Reviewer #4 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

Reviewer #4 (Public review):

Summary:

Here, the authors wish to shed light on factors that contribute to the development of liver disease in what used to be called 'the metabolic syndrome'. This is a human-health problem of considerable significance, and the insights they provide, namely the implication of a defect in mitochondrial cardiolipin (CL) content to the progression from metabolic dysfunction-associated steatotic liver disease to steatohepatitis, are plausible.

Strengths:

The experimental evidence proffered is derived from the observation of lower levels of (CL) in mitochondria from the liver of patients undergoing liver transplant or resection due to end-stage steatohepatitis compared with mitochondria derived from livers of patients with other conditions. This correlation is buttressed by observations made in mice with liver-selective compromise in CL synthesis and which suggest a pathological environment associated with mitochondrial dysfunction and enhanced oxidative stress, features deemed to play a role in the progression from steatotic liver disease to steatohepatitis.

The paper is well written, and the findings are well explained and superficially convincing.

Weaknesses:

It is unclear how much can be learned from compromising a key enzyme that produces a key mitochondrial lipid in a busy metabolic organ like the liver - isn't the discovery of a mitochondrial defect in such a context rather trivial? And how reliably can these findings be related to the human observations? Most importantly, the chain of causality implied by the title is unproven: the key question of whether or not (somehow) preventing the drop in cardiolipin content affects the course of steatohepatitis remains unanswered.

Reviewer #4 (Public review):

Summary:

This study by Gylemo et al. investigates genes that escape X-Chromosome Inactivation (XCI) by analyzing RNA-sequencing data from three female individuals with highly skewed XCI identified in the GTEx database-two newly reported and one previously described. Utilizing these rare non-mosaic XCI cases, the authors assess allelic expression patterns across 30 normal human tissues to classify the XCI status of 380 X-linked genes, including 198 not previously annotated. The study provides a broader and more comprehensive catalog of XCI escape, contributing valuable insights into sex-specific gene expression and the potential implications of X-linked variants in disease.

Strengths:

The primary strength of this work lies in its expanded scope: it doubles the number of tissues and significantly increases the number of X-linked genes with known XCI status compared to previous studies. By focusing on rare individuals with non-random XCI, the authors provide a unique opportunity to observe allelic expression and classify escape status with more confidence. Their findings that escape from XCI is relatively consistent across tissues (rather than tissue-specific) enhance the understanding of XCI mechanisms. The methodology is robust, the data are well-documented, and the supplementary resources are comprehensive. This study thus represents a valuable resource for the XCI field and a promising basis for future investigations.

Weaknesses:

Despite its strengths, the study is limited by its reliance on only three individuals, which restricts statistical power and generalizability. Concerns were raised regarding the comparability of XCI status across tissue types and cell lines, particularly given that previous classifications may have used cancer or immortalized cells. Additionally, more could be done to explore the genetic basis behind the observed skewed XCI, which might affect the conclusions about escape patterns. Finally, the authors are encouraged to expand their approach to additional RNA-seq datasets or single-cell analyses to validate their findings and potentially discover more individuals with skewed XCI, which would deepen understanding of this important biological phenomenon.

Reviewer #4 (Public review):

Summary:

The authors present a follow-up to their initial publication of a volume EM reconstruction of a part of the Octopus vulgaris vertical lobe (VL) (Bidel, Meirovitch et al., eLife 2023). In their previous study, they presented a swath of novel observations pertaining to the neuron types making up the VL and their synaptic connectivity. Here, the authors present an extension of those findings in which they (1) demonstrate that the Complex Amacrine cells (CAMs), which they identified previously, can be grouped into at least 5 distinct subclasses; (2) show that there appears to be distinct compartments in the SFL tract that contain specific synapse types; and (3) present morphological evidence that there may be a neurogenic niche in the VL. The findings are intriguing, advance our understanding of memory circuitry in octopus and across the phylogenetic tree, and open new avenues for deeper investigation.

Strengths:

A deeper dissection of the morphologies of CAMs and their distinct complements of synapse types is valuable. The identification of multiple categories of CAMs makes it clearer how the very simple SFL-to-SAM connectivity is likely enriched by a population of diverse interneurons.

The observation that synapse types may be compartmentalized in the superior frontal lobe tract is an intriguing one, and invites more extensive segmentation and future anatomical studies to further characterize the precise architecture of these compartments.

Finally, the evidence of the possibility of a neurogenic niche in the VL is exciting as it suggests that ongoing neurogenesis may be a common feature of memory circuitry, perhaps contributing to keeping the representation space of the circuit flexible and adequately sparse.

Weaknesses:

A key weakness is the reconstruction and grouping of the CAMs:

(1) CAMs are relatively few in number compared to SAMs, and as such, only 53 are reconstructed in this study. Of those 53 cells, 18 were not classified into one of the 5 categories the authors designate, begging the question of how robust those categories are.

(2) Related to (1), in Figure 1B, the proportions given in the bar graph are given cumulatively across the entire population of each category. The proportions should be presented as means within each category to adequately capture the variability of the small sample sizes.

(3) While the xy dimensions of the serial section EM volume are adequate to capture relatively whole cells and neuronal arbors, the volume is only 27µm thick. Thus, many neurite branches are likely truncated in the z-dimension. This may have contributed to ~1/3 of CAMs eluding categorization. However, it is hard to estimate the effect this may have had without knowing the extent of the truncation. It may be worth the authors' time to count the proportion of CAM neurites that are cut off at the edges of the volume.

(4) The authors state that CAMs appear to have axons and dendrites based on neurite widths. This is an interesting finding, given that amacrine cells are generally thought to possess only one type of neurite, which both send and receive synaptic potentials, and therefore deserves more attention. Is the distribution of neurite widths indeed bimodally distributed? Can the axons and dendrites be differentiated by examining the presence and absence of synaptic vesicle pools, respectively?

In Figure 2, the compartmentalization of synapse types is intriguing; however, due to the 3D nature of the data, it is difficult to appreciate clearly from the panels presented. This is particularly true for the suggestion that glia may be forming a barrier around these compartments. This could be rectified by providing Neuroglancer links for these specific reconstructions (neurites, synapses, and glia).

Lastly, although the identification of a putative neurogenic niche is tantalizing, morphological data alone is only an initial hint. Although the chances are slim, it would be more convincing if the authors could identify any actively dividing cells in the proposed niche. More likely, further work, for instance, immunofluorescence, which the lab has previously shown to be viable in octopus, will be needed to add weight to the claim.

Reviewer #4 (Public review):

Summary

Pinho et al use in vivo calcium imaging and chemogenetic approaches to examine the involvement of hippocampal sub-regions across the different stages of a sensory preconditioning task in mice. They find clear evidence for sensory preconditioning in male but not female mice. They also find that, in the male mice, CaMKII-positive neurons in the dorsal hippocampus: (1) encode the audio-visual association that forms in stage 1 of the task, and (2) retrieve/express sensory preconditioned fear to the auditory stimulus at test. These findings are supported by evidence that ranges from incomplete to convincing. They will be valuable to researchers in the field of learning and memory.

Abstract

Please note that sensory preconditioning doesn't require the stage 1 stimuli to be presented repeatedly or simultaneously.

"Finally, we combined our sensory preconditioning task with chemogenetic approaches to assess the role of these two hippocampal subregions in mediated learning."<br /> This implies some form of inhibition of hippocampal neurons in stage 2 of the protocol, as this is the only stage of the protocol that permits one to make statements about mediated learning. However, it is clear from what follows that the authors interrogate the involvement of hippocampal sub-regions in stages 1 and 3 of the protocol - not stage 2. As such, most statements about mediated learning throughout the paper are potentially misleading (see below for a further elaboration of this point). If the authors persist in using the term mediated learning to describe the response to a sensory preconditioned stimulus, they should clarify what they mean by mediated learning at some point in the introduction. Alternatively, they might consider using a different phrase such as "sensory preconditioned responding".

Introduction

"Low-salience" is used to describe stimuli such as tone, light, or odour that do not typically elicit responses that are of interest to experimenters. However, a tone, light, or odour can be very salient even though they don't elicit these particular responses. As such, it would be worth redescribing the "low-salience" stimuli in some other terms.

"These higher-order conditioning processes, also known as mediated learning, can be captured in laboratory settings through sensory preconditioning procedures2,6-11."<br /> Higher-order conditioning and mediated learning are not interchangeable terms: e.g., some forms of second-order conditioning are not due to mediated learning. More generally, the use of mediated learning is not necessary for the story that the authors develop in the paper and could be replaced for accuracy and clarity. E.g., "These higher-order conditioning processes can be studied in the laboratory using sensory preconditioning procedures2,6-11."

In reference to Experiment 2, it is stated that: "However, when light and tone were separated on time (Unpaired group), male mice were not able to exhibit mediated learning response (Figure 2B) whereas their response to the light (direct learning) was not affected (Figure 2D). On the other hand, female mice still present a lower but significant mediated learning response (Figure 2C) and normal direct learning (Figure 2E). Finally, in the No-Shock group, both male (Figure 2B and 2D) and female mice (Figure 2C and 2E) did not present either mediated or direct learning, which also confirmed that the exposure to the tone or light during Probe Tests do not elicit any behavioral change by themselves as the presence of the electric footshock is required to obtain a reliable mediated and direct learning responses."<br /> The absence of a difference between the paired and unpaired female mice should not be described as "significant mediated learning" in the latter. It should be taken to indicate that performance in the females is due to generalization between the tone and light. That is, there is no sensory preconditioning in the female mice. The description of performance in the No-shock group really shouldn't be in terms of mediated or direct learning: that is, this group is another control for assessing the presence of sensory preconditioning in the group of interest. As a control, there is no potential for them to exhibit sensory preconditioning, so their performance should not be described in a way that suggests this potential.

Methods - Behavior

I appreciate the reasons for testing the animals in a new context. This does, however, raise other issues that complicate the interpretation of any hippocampal engagement: e.g., exposure to a novel context may engage the hippocampus for exploration/encoding of its features - hence, it is engaged for retrieving/expressing sensory preconditioned fear to the tone. This should be noted somewhere in the paper given that one of its aims is to shed light on the broader functioning of the hippocampus in associative processes.

This general issue - that the conditions of testing were such as to force engagement of the hippocampus - is amplified by two further features of testing with the tone. The first is the presence of background noise in the training context and its absence in the test context. The second is the fact that the tone was presented for 30 s in stage 1 and then continuously for 180s at test. Both changes could have contributed to the engagement of the hippocampus as they introduce the potential for discrimination between the tone that was trained and tested.

Results - Behavior

The suggestion of sex differences based on differences in the parameters needed to generate sensory preconditioning is interesting. Perhaps it could be supported through some set of formal analyses. That is, the data in supplementary materials may well show that the parameters needed to generate sensory preconditioning in males and females are not the same. However, there needs to be some form of statistical comparison to support this point. As part of this comparison, it would be neat if the authors included body weight as a covariate to determine whether any interactions with sex are moderated by body weight.

What is the value of the data shown in Figure 1 given that there are no controls for unpaired presentations of the sound and light? In the absence of these controls, the experiment cannot have shown that "Female and male mice show mediated learning using an auditory-visual sensory preconditioning task" as implied by its title. Minimally, this experiment should be relabelled.

"Altogether, this data confirmed that we successfully set up an LTSPC protocol in mice and that this behavioral paradigm can be used to further study the brain circuits involved in higher-order conditioning."<br /> Please insert the qualifier that LTSPC was successfully established in male mice. There is no evidence of LTSPC in female mice.

Results - Brain

"Notably, the inhibition of CaMKII-positive neurons in the dHPC (i.e. J60 administration in DREADD-Gi mice) during preconditioning (Figure 4B), but not before the Probe Test 1 (Figure 4B), fully blocked mediated, but not direct learning (Figure 4D)."<br /> The right panel of Figure 4B indicates no difference between the controls and Group DPC in the percent change in freezing from OFF to ON periods of the tone. How does this fit with the claim that CaMKII-positive neurons in the dorsal hippocampus regulate associative formation during the session of tone-light exposures in stage 1 of sensory preconditioning?

Discussion

"When low salience stimuli were presented separated on time or when the electric footshock was absent, mediated and direct learning were abolished in male mice. In female mice, although light and tone were presented separately during the preconditioning phase, mediated learning was reduced but still present, which implies that female mice are still able to associate the two low-salience stimuli."<br /> This doesn't quite follow from the results. The failure of the female unpaired mice to withhold their freezing to the tone should not be taken to indicate the formation of a light-tone association across the very long interval that was interpolated between these stimulus presentations. It could and should be taken to indicate that, in female mice, freezing conditioned to the light simply generalized to the tone (i.e., these mice could not discriminate well between the tone and light).

"Indeed, our data suggests that when hippocampal activity is modulated by the specific manipulation of hippocampal subregions, this brain region is not involved during retrieval."<br /> Does this relate to the results that are shown in the right panel of Figure 4B, where there is no significant difference between the different groups? If so, how does it fit with the results shown in the left panel of this figure, where differences between the groups are observed?

"In line with this, the inhibition of CaMKII-positive neurons from the dorsal hippocampus, which has been shown to project to the restrosplenial cortex56, blocked the formation of mediated learning."<br /> Is this a reference to the findings shown in Figure 4B and, if so, which of the panels exactly? That is, one panel appears to support the claim made here while the other doesn't. In general, what should the reader make of data showing the percent change in freezing from stimulus OFF to stimulus ON periods?

Reviewer #4 (Public review):

Summary:

The structural mechanism of anion permeation through the open CFTR pore has remained unresolved and is subject to ongoing debate. That is because even in CFTR structures obtained under conditions that normally maximally activate the channel (phosphorylation + ATP + non-hydrolytic mutations + potentiator drugs) a bottleneck region in the pore, too narrow to allow passage of hydrated chloride ions, is observed.

The present study uses molecular dynamics (MD) simulations initiated from such "quasi-open" states to address local conformational dynamics of the pore. The authors conclude that the quasi-open structure stably relaxes to a fully open conformation on the sub-microsecond time scale. They provide a detailed analysis of this fully open structure and of the mechanism of chloride permeation. They conclude that two major exit pathways (a central and a peripheral) exist for chloride ions, and that the ions remain near-fully hydrated throughout the pore: chloride-protein interactions displace only 1-2 waters from the first solvation shell. Furthermore, the simulations provide some hints for conformational changes involved in gating.

Strengths:

The findings are interpreted in the context of the large body of published functional studies on CFTR permeation properties, and caveats are adequately discussed.

Weaknesses:

The conclusions on gating would benefit from further discussions. In particular, a fair comparison of the timescale at which channel gating happens, and that of the MD simulations would strengthen the manuscript.

Reviewer #5 (Public review):

Summary:

The researchers examined selection across multiple levels, including gene expression, biological processes, and regulatory mechanisms, with a particular focus on comparing selection between different environmental conditions. They further explored potential evolutionary mechanisms. This is made possible with a comprehensive dataset comprising gene expression data from 130 accessions with three replicates collected in two environments in the field, genomic data from 125 genotypes, and associated physiological traits. The findings have significant implications for understanding the evolution of stress adaptation, and the identified possible genes and pathways for further investigation.

The researchers began by focusing on the selection of gene expression across two environments, comparing the number of genes under selection and the effect sizes, as well as examining how selection in each environment acts on the same individual genes. They then expanded their analysis to consider selection in biological processes, investigating the relationships between selection acting on individual genes within processes and selection acting among different processes.<br /> Additionally, they explored selection at the organismal level by examining traits.

The study further transitioned from analyzing individual gene expression to investigating gene-gene interactions. They briefly examined correlation variation among gene pairs between the two conditions, identifying pairs with rewired interactions that suggest potential selection on gene regulation or the effect of rewiring on tolerance. The researchers then delved into the genetic architecture underlying these patterns by mapping eQTLs. Their comparison of cis- and trans-eQTLs revealed that trans-eQTLs were more variable across conditions. Notably, they identified hotspots representing master regulators that possibly underlie the greater variability of trans-eQTLs across environments. They further discovered that trans-eQTLs are generally under purifying selection (particularly in salt conditions), while cis-eQTLs are under balancing selection, exhibiting higher nucleotide diversity. As for how cis- and trans-eQTL effects combine at the level of individual genes, more are found to be reinforced and the hypothesis of genetic fixation on cis- and trans-eQTL effects combination is further tested.

Strengths:

A key strength of this study is its comprehensive approach, extending beyond the analysis of gene expression to include gene-gene interactions, genetic architectures, and selections of genetic regulation factors. The exploration of gene expression selection through its connection with fitness, as introduced in the researchers' previous work, provides valuable insights into the role of gene expression in adaptation. The study investigates selection across multiple levels of biological responses, including individual gene expression, genes associated with biological processes, gene-gene interactions, and the underlying genetic architecture. The experimental design enables a direct comparison of selection between control and salinity conditions, which sheds light on the effects of stress on selection and the dynamics of adaptation to stress. Additionally, the manuscript is well-written, with a clear connection to current literature. The discussion effectively integrates findings with broader implications, making it a satisfying read.

