Public review):

Summary:

This manuscript focuses on single-cell RNA sequencing (scRNA-seq) analysis following chronic methamphetamine (METH) treatment in mice. The authors propose two hypotheses:

(1) METH induces neuroinflammation involving T and NKT cells, and (2) METH alters neuronal stem cell differentiation.

Strengths:

The authors provide a substantial dataset with numerous replicates, offering valuable resources to the research community.

Weaknesses:

Concerns persist regarding the interpretation of data and the validation of experiments. First, the presence of T cells, NKT cells, and neutrophils in both the control and METH-treated hippocampi suggests that blood contamination rather than immune cell infiltration is the cause. Since the authors claim that METH disrupts the blood-brain barrier, increasing the infiltration of these immune cells, identifying the source of these immune cells is critical.

Secondly, the pseudotime analysis, which suggests altered neural stem cell (NSC) differentiation, is not conclusively supported by the current data and requires further validation.

Overall, the authors provided comprehensive in vivo data on the impact of methamphetamine on the hippocampus; however, further in vivo and in vitro experimental validation of the key findings is needed.

Reviewer #1 (Public review):

Summary:

This work by Hall et al provides a novel and important new finding about communication between the anterior cingulate cortex (ACC) and the CA1 region of the dorsal hippocampus: there is a clear ability of ACC to predict CA1 activity, and that is modulated by learning/experience. Furthermore, they have some evidence that the modulation differs by whether the CA1 neurons were in the deep versus superficial sub-layer of CA1. The evidence is suggestive of new and exciting findings, but some gaps and weaknesses remain to be addressed before I believe all of the authors' claims can be supported. The figures also need to be slightly better organized, and the discussion is missing a major dimension in my opinion. Overall, this is a strong submission, but with some gaps to fill.

Strengths:

(1) This is a well-written manuscript - the introduction was especially clear, well-cited, and motivating.

(2) The sub-layer specific communication between ACC and CA1 represents the discovery of a novel and functionally impactful piece of neurobiology.

(3) Optogenetics was an important verification of ACC-CA1 communication, as was the analysis of neurons by waveform type.

Weaknesses:

(1) Figure 2: Why are the data separated into two groups from the outset? If all data are combined, is there a general drop in prediction gain from pre to post?

(2) 2b and 2c are important since they are complementary means to show the same thing, and it is important that they cross-validate each other, especially since the non-significant task active neuron difference in 2b appears to be nearly as strong as the significant difference to its left. A more holistic analysis can be done to compare these dimensions.

(3) Sup vs deep neuron definition: Did the authors have any means to validate this anatomical separation using histology or otherwise? I don't believe they described anything like that, and instead use physiology to infer anatomical location. I understand anatomy-based methods may be practically impossible with tetrodes, but this limitation should at least be mentioned, and it should be explained that without something like silicon probes or histological validation, anatomy had to be inferred from physiology.

(4) Superficial vs deep differences in firing rate ratio based on PG: there are many fewer CAdeep neurons, but in 4c, the trends appear to be the same pre-training, top PG lower than others. It seems the lack of difference in CA1deep in 4c may be due to the much lower power/n. This should be discussed or addressed.

(5) In Figure 5, the term "firing rate ratio" is used, and it sounds the same as in previous figures, but this is a different ratio (based on modulation by opto stim, not task).

(6) I would like to learn more about these v-type neurons. I understand we do not yet know about their molecular or morphologic correlate, but more analysis can be done with the current data.

(7) I would like more discussion of ACC-CA1 connectivity.

(8) Some elements may be missing from the discussion, relating baseline functioning versus post-learning function.

Reviewer #1 (Public review):

Summary:

Zhang et al. addressed the question of whether hyperaltruistic preference is modulated by decision context and tested how oxytocin (OXT) may modulate this process. Using an adapted version of a previously well-established moral decision-making task, healthy human participants in this study undergo decisions that gain more (or lose less, termed as context) meanwhile inducing more painful shocks to either themselves or another person (recipient). The alternative choice is always less gain (or more loss) meanwhile less pain. Through a series of regression analyses, the authors reported that hyperaltruistic preference can only be found in the gain context but not in the loss context, however, OXT reestablished the hyperaltruistic preference in the loss context similar to that in the gain context.

Strengths:

This is a solid study that directly adapted a previously well-established task and the analytical pipeline to assess hyperaltruistic preference in separate decision contexts. Context-dependent decisions have gained more and more attention in literature in recent years, hence this study is timely. It also links individual traits (via questionnaires) with task performance, to test potential individual differences. The OXT study is done with great methodological rigor, including pre-registration. Both studies have proper power analysis to determine the sample size.

Weaknesses:

Despite the strengths, multiple analytical decisions have to be explained, justified, or clarified. Also, there is scope to enhance the clarity and coherence of the writing - as it stands, readers will have to go back and forth to search for information. Last, it would be helpful to add line numbers in the manuscript during the revision, as this will help all reviewers to locate the parts we are talking about.

Introduction:<br /> (1) The introduction is somewhat unmotivated, with key terms/concepts left unexplained until relatively late in the manuscript. One of the main focuses in this work is "hyperaltruistic", but how is this defined? It seems that the authors take the meaning of "willing to pay more to reduce other's pain than their own pain", but is this what the task is measuring? Did participants ever need to PAY something to reduce the other's pain? Note that some previous studies indeed allow participants to pay something to reduce other's pain. And what makes it "HYPER-altruistic" rather than simply "altruistic"? Plus, in the intro, the authors mentioned that the "boundary conditions" remain unexplored, but this idea is never touched again. What do boundary conditions mean here in this task? How do the results/data help with finding out the boundary conditions? Can this be discussed within wider literature in the Discussion section? Last, what motivated the authors to examine decision context? It comes somewhat out of the blue that the opening paragraph states that "We set out to [...] decision context", but why? Are there other important factors? Why decision context is more important than studying those others?

Experimental design:<br /> (2) The experiment per se is largely solid, as it followed a previously well-established protocol. But I am curious about how the participants got instructed? Did the experimenter ever mention the word "help" or "harm" to the participants? It would be helpful to include the exact instructions in the SI.

(3) Relatedly, the experimental details were not quite comprehensive in the main text. Indeed, Methods come after the main text, but to be able to guide readers to understand what was going on, it would be very helpful if the authors could include some necessary experimental details at the beginning of the Results section.

Statistical analysis<br /> (3) One of the main analyses uses the harm aversion model (Eq1) and the results section keeps referring to one of the key parameters of it (ie, k). However, it is difficult to understand the text without going to the Methods section below. Hence it would be very helpful to repeat the equation also in the main text. A similar idea goes to the delta_m and delta_s terms - it will be very helpful to give a clear meaning of them, as nearly all analyses rely on knowing what they mean.

(4) There is one additional parameter gamma (choice consistency) in the model. Did the authors also examine the task-related difference of gamma? This might be important as some studies have shown that the other-oriented choice consistency may differ in different prosocial contexts.

