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    1. Reviewer #2 (Public review):

      Summary:

      In this study the authors described a previously developed set of VHH-based PET tracers to track transplants (cancer cells, embryo's) in a murine immune-competent environment.

      Strengths:

      Unique set of PET tracer and mouse strain to track transplanted cells in vivo without genetic modification of the transplanted cells. This is a unique asset and a first-in-kind.

      Weaknesses:

      None

    1. Reviewer #2 (Public review):

      Basson et al. present compelling evidence supporting a gender disparity in article submission to "elite" journals. Most notably, they found that women were more likely to avoid submitting to one of these journals based on advice from a colleague/mentor. Overall, this work is an important addition to the study of gender disparities in the publishing process.

      I thank the authors for addressing my concerns.

    1. Reviewer #2 (Public review):

      Homologous recombination is essential for DNA double-strand break repair, with RAD51-catalyzed strand exchange at its core. This study presents a 2.64 Å resolution cryogenic electron microscopy structure of the RAD51 D-loop complex, achieved through reconstitution of a RAD51 mini-filament. The structure uncovers how specific RAD51 residues drive strand exchange, offering atomic-level insight into the mechanics of eukaryotic HR and DNA repair.

      Comments on revisions:

      Authors acknowledged:

      "We acknowledge that there exists an extensive body of literature that has investigated the polarity of strand exchange by RecA and RAD51 under a variety of experimental conditions, and we have added a brief comment to the text to reflect this, as well as some of the key citations. Undoubtedly, and as we also mention in our reply to the public reviews, further experimental work will be needed for a full reconciliation of the available evidence."

      In the revised manuscript, this is reflected in the statement:

      "Our mechanistic interpretation of static D-loop structures awaits full reconciliation with earlier efforts to determine strand-exchange polarity for RecA and RAD51 measured under a variety of experimental conditions."

      Among the four cited studies, my understanding (as a person who has never studied this subject of polarity) is as follows:<br /> •References 50 (EMBO J. 1997), 51 (Cell. 1995), and 52 (Nature. 2008) suggest that the strand exchange by human RAD51 occurs with a polarity opposite to that of RecA-that is, in the 5′→3′ direction relative to the complementary strand, or 3′→5′ relative to the initiating single-stranded DNA (isDNA).<br /> • In contrast, reference 49 (PNAS 1998) proposed that 5′→3′ polarity (relative to isDNA) is conserved across RecA, human RAD51, and yeast RAD51.

      Given the substantial structural analysis provided in the current manuscript, it would strengthen the work to include a concise description of these earlier biochemical findings, rather than citing them without context. This would benefit readers who are not familiar with the longstanding studies in the field and allow for a more informed interpretation of how the structural observations may reconcile or contrast with previous work.

    1. Reviewer #2 (Public review):

      In this manuscript, the authors investigated how partial loss of SynGap1 affects inhibitory neurons derived from the MGE in the auditory cortex, focusing on their synaptic inputs and excitability. While haplo-insufficiently of SynGap1 is known to lead to intellectual disabilities, the underlying mechanisms remain unclear.

      This is the third revision of the manuscript that has improved further, and the main issues were addressed. Specifically, the Authors addressed the contradiction of mEPSC and sEPSC data of the previous version by new experiments and revision of the manuscript text. While alternative explanations are still possible, the new control experiments provide necessary background for reproducibility and the manuscript text puts the observations in the right context. Furthermore, the manuscript now appropriately emphasizes that anatomical analysis was restricted to somatic excitatory synapses. Thus, the readers will be aware of the potential limitations of these measurements.

      Strengths:

      The questions are novel and relevant. Most of the issues in the experimental design are solved or answered.

      Weaknesses:

      Despite the interesting and novel questions, there are potential alternative interpretations of the observations, but these cannot be addressed within the breadth of a single paper.

    1. Reviewer #3 (Public review):

      Summary:

      Context - this is the 2nd review, of a manuscript that has already undergone some revisions.<br /> The manuscript explores ways to make self-amplifying RNA (saRNA) more silent through the inclusion of genes to inhibit the innate immune response. The readouts are predominantly expression and cell viability. They take a layered approach, adding multiple genes, as well as altering the capping of the anti-immune genes.

      Strengths:

      As described by the other reviewers, the authors take a stepwise approach to demonstrate that they can lead to sustained expression of the transgene.

      Weaknesses:

      The following weaknesses need some consideration

      (1) The data show sustained expression, but do not directly show amplification. The amount of RFP is constantly decreasing over the time course. There is some evidence for the srIκBα-Smad7-SOCS1 construct. But measuring the RNA itself would be beneficial<br /> (2) The end construct is very large - it has 12 genes, this may have manufacturing considerations, affecting the translatability.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript employs serial block‐face electron microscopy (SBEM) and cryofixation to obtain high‐resolution, three‐dimensional reconstructions of Drosophila antennal sensilla containing olfactory receptor neurons (ORNs) that detect CO2. This method has been used previously by the same lab in Gonzales et. al, 2021. (https://elifesciences.org/articles/69896), and Zhang et. al, 2019 Nature Communications. The previous study by Zhang also correlated morphometric measurements from SBEM with asymmetric ephaptic activity for paired neurons using electrophysiology across multiple olfactory sensilla. This manuscript applies the same SBEM method to now characterize the ab1 sensillum which houses the ab1C, CO2 detecting neuron, but stops short of integration neuronal activity with structural variability.

      The SBEM-based morphometric studies do however significantly advance preliminary observations from older two-dimensional TEM-based reports. Previous images of the putative CO2 neuron in Drosophila (Shanbhag et al., 1999) and in mosquitoes (McIver and Siemicki, 1975; Lu et al, 2007) reported that the dendritic architecture of the CO2 neuron was somewhat different (circular and flattened, lamellated) from other olfactory neurons in the antenna of insects. In this study, the authors confirm this different morphology but also classify it into distinct subtypes (loosely curled, fully curled, split, and mixed).

      Strengths:

      The study makes a convincing case that ab1C neurons exhibit a unique, dendritic morphology unlike the canonical cylindrical dendrites found in ab1D neurons. This observation extends previous qualitative TEM findings by not only confirming the presence of flattened lamellae in CO₂ neurons but also quantifying key morphometrics such as dendritic length, surface area, and volume, and calculating surface area-to-volume ratios. The enhanced ratios observed in the flattened segments are speculated to be linked to potential advantages in receptor distribution (e.g., Gr21a/Gr63a) and efficient signal propagation.

      Weaknesses:

      Although this quantitative approach is very robust compared to earlier reports, interpretations are somewhat limited by the absence of direct electrophysiological data to confirm whether ultrastructural differences translate into altered neuronal function. The biggest question remains unanswered: whether structural variation observed in the ab1C dendrites by SBEM have an electrophysiological functional relevance?

      Surveys of ab1 sensillum with single-sensillum recordings (even a few from multiple Drosophila antenna) as they have done for ab2s and others in the past, would have measured spontaneous activity, spike amplitude, and response to CO2. This could have allowed for comparison of frequency of functional variation, if any, to structural variation and a discussion would therefore have strengthened the overall characterization. In the case of ab2 sensilla the authors find very little variance, could the ab1 also be the same? In the absence of this data, it becomes hard to speculate whether structural variation observed in the ab1C dendrites by SBEM have any functional relevance or whether they are simply random variations in dendrite development.

      Additionally, artifacts could be a consideration, even though Cryofixation is superior to chemical fixation. Although this is hard to address, all types of fixations in TEMs cause some artifacts, as does serial sectioning. An understanding of the error rates for the SBEM method would have increased the confidence in the conclusions drawn. For example, what is the structural variation of SBEMs in the ab2 population, which shows very little electrophysiological variation? Can a comparison be done?

    1. Reviewer #2 (Public Review):

      Summary:

      Millet et al. show that C. elegans systematically prefers easy-to-eat bacteria but will switch its choice when harder-to-eat bacteria are offered at higher densities, producing indifference points that fit standard economic discounting models. Detailed kinetic analysis reveals that this bias arises from unchanged patch-entry rates but significantly elevated exit rates on effortful food, and dop-3 mutants lose the preference altogether, implicating dopamine in effort sensitivity. These findings extend effort-discounting behavior to a simple nematode, pushing the phylogenetic boundary of economic cost-benefit decision-making.

      Strengths:

      (1) Extends the well-characterized concept of effort discounting into _C. elegans_, setting a new phylogenetic boundary and opening invertebrate genetics to economic-behavior studies.

      (2) Elegant use of cephalexin-elongated bacteria to manipulate "effort" without altering nutritional or olfactory cues, yielding clear preference reversals and reproducible indifference points.

      (3) Application of standard discounting models to predict novel indifference points is both rigorous and quantitatively satisfying, reinforcing the interpretation of worm behavior in economic terms.

      (4) The three-state patch-model cleanly separates entry and exit dynamics, showing that increased leaving rates-rather than altered re-entry-drive choice biases.

      (5) Investigates the role of dopamine in this behavior to try to establish shared mechanisms with vertebrates.

      (6) Demonstration of discounting in wild strain (solid evidence).

      Weaknesses:

      (1) The kinetic model omits rich trajectory details-such as turning angles or hazard functions-that could distinguish a bona fide roaming transition from other exit behaviors.

      (2) Only _dop-3_ shows an effect, and the statistical validity of this result is questionable. It is not clear if the authors corrected for multiple comparisons, and the effect size is quite small and noisy, given the large number of worms tested. Other mutants do not show effects. Given these two concerns, the role of dopamine in c. elegans effort discounting was unconvincing.

      (3) With only five wild isolates tested (and variable data quality), it's hard to conclude that effort discounting isn't a lab-strain artifact or how broadly it varies in natural populations.

      (4) Detailed analysis of behavior beyond preference indices would strengthen the dopamine link and the claim of effort discounting in wild strains.

      (5) A few mechanistic statements (e.g., tying satiety exclusively to nutrient signals) would benefit from explicit citations or brief clarifications for non-worm specialists.

    1. Reviewer #2 (Public review):

      Summary:

      This study reexamined the idea that action potential broadening serves as a homeostatic mechanism to compensate for changes in network activity. The key finding was that, while action potential broadening does occur in certain neurons - such as CA3 pyramidal cells-it is far from a universal response. This is important because it helps resolve longstanding discrepancies in the field, thereby contributing to a better understanding of network dynamics. The replication of these findings across multiple laboratories further strengthened the study's rigor.

      Strengths:

      Mechanisms of network homeostasis are essential to understand network dynamics.

      Weaknesses:

      No weaknesses were noted by this reviewer.

    1. Reviewer #2 (Public review):

      Summary:

      This manuscript investigates age-related differences in cooperative behavior by comparing adolescents and adults in a repeated Prisoner's Dilemma Game (rPDG). The authors find that adolescents exhibit lower levels of cooperation than adults. Specifically, adolescents reciprocate partners' cooperation to a lesser degree than adults do. Through computational modeling, they show that this relatively low cooperation rate is not due to impaired expectations or mentalizing deficits, but rather a diminished intrinsic reward for reciprocity. A social reinforcement learning model with asymmetric learning rate best captured these dynamics, revealing age-related differences in how positive and negative outcomes drive behavioral updates. These findings contribute to understanding the developmental trajectory of cooperation and highlight adolescence as a period marked by heightened sensitivity to immediate rewards at the expense of long-term prosocial gains.

      Strengths:

      (1) Rigid model comparison and parameter recovery procedure.

      (2) Conceptually comprehensive model space.

      (3) Well-powered samples.

      Weaknesses:

      (1) A key conceptual distinction between learning from non-human agents (e.g., bandit machines) and human partners is that the latter are typically assumed to possess stable behavioral dispositions or moral traits. When a non-human source abruptly shifts behavior (e.g., from 80% to 20% reward), learners may simply update their expectations. In contrast, a sudden behavioral shift by a previously cooperative human partner can prompt higher-order inferences about the partner's trustworthiness or the integrity of the experimental setup (e.g., whether the partner is truly interactive or human). The authors may consider whether their modeling framework captures such higher-order social inferences. Specifically, trait-based models-such as those explored in Hackel et al. (2015, Nature Neuroscience)-suggest that learners form enduring beliefs about others' moral dispositions, which then modulate trial-by-trial learning. A learner who believes their partner is inherently cooperative may update less in response to a surprising defection, effectively showing a trait-based dampening of learning rate.

      (2) This asymmetry in belief updating has been observed in prior work (e.g., Siegel et al., 2018, Nature Human Behaviour) and could be captured using a dynamic or belief-weighted learning rate. Models incorporating such mechanisms (e.g., dynamic learning rate models as in Jian Li et al., 2011, Nature Neuroscience) could better account for flexible adjustments in response to surprising behavior, particularly in the social domain.

      (3) Second, the developmental interpretation of the observed effects would be strengthened by considering possible non-linear relationships between age and model parameters. For instance, certain cognitive or affective traits relevant to social learning-such as sensitivity to reciprocity or reward updating-may follow non-monotonic trajectories, peaking in late adolescence or early adulthood. Fitting age as a continuous variable, possibly with quadratic or spline terms, may yield more nuanced developmental insights.

      (4) Finally, the two age groups compared - adolescents (high school students) and adults (university students) - differ not only in age but also in sociocultural and economic backgrounds. High school students are likely more homogenous in regional background (e.g., Beijing locals), while university students may be drawn from a broader geographic and socioeconomic pool. Additionally, differences in financial independence, family structure (e.g., single-child status), and social network complexity may systematically affect cooperative behavior and valuation of rewards. Although these factors are difficult to control fully, the authors should more explicitly address the extent to which their findings reflect biological development versus social and contextual influences.

    1. Reviewer #2 (Public review):

      Summary:

      This work sought to explore antibody responses in the context of hemorrhagic fever with renal syndrome (HFRS) - a severe disease caused by Hantaan virus infection. Little is known about the characteristics or functional relevance of IgG Fc glycosylation in HFRS. To address this gap, the authors analyzed samples from 65 patients with HFRS spanning the acute and convalescent phases of disease via IgG Fc glycan analysis, scRNAseq, and flow cytometry. The authors observed changes in Fc glycosylation (increased fucosylation and decreased bisection) coinciding with a 4-fold or greater increase in Haantan virus-specific antibody titer. They suggest that these shifts contribute to disease recovery. The study also includes exploratory analyses linking IgG glycan profiles to glycosylation-related gene expression in distinct B cell subsets, using single-cell transcriptomics. Overall, this is an interesting study that combines serological profiling with transcriptomic data to shed light on humoral immune responses in an underexplored infectious disease. The integration of Fc glycosylation data with single-cell transcriptomic data is a strength. However, some improvements could be made in the clarity of both the Results and Materials and Methods sections, and some conclusions would benefit from greater caution, particularly in avoiding overinterpretation of correlative findings.

      Comments:

      (1) While it is great to reference prior publications in the Materials and Methods section, the current level of detail is insufficient to clearly understand the study design and experimental procedures performed. Readers should not be expected to consult multiple previous papers to grasp the core methodological aspects of the present paper. For instance, the categorization of HFRS patients into different clinical subtypes/courses, and the methods for measuring Fc glycosylation should be explicitly described in the Materials and Methods section of this manuscript.

      (2) The authors should explain the nature of their cohort in a bit more detail. While it appears that HFRS cases were identified based on IgM ELISA and/or PCR, these are indicators of the Haantan virus infection. My understanding is that not all Haantan virus infections progress to HFRS. Thus, it is unclear whether all patients in the HFRS group actually had hemorrhagic fever. This distinction is critical for interpreting how the results observed relate to disease severity.

      (3) The authors state that: "A 4-fold or greater increase in HTNV-NP-specific antibody titers usually indicates a protective humoral immune response during the acute phase", but they do not cite any references or provide any context that supports this claim. Given that in their own words, one of the most significant findings in the study is changes in glycosylation coinciding with this 4-fold increase, it is important to ground this claim in evidence. Without this, the use of a 4-fold threshold appears arbitrary and weakens the rationale for using this immune state as a proxy for protective immunity.

      (4) The authors also claim that changes in Fc glycosylation influence recovery from HFRS - a point even emphasized in the manuscript title. However, this conclusion is not well supported by the data for two main reasons. First, the authors appear to measure bulk IgG Fc glycans, not Fc glycans of Hantaan virus-specific antibodies. While reasonable, this is something that should be communicated in the manuscript. Hantaan virus-specific antibodies are likely a very small fraction of total circulating IgG antibodies (perhaps ~1%), even during acute infection. As a result, changes in bulk Fc glycosylation may (or may not) accurately reflect the glycosylation state of Hantaan virus-specific antibodies. Second, even if the bulk Fc glycan shifts do mirror those of Hantaan virus-specific antibodies, it remains unclear whether these changes causally drive recovery or are merely a consequence of the infection being resolved. Thus, while the differences in Fc glycosylation observed are interesting - and it is tempting to speculate on their functional significance - the manuscript treats the observed correlations as causal mechanistic insight without sufficient data or justification.

      (5) Fc glycosylation is known to be influenced by covariates such as age and sex. While it is helpful that the authors stratified the patients by age group and looked for significant differences in glycosylation across them, a more robust approach would be to directly control for these covariates in the statistical analysis - such as by using a linear mixed effects model, in which disease state (e.g., acute vs. convalescent), age, and sex are treated as fixed effects, and subject ID is included as a random effect to account for repeated measures. This would allow the authors to assess whether observed differences in Fc glycosylation remain significant after accounting for potential confounders. This could be important given that some of the reported differences are quite small, for example, 94.29% vs. 94.89% fucosylation.

      (6) The manuscript states that there are limited studies on antibody glycosylation in the context of HFRS, but does not cite any relevant literature. If prior work exists, it should be cited to contextualize the current study. If no prior studies have been conducted/reported, to the author's knowledge, that should be stated explicitly to show the novelty of the work.

    1. Reviewer #2 (Public review):

      The manuscript by Çevrim et al. presents a live-imaging workflow that captures the complete leg regeneration process in the crustacean Parhyale hawaiensis, at a resolution suitable for cell tracking and gene expression analysis. Building on earlier work describing selective stages of leg regeneration (Alwes et al., 2016), the authors recorded 22 confocal time-lapse movies, starting from amputation to full regeneration. They defined three distinct phases of regeneration (wound closure, cell proliferation and morphogenesis, and differentiation) based on cellular and morphological features.

      One movie was used to assess how imaging parameters (z-spacing, time intervals, and image quality) influence tracking reliability and the time required for manual proofreading, with an effort to minimize phototoxicity. Tracking was performed in the upper tissue layers using an improved version of the Mastodon plugin Elephant in Fiji. The same sample was fixed post-imaging for in situ hybridization using an HCR protocol adapted for adult legs, targeting the gene spineless. This enabled the alignment of gene expression with specific cell lineages and the identification of progenitor cells present at the time of amputation.

      In summary, the study provides a proof-of-principle for combining long-term live imaging, cell tracking, and gene expression analysis during regeneration. Given the labor-intensive nature of tracking over a 5-10 day time-lapse movie, the use of a single movie for this study is well justified. The workflow, from imaging to lineage reconstruction and molecular annotation, is successfully demonstrated and well documented with this dataset.

      Although the biological insights from the cell lineage and molecular mapping are still limited, the methodology offers significant potential in regenerative biology to uncover the cellular and molecular contributions to tissue and cell type re-formation.

      Confocal microscopy was used for live imaging, which restricted imaging to the upper 30 µm tissue layer. Light-sheet microscopy could have provided gentler imaging and enabled imaging from multiple angles to image the whole leg. While the authors acknowledge this possibility in the manuscript, they discarded it due to incompatibility between their mounting strategy and available light-sheet microscopes. As a future direction, optimizing the mounting approach for compatibility with light-sheet microscopes could enable more comprehensive tissue imaging.

    1. Reviewer #2 (Public review):

      Summary:

      This is the first study to show how a L-R bias in the relationship between numerical magnitude and space depends on brain lateralisation, and moreover, how this is modulated by in ovo conditions.

      Strengths:

      Novel methodology for investigating the innateness and neural basis of a L-R bias in the relationship between number and space.

      Weaknesses:

      I would query the way the experiment was contextualised. They ask whether culture or innate pre-wiring determines the 'left-to-right orientation of the MNL [mental number line]'.<br /> The term, 'Mental Number Line' is an inference from experimental tasks. One of the first experimental demonstrations of a preference or bias for small numbers in the left of space and larger numbers in the right of space, was more carefully described as the spatial-numerical association of response codes - the SNARC effect (Dehaene, S., Bossini, S., & Giraux, P. (1993). The mental representation of parity and numerical magnitude. Journal of Experimental Psychology: General, 122, 371-396).<br /> This has meant that the background to the study is confusing. First, they note correctly that many other creatures, including insects can show this bias, though in none of these has neural lateralisation been shown to be a cause. Second, their clever experiment shows that an experimental manipulation creates the bias. If it were innate and common to other species, the experimental manipulation shouldn't matter. There would always be a L-R bias. Third, they seem to be asserting that humans have a left-to-right (L-R) MNL. This is highly contentious, and in some studies, reading direction affects it, as the original study by Dehaene et al showed; and in others, task affects direction (e.g. Bachtold, D., Baumüller, M., & Brugger, P. (1998). Stimulus-response compatibility in representational space. Neuropsychologia, 36, 731-735, not cited). Moreover, a very careful study of adult humans, found no L-R bias (Karolis, V., Iuculano, T., & Butterworth, B. (2011), not cited). Mapping numerical magnitudes along the right lines: Differentiating between scale and bias. Journal of Experimental Psychology: General, 140(4), 693-706). Indeed, Rugani et al claim, incorrectly, that the L-R bias was first reported by Galton in 1880. There are two errors here: first, Galton was reporting what he called 'visualised numerals' and are typically referred to now as 'number forms' - spontaneous and habitual conscious visual representations - not an inference from a number line task. Second, Galton reported right-to-left, circular, and vertical visualised numerals, and no simple left-to-right examples (Galton, F. (1880). Visualised numerals. Nature, 21, 252-256.). So in fact did Bertillon, J. (1880). De la vision des nombres. La Nature, 378, 196-198, and more recently Seron, X., Pesenti, M., Noël, M.-P., Deloche, G., & Cornet, J.-A. (1992). Images of numbers, or "When 98 is upper left and 6 sky blue". Cognition, 44, 159-196, and Tang, J., Ward, J., & Butterworth, B. (2008). Number forms in the brain. Journal of Cognitive Neuroscience, 20(9), 1547-1556.

      If the authors are committed to chicks' MN Line they should test a series of numbers showing that the bias to left is greater for 2 and 3 than for 4 etc.

      What does all this mean? I think that the experiment should absolutely be published in eLife, but the paper should be shorn of its misleading contextualisation, including the term 'Mental Number Line'. The authors also speculate, usefully, on why chicks and other species might have a L-R bias. I don't think the speculations are convincing, but at least if there is an evolutionary basis for the bias, it should at least be discussed.

      In fact, I think it would make a very interesting special issue to bring up to date how and why the L-R bias exists, and where and why it does not.

      Karolis, V., Iuculano, T., & Butterworth, B. (2011). Mapping numerical magnitudes along the right lines: Differentiating between scale and bias. Journal of Experimental Psychology: General, 140(4), 693-706. doi:10.1037/a0024255

      Review of the revised version:

      The background and terminology in the text have been significantly altered and clarified: Spatial Numerical Association (SNA) instead of Mental Number Line (MNL) in the text, but with a discussion about how SNA might be the basis of MNL. This entails a link from SNA - a bias - to mental representation of a sequence of numerical magnitudes, which will need to be spelt out in subsequent work with a sequence of numbers rather than a single number, in this case 4. Could the effect be generalised to much larger numbers?

      Although the relationship between number and space seems fundamental, the key question is why the L-R SNA bias should exist at all. The authors take on this challenge and make important arguments for the evolutionary advantage of the bias is (see lines 138ff, 375ff, 444ff), though this is likely still to be controversial.

      Subsequent work may clarify its interaction of brain lateralisation with culture, notably reading and writing direction (e.g. Dehaene, S., Bossini, S., & Giraux, P. (1993). The mental representation of parity and numerical magnitude. Journal of Experimental Psychology: General, 122, 371-396), though this relationship has exceptions and challenges (e.g. Karolis, V., Iuculano, T., & Butterworth, B. (2011). Mapping numerical magnitudes along the right lines: Differentiating between scale and bias. Journal of Experimental Psychology: General, 140(4), 693-706).

      For example, would humans with more lateralised brains show a stronger bias? Would humans with reverse lateralisation show a R-L SNA?

    1. Reviewer #2 (Public review):

      Summary:

      This elegant study by Tolman and colleagues provides fundamental findings that substantially advance our knowledge of the major cell types within the limbus of the mouse eye, focusing on the aqueous humor outflow pathway. The authors used single-cell and single-nuclei RNAseq to very clearly identify 3 subtypes of the trabecular meshwork (TM) cells in the mouse eye, with each subtype having unique markers and proposed functions. The U. Columbia results are strengthened by an independent replication in a different mouse strain at a separate laboratory (Duke). Bioinformatics analyses of these expression data were used to identify cellular compartments, molecular functions, and biological processes. Although there were some common pathways among the 3 subtypes of TM cells (e.g., ECM metabolism), there also were distinct functions. For example:

      • TM1 cell expression supports heavy engagement in ECM metabolism and structure, as well as TGF2 signaling.

      • TM2 cells were enriched in laminin and pathways involved in phagocytosis, lysosomal function, and antigen expression, as well as End3/VEGF/angiopoietin signaling.

      • TM3 cells were enriched in actin binding and mitochondrial metabolism.

      They used high-resolution immunostaining and in situ hybridization to show that these 3 TM subtypes express distinct markers and occupy distinct locations within the TM tissue. The authors compared their expression data with other published scRNAseq studies of the mouse as well as the human aqueous outflow pathway. They used ATAC-seq to map open chromatin regions in order to predict transcription factor binding sites. Their results were also evaluated in the context of human IOP and glaucoma risk alleles from published GWAS data, with interesting and meaningful correlations. Although not discussed in their manuscript, their expression data support other signaling pathways/ proteins/ genes that have been implicated in glaucoma, including: TGF2, BMP signaling (including involvement of ID proteins), MYOC, actin cytoskeleton (CLANs), WNT signaling, etc.

      In addition to these very impressive data, the authors used scRNAseq to examine changes in TM cell gene expression in the mouse glaucoma model of mutant Lmxb1-induced ocular hypertension. In man, LMX1B is associated with Nail-Patella syndrome, which can include the development of glaucoma, demonstrating the clinical relevance of this mouse model. Among the gene expression changes detected, TM3 cells had altered expression of genes associated with mitochondrial metabolism. The authors used their previous experience using nicotinamide to metabolically protect DBA2/J mice from glaucomatous damage, and they hypothesized that nicotinamide supplementation of mutant Lmx1b mice would help restore normal mitochondrial metabolism in the TM and prevent Lmx1b-mediated ocular hypertension. Adding nicotinamide to the drinking water significantly prevented Lmxb1 mutant mice from developing high intraocular pressure. This is a laudable example of dissecting the molecular pathogenic mechanisms responsible for a disease (glaucoma) and then discovering and testing a potential therapy that directly intervenes in the disease process and thereby protects from the disease.

      Strengths:<br /> There are numerous strengths in this comprehensive study including:<br /> • Deep scRNA sequencing that was confirmed by an independent dataset in another mouse strain at another university.<br /> • Identification and validation of molecular markers for each mouse TM cell subset along with localization of these subsets within the mouse aqueous outflow pathway.<br /> • Rigorous bioinformatics analysis of these data as well as comparison of the current data with previously published mouse and human scRNAseq data.<br /> • Correlating their current data with GWAS glaucoma and IOP "hits".<br /> • Discovering gene expression changes in the 3 TM subgroups in the mouse mutant Lmx1b model of glaucoma.<br /> • Further pursuing the indication of dysfunctional mitochondrial metabolism in TM3 cells from Lmx1b mutant mice to test the efficacy of dietary supplementation with nicotinamide. The authors nicely demonstrate the disease modifying efficacy of nicotinamide in preventing IOP elevation in these Lmx1b mutant mice, preventing the development of glaucoma. These results have clinical implications for new glaucoma therapies.

      Weaknesses:<br /> • Occasional over-interpretation of data. The authors have used changes in gene expression (RNAseq) to implicate functions and signaling pathways. For example: they have not directly measured "changes in metabolism", "mitochondrial dysfunction" or "activity of Lmx1b".<br /> • In their very thorough data set, there is enrichment of or changes in gene expression that support other pathways that have been previously reported to be associated with glaucoma (such as TGF2, BMP signaling, actin cytoskeletal organization (CLANs), WNT signaling, ossification, etc. that appears to be a lost opportunity to further enhance the significance of this work.

    1. Reviewer #2 (Public review):

      Summary:

      This study presents an integrated experimental and computational pipeline for high-resolution, quantitative imaging and analysis of gastruloids. The experimental module employs dual-view two-photon spectral imaging combined with optimized clearing and mounting techniques to image whole-mount immunostained gastruloids. This approach enables the acquisition of comprehensive 3D images that capture both tissue-scale and single-cell level information.

      The computational module encompasses both pre-processing of acquired images and downstream analysis, providing quantitative insights into the structural and molecular characteristics of gastruloids. The pre-processing pipeline, tailored for dual-view two-photon microscopy, includes spectral unmixing of fluorescence signals using depth-dependent spectral profiles, as well as image fusion via rigid 3D transformation based on content-based block-matching algorithms. Nuclei segmentation was performed using a custom-trained StarDist3D model, validated against 2D manual annotations, and achieving an F1 score of 85+/-3% at a 50% intersection-over-union (IoU) threshold. Another custom-trained StarDist3D model enabled accurate detection of proliferating cells and the generation of 3D spatial maps of nuclear density and proliferation probability. Moreover, the pipeline facilitates detailed morphometric analysis of cell density and nuclear deformation, revealing pronounced spatial heterogeneities during early gastruloid morphogenesis.

      All computational tools developed in this study are released as open-source, Python-based software.

      Strengths:

      The authors applied two-photon microscopy to whole-mount deep imaging of gastruloids, achieving in toto visualization at single-cell resolution. By combining spectral imaging with an unmixing algorithm, they successfully separated four fluorescent signals, enabling spatial analysis of gene expression patterns.

      The entire computational workflow, from image pre-processing to segmentation with a custom-trained StarDist3D model and subsequent quantitative analysis, is made available as open-source software. In addition, user-friendly interfaces are provided through the open-source, community-driven Napari platform, facilitating interactive exploration and analysis.

      Weaknesses:

      The computational module appears promising. However, the analysis pipeline has not been validated on datasets beyond those generated by the authors, making it difficult to assess its general applicability.<br /> Besides, the nuclei segmentation component lacks benchmarking against existing methods.