Weaknesses:

The lack of formal testing for environment-specific selections (e.g., selection of gene expression specifically in salinity stress, PCs, or traits) is a major limitation, as previous reviewers have flagged. Explicit tests of eQTLs variation between conditions are introduced, so similar formal tests should also be introduced in selection sections. For example, a formal test of selections of gene expression might be helpful to solve variance/mean- standardization concerns between two environments.

Additionally, some aspects of the analysis appear somewhat arbitrary and could benefit from further sensitivity testing. Line 203: The concern about bias in detecting more CN than AP, as mentioned by the authors and previously flagged by reviewers, does not seem fully resolved with the current methods given the arbitrary cut-off. Incorporating additional tests suggesting the conclusion is insensitive to the cutoff would be very helpful. Similar is the classification of genes into compensatory and reinforcing categories based on 60% of individuals as a cutoff.

While this study focuses on gene regulation, its connection with the selection of gene expression and biological pathways is not well integrated. In particular, the discovery of eQTLs is not explicitly linked to gene expression selection or biological pathways, leaving this relationship underexplored. Suggestive comments: Currently the summarization of selection is based on eQTLs. It would be interesting to also summarize the selection patterns identified from previous sections based on genes being cis/trans-regulated. Moreover, it might be interesting to see if there is more loss or gain of eQTLs under salt stress and their functions. The current results mentioned variations of eQTLs but not clear if they are loss or gain. E.g., one way is to identify genes related to cis and trans-eQTLs and see their correlation changes with genes being regulated using CILP (also as a way to informatively narrow down gene pairs for CILP).

Similarly, the section on selection at the organismal trait level appears disconnected from the rest of the analysis (e.g., if it is not tested to be related to other features, mentioning why it might not be related would be helpful). Admittedly, the discussion of how biological processes discovered at different levels integrate together is helpful.

Other comments: given there is no comparison between loss of coherence (correlations) and gain of coherence under salinity stress to show the dominant role of decoherence, maybe need to also discuss the genes and processes related to the gain of coherence? This is because the understanding of activation (gain of coherence) of some regulations/processes under stress conditions could also be interesting. It is not clear if decoherence (e.g., lines 293-296) refers to significant correlation changes or just loss of the correlation in salinity stress.

Reviewer #4 (Public Review):

Summary:

In their revised manuscript "Conformational dynamics of a nicotinic receptor neurotransmitter binding site," Singh and colleagues present molecular docking and dynamics simulations to explore the initial conformational changes associated with agonist binding in the muscle nicotinic acetylcholine receptor, in context with the extensive experimental literature on this system. Their central findings are of a consistently preferred pose for agonists upon initial association with a resting channel, followed by a dramatic rotation of the ligand and contraction of a critical loop over the binding site. Principal component analysis also suggests the formation of an intermediate complex, not yet captured in structural studies. Binding free energy estimates are consistent with the evolution of a higher-affinity complex following agonist binding, with a ligand efficiency notably similar to experimental values. Snapshot comparisons provide a structural rationale for these changes on the basis of pocket volume, hydration, and rearrangement of key residues at the subunit interface.

Strengths:

Docking results are clearly presented and remarkably consistent. Simulations are produced in triplicate with each of four different agonists, providing an informative basis for internal validation. They identify an intriguing transition in ligand pose, not well documented in experimental structures, and potentially applicable to mechanistic or even pharmacological modeling of this and related receptor systems. The paper seems a notable example of integrating quantitative structure-function analysis with systematic computational modeling and simulations, likely applicable to the wider journal audience.

Weaknesses:

The response to the initial review is somewhat disappointing, declining in some places to implement suggested clarifications, and propagating apparent errors in at least one table (Fig 2-source data 1). Some legends (e.g. Fig 2-supplement 4, Fig 3, Fig 4) and figure shadings (e.g. Fig 2-supplement 2, Fig 6-supplement 2) remain unclear. Apparent convergence of agonist-docked simulations towards a desensitized state (l 184) is difficult to interpret in absence of comparative values with other states, systems, etc. In more general concerns, aside from the limited timescales (200 ns) that do not capture global rearrangements, it is not obvious that landscapes constructed on two principal components to identify endpoint and intermediate states (Fig 3) are highly robust or reproducible, nor whether they relate consistently to experimental structures.

Reviewer #3 (Public review):

Summary:

This study investigated motor system adaptation to new environments through modifications in redundant body movements. Utilizing a novel bimanual stick-manipulation task, participants controlled a virtual stick to reach targets, focusing on how tip-movement direction perturbations affected tip movement and stick-tilt adaptation. The findings revealed a consistent strategy among participants who flexibly adjusted the tilt angle of the stick in response to errors. The adaptation patterns were influenced by physical space relationships, which guided the motor system's selection of movement patterns. This study underscores the motor system's adaptability through changes in redundant body movement patterns.

Strengths:

This study introduces an innovative bimanual stick manipulation task to explore motor system adaptation to novel environments through alterations in redundant body movement patterns. It also expands the use of endpoint robots in motor control studies.

Weaknesses:

The generalizability of the findings is limited. Future work may strengthen the present study's findings by examining whether the observed relationships hold for different stick lengths (i.e., varying hand positions along the virtual stick) or when reaching targets to the left and right of the starting position, not just at varying angles along one side. Additionally, a more comprehensive review of the existing literature on redundant systems, rather than primarily focusing on the lack of redundancy in endpoint-reaching tasks, would have strengthened this study. While the novel task expands the use of endpoint robots in motor control studies, its utility in exploring broader aspects of motor control and learning may be constrained.

Reviewer #4 (Public review):

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

I reviewed this paper primarily from a statistical perspective. Evidently, an impressive amount of work has been conducted, and concisely summarised, and great effort has been undertaken to add layers of insight to the findings. I am no stranger to what an undertaking this would have been. My primary concern, however, is that the novel statistical procedure proposed, and applied to identify the gene lists, as far as I can tell offers no statistical error control or quantification. Consequently, we have no sense of what proportion of the highlighted GEAR genes and networks are likely to just be noise.

Major comments:

(1) The main methodology used to identify the GEAR genes, "Multi-gradient Permutation Survival Analysis" does not formally account for multiple testing and offers no formal error control. Meaning we are left with no understanding of what the family-wise (aka type 1) error rate is among the GEAR lists, nor the false discovery rate. I would generally recommend against the use of any feature selection methodology that does not provide some form of error quantification and/or control because otherwise we do not know if we are encouraging our colleagues and/or readers to put resources into lists of genes that contain more noise than not. There are numerous statistical techniques available these days that offer error control, including for lists of p-values from arbitrary sets of tests (see expansion on this and some review references below).

(2) Similarly, no formal significance measure was used to determine which of the strongest "SAS" connections to include as edges in the "Core Survival Network".

(3) There is, as far as I could tell, no validation of any identified gene lists using an independent dataset external to the presently analysed TCGA data.

(4) There are quite a few places in the methods section where descriptions were not clear (e.g. elements of matrices referred to without defining what the columns and rows are), and I think it would be quite challenging to re-produce some aspects of the procedures as currently described (more detailed notes below).

(5) There is a general lack of statistical inference offered. For example, throughout the gene enrichment section of the results, I never saw it stated whether the pathways highlighted are enriched to a significant degree or not.

Reviewer #4 (Public review):

Summary:

The authors have performed endoscopic calcium recordings of individual CeA neuron responses to food and shock, as well as to cues predicting food and shock. They claim that a majority of neurons encode valence, with a substantial minority encoding salience.

Strengths:

The use of endoscopic imaging is valuable, as it provides the ability to resolve signals from single cells, while also being able to track these cells across time. The recordings appear well-executed, and employ a sophisticated circular shifting analysis to avoid statistical errors caused by correlations between neighboring image pixels.

Weaknesses:

My main critique is that the authors didn't fully test whether neurons encode valence. While it is true that they found CeA neurons responding to stimuli that have positive or negative value, this by itself doesn't indicate that valence is the primary driver of neural activity. For example, they report that a majority of CeA neurons respond selectively to either the positive or negative US, and that this is evidence for "type I" valence encoding. However, it could also be the case that these neurons simply discriminate between motivationally relevant stimuli in a manner unrelated to valence per se. A simple test of this would be to check if neural responses generalize across more than one type of appetitive or aversive stimulus, but this was not done. The closest the authors came was to note that a small number of neurons respond to CS cues, of which some respond to the corresponding US in the same direction. This is relegated to the supplemental figures (3 and 4), and it is not noted whether the the same-direction CS-US neurons are also valence-encoding with respect to different USs. For example, are the neurons excited by CS-food and US-food also inhibited by shock? If so, that would go a long way toward classifying at least a few neurons as truly encoding valence in a generalizable way.

A second and related critique is that, although the authors correctly point out that definitions of salience and valence are sometimes confused in the existing literature, they then go on themselves to use the terms very loosely. For example, the authors define these terms in such a way that every neuron that responds to at least one stimulus is either salience or valence-encoding. This seems far too broad, as it makes essentially unfalsifiable their assertion that the CeA encodes some mixture of salience and valence. I already noted above that simply having different responses to food and shock does not qualify as valence-encoding. It also seems to me that having same-direction responses to these two stimuli similarly does not quality a neuron as encoding salience. Many authors define salience as being related to the ability of a stimulus to attract attention (which is itself a complex topic). However, the current paper does not acknowledge whether they are using this, or any other definition of salience, nor is this explicitly tested, e.g. by comparing neural response magnitudes to any measure of attention.

The impression I get from the authors' data is that CeA neurons respond to motivationally relevant stimuli, but in a way that is possibly more complex than what the authors currently imply. At the same time, they appear to have collected a large and high-quality dataset that could profitably be made available for additional analyses by themselves and/or others.

Lastly, the use of 10 daily sessions of training with 20 trials each seems rather low to me. In our hands, Pavlovian training in mice requires considerably more trials in order to effectively elicit responses to the CS. I wonder if the relatively sparse training might explain the relative lack of CS responses?

Reviewer #4 (Public Review):

Summary:

With their 'CMR-replay' model, Zhou et al. demonstrate that the use of spontaneous neural cascades in a context-maintenance and retrieval (CMR) model significantly expands the range of captured memory phenomena.

Strengths:

The proposed model compellingly outperforms its CMR predecessor and, thus, makes important strides towards understanding the empirical memory literature, as well as highlighting a cognitive function of replay.

Weaknesses:

Competing accounts of replay are acknowledged but there are no formal comparisons and only CMR-replay predictions are visualized. Indeed, other than the CMR model, only one alternative account is given serious consideration: A variant of the 'Dyna-replay' architecture, originally developed in the machine learning literature (Sutton, 1990; Moore & Atkeson, 1993) and modified by Mattar et al (2018) such that previously experienced event-sequences get replayed based on their relevance to future gain. Mattar et al acknowledged that a realistic Dyna-replay mechanism would require a learned representation of transitions between perceptual and motor events, i.e., a 'cognitive map'. While Zhou et al. note that the CMR-replay model might provide such a complementary mechanism, they emphasize that their account captures replay characteristics that Dyna-replay does not (though it is unclear to what extent the reverse is also true).

Another important consideration, however, is how CMR replay compares to alternative mechanistic accounts of cognitive maps. For example, Recurrent Neural Networks are adept at detecting spatial and temporal dependencies in sequential input; these networks are being increasingly used to capture psychological and neuroscientific data (e.g., Zhang et al, 2020; Spoerer et al, 2020), including hippocampal replay specifically (Haga & Fukai, 2018). Another relevant framework is provided by Associative Learning Theory, in which bidirectional associations between static and transient stimulus elements are commonly used to explain contextual and cue-based phenomena, including associative retrieval of absent events (McLaren et al, 1989; Harris, 2006; Kokkola et al, 2019). Without proper integration with these modeling approaches, it is difficult to gauge the innovation and significance of CMR-replay, particularly since the model is applied post hoc to the relatively narrow domain of rodent maze navigation.

Reviewer #4 (Public Review):

The authors report the role of the Piruvate Kinase M2 (PKM2) enzyme nuclear translocation as fundamental in the activation of astrocytes in a model of autoimmune encephalitis (EAE). They show that astrocytes, activated through culturing in EAE splenocytes medium, increase their nuclear PKM2 with a consequent activation of NFkB and STAT3 pathways. Prevention of PKM2 nuclear translocation decreases astrocyte counteracts this activation. The authors found that the E3 ubiquitin ligase TRIM21 interacts with PKM2 and promotes its nuclear translocation. In vivo, either silencing of TRIM21 or inhibition of PKM2 nuclear translocation ameliorates the severity of the disease in the EAE model.

Strengths

This work contributes to the knowledge of the complex action of the PKM2 enzyme in the context of an autoimmune-neurological disease, highlighting its nuclear role and a novel partner, TRIM21, promoting its nuclear translocation. In vivo amelioration of the pathological signs through inhibition of either of the two, PKM2 and TRIM21, provides a novel rationale for therapeutic targeting.

Weaknesses

I believe that the major weakness is the fact that TRIM21 is known to have per se many roles in autoimmune and immune pathways and some of the effects observed might be due to a PKM2-independent action. Some of the experiments to link the two proteins, besides their interaction, are not completely clarifying the issue. On top of that, the in vivo experiments address the role of TRIM21 and the nuclear localisation of PKM2 independently, thus leaving the matter unsolved.

In general, the conclusions of the manuscript are supported by the reported results. The points to be addressed in future are the assessment of PKM2 as substrate of TRIM21 ubiquitin ligase activity and the proof of the epistatic relationship of TRIM21 and PKM2 in astrocyte activation. However, the data surely open novel directions to follow for the understanding of multiple sclerosis and related pathologies.

Reviewer #4 (Public Review):

The manuscript examines how patterns of selection on gene expression differ between a normal field environment and a field environment with elevated salinity based on transcript abundances obtained from leaves of a diverse panel of rice germplasm. In addition, the manuscript also maps expression QTL (eQTL) that explains variation in each environment. One highlight from the mapping is that a small group of trans-mapping regulators explains some gene expression variation for large sets of transcripts in each environment. The overall scope of the datasets is impressive, combining large field studies that capture information about fecundity, gene expression, and trait variation at multiple sites. The finding related to patterns indicating increased LD among eQTLs that have cis-trans compensatory or reinforcing effects is interesting in the context of other recent work finding patterns of epistatic selection. However, other analyses in the manuscript are less compelling or do not make the most of the value of collected data. Revisions are also warranted to improve the precision with which field-specific terminology is applied and the language chosen when interpreting analytical findings.

Selection of gene expression:<br /> One strength of the dataset is that gene expression and fecundity were measured for the same genotypes in multiple environments. However, the selection analyses are largely conducted within environments. The addition of phenotypic selection analyses that jointly analyze gene expression across environments and or selection on reaction norms would be worthwhile.

Gene expression trade-offs:<br /> The terminology and possibly methods involved in the section on gene expression trade-offs need amendment. I specifically recommend discontinuing reference to the analysis presented as an analysis of antagonistic pleiotropy (rather than more general trade-offs) because pleiotropy is defined as a property of a genotype, not a phenotype. Gene expression levels are a molecular phenotype, influenced by both genotype and the environment. By conducting analyses of selection within environments as reported, the analysis does not account for the fact that the distribution of phenotypic values, the fitness surface, or both may differ across environments. Thus, this presents a very different situation than asking whether the genotypic effect of a QTL on fitness differs across environments, which is the context in which the contrasting terms antagonistic pleiotropy and conditional neutrality have been traditionally applied. A more interesting analysis would be to examine whether the covariance of phenotype with fitness has truly changed between environments or whether the phenotypic distribution has just shifted to a different area of a static fitness surface.

Biological processes under selection / Decoherence: PCs are likely not the most ideal way to cluster genes to generate consolidated metrics for a selection gradient analysis. Because individual genes will contribute to multiple PCs, the current fractional majority-rule method applied to determine whether a PC is under direct or indirect selection for increased or decreased expression comes across as arbitrary and with the potential for double-counting genes. A gene co-expression network analysis could be more appropriate, as genes only belong to one module and one can examine how selection is acting on the eigengene of a co-expression module. Building gene co-expression modules would also provide a complementary and more concrete framework for evaluating whether salinity stress induces "decoherence" and which functional groups of genes are most impacted.

Selection of traits:<br /> Having paired organismal and molecular trait data is a strength of the manuscript, but the organismal trait data are underutilized. The manuscript as written only makes weak indirect inferences based on GO categories or assumed gene functions to connect selection at the organismal and molecular levels. Stronger connections could be made for instance by showing a selection of co-expression module eigengene values that are also correlated with traits that show similar patterns of selection, or by demonstrating that GWAS hits for trait variation co-localize to cis-mapping eQTL.