(5) I am not fully convinced that the authors included two types of models: the harm aversion model and logistic regression models. Indeed, the models look similar, and the authors have acknowledged that. But I wonder if there is a way to combine them? For example:<br /> Choice ~ delta_V * context * recipient (*Oxt_v._placebo)<br /> The calculation of delta_V follows Equation 1.<br /> Or the conceptual question is, if the authors were interested in the specific and independent contribution of dalta_m and dalta_s to behavior, as their logistic model did, why the authors examine the harm aversion first, where a parameter k is controlling for the trade-off? One way to find it out is to properly run different models and run model comparison. In the end, it would be beneficial to only focus on the "winning" model to draw inferences.

(6) The interpretation of the main OXT results needs to be more cautious. According to the operationalization, "hyperaltruistic" is the reduction of pain of others (higher % of choosing the less painful option) relative to the self. But relative to the placebo (as baseline), OXT did not increase the % of choosing the less painful option for others, rather, it decreased the % of choosing the less painful option for themselves. In other words, the degree of reducing other's pain is the same under OXT and placebo, but the degree of benefiting self-interest is reduced under OXT. I think this needs to be unpacked, and some of the wording needs to be changed. I am not very familiar with the OXT literature, but I believe it is very important to differentiate whether OXT is doing something on self-oriented actions vs other-oriented actions. Relatedly, for results such as that in Fig5A, it would be helpful to not only look at the difference, but also the actual magnitude of the sensitivity to the shocks, for self and others, under OXT and placebo.

Comments on revisions:

I did not change my original public review, as I think it can still be helpful for the field to see the reasoning and argument.

For the revision, the authors have done a thorough job of addressing my previous comments and questions.

The only aspect I would like to ask is that, it would still be great to have a clear definition of hyperaltruism. As it stands, hyperaltruism refers to "people's willingness to pay more to reduce other's pain than<br /> their own pain", ie, this means the "hyper" bit is considered with respect to "self". But shouldn't hyperaltruism be classified contrasting "normal" altruism?

It is fine that it follows a previously published work (Crockett et al., 2014), but it would still be necessary to explain/define the construct being tested in a standalone fashion rather than letting readers to go back to the original work.

Reviewer #1 (Public review):

Summary:

Praegel et al. explore the differences in learning an auditory discrimination task between adolescent and adult mice. Using freely-moving (Educage) and head-fixed paradigms, they compare behavioral performance and neuronal responses over the course of learning. The mice were initially trained for seven days on an easy pure frequency tone Go/No-go task (frequency difference of one octave), followed by seven days of a harder version (frequency difference of 0.25 octave). While adolescents and adults showed similar performance on the easy task, adults performed significantly better on the harder task. Quantifying the lick bias of both groups, the authors then argue that the difference in performance is not due to a difference in perception, but rather to a difference in cognitive control. The authors then used neuropixel recordings across 4 auditory cortical regions to quantify the neuronal activity related to the behavior. At the single cell level, the data shows earlier stimulus-related discrimination for adults compared to adolescents in both the easy and hard tasks. At the neuronal population level, adults displayed a higher decoding accuracy and lower onset latency in the hard task as compared to adolescents. Such differences were not only due to learning, but also to age as concluded from recordings in novice mice. After learning, neuronal tuning properties had changed in adults but not in adolescent. Overall, the differences between adolescent and adult neuronal data correlates with the behavior results in showing that learning a difficult task is more challenging for younger mice.

Strengths:

- The behavioral task is well designed, with the comparison of easy and difficult tasks allowing for a refined conclusion regarding learning across age. The experiments with optogenetics and novice mice are completing the research question in a convincing way.<br /> - The analysis, including the systematic comparison of task performance across the two age groups, is most interesting, and reveals differences in learning (or learning strategies?) that are compelling.<br /> - Neuronal recording during both behavioral training and passive sound exposure is particularly powerful, and allows interesting conclusions.

Weaknesses:<br /> - The presentation of the paper must be strengthened. Inconsistencies, missing information or confusing descriptions should be fixed.<br /> - The recording electrodes cover regions in the primary and secondary cortices. It is well known that these two regions process sounds quite differently (for example, one has tonotopy, the other not), and separating recordings from both regions is important to conclude anything about sound representations. The authors show that the conclusions are the same across regions for Figure 4, but is it also the case for the subsequent analysis? Comparing to the original manuscript, the authors have now done the analysis for AuDp and AUDv separately, and say that the differences are similar in both regions. The data however shows that this is not the case (Fig S7). And even if it were the case, how would it compatible with the published literature?

Reviewer #1 (Public review):

Summary:

Both flies and mammals have D1-like and D2-like dopamine receptors, yet the role of D2-like receptors in Drosophila learning and memory remains underexplored. This paper investigates the role of the D2-like dopamine receptor D2R in single pairs of dopaminergic neurons (DANs) during single-odor aversive learning in the Drosophila larva. First, confocal imaging is used to screen GAL4 driver strains that drive GFP expression in just single pairs of dopaminergic neurons. Next, thermogenetic manipulations of one pair of DANs (DAN-c1) suggest that DAN-c1 activity during larval aversive learning is important. Confocal imaging is then used to reveal expression of D2R in the DANs and mushroom body of the larval brain. Finally, optogenetic activation during training phenocopies D2R knockdown in these neurons: aversive learning is impaired when DAN-c1 is targeted, while appetitive and aversive learning are impaired when the mushroom body is manipulated. Finally, a model is proposed in which D2R limits excessive dopamine release to facilitate successful olfactory learning.

Strengths:

The paper convincingly reproduces prior findings that demonstrated D2R knockdown in DL1 DANs or the mushroom body impairs aversive olfactory learning in Drosophila larvae (Qi and Lee, 2014; doi:10.3390/biology3040831). These previous findings were built upon and extended with a comprehensive confocal imaging screen of 57 GAL4 drivers that identified tools driving GFP expression in individual DANs. One of the drivers, R76F02-AD; R55C10-DBD, was consistently shown to label DAN-c1 neurons and no other DANs in the larval brain. Confocal imaging is also used to demonstrate that GFP-tagged D2R is expressed in most DANs and the mushroom body. Behavioral experiments demonstrate that driving D2R knockdown in DAN-c1 neurons impairs aversive learning, as do other loss-of-function manipulations of DAN-c1 neurons.

Limitations:

(1) The single-odor paradigm used to train larvae does not have the advantages of a more conventional balanced or reciprocal training paradigm. The paper describes how the single-odor experimental design could be controlled for non-associative effects, but does not provide an independent validation of the control experiments performed by a different research group using different odors and genotypes 15 to 20 years ago (see Honjo and Furukubo-Tokunaga, 2005; doi:10.1523/jneurosci.2135-05.2005 and Honjo and Furukubo-Tokunaga, 2009; doi:10.1523/jneurosci.1315-08.2009). Whether the involvement of DAN-c1 for aversive learning generalizes to standard paradigms remains unclear (see Eschbach et al., 2020; doi:10.1038/s41593-020-0607-9 and Weber et al., 2023; doi:10.7554/elife.91387.1).