      Appraisal:

      The authors set out to establish a quantitative imaging and analysis pipeline for gastruloids using dual-view two-photon microscopy, spectral unmixing, and a custom computational framework for 3D segmentation and gene expression analysis. This aim is largely achieved. The integration of experimental and computational modules enables high-resolution in toto imaging and robust quantitative analysis at the single-cell level. The data presented support the authors' conclusions regarding the ability to capture spatial patterns of gene expression and cellular morphology across developmental stages.

      Impact and utility:

      This work presents a compelling and broadly applicable methodological advance. The approach is particularly impactful for the developmental biology community, as it allows researchers to extract quantitative information from high-resolution images to better understand morphogenetic processes. The data are publicly available on Zenodo, and the software is released on GitHub, making them highly valuable resources for the community.

    1. Reviewer #2 (Public review):

      Summary:

      The study employs a specific set of transcription factors to promote lineage conversion of pluripotent stem cells into fetal hair cells. In pluripotent stem cells, an inducible expression system containing SIX1, ATOH1, POU4F3, and GFI1 (SAPG) was inserted into a safe harbor site. The stable cell line allows for doxycycline-inducible expression of transcription factors to generate induced hair cells (iHCs). These changes were observed in gene expression and electrophysiological properties. Comparing the transcriptome with iHCs derived from fibroblasts or primary human inner ear tissue suggested that it is similar to human hair cells. Although the iHCs did not have hair bundles - a key morphological feature of hair cells - the cellular system has immense potential for the field. The defined transcription factors allow for the dissection of gene regulatory networks and provide a molecular handle for the lineage conversion process. The results also suggest that the pluripotent stem cells were not directly converted into iHCs. Instead, there are several transitional cell states. These observations indicate that lineage conversion may still be hampered by yet undefined molecular obstacles and may help identify and overcome these in future work. The stable cell line allows for repeatable and large-scale screening studies, which is not feasible using primary human cells.

      Strengths:

      The cellular system is well-designed, with clearly described expression of the defined factors. Transient expression of the exogenous transcription factors SIX1, ATOH1, POU4F3, and GFI1 (SAPG) upon doxycycline induction is well-documented. Increased expression of endogenous SAPG factors suggests activation of self-regulatory feedback pathways during conversion. The stable iPS cell line provides a tool for the field to study lineage conversion or generate large numbers of iHCs.

      Single-nuclear RNA-seq distinguishes distinct cell clusters and cellular transition states, validating the system's utility. A comparison of previously published data from iHCs and human fetal hair cells also suggested that iHCs are similar to developing human hair cells at the transcriptome level. Whole-cell patch clamp recordings show the generation of excitable cells with heterogeneous ion channel properties, which suggests a change in the cell type.

      Weaknesses:

      The interpretation of the snRNA-seq results could be strengthened by explaining the three distinct clusters for uninduced cells and how they transition into the iHC trajectory.

      Although the analysis focuses on the cell cluster that represents iHCs (R5), a short discussion on what clusters R1-R4 (Figure 3B) represent would be useful. These cells do not express high levels of the SAPG factors even after 21 days of continuous doxycycline induction and may provide insight into hurdles that hamper lineage conversion.

      RNA velocity analysis on single-nuclear RNA-seq is impressive but requires clarification on inferring the pseudotime trajectory. Some rationale and explanation on how the ratio of unspliced to spliced mRNA in the nucleus can be used to infer the differentiation trajectory would strengthen the discussion.

    1. Reviewer #2 (Public review):

      Summary:

      This paper by Wang et al. uses rich brain, behaviour, and genetics data from the ABCD cohort to ask how well cognitive abilities can be predicted from mental health related measures, and how brain and genetics influence that prediction. They obtain an out of sample correlation of 0.4, with neuroimaging (in particular task fMRI) proving the key mediator. Polygenic scores contributed less.

      Strengths:

      This paper is characterized by the intelligent use of a superb sample (ABCD) alongside strong statistical learning methods and a clear set of questions. The outcome - the moderate level of prediction between brain, cognition, genetics and mental health - is interesting, and particularly important is the dissection of which features best mediate that prediction and how developmental and lifestyle factors play a role.

      Weaknesses:

      There are relatively few weaknesses to this paper. It has already undergone review at a different journal, and the authors clearly took the original set of comments into account in revising their paper. Overall, while the ABCD sample is superb for the questions asked, it would have been highly informative to extend the analyses to datasets containing more participants with neurological/psychiatric diagnoses (e.g. HBN, POND) or extending it into adolescent/early adult onset psychopathology cohorts. But it is fair enough that the authors want to leave that for future work.

    1. Reviewer #2 (Public review):

      Summary:

      The authors aim to quantify passive muscle forces in the legs of Drosophila, and test the hypothesis that these forces would be sufficient to support body weight in small insects. They take advantage of the genetic tools available in Drosophila, and use a combination of genetic silencing (optogenetic inactivation of motor neurons), kinematic measurements, and simulations using OpenSim. This integrative toolkit is used to examine the role of passive torques across multiple leg joints. They find that passive forces are weaker than expected - in particular, passive forces were found to be too weak to support the body weight of the fly. This challenges previous scaling assumptions derived from studies in larger insects and has potential implications for our understanding of motor control in small animals.

      Strengths:

      The primary strength of this work lies in its integration of multiple analyses. By pulling together simulations, kinematic measurements from high-resolution videos, and genetic manipulation, they are able to overcome limitations of past studies. In particular, optogenetic manipulation allowed for measurements to be made in whole animals, and the modeling component is valuable because it both validates experimental findings and elucidates the mechanism behind some of the observed dynamic consequences (e.g., the rapid fall after motor inactivation). The conclusions made in the study are well-supported by the data and could have an impact on a number of fields, including invertebrate neurobiology and bioinspired design.

      Weaknesses:

      While (as mentioned above) the study's conclusions are well-supported by the results and modeling, limitations arise because of the assumptions made. For instance, using a linear approximation may not hold at larger joint angles, and future studies would benefit from accounting for nonlinearities. Future studies could also delve into the source of passive forces, which is important for more deeply understanding the anatomical and physical basis of the results in this study. For instance, assessments of muscle or joint properties to correlate stiffness values with physical structure might be an area of future consideration

    1. Reviewer #3 (Public review):

      Summary:

      Perlee et al. present a method for generating cell-type restricted knockouts in zebrafish, focusing on melanocytes. For this method, the authors knock-in a Cas9 encoding sequence into the mitfa locus. This mitfaCas9 line has restricted Cas9 expression, allowing the authors to generate melanocyte-specific knockouts rapidly by follow-up injection of sgRNA expressing transposon vectors.

      The paper presents some interesting vignettes to illustrate the utility of their approach. These include 1) a derivation of albino mutant fish as a demonstration of the method's efficiency, 2) an interrogation and novel description of tuba1a/tuba1c as a potential non-autonomous contributor to melanosome dispersion, and 3) the generation of sox10 deficient melanoma tumors that show "escape" of sox10 loss through upregulation of sox9. The latter two examples highlight the usefulness of cell-type targeted knockouts (Body-wide sox10 and tuba1a loss elicit developmental defects). Additionally, the tumor models involve highly multiplexed sgRNAs for tumor initiation which is nicely facilitated by the stable Cas9.

      Strengths:

      The approach is clever and could prove very useful for studying melanocytes and other cell types. As the authors hint at in their discussion, this approach would become even more powerful with the generation of other Cas9-restricted lineages so a single sgRNA construct can be screened across many lineages rapidly (or many sgRNA and fish lines screened combinatorially).

      The biological findings used to demonstrate the power of the approach are interesting in their own right. The non-autonomous effect of tuba1a/tuba1c loss on melanosome dispersion are striking and demonstrates very nicely how one could use Perlee et al.'s approach to search for similar mechanisms systematically. The dual targeting nature of the tuba1a/tuba1c sgRNA also suggests similar approaches might be explored for knocking out paralogs. The observation of the sox9 escape mechanism with sox10 loss is a beautiful demonstration of the relevance of SOX10/SOX9's reciprocal regulation in vivo. This system would be a very nice model for further interrogating mechanisms/interventions surrounding Sox10 in melanoma.

      Finally, the figure presentation is very nice. This work involves complex genetic approaches, including multiple fish generations and multiplexed construct injections. The vector diagrams and breeding schemes in the paper make everything very clear/"grok-able," and the paper was enjoyable to read.

      Weaknesses:

      The authors' claims are grounded and tested rigorously. The major weaknesses that we raised in the first round of reviews were either addressed experimentally or are now detailed as limitations in the text. Congrats on the beautiful paper!

    1. Reviewer #2 (Public review):

      Garbelli et. al. set out to elucidate the function of two glutamate transporters, EAAT5b and EAAT7, in the functional and behavioral responses to different wavelengths of light. The question is an interesting one because these transporters are well-positioned to affect responses to light, and their distribution in the retina suggests that they could play differential roles in visual behaviors. However, the resolution of the functional and behavioral data presented here means that the conclusions are necessarily a bit vague.

      In Figure 1, the authors show that the double KO has a decreased ERG response to UV/blue and red wavelengths. However, the individual mutations both only affect the response to red light, suggesting that they might affect behaviors such as OMR that typically rely on this part of the visual spectrum. However, there was no significant change in the response to UV/blue light of any intensity, making it unclear whether the mutations could individually play roles in detection of UV prey. Based on the later behavioral data, it seems likely that at least the EAAT7 KO should affect retinal responses to UV light, but it may be that the ERG does not have the spatial or temporal resolution to detect the difference, or that the presence of blue light overwhelmed any effect of the individual knockouts on the response to UV light.

      In Figures 5 and 6, the authors compare the two knockouts to wild-type fish in terms of their sensitivity to UV prey in a hunting assay. The EAAT5b KO showed no significant impairment in UV sensitivity, while the EAAT7 KO fish actually had an increased hunting response to UV prey. However, there is no comparison of the KO and WT responses to different UV intensities, only in bulk, so we cannot conclude that the EAAT7 KO is allowing the fish to detect weaker prey-like stimuli.

      In Figure 7, the EAAT5b KO seems to cause a decrease in OMR behavior to red grating stimuli, but only one stimulus is tested, so it is unclear whether this is due to a change in visual sensitivity or resolution.

      The conclusions made in the manuscript are appropriately conservative; the abstract states that these transporters somehow influence prey detection and motion sensing, and this is likely true.

      In terms of impact on the field, this work highlights the potential importance of these two transporters to visual processing, but further studies will be required to say how important they are and exactly what they are doing.

    1. Reviewer #2 (Public review):

      Summary:

      In this study, the authors have devised a novel assay to measure relative social rank in mice that is aimed at incorporating multiple aspects of social competition while minimizing direct contact between animals. Forming a hierarchy often involves complex social dynamics related to competitive drives for different fundamental resources, including access to food, water, territory and sexual mates. This makes the study of social dominance and its neural underpinnings hard, warranting the development of new tools and methods that can help understand both social function as well as dysfunction.

      Strengths:

      This study showcases an assay called the Food Pellet Competition Test, where cagemate mice compete for food, without direct contact, by pushing a block in a tube from opposite directions. This task ran with stranger mice leads to more variable outcomes, suggesting co-housing helps stabilize outcomes. The authors have attempted to quantify motivation to obtain the food independent of other factors by running the assay under two conditions: one where the food is accessible and one where it isn't. This assay results in high outcome consistency across days for females and males paired housed and for male groups of three. Further, the determined social ranks correlate strongly with two common assays: the tube test and the warm spot test.

      Weaknesses:

      This new assay has limited ethological validity since mice do not compete for food without touching each other with a block in the middle. In addition, the assay may only be valid for a single trial per day, making its utility for recording neural recordings and manipulations limited to a single sample per mouse. The authors claim, as currently stated in results, for the new control experiment in 1H-J is not warranted given that 6/8 mice had majority winning or losing across all strangers.

    1. Reviewer #3 (Public review):

      Summary:

      In this study, Wang et al utilized the available GTEx data to compile a comprehensive analysis that attempt to reveal aging-related sex-dimorphic gene expression as well as alternative splicing changes in human. The key conclusions based upon their analysis are that 1) extensive sex-dimorphisms during aging with distinct patterns of change in gene expression and alternative splicing (AS), and 2) the male-biased age-associated AS events have a stronger association with Alzheimer's disease, and 3) the females-biased events are often regulated by several sex-biased splicing factors that may be controlled by estrogen receptors. They further performed break-point analysis and reveal in males there are two main breakpoints around ages 35 and 50, while in female only one breakpoint at 45.

      Strengths:

      This study sets an ambitious goal, leveraging the extensive GTEx dataset to investigate aging-related, sex-dimorphic gene expression and alternative splicing changes in humans. The research addresses a significant question, as our understanding of sex-dimorphic gene expression in the context of human aging is still in its early stages. Advancing our knowledge of these molecular changes is vital for identifying therapeutic targets for age-related diseases and extending human healthspan. The study is highly comprehensive, and the authors are commendable for their attempted thorough analysis of both gene expression and alternative splicing-an area often overlooked in similar studies.

    1. Reviewer #2 (Public review):

      Summary:

      The authors set out to visualize the molecular architecture of the adult forebrain glutamatergic synapses in a near-native state. To this end, they use a rapid workflow to extract and plunge-freeze mouse synapses for cryo-electron tomography. In addition, the authors use knockin mice expression PSD95-GFP in order to perform correlated light and electron microscopy to clearly identify pre- and synaptic membranes. By thorough quantification of tomograms from plunge- and high-pressure frozen samples, the authors show that the previously reported 'post-synaptic density' does not occur at high frequency and therefore not a defining feature of a glutamatergic synapse.

      Subsequently, the authors are able to reproduce the frequency of post-synaptic density when preparing conventional electron microscopy samples, thus indicating that density prevalence is an artifact of sample preparation. The authors go on to describe the arrangement of cytoskeletal components, membraneous compartments, and ionotropic receptor clusters across synapses.

      Demonstrating that the frequency of the post-synaptic density in prior work is likely an artifact and not a defining feature of glutamatergic synapses is significant. The descriptions of distributions and morphologies of proteins and membranes in this work may serve as a basis for the future of investigation for readers interested in these features.

      Strengths:

      The authors perform a rigorous quantification of the molecular density profiles across synapses to determine the frequency of the post-synaptic density. They prepare samples using two cryogenic electron microscopy sample preparation methods, as well as one set of samples using conventional electron microscopy methods. The authors can reproduce previous reports of the frequency of the post-synaptic density by conventional sample preparation, but not by either of the cryogenic methods, thus strongly supporting their claim.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled the identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

      Strengths:

      The manuscript is well written, and the analysis pipeline is clearly described. Moreover, the dataset used for validation is of high quality, considering the technical challenges associated with longitudinal two-photon recordings in mouse pups. The authors provide a convincing comparison of both manual annotation and "CellReg" to demonstrate the tracking performance of "Track2p". Applying this tracking algorithm, Majnik and colleagues characterized hallmark developmental changes in spontaneous network activity, highlighting the impact of longitudinal imaging approaches in developmental neuroscience. Additionally, the code is available on GitHub, along with helpful documentation, which will facilitate accessibility and usability by other researchers.

      Weaknesses:

      (1) The main critique of the "Track2p" package is that, in its current implementation, it is dependent on the outputs of "Suite2p". This limits adoption by researchers who use alternative pipelines or custom code. One potential solution would be to generalize the accepted inputs beyond the fixed format of "Suite2p", for instance, by accepting NumPy arrays (e.g., ROIs, deltaF/F traces, images, etc.) from files generated by other software. Otherwise, the tool may remain more of a useful add-on to "Suite2p" (see https://github.com/MouseLand/suite2p/issues/933) rather than a fully standalone tool.

      (2) Further benchmarking would strengthen the validation of "Track2p", particularly against "CaIMaN" (Giovannucci et al., eLife, 2019), which is widely used in the field and implements a distinct registration approach.

      (3) The authors might also consider evaluating performance using non-consecutive recordings (e.g., alternate days or only three time points across the week) to demonstrate utility in other experimental designs.

    1. Reviewer #2 (Public review):

      Summary:

      Human histone H3K36 methyltransferase Setd2 has been previously shown to be a tumor suppressor in lung and pancreatic cancer. In this manuscript by Mack et al., the authors first use a mouse KRASG12D-driven lung cancer model to confirm in vivo that Setd2 depletion exacerbates tumorigenesis. They then investigate the enzymatic regulation of the Setd2 SET domain in vitro, demonstrating that H2A, H3, or H4 acetylation stimulates Setd2-SET activity, with specific enhancement by mono-acetylation at H3K14ac or H3K27ac. In contrast, histone ubiquitination has no effect. The authors propose that H3K27ac may regulate Setd2-SET activity by facilitating its binding to nucleosomes. This work provides insight into how cross-talk between histone modifications regulates Setd2 function. However, the manuscript lacks a clear discussion on how Setd2's in vivo tumor suppressor role and the in vitro mechanistic regulation findings are connected. Additionally, some experiments require more controls and better data quality for proper interpretation.

      Specific comments:

      (1) As for Figure 2F, Setd2-SET activity on WT rNuc (H3) appears to be significantly lower compared to what is extensively reported in the literature. This is particularly puzzling given that Figure 2B suggests that using 3H-SAM, H3-nuc are much better substrates than K36me1, whereas in Figure 3F, rH3 is weaker than K36me1. It is recommended for the authors to perform additional experimental repeats and include a quantitative analysis to ensure the consistency and reliability of these findings.

      (2) The additional bands observed in Figure 4B, which appear to be H4, should be accompanied by quantification of the intensity of the H3 bands to better assess K36me3 activity. Additionally, the quantification presented in Figure 4C for SAH does not seem accurate as it potentially includes non-specific methylation activity, likely from H4. This needs to be addressed for clarity and accuracy.

      (3) In Figure 4E, the differences between bound and unbound substrates are not sufficiently pronounced. Given the modest differences observed, authors might want to consider repeating the assay with sufficient replicates to ensure the results are statistically robust.

      (4) Regarding labeling, there are multiple issues that need correction: In the depiction of Epicypher's dNuc, it is crucial to clearly mark H2B as the upper band, rather than ambiguously labeling H2A/H2B together when two distinct bands are evident. In Figure 3B and D, the histones appear to be mislabeled, and the band corresponding to H4 has been cut off. It would be beneficial to refer to Figure 3E for correct labeling to maintain consistency and accuracy across figures.

      (5) There are issues with the image quality in some blots; for instance, Figure 2EF and Figure 2D exhibit excessive contrast and pixelation, respectively. These issues could potentially obscure or misrepresent the data, and thus, adjustments in image processing are recommended to provide clearer, more accurate representations.

      (6) The authors are recommended to provide detailed descriptions of the materials used, including catalog numbers and specific products, to allow for reproducibility and verification of experimental conditions.

      (7) The identification of Setd2 as a tumor suppressor in KrasG12C-driven LUAD is a significant finding. However, the discussion on how this discovery could inspire future therapeutic approaches needs to be more balanced. The current discussion (Page 10) around the potential use of inhibitors is somewhat confusing and could benefit from a clearer explanation of how Setd2's role could be targeted therapeutically. It would be beneficial for the authors to explore both current and potential future strategies in a more structured manner, perhaps by delineating between direct inhibitors, pathway modulators, and other therapeutic modalities.

    1. Reviewer #2 (Public review):

      Summary:

      The authors have used single-particle cryoEM imaging to determine how small-molecule regulators of the SK channel interact with it and modulate their function.

      Strengths:

      The reconstructions are of high quality, and the structural details are well described.

      Weaknesses:

      The electrophysiological data are poorly described. Several details of the structural observations require a mechanistic context, perhaps better relating them to what is known about SK channels or other K channel gating dynamics.

      The most pressing point I have to make, which could help improve the manuscript, relates to the selectivity filter (SF) conformation. Whether the two ion-bound state of SK2-4 (Figure 4A) represents a non-selective, conductive SF occluded by F243 or represents a C-type inactivated SF, further occluded by F243, is unclear. It would be important to discuss this. Reconstructions of Kv1.3 channels also feature a similar configuration, which has been correlated to its accelerated C-type inactivation.

      Furthermore, binding of a toxin derivative to Kv1.3 restores the SF into a conductive form, though occluded by the toxin. It appears that apamin binding to SK2-4 might be doing something similar. Although I am not sure whether SK channels undergo C-type inactivation like gating, classical MTS accessibility studies have suggested that dynamics of the SF might play a role in the gating of SK channels. It would be really useful (if not essential) to discuss the SF dynamics observed in the study and relate them better to aspects of gating reported in the literature.

      The SF of K channels, in conductive states, are usually stabilized by an H-bond network involving water molecules bridged to residues behind the SF (D363 in the down-flipped conformation and Y361). Considering the high quality of the reconstructions, I would suspect that the authors might observe speckles of density (possibly in their sharpened map) at these sites, which overlap with water molecules identified in high-resolution X-ray structures of KcsA, MthK, NaK, NaK2K, etc. It could be useful to inspect this region of the density map.

    1. Reviewer #2 (Public Review):

      Summary:

      Peptidoglycan remodeling, particularly that carried out by enzymes known as amidases, is essential for the later stages of cell division including cell separation. In E. coli, amidases are generally activated by the periplasmic proteins EnvC (AmiA and AmiB) and NlpD (AmiC). The ABC family member, FtsEX, in turn, has been implicated as a modulator of amidase activity through interactions with EnvC. Specifically, how FtsEX regulates EnvC activity in the context of cell division remains unclear.

      Strengths:

      Li et al. make two primary contributions to the study of FtsEX. The first, the finding that ATP binding stabilizes FtsEX in vitro, enables the second, structural resolution of full-length FtsEX both alone (Figure 2) and in combination with EnvC (Figure 3). Leveraging these findings, the authors demonstrate that EnvC binding stimulates FtsEX-mediated ATP hydrolysis approximately two-fold. The authors present structural data suggesting EnvC binding leads to a conformational change in the complex. Biochemical reconstitution experiments (Figure 5) provide compelling support for this idea.

      Weaknesses:

      The potential impact of the study is curtailed by the lack of experiments testing the biochemical or physiological relevance of the model which is derived almost entirely from structural data.

      Altogether the data support a model in which interaction with EnvC, results in a conformational change stimulating ATP hydrolysis by FtsEX and EnvC-mediated activation of the amidases, AmiA and AmiB. However, the study is limited in both approach and scope. The importance of interactions revealed in the structures to the function of FtsEX and its role in EnvC activation are not tested. Adding biochemical and/or in vivo experiments to fill in this gap would allow the authors to test the veracity of the model and increase the appeal of the study beyond the small number of researchers specifically interested in FtsEX.

      Comments after revision:

      Although I appreciate the authors' desire to save future biochemical experiments for a separate publication, the lack of in vitro data verifying their model makes it challenging to reconcile with published studies from other groups. The other reviewer's point about EnvC activating FtsEX ATPase activity resulting in a futile cycle since both are recruited to the septum well before constriction, is a good example of the disconnect between the model presented here and in vivo data.

    1. Reviewer #2 (Public review):

      Summary:

      Mitochondrial DNA (mtDNA) is exclusively maternally transmitted in almost all species. Paternal mitochondria, with their mtDNA, must be rapidly degraded after fertilisation to prevent their transmission to progeny, which could lead to subsequent detrimental mito-nuclear incompatibilities. Multiple layers of mechanisms contribute to blocking the transmission of paternal mitochondria and their mtDNA to progeny. Endonuclease activity and mitophagy form a part of these strategies. However, other key regulatory mechanisms remain to be elucidated, as inactivating endonuclease and mitophagy activity only delays the clearance of paternal mitochondria. In this study, the authors mainly focused on genes involved in endonuclease function (csp-6) and autophagy (allo-1) in C. elegans, demonstrating a synergic genetic interaction that potentialize their activity. They also revealed a contribution by pink-1/pink1, in the absence of allo-1.

      Strengths:

      The majority of data relies on confocal microscopy images and corresponding image analysis and quantification. Images are clear, and quantifications are supported by several biological replicates of >10 n and standard statistical tests. Mutants used were obtained from the Caenorhabditis Genetics Center (CGC) and were previously validated and confirmed by the C. elegans community. The scientific approach is solid and rigorous and in line with state-of-the-art C. elegans methods. Proper controls have been performed to rule out the effect of animal viability on observed results or to confirm the staining validity of TUBES on subcellular structures surrounding paternal mitochondria. Controls validating uaDf5 PCR specificity were conducted.

      Weaknesses:

      However, the embryonic expression of paternally contributing genes in feminised animals cannot be completely ruled out, as RNAi was used to alter gene expression levels. An issue inherent to RNAi approaches. Also, the impact of pink-1/pink1 is significant, but there is a lack of evidence demonstrating its mitophagic function.

      Goal achievements and data supportive of conclusions:

      In the first part of the study, the authors strongly and clearly demonstrate the synergistic interaction between the csp-6 and allo-1 in delaying paternal mitochondria degradation and associated mtDNA in the fertilised egg. In wild-type animals, paternal mitochondria are visible (using a mitochondrial HSP-::GFP marker) until the 4-cell stage embryo. In the csp-6; allo-1 double mutant genetic background, paternal mitochondria very significantly perdures until the 2-fold embryonic stage. The uaDf5 mitochondrial deletion, detectable by PCR, that was introduced by crossing with a male, followed the same trend. In addition, loss of fncd-1/fndc1 and phb-2 did not extend the perdurance of paternal mitochondria. In the second part of the study, the authors demonstrate a contribution of the loss of pink-1/pink1, in the absence of allo-1, in delaying paternal mitochondria degradation until the 100-cell stage. Overall, the conclusions are in accordance with the data shown.

      Impact on the field:

      Endonuclease activity and mitophagy aren't sufficient to prevent the transmission of paternal mitochondria and associated mtDNA to progeny, but they still contribute significantly to regulating the perdurance of paternal mitochondria in early embryos. Understanding how these two functions work in concert to potentialize their activity is important, as they could potentially be manipulated/enhanced to improve paternal mitochondrial degradation in the future. Here, the authors demonstrate a detailed synergistic genetic interaction between these functions. Also, they pointed out a new potential contribution of pink-1/pink1, which may underlie a potentially more complex mitophagic protective function.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript by Jia and Chen addresses the structural basis of voltage-activation of BK channels using computational approaches. Although a number of experimental studies using gating current and patch-clamp recording have analyzed voltage-activation in terms of observed charge movements and the apparent energetic coupling between voltage-sensor movement and channel opening, the structural changes that underlie this phenomenon have been unclear. The present studies use a reduced molecular system comprising the transmembrane portion of the BK channel (i.e. the cytosolic domain was deleted), embedded in a POPC membrane, with either 0 or 750 mV applied across the membrane. This system enabled acquisition of long simulations of 10 microseconds, to permit tracking of conformational changes of the channel. The authors principal findings were that the side chains of R210 and R213 rapidly moved toward the extracellular side of the membrane (by 8 - 10 Å), with greater displacements than any of the other charged transmembrane residues. These movements appeared tightly coupled to movement of the pore-lining helix, pore hydration, and ion permeation. The authors estimate that R210 and R213 contribute 0.25 and 0.19 elementary charges per residue to the gating current, which is roughly consistent with estimates based on electrophysiological measurements that used the full-length channel.

      Strengths:

      The methodologies used in this work are sound, and these studies certainly contribute to our understanding of voltage-gating of BK channels. An intriguing observation is the strongly coupled movement of the S4, S5, and S6 helices that appear to underlie voltage-dependent opening. Based on Fig 2a-d, the substantial movements of the R210 and R213 side chains occur nearly simultaneously to the S6 movement (between 4 - 5 usec of simulation time). This seems to provide support for a "helix-packing" mechanism of voltage gating in the so-called "non-domain-swapped" voltage-gated K channels.

      Weaknesses:

      The main limitation is that these studies used a truncated version of the BK channel, and there are likely to be differences in VSD-pore coupling in the context of the full-length channels that will not be resolved in the present work. Nonetheless, the authors provide a strong rationale for their use of the truncated channel, and the results presented will provide a good starting point for future computational studies of this channel.

    1. Reviewer #2 (Public review):

      Summary:

      The authors developed a computational pipeline named CHROMAS to track and analyze chromatophore dynamics, which provides a wide range of biological analysis tools without requiring the user to write code.

      Strengths:

      (1) CHROMAS is an integrated toolbox that provides tools for different biological tasks such as: segment, classify, track and measure individual chromatophores, cluster small groups of chromatophores, analyze full-body patterns, etc.

      (2) It could be used to investigate different species. The authors have already applied it to analyze the skin of the bobtail squid Euprymna berryi and the European cuttlefish Sepia officinalis.

      (3) The tool is open-source and easy to install. The paper describes in detail the experiment requirements, command to complete each task and provides relevant sample figures, which are easy to follow.

      Weaknesses:

      (1) There are some known limitations for the current version. The users should read the "Discussion" chapter carefully before preparing their experiments and using CHROMAS.

    1. Reviewer #2 (Public review):

      Summary:

      The authors have worked up a ``virtual thymus' using EPISIM, which has already been published. Attractive features of the computational model are stochasticity, cell-to-cell variability, and spatial heterogeneiety. They seek to explore the role of TECs, that release IL-7 which is important in the process of thymocyte division.

      In the model, ordinary clones have IL7R levels chosen from a distribution, while `lesioned' clones have an IL7R value set to the maximum. The observation is that the lesioned clones are larger families, but the difference is not dramatic. This might be called a cell-intrinsic mechanism. One promising cell-extrinsic mechanism is mentioned: if a lesioned clone happens to be near a source of IL-7 and begins to proliferate, the progeny can crowd out cells of other clones and monopolise the IL-7 source. The effect will be more noticeable if sources are rare, so is seen when the TEC network is sparse.

      Strengths:

      Thymic disfunctions are of interest, not least because of T-ALL. New cells are added, one at a time, to simulate the conveyor belt of thymocytes on a background of stationary cells. They are thus able to follow cell lineages, which is interesting because one progenitor can give rise to many progeny.

      There are some experimental results in Figures 4,5 and 6. For example, il7 crispant embryos have fewer thymocytes and smaller thymii; but increasing IL-7 availability produces large thymii.

    1. Reviewer #2 (Public review):

      Summary:

      Here, the Fischer et al. attempt to understand the role of parental care, specifically the transport of offspring, in the development of the amphibian microbiome. The amphibian microbiome is an important study system due to its association with host health and disease outcomes. This study provides vertical transfer of bacteria through parental transport of tadpoles as one mechanism, among others, influencing tadpole microbiome composition. This paper gives insight into the relative roles of the environment, species, and parental care in amphibian microbiome assembly.

      The authors determine the time of bacterial colonization during tadpole development using PCR, observing that tadpoles were not colonized by bacteria prior to hatching from the vitelline membrane. This is an important finding for amphibian microbiome research and I would be curious to see if this is seen broadly across amphibian species. By doing this, the impact of transport can be more accurately assessed in their laboratory experiments. The authors found that caregiver species influenced community composition, with transported tadpoles sharing a greater proportion of their skin communities with the transporting species.