Genetic architecture of gene expression variation:<br /> The descriptive statistics of the eQTL analysis summarize counts of eQTLs observed in each environment, but these numbers are not broken down to the molecular trait level (e.g., what are the median and range of cis- and trans-eQTLs per gene). In addition, genetic architecture is a combination of the numbers and relative effect sizes of the QTLs. It would be useful to provide information about the relative distributions of phenotypic variance explained by the cis- vs. trans- eQTLs and whether those distributions vary by environment. The motivation for examining patterns of cis-trans compensation specifically for the results obtained under high salinity conditions is unclear to me. If the lines sampled have predominantly evolved under low salinity conditions and the hypothesis being evaluated relates to historical experience of stabilizing selection, then my intuition is that evaluating the eQTL patterns under normal conditions provides the more relevant test of the hypothesis.

Reviewer #4 (Public Review):

Summary:

The authors report a novel isomorphism in which the folds of the elephant trunk are recognizably mapped onto the principal sensory trigeminal nucleus in the brainstem. Further, they identify the enlarged nucleus as being situated in this species in an unusual ventral midline position.

Strengths:

The identity of the purported trigeminal nucleus and the isomorphic mapping with the trunk folds is supported by multiple lines of evidence: enhanced staining for cytochrome oxidase, an enzyme associated with high metabolic activity; dense vascularization, consistent with high metabolic activity; prominent myelinated bundles that partition the nucleus in a 1:1 mapping of the cutaneous folds in the trunk periphery; near absence of labeling for the anti-peripherin antibody, specific for climbing fibers, which can be seen as expected in the inferior olive; and a high density of glia.

Weaknesses:

Despite the supporting evidence listed above, the identification of the gross anatomical bumps, conspicuous in the ventral midline, is problematic. This would be the standard location of the inferior olive, with the principal trigeminal nucleus occupying a more dorsal position. This presents an apparent contradiction which at a minimum needs further discussion. Major species-specific specializations and positional shifts are well-documented for cortical areas, but nuclear layouts in the brainstem have been considered as less malleable.

Reviewer #4 (Public Review):

In this manuscript by Sha et al. the authors test the role of TNFa in modulating tumor regression/recurrence under therapeutic pressure from castration (or enzalutamide) in both in vitro and in vivo models of prostate cancer. Using the PTEN-null genetic mouse model, they compare the effect of a TNFα ligand trap, etanercept, at various points pre- and post-castration. Their most interesting findings from this experiment were that etanercept given 3 days prior to castration prevented tumor regression, which is a common phenotype seen in these models after castration, but etanercept given 1 day prior to castration prevented prostate cancer recurrence after castration. They go on to perform RNA sequencing on tumors isolated from either sham or castrate mice from two time points post-castration to study acute and delayed transcriptional responses to androgen deprivation. They found enrichment of gene sets containing TNF-targets which initially decrease post-castration but are elevated by 35 days, the time at which tumors recur. The authors conduct a similar set of experiments using human prostate cancer cell lines treated with the androgen receptor inhibitor enzalutamide and observe that drug treatment leads to cells with basal stem-like features that express high levels of TNF. They noticed that CCL2 levels correlate with changes in TNF levels raising the possibility that CCL2 might be a critical downstream effector for disease recurrence. To this end, they treated PTEN-null and hi-MYC castrated mice with a CCR2-antagonist (CCR2a) because CCR2 is one receptor of CCL2 and monitors tumor growth dynamics. Interestingly, upon treatment with CCR2a, tumors did not recur according to their measurements. They go on to demonstrate that the tumors pre-treated with CCR2a had reduced levels of putative TAMs and increased CTLs in the context of TNF or CCR2 inhibition providing a cellular context associated with disease regression. Lastly, they perform single-cell RNA sequencing to further characterize the tumor microenvironment post-castration and report that the ratio of CTLs to TAMs is lower in a recurrent tumor.

While the concepts behind the study have merit, the data are incomplete and do not fully support the authors' conclusions. The author's definition of recurrence is subjective given that the amount of disease regression after castration is both variable (Figure 8) and relatively limited, particularly in the PTEN loss model. Critical controls are missing. For example, both drug experiments were completed without treating non-castrate plus drug controls which raises the question of how specific these findings are to castration resistance. No validation was performed to ensure that either the TNF ligand trap or the CCR2 agonist was acting on target. The single-cell sequencing experiments were done without replicates which raises concern about its interpretation. At a conceptual level, the authors say that a major cause of disease recurrence in the immunosuppressive TME, but provide little functional data that macrophages and T cells are directly responsible for this phenotype. Statistical analyses were performed on only select experiments. In summary, further work is recommended to support the conclusions of this story.

Reviewer #4 (Public Review):

Summary:

This manuscript claims to provide a new null hypothesis for testing the effects of biodiversity on ecosystem functioning. It reports that the strength of biodiversity effects changes when this different null hypothesis is used. This main result is rather inevitable. That is, one expects a different answer when using a different approach. The question then becomes whether the manuscript's null hypothesis is both new and an improvement on the null hypothesis that has been in use in recent decades.

Strengths:

In general, I appreciate studies like this that question whether we have been doing it all wrong and I encourage consideration of new approaches.

Weaknesses:

Despite many sweeping critiques of previous studies and bold claims of novelty made throughout the manuscript, I was unable to find new insights. The manuscript fails to place the study in the context of the long history of literature on competition and biodiversity and ecosystem functioning. The Introduction claims the new approach will address deficiencies of previous approaches, but after reading further I see no evidence that it addresses the limitations of previous approaches noted in the Introduction. Furthermore, the manuscript does not reproducibly describe the methods used to produce the results (e.g., in Table 1) and relies on simulations, claiming experimental data are not available when many experiments have already tested these ideas and not found support for them. Finally, it is unclear to me whether rejecting the 'new' null hypothesis presented in the manuscript would be of interest to ecologists, agronomists, conservationists, or others. I will elaborate on each of these points below.

The critiques of biodiversity experiments and existing additive partitioning methods are overstated, as is the extent to which this new approach addresses its limitations. For example, the critique that current biodiversity experiments cannot reveal the effects of species interactions (e.g., lines 37-39) isn't generally true, but it could be true if stated more specifically. That is, this statement is incorrect as written because comparisons of mixtures, where there are interspecific and intraspecific interactions, with monocultures, where there are only intraspecific interactions, certainly provide information about the effects of species interactions (interspecific interactions). These biodiversity experiments and existing additive partitioning approaches have limits, of course, for identifying the specific types of interactions (e.g., whether mediated by exploitative resource competition, apparent competition, or other types of interactions). However, the approach proposed in this manuscript gets no closer to identifying these specific mechanisms of species interactions. It has no ability to distinguish between resource and apparent competition, for example. Thus, the motivation and framing of the manuscript do not match what it provides. I believe the entire Introduction would need to be rewritten to clarify what gap in knowledge this proposed approach is addressing and what would be gained by filling this knowledge gap.

I recommend that the Introduction instead clarify how this study builds on and goes beyond many decades of literature considering how competition and biodiversity effects depend on density. This large literature is insufficiently addressed in this manuscript. This fails to give credit to previous studies considering these ideas and makes it unclear how this manuscript goes beyond the many previous related studies. For example, see papers and books written by de Wit, Harper, Vandermeer, Connolly, Schmid, and many others. Also, note that many biodiversity experiments have crossed diversity treatments with a density treatment and found no significant effects of density or interactions between density and diversity (e.g., Finn et al. 2013 Journal of Applied Ecology). Thus, claiming that these considerations of density are novel, without giving credit to the enormous number of previous studies considering this, is insufficient.

Replacement series designs emerged as a consensus for biodiversity experiments because they directly test a relevant null hypothesis. This is not to say that there are no other interesting null hypotheses or study designs, but one must acknowledge that many designs and analyses of biodiversity experiments have already been considered. For example, Schmid et al. reviewed these designs and analyses two decades ago (2002, chapter 6 in Loreau et al. 2002 OUP book) and the overwhelming consensus in recent decades has been to use a replacement series and test the corresponding null hypothesis.

It is unclear to me whether rejecting the 'new' null hypothesis presented in the manuscript would be of interest to ecologists, agronomists, conservationists, or others. Most biodiversity experiments and additive partitions have tested and quantified diversity effects against the null hypothesis that there is no difference between intraspecific and interspecific interactions. If there was no less competition and no more facilitation in mixtures than in monocultures, then there would be no positive diversity effects. Rejecting this null hypothesis is relevant when considering coexistence in ecology, overyielding in agronomy, and the consequences of biodiversity loss in conservation (e.g., Vandermeer 1981 Bioscience, Loreau 2010 Princeton Monograph). This manuscript proposes a different null hypothesis and it is not yet clear to me how it would be relevant to any of these ongoing discussions of changes in biodiversity.

The claim that all previous methods 'are not capable of quantifying changes in ecosystem productivity by species interactions and species or community level' is incorrect. As noted above, all approaches that compare mixtures, where there are interspecific interactions, to monocultures, where there are no species interactions, do this to some extent. By overstating the limitations of previous approaches, the manuscript fails to clearly identify what unique contribution it is offering, and how this builds on and goes beyond previous work.

The manuscript relies on simulations because it claims that current experiments are unable to test this, given that they have replacement series designs (lines 128-131). There are, however, dozens of experiments where the replacement series was repeated at multiple densities, which would allow a direct test of these ideas. In fact, these ideas have already been tested in these experiments and density effects were found to be nonsignificant (e.g., Finn et al. 2013).

It seems that the authors are primarily interested in trees planted at a fixed density, with no opportunity for changes in density, and thus only changes in the size of individuals (e.g., Fig. 1). In natural and experimental systems, realized density differs from the initial planted density, and survivorship of seedlings can depend on both intraspecific and interspecific interactions. Thus, the constrained conditions under which these ideas are explored in this manuscript seem narrow and far from the more complex reality where density is not fixed.

Additional detailed comments:

It is unclear to me which 'effects' are referred to on line 36. For example, are these diversity effects or just effects of competition? What is the response variable?

The usefulness of the approach is overstated on line 52. All partitioning approaches, including the new one proposed here, give the net result of many types of species interactions and thus cannot 'disentangle underlying mechanisms of species interactions.'

The weaknesses of previous approaches are overstated throughout the manuscript, including in lines 60-61. All approaches provide some, but not all insights. Sweeping statements that previous approaches are not effective, without clarifying what they can and can't do, is unhelpful and incorrect. Also, these statements imply that the approach proposed here addresses the limitations of these previous approaches. I don't yet see how it does so.

The definitions given for the CE and SE on line 71 are incorrect. Competition affects both terms and CE can be negative or have nothing to do with positive interactions, as noted in many of the papers cited.

The proposed approach does not address the limitations noted on lines 73 and 74.

The definition of positive interactions in lines 77 and 78 seems inconsistent with much of the literature, which instead focuses on facilitation or mutualism, rather than competition when describing positive interactions.

Throughout the manuscript, competition is often used interchangeably with resource competition (e.g., line 82) and complementarity is often attributed to resource partitioning (e.g., line 77). This ignores apparent competition and partitioning enemy-free niche space, which has been found to contribute to biodiversity effects in many studies.

In what sense are competitive interactions positive for competitive species (lines 82-83)? By definition, competition is an interaction that has a negative effect. Do you mean that interspecific competition is less than intraspecific competition? I am having a very difficult time following the logic.

Results are asserted on lines 93-95, but I cannot find the methods that produced these results. I am unable to evaluate the work without a repeatable description of the methods.

The description of the null hypothesis in the common additive partitioning approach on lines 145-146 is incorrect. In the null case, it does not assume that there are no interspecific interactions, but rather that interspecific and intraspecific interactions are equivalent.

Reviewer #4 (Public Review):

Summary:

The authors report a novel isomorphism in which the folds of the elephant trunk are recognizably mapped onto the principal sensory trigeminal nucleus in the brainstem. Further, they identifiy the enlarged nucleus as being situated in this species in an unusual ventral midline position.

Strengths:

The identity of the purported trigeminal nucleus and the isomorphic mapping with the trunk folds is supported by multiple lines of evidence: enhanced staining for cytochrome oxidase, an enzyme associated with high metabolic activity; dense vascularization, consistent with high metabolic activity; prominent myelinated bundles that partition the nucleus in a 1:1 mapping of the cutaneous folds in the trunk periphery; near absence of labeling for the anti-peripherin antibody, specific for climbing fibers, which can be seen as expected in the inferior olive; and a high density of glia.

Weaknesses:

Despite the supporting evidence listed above, the identification of the gross anatomical bumps, conspicuous in the ventral midline, is problematic. This would be the standard location of the inferior olive, with the principal trigeminal nucleus occupying a more dorsal position. This presents an apparent contradiction which at a minimum needs further discussion. Major species-specific specializations and positional shifts are well-documented for cortical areas, but nuclear layouts in the brainstem have been considered as less malleable.

Reviewer #4 (Public Review):

This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications for understanding how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms. I didn't observe significant weaknesses in the study, but I have several comments that I hope the authors will address.

Reviewer #4 (Public Review):

Summary:

The authors report the role of the Pyruvate Kinase M2 (PKM2) enzyme nuclear translocation as fundamental in the activation of astrocytes in a model of autoimmune encephalitis (EAE). They show that astrocytes, activated through culturing in EAE splenocytes medium, increase their nuclear PKM2 with consequent activation of NFkB and STAT3 pathways. Prevention of PKM2 nuclear translocation decreases astrocyte counteracts this activation. The authors found that the E3 ubiquitin ligase TRIM21 interacts with PKM2 and promotes its nuclear translocation. In vivo, either silencing of TRIM21 or inhibition of PKM2 nuclear translocation ameliorates the severity of the disease in the EAE model.

Strengths:

This work contributes to the knowledge of the complex action of the PKM2 enzyme in the context of an autoimmune-neurological disease, highlighting its nuclear role and a novel partner, TRIM21, and thus adding a novel rationale for therapeutic targeting.

Weaknesses:

Despite the relevance of the work and its goals, some of the conclusions drawn would require more thorough proof:

I believe that the major weakness is the fact that TRIM21 is known to have per se many roles in autoimmune and immune pathways and some of the effects observed might be due to a PKM2-independent action. Some of the experiments to link the two proteins, besides their interaction, do not completely clarify the issue. On top of that, the in vivo experiments address the role of TRIM21 and the nuclear localisation of PKM2 independently, thus leaving the matter unsolved.

Some experimental settings are not described to a level that is necessary to fully understand the data, especially for a non-expert audience: e.g. the EAE model and MOG treatment; action and reference of the different nuclear import inhibitors; use of splenocyte culture medium and the possible effect of non-EAE splenocytes.

The statement that PKM2 is a substrate of TRIM21 ubiquitin ligase activity is an overinterpretation. There is no evidence that this interaction results in ubiquitin modification of PKM2; the ubiquitination experiment is minimal and is not performed in conditions that would allow us to see ubiquitination of PKM2 (e.g. denaturing conditions, reciprocal pull-down, catalytically inactive TRIM21, etc.).

Reviewer #4 (Public Review):<br /> <br /> This study provides direct evidence showing that Sema7a plays a role in the axon growth during the formation of peripheral sensory circuits in the lateral-line system of zebrafish. This is a valuable finding because the molecules for axon growth in hair-cell sensory systems are not well understood. The majority of the experimental evidence is convincing, and the analysis is rigorous. The evidence supporting Sema7a's juxtracrine vs. secreted role and involvement in synapse formation in hair cells is less conclusive. The study will be of interest to cell, molecular and developmental biologists, and sensory neuroscientists.

Reviewer #4 (Public Review):

This study provides direct evidence showing that Sema7a plays a role in the axon growth during the formation of peripheral sensory circuits in the lateral-line system of zebrafish. This is a valuable finding because the molecules for axon growth in hair-cell sensory systems are not well understood. The majority of the experimental evidence is convincing, and the analysis is rigorous. The evidence supporting Sema7a's juxtracrine vs. secreted role and involvement in synapse formation in hair cells is less conclusive. The study will be of interest to cell, molecular and developmental biologists, and sensory neuroscientists.

Reviewer #4 (Public Review):

Summary:

In their revised manuscript "Conformational dynamics of a nicotinic receptor neurotransmitter binding site," Singh and colleagues present molecular docking and dynamics simulations to explore the initial conformational changes associated with agonist binding in the muscle nicotinic acetylcholine receptor, in context with the extensive experimental literature on this system. Their central findings are of a consistently preferred pose for agonists upon initial association with a resting channel, followed by a dramatic rotation of the ligand and contraction of a critical loop over the binding site. Principal component analysis also suggests the formation of an intermediate complex, not yet captured in structural studies. Binding free energy estimates are consistent with the evolution of a higher-affinity complex following agonist binding, with a ligand efficiency notably similar to experimental values. Snapshot comparisons provide a structural rationale for these changes on the basis of pocket volume, hydration, and rearrangement of key residues at the subunit interface.

Strengths:

Docking results are clearly presented and remarkably consistent. Simulations are produced in triplicate with each of four different agonists, providing an informative basis for internal validation. They identify an intriguing transition in ligand pose, not well documented in experimental structures, and potentially applicable to mechanistic or even pharmacological modeling of this and related receptor systems. The paper seems a notable example of integrating quantitative structure-function analysis with systematic computational modeling and simulations, likely applicable to the wider journal audience.