(2) In 11 of 22 larval brains examined in the paper, R76F02-AD; R55C10-DBD appears to drive GFP expression in 1 to 8 additional non-dopaminergic neurons (Figure S1P and Table S3). Of the remaining 11 brains, 4 of their corresponding ventral nerve cords also have expression in 2 to 4 neurons (Table S3). Therefore, experiments involving with the R76F02-AD; R55C10-DBD driver could be manipulating the activity of additional neurons in around 60% of larvae. The conclusions of the paper would be strengthened if key experiments were repeated with other GAL4 drivers that may label DAN-c1 with even greater specificity, such as SS03066 (Truman et al., 2023; doi:10.7554/elife.80594) or MB320C (Hige et al., 2015; doi:10.1016/j.neuron.2015.11.003).

(3) Successful immunostaining with an anti-D2R antibody (Draper et al., 2007; doi:10.1002/dneu.20355 and Love et al., 2023; doi:10.1111/gbb.12836) could validate GFP-tagged D2R expression (Figure 3) in the same way that TH immunostaining was used throughout the paper to determine whether neurons were dopaminergic.

(4) The paper proposes a model in which DAN-c1 activity conveys an aversive teaching signal (Figure 2f) but excessive artificial DAN-c1 activation causes excessive dopamine release that impairs aversive learning (Figures 2i and 5b). According to this model, thermogenetic DAN-c1 activation during training with water or sucrose conveys an aversive teaching signal that reduces performance (Figure 2i) whereas optogenetic DAN-c1 activation does not due to excessive dopamine release (Figures 5c and 5d). The model suggests that optogenetic DAN-c1 activation is strong enough to cause excessive dopamine release by itself whereas thermogenetic DAN-c1 activation can only achieve the same outcome when it occurs in conjunction with natural DAN-c1 activation evoked by quinine. Therefore, an experiment with weaker optogenetic DAN-c1 activation (with lower intensity light or pulsed at a lower frequency) during water or sucrose training would be expected to convey an aversive teaching signal rather than excessive dopamine release, reducing performance. Such an experiment could reconcile the differing thermogenetic and optogenetic results of the paper.

Reviewer #1 (Public review):

Summary:

The topic of nanobody-based PET imaging is important, and holds great potential for real-world applications since nanobodies have many advantages over full sized immunoglobulins and small molecules.

Strengths:

The submitted manuscript contains quite a bit of interesting data from a collaborative team of well-respected researchers. The authors are to be congratulated for presenting results that may not have turned out the way they had hoped, and doing so in a transparent fashion.

Weaknesses:

However, the manuscript could be considered to be a collection of exploratory findings rather than a complete and mature scientific exposition. Most of the sample sizes were 3 per group, which is fine for exploratory work, but insufficient to draw strong, statistically robust conclusions for definitive results.

Overall, the following specific limitations are noted as suggestions for future work:

(1) The authors used DFO, which is well known to leak Zr, rather than the current standard for 89Zr PET which is DFO* (DFO-star)

(2) The brain tissues were not capillary depleted, which limits interpretation. Capillary depletion, with quantitative assessment of the completion of the depletion process, is the standard in the field.

(3) The authors have not experimentally tested the hypothesis that the PEG adduct reduced BBB transcytosis.

(4) The results in Fig. 7 involving the placenta are interesting, but need confirmation using constructs with 18F labeling and without the PEG adduct.

(5) If this line of investigation were to be translated to humans, an important consideration would be the relative safety of 89Zr and 64Cu. It is likely to be quite a bit worse than for 18F, since the 89Zr and 64Cu have longer half-lives, dissociate from their chelators, and lodge in off-target tissues.

(6) A surprising and somewhat disappointing finding was the modest amount of BBB transcytosis. Clearly additional work will be needed before nanobody-based brain PET becomes feasible.

Reviewer #1 (Public review):

Summary:

The authors use analysis of existing data, mathematical modelling and new experiments to explore the relationship between protein expression noise, translation efficiency and transcriptional bursting.

Strengths:

The analysis of the old data and the new data presented is interesting and mostly convincing.

Weaknesses:

My main concern is the analysis presented in Figure 4. This is the core of mechanistic analysis that suggests ribosomal demand can explain the observed phenomenon. Revisions have improved clarity but I am both confused by the assumptions used here in the mathematical modelling of this section. I said before, the authors assumption that the fluctuations of a single gene mRNA levels will significantly affect ribosome demand is puzzling. The author's seem to dismiss this and maybe I am missing something. However, the specific forms used in equations of table S1 seem very phenomenological and I am not sure how these can be taken as good approximations for modelling ribosome demand. Why kc has this specific form, why such a sharp hill number is appropriate. how many total ribosomes per mRNA is assumed here (if this assumption is indeed needed). Again, my intuition is that on average the total level of mRNA across all genes would stay constant and therefore there are not big fluctuations in the ribosome demand due to the burstiness of transcription of individual genes (as this on average is compensated with drop in level of other transcripts). Should not one be considering all transcripts and total ribosomes to be able to model ribosome demand?

Reviewer #1 (Public review):

Summary:

In this study, Floedder et al report that dopamine ramps in both Pavlovian and Instrumental conditions are shaped by reward interval statistics. Dopamine ramps are an interesting phenomenon because at first glance they do not represent the classical reward prediction errors associated with dopamine signaling. Instead, they seem somewhat to bridge the gap between tonic and phasic dopamine, with an intense discussion still being held in the field about what is their actual behavioral role. Here, in tests with head-fixed mice, and dopamine being recorded with a genetically encoded fluorescent sensor in the nucleus accumbens, the authors find that dopamine ramps were only present when intertrial intervals were relatively short and the structure of the task (Pavlovian cue or progression in a VR corridor) contained elements that indicated progression towards the reward (e.g., a dynamic cue). The authors propose that although these findings can be explained by classical theories of dopamine function, they are better explained by their model of Adjusted Net Contingency of Causal Relation (ANCCR). The results of this study provide constraints on future models of dopamine function, and are of high interest to the field.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors recorded activity in the posterior parietal cortex (PPC) of monkeys performing a perceptual decision-making task. The monkeys were first shown two choice dots of two different colors. Then, they saw a random dot motion stimulus. They had to learn to categorize the direction of motion as referring to either the right or left dot. However, the rule was based on the color of the dot and not its location. So, the red dot could either be to the right or left, but the rule itself remained the same. It is known from past work that PPC neurons would code the learned categorization. Here, the authors showed that the categorization signal depended on whether the executed saccade was in the same hemifield as the recorded PPC neuron or in the opposite one. That is, if a neuron categorized the two motion directions such that it responded stronger for one than the other, then this differential motion direction coding effect was amplified if the subsequent choice saccade was in the same hemifield. The authors then built a computational RNN to replicate the results and make further tests by simulated "lesions".

Strengths:

Linking the results to RNN simulations and simulated lesions.