      In a comparison of three sympatric amphibian species that vary in their reproductive strategies, the authors found that tadpole community diversity was not reflective of habitat diversity, but may be associated with the different reproductive strategies of each species. Parental care explained some of the variance of tadpole microbiomes between species, however, transportation by conspecific adults did not lead to more similar microbiomes between tadpoles and adults compared to species that do not exhibit parental transport. This finding is in agreement with the understanding that the amphibian microbiome is distinct between developmental stages (eggs/tadpoles/adults) and also that amphibian microbiome composition is generally species specific.

      When investigating contributions of caretakers to transported offspring, the authors found that tadpole-adult pairs with a history of direct contact were not more similar than tadpole-adult pairs lacking that history. This conclusion was surprising when considering the direct contact between the adults and tadpoles, however if only certain taxa from the adults are capable of colonizing tadpoles, then one could expect that similar ASVs might be donated between tadpole-adult pairs.

      I did not find any major weaknesses in my review of this paper. I think that the data and conclusions here are of value to other researchers looking into the assembly of the amphibian microbiome. This paper offers insight into how tadpole-transport could influence the microbiome and adds to our overall understanding of amphibian microbiome assembly across the varied life histories of frogs.

    1. Reviewer #2 (Public review):

      Summary:

      Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin-mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time, a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation.

      These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis.

      Strengths:

      The 15N,1H, and 13C, methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of the activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin, with both kD and description of the interfaces.

      Weaknesses:

      This reviewer has identified only minor weaknesses with the study.

      (1) Arrestin-2 1-418 resonances all but disappear with CCR5pp6 addition. Are they recovered with Ap2Beta2 addition, and is this what is shown in Supplementary Figure 2D?

      (2) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems.

      (3) At one point, the authors added an excess fully phosphorylated CCR5 phosphopeptide (CCR5pp6). Does the phosphopeptide rescue resolution of arrestin2 (NH or methyl) to the point where interaction dynamics with clathrin (CLTC NTD) are now more evident on the arrestin2 surface?

      (4) Once phosphopeptide activates arrestin-2 and AP2 binds, can phosphopeptide be exchanged off? In this case, would it be possible for the activated arrestin-2 AP2 complex to re-engage a new (phosphorylated) receptor?

      (5) Did the authors ever try SEC measurements of arrestin-2 + AP2beta2+CCR5pp6 with and without PIP2, and with and without clathrin (CLTC NTD? The question becomes what the active complex is and how PIP2 modulates this cascade of complexation events in class B receptors.

    1. Reviewer #3 (Public review):

      Summary:

      In this manuscript, Franco and colleagues describe careful analyses of Salmonella chemotactic behavior in the presence of conflicting environmental stimuli. By doing so, the authors describe that this human pathogen integrates signals from a chemoattractant and a chemorepellent into an intermediate "chemohalation" phenotype.

      Strengths:

      The study was clearly well-designed and well-executed. The methods used are appropriate and powerful. The manuscript is very well written, and the analyses are sound. This is an interesting area of research, and this work is a positive contribution to the field.

      Weaknesses:

      No significant weaknesses noted.

    1. Reviewer #2 (Public review):

      Li et al. describe ambisim, a tool with the goal of creating realistic synthetic single-nucleus RNA/ATAC sequencing datasets. It has become standard to pool multiple genetically distinct donors when using single-cell sequencing followed by genotype-based demultiplexing (i.e., using donor single-nucleotide variants to identify specific donor origin). A plethora of tools exist to accomplish this demultiplexing, but advanced tools to create synthetic datasets, and therefore provide definitive benchmarking, are lacking. Ambisim is a well-thought-out simulator that improves upon previous tools available by allowing for modeling of variable ambient contamination proportions and doing so in a genotype-aware fashion. This provides more realistic synthetic datasets that provide challenging scenarios for future demultiplexing tools. The authors use ambisim to benchmark a large number of available and commonly used genotype-free and -dependent demultiplexing tools. They identify the strengths and weaknesses of these tools. They also go on to define a new metric, variant consistency, to further assess demultiplexing performance across tools. Overall, this manuscript provides a useful framework to more thoroughly evaluate future demultiplexing tools, as well as provides rationale for tool selection depending on a user's experimental conditions.

      The authors provide measured conclusions that are supported by their findings. There are some aspects that are unclear.

      (1) Throughout the manuscript, the figure legends are difficult to understand, and this makes it difficult to interpret the graphs.

      (2) Since this is both a new tool and a benchmark, it would be worthwhile in the Discussion to comment on which demultiplexing tools one may want to choose for their dataset, especially given the warning against ensemble methods. From this extensive benchmarking, one may want to choose a tool based on the number of donors one has pooled, the modalities present, and perhaps even the ambient RNA (if it has been estimated previously).

      (3) What are the minimal computational requirements for running ambisim? What is the time cost?

    1. Reviewer #2 (Public review):

      Summary:

      The authors perform a series of studies to follow up on their previous work, which established a role for dorsal raphe dopamine neurons (DRN) in the regulation of social-isolation-induced rebound in mice. In the present study, Lee et. al, use a combination of modern circuit tools to investigate putatively distinct roles of DRN dopamine transporting containing (DAT) projections to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP). Notably, they reveal that optogenetic stimulation of distinct pathways confers specific behavioral states, with DRNDAT-BLP driving aversion, DRNDAT-BNST regulating non-social exploratory behavior, and DRNDAT-CeA promoting socialability. A combination of electrophysiological studies and in situ hybridization studies reveal heterogenous dopamine and neuropeptide expression and different firing properties, providing further evidence of pathway-specific neural properties. Lastly, the authors combine optogenetics and calcium imaging to resolve social encoding properties in the DRNDAT-CeA pathway, which correlates observed social behavior to socially engaged neural ensembles.

      Collectively, these studies provide an interesting way of dissecting out separable features of a complex multifaceted social-emotional state that accompanies social isolation and the perception of 'loneliness.' The main conclusions of the paper provide an important and interesting set of findings that increase our understanding of these distinct DRN projections and their role in a range of social (e.g., prosocial, dominance), non-social, and emotional behaviors. However, as noted below, the examination of these circuits within a homeostatic framework is limited given that a number of the datasets did not include an isolated condition. The DRNDAT-CeA pathway was investigated with respect to social homeostatic states in the present study for some of the datasets.

      Strengths:

      (1) The authors perform a comprehensive and elegant dissection of the anatomical, behavioral, molecular, and physiological properties of distinct DRN projections relevant to social, non-social, and emotional behavior, to address multifaceted and complex features of social state.

      (2) This work builds on prior findings of isolation-induced changes in DRN neurons and provides a working framework for broader circuit elements that can be addressed across social homeostatic state.

      (3) This work characterizes a broader circuit implicated in social isolation and provides a number of downstream targets to explore, setting a nice foundation for future investigation.

      (4) The studies account for social rank and anxiety-like behavior in several of the datasets, which are important consideration to the interpretation of social motivation states, especially in male mice with respect to dominance behavior.

      Weaknesses:

      (1) The conceptual framework of the study is based on the premise of social isolation and perceived 'loneliness' under the framework of social homeostasis, analogous to hunger. In this framework, social isolation should provoke an aversive state and compensatory social contact behavior. In the authors' prior work, they demonstrate synaptic changes in DRN neurons and social rebound following acute social isolation. Thus, the prediction would be that downstream projections also would show state dependent changes as a function of social isolation state (e.g., grouped/socially engaged vs. isolated). In the current paper, a social isolation condition was included for some but not all experiments, which should be considered in the interpretation of the data, specifically within the context of dynamic isolation states.

      (2) Figure 1 confirms co-laterals in the BNST and CeA via anatomical tracing studies. The goal of the optogenetic studies is to dissociate functional/behavioral roles of distinct projections. One limitation of optogenetic projection targeting is the possibility of back-propagating action potentials (stimulation of terminals in one region may back-propagate to activate cell bodies, and then afferent projections to other regions), and/or stimulation of fibers of passage. However, this is addressed in the discussion and the present data are convincing, which minimizes the concern.

      (3) Sex as a biological variable should be considered in the present data, as included in the discussion.

    1. Reviewer #2 (Public review):

      Summary:

      This paper investigates the neural underpinnings of an interactive speech task requiring verbal coordination with another speaker. To achieve this, the authors recorded intracranial brain activity from the left (and to a lesser extent, the right) hemisphere in a group of drug-resistant epilepsy patients while they synchronised their speech with a 'virtual partner'. Crucially, the authors were able to manipulate the degree of success of this synchronisation by programming the virtual partner to either actively synchronise or desynchronise their speech with the participant, or else to not vary its speech in response to the participant (making the synchronisation task purely one-way). Using such a paradigm, the authors identified different brain regions that were either more sensitive to the speech of the virtual partner (primary auditory cortex), or more sensitive to the degree of verbal coordination (i.e. synchronisation success) with the virtual partner (left secondary auditory cortex and bilateral IFG). Such sensitivity was measured by (1) calculating the correlation between the index of verbal coordination and mean power within a range of frequency bands across trials, and (2) calculating the phase-amplitude coupling between the behavioural and brain signals within single trials (using the power of high-frequency neural activity only). Overall, the findings help to elucidate some of the brain areas involved in interactive speaking behaviours, particularly highlighting high-frequency activity of the bilateral IFG as a potential candidate supporting verbal coordination.

      Strengths:

      This study provides the field with a convincing demonstration of how to investigate speaking behaviours in more complex situations that share many features with real-world speaking contexts e.g. simultaneous engagement of speech perception and production processes, the presence of an interlocutor and the need for inter-speaker coordination. The findings thus go beyond previous work that has typically studied solo speech production in isolation, and represent a significant advance in our understanding of speech as a social and communicative behaviour. It is further an impressive feat to develop a paradigm in which the degree of cooperativity of the synchronisation partner can be so tightly controlled; in this way, this study combines the benefits of using pre-recorded stimuli (namely, the high degree of experimental control) with the benefits of using a live synchronisation partner (allowing the task to be truly two-way interactive, an important criticism of other work using pre-recorded stimuli). A further key strength of the study lies in its employment of stereotactic EEG to measure brain responses with both high temporal and spatial resolution, an ideal method for studying the unfolding relationship between neural processing and this dynamic coordination behaviour.

      Weaknesses:

      One limitation of the current study is the relatively sparse coverage of the right hemisphere by the implanted electrodes (91 electrodes in the right compared to 145 in the left). Of course, electrode location is solely clinically motivated, and so the authors did not have control over this. In a previous version of this article, the authors therefore chose not to include data from the right hemisphere in reported analyses. However, after highlighting previous literature suggesting that the right hemisphere likely has high relevance to verbal coordination behaviours such as those under investigation here, the authors have now added analyses of the right hemisphere data to the results. These confirm an involvement of the right hemisphere in this task, largely replicating left hemisphere results. Some hemispheric differences were found in responses within the STG; however, interpretation should be tempered by an awareness of the relatively sparse coverage of the right hemisphere meaning that some regions have very few electrodes, resulting in reduced statistical power.

    1. Reviewer #2 (Public review):

      Summary:

      Microbial population abundances are regularly estimated by multiplying plate counts by dilution factors, with inferences made about sample heterogeneity without taking into account heterogeneity generated through dilution and plating methods. The authors have developed REPOP, a method for disentangling methodological stochasticity from ecological heterogeneity using a Bayesian framework. They present three models: a unimodal distribution, a multimodal distribution, and a multimodal distribution that incorporates a colony count cutoff. They use a combination of simulated and experimental data to show the effectiveness of the REPOP method in resolving true microbial population distributions.

      Strengths:

      Overall, this paper addresses a significant issue in microbial ecology and reliably demonstrates that the REPOP method improves upon current methods of estimating microbial population heterogeneity, particularly with simulation data. The three models presented build upon each other and are discussed in a way that is fairly accessible to a broad audience. The authors also show that leveraging the information provided by non-countable plates is important. Additionally, the authors address the potential for extending this method to other sources of methodological stochasticity that may occur in microbial plating. However, it does seem that they could extend this further by discussing ways that this method could be applied to non-microbial systems, allowing this work to appeal to a broader audience.

      Weaknesses:

      A more thorough discussion of when and by how much estimated microbial population abundance distributions differ from the ground truth would be helpful in determining the best practices for applying this method. Not only would this allow researchers to understand the sampling effort necessary to achieve the results presented here, but it would also contextualize the experimental results presented in the paper. Particularly, there is a disconnect between the discussion of the large sample sizes necessary to achieve accurate multimodal distribution estimates and the small sample sizes used in both experiments.

    1. Reviewer #2 (Public review):

      Summary:

      The study in question utilizes functional magnetic resonance imaging (fMRI) to dynamically estimate the locus and extent of covert spatial attention from visuocortical activity. The authors aim to address an important gap in our understanding of how the size of the attentional field is represented within the visual cortex. They present a novel paradigm that allows for the estimation of the spatial tuning of the attentional field and demonstrate the ability to reliably recover both the location and width of the attentional field based on BOLD responses.

      Strengths:

      (1) Innovative Paradigm: The development of a new approach to estimate the spatial tuning of the attentional field is a significant strength of this study. It provides a fresh perspective on how spatial attention modulates visual perception.

      (2) Refined fMRI Analysis: The use of fMRI to track the spatial tuning of the attentional field across different visual regions is methodologically rigorous and provides valuable insights into the neural mechanisms underlying attentional modulation.

      (3) Clear Presentation: The manuscript is well-organized, and the results are presented clearly, which aids in the reader's comprehension of the complex data and analyses involved.

      Weaknesses:

      (1) Lack of Neutral Cue Condition: The study does not include a neutral cue condition where the cue width spans 360{degree sign}, which could serve as a valuable baseline for assessing the BOLD response enhancements and diminishments in both attended and non-attended areas.

      (2) Clarity on Task Difficulty Ratios: The explicit reasoning for the chosen letter-to-number ratios for various cue widths is not detailed. Ensuring clarity on these ratios is crucial, as it affects the task difficulty and the comparability of behavioral performance across different cue widths. It is essential that observed differences in behavior and BOLD signals are attributable solely to changes in cue width and not confounded by variations in task difficulty.

      Comments on revisions:

      (1) Please standardize the naming of error metrics across Figures 4-6 to improve clarity (e.g., "angular error" (Figure 4), "|angular error|" (Figure 5), and "absolute error" (Figure 6) appear to refer to the same measure). This inconsistency is also present in the main text.

      (2) Consider briefly mentioning the baseline offset in Lines 179-186. It is included in Figures 4-7 and serves as a reference for interpreting attentional modulation alongside gain. Introducing it with other model parameters would improve clarity.

      (3) It may be valuable to examine BOLD responses in unattended visual regions. As shown in Figure 2a, suppression patterns (e.g., the most negative responses) appear to vary in extent and distribution with attentional cue width. Analyzing these unattended regions may offer a more complete view of how attention shapes the spatial profile of cortical activity.

    1. Reviewer #2 (Public review):

      Summary:

      The authors were trying to establish the role of Plasmodium falciparum surface protein 2 in merozoite biology, specifically the process of erythrocyte invasion.

      Strengths:

      The major strengths of the manuscript are in the Plasmodium falciparum genetic and phenotyping approaches. PfMSP2 knockouts are made in two different strains, which is important as it is known that invasion pathways can vary between strains, but is a level of comprehensiveness that is not always delivered in P. falciparum genetic studies. The knockout strains are characterised very thoroughly using multiple different assays, and the authors should be commended for publishing a good deal of negative data, where no phenotype was detected. This is not always done, but is very helpful for the field and reduces the potential for experimental redundancy, i.e., others repeating work that has already been performed but never published. The quality of the writing, referencing, and figures is also generally strong, although a few minor typos and technical comments on presentation have been communicated to the authors.

      Weaknesses:

      There are, however, some areas that are weaker.

      (1) The section describing Laverania and avian Plasmodium MSP2 comparison is a lengthy section and could be told much more concisely for clarity in delivering the key message, i.e., that conservation in distantly related Plasmodium species could indicate an important function. The identification of MSP2-like genes in avian Plasmodium species was highlighted previously in the referenced Escalante paper, so it is not entirely novel, although this paper goes into more detailed characterisation of the extent of conservation. Overall, this section takes up much more space in the manuscript than is merited by the novelty and significance of the findings.

      (2) Characterisation of the knockout strains is generally thorough, though relatively few interactions were followed by live microscopy (Figures 3E-H). A minimum of 30 merozoites were followed in each assay (although the precise number is not specified in the figure or legend), but there are intriguing trends in the data that could potentially have become significant if n was increased.

      (3) The comparative RNAseq data is interesting, but is not followed up to any significant degree. Multiple transcripts are up-regulated in the absence of PfMSP2, but they are largely dismissed because they are genes of unknown function, not previously linked to invasion, or lack an obvious membrane anchor. Having gone to the lengths of exploring potentially compensatory changes in gene expression, it is disappointing not to validate or explore the hits that result.

      (4) Given the abundance of PfMSP2 on the merozoite surface, it would have been interesting to see whether the knockout lines have any noticeable difference in surface composition, as viewed by electron microscopy, although, of course, this experiment relies on access to the appropriate facilities.

      (5) One of the key findings is that deletion of PfMSP2 increases inhibition by some antibodies/nanobodies (some anti-CSS2, some anti-AMA1) but not others (anti-EBA/RH, anti-EBA175, anti-Rh5, anti-TRAMP, some anti-CSS2, some anti-AMA1). The data supporting these changes in inhibition are solid, but the selectivity of the effect (only a few antibodies, and generally those targeting later stages in invasion) is not really discussed in any detail. Do the authors have a hypothesis for this selectivity? The authors make attempts to explore the mechanisms for this antibody-masking (Figure 7), but the data is less solid. Surface Plasmon Resonance was non-conclusive, while an ELISA approach co-incubating MSP2 and anti-AMA1 antibodies to wells coated with AMA1 lacks appropriate controls (eg, including other merozoite proteins in similar experiments).

      Overall, the claim that PfMSP2 is non-essential for in vitro growth is well justified and is an important contribution to the field. The impact of PfMSP2 deletion on antibody inhibition (which is highlighted in the title of the manuscript) and the mechanism behind it is much less definitive, but does open up an interesting area for further investigation, with more work to be done.

    1. Reviewer #2 (Public review):

      Summary:

      Guo et al. benchmarked and optimized methods for detecting Identity-By-Descent (IBD) segments in Plasmodium falciparum (Pf) genomes, which are characterized by high recombination rates and low marker density. Their goal was to address the limitations of existing IBD detection tools, which were primarily developed for human genomes and do not perform well in the genomic context of highly recombinant genomes. They first analysed various existing IBD callers, such as hmmIBD, isoRelate, hap-IBD, phased-IBD, and refinedIBD. They focused on the impact of recombination on the accuracy, which was calculated based on two metrics, the false negative rate and the false positive rate. The results suggest that high recombination rates significantly reduce marker density, leading to higher false negative rates for short IBD segments. This effect compromises the reliability of IBD-based downstream analyses, such as effective population size (Ne) estimation.<br /> They showed that the best tool for IBD detection in Pf is hmmIBD, because it has relatively low FN/FP error rates and is less biased for relatedness estimates. However, this method is less computationally efficient.<br /> Their suggestion is to optimize human-oriented IBD methods and use hmmIBD only for the estimation of Ne.

      Strengths:

      Although I am not an expert on Plasmodium falciparum genetics, I believe the authors have developed a valuable benchmarking framework tailored to the unique genomic characteristics of this species. Their framework enables a thorough evaluation of various IBD detection tools for non-human data, such as high recombination rates and low marker density, addressing a key gap in the field.

      This study provides a comparison of multiple IBD detection methods, including probabilistic approaches (hmmIBD, isoRelate) and IBS-based methods (hap-IBD, Refined IBD, phased IBD). This comprehensive analysis offers researchers valuable guidance on the strengths and limitations of each tool, allowing them to make informed choices based on specific use cases. I think this is important beyond the study of Pf.

      The authors highlight how optimized IBD detection can help identify signals of positive selection, infer effective population size (Ne), and uncover population structure.

      They demonstrate the critical importance of tailoring analytical tools to suit the unique characteristics of a species. Moreover, the authors provide practical recommendations, such as employing hmmIBD for quality-sensitive analyses and fine-tuning parameters for tools originally designed for non-P. falciparum datasets before applying them to malaria research.

      Overall, this study represents a meaningful contribution to both computational biology and malaria genomics, with its findings and recommendations likely to have an impact on the field.

      Weaknesses:

      One weakness of the study is the lack of emphasis on the broader importance of studying Plasmodium falciparum as a critical malaria-causing organism. Malaria remains a significant global health challenge, causing hundreds of thousands of deaths annually.

      While the study provides a thorough technical evaluation of IBD detection methods and their application to Pf, it does not adequately connect these findings to the broader implications for malaria research and control efforts. Additionally, the discussion on malaria and its global impact could have framed the study in a more accessible and compelling way, making the importance of these technical advances clearer to a broader audience, including researchers and policymakers in the fight against malaria. In the revised version, the authors have placed greater emphasis on this aspect, while still maintaining the methodological focus of the paper.

    1. Reviewer #2 (Public review):

      Gekko et al investigate the impact of perturbing mitochondrial during early embryo development, through modulation of the mitochondrial fission protein Drp1 using Trim-Away technology. They aimed to validate a role for mitochondrial dynamics in modulating chromosomal segregation, mitochondrial inheritance and embryo development and achieve this through the examination of mitochondrial and endoplasmic reticulum distribution, as well as actin filament involvement, using targeted plasmids, molecular probes and TEM in pronuclear stage embryos through the first cleavages divisions. Drp1 deletion perturbed mitochondrial distribution, leading to asymmetric partitioning of mitochondria to the 2-cell stage embryo, prevented appropriate chromosomal segregation and culminated in embryo arrest. Resultant 2-cell embryos displayed altered ATP, mtDNA and calcium levels. Microinjection of Drp1 mRNA partially rescued embryo development. A role for actin filaments in mitochondrial inheritance is described, however the actin-based motor Myo19 does not appear to contribute.

      Overall, this study builds upon their previous work and provides further support for a role of mitochondrial dynamics in mediating chromosomal segregation and mitochondrial inheritance. In particular, Drp1 is required for the redistribution of mitochondria to support symmetric partitioning and ongoing development.

      Strengths:

      The study is well designed, the methods are appropriate and the results are clearly presented. The findings are nicely summarised in a schematic.

      The addition of further quantification, including mitochondrial cluster size, elongation/aspect ratio and ROS, as requested by the reviewers, has provided further evidence for the impact of Drp1 depletion on mitochondrial morphology and function.

      Understanding the role of mitochondria in binucleation and mitochondrial inheritance is of clinical relevance for patients undergoing infertility treatment, particularly those undergoing mitochondrial replacement therapy.

      Weaknesses:

      The only remaining weakness is that the authors have not undertaken additional experiments to clarify any role for mitochondrial transport following Drp1 depletion.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript by Chuah et al. reports the experimental results that suggest the occupancy of the HbYX pockets suffices for proteasome gate opening. The authors conducted cryo-EM reconstructions of two mutant archaeal proteasomes. The work is technically sound and may be of special interest in the field of structural biology of the proteasomes.

      Strengths:

      Overall, the work incrementally deepens our understanding of the proteasome activation and expands the structural foundation for therapeutic intervention of proteasome function. The evidence presented appears to be well aligned with the existing literature, which adds confidence in the presentation.

      Comments on revisions:

      The authors have addressed all my questions.

    1. Reviewer #2 (Public review):

      This work describes the single-cell expression profiling of thousands of cells of recombinant genotypes from a model natural-variation system, a cross between two divergent yeast strains.

      I appreciate the addition of lines 282-291, which now makes the authors' point about one advantage of the single-cell technique for eQTL mapping clearly: the authors don't need to normalize for culture-to-culture variation the way standard bulk methods do (e.g. in Albert et al., 2018 for the current yeast cross), and without this normalization, they can integrate analyses of expression with those of estimates of growth behaviors from the abundance of a genotype in the pool. The main question the manuscript addresses with the latter, in Figure 3, is how much variation in growth appears to have nothing to do with expression, for which the answer the authors given is 30%. I agree that this represents a novel finding. The caveats are (1) the particular point will perhaps only be interesting to a small slice of the eQTL research community; (2) the authors provide no statistical controls/error estimate or independent validation of the variance partitioning analysis in Figure 3, and (3) the authors don't seem to use the single-cell growth/fitness estimates for anything else, as Figure 4 uses loci mapped to growth from a previously published, standard culture-by-culture approach.

    1. Reviewer #2 (Public review):

      Summary:

      This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

      Strengths:

      (1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.

      (2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with thalamus, as well as migration of cINs from CGE and MGE to cortex.

      Weaknesses:

      (1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

      (2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same mice. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

      (3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

      (4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

      Comments on revisions:

      The authors have made significant and thoughtful responses as well as experimental additions to the authors comments. Their efforts are appreciated and the manuscript is much improved.

    1. Reviewer #2 (Public review):

      This study investigates the role of cDC1 in atherosclerosis progression using Xcr1Cre-Gfp Rosa26LSL-DTA ApoE-/- mice. The authors demonstrate that selective depletion of cDC1 reduces atherosclerotic lesions in hyperlipidemic mice. While cDC1 depletion did not alter macrophage populations, it suppressed T cell activation (both CD4+ and CD8+ subsets) within aortic plaques. Further, targeting the chemokine Xcl1 (ligand of Xcr1) effectively inhibits atherosclerosis. The manuscript is well-written, and the data are clearly presented. However, several points require clarification:

      (1) In Figure 1C (upper plot), it is not clear what the Xcr1 single-positive region in the aortic root represents, or whether this is caused by unspecific staining. So I wonder whether Xcr1 single-positive staining can reliably represent cDC1. For accurate cDC1 gating in Figure 1E, Xcr1+CD11c+ co-staining should be used instead.

      (2) Figure 4D suggests that cDC1 depletion does not affect CD4+/CD8+ T cells. However, only the proportion of these subsets within total T cells is shown. To fully interpret effects, the authors should provide:<br /> a) Absolute numbers of total T cells in aortas.<br /> b) Absolute counts of CD4+ and CD8+ T cells.

      (3) How does T cell activation mechanistically influence atherosclerosis progression? Why was CD69 selected as the sole activation marker? Were other markers (e.g., KLRG1, ICOS, CD44) examined to confirm activation status?

      (4) Figure 7B: Beyond cDC1/2 proportions within cDCs, please report absolute counts of: Total cDCs,cDC1, and cDC2 subsets. Figure 7D: In addition to CD4+/CD8+ T cell proportions, the following should be included:<br /> a) Total T cell numbers in aortas<br /> b) Absolute counts of CD4+ and CD8+ T cells.

      (5) cDC1 depletion reduced CD69+CD4+ and CD69+CD8+ T cells, whereas Xcl1 depletion decreased Xcr1+ cDC1 cells without altering activated T cells. How do the authors explain these different results? This discrepancy needs explanation.

    1. Reviewer #2 (Public review):

      Summary:

      Wang et al. studied the therapeutic potential of focused ultrasound noninvasively stimulating the spleen (FUS sti. spleen) to modulate the splenic immunity, with an aim to exert anti-tumor effect. They found that the treatment enhanced antitumor capability in CD8 T/ NK cells and reduced the immunosuppression, facilitating the inhibition of HCC growth in vivo.

      Strengths:

      They have utilized bulk RNA sequencing, single cell RNA sequencing, and flow cytometry to investigate the immune and tumor cell profiling in the mouse models upon FUS sti. spleen. Moreover, they highlighted the importance of combining FUS with spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STND@Ca2+), which facilitated the calcium influx into the murine spleen and further enhanced the therapeutic efficacy of FUS.

      Weaknesses:

      While the study is interesting and potentially clinically impactful, the mechanism of action of the therapy is not fully elucidated. It would benefit from more rigorous approaches. With the theoretical part strengthened, this paper would be of interest to cancer cell biologists and clinician scientists working on the oncology field.

    1. Reviewer #2 (Public review):

      Summary:

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

      Strengths:

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

      Weaknesses/Suggestions:

      Although the design and analysis choices are generally solid, there are a few finer details/nuances that merit further clarification or consideration in order to strengthen the readers' confidence in the authors' interpretation of their data.

      (1) Given the known behavioural and neural effects of boundaries in sequence memory, I was wondering whether the number of traversed context boundaries (i.e., between morning-afternoon, and afternoon-evening) was controlled for across sequential length in the internal perspective condition? Or, was it the case that reference-target event pairs with higher sequential numbers were more likely to span across two parts of the day compared to lower sequential numbers? Similarly, did the authors examine any potential differences, whether behaviourally or neurally, for day part same vs. day part different external task trials?

      (2) I would appreciate further insight into the authors' decision to model their task trials as stick functions with duration 0 in their GLMs, as opposed to boxcar functions with varying durations, given the potential benefits of the latter (e.g., Grinband et al., 2008). I concur that in certain paradigms, RT is considered a potential confound and is taken into account as a nuisance covariate (as the authors have done here). However, given that RTs appear to be critical to the authors' interpretation of participant behavioural performance, it would imply that variations in RT actually reflect variations in cognitive processes of interest, and hence, it may be worth modelling trials as boxcar functions with varying durations.

      (3) The activity pattern across tasks and sequential distance in the posterior parietal cortex appears to parallel the RT data. Have the authors examined potential relationships between the two (e.g., individual participant slopes for RT across sequential distance vs. activity betas in the posterior parietal cortex)?

      (4) There were a few places in the manuscript where the writing/discussion of the wider literature could perhaps be tightened or expanded. For instance:

      i) On page 16, the authors state 'The negative correlation between the activation level in the right PPC and sequential distance has already been observed in a previous fMRI study (Gauthier & van Wassenhove, 2016b). The authors found a similar region (the reported MNI coordinate of the peak voxel was 42, -70, 40, and the MNI coordinate of the peak voxel in the present study was 39, -70, 35), of which the activation level went up when the target event got closer to the self-positioned event. This finding aligns with the evidence suggesting that the posterior parietal cortex implements egocentric representations.' Without providing a little more detail here about the Gauthier & van Wassenhove study and what participants were required to do (i.e., mentally position themselves at a temporal location and make 'occurred before' vs. 'occurred after' judgements of a target event), it could be a little tricky for readers to follow why this convergence in finding supports a role for the posterior parietal cortex in egocentric representations.

      ii) Although the authors discuss the Lee et al. (2020) review and related studies with respect to retrospective memory, it is critical to note that this work has also often used prospective paradigms, pointing towards sequential processing being the critical determinant of hippocampal involvement, rather than the distinction between retrospective vs. prospective processing.

      iii) The authors make an interesting suggestion with respect to hippocampal longitudinal differences in the representation of event sequences, and may wish to relate this to Montagrin et al. (2024), who make an argument for the representation of distant goals in the anterior hippocampus and immediate goals in the posterior hippocampus.