Weaknesses:

The response to initial review is somewhat disappointing, declining in some places to implement suggested clarifications, and propagating apparent errors in at least one table (Fig 2-source data 1). Some legends (e.g. Fig 2-supplement 4, Fig 3, Fig 4) and figure shadings (e.g. Fig 2-supplement 2, Fig 6-supplement 2) remain unclear. Apparent convergence of agonist-docked simulations towards a desensitized state (l 184) is difficult to interpret in absence of comparative values with other states, systems, etc. In more general concerns, aside from the limited timescales (200 ns) that do not capture global rearrangements, it is not obvious that landscapes constructed on two principal components to identify endpoint and intermediate states (Fig 3) are highly robust or reproducible, nor whether they relate consistently to experimental structures.

Reviewer #3 (Public Review):

Summary:

The molecular mechanism of regulated exocytosis has been extensively studied in the context of synaptic transmission. However, in addition to neurotransmitters, neurons also secrete neuropeptides and neurotrophins, which are stored in dense core vesicles (DCVs). These factors play a crucial role in cell survival, growth, and shaping the excitability of neurons. The mechanism of release for DCVs is similar, but not identical, to that used for SV exocytosis. This results in slow kinetic and low release probabilities for DCV compared to SV exocytosis. There is a limited understanding of the molecular mechanisms that underlie these differences. By investigating the role of rabphilin-3A (RPH3A), Hoogstraaten et al. uncovered for the first time a protein that inhibits DCV exocytosis in neurons.

Strengths:

In the current work, Hoogstraaten et al. investigate the function of rabphilin-3A (RPH3A) in DVC exocytosis. This RAB3 effector protein has been shown to possess a Ca2+ binding site and an independent SNAP25 binding site. Using colocalization analysis of confocal imaging the authors show that in hippocampal neurons RPH3A is enriched at pre- and post-synaptic sites and associates specifically with immobile DCVs. Using site-specific RPH3A mutants they found that the synaptic location was due to its RAB3 interaction site. They further could show that RPH3A inhibits DCV exocytosis due to its interaction with SNAP25. They came to that conclusion by comparing NPY-pHluorin release in WT and RPH3A KO cells and by performing rescue experiments with RPH3A mutants. Finally, the authors showed that by inhibiting stimulated DCV release, RPH3A controlled the axon and dendrite length possibly through the reduced release of neurotrophins. Thereby, they pinpoint how the proper regulation of DCV exocytosis affects neuron physiology.

Weaknesses:

Data context<br /> One of the findings is that RPH3A accumulates at synapses and is mainly associated with immobile DCVs. However, Farina et al. (2015) showed that 66% of all DCVs are secreted at synapses and that these DCVs are immobile prior to secretion. To provide additional context to the data, it would be valuable to determine if RPH3A KO specifically enhances secretion at synapses. Additionally, the authors propose that RPH3A decreases DCV exocytosis by sequestering SNAP25 availability. At first glance, this hypothesis appears suitable. However, due to RPH3A synaptic localization, it should also limit SV exocytosis, which it does not. In this context, the only explanation for RPH3A's specific inhibition of DCV exocytosis is that RPH3A is located at a synapse site remote from the active zone, thus protecting the pool of SNAP25 involved in SV exocytosis from binding to RPH3A. This hypothesis could be tested using super-resolution microscopy.

Technical weakness<br /> One technical weakness of this work consists in the proper counting of labeled DCVs. This is significant since most findings in this manuscript rely on this analysis. Since the data was acquired with epi-fluorescence or confocal microscopy, it doesn't provide the resolution to visualize individual DCVs when they are clumped. The authors use a proxy to count the number of DCVs by measuring the total fluorescence of individual large spots and dividing it by the fluorescence intensity of discrete spots assuming that these correspond to individual DCVs. This is an appropriate method but it heavily depends on the assumption that all DCVs are loaded with the same amount of NPY-pHluorin or chromogranin B (ChgB ). Due to the importance of this analysis for this manuscript, I suggest that the authors show that the number of DCVs per µm2 is indeed affected by RPH3A KO using super-resolution techniques such as dSTORM, STED, SIM, or SRRF.

Reviewer #4 (Public Review):

Summary:<br /> Recent progress in root economics has revealed global-scale axes of covaried root traits that reflect various root resource acquisition strategies. These covariance patterns are powerful tools for understanding root functional diversity. However, roots do not function in isolation for below-ground resource acquisition. Rather, symbiotic fungi and rhizosphere microorganisms often collaborate with plant roots, forming a root-microbial-soil continuum. This study seeks to provide novel insights into this continuum by extending the existing framework of root economics to include the structures of root-associated microorganisms. I find this topic highly relevant. Considering the role of soil microorganisms is undoubtedly crucial for a more comprehensive understanding of below-ground resource strategies.

Major comments:<br /> A key finding of this study is a relationship between root N and the tendency for roots to associate with particular types of mycorrhizal associations (Line 27, Fig. 2). The authors concluded that this indicates "a linkage from simple root traits to fungal-mediated carbon nutrient cycling" (line 27) and integrates "microbial functions into the root economics framework," (line 32). If substantiated, this correlation could represent a significant discovery about the connection between root functional traits and root-associated fungi. It suggests that low root N, indicative of low metabolic activity within the root economics framework, is linked with forming EcM associations. However, I am not fully convinced this is the case based on the current data presentation and interpretation.

First, there is no biological interpretation of this relationship between root N and mycorrhizal type. It merely noted that root N is indicative of root metabolic activity, and thus by relating root N to fungal composition, "the trait-related root economics and fungal-driven nutrient economics may be integrated into a unified framework" (lines 221-224). Why would roots with low N and low metabolic activity tend to favor EcM associations? What are the potential mechanisms? Biological interpretation is essential for understanding whether a statistical correlation reflects a causal and meaningful relationship or is coincidental.

I am also concerned that this relationship may be spurious, especially when it lacks biological interpretation. EcM is underrepresented in this study (8 EcM species, of which more than half are conifers and oaks vs. 44 AM) and seems to cluster at higher elevations (line 231). Thus, the tree species/individual data points are not independent, but phylogenetically and geographically clustered. The unique properties at higher elevations (e.g., distinct plant community structures, low levels of mineral N) may drive both the lower root N and the prevalence of EcM associations. This scenario aligns with the observation that at higher elevations, AM roots also exhibited low root N (Line 231). In this case, root N may not directly relate to mycorrhizal type but is characteristic of certain locations (or closely related species), and it would be misleading to suggest that low root N/metabolic activity, a proxy in fast-slow root economics, is directly linked to the preference for a particular mycorrhizal type (lines 27-28, 220 - 224). In summary, because the studied tree species appear to be clustered both phylogenetically and geographically, these factors need to be carefully taken into account in the statistical analysis and data interpretation to understand the underlying causes of the apparent relationship and prevent overinterpretation. I also recommend, if possible, providing a visual presentation of the geographical and phylogenetic distribution of the studied tree species.

That being said, this dataset is undoubtedly valuable in revealing the shifts in the compositional structures of root-associated soil microorganisms. However, integrating the traits of microbial composition to root trait economics would require more caution and careful examination of the potential driving causes.

Reviewer #4 (Public Review):

The members of the Kimmins lab perform a dietary study in mice to investigate the impact of obesity of fathers on the development of their offspring. To do so, they expose male mice to a high fat diet and determine the distribution and occupancy levels of the histone H3 lysine 4 trimethylation (H3K4me3) mark in spermatozoa and perform gene expression studies on placenta tissue obtained from mouse embryos during mid-gestation development. The authors report changes in H3K4me3 occupancy in sperm as well as in transcriptomes of placentas of male and female embryonic offspring. While the authors perform extensive computational analysis of the transcriptomic and chromatin immunoprecipitation data, the authors do not go much beyond making correlative statements at mainly the genome wide level between changes for H3K4me3 in sperm and transcriptional changes in placenta, the latter of which are in part related to changes in cellular composition (as deduced from transcriptional data). Given that both parental mice had the same genetic background, it was not possible to deduce parental specific contributions to transcriptional changes as observed in placentas of offspring. In all, the study falls short in increasing mechanistic insights into this important biological phenomenon.

Reviewer #4 (Public Review):

Summary:<br /> The authors set out to address whether TTX resistance in a subset of snakes is due to mutations near the selectivity filters of their Nav1.4 channels. They present an investigation of the properties of two heterologously expressed Nav1.4 channels, bearing the Nav1.4EPN and Nav1.4LVNV mutations found in TTX-resistant snakes. After assessing their sensitivity to TTX, they have studied the biophysical properties of these mutants by electrophysiological methods and discovered that the voltage dependence of their activation and inactivation remains unchanged compared to the TTX-sensitive Nav1.4. These experiments revealed some kinetic differences in Nav1.4LVNV and that both Nav1.4EPN and Nav1.4LVNV show a reduced unitary conductance. The authors also assessed muscle properties (resistance, force development, and contraction timing) of two groups of snakes (in vivo and in dissected muscles) with Nav1.4EPN and Nav1.4LVNV mutations. These experiments showed a reduced performance for the skeletal muscles of snakes bearing Nav1.4EPN and Nav1.4LVNV background. Finally, the authors have built homology models of Nav1.4EPN and Nav1.4LVNV channels to hypothesize a molecular explanation of the altered properties.

Strengths:<br /> • Three levels of analysis are performed in this study: 1) functional characterization of mutated Nav1.4 channels through electrophysiology; 2) molecular level comparisons between human and snake Nav1.4 channels structures through homology modelling; 3) organismal performance/muscle strength experiments on snakes that carry Nav1.4 mutants that render them virtually TTX resistant.

Weaknesses:<br /> • While there is reason to believe that there is a causal link between the observed changes in Nav1.4 and the changes on the organismal level, the evidence presented is not definitive. Specifically, the conclusions from the biophysical/electrophysiological experiments are extrapolated to be causal for the altered muscle performance in TTX-resistant snakes, although there might be alternative explanations. First, the reduction in muscle force could also originate from changes in the calcium release apparatus or other alterations in the electrical properties of the muscle (are there changes in length or duration of muscle action potentials? Is there a change in the fraction of muscle cells that fail action potentials, as would be expected for a significant reduction in conductance?). Second, it remains unclear if, among the different snake Nav channels (e.g. Nav1.6 in motor neurons), Nav1.4 is the only one to display side chain alterations in these TTX-resistant snakes.

• Some of the data presented as part of the NSNA is not sufficiently convincing and should be supplemented with additional evidence or carefully discussed with regard to its limitations.

• The mutations studied are located close to the selectivity filter of Nav1.4. This means that the most likely consequence of the mutations is altered sodium selectivity, possibly along with changes to block extracellular calcium. But these possibilities are not currently addressed.

• The description and accuracy of the homology model remains somewhat unclear, as no validation of the modeled channel has been presented. Therefore, the accuracy of the homology model remains vague, which calls into question to what degree the molecular features of this model can be linked to the electrophysiological findings.

Reviewer #4 (Public Review)

Summary:<br /> Cav1.4 voltage-gated calcium channels play an important role in neurotransmission at mammalian photoreceptor synapses. Mutations in the CACNA1f gene lead to congenital stationary night blindness that particularly affects the rod pathway. Mouse Cav1.4 knockout and Cav1.4 knockin models suggest that Cav1.4 is also important for the cone pathway. Deletion of Cav1.4 in the knockout models leads to signaling malfunctions and to abundant morphological re-arrangements of the synapse suggesting that the channel not only has a role in the influx of Ca2+ but also in the morphological organization of the photoreceptor synapse. Of note, also additional Cav-channels have been previously detected in cone synapses by different groups, including L-type Cav1.3 (Wu et al., 2007; pmid; Kersten et al., 2020; pmid), and also T-type Cav3.2 (Davison et al., 2021; pmid 35803735).

In order to study a conductivity-independent role of Cav1.4 in the morphological organization of photoreceptor synapses, the authors generated the knockin (KI) mouse Cav1.4 G369i in a previous study (Maddox et al., eLife 2020; pmid 32940604). The Cav1.4 G369i KI channel no longer works as a Ca2+-conducting channel due to the insertion of a glycine in the pore-forming unit (Madox et al. elife 2020; pmid 32940604). In this previous study (Madox et al. elife 2020; pmid 32940604), the authors analyzed Cav1.4 G369i in rod photoreceptor synapses. In the present study, the authors analyzed cone synapses in this KI mouse.

For this purpose, the authors performed a comprehensive set of experimental methods including immunohistochemistry with antibodies (also with quantitative analyses), electrophysiological measurements of presynaptic Ca2+ currents from cone photoreceptors in the presence/absence of inhibitors of L-type- and T-type- calcium channels, electron microscopy (FIB-SEM), ERG recordings and visual behavior tests of the Cav G369i KI in comparison to the Cav1.4 knockout and wild-type control mice.

The authors found that the non-conducting Cav channel is properly localized in cone synapses and demonstrated that there are no gross morphological alterations (e.g., sprouting of postsynaptic components that are typically observed in the Cav1.4 knockout). These findings demonstrate that cone synaptogenesis relies on the presence Cav1.4 protein but not on its Ca2+ conductivity. This result, obtained at cone synapses in the present study, is similar to the previously reported results observed for rod synapses (Maddox et al., eLife 2020, pmid 32940604). No further mechanistic insights or molecular mechanisms were provided that demonstrated how the presence of the Cav channels could orchestrate the building of the cone synapse.

Strengths:<br /> The study has been expertly performed. A comprehensive set of experimental methods including immunohistochemistry with antibodies (also with quantitative analyses), electrophysiological measurements of presynaptic Ca2+ currents from cone photoreceptors in the presence/absence of inhibitors of L-type- and T-type- calcium channels, electron microscopy (FIB-SEM), ERG recordings and visual behavior tests of the Cav G369i KI in comparison to the Cav1.4 knockout and wild-type control mice.

Weaknesses:<br /> The study has been expertly performed but remains descriptive without deciphering the underlying molecular mechanisms of the observed phenomena, including the proposed homeostatic switch of synaptic calcium channels. Furthermore, a relevant part of the data in the present paper (presence of T-type calcium channels in cone photoreceptors) has already been identified/presented by previous studies of different groups (Macosko et al., 2015; pmid 26000488; Davison et al., 2021; pmid 35803735; Williams et al., 2022; pmid 35650675). The degree of novelty of the present paper thus appears limited.

Reviewer #4 (Public Review):

Summary:<br /> In their manuscript "Conformational dynamics of a nicotinic receptor neurotransmitter binding site," Singh and colleagues present cogent molecular docking and dynamics simulations to explore the initial conformational changes associated with agonist binding in the muscle nicotinic acetylcholine receptor, aligned with the extensive experimental literature on this system. Their central findings are of a consistently preferred pose for agonists upon initial association with a resting channel, followed by a dramatic rotation of the ligand and contraction of a critical loop over the binding site. Principal component analysis also suggests the formation of an intermediate complex, not yet captured in structural studies. Binding free energy calculations are consistent with the evolution of a higher-affinity complex following agonist binding, with a ligand efficiency notably similar to experimental values. Snapshot comparisons provide a structural rationale for these changes on the basis of pocket volume, hydration, and rearrangement of key residues at the subunit interface.

Strengths:<br /> Docking results are clearly presented and remarkably consistent. Simulations are produced in triplicate with each of four different agonists, providing an informative basis for internal validation. They identify an intriguing transition in ligand pose, not well documented in experimental structures, and potentially applicable to mechanistic or even pharmacological modeling of this and related receptor systems. The paper seems a notable example of integrating quantitative structure-function analysis with systematic computational modeling and simulations, likely applicable to the wider journal audience.

Weaknesses:<br /> Timescales (200 ns) do not capture global rearrangements of the extracellular domain, let alone gating transitions of the channel pore, though this work may provide a launching point for more extended simulations. A more general concern is the reproducibility of the simulations, and how representative states are defined. It is not clear whether replicates were included in principal component analysis or subsequent binding energy calculations, nor how simulation intervals were associated with specific states. Structural analysis largely focuses on snapshots, with limited direct evidence of consistency across replicates or clusters. Figure legends and tables could be clarified.

Reviewer #4 (Public Review):

Overview:

The present manuscript by Zhou and colleagues investigates the impact of a new combination of compounds termed CHIR99021 and A-485 on stimulating cardiac cell regeneration. This manuscript fits the journal and addresses an important contribution to scientific knowledge. However, the following major revisions need to be addressed as stated below.