Weaknesses:

Potential interpretational issues due to a lack of explicit evidence on the sizes and locations of the response fields of the neurons. For example, is the contra/ipsi effect explained by the fact that in the contra condition, the response target and the saccade might have infringed on the outer edges of the response fields?

Reviewer #1 (Public review):

Summary:

Early and accurate diagnosis is critical to treating N. fowleri infections, which often lead to death within 2 weeks of exposure. Current methods are based on sampling cerebrospinal fluid, and are invasive, slow, and sometimes unreliable. Therefore, there is a need for a new diagnostic method. Russell et al. address this need by identifying small RNAs secreted by Naegleria fowleri (Fig. 1) that are detectable by RT-qPCR in multiple biological fluids including blood and urine. SmallRNA-1 and smallRNA-2 were detectable in plasma samples of mice experimentally infected with 6 different N. fowleri strains, and were not detected in uninfected mouse or human samples (Fig. 4). Further, smallRNA-1 is detectable in the urine of experimentally infected mice as early as 24 hours post infection (Fig. 5). The study culminates with testing human samples (obtained from the CDC) from patients with confirmed N. fowleri infections; smallRNA-1 was detectable in cerebrospinal fluid in 6 out of 6 samples (Fig. 6B), and in whole blood from 2 out of 2 samples (Fig. 6C). These results suggest that smallRNA-1 could be a valuable diagnostic marker for N. fowleri infection, detectable in cerebrospinal fluid, blood, or potentially urine.

Strengths:

This study investigates an important problem, and comes to a potential solution with a new diagnostic test for N. fowleri infection that is fast, less invasive than current methods, and seems robust to multiple N. fowleri strains. The work in mice is convincing that smallRNA1 is detectable in blood and urine early in infection. Analysis of patient blood samples shows that whole blood could be tested for smallRNA-1 to diagnose N. fowleri infections. The potential for human blood or urine to be tested for N. fowleri could lead to critical early interventions.

Weaknesses:

There are not many N. fowleri cases, so the authors were limited in the human samples available for testing. It is difficult to know how robust this biomarker is in whole blood, serum, or human urine due to little to no sample material being available for testing. This limitation is examined thoroughly in the discussion section, and additional tests are beyond the scope of this work.

Reviewer #1 (Public review):

Summary

This paper summarises responses from a survey completed by around 5,000 academics on their manuscript submission behaviours. The authors find several interesting stylised facts, including (but not limited to):

- Women are less likely to submit their papers to highly influential journals (e.g., Nature, Science and PNAS).<br /> - Women are more likely to cite the demands of co-authors as a reason why they didn't submit to highly influential journals.<br /> - Women are also more likely to say that they were advised not to submit to highly influential journals.

The paper highlights an important point, namely that the submission behaviours of men and women scientists may not be the same (either due to preferences that vary by gender, selection effects that arise earlier in scientists' careers or social factors that affect men and women differently and also influence submission patterns). As a result, simply observing gender differences in acceptance rates - or a lack thereof - should not be automatically interpreted as as evidence for or against discrimination (broadly defined) in the peer review process.

Major comments

What do you mean by bias?

In the second paragraph of the introduction, it is claimed that "if no biases were present in the case of peer review, then we should expect the rate with which members of less powerful social groups enjoy successful peer review outcomes to be proportionate to their representation in submission rates." There are a couple of issues with this statement.

First, the authors are implicitly making a normative assumption that manuscript submission and acceptance rates *should* be equalised across groups. This may very well be the case, but there can also be valid reasons - even when women are not intrinsically better at research than men - why a greater fraction of female-authored submissions are accepted relative to male-authored submissions (or vice versa). For example, if men are more likely to submit their less ground-breaking work, then one might reasonably expect that they experience higher rejection rates compared to women, conditional on submission.

Second, I assume by "bias", the authors are taking a broad definition, i.e., they are not only including factors that specifically relate to gender but also factors that are themselves independent of gender but nevertheless disproportionately are associated with one gender or another (e.g., perhaps women are more likely to write on certain topics and those topics are rated more poorly by (more prevalent) male referees; alternatively, referees may be more likely to accept articles by authors they've met before, most referees are men and men are more likely to have met a given author if he's male instead of female). If that is the case, I would define more clearly what you mean by bias. (And if that isn't the case, then I would encourage the authors to consider a broader definition of "bias"!)

Identifying policy interventions is not a major contribution of this paper

I would take out the final sentence in the abstract. In my opinion, your survey evidence isn't really strong enough to support definitive policy interventions to address the issue and, indeed, providing policy advice is not a major - or even minor - contribution of your paper. (Basically, I would hope that someone interested in policy interventions would consult another paper that much more thoughtfully and comprehensively discusses the costs and benefits of various interventions!) While it's fine to briefly discuss them at the end of your paper - as you currently do - I wouldn't highlight that in the abstract as being an important contribution of your paper.

Minor comments

- What is the rationale for conditioning on academic rank and does this have explanatory power on its own - i.e., does it at least superficially potentially explain part of the gender gap in intention to submit?

Joint Public Review:

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using innovative imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. The authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The single cell voltage imaging used in this study is a highly novel method that may allow recordings that were not previously possible using existing methods.

Weaknesses:

The strength of evidence remains incomplete because of the main claim that synchronous events are not associated with ripples. As was mentioned in previous rounds of review, ripples emerge locally and independently in the two hemispheres. Thus, obtaining ripple recordings from the contralateral hemisphere does not provide solid evidence for this claim. The papers the authors are citing to make the claim that "Additionally, we implanted electrodes in the contralateral CA1 region to monitor theta and ripple oscillations, which are known to co-occur across hemispheres (29-31)" do not support this claim. For example, reference 29 contains the following statement: "These findings suggest that ripples emerge locally and independently in the two hemispheres".

Reviewer #1 (Public review):

The authors of this study use electron microscopy and 3D reconstruction techniques to study the morphology of distinct classes of Drosophila sensory neurons *across many neurons of the same class.* This is a comprehensive study attempting to look at nearly all the sensory neurons across multiple sensilla in the same animal to determine a) how much morphological variability exists between and within neurons of different and similar sensory classes and b) identify dendritic features that may have evolved to support particular sensory functions. This study builds upon the authors' previous work which allowed them to identify and distinguish sensory neuron subtypes in the EM volumes without additional staining so that reconstructed neurons could reliably be placed in the appropriate class. This work is unique in looking at a large number of individual neurons of the same class to determine what is consistent and what is variable about their class-specific morphologies.

This means that in addition to providing specific structural information about these particular cells, the authors explore broader questions of how much morphological diversity exists between sensory neurons of the same class. This then informs our conceptualization about how different dendritic morphologies might affect specific sensory and physiological properties of neurons.