    1. Reviewer #2 (Public review):

      Summary:

      In this paper, the authors present a detailed computational model and experimental data concerning overground locomotion in rats before and after recovery from spinal cord injury. They are able to manually tune the parameters of this physiologically based, detailed model to reproduce many aspects of the observed animals' locomotion in the naive case and in two distinct injury cases.

      Strengths:

      The strengths are that the model is driven to closely match clean experimental data, and the model itself has detailed correspondence to proposed anatomical reality. As such, this makes the model more readily applicable to future experimental work. It can make useful suggestions. The model reproduces a large number of conditions across frequencies, and with the model structure changed by injury and recovery. The model is extensive and is driven by known structures, with links to genetic identities, and has been extensively validated across multiple experiments and manipulations over the years. It models a system of critical importance to the field, and the tight coupling to experimental data is a real strength.

      Weaknesses:

      A downside is that, scientifically, here, the only question tackled is one of sufficiency. By manually tuning parameters in a manner that aligns with the field's understanding from experimental work, the detailed model can accurately reproduce the experimental findings. One of the benefits of computational models is that the counterfactual can be tested to provide evidence against alternative hypotheses. That isn't really done here. I'm fairly certain that there are competing theories regarding what happens during recovery from a hemi-section injury and a contusion injury. The model could be used to make predictions for some alternative hypotheses, supporting or rejecting theories of recovery. This may be part of future plans. Here, the focus is on showing that the model is capable of reproducing the experimental results at all, for any set of parameters, however tuned.

  2. Jul 2025
    1. Reviewer #2 (Public review):

      Significance:

      TREM2 is an immunomodulatory receptor expressed on myeloid cells and microglia in the brain. TREM2 consists of a single immunoglobular (Ig) domain that leads into a flexible stalk, transmembrane helix, and short cytoplasmic tail. Extracellular proteases can cleave TREM2 in its stalk and produce a soluble TREM2 (sTREM2). TREM2 is genetically linked to Alzheimer's disease (AD), with the strongest association coming from an R47H variant in the Ig domain. Despite intense interest, the full TREM2 ligand repertoire remains elusive, and it is unclear what function sTREM2 may play in the brain. The central goal of this paper is to assess the ligand-binding role of the flexible stalk that is generated during the shedding of TREM2. To do this, the authors simulate the behavior of constructs with and without stalk. However, it is not clear why the authors chose to use the isolated Ig domain as a surrogate for full-length TREM2. Additionally, experimental binding evidence that is misrepresented by the authors contradicts the proposed role of the stalk.

      Summary and strengths:

      The authors carry out MD simulations of WT and R47H TREM2 with and without the flexible stalk. Simulations are carried out for apo TREM2 and for TREM2 in complex with various lipids. They compare results using just the Ig domain to results including the flexible stalk that is retained following cleavage to generate sTREM2. The computational methods are well-described and should be reproducible. The long simulations are a strength, as exemplified in Figure 2A where a CDR2 transition happens at ~400-600 ns. The stalk has not been resolved in structural studies, but the simulations suggest the intriguing and readily testable hypothesis that the stalk interacts with the Ig domain and thereby contributes to the stability of the Ig domain and to ligand binding. I suspect biochemists interested in TREM2 will make testing this hypothesis a high priority.

      Comments on latest version:

      The authors have addressed my critiques and carried out additional simulations, as requested. I would upgrade my assessment of the evidence to "solid."

    1. Reviewer #3 (Public review):

      Summary:

      Using flies, Kazama et al. combined behavioral analysis, electrophysiological recordings, and calcium imaging experiments to elucidate how odors activate gustatory receptor neurons (GRNs) and elicit a proboscis extension response, which is interpreted as a feeding response.

      The authors used DeepLabCut v2.0 to estimate the extension of the proboscis, which represents an unbiased and more precise method for describing this behavior compared to manual scoring.

      They demonstrated that the probability of eliciting a proboscis extension increases with higher odor concentrations. The most robust response occurs at a 0.5 v/v concentration, which, despite being diluted in the air stream, remains a relatively high concentration. Although the probability of response is not particularly high it is higher than control stimuli. Notably, flies respond with a proboscis extension to both odors that are considered positive and those regarded as negative.

      The authors used various transgenic lines to show that the response is mediated by GRNs. Specifically, inhibiting Gr5a reduces the response, while inhibiting Gr66a increases it in fed flies. Additionally, they find that odors induce a strong positive response in both types of GRNs, which is abolished when the labella of the proboscis are covered. This response was also confirmed through electrophysiological tip recordings.

      Finally, the authors demonstrated that the response increases when two stimuli of different modalities, such as sucrose and odors, are presented together, suggesting clear multimodal integration

      Strengths:

      The integration of various techniques, which collectively supports the robustness of the results.<br /> The assessment of electrophysiological recordings in intact animals, preserving natural physiological conditions.

      Weaknesses:

      Only highly concentrated odours are capable of evoking positive responses and, even then, the proportion remains relatively low.

      The authors have incorporated my suggestions.

    1. Reviewer #3 (Public review):

      Summary:

      A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding additional cellular alterations during HPV oncogenesis. Authors used the advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increase RAS signaling, especially phosphorylation of Akt and Erk1/2.

      Strengths:

      (1) HPV E6E7 are required for full immortalization and maintenance of malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further the understanding of other cellular alterations in HPV oncogenesis.<br /> (2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines and HPV transduced cell lines.<br /> (3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.<br /> (4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2 and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.<br /> (5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.<br /> (6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.

      Comments on revisions:

      The revised manuscript has been significantly improved. The authors addressed all my concerns.

    1. Reviewer #2 (Public review):

      The paper deals with the important question of gene epistasis, focusing on asking what is the correct null model for which we should declare no epistasis.

      In the first part, they use the Synthetic Genetic Array dataset to claim that the effects of a double mutation on growth rate is well predicted by the product of the individual effects (much more than e.g. the additive model). The second (main) part shows this is also the prediction of two simple, coarse-grained models for cell growth.

      I find the topic interesting, the paper well written, and the approach innovative.

      Comments on revisions:

      The authors have adequately addressed the comments raised in the review below, and I find that the paper has improved.

    1. Reviewer #2 (Public review):

      Summary:

      The authors have tried to determine the regulatory role of Phosphoglycerate mutate (PGAM), an enzyme involved in converting 3-phosphoglycerate to 2-phosphoglycerate in glycolysis, in differentiation and suppressive function of regulatory CD4 T cells through de novo serine synthesis. This is done by contributing one carbon metabolism and eventually epigenetic regulation of Treg differentiation.

      Strengths:

      The authors have rigorously used inhibitors and antisense RNA to verify the contribution of these pathways in Treg differentiation in-vitro. This has also been verified in an in-vivo murine model of autoimmune colitis. This has further clinical implications in autoimmune disorders and cancer.

      [Editors' note: The authors addressed important comments by the reviewers.]

    1. Reviewer #2 (Public review):

      Summary:

      The authors set out to characterise the GPCR family in choanoflagellates (and other unicellular holozoans). GPCRs are the most abundant gene family in many animal genomes, playing crucial roles in a wide range of physiological processes. Although they are known to evolve rapidly, GPCRs are an ancient feature of eukaryotic biology. Identifying conserved elements across the animal-protist boundary is therefore a valuable goal, and the increasing availability of genomes from non-animal holozoans provides new opportunities to explore evolutionary patterns that were previously obscured by limited taxon sampling. This study presents a comprehensive re-examination of GPCRs in choanoflagellates, uncovering examples of differential gene retention and revealing the dynamic nature of the GPCR repertoire in this group. As GPCRs are typically involved in environmental sensing, understanding how these systems evolved may shed light on how our unicellular ancestors adapted their signalling networks in the transition to complex multicellularity.

      Strengths:

      The paper combines a broad taxonomic scope with the use of both established and recently developed tools (e.g., Foldseek, AlphaFold), enabling a deep and systematic exploration of GPCR diversity. Each family is carefully described, and the manuscript also functions as an up-to-date review of GPCR classification and evolution. Although similar attempts to understand GPCR evolution were made over the last decade, the authors build on this foundation by identifying new families and applying improved computational methods to better predict structure and function. Notably, the presence of Rhodopsin-like GPCRs in some choanoflagellates and ichthyosporeans is intriguing, even though they do not fall within known animal subfamilies. The computational framework presented here is broadly applicable, offering a blueprint for surveying GPCR diversity in other non-model eukaryotes (and even in animal lineages), potentially revealing novel families relevant to drug discovery or helping revise our understanding of GPCR evolution beyond model systems.

      Weaknesses:

      While the study contributes several interesting observations, it does not radically revise the evolutionary history of the GPCR family. However, in an era increasingly concerned with the reproducibility of scientific findings, this is arguably a strength rather than a weakness. It is encouraging to see that previously established patterns largely hold, and that with expanded sampling and improved methods, new insights can be gained, especially at the level of specific GPCR subfamilies. Then, no functional follow-ups are provided in the model system Salpingoeca rosetta, but I am sure functional work on GPCRs in choanoflagellates is set to reveal very interesting molecular adaptations in the future.

    1. Reviewer #2 (Public review):

      Summary:

      This important theoretical and computational study by Burger and Gerland attempts to set environmental, compositional, kinetic, and thermodynamic constraints on the proposed virtual circular genome (VCG) model for the early non-enzymatic replication of RNA. The authors create a solid kinetic model using published kinetic and thermodynamic parameters for non-enzymatic RNA ligation and (de)hybridization, which allows them to test a variety of hypotheses about the VCG. Prominently, the authors find that the length (longer is better) and concentration (intermediate is better) of the VCG oligos have an outsized impact on the fidelity and yield of VCG production with important implications for future VCG design. They also identify that activation of only RNA monomers, which can be achieved using environmental separation of the activation and replication, can relax the constraints on the concentration of long VCG component oligos by avoiding the error-prone oligo-oligo ligation. Finally, in a complex scenario with multiple VCG oligo lengths, the authors demonstrate a clear bias for the extension of shorter oligos compared to the longer ones. This effect has been observed experimentally (Ding et al., JACS 2023) but was unexplained rigorously until now. Overall, this manuscript will be of interest to scientists studying the origin of life and the behavior of complex nucleic acid systems.

      Strengths:

      - The kinetic model is carefully and realistically created, enabling the authors to probe the VCG thoroughly.<br /> - Fig. 6 outlines important constraints for scientists studying the origin of life. It supports the claim that the separation of activation and replication chemistry is required for efficient non-enzymatic replication. One could easily imagine a scenario where activation of molecules occurs, followed by their diffusion into another environment containing protocells that encapsulate a VCG. The selective diffusion of activated monomers across protocell membranes would then result in only activated monomers being available to the VCG, which is the constraint outlined in this work. The proposed exclusive replication by monomers also mirrors the modern biological systems, which nearly exclusively replicate by monomer extension.<br /> - Another strength of the work is that it explains why shorter oligos extend better compared to the long ones in complex VCG mixtures. This point is independent of the activation chemistry used (it simply depends on the kinetics and thermodynamics of RNA base-pairing) so it should be very generalizable.

    1. Reviewer #2 (Public review):

      Summary:

      The research identifies two main SiNET subtypes (epithelial-like and neuronal-like) and reveals heterogeneity in non-neuroendocrine cells within the tumor microenvironment. The study validates findings using external datasets and explores unexpected proliferation patterns. While it contributes to understanding SiNET oncogenic processes, the limited sample size and depth of analysis present challenges to the robustness of the conclusions.

      Strengths:

      The studies effectively identified two subtypes of SiNET based on epithelial and neuronal markers. Key findings include the low proliferation rates of neuroendocrine (NE) cells and the role of the tumor microenvironment (TME), such as the impact of Macrophage Migration Inhibitory Factor (MIF).

      Weaknesses:

      However, the analysis faces challenges such as a small sample size and lack of clear biological interpretation in some analyses.

    1. Reviewer #2 (Public review):

      Summary:

      This paper investigates the evolution of pesticide resistance in the two-spotted spider mite following the introduction of an SDHI acaricide, cyatpyrafen, in China. The authors make use of cyatpyrafen-naive populations collected before that pesticide was first used, as well as more recent populations (both sensitive and resistant) to conduct comparative population genomics. They report 15 different mutations in the insecticide target site from resistant populations, many reported here for the first time, and look at the mutation and selection processes underlying the evolution of resistance, through GWAS, haplotype mapping, and testing for loss of diversity indicating selective sweeps. None of the target site mutations found in resistant populations was found in pre-exposure populations, suggesting that the mutations may have arisen de novo rather than being present as standing variation, unless initially present at very low frequencies; a de novo origin is also supported by evidence of selective sweeps in some resistant populations. Furthermore, there is no significant evidence of migration of resistant genotypes between the sampled field populations indicating multiple origins of common mutations. Overall, this indicates a very high mutation rate and a wide range of mutational pathways to resistance for this target site in this pest species. The series of population genomic analyses carried out here, in addition to the evolutionary processes that appear to underly resistance development in this case, could have implications for the study of resistance evolution more widely.

      Strengths:

      This paper combines phenotypic characterisation with extensive comparative population genomics, made possible by the availability of multiple population samples (each with hundreds of individuals) collected before as well as after then introduction of the pesticide cyatpyrafen, as well as lab-evolved lines. This resuts in findings of mutation and selection processes that can be related back to the pesticide resistance trait of concern. Large numbers of mites were tested phenotypically to show the levels of resistance present, and the authors also made near-isogenic lines to confirm the phenotypic effects of key mutations. The population genomic analyses consider a range of alternative hypotheses, including mutations arising by de novo mutation or selection from standing genetic variation; and mutations in different populations arising independently or arriving by migration. The claim that mutations most likley arose by multiple repeated de novo mutations is therefore supported by multiple lines of evidence: the direct evidence of none of the mutations being found in over 2000 individuals from naive populations, and the indirect evidence from population genomics showing evidence of selective sweeps but not of significant migration between the sampled populations.

      Weaknesses:

      As acknowledged within the discussion, whilst evidence supports a de novo origin of the resistance associated mutations, this cannot be proven definitively as mutations may have been present at a very low frequency and therefore not found within the tested pesticide-naive population samples.

      Near-isofemale lines were made to confirm the resistance levels associated with five of the 15 mutations, but otherwise the genotype-phenotype associations are correlative as confirmation by functional genetics was beyond the scope of this study.

      Comments on revisions:

      My recommendations have all been addressed in the revised version.

    1. Reviewer #2 (Public review):

      In this study, the authors identified CG14545 (named it sakura), as a key gene essential for Drosophila oogenesis. Genetic analyses revealed that Sakura is vital for both oogenesis progression and ultimate female fertility, playing a central role in the renewal and differentiation of germ stem cells (GSC).

      The absence of Sakura disrupts the Dpp/BMP signaling pathway, resulting in abnormal bam gene expression, which impairs GSC differentiation and leads to GSC loss. Additionally, Sakura is critical for maintaining normal levels of piRNAs. Also, the authors convincingly demonstrate that Sakura physically interacts with Otu, identifying the specific domains necessary for this interaction, suggesting a cooperative role in germline regulation. Importantly, the loss of otu produces similar defects to those observed in sakura mutants, highlighting their functional collaboration.

      The authors provide compelling evidence that Sakura is a critical regulator of germ cell fate, maintenance, and differentiation in Drosophila. This regulatory role is mediated through modulation of pMad and Bam expression. However, the phenotypes observed in the germarium appear to stem from reduced pMad levels, which subsequently trigger premature and ectopic expression of Bam. This aberrant Bam expression could lead to increased CycA levels and altered transcriptional regulation, impacting piRNA expression. In this revised manuscript, the authors further investigated whether Sakura affects the function of Orb, a binding partner they identified, in deubiquitinase activity when Orb interacts with Bam.

      This elaborate study will be embraced by both germline-focused scientists and the developmental biology community.

      Latest comments:

      The authors answered all my persistent concerns and made changes according to the recommendations I incorporated for the revised version of the manuscript.

    1. Reviewer #2 (Public review):

      Summary:

      The aim of the study was to understand how cells of the skin communicate across dermal layers. The research group has previously demonstrated that cellular connections called airinemes contribute to this communication. The current work builds upon this knowledge by showing that differentiated keratinocytes also use cytonemes, specialized signaling filopodia, to communicate with undifferentiated keratinocytes. They show that cytonemes are the more abundant type of cellular extension used for communication between the differentiated keratinocyte layer and the undifferentiated keratinocytes. Disruption of cytoneme formation led to expansion of the undifferentiated keratinocytes into the periderm, mimicking skin diseases like psoriasis. The authors go on to show that disruption of cytonemes results in perturbations in Notch signaling between the differentiated keratinocytes of the periderm and the underlying proliferating undifferentiated keratinocytes. Further the authors show that Interleukin-17, also known to drive psoriasis, can restrict formation of periderm cytonemes, possibly through the inhibition of Cdc42 expression. This work suggests that cytoneme mediated Notch signaling plays a central role in normal epidermal regulation. The authors propose that disruption of cytoneme function may be an underlying cause of various human skin diseases.

      Strengths:

      The authors provide strong evidence that periderm keratinocytes cytonemes contain the notch ligand DeltaC to promote Notch activation in the underlying intermediate layer to regulate accurate epidermal maintenance.

      Weaknesses:

      The impact of the study would be increased if the mechanism by which Interlukin-17 and Cdc42 collaborate to regulate cytonemes was defined. Experiments measuring Cdc42 activity, rather than just measuring expression, would strengthen the conclusions.

      Comments on revisions:

      The authors have sufficiently addressed my critiques from the initial round of evaluation. They have included useful representative images, clarified how they scored cytonemes and provided additional controls/experimental conditions that improve the rigor of the study. The results provided now support the key conclusions of the study.

    1. Reviewer #2 (Public review):

      Summary:

      The function of neural circuits relies heavily on the balance of excitatory and inhibitory inputs. Particularly, inhibitory inputs are understudied when compared to their excitatory counterparts due to the diversity of inhibitory neurons, their synaptic molecular heterogeneity, and their elusive signature. Thus, insights into these aspects of inhibitory inputs can inform us largely on the functions of neural circuits and the brain.

      Endophilin A1, an endocytic protein heavily expressed in neurons, has been implicated in numerous pre- and postsynaptic functions, however largely at excitatory synapses. Thus, whether this crucial protein plays any role in inhibitory synapse, and whether this regulates functions at the synaptic, circuit, or brain level remains to be determined.

      New Findings:

      (1) Endophilin A1 interacts with the postsynaptic scaffolding protein gephyrin at inhibitory postsynaptic densities within excitatory neurons.

      (2) Endophilin A1 promotes the organization of the inhibitory postsynaptic density and the subsequent recruitment/stabilization of GABA A receptors via Endophilin A1's membrane binding and actin polymerization activities.

      (3) Loss of Endophilin A1 in CA1 mouse hippocampal pyramidal neurons weakens inhibitory input and leads to susceptibility to epilepsy.

      (4) Thus the authors propose that via its role as a component of the inhibitory postsynaptic density within excitatory neurons, Endophilin A1 supports the organization, stability, and efficacy of inhibitory input to maintain the excitatory/inhibitory balance critical for brain function.

      (5) The conclusion of the manuscript is well supported by the data but will be strengthened by addressing our list of concerns and experiment suggestions.

      Comments on revised version:

      The authors addressed the concerns adequately. The three remaining concerns are:

      (1) The use of one-way ANOVA is not well justified.

      (2) The use of superplots to show culture to culture variability would make it more transparent.

      (3) Change EEN1 in Figure 8B to EndoA1.

    1. Reviewer #2 (Public review):

      In this manuscript, Ross and Miscik et. al described the phenotypic discrepancies between F0 zebrafish mosaic mutant ("CRISPants") and morpholino knockdown (Morphant) embryos versus a set of 5 different loss-of-function (LOF) stable mutants in one particular gene involved in hepatic stellate cells development: podxl. While transient LOF and mosaic mutants induced a decrease of hepatic stellate cells number stable LOF zebrafish did not. The authors analyzed the molecular causes of these phenotypic differences and concluded that LOF mutants are genetically compensated through the upregulation of the expression of many genes. Additionally, they ruled out other better-known and described mechanisms such as the expression of redundant genes, protein feedback loops, or transcriptional adaptation.

      While the manuscript is clearly written and conclusions are, in general, properly supported, there are some aspects that need to be further clarified and studied.

      (1) It would be convenient to apply a method to better quantify potential loss-of-function mutations in the CRISPants. Doing this it can be known not only percentage of mutations in those embryos but also what fraction of them are actually generating an out-of-frame mutation likely driving gene loss of function (since deletions of 3-6 nucleotides removing 1-2 aminoacid/s will likely not have an impact in protein activity, unless that this/these 1-2 aminoacid/s is/are essential for the protein activity). With this, the authors can also correlate phenotype penetrance with the level of loss-of-function when quantifying embryo phenotypes that can help to support their conclusions.

      (2) It is unclear that 4.93 ng of morpholino per embryo is totally safe. The amount of morpholino causing undesired effects can differ depending on the morpholino used. I would suggest performing some sanity check experiments to demonstrate that morpholino KD is not triggering other molecular outcomes, such as upregulation of p53 or innate immune response.

      (3) Although the authors made a set of controls to demonstrate the specificity of the CRISPant phenotypes, I believe that a rescue experiment could be beneficial to support their conclusions. Injecting an mRNA with podxl ORF (ideally with a tag to follow protein levels up) together with the induction of CRISPants could be a robust manner to demonstrate the specificity of the approach. A rescue experiment with morphants would also be good to have, although these are a bit more complicated, to ultimately demonstrate the specificity of the approach.

      (4) In lines 314-316, the authors speculate on a correlation between decreased HSC and Podxl levels. It would be interesting to actually test this hypothesis and perform RT-qPCR upon CRISPant induction or, even better and if antibodies are available, western blot analysis.

      (5) Similarly, in lines 337-338 and 342-344, the authors discuss that it could be possible that genes near to podxl locus could be upregulated in the mutants. Since they already have a transcriptomic done, this seems an easy analysis to do that can address their own hypothesis.

      (6) Figures 4 and 5 would be easier to follow if panels B-F included what mutants are (beyond having them in the figure legend). Moreover, would it be more accurate and appropriate if the authors group all three WT and mutant data per panel instead of showing individual fish? Representing technical replicates does not demonstrate in vivo variability, which is actually meaningful in this context. Then, statistical analysis can be done between WT and mutant per panel and per set of primers using these three independent 3-month-old zebrafish.

    1. Reviewer #2 (Public review):

      Summary:

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

      My comments are on the MD part.

      Strengths:

      The study

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

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

      Weaknesses:

      Unphysiological membrane potential. A field of 0.1 V nm⁻¹ (~1 V across the bilayer) was applied to accelerate translocation. From the traces (Figure 1c), it can be seen that the translocation occurred really quickly through the channel, suggesting that the field might have introduced some large changes in the protein. The authors state that they checked visually for this, but some additional analysis, especially of the residues next to the drug, would be welcome.

      Based on applied voltage simulations, the authors argue that the membrane potential would help get the drug into the cell, and that a high value of the potential was applied merely to speed up the simulation. At the same time, the barrier for translocation from PMF calculations is ~40 kJ/mol for WT. Is the physiological membrane voltage enough to overcome this barrier in a realistic time? In this context, I do not see how much value the applied voltage simulations have, as one can estimate the work needed to translocate the substrate on PMF profiles alone. The authors might want to tone down their conclusions about the role of membrane voltage in the drug translocation.

      Pentamidine charge state and protonation. The ligand was modeled as +2, yet pKa values might change with the micro-environment. Some justification of this choice would be welcome.

      I don't follow the RMSD calculations. The authors state that this RMSD is small for the substrate and show plots in Figure S7a, with the bottom plot being presumably done for the substrate (the legends are misleading, though), levelling off at ~0.15 nm RMSD. However, in Figure S7a, we see one trace (light blue) deviating from the initial position by more than 0.2 nm - that would surely result in an RMSD larger than 0.15, but this is somewhat not reflected in the RMSD plots.

    1. Reviewer #2 (Public review):

      Summary:

      Using a genetic model of beta-pix conditional trap, the authors are able to regulate the spatio-temporal depletion of beta-pix, a gene with an established role in maintaining vascular integrity (shown elsewhere). This study provides strong in vivo evidence that glial beta-pix is essential to the development of the blood-brain barrier and maintaining vascular integrity. Using genetic and biochemical approaches, the authors show that PAK1 and Stathmins are in the same signaling axis as beta-pix, and act downstream to it, potentially regulating cytoskeletal remodeling and controlling glial migration. How exactly the glial-specific (beta-pix driven-) signaling influences angiogenesis or vascular integrity is not clear.

      Strengths:

      (1) Developing a conditional gene-trap genetic model which allows for tracking knockin reporter driven by endogenous promoter, plus allowing for knocking down genes. This genetic model enabled the authors to address the relevant scientific questions they were interested in, i.e., a) track expression of beta-pix gene, b) deletion of beta-pix gene in a cell-specific manner.

      (2) The study reveals the glial-specific role of beta-pix, which was unknown earlier. This opens up avenues for further research. (For instance, how do such (multiple) cell-specific signaling converge onto endothelial cells which build the central artery and maintain the blood-brain barriers?)

      Weaknesses:

      Major:

      (1) The study clearly establishes a role of beta-pix in glial cells, which regulates the length of the central artery and keeps the hemorrhages under control. Nevertheless, it is not clear how this is accomplished.<br /> a. Is this phenotype (hemorrhage) a result of the direct interaction of glial cells and the adjacent endothelial cells? If direct, is the communication established through junctions or through secreted molecules?<br /> b. The authors do not exclude the possibility that the effects observed on endothelial cells (quantified as length of central artery) could be secondary to the phenotype observed with deletion of glial beta-pix. For instance, can glial beta-pix regulate angiogenic factors secreted by peri-vascular cells, which consequently regulate the length of the central artery or vascular integrity?<br /> c. The pictorial summary of the findings (Figure 7) does not include Zfhx or Vegfa. The data do not provide clarity on how these molecules contribute (directly or indirectly) to endothelial cell integrity. Vegfaa is expressed in the central artery, but the expression of the receptor in these endothelial cells is not shown. Similarly, all other experimental analyses for Zfhx and Vegfa expression were performed in glial cells. More experimental evidence is necessary to show the regulation of angiogenesis (of endothelial cells) by glial beta-pix. Is the Vegfaa receptor present on central arteries, and how does glial depletion of beta-pix affect its expression or response of central artery endothelial cells (both pertaining to angiogenesis and vascular integrity).

      (2) Microtubule stabilization via glial beta-pix, claimed in Figure 5M, is unclear. Magnified images for h-betapix OE and h-stmn-1 glial cells are absent. Is this migration regulated by beta-pix through its GEF activity for Cdc42/Rac?

      (3) Hemorrhages are caused by compromised vascular integrity, which was not measured (either qualitatively or quantitatively) throughout the manuscript. The authors do measure the length of the central artery in several gene deletion models (2I, 3C. 5F/J, 6G/K), which is indicative of artery growth/ angiogenesis. How (if at all) defects in angiogenesis are an indication of hemorrhage should be explained or established. Do these angiogenic growth defects translate into junctional defects at later developmental timepoints? Formation and maintenance of endothelial cell junctions within the hemorrhaging arteries should be assessed in fish with deleted beta-pix from astrocytes.

      (4) More information is required about the quality control steps for 10X sequencing (Figure 4, number of cells, reads, etc.). What steps were taken to validate the data quality? The EC groups, 1 and 2-days post-KO are not visible in 4C. One appreciates that the progenitor group is affected the most 2 days post-KO. But since the effects are expected to be on the endothelial cell group as well (which is shown in in vivo data), an extensive analysis should be done on the EC group (like markers for junctional integrity, angiogenesis, mesenchymal interaction, etc.). Are Stathmins limited to glial cells? Are there indicators for angiogenic responses in endothelial cells?

    1. Reviewer #2 (Public review):

      This study by M. Blatkiewicz et al. seeks to define the spatial gene expression pattern of the adult male mouse adrenal gland using current spatial transcriptomic techniques. They propose new zone-specific gene markers and specific intra- and inter-zonal signaling pathways based on receptor-ligand expression patterns. Their web tool is user-friendly and will be helpful for adrenal scientists. The manuscript is easy to follow, but validation of crucial results of the large dataset is missing. There are also several contradictory results/interpretations, and the opportunity to dissect the sexually dimorphic gene expression pattern and mouse-human interspecies differences is a missed opportunity.

      (1) The authors used 10-week-old CD1 male mouse adrenal glands to assess the spatial transcriptomics of the adrenal gland. As they also mentioned, male mice typically lose their zone-X after puberty (around 6-8 weeks of age). However, their analysis in 10-week-old mice suggests that zone-X covers most of the adrenal cortex. As shown in Figure 3A, the dots between the zona glomerulosa and the medulla are mostly positive for zone-X, which would suggest that the zona fasciculata represents a relative minority of the overall adult adrenal cortex. Is this correct? Is the presence of zone-X in sexually mature adult male mice unique to the CD1 strain? Providing histology data in support of this conclusion, using zone-specific markers combined with RNA in situ hybridization or immunofluorescence techniques in the CD1 male adrenal gland, would help to interpret these data further. Given the relatively low resolution of their gene expression profiles, it is possible there is overlap between the zona fasciculata and the zone-X.

      (2) The pseudotime trajectory analysis confirms prior reports in the literature showing zonal transdifferentiation but does not provide novel insight. It would be nice to know what gene expression patterns correlate (positively or negatively) based on an unbiased analysis.

      (3) The authors suggest that they identified new zonal markers, but it would be nice to see confirmation of some of these markers (e.g., Frmpd4, Oca2, Sphkap for the ZG or Cited1, Nat8f5 for the ZF, etc. ) with in situ or immunofluorescence combined with known markers such as Dab2, Cyp11b2, or Cyp11b1.

      (4) The authors mention a gradual transition between the zones. It would be interesting to know whether transition zones exist between the zona glomerulosa and the zona fasciculata or the zona fasciculata and the zone-X.

      (5) The authors note using Visium cyst assist, but they do not discuss the advantages of this system compared to other systems. Explanation of the approximate resolution of their analysis (e.g., how many cells were pooled in the wells) would help readers to interpret their data. It would also be nice to compare it to other spatial transcriptomic analyses of human adrenals, given the differences between the zonation of human and mouse adrenals.

      (6) Interestingly, CellChat analysis suggests possible communication between the medulla and the zona fasciculata and zona glomerulosa. How do the authors explain the transfer of these molecules from the medulla to the outer zones given centripetal blood flow in the adrenal? Also, how does the fact that Igf2 expression has been shown to be expressed in the capsule (PMID: 22266195) affect the interpretation of their data?