Major comments:

-The authors should include more information that clarifies and justifies their hypothesis.<br /> -The story line is not well developed and thus not convincing since the results from different sections are not well connected.<br /> -The main text needs to be improved, and authors should explain their purpose in choosing to study ISL1-CMs. Also, to well argument why they conducted this study and its significance.<br /> -Page 3, row 57-58: Please add the references.<br /> -Page 3-4, row 67-68, authors stated "When CMs resumed contraction, they were treated with individual small molecules from a collection of over 4,000 compounds for 3 days (SI Appendix, Fig. S1C and Table S1), and then fixed and immunostained with ISL1". Please explain better, and show the results of the selected screening compounds.<br /> -Authors must make an effort to discuss their findings in a bold way in order to provide a comprehensive and articulate explanation of their results to the readers. There is much information missing from this section. This should also propose new research avenues and foresee the challenges in future investigations.<br /> -Authors must include a conclusion and future perspectives of this study.<br /> - Page 4, row 73, the authors stated that the unique compound combination 'CHIR99021 and A-485' was found to be the most efficient in promoting ISL1 expression with a healthy cell state. However, the authors should prove that by showing at least the cell viability of these compound combinations at different concentrations and timings as a supplementary figure.<br /> -There is some missing information in the methods part, for example, "Images were captured using a confocal Zeiss LSM710 and Olympus IX83 inverted microscope"; authors should include the objective used and the image size, and should include which method they used to analyze the acquired images.<br /> -Figure S3A shows that the TNNT2 mRNA expression was completely absent after 60 hours of 2C administration. Authors should explain this further.<br /> -Figure 3J, there is high variability in the graph of mCherry cells (%). Please choose a better graph, or increase the independent experiment.<br /> -Authors did not explain/discuss their results of the DNA-binding motif analysis of ISL1 in the cells treated with A-485 or 2C (Figure 7K).<br /> -Figure S1B and D: the image's labeling is not clear. In the exact same figure S1B, how can the authors explain the reduction of ISL cells? Do the authors make the treatment with the compound CHIR99021 as shown in figure S1A? If so, the authors should clarify the ISL reduction in Figure S1B.<br /> -Figure 1H: please improve the immunoblot, the level of B-actin does not match among the different conditions, or provide a relative quantification of the proteins.<br /> -Please indicate further information in the methodology part about the compounds used in this study.<br /> -Figures are not well justified and figure legends are not sufficient enough to explain the figures.<br /> -Please improve the figure legends by including more further information; for example, in Figure 2SH it is highlighted only the "DAPI (4′,6-diamidino-2- phenylindole) staining labeled nuclei as blue" but how about the other markers?<br /> -Figure 2F: the graph shows some high variations in "ns" between NC at 2C and in 60h+3d. I would recommend increasing the independent experiments. Similar observation goes also for figure 2E.<br /> -Authors should provide limitations of this study.

Reviewer #4 (Public Review):

The study compares the number of sporozoites expelled by mosquitoes with different Plasmodium infection burden. To my knowledge this is the first report comparing the number of expelled P. falciparum sporozoites and their relation to oocyst burden (intact and ruptured) and residual sporozoites in salivary glands. The study provides important evidence on malaria transmission biology although conclusions cannot be drawn on direct impact on transmission.

Although there is some evidence from malaria challenge studies that the burden of sporozoites injected into a host is directly correlated with the likelihood of infection, this has been done using experimental infection models which administer sporozoites intravenously. It is unclear whether the same correlation occurs with natural infections and what the actual threshold for infection may be. Host immunity and other host related factors also play a critical role in transmission and need to be taken into consideration; these have not been mentioned by the authors. This is of particular importance as host immunity is decreasing with reduction in transmission intensity.

The natural infections reported in the study were not natural as the authors described. Gametocyte enrichment was done to attain high oocyst infection numbers. Studying natural infections would have been better without the enrichment step. The infected mosquitoes have much larger infection burden than what occurs in the wild.<br /> Nevertheless, the findings support the same results as in the experiments conducted in the Netherlands and therefore are of interest. I suggest the authors change the wording. Rather than calling these "natural" infections, they could be called, for example, "experimental infections with wild parasite strains".

I do not believe the study results generate sufficient evidence to conclude that lower infection burden in mosquitoes is likely to result in changes to transmission potential in the field. In study limitations section, the authors say "In addition, our quantification of sporozoite inoculum size is informative for comparisons between groups of high and low-infected mosquitoes but does not provide conclusive evidence on the likelihood of achieving secondary infections. Given striking differences in sporozoite burden between different Plasmodium species - low sporozoite densities appear considerably more common in mosquitoes infected with P. yoelli and P. Berghei the association between sporozoite inoculum and the likelihood of achieving secondary infections may be best examined in controlled human infection studies. However, in the abstract conclusion the authors state "Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is associated with the number of expelled sporozoites and may need to be considered in estimations of transmission potential." Kindly consider ending the sentence at "expelled sporozoites." Future studies on CHMI can be recommended as a conclusion if authors feel fit.

Reviewer #4 (Public Review):

This work by Fleck et al. and colleagues documented the auxin feeding-induced effects in adult flies, since auxin could be used in temporally controlled gene expression using a modified Gal4/Gal80 system. Overall, the experiments were well-designed and carefully executed. The results were quantified with appropriate statistical analyses. The paper was also well-written and the results were presented logically. The findings demonstrate that auxin-fed flies have significantly lower triglyceride levels than the control flies using Ultra High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)-based metabolomics assays. Further transcriptome analyses using the whole flies show changes in genes involved in fatty acid metabolism. However, female oogenesis and fecundity do not seem to be affected, at least using the current assays. These results indicate that auxin may not be used in experiments involving lipid-related metabolism, but could be appropriate to be applied for other biological processes.

Reviewer #4 (Public Review):

This is an admirable piece of work. The authors build on a previous dataset they assembled, but expand it to include all stages of early development in the nematode Caenorhabditis elegans. Cell collection was done manually, which is very impressive, and is clearly far better than pooled unidentified cells. I will not comment on the specific sequencing and analysis, since this is not my expertise, but will comment on the general conclusions and comparative framework in which the authors place their results.

While the Introduction and Discussion sections are actually fairly short, much of the presentation of the results is based on a certain comparative framework, which is explicitly a comparison between C. elegans and Drosophila melanogaster. This is an important perspective, but I feel the authors' interpretation is in some places exaggerated and in other places almost trivial.

Drosophila and C. elegans are two of the main models for developmental biology. However, it has been clear for over two decades that both species are highly derived and specialized and therefore, treating them as representative for their taxa is problematic. Much of the authors' discussion hinges on the question of comparing syncytial and lineage-dependent development. The syncytial early development of Drosophila is very specific and is clearly a recent innovation within a restricted group of flies. The canonical Drosophila segmentation cascade is mostly a novelty and most elements within the cascade are recent. Specifically, the expression of gap genes in regional stripes is not found very broadly. Conversely, the polarizing role of Caudal is very ancient and is probably found in all Bilateria. When making comparisons with a distantly related species, it is important to keep this in mind. Not as much is known about development of other nematodes, but the little that is known indicates that C. elegans is also unusual, and specifically the eutelic development (conserved cell lineages in development) is not found in all nematodes.

The authors suggest that regional expression of transcription factors in stripes is a conserved characteristic of development. This is true for Hox genes and has been known for decades. The regional expression they show for other genes is not convincing as "stripes". It is no surprise that developmental transcription factors are regionalized, but linking this to the stripes of Drosophila gap genes and even more so to Drosophila pair-rule and segment-polarity genes is a bit far-fetched. Yes, many genes are expressed in restricted domains along the A-P axis, but that is all that can be said based on the data. Calling them "Drosophila-like" is unfounded.

Reviewer #4 (Public Review):

Summary:<br /> The work by Dasgupta et al identifies Sema7a as a novel guidance molecule in hair cell sensory systems. The authors use the both genetic and imaging power of the zebrafish lateral-line system for their research. Based on expression data and immunohistochemistry experiments, the authors demonstrate that Sema7a is present in lateral line hair cells. The authors then examine a sema7a mutant. In this mutant, Sema7a proteins levels are nearly eliminated. Importantly, the authors show that when Sema7a is absent, afferent terminals show aberrant projections and fewer contacts with hair cells. Lastly the authors show that ectopic expression of the secreted form of Sema7a is sufficient to recruit aberrant terminals to non-hair cell targets. The sema7a innervation defects are well quantified. Overall, the paper is extremely well written and easy to follow.

Strengths:<br /> 1. The axon guidance phenotypes in sema7a mutants are novel, striking and thoroughly quantified.<br /> 2. By combining both loss of function sema7a mutants and ectopic expression of the secreted form of Sema7a the authors demonstrate the Sema7a is both necessary and sufficient to guide sensory axons

Weaknesses:<br /> 1. Control. There should be an uninjected heatshock control to ensure that heatshock itself does not cause sensory afferents to form aberrant arbors. This control would help support the hypothesis that exogenously expressed Sema7a (via a heatshock driven promoter) is sufficient to attract afferent arbors.<br /> 2. Synapse labeling. The numbers obtained for postsynaptic labeling in controls do not match up with the published literature - they are quite low. Although there are clear differences in postsynaptic counts between sema7a mutants and controls, it is worrying that the numbers are so low in controls. In addition, the authors do not stain for complete synapses (pre- and post-synapses together). This staining is critical to understand how Sema7a impacts synapse formation.<br /> 3. Hair cell counts. The authors need to provide quantification of hair cell counts per neuromast in mutant and control animals. If the counts are different, certain quantification may need to be normalized.<br /> 4. Developmental delay. It is possible that loss of Sema7a simply delays development. The latest stage examined was 4 dpf, an age that is not quite mature in control animals. The authors could look at a later age, such as 6 dpf to see if the phenotypes persist or recover.

Reviewer #4 (Public Review):

This manuscript describes a complex, highly ambitious set of modeling and experimental studies that appear designed to compare the structural and functional properties of beta cell subpopulations within the islet network in terms of their influence on network synchronization. The authors conclude that the most functionally coupled cell subpopulations in the islet network are not those that are most structurally coupled via gap junctions but those that are most metabolically active.

Strengths of the paper include (1) its use of an interdisciplinary collection of methods including computer simulations, FRAP to monitor functional coupling by gap junctions, the monitoring of Ca2+ oscillations in single beta cells embedded in the network, and the use of sophisticated approaches from probability theory. Most of these methods have been used and validated previously. Unfortunately, however, it was not clear what the underlying premise of the paper actually is, despite many stated intentions, nor what about it is new compared to previous studies, an additional weakness.

Although the authors state that they are trying to answer 3 critical questions, it was not clear how important these questions are in terms of significance for the field. For example, they state that a major controversy in the field is whether network structure or network function mediates functional synchronization of beta cells within the islet. However, this question is not much debated. As an example, while it is known that there can be long-range functional coupling in islets, no workers in the field believe there is a physical structure within islets that mediates this, unlike the case for CNS neurons that are known to have long projections onto other neurons. Beta cells within the islets are locally coupled via gap junctions, as stated repeatedly by the authors but these mediate short-range coupling. Thus, there are clearly functional correlations over long ranges but no structures, only correlated activity. This weakness raises questions about the overall significance of the work, especially as it seems to reiterate ideas presented previously.

Specific Comments

1. The authors state it is well accepted that the disruption of gap junctional coupling is a pathophysiological characteristic of diabetes, but this is not an opinion widely accepted by the field, although it has been proposed. The authors should scale back on such generalizations, or provide more compelling evidence to support such a claim.<br /> 2. The paper relies heavily on simulations performed using a version of the model of Cha et al (2011). While this is a reasonable model of fast bursting (e.g. oscillations having periods <1 min.), the Ca2+ oscillations that were recorded by the authors and shown in Fig. 2b of the manuscript are slow oscillations with periods of 5 min and not <1 min, which is a weakness of the model in the current context. Furthermore, the model outputs that are shown lack the well-known characteristics seen in real islets, such as fast-spiking occurring on prolonged plateaus, again as can be seen by comparing the simulated oscillations shown in Fig. 1d with those in Fig. 2b. It is recommended that the simulations be repeated using a more appropriate model of slow oscillations or at least using the model of Cha et al but employed to simulate in slower bursting.<br /> 3. Much of the data analyzed whether obtained via simulation or through experiment seems to produce very small differences in the actual numbers obtained, as can be seen in the bar graphs shown in Figs. 1e,g for example (obtained from simulations), or Fig. 2j (obtained from experimental measurements). The authors should comment as to why such small differences are often seen as a result of their analyses throughout the manuscript and why also in many cases the observed variance is high. Related to the data shown, very few dots are shown in Figs. 1e-g or Fig 4e and 4h even though these points were derived from simulations where 100s of runs could be carried out and many more points obtained for plotting. These are weaknesses unless specific and convincing explanations are provided.<br /> 4. The data shown in Fig. 4i,j are intended to compare long-range synchronization at different distances along a string of coupled cells but the difference between the synchronized and unsynchronized cells for gcoup and gKglyc was subtle, very much so.<br /> 5. The data shown in Fig. 5 for Cx36 knockout islets are used to assess the influence of gap junctional coupling, which is reasonable, but it would be reassuring to know that loss of this gene has no effects on the expression of other genes in the beta cell, especially genes involved with glucose metabolism.<br /> 6. In many places throughout the paper, it is difficult to ascertain whether what is being shown is new vs. what has been shown previously in other studies. The paper would thus benefit strongly from added text highlighting the novelty here and not just restating what is known, for instance, that islets can exhibit small-world network properties. This detracts from the strengths of the paper and further makes it difficult to wade through. Even the finding here that metabolic characteristics of the beta cells can infer profound and influential functional coupling is not new, as the authors proposed as much many years ago. Again, this makes it difficult to distill what is new compared to what is mainly just being confirmed here, albeit using different methods.

Reviewer #4 (Public Review):

This manuscript covers an important topic of gender biases in the authorship of scientific publications. Specifically, it investigates potential mechanisms behind these biases, using a solid approach, based on a survey of researchers.

Main strengths

The topic of the MS is very relevant given that across sciences/academia representation of genders is uneven, and identified as concerning. To change this, we need to have evidence on what mechanisms cause this pattern. Given that promotion and merit in academia are still largely based on the number of publications and impact factor, one part of the gap likely originates from differences in publication rates of women compared to men.

Women are underrepresented compared to men in journals with high impact factor. While previous work has detected this gap, as well as some potential mechanisms, the current MS provides strong evidence, based on a survey of close to 5000 authors, that this gap might be due to lower submission rates of women compared to men, rather than the rejection rates. The data analysis is appropriate to address the main research aims. The results interestingly show that there is no gender bias in rejection rates (desk rejection or overall) in three high-impact journals (Science, Nature, PNAS). However, submission rates are lower for women compared to men, indicating that gender biases might act through this pathway. The survey also showed that women are more likely to rate their work as not groundbreaking, and be advised not to submit to prestigious journals

With these results, the MS has the potential to inform actions to reduce gender bias in publishing, and actions to include other forms of measuring scientific impact and merit.

Main weakness and suggestions for improvement

1) The main message/further actions: I feel that the MS fails to sufficiently emphasise the need for a different evaluation system for researchers (and their research). While we might act to support women to submit more to high-impact journals, we could also (and several initiatives do this) consider a broader spectrum of merits (e.g. see https://coara.eu/ ). Thus, I suggest more space to discuss this route in the Discussion. Also, I would suggest changing the terms that imply that prestigious journals have a better quality of research or the highest scientific impact (line 40: journals of the highest scientific impact) with terms that actually state what we definitely know (i.e. that they have the highest impact factor). And think this could broaden the impact of the MS

2) Methods: while methods are all sound, in places it is difficult to understand what has been done or measured. For example, only quite late (as far as I can find, it's in the supplement) we learn the type of authorship considered in the MS is the corresponding authorship. This information should be clear from the very start (including the Abstract).

Second, I am unclear about the question on the perceived quality of research work. Was this quality defined for researchers, as quality can mean different things (e.g. how robust their set-up was, how important their research question was)? If researchers have different definitions of what quality means, this can cause additional heterogeneity in responses. Given that the survey cannot be repeated now, maybe this can be discussed as a limitation.

I was surprised to see that discipline was considered as a moderator for some of the analyses but not for the main analysis on the acceptance and rejection rates.

I was also suppressed not to see publication charges as one of the reasons asked for not submitting to selected journals. Low and middle-income countries often have more women in science but are also less likely to support high publication charges.

Finally, academic rank was asked of respondents but was not taken as a moderator.

Reviewer #3 (Public Review):

In this manuscript, the authors challenge the fundamental concept that all neurons are derived from ectoderm. Specifically, they aim to show that while the early ENS arises embryologically from neural crest (NENs), with age it is slowly replaced by mesoderm-derived neurons (MENs). This claim is based on an array of transgenic reporter mice, immunofluorescence, and transcriptomics. They further propose that the transition from NENs to MENs is regulated by a changing balance in GDNF-RET versus HGF-MET signaling, respectively.

This is a provocative and potentially paradigm-changing proposal, but the data presented and the interpretation of that data fall short of establishing it.

1) MENs share more common characteristics with fibroblasts. The authors interpret this as representing neurons with fibroblast characteristics. Why not fibroblasts with neuronal characteristics? The ability to express neurotransmitter receptors and calcium channels is common in fibroblasts, but that isn't sufficient to characterize a neuron. For example, many cell types express neurotransmitters (CGRP in ILCs, Penk in fibroblasts). Expressing one of the Hu proteins (Elavl2) probably isn't enough to call these "neurons," especially when neurons usually express Elavl3-4 (HuC/D). Including calcium imaging and showing presence of action potentials would strengthen the argument that these are in fact neurons.