The authors found that CO2 sensing neurons have an unusual, sheet-like morphology in contrast to the thin branches of odor-sensing neurons. They show that this morphology greatly increases the surface area to volume ratio above what could be achieved by modest branching of thin dendrites, and posit that this might be important for their sensory function, though this was not directly tested in their study due to technical limitations. The study is mainly descriptive in nature, but thorough, and provides a nice jumping off point for future functional studies. One interesting future analysis could be to examine all four cell types within a single sensilla together to see if there are any general correlations that could reveal insights about how morphology is determined and relative contributions of intrinsic mechanisms vs interactions with neighboring cells. For example, if higher-than-average branching in one cell type correlated with higher-than-average branching in another type when within the same sensilla, it might suggest differential amounts of extracellular growth or branching cues within a given sensillum drive any heterogeneity observed within a class across sensilla. Conversely, if higher branching in one cell type consistently leads to reduced length or branching of the other neurons within its sensillum, this might point to dendrite-dendrite interactions between cells undergoing competitive or repulsive interactions to define territories within each sensillum as a major determinant of the variability.

Strengths:

This work provides a thorough morphometric analysis of the neurons of the *majority of all ab1 sensilla* across a single antenna. The authors use this analysis to 1) characterize the unique dendritic architecture of ab1C neurons relative to other ORNs including ab1D and 2) provide evidence of substantial morphological diversity even within a single subclass of neuron.

Weaknesses:

This is primarily a descriptive paper due to technical limitations since it is not currently technically feasible to determine individual ORN response properties and tie them to identified neurons with detailed EM-based ultrastructural analyses, nor to predictably alter dendritic morphology of these cells to directly test how different morphologies affect sensory function. However, the quantitative descriptive findings presented here will shape these future questions and are necessary for any such future work.

Reviewer #1 (Public Review):

Summary:

Here, Millet et al. consider whether the nematode C. elegans 'discounts' the value of reward due to effort in a manner similar to that shown in other species, including rodents and humans. They designed a T-maze effort choice paradigm inspired by previous literature, but manipulated how effortful the food is to consume. C. elegans worms were sensitive to this novel manipulation, exhibiting effort-discounting-like behaviour that could be shaped by varying the density of food at each alternative in order to calculate an indifference point. This discounting-like behaviour was related to worms' rates of patch leaving, which differed between the low and high effort patches in isolation. The authors also found a potential relationship to dopamine signalling, and also that this discounting behaviour was not specific to lab-based strains of C. elegans.

Strengths:

The question is well-motivated, and the approach taken here is novel. The authors are careful in their approach to altering and testing the properties of the effortful, elongated bacteria. Similarly, they go to some effort to understand what exactly is driving behavioural choices in this context, both through the application of simple standard models of effort discounting and a kinetic analysis of patch leaving. The comparisons to various dopamine mutants further extend the translational potential of their findings. I also appreciate the comparison to natural isolate strains, as the question of whether this behaviour may be driven by some sort of strain-specific adaptation to the environment is not regularly addressed in mammalian counterparts. The manuscript is well-written, and the figures are clear and comprehensible.

Weaknesses:

Discounting is typically defined as the alteration of a subjective value by effort (or time, risk, etc.), which is then used to guide future decision-making. By adapting the standard t-maze task for C. elegans as a patch-leaving paradigm, the authors observe behaviour strongly consistent with discounting models, but that is likely driven by a different process, in particular by an online estimate of the type of food in the current patch, which then influences patch-leaving dynamics (Figure 3). This is fundamentally different from decision-making strategies relating to effort that have been described in the rodent and human literatures. Similarly, the calculation of indifference points at the group instead of at the individual level also suggests a different underlying process and limits the translational potential of their findings. The authors do not discuss the implications of these differences or why they chose not to attempt a more analogous trial-based experiment.

In the case of both the dopamine and natural isolate experiments, the data are very noisy despite large (relative to other C. elegans experiments) sample sizes. In the dopamine experiment, disruption of dop-1, dop-2, and cat-2 had no statistically significant effect. There do not appear to be any corrections for multiple comparisons, and the single significant comparison, for dop-3, had a small effect size. More detailed behavioural analyses on both these and the wild isolate strains, for example by applying their kinetic analysis, would likely give greater insight as to what is driving these inconsistent effects.

Reviewer #1 (Public review):

Summary:

Ritzau-Jost et al. investigate the potential contribution of AP broadening in homeostatic upregulation of neuronal network activity with a specific focus on dissociated neuronal cultures. In cultures obtained from a few brain regions from mice or rats using different culture conditions and examined by different laboratories, AP half-width remained stable despite chronic activity block with TTX. The finding suggests that AP width is not significantly modulated by changes in sodium channel activity.

Strengths:

The collaborative nature of the study amongst the neuronal culture experts and the rigorous electrophysiological assessments provides for a compelling support of the main conclusion.

Weaknesses:

Given the negative nature of the results, a couple of remaining issues (such as the cell density of cultures and the presentation of imaging experiments with a voltage sensor) warrant further consideration. In addition, a discussion of the reasons for the seeming stability of AP half-width to sodium channel modulation might help extend the scope of the study beyond the presentation of a negative conclusion.

Reviewer #1 (Public review):

Summary:

Wu and colleagues aimed to explain previous findings that adolescents, compared to adults, show reduced cooperation following cooperative behaviour from a partner in several social scenarios. The authors analysed behavioural data from adolescents and adults performing a zero-sum Prisoner's Dilemma task and compared a range of social and non-social reinforcement learning models to identify potential algorithmic differences. Their findings suggest that adolescents' lower cooperation is best explained by a reduced learning rate for cooperative outcomes, rather than differences in prior expectations about the cooperativeness of a partner. The authors situate their results within the broader literature, proposing that adolescents' behaviour reflects a stronger preference for self-interest rather than a deficit in mentalising.

Strengths:

The work as a whole suggests that, in line with past work, adolescents prioritise value accumulation, and this can be, in part, explained by algorithmic differences in weighted value learning. The authors situate their work very clearly in past literature, and make it obvious the gap they are testing and trying to explain. The work also includes social contexts that move the field beyond non-social value accumulation in adolescents. The authors compare a series of formal approaches that might explain the results and establish generative and model-comparison procedures to demonstrate the validity of their winning model and individual parameters. The writing was clear, and the presentation of the results was logical and well-structured.

Weaknesses:

I also have some concerns about the methods used to fit and approximate parameters of interest. Namely, the use of maximum likelihood versus hierarchical methods to fit models on an individual level, which may reduce some of the outliers noted in the supplement, and also may improve model identifiability.

There was also little discussion given the structure of the Prisoner's Dilemma, and the strategy of the game (that defection is always dominant), meaning that the preferences of the adolescents cannot necessarily be distinguished from the incentives of the game, i.e. they may seem less cooperative simply because they want to play the dominant strategy, rather than a lower preferences for cooperation if all else was the same.

Appraisal & Discussion:

The authors have partially achieved their aims, but I believe the manuscript would benefit from additional methodological clarification, specifically regarding the use of hierarchical model fitting and the inclusion of Bayes Factors, to more robustly support their conclusions. It would also be important to investigate the source of the model confusion observed in two of their models.