      (7) The study misses the opportunity to dissect sexually dimorphic gene expression patterns in the mouse adrenal. For example, the authors could have focused on the role of stem cells between male and female mouse adrenals, which have been reported to differ (PMID: 31104943). In addition, the authors could have focused on the sexually dimorphic zone-X and its regulation by sex hormone signaling.

      (8) The capsule is classified as a connective tissue, which may be misleading given its important role as a signaling center in the adrenal. Genes enriched in typical connective tissues do not include many of the genes that seem to define the adrenal capsule. Also, some of the capsule markers appear to be found in the zona glomerulosa. Is this a result of not being able to fully resolve the small layer of zG cells and the even smaller layer of capsular cells? Guided reclustering of the cells based on known markers and separation of capsule and connective tissue might help to present their data on adrenal zonation more clearly.

    1. Reviewer #2 (Public review):

      Summary:

      The current paper consists of two parts. The first part is the rigorous feature optimization of the MEG signal to decode individual finger identity performed in a sequence (4-1-3-2-4; 1~4 corresponds to little~index fingers of the left hand). By optimizing various parameters for the MEG signal, in terms of (i) reconstructed source activity in voxel- and parcel-level resolution and their combination, (ii) frequency bands, and (iii) time window relative to press onset for each finger movement, as well as the choice of decoders, the resultant "hybrid decoder" achieved extremely high decoding accuracy (~95%).

      In the second part of the paper, armed with the successful 'hybrid decoder,' the authors asked how neural representation of individual finger movement that is embedded in a sequence, changes during a very early period of skill learning and whether and how such representational change can predict skill learning. They assessed the difference in MEG feature patterns between the first and the last press 4 in sequence 41324 at each training trial and found that the pattern differentiation progressively increased over the course of early learning trials. Additionally, they found that this pattern differentiation specifically occurred during the rest period rather than during the practice trial. With a significant correlation between the trial-by-trial profile of this pattern differentiation and that for accumulation of offline learning, the authors argue that such "contextualization" of finger movement in a sequence (e.g., what-where association) underlies the early improvement of sequential skill. This is an important and timely topic for the field of motor learning and beyond.

      Strengths:

      The use of temporally rich neural information (MEG signal) has a significant advantage over previous studies testing sequential representations using fMRI. This allowed the authors to examine the earliest period (= the first few minutes of training) of skill learning with finer temporal resolution. Through the optimization of MEG feature extraction, the current study achieved extremely high decoding accuracy (approx. 94%) compared to previous works. The finding of the early "contextualization" of the finger movement in a sequence and its correlation to early (offline) skill improvement is interesting and important. The comparison between "online" and "offline" pattern distance is a neat idea.

      Weaknesses:

      One potential weakness, in terms of the generality, is that the study assessed the single sequence, the "41324" across all participants. Future confirmation test of using different sequences would be important.

  3. Jun 2025
    1. Reviewer #2 (Public review):

      Summary:

      In this work, the investigators isolated one Lacticaseibacillus rhamnosus strain (P118), and determined this strain worked well against Salmonella Typhimurium infection. Then, further studies were performed to identify the mechanism of bacterial resistance, and a list of confirmatory assays were carried out to test the hypothesis.

      Strengths:

      The authors provided details regarding all assays performed in this work, and this reviewer trusted that the conclusion in this manuscript is solid. I appreciate the efforts of the authors to perform different types of in vivo and in vitro studies to confirm the hypothesis.

    1. Reviewer #2 (Public review):

      This manuscript by Walton et al. suggests that they have identified a new bacteriophage that uses the exopolysaccharide Psl from Pseudomonas aeruginosa (PA) as a receptor. As Psl is an important component in biofilms, the authors suggest that this phage (and others similarly isolated) may be able to specifically target biofilm-growing bacteria.

      Comments on revised version:

      The authors have generally responded well to the reviewers' comments. This has served to improve this manuscript that has identified a new bacteriophage that uses the exopolysaccharide Psl from Pseudomonas aeruginosa as a receptor.

    1. Reviewer #2 (Public review):

      Summary:

      The authors attempted to investigate the pangenome of MTBC by using a selection of state-of-the-art bioinformatic tools to analyse 324 complete and 11 new genomes representing all known lineages and sublineages. The aim of their work was to describe the total diversity of the MTBC and to investigate the driving evolutionary force. By using long read and hybrid approaches for genome assembly, an important attempt was made to understand why the MTBC pangenome size was reported to vary in size by previous reports. This study provides strong evidence that the MTBC pangenome is closed and that genome reduction is the main driver of this species evolution.

      Strengths:

      A stand-out feature of this work is the inclusion of non-coding regions as opposed to only coding regions which was a focus of previous papers and analyses which investigated the MTBC pangenome. A unique feature of this work is that it highlights sublineage-specific regions of difference (RDs) that was previously unknown. Another major strength is the utilisation of long-read whole genomes sequences, in combination with short-read sequences when available. It is known that using only short reads for genome assembly has several pitfalls. The parallel approach of utilizing both Panaroo and Pangraph for pangenomic reconstruction illuminated limitations of both tools while highlighting genomic features identified by both. This is important for any future work and perhaps alludes to the need for more MTBC-specific tools to be developed. Lastly, ample statistical support in the form of Heaps law and genome fluidity calculations for each pangenome to demonstrate that they are indeed closed.

      Weaknesses:

      There are no major weaknesses in the revised version of this manuscript.

    1. Reviewer #3 (Public review):

      Summary:

      The authors attempted to study connections with the inferior olive to the cerebellar cortex and analyze impacts on optokinetic reflex using optogenetics to perturb the pathway. This is a commendable effort as these methods are very challenging due to the location of the inferior olive and recording methods.

      Strengths:

      The authors have shown that climbing fiber activity was altered due to the optogenetic perturbation. They have added an additional figure to show that complex spikes disappear with inhibitory optogenetics and the impacts on behavior are interesting.

      Weaknesses:

      The images provided to show injection region are difficult to see and specific cell types are not co-labeled. The data and strength of the results would benefit from high-resolution images demonstrating selectivity and expression, in particular for Figure 2A and 3A. In addition, while the processed recording data looks very striking, including the raw data, as done in Figure 2, would again support the conclusions.

      One major concern is that the viruses chosen are non-specific to the cell targets and a cre-based approach is lacking to draw conclusions on only the targeted pathway of interest. It is unclear based on the figures provided if the AAVs labeled only the pathway of interest. It would be interesting to know if typical memory acquisition returns in the same animals if inhibition stops and if animal movement was impacted by the perturbation.

    1. Reviewer #2 (Public review):

      Summary:

      The study introduces new tools for measuring intracellular Ca2+ concentration gradients around retinal rod bipolar cell (rbc) synaptic ribbons. This is done by comparing the Ca2+ profiles measured with mobile Ca2+ indicator dyes versus ribbon-tethered (immobile) Ca2+ indicator dyes. The Ca2+ imaging results provide a straightforward demonstration of Ca2+ gradients around the ribbon and validate their experimental strategy. This experimental work is complemented by a coherent, open-source, computational model that successfully describes changes in Ca2+ domains as a function of Ca2+ buffering. In addition, the authors try to demonstrate that there is heterogeneity among synaptic ribbons within an individual rbc terminal.

      Strengths:

      The study introduces a new set of tools for estimating Ca2+ concentration gradients at ribbon AZs, and the experimental results are accompanied by an open-source, computational model that nicely describes Ca2+ buffering at the rbc synaptic ribbon. In addition, the dissociated retinal preparation remains a valuable approach for studying ribbon synapses. Lastly, excellent EM.

      Comments on revisions:

      Specific minor comments:

      (1) Rewrite the final sentence of the Abstract. It is difficult to understand.

      (2) Add a definition in the Introduction (and revisit in the Discussion) that delineates between micro- and nano-domain. A practical approach would be to round up and round down. If you round up from 0.6 um, then it is microdomain which means ~ 1 um or higher. Likewise, round down from 0.3 um to nanodomain? If you are using confocal, or even STED, the resolution for Ca imaging will be in the 100 to 300 nm range. The point of your study is that your new immobile Ca2-ribbon indicator may actually be operating on a tens of nm scale: nanophysiology. The Results are clearly written in a way that acknowledges this point but maybe make such a "definition" comment in the intro/discussion in order to: 1) demonstrate the power of the new Ca2+ indicator to resolve signals at the base of the ribbon (effectively nano), and 2) (Discussion) to acknowledge that some are achieving nanoscopic resolution (50 to 100nm?) with light microscopy (as you ref'd Neef et al., 2018 Nat Comm).

      (3) Suggested reference: Grabner et al. 2022 (Sci Adv, Supp video 13, and Fig S5). Here rod Cav channels are shown to be expressed on both sides the ribbon, at its base, and they are within nanometers from other AZ proteins. This agrees with the conclusions from your imaging work.

      (4) In the Discussion, add a little more context to what is known about synaptic transmission in the outer and inner retina.. First, state that the postsynaptic receptors (for example: mGluR6-OnBCs vs KARs-Off-BCs, vs. AMPAR-HCs), and possibly the synaptic cleft (ground squirrel), are known to have a significant impact on signaling in the outer retina. In the inner retina, there are many more unknowns. For example, when I think of the pioneering Palmer JPhysio study, which you sight, I think of NMDAR vs AMPAR, and uncertainty in what type postsynaptic cell was patched (GC or AC....). Once you have informed the reader that the postsynapse is known to have a significant impact on signaling, then promote your experimental work that addresses presynaptic processes: "...the new tool and results allow us to explore release heterogeneity, ribbon by ribbon in dissociated preps, which we eventually plan to use at ribbon synapses within slices......to better understand how the presynapse shapes signaling......".

    1. Reviewer #2 (Public review):

      Summary:

      The authors show that co-expression of bassoon, RIBEYE, Cav1.3-alpha1, Cav-beta3, Cav-alpha2delta1, and RBP2 in a heterologus system (HEK293 cells) is sufficient to generate a protein complex resembling a presyanptic ribbon-type active zone both in morphology and in function (in clustering voltage-gated Ca channels and creating sites for localized Ca2+ entry). If the 3 separate Cav gene products are taken as a single protein (i.e. a Ca channel), the conclusion is that the core of a ribbon synapse comprises 4 proteins: bassoon holds the RIBEYE-containing ribbon to the plasma membrane, and RPB2 binds to bassoon and Ca channels, tethering the Ca channels to the presynaptic active zone.

      Strengths:

      (1) Good use of a heterologous system with generally appropriate controls provides convincing evidence that a presynaptic ribbon-type active zone (without the ability to support exocytosis), with the ability to support localized Ca2+ entry (a key feature of ribbon-type pre-synapses) can be assembled from a few proteins.<br /> (2) In the revised manuscript, the authors do a good job of addressing the limitations of their cultured cell-system.

      Weaknesses:

      (1) Relies on over-expression, which almost certainly diminishes the experimentally-measured parameters (e.g. pre-synapse clustering, localization of Ca2+ entry).<br /> (2) Are HEK cells the best model? HEK cells secrete substances and have a studied-endocytitic pathway, but they do not create neurosecretory vesicles. Initially, I asked why didn't the authors did not try to reconstitute a ribbon synapse in a cell that makes neurosecretory vesicles like a PC12 cell, and the authors addressed this question in their revision.<br /> (3) Related to 1 and 2: the Ca channel localization observed is significant but not so striking given the presence of Cav protein and measurements of Ca2+ influx distributed across the membrane. Presumably, this is the result of overexpression and an absence of pathways for pre-synaptic targeting of Ca channels. But, still, it was surprising that Ca channel localization was so diffuse. I suppose that the authors tried to reduce the effect of over-expression by using an inducible Cav1.3? Even so, the accessory subunits were constitutively over-expressed.

    1. Reviewer #1 (Public review):

      Summary:

      The authors, Dalal, et. al., determined cryo-EM structures of open, closed, and desensitized states of the pentameric ligand-gated ion channel ELIC reconstituted in liposomes, and compared them to structures determined in varying nanodisc diameters. They argue that the liposomal reconstitution method is more representative of functional ELIC channels, as they were able to test and recapitulate channel kinetics through stopped-flow thallium flux liposomal assay. The authors and others have described channel interactions with membrane scaffold proteins (MSP), initially thought to be in a size-dependent manner. However, the authors reported their cryo-EM ELIC structure interacts with the large nanodisc spNW25, contrary to their original hypotheses. This suggests that the channels interactions with MSPs might alter its structure, possibly influencing the functional states of the channel. Thus, the authors describe reconstitution in liposomes are more representative of the native structure and can recapitulate all channel states.

      Strengths:

      Cryo-EM structural determination from proteoliposomes is promising methodology within the ion channel field due to their large surface area and lack of MSP or other membrane memetics that could alter channel structure. The authors succeeded in comparing structures determined in liposomes to those in a wide range of nanodisc diameters. This comparison gives rise to important discussions for other membrane protein structural studies when deciding the best method for individual circumstances.

      Weaknesses:

      As the overarching goal of the study was to determine structural differences of ELIC in detergent nanodiscs and liposomes. The authors stated they determined open, closed, and desensitized states of ELIC reconstituted in liposomes and suggest the desensitization gate is at the 9' region of the pore. However, limited functional data was provided when determining the functional states of the channel with most of the evidence deriving from structures, which only provides snapshots of channels.

    1. Reviewer #2 (Public review):

      Summary:

      In this paper, the authors test whether controllability beliefs and associated actions/resource allocation are modulated by things like time, effort, and monetary costs (what they call "elastic" as opposed to "inelastic" controllability). Using a novel behavioral task and computational modeling, they find that participants do indeed modulate their resources depending on whether they are in an "elastic," "inelastic," or "low controllability" environment. The authors also find evidence that psychopathology is related to specific biases in controllability.

      Strengths:

      This research investigates how people might value different factors that contribute to controllability in a creative and thorough way. The authors use computational modeling to try to dissociate "elasticity" from "overall controllability," and find some differential associations with psychopathology. This was a convincing justification for using modeling above and beyond behavioral output and yielded interesting results. Notably, the authors conclude that these findings suggest that biased elasticity could distort agency beliefs via maladaptive resource allocation. Overall, this paper reveals important findings about how people consider components of controllability.

      Weaknesses:

      The authors have gone to great lengths to revise the manuscript to clarify their definitions of "elastic" and "inelastic" and bolster evidence for their computational model, resulting in an overall strong manuscript that is valuable for elucidating controllability dynamics and preferences. One minor weakness is that the justification for the analysis technique for the relationships between the model parameters and the psychopathology measures remains lacking given the fact that simple correlational analyses did not reveal any significant associations.

    1. Reviewer #2 (Public review):

      Summary:

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

      Strengths:

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

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

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

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

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

      Weaknesses:

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

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

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

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

    1. Reviewer #2 (Public review):

      Summary:

      Tsurumi et al. show that recurrent neural networks can learn state and value representations in simple reinforcement learning tasks when trained with random feedback weights. The traditional method of learning for recurrent network in such tasks (backpropogation through time) requires feedback weights which are a transposed copy of the feed-forward weights, a biologically implausible assumption. This manuscript builds on previous work regarding "random feedback alignment" and "value-RNNs", and extends them to a reinforcement learning context. The authors also demonstrate that certain non-negative constraints can enforce a "loose alignment" of feedback weights. The author's results suggest that random feedback may be a powerful tool of learning in biological networks, even in reinforcement learning tasks.

      Strengths:

      The authors describe well the issues regarding biologically plausible learning in recurrent networks and in reinforcement learning tasks. They take care to propose networks which might be implemented in biological systems and compare their proposed learning rules to those already existing in literature. Further, they use small networks on relatively simple tasks, which allows for easier intuition into the learning dynamics.

      Weaknesses:

      The principles discovered by the authors in these smaller networks are not applied to larger networks or more complicated tasks with long temporal delays (>100 timesteps), so it remains unclear to what degree these methods can scale or can be used more generally.

      Comments on revisions: I would still want to see how well the network learns tasks with longer time delays (on the order of 100 or even 1000 timesteps). Previous work has shown that random feedback struggles to encode longer timescales (see Murray 2019, Figure 2), so I would be interested to see how that translates to the RL context in your model.

    1. But scientists have also identified at least

      The climate crisis Is approaching Irreversible tipping points

    2. Even at the current level of about 1.5C, the impacts of warming are emerging on the worst side of the range of possible outcomes and there is growing concern of tipping points for the

      Scientists warn that we are nearing or have already crossed several environmental tipping points (e.g. AMOC, Antarctic sea ice, coral reefs).

    1. Reviewer #3 (Public review):

      Summary:

      The study uses publicly available sequences of classical and non-classical genes from a number of primate species to assess the extent and depth of TSP across the primate phylogeny. The analyses were carried out in a coherent and, in my opinion, robust inferential framework and provide evidence for ancient (even > 30 million years) TSP at several classical class I and class II genes. The authors also characterise evolutionary rates at individual codons, map these rates onto MHC protein structures, and find that the fastest evolving codons are extremely enriched for autoimmune and infectious disease associations.

      Strengths:

      The study is comprehensive, relying on a large data set, state-of-the-art phylogenetic analyses and elegant tests of TSP. The results are not entirely novel, but a synthesis and re-analysis of previous findings is extremely valuable and timely.

      Weaknesses:

      Following the revision by the Authors I see mostly one weakness - Older literature on the subject is duly cited, but the discussion of the findings the context of this literature is limited.

      Comments on revisions:

      Lines 441-452 - In this section, you discuss an apparent paradox between long-lived balancing selection and strong directional selection, referencing elevated substitution rates. However, this issue is more nuanced and may not be best framed in terms of substitution rates. That terminology is common in phylogenetic analyses, where differences between sequences-or changes along phylogenetic branches-are often interpreted as true substitutions in the population genetic sense. In the case of MHC trees and the rates you're discussing here, the focus is more accurately on the rate at which new mutations become established within particular allelic lineages. So while this still concerns evolutionary rates at specific codons, equating them directly with substitution rates may be misleading. A more precise term or framing might be warranted in this context.

    1. Reviewer #2 (Public review):

      Summary:

      In their manuscript, Gouirand et al. report on the role of Layilin expression for the motility and suppressive capacity of regulatory T cells (Tregs). In previous studies, the authors had already demonstrated that Layilin is expressed on Tregs, that it acts as a negative regulator of their suppressive capacity, that it functions to anchor Tregs in non-lymphoid tissues, and that it enhances the adhesive properties of Layilin-expressing cells by co-localization with the integrin αLβ2 (LFA-1). Building on these published data, the authors now show that Layilin is highly expressed on a subset of clonally expanded effector Tregs in both healthy and psoriatic skin and that deletion of Layilin in Tregs in vivo resulted in significantly attenuated skin inflammation. Furthermore, the authors addressed the molecular mechanism by which Layilin affects the suppressive capacity of Tregs and showed that Layilin increased Treg adhesion via modulation of LFA-1, resulting in distinct cytoskeletal changes.

      Strengths:

      Certainly, the strength of this study lies in the combination of data from mouse and human models.

      Weaknesses:

      Some of the conclusions drawn by the authors must be treated with caution, as the experimental conditions were not always appropriate, leading to a risk of misinterpretation.

    1. Reviewer #2 (Public review):

      Summary:

      Microglia have been implicated in brain development, homeostasis, and diseases. "Microglia replacement" has gain tractions in recent years, using primary microglia, bone marrow or blood-derived myeloid cells, or human iPSC-induced microglia. Here, the authors extended their previous work in the area and provide evidence to support: (1) Estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages from bone marrow can serve as stable, genetically manipulated cell lines. These cells are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells (MLCs). Taking advantage of this model system, the authors created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières Syndrome (AGS) disease mechanism.

      Strengths:

      The studies are carefully designed and well-conducted. The imaging data and gene expression analysis are carried out at a high level of technical competences and the studies provide strong evidence that ER-Hoxb8 immortalized macrophages from bone marrow are a reasonable source for "microglia replacement" exercise. The findings are clearly presented, and the main message will be of general interest to the neuroscience and microglia communities.

    1. Reviewer #2 (Public review):

      The authors aimed to develop an animal model of temporal lobe epilepsy (TLE) that will generate "on-demand" seizures and an improved platform to advance our ability to find new anti-seizure drugs (ASDs) for drug-resistant epilepsy (DRE). Unlike some of the work in this field, the authors are studying actual seizures, and hopefully events that are similar to actual epileptic seizures. To develop an optimized screening tool, however, one also needs high-throughput systems with actual seizures as a quantitative, rigorous, and reproducible outcome measures. The authors aim to provide such a model; however, this approach may be over-stated here and seems unlikely to address the critical issue of drug resistance, which is their most important claim.

      Strengths:

      - The authors have generated an animal model of "on demand" seizures, which could be used to screen new ASDs and potentially other therapies. The authors and their model make a good-faith effort to emulate the epileptic condition and to use seizure susceptibility or probability as a quantitative output measure.

      - The events considered to be seizures appear to be actual seizures, with some evidence that the seizures are different from seizures in the naïve brain. Their effort to determine how different ASDs raise seizure probability or threshold to an optogenetic stimulus to the CA1 area of the rodent hippocampus is focused on an important problem, as many if not most ASD screening uses surrogate measures that may not be as well linked to actual epileptic seizures.

      - Another concern is their stimulation of dorsal hippocampus, while ventral hippocampus would seem more appropriate.

      - Use of optogenetic techniques allows specific stimulation of the targeted CA1 pyramidal cells, and it appears that this approach is reproducible and reliable with quantitative rigor.

      - The authors have taken on a critically important problem, and have made a good-faith effort to address many of the technical concerns raised in the reviews, but the underlying problem of DRE remains.

      Weaknesses:

      - Although the model has potential advantages, it also has disadvantages. As stated by the authors, the pre-test work-load to prepare the model may not be worth the apparent advantages. And most important, the paper frequently mentions DRE but does not directly address it, and yet drug resistance is the critical issue in this field.

      - Although the paper shows examples of actual seizures, there remains some concern that some of the events might not be seizures - or a homogeneous population of seizures. More quantitative assessment of the electrical properties (e.g., duration) of the seizures and their probability is likely to be more useful than the proposed quantification in the future of the behavioral seizure stages, because the former could be both more objective and automated, while the behavioral analysis of the seizures will likely be more subjective and less reliable (and also fraught with subjectivity and analytical problems). Nonetheless, the authors point that the presence of "Racine 3 or above" behavioral seizures (in addition to their electrical data) is a good argument that many (if not all) of the "seizures" are actual epileptic seizures.

      - Optogenetic stimulation of CA1 provides cell-specificity for the stimulation, but it is not clear that this method would actually be better than electrical stimulation of a kindled rodent with superimposed hippocampal injury. The reader is unfortunately left with the concern of whether this model would be easier and more efficacious than kindling.

      - Although the authors have taken on a critically important problem, and have combined a variety of technologies, this approach may facilitate more rapid screening of ASDs against actual seizures (beneficial), but it does not really address the fundamentally critical yet difficult problem of DRE. A critical issue for DRE that is not well-addressed relates to adverse effects, which is often why many ASDs are not well tolerated by many patients (e.g., LEV). Thus, we are left with: how does this address anti-seizure DRE?

      - The focus of this paper seems to be more on seizures more than on epilepsy. In the absence of seizure spontaneity, the work seems to primarily address the issues of seizure spread and duration. Although this is useful, it does not seem to be addressing the question of what trips the system to generate a seizure.

      An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

      - The authors seem to have developed a new and useful model; however, it is not clear how this will address that core problem of DRE, which was their stated aim.

      - A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.

      - As stated before in the original review, the potential impact would primarily be aimed at the ETSP or a drug-testing CRO; however, much more work will be required to convince the epilepsy community that this approach will actually identify new ASDs for DRE. The approach is potentially time-consuming with a steep and potentially difficult optimization curve, and thus may not be readily adaptable to the typical epilepsy-models neuroscience laboratory.

      Any additional context you think would help readers interpret or understand the significance of the work:

      - The problem of DRE is much more complicated than described by the authors here; however, the paper could end up being more useful than is currently apparent. Although this work could be seen as technically - and maybe conceptually - elegant and a technical tour de force, will it "deliver on the promise"? Is it better than kindling for DRE? In attempting to improve the discovery process, how will the model move us to another level? Will this model really be any better than others, such as kindling?

    1. Reviewer #2 (Public review):

      Summary:

      This manuscript by Hosack and Arce-McShane examines the directional tuning of neurons in macaque primary motor (MIo) and somatosensory (SIo) cortex. The neural basis of tongue control is far less studied than, for example, forelimb movements, partly because the tongue's kinematics and kinetics are difficult to measure. A major technical advantage of this study is using biplanar video-radiography, processed with modern motion tracking analysis software, to track the movement of the tongue inside the oral cavity. Compared to prior work, the behaviors are more naturalistic behaviors (feeding and licking water from one of three spouts), although the animals were still head-fixed.

      The study's main findings are that:

      • A majority of neurons in MIo and a (somewhat smaller) percentage of SIo modulated their firing rates during tongue movements, with different modulation depending on the direction of movement (i.e., exhibited directional tuning). Examining the statistics of tuning across neurons, there was anisotropy (e.g., more neurons preferring anterior movement) and a lateral bias in which tongue direction neurons preferred that was consistent with the innervation patterns of tongue control muscles (although with some inconsistency between monkeys).<br /> • Consistent with this encoding, tongue position could be decoded with moderate accuracy even from small ensembles of ~28 neurons.<br /> • There were differences observed in the proportion and extent of directional tuning between the feeding and licking behaviors, with stronger tuning overall during licking. This potentially suggests behavioral context-dependent encoding.<br /> • The authors then went one step further and used a bilateral nerve block to the sensory inputs (trigeminal nerve) from the tongue. This impaired the precision of tongue movements and resulted in an apparent reduction and change in neural tuning in Mio and SIo.

      Strengths:

      The data are difficult to obtain and appear to have been rigorously measured, and provide a valuable contribution to this under-explored subfield of sensorimotor neuroscience. The analyses adopt well-established methods especially from the arm motor control literature, and represent a natural starting point for characterizing tongue 3D direction tuning.

      Weaknesses:

      There are alternative explanations from some of the interpretations, but those interpretations are described in a way that clearly distinguishes results from interpretations, and readers can make their own assessments. Some of these limitations are described in more detail below.

      One weakness of the current study is that there is substantial variability in results between monkeys.

      This study focuses on describing directional tuning using the preferred direction (PD) / cosine tuning model popularized by Georgopoulous and colleagues for understanding neural control of arm reaching in the 1980s. This is a reasonable starting point and a decent first order description of neural tuning. However, the arm motor control field has moved far past that viewpoint, and in some ways an over-fixation on static representational encoding models and PDs held that field back for many years. The manuscript benefit from drawing the readers' attention (perhaps in their Discussion) that PDs are a very simple starting point for characterizing how cortical activity relates to kinematics, but that there is likely much richer population-level dynamical structure and that a more mechanistic, control-focused analytical framework may be fruitful. A good review of this evolution in the arm field can be found in Vyas S, Golub MD, Sussillo D, Shenoy K. 2020. Computation Through Neural Population Dynamics. Annual Review of Neuroscience. 43(1):249-75. A revised version of the manuscript incorporates more population-level analyses, but with inconsistent use of quantifications/statistics and without sufficient contextualization of what the reader is to make of these results.

      The described changes in tuning after nerve block could also be explained by changes in kinematics between these conditions, which temper the interpretation of these interesting results.

      I am not convinced of the claim that tongue directional encoding fundamentally changes between drinking and feeding given the dramatically different kinematics and the involvement of other body parts like the jaw (e.g., the reference to Laurence-Chasen et al. 2023 just shows that there is tongue information independent of jaw kinematics, not that jaw movements don't affect these neurons' activities). I also find the nerve block results inconsistent (more tuning in one monkey, less in the other?) and difficult to really learn something fundamental from, besides that neural activity and behavior both change - in various ways - after nerve block (not at all surprising but still good to see measurements of).

      The manuscript states that "Our results suggest that the somatosensory cortex may be less involved than the motor areas during feeding, possibly because it is a more ingrained and stereotyped behavior as opposed to tongue protrusion or drinking tasks". An alternative explanation be more statistical/technical in nature: that during feeding, there will be more variability in exactly what somatosensation afferent signals are being received from trial to trial (because slight differences in kinematics can have large differences in exactly where the tongue is and the where/when/how of what parts of it are touching other parts of the oral cavity)? This variability could "smear out" the apparent tuning using these types of trial-averaged analyses. Given how important proprioception and somatosensation are for not biting the tongue or choking, the speculation that somatosensory cortical activity is suppressed during feedback is very counter-intuitive to this reviewer. In the revised manuscript the authors note these potential confounds and other limitations in the Discussion.

    1. Reviewer #2 (Public review):

      The manifestation and progression of neurodegenerative disorders is poorly understood. Many of the neuronal disorders start by presenting subtle changes in neuronal circuit and quantification and measurement of these subtle behavior responses could help one delineate the mechanisms involved. The present study very nicely uses the flies' behavioral response to predator-mimicking passing shadows to measure subtle changes in their behavior. The data from various fly genetic models of Parkinson's disease supports their claim. This single trial method is useful to capture the individual animal's response to the threatening stimuli but stops short of capturing the fine ambulatory responses which could provide further information on an individual's behavioral response. By capturing the fine features, the authors could get detailed observations, such as posture, gait or wing positioning for a better understanding the behavioral response to the passing shadow.

    1. Reviewer #2 (Public review):

      Summary:

      This is a well written manuscript describing studies directed at identifying the cell type responsible for pacemaking in murine collecting lymphatics. Using state of the art approaches, the authors identified a number of different cell types in the wall of these lymphatics and then using targeted expression of Channel Rhodopsin and GCaMP, the authors convincingly demonstrate that only activation of lymphatic muscle cells produces coordinated lymphatic contraction and that only lymphatic muscle cells display pressure-dependent Ca2+ transients as would be expected of a pacemaker in these lymphatics.

      Strengths:

      The use of targeted expression of channel rhodopsin and GCaMP to test the hypothesis that lymphatic muscle cells serve as the pacemakers in musing lymphatic collecting vessels.

      Weaknesses:

      The only significant weakness was the lack of quantitative analysis of most of the imaging data shown in Figures 1-11. In particular the colonization analysis should be extended to show cells not expected to demonstrate colocalization as a negative control for the colocalization analysis that the authors present. These weaknesses have been resolved by revision and addition of new and novel RNAseq data, additional colocalization data and membrane potential measurements.

      Comments on revisions: No additional concerns.

    1. Reviewer #3 (Public review):

      This communication from Sukhina et al argues that a period of malnutrition (modeled by caloric restriction) causes lasting immune deficiencies (myelopoesis) not rescued by re-feeding. This is a potentially important paper exploring the effects of malnutrition on immunity, which is a clinically important topic. The revised study adds some details with respect to kinetics of immune compartment and body weight changes, but most aspects raised by the referees were deferred experimentally. Several textual changes have been made to avoid over-interpreting their data. My overall assessment of this revised study is similar to my impression before, which is that while the observations are interesting, there is both a lack of mechanistic understanding of the phenomena and a lack of resolution/detail about the phenomena itself.