2) The scRNA-seq is unconvincing. There are several technical issues and the analysis omits important information required to make an unbiased assessment.

a. One issue in the interpretation is that MENs are shown by IHC to constitute half the neuronal population, with NENs making up the other half. The authors state that they performed an unbiased approach, sequencing all cells in the muscularis. If it were truly unbiased, then why do they detect a 28-fold increase in MENs in the single cell data? This does not reflect the IHC findings and points to an issue in technique that needs to be addressed.

b. Cell populations annotated by the author are confusing. The "unknown" population expresses many genes that are epithelial markers. This is puzzling because the authors state that they only sequenced the muscularis. This leads to questions regarding the initial samples and whether they were dissected appropriately or contaminated by another population.

c. The authors report a population of ICCs at P21 which is not identified at 6-months. Closer inspection of their data shows bona fide ICC markers, Ano1 and Kit, in their SMC cluster at 6-months, with failure to identify ICC clusters, raising questions about whether they have identified a new cell type.

d. While the authors critically examine other scRNA-seq datasets and claim that those groups mislabeled their populations, the above does not instill confidence in their ability to counter the unified literature.

3) MENs are identified based on genes that could be related to neurons, including calcium channels, neurotransmitter receptors, etc. It is worth noting that mesenchymal cells, ICCs, and smooth muscle also possess these characteristics. Therefore, it hard to justify why these MENs are considered "neurons." The authors should perform an analysis to examine homology between clusters in order to show which clusters the MENs are more similar to, neurons or otherwise.

4) Several issues raise questions about the quality of the scRNA-seq data, making interpretations very difficult:

a. MENs are identified to have higher UMI counts than other cells, which the authors interpret as the cells being bigger than others. If this is the case, why is this only observed in the P21 dataset and not at 6 months. Notably, high UMIs are also a sign of doublet contamination.

b. Authors include data from RBCs. As they do not have a nucleus, RNA abundance is low as expected. However, markers for RBCs include smooth muscle specific markers, MYH11 (an MEN marker) and Acta2. The presence of these markers can indicate high levels of "ambient RNA" which enters droplets from other cells lysed during digestion. Interestingly, MENs appear to cluster close to RBCs.

c. In light of the above possible evidence of doublet contamination and high levels of ambient RNA, the markers of MENs need to be reconsidered. MENs are stated to express markers that were previously (up until this manuscript) accepted markers of intestinal mesothelium (Ukp3b Krt19, WT1), smooth muscle cells (Myh11), and fibroblasts (Dcn, C3, Col6a1), raising the possibility that MENs are an erroneous cluster containing RNA from all these cell types.

5) The MEN population appears to be the largest cell population in the gut, which is unprecedented. The authors compare their scRNA-seq data to several other studies that have not made similar observations. Such analysis of other datasets is used to inform on the new data being generated. In the current manuscript, however, this takes the reverse approach and the authors analyze other data based on the assumption that they all mislabeled the MEN population.

a. In their assessment of Drokhlyansky et al., the authors claim that their mesothelium annotation is wrong despite expressing known mesothelial markers. This includes the gene Upk3b which is a bona fide mesothelial marker in the gut but is also expressed by "MENs." They proceed to analyze the Elmentaite et al. dataset and state that their "transitional fibroblast" population are actually MENs. That paper also has a population of Upk3b+ mesothelial cells and it is unclear why those are not actually MENs like in the Drokhlyansky et al. study.

b. The authors often refer to the study of May-Zhang et al. and their cluster annotated as "mesenchymal neurons" in the gut. It should be known that the original authors never made this claim. Rather, they acknowledge that the clusters in their study with poor correlation to neuronal profiles exhibit strong predictions for mesenchymal and vascular/immune cell types. They state: "We considered the possibility that these clusters might be non-neuronal." If these are "mesenchymal neurons" then the same logic would indicate that there are vascular neurons and immune cell neurons, and therefore this does not make a very compelling case.

6) A weakness of this study is that a lot of the data relies on reporter gene expression. The authors need to acknowledge several weaknesses of this approach. First, Wnt1-tdT recombination may be incomplete or one can have "Cre mosaicism" and therefore the lack of tdT is not sufficient evidence to say that those neurons are not neural crest-derived. Second, one can have off-target or leaky Cre expression, leading to low-level tdT expression, as seen in many of the images in this study. Third, Cre can exhibit toxicity and this may be more problematic in older mice given the long-term continuous expression of Cre (He et al, Am J Pathology, 2014;184:1660; Loonstra et al, PNAS, 2001;98:9209; Forni et al, J Neurosci, 2006;26:9593; Rehmani et al, Molecules, 2019;24:1189; Gillet et al, Sci Rep, 2019;9:19422; Stifter and Greter, Eur J Immunol, 2020;50:338).

Reviewer #4 (Public Review):

This is potentially a landmark study with far-reaching consequences for archaeology, palaeoanthropology, and more widely. The antiquity of intentional human mark marking is a hot topic but this study – understood as initial – has as yet incomplete sources of evidence and methods; and it will be interesting to follow how the study develops in subsequent studies.

Strengths and points to build on:

* Heuristic potential: As knowledge advances it poses a risk to accepted knowledge – and we should accept that one such risk is moving on from long-held disciplinary tenets. In this case, there has been a growing quantum of evidence – all hotly debated – for the deep antiquity of mark-making and even symbolism by species other than ourselves. Most researchers now accept Neanderthal symbolic capacity actualised in burials, intentional mark-making and the like. The evidence here presented is not unequivocal but is very suggestive and an ideal test case for applying multi-disciplinary techniques of analysis and interpretation beyond the expertise of the listed authors *see comments in 'weaknesses'). This work by itself may be equivocal but when taken together with other such work, points to a 'human' sensu lato past that is as complex as it is long. This work then helps all researchers to at least be alive to the possibility of things like anthropic marks and residues in a context not normally thought to have it.

* Decentering speciesism: As per the above comment, I appreciate empirical studies that erode speciesism – in particular studies that open up our minds to the possibility that multiple members of the Genus Homo were capable of intentional mark-making and even 'symbolic' behaviour, though this latter term is not well understood or uniformly used. This is probably because of continuous unconscious bias on our part as currently the only exemplar of our genus living - in contrast to most of the past in which different species and genera co-existed - if not on the same landscape and/or at exactly the same time, then with enough overlap that people would have realised 'others' were about either by sight and/or by encountering their physical remains and artefacts.

* Problematising 'firsts' and deep time: A strength – but which needs to be developed in this manuscript – is our understanding of time and change. We have a plethora of dating techniques but relatively few substantive monographs, articles, and think tanks on time – and especially on how change comes about and what causes it. This leads us to privilege 'firsts' and the 'oldest' finds in 'deep' time above those that are more recent and in 'shallow' time. I would suggest in addition to the claims for the oldest of the reported marks, the authors develop nascent remarks on the possibility the suite of marks may have been made over time. This will help counter criticism that these marks – if established to be anthropic – were not just a singularity, but part of patterned behaviour, which would move it towards the realm of 'symbolic' cognitive behaviour. And indeed, it would be good to hear more about why in this place, these marks were made to establish a replicable model for identifying early anthropic marks.

Ultimately, this manuscript presents evidence that those who are pro the deep antiquity of intentional mark-making by Homo (and possibly even other genera) will find enough evidence to support; while those sceptical of such claims will find enough methodological flaws and evidential limits to refute those claims. The next decade of work will likely be definitive and this article makes a key contribution to the debate.

Weaknesses and points to attend to:

* Definitions: The term 'rock engraving' is used rather uncritically and also the term 'etching' – and it would be useful to have a short definition of how the authors understand the term. Rock art scholars regularly debate these terms and whether they are or are not 'rock art' with its overwhelmingly visual bias; which this discovery may usefully help overthrow and advance.

* Dating: There is no evidence provided for dating the marks found in the cave system. They could, for example, have been made more recently than the dates claimed – and by another species (if we accept their anthropogenic authorship). This is a perennial problem of much rock art research – especially when it comes to understanding the wider archaeological/palaeoanthropological context. More crucially, accurate dating allows a more reliable understanding of authorship and who/what was responsible for a particular artefact or feature. This has not been demonstrated in this case, though we do have fossil evidence of Homo naledi in the cave system. The article title is this incorrect / and unsupported claim as the marks, if they are anthropic, have not been dated and are of unknown age. The authors allow that there may have been multiple episodes, but not that the marks can belong to a time other than they posit – either earlier, later, or distributed over a long period as the authors allow for in their concluding remarks.

* Authorship: The study does not utilise either a geoscientist as one of the authorial team, or a rock art specialist. These are key oversights as the former would help better contextualise the dating of the marks reported on, as well as explore alternative non-anthropogenic agents that may have created the marks reported on. For example, the marks and 'pitting' etc may be the result of water bringing abrasive agents during times of flooding, hitting prominent rock features in the cave system. Some explanation is given from lines 114-124, but are uncited. The overlying 'sediment' may be similar to the mondmilch found in cave systems and which is of natural origin. It may be that these non-anthropogenic causes are easy to discount; but the arguments do need to be made. Or, that the polishing was made by Homo naledi brushing against the surfaces as they moved in the cave system, independent of any mark-making. A Table showing the pros and cons of intentional anthropic versus natural authorship would be very effective - as well as showing some of the natural linear marks in the cave system to avoid any confirmation or similar bias. FTIR analysis of the panel A-C would be more than useful to determine whether an additional layer of material has been added. This is mentioned for future work, but this seems a rather post-hoc research programme.

* Use-wear analysis: If the marks are anthropic in origin; they are likely to have been made by a stone tool, which would leave characteristic marks, directionality and sequencing, distinct from natural causes. It is vital this work – such as was done on the Blombos engraved ochre – is done here – for example, linking to the chert and other tools described on lines 152-158. Note Figure 19, of such a tool, is very hard to make out. The Blombos – and Klasies River Mouth engraved ochres (curiously not referenced) – have very similar geometric markings and there is a real opportunity to compare these in securely dated contexts of 70-120 kya –which could support the argument made here for Homo naledi's cognitive capacity. On figure 16 it would be good to know on what basis some marks were selected as anthropic – and why others were not; this would help demonstrate the methodology and ability to distinguish between the two kinds of marks.

* Viewshed: The rock art specialist would have added essential expertise on how to study anthropic marks. For example, the images of the marks shown are all of individual or small collections of motifs rather than showing each panel as well as all panels together, to help understand the iconographic context as an ensemble – a 'feature' rather than isolated 'artefacts' or 'motifs'. Line 60 mentions being able to see these as a 'triptych' but the reader is not able to have this view in this manuscript. From the cave map, it is not clear whether all three 'panels' (an unfortunate art historical term that suggests a framed entity - better to use a term like 'cluster') can be viewed simultaneously or in sequence. The view shed in relation to the area where the bodies were recovered is vaguely stated as 'only a few metres away' and is worth developing. I understand 3D scans have been made so it would be useful to have a version showing the marks in relation to where the bodies were recovered and as a 3-cluster ensemble.

* Image enhancements: Also, in addition to polarised images, have colour enhancement tools like DStretch been tried to see if, for example, attempts at colouring with different coloured sands were made? Similarly, a 3D scan of the motif and panel – (Metashape is mentioned but not shown) – might assist in understanding how the marks and the rock they are on might relate to each other- as research in European upper Palaeolithic contexts has shown. Here, experimenting with different kinds of lighting - or in the absence of lighting, of tactility and how these marks and their rock support may have been experienced by those who may have made and interacted with them? As a note, it would be useful to have a scale in each image of the 'engravings' and it is a pity the one in situ photograph with the scale is not a standard rock art colour-corrected scale as is commonly used in rock art research.

Reviewer #4 (Public Review):

Berger et al. 2023a argues that Homo naledi intentionally buried their dead within the Rising Star cave system by digging pits and covering the bodies with infilled sediment. The authors identified two burials: Dinaledi Feature 1 from the Dinaledi Chamber, and the Hill Antechamber Feature from the Hill Antechamber. The evolutionary and behavioral implications for such behavior are highly significant and would be the first instance of a relatively small-brained hominin engaging is complex behavior that is often found in association with Homo sapiens and Homo neanderthalensis. Thus, the scientific rigor to validate these findings should be of the highest quality, and thus, provide clear documentation of intentional burial. In an attempt to meet these standards, the authors stated a series of tests that would support their hypothesis of intentional burials in the Rising Star Cave system:

"The key observations are (1) the difference in sediment composition within the feature compared to surrounding sediment; (2) the disruption of stratigraphy; (3) the anatomical coherence of the skeletal remains; (4) the matrix-supported position of some skeletal elements; and (5) the compatibility of non-articulated material with decomposition and subsequent collapse." (page 5)

To find support for the first (1) test, the authors collected sediment samples from various locations within the Rising Star Cave system, including sediment from within and outside Dinaledi Feature 1. However:

• The authors did not select sediment samples from within the Hill Antechamber Feature, so this test was only used to assess Dinaledi Feature 1.

• The sediment samples were analyzed using x-ray diffraction (XRD) and x-ray fluorescence (XRF) to test the mineralogy and chemistry of the samples from within and outside the feature. The XRF results were presented as weighted percentages (not intensities) with no control source reported. The weighted percentages were analyzed using a principal components analysis (PCA) while the particle-size distribution was analyzed using GRADISTAT statistics package and the Folk and Ward Method to summarize "mean grain size, sorting, skewness and kurtosis in addition to the percentages of clay, silt and sand in each sample." (page 28).

• The PCA results were reported solely as a biplot without showing the PC scores projected into the loading space, which is unusual and does not present the data accurately. Instead, the authors present the scores of a single component (PC2, figure 3) because the authors interpreted this component as "distinctly delineates fossil-bearing sediments from sterile sediments based on the positive loadings of P and S" (Page 6). However, the supplementary table that reports XRF bulk chemistry results as a weighted percentage of minerals within each sample (SI Table 1) shows mostly an absence of data for both Na and S. Since Na is at the lower end of detection limits for the method, and S seems to just be absent from the list, the intentions of the authors for showing the inclusion of these elements in their PCA results is unclear. Given that this is the author's primary method for demonstrating a burial, this issue is particularly concerning and requires additional attention.

• Regardless of the missing data, this reviewer attempted to replicate the XRF PCA results using the data provided in SI Table 1 and was unsuccessful. The samples that were collected from within the feature (SB) cluster with samples collected from sterile sediments and other locations around the cave system. Thus, these results are not replicable as currently reported.

• Visual comparisons of sediment grain size, shape, and composition were qualitatively summarized. Grain size was plotted as a line graph and is buried as supplemental Figure S13 showing sample by color and area, but these results do not distinguish samples from WITHIN the burial compared to OUTSIDE the burial as the authors state in the methods as a primary goal.

To test the second (2) aim, the "stratigraphy" was primarily described in text.

• For Dinaledi Feature 1, the authors state that the layer around Feature 1 "is continuous in the profile immediately to the east of the feature; it is disrupted in the sediment profile at the southern extent of the feature (fig. 3b)." Upon examination of figure 3b, the image shows an incredibly small depiction of the south (?) profile view with an extremely large black box overlaying a large portion of the photograph containing a small 5 cm scale. Visually, there is no difference in the profile that would suggest a disruption in the form of a pit. The LORM (orange-red mud layer) does seem to become fragmentary, but no micromorphological analysis was conducted on this section to provide an evaluation of stratigraphic composition. Also, by only excavating a portion of the feature, the authors were unable to adequately demonstrate the full extent of this feature.

• The authors attempt to describe "a bowl-shaped concave layer of clasts and sediment-free voids make up the bottom of the feature" (page 13) and refer to figures and supplementary information that do not depict any stratigraphic profile. Moreover, the authors state that "the leg, foot, and adjacent [skeletal?] material cut across stratigraphy" indicating that the skeleton is orientated on a flat plane against the surrounding stratigraphy that is "30{degree sign} slope of floor and underlying strata" (page 51, fig. 10c captions). There is no mention of infilled sediment from a pit and how this relates to the skeleton or the slope of the floor. It is therefore extremely unclear what the authors are meaning to describe without any visual or micromorphological supplementation to demonstrate a "bowl-shaped concave layer".

The third (3) test was to evaluate the anatomical coherence of the skeletal remains using macro- and micro-CT (computed tomography) of the Hill Antechamber Feature that was removed during excavation. To visually assess the anatomy of the Dinaledi Feature 1 burial, the authors describe the spatial relationship of skeletal elements as they were being excavated but halted partway through the excavation.

• The authors do not provide any documentation (piece-plotting, 3D rendering of stages of excavation, etc.) of the elements that were removed from the Dinaledi Feature. Figure 4 and SI Fig. S22 show the spatial relationship between identifiable skeletal elements that remain in the Feature. However, in Fig. 4, it is unclear why the authors chose to plot 2023-2014 excavated material along with material reported here, and it's even more difficult to understand the anatomical positioning of the elements given their color and point size choices. Although, the authors do provide a 3D rendering of the unexcavated remains showing some skeletal cohesion, apart from the mandible and teeth being re-located near the pelvis (Fig. 9). That said, it is very difficult to visually confirm the elements from this model or understand the original placement of the skeleton.