I am unconvinced by the claim that failures in mentalising have been empirically ruled out, even though I am theoretically inclined to believe that adolescents can mentalise using the same procedures as adults. While reinforcement learning models are useful for identifying biases in learning weights, they do not directly capture formal representations of others' mental states. Greater clarity on this point is needed in the discussion, or a toning down of this language.

Additionally, a more detailed discussion of the incentives embedded in the Prisoner's Dilemma task would be valuable. In particular, the authors' interpretation of reduced adolescent cooperativeness might be reconsidered in light of the zero-sum nature of the game, which differs from broader conceptualisations of cooperation in contexts where defection is not structurally incentivised.

Overall, I believe this work has the potential to make a meaningful contribution to the field. Its impact would be strengthened by more rigorous modelling checks and fitting procedures, as well as by framing the findings in terms of the specific game-theoretic context, rather than general cooperation.

Reviewer #1 (Public review):

Summary:

The authors investigated the potential role of IgG N-glycosylation in Haemorrhagic Fever with Renal Syndrome (HFRS), which may offer significant insights for understanding molecular mechanisms and for the development of therapeutic strategies for this infectious disease. However, several issues need to be addressed.

Major Points:

(1) The authors should provide a detailed description of the pathogenesis of Haemorrhagic Fever with Renal Syndrome (HFRS) and elaborate on the crucial role of IgG proteins in the disease's progression (line 65).

(2) An additional discussion on the significance of glycosylation, particularly IgG N-glycosylation, in viral infections should be included in the Introduction section.

(3) In the Abstract section, the authors state that HTNV-specific IgG antibody titers were detected and IgG N-glycosylation was analyzed. However, the analysis of plasma IgG N-glycans is described in the Methods section. Therefore, the authors should clarify the glycome analysis process. Was the specific IgG glycome profile similar to the total IgG N-glycome? Given the biological relevance of specific IgG in immunological diseases, characterizing the specific IgG N-glycome profile would be more significant than analyzing the total plasma IgG.

(4) Further details regarding the N-glycome analysis should be provided, including the quantity of IgG protein used and the methodology employed for analyzing IgG N-glycans (lines 286-287).

(5) Additional statistical analyses should be performed, including multiple comparisons with p-value adjustment, false discovery rate (FDR) control, and Pearson correlation (line 291).

(6) Quality control should be conducted prior to the IgG N-glycome analysis. Additionally, both biological and technical replicates are essential to assess the reproducibility and robustness of the methods.

(7) Multiple regression analysis should be conducted to evaluate the influence of genetic and environmental factors on the IgG N-glycome.

(8) Line 196. Additional discussions should be included, focusing on the underlying correlation between the differential expression of B-cell glycogenes and the dysregulated IgG N-glycome profile, as well as the potential molecular mechanisms of IgG N-glycosylation in the development of HFRS.

Reviewer #1 (Public review):

Building upon their previous work, the authors present an enhanced method for confocal live imaging of leg regeneration in the crustacean Parhyale hawaiensis. Parhyale is an emerging and tractable model system that offers insights into the evolution and mechanisms of development and regeneration. Çevrim et al. demonstrate the ability to image the complete leg regeneration process, spanning several days, with 10-20 minute time intervals and cellular resolution. They have concurrently optimized imaging conditions to enable cell tracking while minimizing phototoxicity. Additionally, they report successfully implementing HCR in situ hybridization in Parhyale, allowing for specific gene transcript staining at the endpoint of live imaging. This opens the possibility of assigning molecular identities to tracked cells.

A key challenge in many regeneration models is achieving continuous imaging throughout the entire regenerative process, as many organisms are difficult to immobilize or cannot tolerate extended imaging without stress. This manuscript's major strength lies in providing practical solutions to these challenges in Parhyale, a compelling and accessible arthropod model for limb regeneration. The authors also employ complementary tools to analyze time-lapse movies and correlate them with endpoint staining. Together, these advances will serve as a useful resource for researchers studying regeneration in Parhyale or in other systems where parts of this workflow can be adapted.

While the data demonstrating the methodological advancement and technical feasibility are solid, much of the benchmarking and regeneration characterization remains qualitative. This does not undermine the validity of the proof-of-principle, but limits the study's broader appeal.

Reviewer #1 (Public review):

Functional lateralization between the right and left hemispheres is reported widely in animal taxa, including humans. However, it remains largely speculative as to whether the lateralized brains have a cognitive gain or a sort of fitness advantage. In the present study, by making use of the advantages of domestic chicks as a model, the authors are successful in revealing that the lateralized brain is advantageous in the number sense, in which numerosity is associated with spatial arrangements of items. Behavioral evidence is strong enough to support their arguments. Brain lateralization was manipulated by light exposure during the terminal phase of incubation, and the left-to-right numerical representation appeared when the distance between items gave a reliable spatial cue. The light-exposure induced lateralization, though quite unique in avian species, together with the lack of intense inter-hemispheric direct connections (such as the corpus callosum in the mammalian cerebrum), was critical for the successful analysis in this study. Specification of the responsible neural substrates in the presumed right hemisphere is expected in future research. Comparable experimental manipulation in the mammalian brain must be developed to address this general question (functional significance of brain laterality) is also expected.

Reviewer #1 (Public review):

Summary:

The image analysis pipeline is tested in analysing microscopy imaging data of gastruloids of varying sizes, for which an optimised protocol for in toto image acquisition is established based on whole mount sample preparation using an optimal refractive index matched mounting media, opposing dual side imaging with two-photon microscopy for enhanced laser penetration, dual view registration, and weighted fusion for improved in toto sample data representation. For enhanced imaging speed in a two-photon microscope, parallel imaging was used, and the authors performed spectral unmixing analysis to avoid issues of signal cross-talk.

In the image analysis pipeline, different pre-treatments are done depending on the analysis to be performed (for nuclear segmentation - contrast enhancement and normalisation; for quantitative analysis of gene expression - corrections for optical artifacts inducing signal intensity variations). Stardist3D was used for the nuclear segmentation. The study analyses into properties of gastruloid nuclear density, patterns of cell division, morphology, deformation, and gene expression.

Strengths:

The methods developed are sound, well described, and well-validated, using a sample challenging for microscopy, gastruloids. Many of the established methods are very useful (e.g. registration, corrections, signal normalisation, lazy loading bioimage visualisation, spectral decomposition analysis), facilitate the development of quantitative research, and would be of interest to the wider scientific community.

Weaknesses:

A recommendation should be added on when or under which conditions to use this pipeline.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rainey et al investigated the effects of transcription factors, ATOH1, POU4F3, GFI1, and SIX1 on the induction of hair cells from human pluripotent stem cells. The authors used a doxycycline-inducible system to control transgene expression and demonstrated significant improvement in the efficiency of MYO7A+ hair cell differentiation compared to the retrovirus-mediated approach. Next, they characterized differentiated cells using single-cell RNA-seq and identified a population of hair cell-like cells with gene expression profiles similar to the fetal human vestibular hair cells. Finally, they revealed the electrophysiological properties of induced cells consistent with those of mechanosensitive hair cells.