    1. Reviewer #2 (Public review):

      The manuscript by Desingu et al., explores the role of SIRT2 in regulating Japanese Encephalitis Virus (JEV) replication and disease progression in rodent models. Using both an in vitro and an in vivo approach, the authors demonstrate that JEV infection leads to decreased SIRT2 expression, which they hypothesize is exploited by JEV for viral replication. To test this hypothesis, the authors utilize SIRT2 inhibition (via AGK2 or genetic knockout) and demonstrate that it leads to increased viral load and worsens clinical outcomes in JEV-infected mice. Conversely, SIRT2 overexpression via an AAV delivery system reduces viral replication and improves survival among infected mice. The study proposes a mechanism in which SIRT2 suppresses JEV-induced autophagy and inflammation by deacetylating NF-κB, thereby reducing Beclin-1 expression (an NF-κB-dependent gene) and autophagy, which the authors consider a pathway that JEV exploits for replication. Transcriptomic analysis further supports that SIRT2 deficiency leads to NF-κB-driven cytokine hyperactivation. Additionally, pharmacological inhibition of NF-κB using Bay 11 (an IKK inhibitor) results in reduced viral load and improved clinical pathology in WT and SIRT2 KO mice. Overall, the findings from Desingu et al. are generally supported by the data and suggest that targeting SIRT2 may serve as a promising therapeutic approach for JEV infection and potentially other RNA viruses that SIRT2 helps control. However, the paper does fall short in some areas. Please see below for our comments to help improve the paper.

    1. Reviewer #2 (Public review):

      Summary:

      An intensification study with a double dose of 2nd generation integrase inhibitor with a background of nucleoside analog inhibitors of the HIV retrotranscriptase in 2, and inflammation is associated with the development of co-morbidities in 20 individuals randomized with controls, with an impact on the levels of viral reservoirs and inflammation markers. Viral reservoirs in HIV are the main impediment to an HIV cure, and inflammation is associated with co-morbidities.

      Strengths:

      The intervention that leads to a decrease of viral reservoirs and inflammation is quite straightforward forward as a doubling of the INSTI is used in some individuals with INSTI resistance, with good tolerability.

      This is a very well documented study, both in blood and tissues, which is a great achievement due to the difficulty of body sampling in well-controlled individuals on antiretroviral therapy. The laboratory assays are performed by specialists in the field with state-of-the art quantification assays. Both the introduction and the discussion are remarkably well presented and documented.

      The findings also have a potential impact on the management of chronic HIV infection.

      Weaknesses:

      I do not think that the size of the study can be considered a weakness, nor the fact that it is open-label either.

    1. Reviewer #2 (Public review):

      Summary and strengths:

      In this manuscript, Karjee and colleagues used coalescent-based effective population size reconstruction (PSMC) from single genomes to understand past population trends in island birds and related this to life history traits and glacial patterns. This concept is fairly new, as there are still relatively few multiple PSMC synthesis studies. I also thought that the focus on island endemics was unique and adds value to this paper. I enjoyed seeing a paper focused on South East Asia and think that this could help contribute to our knowledge of the important biodiversity within this region.

      Major weaknesses:

      My biggest concern with this paper is that the analyses are limited to 20-30 species, and significant taxonomic bias is present (there are multiple species of passerine but only 1-2 representatives of other groups). While this is not an issue alone, many of the life history traits or geographical traits are conflated with phylogenetic diversity (e.g., there are no large-bodied passerines). Thus, it is my opinion that the impact of these drivers of past population size is conflated and cannot be disentangled with the current data. The authors themselves state that the core hypothesis surrounding Ne and habitat availability is not supported by their entire dataset (only seen in Passerines). This was not clear enough in the abstract, and conclusions cannot be drawn here as the impact of taxonomy cannot be separated from data richness, traits, etc. The PSMC analysis was done according to the most recent recommendations, and this part of the manuscript is fairly robust. However, in several places, it is incorrectly stated that the PSMC measures or can infer genetic diversity; PSMC only infers past effective population size. It cannot measure genetic diversity in the past. I cannot review the habitat reconstruction modelling as I am a conservation genomics specialist.

      Appraisal:

      I am not convinced about the findings within the paper. I do not think that the results are sufficiently supported at this time, largely due to the conflation of taxonomy with other variables. As this type of comparison is new, I do think that there is a chance for reasonable impact on the field of genomics and island biogeography if the manuscript's constraints are addressed. I do not see scope for impact on conservation at this time and find the conclusions in the abstract regarding conservation relevance to be unfounded.

    1. Reviewer #2 (Public review):

      In the manuscript "Cancer cells differentially modulate mitochondrial respiration to alter redox state and enable biomass synthesis in nutrient-limited environments", Chang et al investigate how cancer cells respond to the limitation of certain environmental nutrients by regulating the cellular NAD+/NADH ratio. They focus on serine and lipid metabolism, pathways known to be controlled by the NAD+/NADH ratio, and propose that changes in mitochondrial respiration in response to deprivation of these nutrients can influence the NAD+/NADH ratio, thereby impacting biomass synthesis.

      While the study is descriptive in nature and does not investigate specific molecular mechanisms that explain the crosstalk between nutrient availability and mitochondrial redox changes, the experimental component is robust, and the conclusions are well supported by the results. Some suggestions could further refine the conclusions and enhance the quality of the manuscript.

      Main critiques:

      (1) Throughout the manuscript, the authors utilise the number of cell doublings per day as an endpoint readout of cell proliferation. It would be advisable to include a quantification of the cell number and to display the proliferation rate over time. This would provide valuable insights into the timeline of cellular responses and avoid potential confounding effects associated with the use of Sulforhodamine B dye, an indirect measure of cell proliferation based on protein content, which may be influenced by some of the interventions. Furthermore, it will help determine whether specific treatments reduce cellular doublings resulting from cell death. This concern is particularly evident in treatments with rotenone, e.g., Fig. 1G, where the increase in doublings could be attributed to cell death.

      (2) The authors propose a model in which the deprivation of extracellular nutrients impacts mitochondrial respiration, which in turn increases the NAD+/NADH ratio and ultimately affects metabolic biosynthetic pathways that occur in the cytosol, such as serine biosynthesis. The mechanism by which nutrient availability is sensed and transmitted across different cellular compartments to regulate mitochondrial redox status remains unclear. This concern is particularly relevant for serine metabolism, as its synthesis occurs in the cytosol, but the authors connect it to mitochondrial respiration. Compartment-specific measurements of NAD+/NADH ratio would help to understand to what extent the redox state is affected by nutrients in the mitochondria and in the cytoplasm (see also minor critiques point 2). Moreover, the use of the genetic tool LbNox could be employed to manipulate the NAD+/NADH ratio in a compartment-specific manner, while also avoiding the toxicity of certain compounds, such as rotenone. This set of experiments would add depth to the investigation, which might otherwise appear too descriptive.

    1. Reviewer #2 (Public review):

      Summary:

      The authors set out to dissect the mechanistic basis of their previously published finding that encountering cutaneous antigen augments the persistence of CD8+ memory T cells that enter skin (TRM) (Hirai et al., 2021, Immunity). Here they use the same murine model to study the fate of CD8+ T cells after antigen-priming in the lymph nodes, (1) those that re-encounter antigen in the skin via vaccinia virus (VV) versus (2) those that do not encounter antigen in skin but rather are recruited via topical dinitrofluorobenzene (DNFB) (so-called "bystander TRM"). The authors' previous publication establishes that this first group of CD8+ TRM has a persistence advantage over bystander TRM under TGFb-limiting conditions. The current paper advances this finding by elucidating the role of TGFBR3 in regulating CD8+ TRM skin persistence upon topical antigen exposure. Key novelty of the work lies in the generation and use of the CD8+ T cell-specific TGFBR3 knockout model, which allows them to demonstrate the role of TGFBR3 in fine-tuning the degree of CD8+ T cell skin persistence and that TGFBR3 expression is promoted by CD8+ TRM encountering their cognate antigen upon initial skin entry. Future work directly measuring active TGFb in the skin under different conditions would help identify physiologic scenarios that yield active TGFb-limiting conditions, thus establishing physiologic relevance.

      Strengths:

      Technical strengths of the paper include (1) complementary imaging and flow cytometry analyses, (2) integration of their scRNA-seq data with the existing CD8+ TRM literature via pathway analysis, and (3) use of orthogonal models where possible. Using a vaccina virus (VV) model, with and without ovalbumin (OVA), the authors investigate how topical antigen exposure and TCR strength regulate CD8+ TRM skin recruitment and retention. The authors use both FTY720 and a Thy1.1 depleting antibody to demonstrate that skin CD8+ TRM expand locally following both a primary and secondary recall response to topical OVA application.

      A conceptual strength of the paper is the authors' observation that TCR signal strength upon initial TRM tissue entry helps regulate the extent of their local re-expansion on subsequent antigen re-exposure. They achieved this by applying peptides of varying affinity for the OT-I TCR on the DNFB-exposed flank in tandem with initial VV-OVA + DNFB treatment. They then measured TRM expansion after OVA peptide rechallenge, revealing that encountering a higher-affinity peptide upon skin entry leads to greater subsequent re-expansion. Additionally, by generating an OT-I Thy1.1+ E8i-creERT2 huNGFR Tgfbr3fl/fl (Tgfbr3∆CD8) mouse, the authors were able to elucidate a unique role for TGFBR3 in CD8+TRM persistence when active TGFb in skin is limited.

      Weaknesses:

      Overall, the authors' conclusions are well supported, although there are some instances where additional controls, experiments, or clarifications would add rigor. The conclusions regarding skin-localized TCR signaling leading to increased skin CD8+ TRM proliferation in-situ and increased TGFBR3 expression would be strengthened by assessing skin CD8+ TRM proliferation and TGFBR3 expression in models of high versus low avidity topical OVA-peptide exposure. The authors could further increase the novelty of the paper by exploring whether TGFBR3 is regulated at the RNA or protein level. To this end, they could perform analysis of their single-cell RNA sequencing data (Figure 1), comparing Tgfbr3 mRNA in DNFB versus VV-treated skin.

      For clarity, when discussing antigen exposure throughout the paper, it would be helpful for the authors to be more precise that they are referring to the antigen in the skin rather than in the draining lymph node. A more explicit summary of some of the lab's previous work focused on CD8+ TRM and the role of TGFb would also help readers better contextualize this work within the existing literature on which it builds.

      For rigor, it would be helpful where possible to pair flow cytometry quantification with the existing imaging data. Additional controls, namely enumerating TRM in the opposite, untreated flank skin of VV-only-treated mice and the treated flank skin of DNFB-only treated mice, would help contextualize the results seen in dually-treated mice in Figure 1. In figure legends, we suggest clearly reporting unpaired T tests comparing relevant metrics within VV or DNFB-treated groups (for example, VV-OVA PBS vs VV-OVA FTY720 in Figure 3F). Finally, quantifying right and left skin draining lymph node CD8+ T cell numbers would clarify the skin specificity and cell trafficking dynamics of the authors' model.

    1. Reviewer #2 (Public review):

      This paper introduces a framework for modeling individual differences in decision-making by learning a low-dimensional representation (the "individuality index") from one task and using it to predict behaviour in a different task. The approach is evaluated on two types of tasks: a sequential value-based decision-making task and a perceptual decision task (MNIST). The model shows improved prediction accuracy when incorporating this learned representation compared to baseline models.

      The motivation is solid, and the modelling approach is interesting, especially the use of individual embeddings to enable cross-task generalization. That said, several aspects of the evaluation and analysis could be strengthened.

      (1) The MNIST SX baseline appears weak. RTNet isn't directly comparable in structure or training. A stronger baseline would involve training the GRU directly on the task without using the individuality index-e.g., by fixing the decoder head. This would provide a clearer picture of what the index contributes.

      (2) Although the focus is on prediction, the framework could offer more insight into how behaviour in one task generalizes to another. For example, simulating predicted behaviours while varying the individuality index might help reveal what behavioural traits it encodes.

      (3) It's not clear whether the model can reproduce human behaviour when acting on-policy. Simulating behaviour using the trained task solver and comparing it with actual participant data would help assess how well the model captures individual decision tendencies.

      (4) Figures 3 and S1 aim to show that individuality indices from the same participant are closer together than those from different participants. However, this isn't fully convincing from the visualizations alone. Including a quantitative presentation would help support the claim.

      (5) The transfer scenarios are often between very similar task conditions (e.g., different versions of MNIST or two-step vs three-step MDP). This limits the strength of the generalization claims. In particular, the effects in the MNIST experiment appear relatively modest, and the transfer is between experimental conditions within the same perceptual task. To better support the idea of generalizing behavioural traits across tasks, it would be valuable to include transfers across more structurally distinct tasks.

      (6) For both experiments, it would help to show basic summaries of participants' behavioural performance. For example, in the MDP task, first-stage choice proportions based on transition types are commonly reported. These kinds of benchmarks provide useful context.

      (7) For the MDP task, consider reporting the number or proportion of correct choices in addition to negative log-likelihood. This would make the results more interpretable.

      (8) In Figure 5, what is the difference between the "% correct" and "% match to behaviour"? If so, it would help to clarify the distinction in the text or figure captions.

      (9) For the cognitive model, it would be useful to report the fitted parameters (e.g., learning rate, inverse temperature) per individual. This can offer insight into what kinds of behavioural variability the individuality index might be capturing.

      (10) A few of the terms and labels in the paper could be made more intuitive. For example, the name "individuality index" might give the impression of a scalar value rather than a latent vector, and the labels "SX" and "SY" are somewhat arbitrary. You might consider whether clearer or more descriptive alternatives would help readers follow the paper more easily.

      (11) Please consider including training and validation curves for your models. These would help readers assess convergence, overfitting, and general training stability, especially given the complexity of the encoder-decoder architecture.

    1. Reviewer #2 (Public review):

      Summary:

      The paper is a methodological contribution to multivariate pattern analysis and, in particular, the analysis of representational geometry via pairwise representational distances, sometimes called representational dissimilarity analysis (RDA). The authors investigate through theoretical analysis and simulations how true representational distances (defined on the neural level) give rise to representational distances estimated from neurophysiological data, including fMRI and cell recordings. They demonstrate that, due to the way measurements sample neural activity, the activity common to all sampled neurons can be amplified in the representational geometry derived from these measurements, and therefore, an empirical representational geometry may deviate substantially from the true representational geometry. The authors propose to modify the obtained representational structure by removing the dimension corresponding to that common activity, and argue that such a removal of a single dimension does not relevantly affect the representational structure, again underpinned by mathematical analysis and simulation.

      Importance:

      The paper may at first sight be tackling a specific problem within a specific subfield of cognitive neuroscience methods. However, understanding the structure of representations is a fundamental goal of cognitive psychology and cognitive neuroscience, and the fact that methods of representational geometry are not yet routinely used by the wider community may at least partially be due to uncertainty regarding the reliability of these methods. This paper is an important step towards clarifying and improving reliability, and therefore towards more widespread adoption of representational geometry methods.

      Strengths:

      The paper makes its argument generally well, relying on previous work by the authors as well as others to support assumptions about neural sampling by neurophysiological measurements. Their main points are underpinned by both detailed mathematical analysis and simulations, and the latter also produces intuitively accessible illustrations of the authors' argument. The authors discuss in detail under which exact circumstances common neural activity distorts the representational geometry, and therefore, when exactly the removal of the common dimension is necessary to minimize that distortion.

      Weaknesses:

      (1) The argument around the Johnson-Lindenstrauss lemma on pages 5 & 6 is somewhat confused, and also not really convincing.

      First, the correct reference for the lemma seems to be not [20] = Johnson et al. (1986), but Johnson & Lindenstrauss (1984). Moreover, as far as I can tell, Johnson et al. (1986) do not discuss random projections, and while they play a role in Johnson & Lindenstrauss (1984), that is only as a proof device. The paper text suggests that the lemma itself is probabilistic, while actually it is a statement of existence.

      Second, the authors correctly state that the lemma implies that "the number of measurement channels required for a good approximation does not depend on the number of neurons and grows only logarithmically with the number of stimuli", but it is not clear what the relevance of this statement for this paper is, considering that distances between N points can be exactly preserved within an N − 1 dimensional subspace, irrespective of the number of dimensions of the original space, and since in cognitive neuroscience the number of measurement channels is usually (much) larger than the number of experimental stimuli.

      The actually centrally important statement is not the Johnson-Lindenstrauss lemma, but one about the metric-preserving properties of random projections with zero-mean weights. It is this statement that needs to be backed up by the correct references, which, as far as I can tell, are neither the cited Johnson et al. (1986) nor even Johnson & Lindenstrauss (1984) for the lemma.

      (2) The detailed mathematical analyses and simulations focus on the effect of non-zero-mean sampling weights, and that is justified by the result that such sampling leads to a distorted representational geometry. However, there is another assumption which seems to be used almost everywhere in both mathematical analyses and simulations, and which I suspect may have a relevant effect on the observed representational geometry: statistical independence between weights. In particular, in fMRI, the existence of a naturally limited spatial resolution (due to MRI technology or vasculature) makes it unlikely that the weights with which a given neuron affects different voxels are independent.

    1. Reviewer #2 (Public review):

      This paper by Kaller and colleagues combines an interesting replication of findings on the importance of maternal behavior on brain development in the offspring with a state-of-the-art MRI analysis and a novel comparison between such perinatal and early postnatal enrichment via the activity of the mother and a classical enriched environment in the adult. In general, the observations are as one would have expected. Early postnatal enrichment and adult enrichment have differential effects, which is plausible because, as the source of these changes is environmental, and environmental means very different things at these different stages. The three data sets presented are really interesting, and while the comparison between them might not always be as straightforward as it seems, the cross-sectional phenotyping with MRI already provides very important material and allows for interesting insight. Most interesting is possibly the massive effect of housing conditions at P7.

      In particular, the role of individual behavior differs. The authors highlight this role of the interaction with the environment, rather than the environment alone. Maternal care is a process that involves the pup.

      Importantly, the study shows that being born into an enriched environment predates certain changes that are still available after exposure at a later stage, but that there are also important differences. Detailed interpretation of these effects is not easy, however.

      Notably, the study does not include a condition of enrichment from birth into adulthood, and no analysis of the perinatal enrichment effects at an adult age. The timeline can be guessed from Figure 1b, but the authors might in places be more explicit about the fact that, indirectly and sometimes directly, animals of different ages (young adult versus adult) are compared. There is obviously no experience of maternal care in adulthood and no active exploration, etc in childhood. In part, this is what this paper is about, but it requires some thought for the reader to separate the more trivial from the more profound conclusions. Some more guidance would probably be welcome here. In general, Figure 4 is a great idea (and visually very appealing), but the content is not quite clear. "Adults born in EE vs. switched to EE in adulthood": this has, as far as I can tell, not been studied. What is compared are EE effects at two different time-points with two supposedly different mechanisms.

      From such a more mechanistic side, the authors might, for example, want to relate the observed patterns to what is known about the developmental (and plastic) dynamics in the respective brain regions at the given time. But age is a confounder here.

      There is another interesting point that the authors might discuss more prominently. The inter-individual differences in Z-score are dramatic within essentially all groups. So while the mean effects might still be statistically different, a large proportion of animals are within a range of values that could be found in either experimental group. The same is also true for the effects of maternal care, as depicted in Figure 3. While there is, for this ROI, a clear trend that overall relative volume decreases with maternal contact time at each time point, there is a large range of values for each maternal contact time bin. Consequently, neither genetics nor maternal care per se can be the driver of this variation. Part of it will be technical, but the trend in the data indicates that certainly not all of this is noise and technical error.

      This study has some open ends but also provides a very important and interesting direction for future study, corroborating the idea that behavior, maternal and own, does matter.

    1. Reviewer #2 (Public review):

      Summary:

      In this study, the authors aimed to investigate how humans learn and adapt their behavior in dynamic environments characterized by two distinct types of uncertainty: volatility (systematic changes in outcomes) and noise (random variability in outcomes). Specifically, they sought to understand how participants adjust their learning rates in response to changes in these forms of uncertainty.

      To achieve this, the authors employed a two-step approach:

      Reinforcement Learning (RL) Model:<br /> They first used an RL model to fit participants' behavior, revealing that the learning rate was context-dependent-it varied based on the levels of volatility and noise. However, the RL model showed that participants misattributed noise as volatility, leading to higher learning rates in noisy conditions, where the optimal strategy would be to be less sensitive to random fluctuations.

      Bayesian Observer Model (BOM):<br /> To better account for this context dependency, they introduced a Bayesian Observer Model (BOM), which models how an ideal Bayesian learner would update their beliefs about environmental uncertainty. They found that a degraded version of the BOM, where the agent had a coarser representation of noise compared to volatility, best fit the participants' behavior. This suggested that participants were not fully distinguishing between noise and volatility, instead treating noise as volatility and adjusting their learning rates accordingly.

      The authors also aimed to use pupillometry data (measuring pupil dilation) as a physiological marker to arbitrate between models and understand how participants' internal representations of uncertainty influenced both their behavior and physiological responses. Their objective was to explore whether the BOM could explain not just behavioral choices but also these physiological responses, thereby providing stronger evidence for the model's validity.

      Overall, the study sought to reconcile approximate rationality in human learning by showing that participants still follow a Bayesian-like learning process, but with simplified internal models that lead to suboptimal decisions in noisy environments.

      Strengths:

      The generative model presented in the study is both innovative and insightful. The authors first employ a Reinforcement Learning (RL) model to fit participants' behavior, revealing that the learning rate is context-dependent-specifically, it varies based on the levels of volatility and noise in the task. They then introduce a Bayesian Observer Model (BOM) to account for this context dependency, ultimately finding that a degraded BOM-in which the agent has a coarser representation of noise compared to volatility-provides the best fit to the participants' behavior. This suggests that participants are not fully distinguishing between noise and volatility, leading to misattribution of noise as volatility. Consequently, participants adopt higher learning rates even in noisy contexts, where an optimal strategy would involve being less sensitive to new information (i.e., using lower learning rates). This finding highlights a rational but approximate learning process, as described in the paper.

      Weaknesses:

      While the RL and Bayesian models both successfully predict behavior, it remains unclear how to fully reconcile the two approaches. The RL model captures behavior in terms of a fixed or context-dependent learning rate, while the BOM provides a more nuanced account with dynamic updates based on volatility and noise. Both models can predict actions when fit appropriately, but the pupillometry data offers a promising avenue to arbitrate between the models. However, the current study does not provide a direct comparison between the RL framework and the Bayesian model in terms of how well they explain the pupillometry data. It would be valuable to see whether the RL model can also account for physiological markers of learning, such as pupil responses, or if the BOM offers a unique advantage in this regard. A comparison of the two models using pupillometry data could strengthen the argument for the BOM's superiority, as currently, the possibility that RL models could explain the physiological data remains unexplored.

      The model comparison between the Bayesian Observer Model and the self-defined degraded internal model could be further enhanced. Since different assumptions about the internal model's structure lead to varying levels of model complexity, using a formal criterion such as Bayesian Information Criterion (BIC) or Akaike Information Criterion (AIC) would allow for a more rigorous comparison of model fit. Including such comparisons would ensure that the degraded BOM is not simply favored due to its flexibility or higher complexity, but rather because it genuinely captures the participants' behavioral and physiological data better than alternative models. This would also help address concerns about overfitting and provide a clearer justification for using the degraded BOM over other potential models.

      Comments on revisions:

      The authors have addressed all my questions. Congratulations on the impressive work accomplished by the authors!

    1. Reviewer #2 (Public review):

      Summary:

      The authors report describes a novel vaccine platform derived from a newly discovered organelle called a migrasome. First, the authors address a technical hurdle for using migrasomes as a vaccine platform. Natural migrasome formation occurs at low levels and is labor intensive, however, by understanding the molecular underpinning of migrasome formation, the authors have designed a method to make engineered migrasomes from cultures cells at higher yields utilizing a robust process. These engineered migrasomes behave like natural migrasomes. Next, the authors immunized mice with migrasomes that either expressed a model peptide or the SARS-CoV-2 spike protein. Antibodies against the spike protein were raised that could be boosted by a 2nd vaccination and these antibodies were functional as assessed by an in vitro pseudoviral assay. This new vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA that require very stringent storage conditions.

      Strengths:

      The authors present very robust studies detailing the biology behind migrasome formation and this fundamental understanding was used to from engineered migrasomes, which makes it possible to utilize migrasomes as a vaccine platform. The characterization of engineered migrasomes is thorough and establishes comparability with naturally occurring migrasomes. The biophysical characterization of the migrasomes is well done, including thermal stability and characterization of the particle size (important characterizations for a good vaccine).

      Weaknesses:

      With a new vaccine platform technology, it would be nice to compare them head-to-head against a proven technology. The authors would improve the manuscript if they made some comparisons to other vaccine platforms such as a SARS-CoV-2 mRNA vaccine or even an adjuvanted recombinant spike protein. This would demonstrate a migrasome based vaccine could elicit responses comparable to a proven vaccine technology. Additionally, understanding the integrity of the antigens expressed in their migrasomes could be useful. This could be done by looking at functional monoclonal antibody binding to their migrasomes in a confocal microscopy experiment.

      Updates after revision:

      The revised manuscript has additional experiments that I believe improve the strength of evidence presented in the manuscript and address the weaknesses of the first draft. First, they provide a comparison to the antibody responses induced by their migrasome based platform to recombinant protein formulated in an adjuvant and show the response is comparable. Second, they provide evidence that the spike protein incorporated into their migrasomes retains structural integrity by preserving binding to monoclonal antibodies. Together, these results strengthen the paper significantly and support the claims that the novel migrasome based vaccine platform could be a useful in the vaccine development field.

    1. Reviewer #2 (Public review):

      Summary:

      This work reports the discovery of a new rhodopsin from reef-building corals that is characterized experimentally, spectroscopically, and by simulation. This rhodopsin lacks a carboxylate-based counterion, which is typical for this family of proteins. Instead, the authors find that a chloride ion stabilizes the protonated Schiff base and thus serves as a counterion.

      Strengths:

      This work focuses on the rhodopsin Antho2a, which absorbs in the visible spectrum with a maximum at 503 nm. Spectroscopic studies under different pH conditions, including the mutant E292A and different chloride concentrations, indicate that chloride acts as a counterion in the dark. In the photoproduct, however, the counterion is identified as E292.

      These results lead to a computational model of Antho2a in which the chloride is modeled in addition to the Schiff base. This model is improved using the hybrid QM/MM simulations. As a validation, the absorption maximum is calculated using the QM/MM approach for the protonated and deprotonated E292 residue as well as the E292A mutant. The results are in good agreement with the experiment. However, there is a larger deviation for ADC(2) than for sTD-DFT. Nevertheless, the trend is robust since the wt and E292A mutant models have similar excitation energies. The calculations are performed at a high level of theory that includes a large QM region.

    1. Reviewer #2 (Public review):

      The manuscript by Eggan et al. investigates the energetics of conformational transitions in the cyclic nucleotide-gated (CNG) channel SthK. This lab pioneered transition metal FRET (tmFRET), which has previously provided detailed insights into ion channel conformational changes. Here, the authors analyze tmFRET fluorescence lifetime measurements in the time domain, yielding detailed insights into conformational transitions within the cyclic nucleotide binding domains (CNBDs) of the channel. The integration of tmFRET with time-correlated single-photon counting (TCSPC) represents an advancement of this technique.

    1. Reviewer #2 (Public review):

      Summary:

      The authors provide an important summary of ten years of Brain Initiative funding including a description of the historical development of the initiative, the specific funding mechanisms utilized, and examples of grants funded and work produced. The authors also conduct analyses of the impact on overall funding in Systems and Computational Neuroscience, the raw and field normalized bibliographic impact of the work, the social media impact of the funded work, and the popularity of some tools developed.

      The authors have improved the presentation by integrating the weaker of the two manuscripts with the stronger, by clarifying terminology and by performing additional analyses.

    1. Reviewer #2 (Public review):

      Summary:

      Nishi et al, investigate the well-known and previously described phenomenon of age-associated myeloid-biased hematopoiesis. Using a previously established HoxB5mCherry mouse model, they used HoxB5+ and HoxB5- HSCs to discriminate cells with long-term (LT-HSCs) and short-term (ST-HSCs) reconstitution potential and compared these populations to immunophenotypically defined 'bulk HSCs' that consists of a mixture of LT-HSC and ST-HSCs. They then isolated these HSC populations from young and aged mice to test their function and myeloid bias in non-competitive and competitive transplants into young and aged recipients. Based on quantification of hematopoietic cell frequencies in the bone marrow, peripheral blood, and in some experiments the spleen and thymus, the authors argue against the currently held belief that myeloid-biased HSCs expand with age.

      While aspects of their work are fascinating and might have merit, several issues weaken the overall strength of the arguments and interpretation. Multiple experiments were done with a very low number of recipient mice, showed very large standard deviations, and had no statistically detectable difference between experimental groups. While the authors conclude that these experimental groups are not different, the displayed results seem too variable to conclude anything with certainty. The sensitivity of the performed experiments (e.g. Fig 3; Fig 6C, D) is too low to detect even reasonably strong differences between experimental groups and is thus inadequate to support the author's claims. This weakness of the study is not acknowledged in the text and is also not discussed. To support their conclusions the authors need to provide higher n-numbers and provide a detailed power analysis of the transplants in the methods section.

      As the authors attempt to challenge the current model of the age-associated expansion of myeloid-biased HSCs (which has been observed and reproduced by many different groups), ideally additional strong evidence in the form of single-cell transplants is provided.<br /> It is also unclear why the authors believe that the observed reduction of ST-HSCs relative to LT-HSCs explains the myeloid-biased phenotype observed in the peripheral blood. This point seems counterintuitive and requires further explanation.

      Based on my understanding of the presented data, the authors argue that myeloid-biased HSCs do not exist, as:<br /> a) they detect no difference between young/aged HSCs after transplant (mind low n-numbers and large std);<br /> b) myeloid progenitors downstream of HSCs only show minor or no changes in frequency and c) aged LT-HSCs do not outperform young LT-HSC in myeloid output LT-HScs in competitive transplants (mind low n-numbers and large std!!!).<br /> However, given the low n-numbers and high variance of the results, the argument seems weak and the presented data does not support the claims sufficiently. That the number of downstream progenitors does not change could be explained by other mechanisms, for instance, the frequently reported differentiation short-cuts of HSCs and/or changes in the microenvironment.

      Strengths:

      The authors present an interesting observation and offer an alternative explanation of the origins of aged-associated myeloid-biased hematopoiesis. Their data regarding the role of the microenvironment in the spleen and thymus appears to be convincing.