• 3D renderings of the Hill Antechamber feature skeletal material is clearly shown in SI Fig. S26. Contrary to what the authors state in text, there is a rather wide dispersal and rearrangement of elements for a "burial" that is theoretically protected from scavengers and other agents that would aid in dispersing bone from the surface. The authors do not offer any alternatives to explain disturbance, such as human activity, which clearly took place.

• Moreover, there does not appear to be any intentional arrangement of limbs that may suggest symbolic orientation of the dead (another line of evidence often used to support intentional burial but omitted by the authors). Thus, skeletal cohesion is not enough evidence to support the hypothesis of an intentional burial.

The fourth (4) test was attempted by evaluating whether some elements were vertically aligned from 3D reconstructed models of Hill Antechamber Feature and a photogrammetric model of the Dinaledi Feature 1. The authors state that "the spatial arrangement of the skeletal remains is consistent with primary burial of the fleshed body" (page 8 in reference to Dinaledi Feature 1) without providing any evidence, qualitative or quantitative, that this is the case for either burial.

Since this reviewer was unable to understand the fifth (5) test as it was written by the authors, I am unable to comment on the evidence to support this test and will default to the other reviewers for evaluation of this claim.

In addition to a lack of evidence to support the claims of intentional burial, this paper was also written extremely poorly. For example, the authors often overused 'persuasive communication devices' (see eLife article, https://elifesciences.org/articles/88654) to mislead readers:

"During this excavation, we recognized that the developing evidence was suggestive of a burial, due to the spatial configuration of the feature and the evidence that the excavated material seemed to come from a single body." (page 5)

As an opening statement to introduce Dinaledi Feature 1, the authors state the interpretation and working hypothesis as fact before the authors present any evidence. This is known as "HARKing" and "gives the impression that a hypothesis was formulated before data were collected" (Corneille et al. 2023). This type of writing is pervasive throughout the manuscript and requires extensive editing. I recommend that the authors review the article provided by eLife (https://elifesciences.org/articles/88654) and carefully review the manuscript. Moreover, as this text demonstrates, the authors’ word choice is indicative of storytelling for a popular news article instead of a scientific paper. I highly suggest that the authors review the manuscript carefully and present the data prior to giving conclusions in a clear and concise manner.

Moreover, the writing structure is inconsistent. Information that should be included in results is included in the methods, text in the results should be in discussions, and so forth. This inconsistency is pervasive throughout the entire manuscript, making it incredibly difficult to adequately understand what the authors had done and how the results were interpreted.

Finally, the "artifact" that was described and visualized using CT models is just that - a digitally colored model. The object in question has not been analyzed. Until this object is removed from the dirt and physically analyzed, this information needs to be removed from the manuscript as there is nothing to report before the object is physically examined.

Overall, there is not enough evidence to support the claim that Homo naledi intentionally buried their dead inside the Rising Star Cave system. Unfortunately, the manuscript in its current condition is deemed incomplete and inadequate, and should not be viewed as finalized scholarship.

Reviewer #4 (Public Review):

This work aims at analyzing the impact of histone variants and histone modifications on chromatin states of the Arabidopsis genome. Authors claim that histone variants are as significant as histone modifications in determining chromatin states. They also study the effect of mutations in the DDM1 gene on the exchange of H2A.Z to H2A.W, which convert the silent state of transposons into a chromatin state normally found on protein coding genes.

This is an interesting and well done study on the organization of the Arabidopsis genome in different chromatin states, adding to the previous reports on this issue.

Reviewer #4 (Public Review):

A combination of optogenetic behavioral experiments and functional imaging are employed to identify the role of mechanosensory neurons in food swallowing in adult Drosophila. While some of the findings are intriguing and the overall goal of mapping a sensory to motor circuit for this rhythmic movement are admirable, the data presented could be improved.

The circuit proposed (and supported by GRASP contact data) shows these multi-dendritic neurons connecting to pharyngeal motor neurons. This is pretty direct - there is no evidence that they affect the hypothetical central pattern generator - just the execution of its rhythm. The optogenetic activation and inhibition experiments are constitutive, not patterned light, and they seem to disrupt the timing of pumping, not impose a new one. A slight slowing of the rhythm is not consistent with the proposed function.

The mechanosensory channel mutants nompC, piezo, and TMC have a range of defects. The role of these channels in swallowing may not be sufficiently specific to support the interpretation presented. Their other defects are not described here and their overall locomotor function is not measured. If the flies have trouble consuming sufficient food throughout their development, how healthy are they at the time of assay? The level of starvation or water deprivation can affect different properties of feeding - meal size and frequency. There is no description of how starvation state was standardized or measured in these experiments.

The brain is likely to move considerably during swallow, so the GCaMP signal change may be a motion artifact. Sometimes this can be calculated by comparing GCaMP signal to that of a co-expressed fluorescent protein, but there is no mention that this is done here. Therefore, the GAaMP data cannot be interpreted.

Reviewer #4 (Public Review):

The authors have collected an impressive array of physiological data and provided some beautiful 3D images of SBCs with dendrites. These are clearly strengths. The computational models for mechanisms of SBC responses, however, are made to fit what may be inadequate anatomical data. Instead of conclusions, perhaps they need to reword their discussions to refer to the anatomy as hypothetical substrates.

Reviewer #4 (Public Review):

In this manuscript, Clary and colleagues use two-photon imaging to visualize the dynamics of Merkel cells and their innervating sensory axons using a combination of transgenic lines, where these parts of the mechanosensory organs of the skin are labelled with distinct fluorescent proteins. It is noteworthy that this study does not stand alone, but should be compared to prior published work cited by the authors, such as Wright et al., Developmental Biology 422 (2017) 4-13.

The study demonstrates a comparably high degree of remodelling, with a large fraction of Merkel cells (50% in three weeks) and a similar fraction of elaborated (cup-like) axons endings disappearing. It appears by timing and correlation that changes in Merkel cells can clearly drive axonal remodelling, while axons can still remodel even if the Merkel cells remain stable by the parameters measured here. Moreover, changes in Merkel cells partially relate to the hair growth cycle.

The imaging approach chosen is straightforward and clearly suited in principle to reveal the dynamism of the studied cellular structures. To co-visualize two synaptic partners in a vertebrate sensory organ in vivo - while not unprecedented - certainly remains quite challenging, and represents a strength of the paper. Similarly, understanding how stable structures in the nervous system are under homeostatic (rather than developmental) conditions, remains an understudied topic. I also found some of the correlative analysis in the later parts of the study quite interesting, albeit not always straightforward to interpret.

My central concern is the very high disappearance rate of Merkel cells. This, in my view is not compatible with a steady state situation in an adult animal - and not with the prior literature (especially the similar study by Wright et al. cited above). Obviously, if this rate were to continue, Merkel cells would all be lost in early adulthood in mice. Whether this is the case in the specific anatomical location was not examined in the study - but it would also imply that the study really addresses a dynamic developmental remodelling situation and should be written up accordingly. I am more suspicious of the depilation agent (plus the shaving). As Wright et al. already show that shaving causes some changes in Merkel cell dynamics (but, as far as I can tell, did not chemically depilate), I would not be surprised that we see an artificially high remodelling rate. Such skin treatment-related biology is probably less relevant in the context of neurobiology (albeit probably quite interesting to other audiences). So, my recommendation to the authors would be to invest some energy to find out, what causes the swift Merkel cell loss.

Another technical point that warrants discussion is the axonal labelling - first, I do not find the innervation patterns always easy to discern in the images provided, so I am not always sure how reliable this part is. Any artefact here creates the impression of dynamics, as during in vivo imaging stability is more reassuring than change. There are many ways not to see or recognize something, while there are few options to explain by an artefact why something did not change. Additionally, it might be good to explicitly mention that the TrkC mice are knock-in/knock-out (this is how I understood the JAX entry) - so the observations were made under reduced TrkC expression. It would help to explain, why this cannot affect axonal dynamics or Merkel cell-axon interactions.

Overall, while I feel that the authors performed an interesting in vivo imaging study, I think technical aspects make it difficult to conclude with confidence, whether we are watching a normal and physiological process here or dynamics that are induced by specific interventions. While these interventions might represent conditions that can occur also outside the laboratory, it would be important to clarify how the reader should contextualize this study.

Reviewer #4 (Public Review):

This work aims at analyzing the impact of histone variants and histone modifications on chromatin states of the Arabidopsis genome. Authors claim that histone variants are as significant as histone modifications in determining chromatin states. They also study the effect of mutations in the DDM1 gene on the exchange of H2A.Z to H2A.W, which convert the silent state of transposons into a chromatin state normally found on protein coding genes.

This is an interesting and well done study on the organization of the Arabidopsis genome in different chromatin states, adding to the previous reports on this issue.

Reviewer #4 (Public Review):

This is an admirable piece of work. The authors build on a previous dataset they assembled, but expand it to include all stages of early development in the nematode Caenorhabditis elegans. Cell collection was done manually, which is very impressive, and is clearly far better than pooled unidentified cells. I will not comment on the specific sequencing and analysis, since this is not my expertise, but will comment on the general conclusions and comparative framework in which the authors place their results.

While the Introduction and Discussion sections are actually fairly short, much of the presentation of the results is based on a certain comparative framework, which is explicitly a comparison between C. elegans and Drosophila melanogaster. This is an important perspective, but I feel the authors' interpretation is in some places exaggerated and in other places almost trivial.

Drosophila and C. elegans are two of the main models for developmental biology. However, it has been clear for over two decades that both species are highly derived and specialized and therefore, treating them as representative for their taxa is problematic. Much of the authors' discussion hinges on the question of comparing syncytial and lineage-dependent development. The syncytial early development of Drosophila is very specific and is clearly a recent innovation within a restricted group of flies. The canonical Drosophila segmentation cascade is mostly a novelty and most elements within the cascade are recent (the authors are invited to browse my 2020 review in Curr. Top. Dev. Biol.) Specifically, the expression of gap genes in regional stripes is not found very broadly. Conversely, the polarizing role of Caudal is very ancient and is probably found in all Bilateria. When making comparisons with a distantly related species, it is important to keep this in mind. Not as much is known about development of other nematodes, but the little that is known indicates that C. elegans is also unusual, and specifically, the eutelic development (conserved cell lineages in development) is not found in all nematodes.

The authors suggest that regional expression of transcription factors in stripes is a conserved characteristic of development. This is true for Hox genes and has been known for decades. The regional expression they show for other genes is not convincing as "stripes". It is no surprise that developmental transcription factors are regionalized, but linking this to the stripes of Drosophila gap genes and even more so to Drosophila pair-rule and segment-polarity genes is a bit far-fetched. Yes, many genes are expressed in restricted domains along the A-P axis, but that is all that can be said based on the data. Calling them "Drosophila-like" is unfounded.

Beyond these broad homology statements, the rest of the presentation is fine and I have no major comments.

Reviewer #4 (Public Review):

In this manuscript, Tornini and colleagues identify two previously un-characterized micropeptides encoded by linc-mipep and linc-wrb as important modulators of day-time activity in zebrafish larvae. The authors demonstrate that each single mutant shows an increase in day-time activity and that double mutants show a more pronounced effect. Of interest, ubiquitous overexpression of the ORF encoding the linc-mipep-derived peptide can rescue the day-time over-activity phenotype of linc-mipep mutant larvae, establishing that linc-mipep acts indeed as a protein and not at the level of RNA. Using a series of experimental approaches, including ATAC-Seq from double mutant brains and scRNA-Seq and scATAC-seq analyses from linc-mipep mutants as well as linc-mipep and linc-wrb CHIP analyses, the authors furthermore identify differences in chromatin accessibility and gene expression in specific cell types of the larval brain in the absence of linc-mipep (and in case of globale ATAC-Seq, in the absence of both peptides). They conclude that the micropeptides regulate behavior and neuronal states by modulating chromatin accessibility, revealing functional similarities to their known vertebrate homolog HMGN1.

Overall, the key finding of this paper, namely the identification of two functional microproteins that had previously been misannotated as lincRNAs but have homology to HMGN1 both based on their sequence and function is an exciting discovery since relatively few newly predicted micropeptides have been functionally characterized to date, and because it advances our understanding of the molecular mechanisms underlying vertebrate-specific neuronal function and diversity. The F0 screen leading to the identification of 2 functional micropeptides provides a major advance to the field since so far screens in the F0 generation have not been typically done (rather germline-transmission). Thus, this work provides a major step forward in this regard. In addition, it includes a series of scRNA- and scATAC analyses that are technologically at the forefront and not easy to conduct and analyse.

The weakest part of the paper in its current form is on the one hand missing the link between the behavioral phenotype in mutants and the molecular phenotypes in the larval brain. It remains unclear how one can reconcile the broad neuronal expression (in the case of linc-mipep preferentially in Purkinje cells) and linc-wrb with the cell-specific effects. Moreover, it is not clear whether both peptides act redundantly or in parallel but distinct pathways since the rescue is only shown for the single linc-mipep mutant by linc-mipep overexpression (and no rescue is shown for linc-wrb or the double mutant). While the authors suggest throughout the manuscript that both peptides have similar functions (act redundantly), no clear data is provided for this, and the use of either single linc-mipep mutants (all single-cell analyses in the last Figure) or double linc-mipep/linc-wrb mutants (global brain ATAC-Seq analyses) for different brain analyses makes the molecular analyses inconsistent and not easy to interpret. While the overall finding(s) of the paper is really interesting, to make this paper really solid, additional controls and analyses will be needed.

Reviewer #4 (Public Review):

In the manuscript the author tried to find the cellular level mechanism that causes sudden cardiac death in elite athletes. They found that there are more ventricular fibrosis, ventricular extrasystole burden, longer action potential duration, higher ventricular fibrillation (VF) inducibility, higher HCN4 expression and decreased Ito in sustained trained dog model.

The author successfully conducted large animal training model, showed bradycardia and ventricular fibrosis as a finding similar in athletes and demonstrated the increased ventricular arrhythmia susceptibility to electrical stimulation. The finding of increased action potential duration can be postulated to be a factor of sudden cardiac death in these athletes. However, the interpretation of these findings should be cautious just like all the animal studies. Human has a more complex interaction with the environment and individual variabilities. Will the higher susceptibility of VF to electrical stimulation be the same in athletes is still hard to answer.

Still, it is the first study to provide a large animal model of sustained training mimicking trained athletes and to give insights into the cellular level of change in an athlete's heart. The young death of this special group is a tragedy and the importance of these studies cannot be overemphasized.

Reviewer #4 (Public Review):

Hu et al introduced the MS2-Suntag system into C. elegans to tag and image the dynamics of individual mRNAs in a live animal. The system involves CRISPR-based integration of 8x MS2 motifs into the target gene, and two transgene constructs (MCP-Suntag; scFv-sfGFP) that can potentially recruit up to 384 GFP molecule to an mRNA to amplify the fluorescent signal. The images show very high signal to background ratio, indicating a large range of optimization to control phototoxicity for live imaging and/or artifacts caused by excessive labeling. The use of epidermal wound repair as a case study provides a simplified temporal context to interpret the results, such as the initiation of transcription upon wounding. The preliminary results also reveal potentially novel biology such as localization of mRNAs and dynamic RNP complexes in wound response and repair. On the other hand, the system recruits a large protein complex to an mRNA molecule, an immediate question is to what extent it may interfere with in vivo regulation. Phenotypic assays, e.g., in development and wound repair, would have been a powerful argument but are not explored. In all, C. elegans is powerful system for live imaging, and the genome is rich in RNA binding proteins as well as miRNAs and other small RNAs for rich posttranscriptional regulation. The manuscript provides an important technical progress and valuable resource for the field to study posttranscriptional regulation in vivo.

Reviewer #4 (Public Review):

This is an extraordinary study that will serve as key resource for all researchers in the field of Drosophila testis development. The lineages that derive from the germline stem cells and somatic stem cells are described in a detail that has not been previously achieved. The RNAseq approaches have permitted the description of cell states that have not been inferred from morphological analyses, although it is the combination of RNAseq and morphological studies that makes this study exceptional. The field will now have a good understanding of interactions between specific cell states in the somatic lineage with specific states in the germ cell lineage. This resource will permit future studies on precise mechanisms of communication between these lineages during the differentiation process, and will serve as a model for studies of co-differentiation in other stem cell systems. The combination of snRNAseq and scRNAseq has conclusively shown differences in transcriptional activation and RNA storage at specific stages of germ cell differentiation and is a unique study that will inform other studies of cell differentiation.

Could the authors please describe whether genes on the Y chromosome are expressed outside of the male germline. For example, what is represented by the spots of expression within the seminal vesicle observed in Figure 3D?

I would appreciate some discussion of the "somatic factors" that are observed to be upregulated in spermatocytes (e.g. Mhc, Hml, grh, Syt1). Is there any indication of functional significance of any of these factors in spermatocytes?