Strengths:

A key finding in this study is the rapid induction of cells expressing multiple hair cell markers that takes place within 21 days after overexpression of the four transcription factors. Additionally, the authors demonstrate that doxycycline-mediated gene overexpression outperforms retroviral-mediated gene transfer in terms of both the efficiency and reproducibility of hair cell induction. Furthermore, the authors demonstrate that these induced hair cells can be used to study hair cell protection from cisplatin ototoxicity.

Weaknesses:

The authors conclude that the induced cells lack distinct hair cell subtypes. However, the characterization of generated hair cells in single-cell RNA-seq data is insufficient. Additional vestibular or cochlear hair cell-enriched marker gene and protein expression should be analyzed. Moreover, the morphological features and mechanotransduction channel activity of the induced hair cells have not been analyzed.

Reviewer #1 (Public review):

Summary:

This work integrates two timepoints from the Adolescent Brain Cognitive Development Study to understand how neuroimaging, genetic and environmental data contribute to the predictive power of mental health variables in predicting cognition in a large early adolescent sample. Their multimodal and multivariate prediction framework involves a novel opportunistic stacking model to handle complex types of information to predict variables that are important in understanding mental health-cognitive performance associations.

Strengths:

The authors are commended for incorporating and directly comparing the contribution of multiple imaging modalities (task fMRI, resting state fMRI, diffusion MRI, structural MRI), neurodevelopmental markers, environmental factors and polygenic risk scores in a novel multivariate framework (via opportunistic stacking), as well as interpreting mental health-cognition associations with latent factors derived from Partial Least Squares. The authors also use a large well-characterized and diverse cohort of adolescents from the Adolescent Brain Cognitive Development (ABCD) Study. The paper is also strengthened by commonality analyses to understand the shared and unique contribution of different categories of factors (e.g., neuroimaging vs mental health vs polygenic scores vs sociodemographic and adverse developmental events) in explaining variance in cognitive performance

Weaknesses:

The paper is framed with an over-reliance on the RDoC framework in the introduction, despite deviations from the RDoC framework in the methods. The field is also learning more about RDoC's limitations when mapping cognitive performance to biology. The authors also focus on a single general factor of cognition as the core outcome of interest as opposed to different domains of cognition. The authors could consider predicting mental health rather than cognition. Using mental health as a predictor could be limited by the included 9-11 year age range at baseline (where mental health concerns are likely to be low or not well captured), as well as the nature of how the data was collected, i.e., either by self-report or from parent/caregiver report.

Comments on revisions:

The authors have done an excellent job of addressing my comments. I have no other suggestions to add. Great work!

Reviewer #1 (Public review):

Summary:

Perlee et al. sought to generate a zebrafish line where CRISPR-based gene editing is exclusively limited to the melanocyte lineage, allowing assessment of cell-type restricted gene knockouts. To achieve this, they knocked in Cas9 to the endogenous mitfa locus, as mitfa is a master regulator of melanocyte development. The authors use multiple candidate genes - albino, sox10, tuba1a, ptena/ptenb, tp53 - to demonstrate that their system induces lineage-restricted gene editing. This method allows researchers to bypass embryonic lethal and non-cell autonomous phenotypes emerging from whole body knockout (sox10, tuba1a), drive directed phenotypes, such as depigmentation (albino), and induce lineage-specific tumors, such as melanomas (ptena/ptenb, tp53, when accompanied with expression of BRAFV600E). The main weakness of the manuscript is that the mechanistic explanations proposed to underlie the presented phenotypes are minimally interrogated, but nonetheless interesting and motivating for future experimentation. Overall, there is a clear use for this genetic methodology, and its implementation will be of value to many in vivo researchers.

Strengths:

The strongest component of this manuscript is the genetic control offered by the mitfa:Cas9 system and the ability to make stable, lineage-specific knockouts in zebrafish. This is exemplified by the studies of tuba1a, where the authors nicely show non-cell autonomous mechanisms have obfuscated the role of this gene in melanocyte development. In addition, the mitfa:Cas9 system is elegantly straightforward and can be easily implemented in many labs. Mostly, the figures are clean, controls are appropriate, and phenotypes are reproducible. The invented method is a welcome addition to the arsenal of genetic tools used in zebrafish. The authors kindly and honestly responded to reviewer criticism, which has led to an improved manuscript and a pleasant review process.

Weaknesses:

The authors argue that the benefit of their system is the maintenance of endogenous regulatory elements. However, no direct comparison is made with other tools that offer similar genetic control, such as MAZERATI. This is a missed opportunity to provide researchers the ability to evaluate these two similar genetic approaches. There is a slight concern that tumor onset with this system is hindered by the heterozygous state it imparts to the lineage master regulator (here, mitfa). The authors do a good job at addressing these issues in the Discussion, but experimentation would have been appreciated. Additionally, the authors claim 86% of mitfa+ cells express Cas9. The image shown in Figure 1C does not do a convincing job at showing this percentage.

Another weakness of the manuscript regards minimally investigated mechanistic explanations for each biological vignette. Detailed mechanistic information is indeed out-of-scope for this manuscript, which intends to prove the efficacy of a genetic tool. Readers are cautioned to use the mechanistic insights from these vignettes as inspiration rather than bona fide truth.

The authors performed the necessary experiments to address each of the reviewers' concerns and thereby quell any substantial issues raised during the first review. They have additionally edited their language appropriately to make their claims more accurate. Their efforts during the review process are appreciated.

Conclusion:

The authors were highly receptive to reviewer comments and improved their manuscript from the first submission. The authors were successful in their goal of creating a rapid genetic approach to study cell-type specific genetic insults in vivo. They have presented multiple interesting and convincing stories to support the power of their invented methodology. The refined mechanisms underlying their observed phenotypes may be lacking but this does not take away from the methodological benefit this manuscript provides to the large field of in vivo researchers.

Reviewer #1 (Public review):

Summary:

This manuscript by Garbelli et al. investigates the roles of excitatory amino acid transporters (EAATs) in retinal bipolar cells. The group previously identified that EAAT5b and EAAT7 are expressed at the dendritic tips of bipolar cells, where they connect with photoreceptor terminals. The previous study found that the light responses of bipolar cells, measured by electroretinogram (ERG) in response to white light, were reduced in double mutants, though there was little to no reduction in light responses in single mutants of either EAAT5b or EAAT7.

The current study further explores the roles of EAAT5b and EAAT7 in bipolar cells' chromatic responses. The authors found that bipolar cell responses to red light, but not to green or UV-blue light, were reduced in single mutants of both EAAT5b and EAAT7. In contrast, UV-blue light responses were reduced in double mutants. Additionally, the authors observed that EAAT5b, but not EAAT7, is strongly localized in the UV cone-enriched area of the eye, known as the "Strike Zone (SZ)." This led them to investigate the impact of the EAAT5b mutation on prey detection performance, which is mediated by UV cones in the SZ. Surprisingly, contrary to the predicted role of EAAT5b in prey detection, EAAT5b mutants did not show any changes in prey detection performance compared to wild-type fish. Interestingly, EAAT7 mutants exhibited enhanced prey detection performance, though the underlying mechanisms remain unclear.