      Weaknesses:

      "Then, we found that the myeloid lineage proportions from young and aged LT-HSCs were nearly comparable during the observation period after transplantation (Fig. 3, B and C)."<br /> [Comment to the authors]: Given the large standard deviation and low n-numbers, the power of the analysis to detect differences between experimental groups is very low. Experimental groups with too large standard deviations (as displayed here) are difficult to interpret and might be inconclusive. The absence of clearly detectable differences between young and aged transplanted HSCs could thus simply be a false-negative result. The shown experimental results hence do not provide strong evidence for the author's interpretation of the data. The authors should add additional transplants and include a detailed power analysis to be able to detect differences between experimental groups with reasonable sensitivity.

      Line 293: "Based on these findings, we concluded that myeloid-biased hematopoiesis observed following transplantation of aged HSCs was caused by a relative decrease in ST-HSC in the bulk-HSC compartment in aged mice rather than the selective expansion of myeloid-biased HSC clones."

      [Comment to the authors]: Couldn't that also be explained by an increase in myeloid-biased HSCs, as repeatedly reported and seen in the expansion of CD150+ HSCs? It is not intuitively clear why a reduction of ST-HSCs clones would lead to a myeloid bias. The author should try to explain more clearly where they believe the increased number of myeloid cells comes from. What is the source of myeloid cells if the authors believe they are not derived from the expanded population of myeloid-biased HSCs?

      New comment for the authors:

      While the authors provide new evidence, clarify the text, and adjust their interpretation, the presented data remain weak and do not convincingly challenge the current paradigm. As myeloid-biased HSC expansion with age has been observed and published by many different groups, the authors need to provide much stronger evidence to challenge the observations of others. Key experiments that might support their claims had been suggested, but as indicated, the authors plan to provide these much more rigorous experiments in future studies. As it stands, the overall conclusions of this manuscript thus remain weak and preliminary.

      In an attempt to quantify the absolute cell number of HSPC subpopulations, the authors use a usual readout and quantify "Number of cells per minute of analysis time". This appears to be a quick and dirty reanalysis of already existing flow cytometry data. Unfortunately, this quantification cannot count the absolute number of cells reliably, as the number of cells per minute recorded is heavily influenced by the abundance of other cell populations. Instead, the author should have counted the absolute number of HSCs, MPPs, GMPs, etc. per femur, which is typically done to address this question.

      At this point, as authors are seemingly not willing to provide additional hard evidence to support their claims in this study and are instead in the process of preparing additional data for a future manuscript, I believe this study, as it stands (although weak), suggests an interesting alternative model. Despite being highly controversial, this alternative model warrants future investigations and discussions in the field. As always, it will also be important to reproduce these findings independently in other labs. As my concerns and the concerns of the other reviewers are documented and available to read by others, I believe the manuscript should be published in its current form to stimulate critical discussion and future investigations of the current model.

    1. Reviewer #2 (Public review):

      Summary:

      Mutations in the prolyl hydroxylase, PHD2, cause erythrocytosis and, in some cases, can result in tumorigenesis. Taber and colleagues test the structural and functional consequences of seven patient-derived missense mutations in PHD2 using cell-based reporter and stability assays, and multiple biophysical assays, and find that most mutations are destabilizing. Interestingly, they discover a PHD2 mutant that can hydroxylate the C-terminal ODD, but not the N-terminal ODD, which suggests the importance of N-terminal ODD for biology. A major strength of the manuscript is the multidisciplinary approach used by the authors to characterize the functional and structural consequences of the mutations. However, the manuscript had several major weaknesses, such as an incomplete description of how the NMR was performed, a justification for using neighboring residues as a surrogate for looking at prolyl hydroxylation directly, or a reference to the clinical case studies describing the phenotypes of patient mutations. Additionally, the experimental descriptions for several experiments are missing descriptions of controls or validation, which limits their strength in supporting the claims of the authors.

      Strengths:

      (1) This manuscript is well-written and clear.

      (2) The authors use multiple assays to look at the effects of several disease-associated mutations, which support the claims.

      (3) The identification of P317R as a mutant that loses activity specifically against NODD, which could be a useful tool for further studies in cells.

      Weaknesses:

      Major:

      (1) The source data for the patient mutations (Figure 1) in PHD2 is not referenced, and it's not clear where this data came from or if it's publicly available. There is no section describing this in the methods.

      (2) The NMR hydroxylation assay.

      A. The description of these experiments is really confusing. The authors have published a recent paper describing a method using 13C-NMR to directly detect proly-hydroxylation over time, and they refer to this manuscript multiple times as the method used for the studies under review. However, it appears the current study is using 15N-HSQC-based experiments to track the CSP of neighboring residues to the target prolines, so not the target prolines themselves. The authors should make this clear in the text, especially on page 9, 5th line, where they describe proline cross-peaks and refer to the 15N-HSQC data in Figure 5B.<br /> B. The authors are using neighboring residues as reporters for proline hydroxylation, without validating this approach. How well do CSPs of A403 and I566 track with proline hydroxylation? Have the authors confirmed this using their 13C-NMR data or mass spec?<br /> C. Peak intensities. In some cases, the peak intensities of the end point residue look weaker than the peak intensities of the starting residue (5B, PHD2 WT I566, 6 ct lines vs. 4 ct lines). Is this because of sample dilution (i.e., should happen globally)? Can the authors comment on this?

      (3) Data validating the CRISPR KO HEK293A cells is missing.

      (4) The interpretation of the SEC data for the PHD2 mutants is a little problematic. Subtle alterations in the elution profiles may hint at different hydrodynamic radii, but as the samples were not loaded at equal concentrations or volumes, these data seem more anecdotal, rather than definitive. Repeating this multiple times, using matched samples, followed by comparison with standards loaded under identical buffer conditions, would significantly strengthen the conclusions one could make from the data.

      Minor:

      (1) Justification for picking the seven residues is not clearly articulated. The authors say they picked 7 mutants with "distinct residue changes", but no further rationale is provided.

      (2) A major finding of the paper is that a disease-associated mutation, P317R, can differentially affect HIF1 prolyhydroxylation, however, additional follow-up studies have not been performed to test this in cells or to validate the mutant in another method. Is it the position of the proline within the catalytic core, or the identity of the mutation that accounts for the selectivity?

    1. Reviewer #2 (Public review):

      In the present manuscript, Li et al. use biochemical fractionation of "RNA granules" from P5 wildtype and FMR1 knock-out mouse brains to analyze their protein/RNA content, determine a single particle cryo-EM structure of contained ribosomes, and perform ribo-seq analysis of ribosome-protected RNA fragments (RPFs). The authors conclude from these that neither the composition of the ribosome granules, nor the state of their contained ribosomes, nor the mRNA positions with high ribosome occupancy change significantly. Besides minor changes in mRNA occupancy, the one change the authors identified is a decrease in puromycylated punctae in distal neurites of cultured primary neurons of the same mice, and their enhanced resistance to different pharmacological treatments. These results directly build on their earlier work (Anadolu et al., 2023) using analogous preparations of rat brains; the authors now perform a very similar study using WT and FMR1-KO mouse brains. This is an important topic, aiming to identify the molecular underpinnings of the FMRP protein, which is the basis of a major neurological disease. Unfortunately, several limitations of this study prevent it from being more convincing in its present form.

      In order to improve this study, our main suggestions are as follows:

      (1) The authors equate their biochemically purified "RG" fraction with their imaging-based detection of puromycin-positive punctae. They claim essentially no differences in RGs, but detect differences in the latter (mostly their abundance and sensitivity to DHPG/HHT/Aniso). In the discussion the authors acknowledge the inconsistency between these two modalities: "An inconsistency in our findings is the loss of distal RPM puncta coupled with an increase in the immunoreactivity for S6 in the RG." and "Thus, it may be that the RG is not simply made up of ribosomes from the large liquid-liquid phase RNA granules."

      How can the authors be sure that they are analysing the same entities in both modalities? A more parsimonious explanation of their results would be that, while there might be some overlap, two different entities are analyzed. Much of the main message rests on this equivalence, and I believe the authors should show its validity.

      (2) The authors show that increased nuclease digestion (and magnesium concentration) led to a reduction of their RPF sizes down to levels also seen by other researchers. Analyzing these now properly digested RPFs, the authors state that the CDS coverage and periodicity drastically improved, and that spurious enrichments of secretory mRNAs, which made up one of the major fractions in their previous work, are now reduced. In my opinion, this would be more appropriately communicated as a correction to their previous work, not as a main Figure in another manuscript.

      (3) The fold changes reported in Figure 7 (ranging between log2(-0.2) and log2(+0.25)) are all extremely small and in my opinion should not be used to derive claims such as "The loss of FMRP significantly affected the abundance and occupancy of FMRP-Clipped mRNAs in WT and FMR1-KO RG (Fig 7A, 7B), but not their enrichment between RG and RCs".

      (4) Figure 8 / S8-1 - The authors show that ~2/3 of their reads stem from PCR duplicates, but that even after removing those, the majority of peaks remain unaltered. At the same time, Figure S8-1 shows the total number of peaks to be 615 compared with 1392 before duplicate removal. Can the authors comment on this discrepancy? In addition, the dataset with properly removed artefacts should be used for their main display item instead of the current Figure 8.

      (5) Figure 9 / S9-1, the density of punctae in both WT and FMR1-KO actually increases after treatment of HHT or Anisomycin (Figure S9-1 B-C). Even if a large fraction would now be "resistant to run-off", there should not be an increase. While this effect is deemed not significant, a much smaller effect in Figure 9C is deemed significant. Can the authors explain this? Given how vastly different the sample sizes are (ranging from 23 neurites in Figures S9-1 to 5,171 neurites in Figure 9), the authors should (randomly) sample to the same size and repeat their statistical analysis again, to improve their credibility.

    1. Reviewer #2 (Public review):

      Summary:

      The study elucidates the role of the recently discovered mediator of p53 tumor suppressive activity, ZMAT3. Specifically, the authors find that ZMAT3 negatively regulates HKDC1, a gene involved in the control of mitochondrial respiration and cell proliferation.

      Strengths:

      Mechanistically, ZMAT3 suppresses HKDC1 transcription by sequestering JUN and preventing its binding to the HKDC1 promoter, resulting in reduced HKDC1 expression. Conversely, p53 mutation leads to ZMAT3 downregulation and HKDC1 overexpression, thereby promoting increased mitochondrial respiration and proliferation. This mechanism is novel; however, the authors should address several points.

      Weaknesses:

      The authors conduct mechanistic experiments (e.g., transcript and protein quantification, luciferase assays) to demonstrate regulatory interactions between p53, ZMAT3, JUN, and HKDC1. These findings should be supported with functional assays, such as proliferation, apoptosis, or mitochondrial respiration analyses.

    1. Reviewer #2 (Public review):

      Neiswender et al. investigated the interactomes between wild-type BICD2 and BICD2 mutants that are associated with Spinal Muscular Atrophy with Lower Extremity Predominance (SMALED2). Although BICD2 has previously been implicated in SMALED2, it is unclear how mutations in BICD2 may contribute to disease symptoms. In this study, the authors characterize the interactome of wild-type BICD2 and identify potential new cargos, including the HOPS complex. The authors then chose three SMALED2-associated BICD2 mutants and compared each mutant interactome to that of wild-type BICD2. Each mutant had a change in the interactome, with the most drastic being BICD2_R747C, a mutation in the cargo binding domain of BICD2. This mutant displayed less interaction with a potential new BICD2 cargo, the HOPS complex. Additionally, it displayed more interaction with an ER protein, GRAMD1A.

      The data in the paper is generally strong, but the major conclusions of this paper need more evidence to be better supported.

      (1) The authors use cells that have been engineered to express the different BICD2 constructs. As shown in Figure 4B, the authors see wide expression of BICD2_WT throughout the cell. However, WT BICD2 usually localizes to the TGN. This widespread localization introduces some uncertainty about the interactome data. The authors should either try to verify the interaction data (specifically with the HOPS complex and GRAMD1A) by immunoprecipitating endogenous BICD2 or by repeating their interactome experiment in Figure 1 using BICD2 knockout cells that express the BICD2_WT construct. This should also be done to verify the immunoprecipitation and microscopy data shown in Figure 7.

      (2) The authors conclude that cargo transport defects resulting from BICD2 mutations may contribute to SMALED2 symptoms. However, the authors are unable to determine if BICD2 directly binds to the potential new cargo, the HOPS complex. To address this, the authors could purify full-length WT BICD2 and perform in vitro experiments. Furthermore, the authors were unable to identify the minimal region of BICD2 needed for HOPS interaction. The authors could expand on the experiment attempted with the extended BICD2 C-terminal using a deltaCC1 construct, which could also be used for in vitro experiments.

      (3) Again, the authors conclude that BICD2 mutants cause cargo transport defects that are likely to lead to SMALED2 symptoms. This would be better supported if the authors are able to find a protein relevant to SMALED2 and examine if/how its localization is changed under expression of the BICD2 mutants. The authors currently use the HOPS complex and GRAMD1A as indicators of cargo transport defects, but it is unclear if these are relevant to SMALED2 symptoms.

    1. Reviewer #2 (Public review):

      This study seeks to understand how the Rhino factor knows how to localize to specific transposon loci and to specific piRNA clusters to direct the correct formation of specialized heterochromatin that promotes piRNA biogenesis in the fly germline. In particular, these dual-strand piRNA clusters with names like 42AB, 38C, 80F, and 102F generate the bulk of ovarian piRNAs in the nurse cells of the fly ovary, but the evolutionary significance of these dual-strand piRNA clusters remains mysterious since triple null mutants of these dual-strand piRNA clusters still allows fly ovaries to develop and remain fertile. Nevertheless, mutants of Rhino and its interactors Deadlock, Cutoff, Kipferl and Moonshiner, etc, causes more piRNA loss beyond these dual-strand clusters and exhibit the phenotype of major female infertility, so the impact of proper assembly of Rhino, the RDC, Kipferl etc onto proper piRNA chromatin is an important and interesting biological question that is not fully understood.

      This study tries to first test ectopic expression of Rhino via engineering a Dox-inducible Rhino transgene in the OSC line that only expresses the primary Piwi pathway that reflects the natural single pathway expression the follicle cells and is quite distinct from the nurse cell germline piRNA pathway that is promoted by Rhino, Moonshiner, etc. The authors present some compelling evidence that this ectopic Rhino expression in OSCs may reveal how Rhino can initiate de novo binding via ADMA histone marks, a feat that would be much more challenging to demonstrate in the germline where this epigenetic naïve state cannot be modeled since germ cell collapse would likely ensue. In the OSC, the authors have tested the knockdown of four of the 11 known Drosophila PRMTs (DARTs), and comparing to ectopic Rhino foci that they observe in HP1a knockdown (KD), they conclude DART1 and DART4 are the prime factors to study further in looking for disruption of ADMA histone marks. The authors also test KD of DART8 and CG17726 in OSCs, but in the fly, the authors only test Germ Line KD of DART4 only, they do not explain why these other DARTs are not tested in GLKD, the UAS-RNAi resources in Drosophila strain repositories should be very complete and have reagents for these knockdowns to be accessible.

      The authors only characterize some particular ADMA marks of H3R17me2a as showing strong decrease after DART4 GLKD, and then they see some small subset of piRNA clusters go down in piRNA production as shown in Figure 6B and Figure 6F and Supplementary Figure 7. This small subset of DART4-dependent piRNA clusters does lose Rhino and Kipferl recruitment, which is an interesting result.

      However, the biggest issue with this study is the mystery that the set of the most prominent dual-strand piRNA clusters. 42AB, 38C, 80F, and 102F, are the prime genomic loci subjected to Rhino regulation, and they do not show any change in piRNA production in the GLKD of DART4. The authors bury this surprising negative result in Supplementary Figure 5E, but this is also evident in no decrease (actually an n.s. increase) in Rhino association in Figure 5D. Since these main piRNA clusters involve the RDC, Kipferl, Moonshiner, etc, and it does not change in ADMA status and piRNA loss after DART4 GLKD, this poses a problem with the model in Figure 7C. In this study, there is only a GLKD of DART4 and no GLKD of the other DARTs in fly ovaries.

      One way the authors rationalize this peculiar exception is the argument that DART4 is only acting on evolutionarily "young" piRNA clusters like the bx, CG14629, and CG31612, but the lack of any change on the majority of other piRNA clusters in Figure 6F leaves upon the unsatisfying concern that there is much functional redundancy remaining with other DARTs not being tested by GLKD in the fly that would have a bigger impact on the other main dual-strand piRNA clusters being regulated by Rhino and ADMA-histone marks.

      Also, the current data does not provide convincing enough support for the model Figure 7C and the paper title of ADMA-histones being the key determinant in the fly ovary for Rhino recognition of the dual-strand piRNA clusters. Although much of this study's data is well constructed and presented, there remains a large gap that no other DARTs were tested in GLKD that would show a big loss of piRNAs from the main dual-strand piRNA clusters of 42AB, 38C, 80F, and 102F, where Rhino has prominent spreading in these regions.

      As the manuscript currently stands, I do not think the authors present enough data to conclude that "ADMA-histones [As a Major new histone mark class] does play a crucial role in the initial recognition of dual-strand piRNA cluster regions by Rhino" because the data here mainly just show a small subset of evolutionarily young piRNA clusters have a strong effect from GLKD of DART4. The authors could extensively revise the study to be much more specific in the title and conclusion that they have uncovered this very unique niche of a small subset of DART4-dependent piRNA clusters, but this niche finding may dampen the impact and significance of this study since other major dual-strand piRNA clusters do not change during DART4 GLKD, and the authors do not show data GLKD of any other DARTs. The niche finding of just a small subset of DART-4-dependent piRNA clusters might make another specialized genetics forum a more appropriate venue.

    1. Reviewer #2 (Public review):

      The manuscript from Shan et al., sets out to investigate the role of Chi3l1 in different hepatic macrophage subsets (KCs and moMFs) in MASLD following their identification that KCs highly express this gene. To this end, they utilise Chi3l1KO, Clec4f-CrexChi3l1fl, and Lyz2-CrexChi3l1fl mice and WT controls fed a HFHC for different periods of time.

      Firstly, the authors perform scRNA-seq, which led to the identification of Chi3l1 (encoded by Chil1) in macrophages. However, this is on a limited number of cells (especially in the HFHC context), and hence it would also be important to validate this finding in other publicly available MASLD/Fibrosis scRNA-seq datasets. Similarly, it would be important to examine if cells other than monocytes/macrophages also express this gene, given the use of the full KO in the manuscript. Along these lines, utilisation of publicly available human MASLD scRNA-seq datasets would also be important to understand where the increased expression observed in patients comes from and the overall relevance of macrophages in this finding.

      Next, the authors use two different Cre lines (Clec4f-Cre and Lyz2-Cre) to target KCs and moMFs respectively. However, no evidence is provided to demonstrate that Chil1 is only deleted from the respective cells in the two CRE lines. Thus, KCs and moMFs should be sorted from both lines, and a qPCR performed to check the deletion of Chil1. This is especially important for the Lyz2-Cre, which has been routinely used in the literature to target KCs (as well as moMFs) and has (at least partial) penetrance in KCs (depending on the gene to be floxed). Also, while the Clec4f-Cre mice show an exacerbated MASLD phenotype, there is currently no baseline phenotype of these animals (or the Lyz2Cre) in steady state in relation to the same readouts provided in MASLD and the macrophage compartment. This is critical to understand if the phenotype is MASLD-specific or if loss of Chi3l1 already affects the macrophages under homeostatic conditions.

      Next, the authors suggest that loss of Chi3l1 promotes KC death. However, to examine this, they use Chi3l1 full KO mice instead of the Clec4f-Cre line. The reason for this is not clear, because in this regard, it is now not clear whether the effects are regulated by loss of Chi3l1 from KCs or from other hepatic cells (see point above). The authors mention that Chi3l1 is a secreted protein, so does this mean other cells are also secreting it, and are these needed for KC death? In that case, this would not explain the phenotype in the CLEC4F-Cre mice. Here, the authors do perform a basic immunophenotyping of the macrophage populations; however, the markers used are outdated, making it difficult to interpret the findings. Instead of F4/80 and CD11b, which do not allow a perfect discrimination of KCs and moMFs, especially in HFHC diet-fed mice, more robust and specific markers of KCs should be used, including CLEC4F, VSIG4, and TIM4.

      Additionally, while the authors report a reduction of KCs in terms of absolute numbers, there are no differences in proportions. This, coupled with a decrease also in moMF numbers at 16 weeks (when one would expect an increase if KCs are decreased, based on previous literature) suggests that the differences in KC numbers may be due to differences in total cell counts obtained from the obese livers compared with controls. To rule this out, total cell counts and total live CD45+ cell counts should be provided. Here, the authors also provide tunnel staining in situ to demonstrate increased KC death, but as it is typically notoriously difficult to visualise dying KCs in MASLD models, here it would be important to provide more images. Similarly, there appear to be many more Tunel+ cells in the KO that are not KCs; thus, it would be important to examine this in the CLEC4F-Cre line to ascertain direct versus indirect effects on cell survival.

      Finally, the authors suggest that Chi3l1 exerts its effects through binding glucose and preventing its uptake. They use ex vivo/in vitro models to assess this with rChi3l1; however, here I miss the key in vivo experiment using the CLEC4F-Cre mice to prove that this in KCs is sufficient for the phenotype. This is critical to confirm the take-home message of the manuscript.

    1. Reviewer #2 (Public review):

      Summary:

      In the manuscript by Walter-McNeill, Kruglyak, and team, the authors provide solid evidence of another toxin-antidote (TA) system in C. elegans. Generally, TA systems involve selfish and linked genetic elements, one encoding a toxin that kills progeny inheriting it, unless an antidote (the second element) is also present. Currently, only two TA systems have been characterized in this species, pointing to the importance of identifying new instances of such systems to understand their transmission dynamics, prevalence, and functions in shaping worm populations.

      Strengths:

      This novel TA system (mll-1/smll-1) was identified on LGV in wild C. elegans isolates from the Hawaiian islands, by crossing divergent strains and observing allele frequency distortions by high-throughput genome sequencing after 10 generations. These allele frequency distortions were subsequently confirmed in another set of crosses with a separate divergent strain, and crosses of heterozygous males or hermaphrodites resulted in a pattern of L1 lethality in progeny (with a rod arrest phenotype) that suggested the maternal transmission of this TA system from the XZ1516 genetic background. By elegantly combining the use of near-isogenic lines, CRISPR editing to generate knock-outs, and a transgene rescue of the antidote gene, the authors identified the genes encoding the toxin and the antidote, which they refer to as mll-1 and smll-1. Moreover, the specific mll-1 isoform responsible for the production of the toxin was identified and mll-1 transcripts were observed by FISH in early and late embryos, as well as in larvae. Inducible expression of the toxin in various strains resulted in larval arrest and rod phenotypes. The authors then characterized the genetic variation of 550 wild isolates at the toxin/antidote region on LGV and distinguished three clades: (1) one with the conserved TA system, (2) one having lost the toxin and retaining a mostly functional antidote, and (3) one having lost the antidote and retaining a divergent yet coding toxin (this includes the reference strain Bristol N2, in which the homologous toxin gene has acquired mutations and is known as B0250.8). Further, the authors show that this region is under positive selection. These data are compelling and provide very strong evidence of a new TA system in this species.

      Weaknesses:

      The question remained as to how one clade, including N2, could retain the toxin gene but not possess a functional antidote. In the second part of the manuscript, the authors hypothesized that small RNA targeting (RNAi) of the toxin transcript could provide the necessary repression to allow worms to survive without the antidote. Through a meta-analysis of multiple small RNA datasets from the literature, the authors found evidence to support this idea, in which the toxin transcript is targeted by 22G siRNAs whose biogenesis is dependent on the Mutator foci protein, MUT-16. They note that from previous studies, mut-16 null mutants displayed a varied penetrance of larval arrest. In their own hands, mut-16 mutants displayed 15% varied larval arrest and 2% rod phenotypes. In an attempt to link B0250.8 to mut-16/siRNAs, they made a double mutant and examined body length as a proxy for developmental stage. Here, they observed a partial rescue of the mut-16 size defect by B0250.8 mutation. Finally, the authors also highlight data from further meta-analysis, which predicts the recognition of B0250.8 by several piRNAs. Also based on existing data from the literature, the authors link loss of Piwi (PRG-1), which binds piRNAs, to a depletion of 22G-RNAs targeting B0250.8 and an upregulation of B0250.8 expression in gonads, suggesting that piRNAs are the primary small RNAs that target B0250.8 for downregulation. The data in this portion of the manuscript are intriguing, but somewhat preliminary and incomplete, as they are based on little primary experimentation and a collection of different datasets (which have been acquired by slightly different methods in most cases). This portion of the study would require subsequent experimentation to firmly establish this mechanistic link. For example, to be able to claim that "the N2 toxin allele has acquired mutations that enable piRNA binding to initiate MUT-16-dependent 22G small RNA amplification that targets the transcript for degradation" the identified piRNA sites should be mutated and protein and transcript levels analysed in wild-type and in the strain with mutated piRNA sites. At a minimum, the protein levels in wild-type and mut-16, prg-1, and/or wago-1 mutants should be measured by western blot and/or by live imaging (introducing a GFP or some other tag to the endogenous protein via CRISPR editing) to show that the toxin is not accumulated as a protein in wt, but increases in levels in these mutants. mRNA levels in Figure S5A suggest there is still some expression of the B0250.8 transcript in a wild-type situation.

    1. Reviewer #2 (Public review):

      Summary:

      The authors' aim in this study is to assess the factors that can shift competitive incentives against higher- or lower-ranking groupmates in two gorilla species.

      Strengths:

      This is a relevant topic, where important insights could be gained. The authors brought together a substantial dataset: a long-term behavioral dataset representing two gorilla species from five social groups.

      Weaknesses:

      The authors have not fully shown the data used in the model and explored the potential of the model. Therefore, I remain cautious about the current results and conclusions.

      Some specific suggestions that require attention are

      (1) The authors described how group size can affect aggression patterns in some species (line 54), using a whole paragraph, but did not include it as an explanation variable in their model, despite that they stated the overall group size can "conflate opposing effects of females and males" (line 85). I suggest underlining the effects of numbers of males or/and females here and de-emphasizing the effect of group size in the Introduction.

      (2) There should be more details given about how the authors calculated individual Elo-ratings (line 98). It seems that authors pooled all avoidance/displacement behaviors throughout the study period. But how often was the Elo-rating they included in the model calculated? By the day or by the month? I guess it was by the day, as they "estimate female reproductive state daily" (line 123). If so, it should be made clear in the text.

      In addition, all groups were long-term studied, and the group composition seems fluctuant based on the Table 1 in Reference 11. When an individual enters/leaves the group with a stable hierarchy, it takes time before the hierarchy turns stable again. If the avoidance/displacement behaviors used for the rank relationship were not common, it would take a few days or maybe longer. Also, were the aggressive behaviors more common during rank fluctuations? In other words, if avoidance/displacement behaviors and aggressive behaviors occur simultaneously during rank fluctuations, how did the authors deal with it and take it into consideration in the analysis?

      The authors emphasized several times in the text that gorillas "form highly stable hierarchical relationships". Also, in Reference 25, they found very high stabilities of each group's hierarchy. However, the number of females involved in that analysis was different from that used here. They need to provide more basic info on each group's dominance hierarchy and verify their statement. I strongly suggest that the authors display Elo-rating trajectories and necessary relevant statistics for each group throughout the study period as part of the supplementary materials.

      (3) The authors stated why they differentiated the different stages based on female reproductive status. They also referred to the differences in energetic needs between stages of pregnancy and lactation (lines 127-128). However, in the mixed model, they only compared the interaction score between the female cycling stage and other stages. The model was not well explained, and the results could be expanded. I suggest conducting more pairwise comparisons in the model and presenting the statistics in the text, if there are significant results. If all three pregnancy stages differed significantly from cycling and lactating stages but not from each other, they may be merged as one pregnancy stage. More in-depth analysis would help provide better answers to the research questions.

    1. Reviewer #3 (Public review):

      The revised manuscript demonstrates a marked improvement over the previous version. The authors have successfully incorporated feedback, and have moreover expanded their analyses.

      The Methods section is now more detailed and meets the requirements for reproducible research. Authors have reprocessed the data, creating an integrated dataset using a previously published single-cell RNA-Seq atlas, which includes both healthy donors and individuals with chronic HIV-1 infection. An additional batch correction step was included into the processing pipeline after the explicit analysis of inter-donor variability within immune subsets, as was suggested.

      Several supplementary figures were added, which both improve the understanding of data and address questions raised by the reviewers. The manuscript also provides additional analysis of cell communication inference, as suggested. The study of interactions between NK cells and infected CD4+ T cells, as well as between monocytes and infected CD4+ T cells, is valuable for understanding the influence of cell signaling on antiviral response and the production of HIV-1 transcripts in infected cells.

      The authors have addressed all the reviewers' suggestions, and the current version of the manuscript is both more comprehensive and more informative. Additional analysis has strengthened the narrative and the reproducibility of the research.

      The resulting manuscript is both more robust and more informative.

    1. Reviewer #3 (Public review):

      Summary:

      This paper explores the phenomenon whereby some V1 neurons can respond to stimuli presented far outside their receptive field. It introduces three possible explanations for this phenomenon and it presents experiments that it argues favor the third explanation, which is based on figure/ground segregation.

      Strengths:

      I found it useful to see that there are three possible interpretations of this finding (prediction error, interpolation, and figure/ground). I also found it useful to see a comparison with LGN responses and to see that the effect there is not only absent but actually opposite: stimuli presented far outside the receptive field suppress rather than drive the neurons. Other experiments presented here may also be of interest to the field.

      Weaknesses:

      Though the paper has markedly improved, and now has a clearer statement of the hypotheses, it could be streamlined further, to tighten the relation between hypotheses and analyses, and to draw conclusions from those analyses in terms of the hypotheses.

    1. Reviewer #2 (Public review):

      Summary

      Le Roy et al quantify wing morphology and wing kinematics across twenty eight and eight hoverfly species, respectively; the aim is to identify how weight support during hovering is ensured across body sizes. Wing shape and relative wing size vary non-trivially with body mass, but wing kinematics are reported to be size-invariant. On the basis of these results, it is concluded that weight support is achieved solely through size-specific variations in wing morphology, and that these changes enabled hoverflies to decrease in size. Adjusting wing morphology may be preferable compared to the alternative strategy of altering wing kinematics, because kinematics may be subject to stronger evolutionary and ecological constraints, dictated by the highly specialised flight and ecology of the hoverflies.

      Strengths

      The study deploys a vast array of challenging techniques, including flight experiments, morphometrics, phylogenetic analyses, and numerical simulations; it so illustrates both the power and beauty of an integrative approach to animal biomechanics. The question is well motivated, the methods appropriately designed, and the discussion elegantly places the results in broad biomechanical, ecological, and evolutionary context.