In the discussion of cyst cell lineage differentiation following cluster 74 the authors state that neither the HCC or TCC lineages were enriched for eya (Figure 6V). It seems in this panel that cluster 57 shows some enrichment for eya - is this regarded as too low expression to be considered enriched?

Reviewer #4 (Public Review):

The study employs a number of methods, including TEM morphometric analysis, immunochemistry, western blotting, genomics, genetically modified models, whole heart measurements.

However, the manuscript seems to be a collection of two unfinished works: one on the transition p20-p60 in post-natal development of the heart, second about the role of ephrinB1 in the maturation of the crests of the sarcolemma. Otherwise, it is not clear why in the first figure there is no staining for ephB1, and why there is staining for claudin 5 instead.<br /> The authors are trying to defend the idea that development of the heart in rats doesn't finish on postnatal day 20 and goes on for up to day 60. However, it is not convincing.<br /> It is no surprise transcription profile is different between day 20 and day 60, I am sure as life goes on development continues into aging and any comparison of samples collected with sufficient time lapse will give transcriptional differences. Whether these differences represent a truly separate development stage is not a clear-cut story.<br /> Most of the argument is based on morphometric study of TEM images. However, the method is not described at all. There is reference to another paper by the authors, but this paper doesn't provide a concise description of the morphometry either. It is unclear how randomisation of images and fields of view has been achieved and what statistical methods has been implemented. In TEM it is often possible to find all sorts of oddities depending on how you choose the images.<br /> Why didn't the authors use microscopy of live isolated cells, which may be more relevant to study crest hight?<br /> Both claudin5 and EphrinB1 seem to be expressed highly after p5, which doesn't correlate with the proposed maturation of crests at days 20 to 60.<br /> There is no causative relationship between the lack of ephrinb1 and crest maturing, at least to my mind.

Reviewer #4 (Public Review):

This retrospective study addresses an important aspect of breast cancer treatment for prolonging survival and minimizing adverse events. Moreover, the findings could improve the treatment response of HR+ DCIS patients, which is very promising for the treatment. The article is mostly well-written and supported by encouraging data. The major strength of the study is the finding that ET after mastectomy should not be used for the treatment of HR+ DCIS patients. Nevertheless, only the Chinese population was analyzed, and that is the only limitation. This study would greatly help in the management of HR+ DCIS patients and in clinical decision-making.

Reviewer #4 (Public Review):

In this paper, Zhou et al. propose a polarization microscope for measuring the emission polarization of bifunctional rhodamine molecules attached to AdiC transporters. The polarization is used to resolve the orientation of the fluorophores, which allows the authors to successfully resolve the four conformations of AdiC at a temporal resolution of tens of milliseconds. The measured orientation for each conformation is validated with the results using crystallography.

Overall, I believe the paper is well written and demonstrates a great application for orientation imaging using polarized microscopes. Detailed experimental procedures, calibrations, and mathematical frameworks are included. I have the following recommendations to improve the manuscript.

1) On page 20, the authors note that they set a threshold to filter out molecules whose total intensity varies during the measurements. The statement that "while fluorescence intensity is expected to vary among different polarization directions, the total intensity should be essentially invariant" is not true. Since the authors use TIRF illumination to excite the molecules, the excitation polarization component along the tilting direction (e.g., along the y-axis) of the excitation is 0, i.e., molecules oriented along that direction (e.g., y-oriented) will be excited less effectively compared to other orientations.

2) Could the authors provide more details regarding how the clusters are ranked? The authors note that C1-C4 are "ranked according to the values of both angles". It is not clear to me how this is done. Also, what is the range of the measured theta_L and phi_L? And how is the warping of the spherical coordinates handled in the ranking process, e.g., a change from 350 deg to 10 deg is +20 deg or -340 deg.

3) Is the k-means clustering also based on the distance in the Cartesian space, similar to the state identification?

Reviewer #4 (Public Review):

This manuscript investigates the process of neurotransmitter release from hair cell synapses using electron microscopy of tissue rapidly frozen after optogenetic stimulation. The primary finding is that in the absence of a stimulus very few vesicles appear docked at the membrane, but upon stimulation vesicles rapidly associate with the membrane. In contrast, the number of vesicles associated with the ribbon and within 50 nm of the membrane remains unchanged. Additionally, the authors find no changes in vesicle size that might be predicted if vesicles fuse to one-another prior to fusing with the membrane. The paper claims that these findings argue for rapid replenishment and against a mechanism of multi-vesicular release, but neither argument is that convincing. Nonetheless, the work is of high quality, the results are intriguing, and will be of interest to the field.

1) The abstract states that their results "argue against synchronized multiquantal release". While I might agree that the lack of larger structures is suggestive that homotypic fusion may not be common, this is far from an argument against any mechanisms of multi-quantal release. At least one definition of synchronized multiquantal release posits that multiple vesicles are fusing at the same time through some coordinated mechanism. Given that they do not report evidence of fusion itself, I fail to see how these results inform us one way or the other.

2) The complete lack of docked vesicles in the absence of a stimulus followed by their appearance with a stimulus is a fascinating result. However, since there are no docked vesicles prior to a stimulus, it is really unclear what these docked vesicles represent - clearly not the RRP. Are these vesicles that are fusing or recently fused or are they ones preparing to fuse? It is fine that it is unknown, but it complicates their interpretation that the vesicles are "rapidly replenished". How does one replenish a pool of docked vesicles that didn't exist prior to the stimulus?

Reviewer #4 (Public Review):

This manuscript reports combining recently developed and described in the accompanying paper nanobodies against Sallimus and Projectin with DNA-Paint technology that allows super-resolution imaging. Presented data prove that such a combination provides a powerful system for imaging at a nano-scale the large and protein-dense structures such as Drosophila flight muscle. The main outcome is the observation that in flight muscle sarcomeres Salimus and Projectin overlap at the I/A band border. This was elegantly achieved using double color DNA-Paint with Sls and Projectin nanobodies.

Overall, as it stands, this manuscript even if of high technological value, remains entirely descriptive and short in providing new insights into muscle structure and architecture.<br /> The main finding, an overlap between short Sls isoform and Proj in flight muscle sarcomeres, is redundant with the author's observation (described in the companion paper "A nanobody toolbox to investigate localisation and dynamics of Drosophila titins") that in larval muscles expressing a long Sls isoform, Sls and Proj overlap as well.<br /> Alternatively, combination of Sls and Proj nanobodies with DNA-Paint represents an interesting example of technological development that could strengthen the accompanying nanobodies toolkit manuscript.

Reviewer #4 (Public Review):

This study revealed the structure of TMEM87A for the first time. Unexpectedly, the authors found that TMEM87A shared high structural similarity with WLS that mediates Wnt secretion and trafficking. Particularly, these two proteins share a similar extracellular GOLD domain and a large cavity that is accessible from both the extracellular side and the membrane. Through structural comparison, the authors have also identified a few other membrane proteins that share similar architecture with TMEM87A/WLS. These findings define a new membrane protein family that may play important roles in membrane-associated protein trafficking.

The authors also provided structural analyses and functional characterizations that suggest TMEM87A might function differently from GPCRs or ion channels. This proposal is reasonable. More experimental evidence is needed in either this study or future studies.

Overall, the findings from this study are highly interesting. This work provides a molecular framework for future elucidation of TMEM87A's functional roles and provides important and novel insights into this newly defined family of membrane proteins, and more broadly protein trafficking process.

Reviewer #4 (Public Review):

In "A Toxin-Antidote Selfish Element Increases Fitness of its Host", Long et al. attempt to address an outstanding question in the evolution of toxin-antidote (TA) systems in primarily selfing species: How do TA systems escape drift and spread in a primarily selfing species? The authors use simulations to show that at outcrossing rates similar to that observed in C. elegans a TA element, like the peel-1/zeel-1 element, has a high probability of being lost to genetic drift. However, the authors show that the peel-1 gene provides a fitness advantage to strains harboring it, providing evidence for a dual role for this gene and insights into how this element might have escaped being lost to genetic drift.

Strengths:

The experiments in this paper are well-framed. The authors use simulations to show that the observed frequency of the peel-1/zeel-1 TA element in the C. elegans population is highly unlikely given the inferred outcrossing rates of species.

The authors clearly show that the 140-370kb CB4856 introgression into N2 lowers relative fitness, number of eggs laid, and animal size, relative to N2.

The authors generated null alleles of peel-1 and zeel-1 and showed that a truncated version of PEEL-1 confers a detrimental fitness effect when compared to N2. Furthermore, the authors show that the fitness effect associated with peel-1 is independent of the antidote (zeel-1) component of this TA element.

Weaknesses:

1) The reference N2 strain has been cultivated in the lab for decades and many different versions of this strain exist. The different versions of N2, which might have slightly different genomes, are likely to have different fitness in laboratory conditions. It is unclear whether the N2 strain used to construct QX1198 is the same N2 strain used to construct CX12311, PTM229, and PTM377 (and others derived from these). The potential difference in the N2 strain used for the construction of these strains might contribute to the large discrepancy between the relative fitness shown in Figure 2A (~0.25) and Figure 3E-F (~0.07). Alternatively, the other CB4856-specific variants present in the 140-370 kb introgression in the QX1198 strain might cause this large discrepancy.<br /> Regardless of the potential discrepancy among N2 strains used as the genetic background, the claim that the presence of peel-1 confers higher relative fitness is supported by Figure 3E because PTM377/409 were presumably derived from the same N2 strain.

2) For Figures 2B and 3C, the authors report the number of eggs laid per animal. C. elegans strains can lay embryos that do not hatch and therefore fail to develop into reproductive adults. Does the difference between N2 and N2(peel-1(0)) remain when considering the number of reproductively mature progeny? Presumably, eggs laid translate to reproductive adults because a relative fitness increase is observed when peel-1 is present.

3) The authors did not perform whole-genome sequencing of the peel-1 and zeel-1 CRISPR edited strains or mention any backcrossing done to eliminate potential off-target editing events. Therefore it is difficult to conclude whether off-target effects might influence the quantified traits presented in Figure 3. This concern is somewhat alleviated by the reciprocal competition assay presented in Figure 3E (4th boxplot), but a potential off-target editing event that lowers fitness could have segregated with the silent dpy-10 and peel-1 edits.<br /> The same concern is present with the zeel-1-independence experiment, however, this experiment does not have reciprocal competition experiments.

4) In Figure 3C-D, the authors show that a homozygous truncated version of PEEL-1 confers a reduction in eggs laid per animal (proxy for brood size) and animal length (proxy for developmental speed). However, the authors do not show whether a heterozygous truncated PEEL-1 strain (N2 peel-1/peel-1(kah126)) confers the same reduction in eggs laid or animal size. Would the allele frequency dynamics derived from the simulations be affected by a fitness advantage only being conferred by the presence of two copies of peel-1?

5) The authors show a fitness advantage associated with peel-1 in laboratory conditions. It is obviously extremely difficult to extend these observations to the wild, however, the authors do not take their observations that peel-1 confers a fitness advantage in the lab and apply their empirical observations to the simulation framework. If the laboratory fitness advantage of peel-1 did extend to the wild, one might expect the element would fix in the population in the simulation framework.

6) It seems possible that a truncated version of the PEEL-1 protein might have unknown deleterious fitness consequences that are independent of any beneficial effect the full-length protein might have. The same is true for the truncated ZEEL-1 protein, though potentially less concerning because there are only 5 amino acids.

Reviewer #4 (Public Review):

The manuscript reports an experiment testing how the distribution of rewards and costs influences perceived reward value in ants. Using a bundling manipulation where rewards and costs were presented either in small separated amounts (segregated) or together in a larger amount (bundled), the results show that ants deposited a greater quantity of pheromones (which was used as an index of "liking") when rewards were segregated and costs bundled compared to when both rewards and costs were bundled (although that difference was statistically significant only in ants experiencing the segregated reward condition first during training) and when both rewards and costs were segregated. By contrast, no evidence was found for a bundling effect in terms of choice behaviour (which was used as an index of "wanting"). The authors suggest that these findings demonstrate a bundling effect and a dissociation between "wanting" and "liking" in ants.

Overall, the experiment provides a worthy contribution to the study of the biases that affect the perceived value of rewards in a translational perspective from humans to invertebrate animals. The experimental manipulation is clever, and the results clearly indicate that manipulating bundling affected pheromone deposition in ants. However, the data reported do not appear to fully support the conclusions of an increased "liking" of the segregated rewards and bundled costs compared to bundled rewards and costs. In addition, more evidence (along with stronger justifications) would be needed to establish that choice behaviour and pheromone deposition are appropriate and sensitive measures of "wanting" and "liking", respectively. This aspect renders any claim of a dissociation between "wanting" and "liking" in ants somewhat premature and speculative at this stage. I describe these concerns in more detail below.

1) The main hypothesis tested is that segregated rewards with bundled costs should be the most "liked" option relative to bundled rewards and costs and segregated rewards and costs. The results are interpreted as fully in line with this hypothesis. However, the data reported do not suggest this is the case: The difference between the 'segregated rewards' condition and the 'bundled' condition is not statistically significant when all ants are considered (that difference being statistically significant only for ants that first experienced the 'segregated rewards' condition during training). Although this point is briefly acknowledged in the discussion, more nuance and extra caution are needed in the overall interpretation of the findings, so that this statistically nonsignificant result does not appear as being treated as if it were statistically significant.

2) An important requirement to adequately evaluate the findings from the choice behaviour test is to ensure that ants did learn the associations between the reward conditions and the runway scents. Ruling out potential learning confounds is in fact essential to interpret the results as reflecting the operation of motivational processes such as "wanting". Whereas the results from the pilot experiment suggest that ants learned the contingencies between the runway length and its associated scent, the pilot experiment and the main experiment differ in significant ways. Therefore, it is unclear whether the ants learned the contingencies in the main experiment, which could be advanced as an alternative explanation for the lack of preferences between the two scented arms of the Y-maze during the choice test. Another important aspect to consider is that the reward still has to be valued by the organism to appropriately assess "wanting" processes. Indeed, "wanting" is generally conceptualised as conjointly determined by the associative history between the cue or context (scent) and the reward (sucrose solution) on one hand, and the organism's homeostatic or physiological needs such as hunger on the other hand (e.g., Zhang et al., 2009. https://doi.org/10.1371/journal.pcbi.1000437). In the main experiment, the question arises as to whether reward devaluation could have occurred-resulting in the reward having a diminished value as the ants were able to consume the sucrose solution to satiation multiple times across the experiment. For these reasons, it would be important to provide information showing that (a) the ants learned with which condition the scent was associated and (b) that the reward was still valued during the choice test. These points are key preconditions that need to be fulfilled for ruling out potential confounds that could explain the findings of the choice test as well as for suggesting a dissociation between "wanting" and "liking".

3) Relatedly, a strong justification needs to be formulated to substantiate that the choice test provides a reliable indicator of "wanting". This is critical to conclude that the results can be interpreted as reflecting a dissociation between "wanting" and "liking". In rodents and humans, "wanting" is typically measured as an increased effort mobilisation during the presentation of a cue associated with a reward (e.g., Pool et al., 2016. https://doi.org/10.1016/j.neubiorev.2016.01.006). It remains however unclear how choice can capture such effects. This questions the extent to which choice represents an adequate operationalisation and measure of "wanting" as described in the incentive salience hypothesis (Berridge & Robinson, 2016. https://doi.org/10.1037/amp0000059). Moreover, it should be clearly explained and motivated whether, and if so how, choice purely measures "wanting" without being contaminated or influenced by liking-based processes, such as preferences or expected pleasantness for instance.

4) Little information is provided on how pheromone deposition was measured and on the specificities of this measure, such as its physiological bases, timing properties, and granularity. However, detailed information about this measure is of high relevance to be able to assess if pheromone deposition represents a sensitive measure of "liking". "Liking" is typically measured as hedonic reactions during reward consumption across the rodent and human literature (e.g., Pool et al., 2016. https://doi.org/10.1016/j.neubiorev.2016.01.006). Accordingly, a good index of "liking" should be specifically responsive to reward consumption. By extension, an increased pheromone deposition should be particularly evident after the ants consumed the sucrose drop. As it stands, it is unclear whether this is the case as the pilot experiment showed no statistically significant difference in pheromone deposition between the way towards the sucrose drop or back. If the measure of pheromone deposition allows for distinguishing between pheromones deposited on the way towards the drop and pheromones deposited on the way back in the main experiment, a further test that could be run would be to compare the pheromone deposition on the way towards the drop in the 'segregated all' condition versus the 'segregated rewards' and 'bundled' conditions. A higher pheromone deposition on the way towards the sucrose drop in the 'segregated all' condition could provide converging evidence that pheromone deposition is a sensitive indicator of "liking".

Reviewer #4 (Public Review):

Overall the paper is clear and well-written. The experimental design is elegant and powerful, and it's a stimulating read. Most QTL mapping has focused on directly measurable phenotypes such as expression or drug response; I really like this paper's distinctive approach of placing bespoke functional assays for a specific molecular mechanism into the classical QTL framework.