The distribution of EAAT7 protein in the outer plexiform layer across the eye correlates with the distribution of red cones. Based on this, the authors tested the behavioral performance driven by red light in EAAT5b and EAAT7 mutants. The results here were again somewhat contrary to predictions based on ERG findings and protein localization: the optomotor response was reduced in EAAT5b mutants, but not in EAAT7 mutants.

Strengths:

Although the paper lacks cohesive conclusions, as many results contradict initial predictions as mentioned above, the authors discuss possible mechanisms for these contradictions and suggest future avenues for study. Nevertheless, this paper demonstrates a novel mechanism underlying chromatic information processing.<br /> The manuscript is well-written, the data are well-presented, and the analysis is thorough.

Weaknesses:

I have only a minor comment. The authors present preliminary data on mGluR6b distribution across the eye. Since this result is based on a single fish, I recommend either adding more samples or removing this data, as it does not significantly impact the paper's main conclusions.

Comments on revisions:

The authors addressed all of the concerns that I had in the original manuscript.

Reviewer #1 (Public review):

Summary

The authors present a new protocol to assess social dominance in pairs and triads of C57BL/6j mice, based on a competition to access a hidden food pellet. Using this new protocol, the authors have been able to identify stable ranking among male and female pairs, while reporting more fluctuant hierarchies among triads of males. Ranking readout identified with this new apparatus was compared to the outcome obtained with the same animals competing in the tube and in the warm spot tests, which have been both commonly used during the last decade to identify social ranks in rodents under laboratory conditions.

Strengths

FPCT allows for an easy and fast identification of a winner and loser in a context of food competition. The apparatus and the protocol are relatively easy and quick to implement in the lab and free from any complex post processing/analysis, which qualifies it for wide distribution, particularly within laboratories that do not have the resources to implement more sophisticated protocols. Hierarchical readout identified through the FPCT correlates with social ranks identified with the tube and the warm spot tests, which have been widely adopted during the last decade and allow for study comparison.

Weaknesses

While the FPCT is validated by the tube and the warm spot test, this paper would have gained strength by providing a more ethologically based validation. Tube and warm spot tests have been shown to provide conflicting results and might not be a sufficient measurement for social ranking (see Varholik et al, Scientific Reports, 2019; Battivelli et al, Biological Psychiatry, 2024). Instead, a general consensus pushing toward more ethological approaches for neuroscience studies is emerging.<br /> Other papers already successfully identified social ranks dyadic food competition, using relatively simple scoring protocol (see, for example, Merlot et al., 2006), within a more naturalistic set-up, allowing the 2 opponents to directly interact while competing for the food. A potential issue with the FPCT, is that the opponents being isolated from each other, the normal inhibition expected to appear in subordinates in presence of a dominant to access food, could be diminished, and usually avoiding subordinates could be more motivated to push for the access to the food pellet.

Comments on revisions:

We thank the authors for the significant improvement of the English in the revised version and for the replacement of some conceptual terms that now seem more relevant and appropriate. We only noticed that the term "society" remains in use, although it might not be appropriate to describe a mouse colony (see previous review).

Conclusive remarks

Although this protocol aims to provide a novel approach to evaluate social ranks in mice, it is not clear how it really brings a significant advance in neuroscience research. The FPCT dynamic is very similar to the one observed in the tube test, where mice compete to navigate forward in a narrow space, constraining the opponent to go backwards. The main difference between the FPCT and the tube test is the presence of food between the opponents. In the tube test, food reward was initially used to increase motivation to cross the tube and push the opponent upon the testing day. This component has been progressively abandoned, precisely because it was not necessary for the mice to compete in the tube.<br /> This paper would really bring a significant contribution to the field by providing a neuronal imaging or manipulation correlate to the behavioral outcome obtained by the application of the FPCT.

Reviewer #2 (Public review):

Summary:

In this manuscript, Wang et al analyze ~17,000 transcriptomes from 35 human tissues from the GTEx database and address transcriptomic variations due to age and sex. They identified both gene expression changes as well as alternative splicing events that differ among sexes. Using breakpoint analysis, the authors find sex dimorphic shifts begin with declining sex hormone levels with males being affected more than females. This is an important pan-tissue transcriptomic study exploring age and sex-dependent changes although not the first one.

Strengths:

(1) The authors use sophisticated modeling and statistics for differential, correlational and predictive analysis.

(2) The authors consider important variables such as genetic background, ethnicity, sampling bias, sample sizes, detected genes etc.

(3) This is likely the first study to evaluate alternative splicing changes with age and sex at a pan-tissue scale.

(4) Sex dimorphism with age is an important topic and is thoroughly analyzed in this study.

Reviewer #1 (Public review): <br /> The authors survey the ultrastructural organization of glutamatergic synapses by cryo-ET and image processing tools using two complementary experimental approaches. The first approach employs so-called "ultra-fresh" preparations of brain homogenates from a knock-in mouse expressing a GFP-tagged version of PSD-95, allowing Peukes and colleagues to specifically target excitatory glutamatergic synapses. In the second approach, direct in-tissue (using cortical and hippocampal regions) targeting of the glutamatergic synapses employing the same mouse model is presented. In order to ascertain whether the isolation procedure causes any significant changes in the ultrastructural organization (and possibly synaptic macromolecular organization) the authors compare their findings using both of these approaches. The quantitation of the synaptic cleft height reveals an unexpected variability, while the STA analysis of the ionotropic receptors provides insights into their distribution with respect to the synaptic cleft.

The main novelty of this study lies in the continuous claims by the authors that the sample preservation methods developed here are superior to any others previously used. This leads them as well to systematically downplay or directly ignore a substantial body of previous cryo-ET studies of synaptic structure. Without comparisons with the cryo-ET literature, it is very hard to judge the impact of this work in the field. Furthermore, the data does not show any better preservation in the so-called "ultra-fresh" preparation than in the literature, perhaps to the contrary as synapses with strangely elongated vesicles are often seen. Such synapses have been regularly discarded for further analysis in previous synaptosome studies (e.g. Martinez-Sanchez 2021). Whilst the targeting approach using a fluorescent PSD95 marker is novel and seems sufficiently precise, the authors use a somewhat outdated approach (cryo-sectioning) to generate in-tissue tomograms of poor quality. To what extent such tomograms can be interpreted in molecular terms is highly questionable. The authors also don't discuss the physiological influence of 20% dextran used for high-pressure freezing of these "very native" specimens.

Lastly, a large part of the paper is devoted to image analysis of the PSD which is not convincing (including a somewhat forced comparison with the fixed and heavy-metal staining room temperature approach). Despite being a technically challenging study, the results fall short of expectations.