      Weaknesses

      (1) In assessing evolutionary allometry, it is key to pinpoint the variation expected from changes in size alone. The null hypothesis for wing morphology is well-defined (isometry), but the equivalent predictions for kinematic parameters, although specified, are insufficiently justified, and directly contradict classic scaling theory. A detailed justification of the "kinematic similarity" assumption, or a change in the null hypothesis, would substantially strengthen the paper, and clarify its evolutionary implications.

      (2) By relating the aerodynamic output force to wing morphology and kinematics, it is concluded that smaller hoverflies will find it more challenging to support their body mass--a scaling argument that provides the framework for this work. This hypothesis appears to stand in direct contrast to classic scaling theory, where the gravitational force is thought to present a bigger challenge for larger animals, due to their disadvantageous surface-to-volume ratios. The same problem ought to occur in hoverflies, for wing kinematics must ultimately be the result of the energy injected by the flight engine: muscle. Much like in terrestrial animals, equivalent weight support in flying animals thus requires a positive allometry of muscle force output. In other words, if a large hoverfly is able to generate the wing kinematics that suffice to support body weight, an isometrically smaller hoverfly should be, too (but not vice versa). Clarifying the relation between the scaling of muscle mechanical input, wing kinematics, and weight support would help resolve the conflict between these two contrasting hypotheses, and considerably strengthen the biomechanical motivation and evolutionary interpretation.

      (3) One main conclusion-- that miniaturization is enabled by changes in wing morphology--is insufficiently supported by the evidence. Is it miniaturization or "gigantism" that is enabled by (or drives) the non-trivial changes in wing morphology? To clarify this question, the isolated treatment of constraints on the musculoskeletal system vs the "flapping-wing based propulsion" system needs to be replaced by an integrated analysis: the propulsion of the wings, is, after all, due to muscle action. Revisiting the scaling predictions by assessing what the engine (muscle) can impart onto the system (wings) will clarify whether non-trivial adaptations in wing shape or kinematics are necessary for smaller or larger hovering insects (if at all!).

      In many ways, this work provides a blueprint for work in evolutionary biomechanics; the breadth of both the methods and the discussion reflects outstanding scholarship.

    1. Reviewer #3 (Public review):

      Summary:

      Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of brain-derived estrogens in the control of sexual and aggressive behavior in medaka. The constitutive deletion of Cyp19a1b markedly reduced brain estrogen content in males and to a lesser extent in females. These effects are accompanied by reduced sexual and aggressive behavior in males and reduced preference for males in females. These effects are reversed by adult treatment with supporting a role for estrogens. The deletion of Cyp19a1b is associated with a reduced expression of the genes coding for the two androgen receptors, ara and arb, in brain regions involved in the regulation of social behavior. The analysis of the gene expression and behavior of mutants of estrogen receptors indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. These results provide valuable insight into the role of estrogens in social behavior in the most abundant vertebrate taxon, however the conclusion of brain-derived estrogens awaits definitive confirmation.

      Strengths:

      • Evaluation of the role of brain "specific" Cyp19a1 in male teleost fish, which as a taxon are more abundant and yet proportionally less studied that the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. This approach also offers great potential to study the role of brain estrogen production in females, an understudied question in all taxa.

      • Results obtained from multiple mutant lines converge to show that estrogen signaling, likely synthesized in the brain drives aspects of male sexual behavior.

      • The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.

      • The authors have made important corrections to tone down some of the conclusions which are more in line with the results.

      Weaknesses:

      • No evaluation of the mRNA and protein products of Cyp19a1b and ESR2a are presented, such that there is no proper demonstration that the mutation indeed leads to aromatase reduction. The conclusion that these effects dependent on brain derived estrogens is therefore only supported by measures of E2 with an EIA kit that is not validated. No discussion of these shortcomings is provided in the discussion thus further weakening the conclusion manuscript.

      • Most experiments are weakly powered (low sample size).

      • The variability of the mRNA content for a same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).

      Conclusions:

      Overall, the claims regarding role of estrogens originating in the brain on male sexual behavior is supported by converging evidence from multiple mutant lines. The role of brain-derived estrogens on gene expression in the brain is weaker as are the results in females.

    1. Reviewer #2 (Public review):

      Summary:

      Malboubi et al. present a novel experimental framework to investigate the rheological properties of the cell cytoplasm. Their findings support a model where the cytoplasm behaves as a poroelastic material governed by Darcy's law - a property overlooked in previous literature. They demonstrate that this poroelastic behavior delays the equilibration of hydrostatic pressure gradients within the cytoplasm over timescales of 1 to 10 seconds following a perturbation, likely due to fluid-solid friction within the cytoplasmic matrix. Furthermore, under sustained perturbations such as depressurization, they reveal that pressure gradients can persist for minutes, which they propose might potentially influence physiological processes like mechanotransduction or cell migration typically happening on these timescales.

      Strengths:

      This article holds significant value within the ongoing efforts of the cell biology and biophysics communities to quantitatively characterize the mechanical properties of cells. The experiments are innovative and thoughtfully contextualized with quantitative estimates and a finite element model that supports the authors' hypotheses.

      Comments & Questions:

      While the hypothesis of a poroelastic cytoplasm is insightful and supported by the results, certain parts of the paper (detailed below) rely on qualitative arguments. Given the experimental approaches and accompanying modeling, the study has the potential for more in-depth discussions and stronger quantitative evidence. Placing greater emphasis on quantifications and direct comparisons between the model and experimental data would enhance the work. Additionally, exploring the limitations of the proposed model would add valuable depth to the paper.

      The authors state, "Next, we sought to quantitatively understand how the global cellular response to local indentation might arise from cellular poroelasticity." However, the evidence presented in the following paragraph appears more qualitative than strictly quantitative. For instance, the length scale estimate of ~7 μm is only qualitatively consistent with the observed ~10 μm, and the timescale 𝜏𝑧 ≈ 500 ms is similarly described as "qualitatively consistent" with experimental observations. Strengthening this point would benefit from more direct evidence linking the short timescale to cell surface tension. Have you tried perturbing surface tension and examining its impact on this short-timescale relaxation by modulating acto-myosin contractility with Y-27632, depolymerizing actin with Latrunculin, or applying hypo/hyperosmotic shocks?

      The authors demonstrate that the second relaxation timescale increases (Figure 1, Panel D) following a hyperosmotic shock, consistent with cytoplasmic matrix shrinkage, increased friction, and consequently a longer relaxation timescale. While this result aligns with expectations, is a seven-fold increase in the relaxation timescale realistic based on quantitative estimates given the extent of volume loss?

      If the authors' hypothesis is correct, an essential physiological parameter for the cytoplasm could be the permeability k and how it is modulated by perturbations, such as volume loss or gain. Have you explored whether the data supports the expected square dependency of permeability on hydraulic pore size, as predicted by simple homogeneity assumptions? Additionally, do you think that the observed decrease in k in mitotic cells compared to interphase cells is significant? I would have expected the opposite naively as mitotic cells tend to swell by 10-20 percent due to the mitotic overshoot at mitotic entry (see Son Journal of Cell Biology 2015 or Zlotek Journal of Cell Biology 2015).

      Based on your results, can you estimate the pore size of the poroelastic cytoplasmic matrix? Is this estimate realistic? I wonder whether this pore size might define a threshold above which the diffusion of freely diffusing species is significantly reduced. Is your estimate consistent with nanobead diffusion experiments reported in the literature?

      Do you have any insights into the polymer structures that define this pore size? For example, have you investigated whether depolymerizing actin or other cytoskeletal components significantly alters the relaxation timescale?

      There are no quantifications in Figure 6, nor is there a direct comparison with the model. Based on your model, would you expect the velocity of bleb growth to vary depending on the distance of the bleb from the pipette due to the local depressurization? Specifically, do blebs closer to the pipette grow more slowly?

      I find it interesting that during depressurization of the interphase cells, there is no observed volume change, whereas in pressurization of metaphase cells, there is a volume increase. I assume this might be a matter of timescale, as the microinjection experiments occur on short timescales, not allowing sufficient time for water to escape the cell. Do you observe the radius of the metaphase cells decreasing later on? This relaxation could potentially be used to characterize the permeability of the cell surface.

      I am curious about the saturation of the time lag at 30 microns from the pipette in Figure 4, Panel E for the model's prediction. A saturation which is not clearly observed in the experimental data. Could you comment on the origin of this saturation and the observed discrepancy with the experiments (Figure E panel 2)? Naively, I would have expected the time lag to scale quadratically with the distance from the pipette, as predicted by a poroelastic model and the diffusion of displacement. It seems weird to me that the beads start to move together at some distance from the pipette or else I would expect that they just stop moving. What model parameters influence this saturation? Does membrane permeability contribute to this saturation?

    1. Reviewer #2 (Public review):

      Summary:

      Pablo Ruiz Cuenca et al. conducted a GPS logger study with 124 adult participants across four different slum areas in Salvador, Brazil, recording GPS locations every 35 seconds for 48 hours. The aim of their study was to investigate step-selection models, a technique widely used in movement ecology to quantify contact with environmental risk factors for exposure to leptospires (open sewers, community streams, and rubbish piles). The authors built two different types of models based on distance and based on buffer areas to model human environmental exposure to risk factors. They show differences in movement/contact with these risk factors based on gender and seropositivity status. This study shows the existence of modest differences in contact with environmental risk factors for leptospirosis at small spatial scales based on socio-demographics and infection status.

      Strengths:

      The authors assembled a rich dataset by collecting human GPS logger data, combined with field-recorded locations of open sewers, community streams, and rubbish piles, and testing individuals for leptospirosis via serology. This study was able to capture fine-scale exposure dynamics within an urban environment and shows differences by gender and seropositive status, using a method novel to epidemiology (step selection).

      Weaknesses:

      Due to environmental data being limited to the study area, exposure elsewhere could not be captured, despite previous research by Owers et al. showing that the extent of movement was associated with infection risk. Limitations of step selection for use in studying human participants in an urban environment would need to be explicitly discussed.

    1. Reviewer #2 (Public review):

      Summary:

      In this paper, Selberg et al present an extension of their widely used BUSTED family of codon models for the detection of episodic ("site-branch") positive selection from coding gene sequences. The extension adds an "error component" to ω (dN/dS) to capture misaligned codons. This ω component is set to an arbitrarily high value to distinguish it from positive selection, which is characterised by ω > 1 but assumed not to be so high.

      The new method is tested on several datasets of comparative genomes, characterised by their size and the fact that the authors scanned for positive selection and/or provided filtering of alignment quality. It is also tested on simple simulations.

      Overall, the new method appears to capture relatively little of the ω variability in the alignments, although it is often significant. Given the complexity of codon evolution, adding a new parameter is more or less significant, and the question is whether it captures the signal that is intended, preferably in an unbiased manner.

      Strengths:

      This is an important issue, and I am enthusiastic to see it explicitly modeled within the codon modeling framework, rather than externalised to ad hoc filtering methods. The promise of quantifying the divergence signal from alignment error vs selection is exciting.

      The BUSTED family of models is widely used and very powerful for capturing many aspects of codon evolution, and it is thus an excellent choice for this extension.

      Weaknesses:

      (1) The definition of alignment error by a very large ω is not justified anywhere in the paper. There are known cases of bona fide positive selection with many non-synonymous and 0 synonymous substitutions over branches. How would they be classified here? E.g., lysosyme evolution, bacterial experimental evolution.

      Using the power of the model family that the authors develop, I would suggest characterising a more specific error model. E.g., radical amino-acid "changes" clustered close together in the sequence, proximity to gaps in the alignment, correlation of apparent ω with genome quality.

      Also concerning this high ω, how sensitive is its detection to computational convergence issues?

      (2) The authors should clarify the relation between the "primary filter for gross or large-scale errors" and the "secondary filter" (this method). Which sources of error are expected to be captured by the two scales of filters? What is their respective contribution to false positives of positive selection?

      Sources of error in the alignment of coding genes include:

      a) Errors in gene models, which may differ between species but also propagate among close species (i.e., when one species is used as a reference to annotate others).

      b) Inconsistent choice of alternative transcripts/isoforms.

      Both of these lead to asking an alignment algorithm to align non-homologous sequences, which violates the assumptions of the algorithms, yet both are common issues in phylogenomics.

      c) Sequencing errors, but I doubt they affect results much here.

      d) Low complexity regions of proteins.

      e) Aproximations by alignment heuristics, sometimes non-deterministic or dependent on input order.

      f) Failure to capture aspects of protein or gene evolution in the optimality criteria used.

      For example, Figure 1 seems to correspond to a wrong or inconsistent definition of the final exon of the gene in one species, which I would expect to be classified as "gross or large-scale error".

      (3) The benchmarking of the method could be improved both for real and simulated data.

      For real data, the authors only analysed sequences from land vertebrates with relatively low Ne and thus relatively low true positive selection. I suggest comparing results with e.g. Drosophila genomes, where it has been reported that 50% of all substitutions are fixed by positive selection, or with viral evolution.

      For simulations, the authors should present simulations with or without alignment errors (e.g., introduce non-homologous sequences, or just disturb the alignments) and with or without positive selection, to measure how much the new method correctly captures alignment errors and incorrect positive selection.

      I also recommend simulating under more complex models, such as multinucleotide mutations or strong GC bias, and investigating whether these other features are captured by the alignment error component.

      Finally, I suggest taking true alignments and perturbing them (e.g., add non-homologous segments or random gaps which shift the alignment locally), to verify how the method catches this. It would be interesting to apply such perturbations to genes which have been reported as strong examples of positive selection, as well as to genes with no such evidence.

      (4) It would be interesting to compare to results from the widely used filtering tool GUIDANCE, as well as to the Selectome database pipeline (https://doi.org/10.1093/nar/gkt1065). Moreover, the inconsistency between BUSTED-E and HMMCleaner, and BMGE is worrying and should be better explained.

      (5) For a new method such as this, I would like to see p-value distributions and q-q plots, to verify how unbiased the method is, and how well the chi-2 distribution captures the statistical value.

      (6) I disagree with the motivation expressed at the beginning of the Discussion: "The imprimatur of "positive selection" has lost its luster. Researchers must further refine prolific candidate lists of selected genes to confirm that the findings are robust and meaningful." Our goal should not be to find a few impressive results, but to measure accurately natural selection, whether it is frequent or rare.

    1. Reviewer #2 (Public review):

      Summary:

      Franz and colleagues combined proteomics analysis of OVSAHO cell lines treated with 6 individual drugs. The quantitative proteomics data were then used for computational analysis to identify candidates/modules that could be used to predict combination treatments for specific drugs.

      Strengths:

      The authors present solid proteomics data and computational analysis to effectively repeat at the proteomics level analysis that have previously been done predominantly with transcriptional profiling. Since most drugs either target proteins and/or proteins are the functional units of cells, this makes intuitive sense.

      Weaknesses:

      Considering the available resources of the involved teams, performing the initial analysis in a single HGSC cell is certainly a weakness/limitation.

      The data also shows how challenging it is to correctly predict drug combinations. In Table 2 (if I read it correctly), the majority of the drug combinations predicted for the initial cell line OVSAHO did not result in the predicted effect. It also shows how variable the response was in the different HGSC cell lines used for the combination treatment. The success rate will most likely continue to drop as more sophisticated models are being used (i.e., PDX). Human patients are even more challenging.

      It would most likely be useful to more directly mention/discuss these caveats in the manuscript.

    1. Reviewer #2 (Public review):

      Summary:

      The authors set out to identify cell-free HPV breakpoint junctions and assess their utility in identifying cervical cancer recurrence as a surrogate, tumor-specific assay. They added unrelated findings about a potential relationship between various viral types and cancer recurrence frequencies, concluding that clade alpha 9 types recurred at a lower rate than did non-alpha 9 viral types.

      Strengths:

      The authors analyzed 16 cervical cancer samples and matched serum samples collected initially or upon clinical treatments. An association between virus types and cancer recurrence frequencies is a novel finding that will likely induce further insights into HPV pathogenic mechanisms.

      Weaknesses:

      The main claims of this manuscript are only partially supported by the data as presented, because the sequencing data are not quantified and were not analyzed in a statistically adequate way. First, only one or at most two breakpoints are presented per tumor (Table 1). This finding is discrepant from many extensive, published genomics studies of HPV-positive cancers, in which many unique breakpoints are found frequently in individual cancers, ranging from 1 or 2 up to more than 100. Second, no information is provided about likely correlations between genomic DNA copy number at rearranged loci and breakpoint-identifying sequencing read counts. Third, no direct comparison is presented between supporting read counts from cancer samples and read counts from circulating cell-free DNA samples. Fourth, many of the initial cancer samples harbored no insertional breakpoints, so no correlation with breakpoints in the serum samples would be possible. Fifth, no mention was made about tumor heterogeneity, where a given breakpoint may not be present in every cell of the tumor. Previous literature about the general topic of using cell-free DNA breakpoints as a surrogate for cancer cells is not cited adequately. Findings about potential correlations between various viral types and variable recurrence rates are not well-supported by the authors' own data, because of the limited sample numbers studied. This section of the paper is relatively unrelated to the main thrust, which is about breakpoint detection.

    1. Reviewer #2 (Public review):

      The authors of the present study are responsible for a previous study, which also showed that in response to DNA damage, Werner syndrome protein WRN, WRN interacting protein WRNIP1, and Rev1 assemble together with Y-family Pols (Polη, Polι, or Polκ), and that they are indispensable for Trans-Lesion-Synthesis (TLS) (Genes Dev 2024). They also identified a role of WRN's 3'→5' exonuclease activity in the high in vivo fidelity of TLS by Y-family, through UV-induced CPDs by Polη, through N6 ethenodeoxyadenosine (εdA) by Polι, through thymine glycol by Polκ, and through UV-induced (6-4) photoproducts by Polη and Polι. Thus, by removing nucleotides misinserted opposite DNA lesions by the Y-family Pols, WRN's 3'→5' exonuclease activity improves the fidelity of TLS by these Pols. The present work, which follows up on this previous work, reports the crucial role also of the ATPase activities of WRN and WRNIP1 in raising the fidelity of TLS by Y family Pols, in addition to the exonuclease activity, with an entirely different mechanism, which normally consists in unwinding of DNA containing secondary structures.

      By using adequate cell line models and methodologies, notably DNA fiber, TLS, and mutation analyses assays, as well as specific ATPase point mutations, they found that progression of the replication forks through UV lesions was not affected in cells lacking the WRN exonuclease activity as well as the WRN and WRNIP1 ATPase activities, but occurs with a vast increase in error-prone TLS, notably through CPDs by Polη, with differential impacts on the nature of mutations between WRN ATPase and WRNIP1 ATPase. The relative contributions of these activities (exonuclease and ATPase) to the fidelity of TLS Pols, however, vary, depending upon the DNA lesion and the TLS Pol involved. Additionally, defects in these ATPase activities cause mutational hot spot formation in different sequence contexts. The authors provide evidence that the combined action of WRN and WRNIP1 ATPases, along with WRN 3' to 5' exonuclease, confers an enormous rise in the fidelity of TLS by Y-family Pols. They identify the means by which these otherwise highly error-prone TLS Pols have been adapted to function in an error-free manner. They suggest that WRNIP1 ATPases prevent misincorporations while WRN exonuclease removes misinserted nucleotides. This combination confers a vast increase in the fidelity of Y-family Pols, essential for genome stability.

      Overall, this is a comprehensive and thoughtful manuscript, and all the findings reported are convincing and well supported. The data cannot be considered as entirely novel, as they follow-up on the recent 2024 publication by the same authors who unveiled that the exonuclease activity of WRN and WRNIP1 confers accuracy of TLS. The experimental methods are multiple and rigorous.

    1. Reviewer #2 (Public review):

      Summary

      Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight. The authors linked their behavioral results to visual scene statistics in the hawkmoths' natural environment. The partition of ventral and dorsal visuomotor pathways is well in line with differences in visual cue frequencies. The response hierarchy, however, seems to be dominated by dorsal features, that are less frequent, but presumably highly relevant for the animals' flight safety.

      Strengths

      The data are very interesting and unique. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.

      Weaknesses

      While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?

      I find the majority of the data, which are also the data supporting the main claims of the paper, compelling. However, the measurements of flight height are less solid than the rest and I think these data should be interpreted more carefully.

    1. Reviewer #2 (Public review):

      Summary:

      In the present study, the authors investigated the nature of attentional templates during preparatory period of goal-directed attention. By combing the use of 'pinging' the neural activity with a visual impulse and fMRI-based multivariate decoding, the authors found that the nature of the neural representations of the prospective feature target during preparatory period was contingent on the presence of the 'pinging' impulse. While the preparatory representations contained highly similar information content as the perceptual representations when the pinging impulse was introduced, they fundamentally differed from perceptual representations in the absence of the pinging impulse. Based on these findings, the authors proposed a dual-format mechanism in which both a "non-sensory" template and a latent "sensory" template coexisted during attentional preparation. The former actively guides activity in the preparatory state, and the latter is utilized for future stimulus processing.

      Strengths:

      Overall, I think that the authors' revision has addressed most, if not all, of my major concerns noted in my previous comments.

      Weaknesses:

      The results appear convincing and I do not have additional comments.

    1. Reviewer #2 (Public review):

      Summary:

      This paper addresses the bottom-up and top-down causes of hearing difficulties in middle-aged adults with clinically-normal audiograms using a cross-species approach (humans vs. gerbils, each with two age groups) mixing behavioral tests and electrophysiology.. The study is not only a follow-up of Parthasarathy et al (eLife 2020), since there are several important differences. Parthasarathy et al. (2020) only considered a group of young normal-hearing individuals with normal audiograms yet with high complaints for hearing in noisy situations. Here, this issue is considered specifically regarding aging, using a between-subject design comparing young NH and older NH individuals recruited from the general population, without additional criterion (i.e. no specifically high problems of hearing in noise). In addition, this is a cross-species approach, with the same physiological EFR measurements with the same stimuli deployed on gerbils.

      This article is of very high quality. It is extremely clear, and the results show clearly a decrease of neural phase-locking to high modulation frequencies in both middle-aged humans and gerbils, compared to younger groups/cohorts. In addition, pupillometry measurements conducted during the QuickSIN task suggest increased listening efforts in middle-aged participants, and a statistical model including both EFRs and pupillometry features suggest that both factors contribute to reduced speech-in-noise intelligibility evidenced in middle-aged individuals, beyond their slight differences in audiometric thresholds (although they were clinically normal in both groups).

      These provide strong support to the view that normal aging in humans leads to auditory nerve synaptic loss (cochlear neural degeneration - CND- or, put differently, cochlear synaptopathy) as well as increased listening effort, before any clearly visible audiometric deficits as defined in current clinical standards. This result is very important for the community, since we are still missing direct evidence that cochlear synaptopathy might likely underly a significant part of hearing difficulties in complex environments for listeners with normal thresholds, such as middle-aged and senior listeners. This paper shows that these difficulties can be reasonably well accounted for by this sensory disorder (CND), but also that listening effort, i.e. a top-down factor, further contributes to this problem. The methods are sound, well described and I would like to emphasize that they are presented concisely yet in a very precise manner, so that they can be understood very easily - even for a reader that is not familiar with the employed techniques. I believe this study will be of interest to a broad readership. I have some comments and questions which I think would make the paper even stronger once addressed.

      Main comments:

      (1) Presentation of EFR analyses / Interpretation of EFR differences found in both gerbils and humans

      a) Could you comment further on why you think you found a significant difference only at the highest mod. frequency of 1024 Hz in your study? Indeed, previous studies employing SAM or RAM tones very similar to the ones employed here were able to show age effects already at lower modulation freqs. of ~100H; e.g. there are clear age effects reported in human studies of Vasilikov et al. (2021) or Mepani et al. (2021), and also in animals ( see Garrett et al. bioRxiv : https://www.biorxiv.org/content/biorxiv/early/2024/04/30/2020.06.09.142950.full.pdf)

      Furthermore, some previous EEG experiments in humans that SAM tones with modulation freqs. of ~100Hz showed that EFRs do not exhibit a single peak, i.e. there are peaks not only at fm but also for the first harmonics (e.g. 2fm or 3fm) see e.g. Garrett et al. bioXiv https://www.biorxiv.org/content/biorxiv/early/2024/04/30/2020.06.09.142950.full.pdf

      Did you try to extract EFR strength by looking at the summed amplitude of multiple peaks (Vasilikov Hear Res. 2021), in particular for the lower modulation frequencies? (Indeed, there will be no harmonics for the higher mod. freqs).

      b) How the present EFR results relate to FFR results, where effects of age are already at low carrier freqs? (e.g. Märcher-Rørsted et al., Hear. Res., 2022 for pure tones with freq < 500 Hz) Do you think it could be explained by the fact that this is not the same cochlear region, and that synapses die earlier in higher compared to lower CFs. This should be discussed. Beyond the main group effect of age, there were no negative correlations of EFRs with age in your data?

      (2) Size of the effects / comparing age effects between two species: Although the size of the age effect on EFRs cannot be directly compared between humans and gerbils - the comparison remains qualitative - could you a least provide references regarding the rate of synaptic loss with aging in both humans and gerbils, so that we understand that the yNH/MA difference can be compared between the two age groups used for gerbils; it would have been critical in case of a non-significant age effect in one species.

      Equalization / control of stimuli differences across the two species: For measuring EFRs, SAM stimuli were presented at 85 dB SPL for humans vs. 30 dB above detection threshold (inferred from ABRs) for gerbils - I do not think the results strongly depend on this choice, but it would be good to comment on why you did not choose also to present stimuli 30 dB above thresholds in humans.

      Simulations of EFRs using functional models could have been used to understand (at least in humans) how the differences in EFRs obtained between the two groups are quantitatively compatible with the differences in % of remaining synaptic connections known from histopathological studies for their age range (see the approach in Märcher-Rørsted et al., Hear. Res., 2022)

      (3) Synergetic effects of CND and listening effort Could you test whether there is an interaction between CNR and listening effort? (e.g. one could hypothesize that MA subjects with largest CND have also the higher listening effort)

      Comments on revised version:

      The authors did well to address all the points raised in my review. This paper will make an important contribution to our assessment of the sources of age-related auditory processing deficits beyond the cochlea that impair speech intelligibility.

    1. Reviewer #2 (Public review):

      Summary:

      The authors exposed naïve male flies to different groups of females, either mated or virgin. Male flies can successfully copulate with virgin females; however, they are rejected by mated females. This rejection reduces sugar preference and sensitivity in males. Investigating the underlying neural circuits, the authors show that dopamine signaling onto GR5a sensory neurons is required for reduced sugar preference. GR5a sensory neurons respond less to sugar exposure when they lack dopamine receptors.

      Strengths:

      The findings add another strong phenotype to the existing dataset about brain-wide neuromodulatory effects of mating. The authors use several state-of-the-art methods, such as activity-dependent GRASP, to decipher the underlying neural circuitry. They further perform rigorous behavioral tests and provide convincing evidence for the local labellar circuit.

      Weaknesses:

      The authors focus on the circuit connection between dopamine and gustatory sensory neurons in the male SEZ. Therefore, it is still unknown how mating modulates dopamine signaling and what possible implications on other behaviors might result from a reduced sugar preference.

      The authors updated missing literature in the manuscript and performed additional experiments regarding behavior, but also to further prove the functional connectivity between TH neurons and GR5a neurons.

      I have no further recommendations.

    1. Reviewer #2 (Public review):

      Strengths:

      Fallah et al carefully dissect projections from SNr and GPe - two key basal ganglia nuclei - to the PPN, an important brainstem nucleus for motor control. They consider inputs from these two areas onto 3 types of downstream PPN neurons: GABAergic, glutamatergic, and cholinergic neurons. They also carefully map connectivity along the rostrocaudal axis of the PPN. They provide important and convincing data on PPN connectivity with two important input structures, which will provide a foundation for many future studies. They also consider the behavioral relevance of these different PPN inputs for controlling movement and reinforcement, showing convincing evidence that SNr and GPe inputs have opposing effects on behavior.

      Weaknesses:

      The optogenetics and behavioral studies are intriguing, although more work will be required to fit these data together into a specific model of circuit function and to distinguish the locomotor and reinforcement effects. Interestingly, stimulation of SNr axons in the rostral vs caudal PPN likely differs (as predicted by slice experiments), indicating an area for future investigation and dissection of pathways.

    1. Reviewer #2 (Public review):

      Summary:

      The authors proposed a neural network model to explore the spatial representations of the hippocampal CA1 and entorhinal cortex (EC) and the remapping of these representations when multiple environments are learned. The model consists of a recurrent network and output units (a decoder) mimicking the EC and CA1, respectively. The major results of this study are: the EC network generates cells with their receptive fields tuned to a border of the arena; the decoder develops neuron clusters arranged in a hexagonal lattice. Thus, the model accounts for entrohinal border cells and CA1 place cells. It suggests that the remapping of place cells occurs between different environments through state transitions corresponding to unstable dynamical modes in the recurrent network.

      Strengths:

      The authors found a spatial arrangement of receptive fields similar to their model's prediction in experimental data recorded from CA1. Thus, the model proposes plausible mechanisms to generate hippocampal spatial representations without relying on grid cells. The model also suggests an interesting possibility that path integration is not the speciality of grid cells.

      Weaknesses:

      The role of grid cells in the proposed view, i.e., the boundary-to-place-to-grid model, remains elusive. The model can generate place cells without generating entorhinal grid cells. Moreover, the model can generate hexagonal grid patterns of place cells in a large arena. Whether and how the proposed model is integrated into the entire picture of the hippocampal-entorhinal memory processing remains elusive.

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript, the authors performed the functional analysis of odorant receptors (ORs) of the termite Prorhinotermes simplex to identify the receptor of trail-following pheromone. The authors performed single-sensillum recording (SSR) using the transgenic Drosophila flies expressing a candidate of the pheromone receptor and revealed that PsimOR14 strongly responds to neocembrene, the major component of the pheromone. Also, the authors found that one sensillum type (S I) detects neocembrene and also performed SSR for S I in the wild termite workers. Furthermore, the authors revealed the gene, transcript, and protein structures of PsimOR14, predict the 3D model and ligand docking of PsimOR14, and demonstrated that PsimOR14 is higher expressed in workers than soldiers using RNA-seq for heads of workers and soldiers of P. simplex and that EAG response to neocembrene is higher in workers than soldiers. I considered that this study will contribute to further understanding of the molecular and evolutionary mechanisms of chemoreception system in termites.

      Strength:

      The manuscript is well written. As far as I know, this study is the first study that identified a pheromone receptor in termites. The authors not only present a methodology for analyzing the function of termite pheromone receptors but also provide important insights in terms of the evolution of ligand selectivity of termite pheromone receptors.

      Weakness:

      This revised manuscript appears to me to have no major weaknesses.

    1. Reviewer #2 (Public review):

      Summary:

      Kirschner and colleagues test whether methamphetamine (MA) alters learning rate dynamics in a validated reversal learning task. They find evidence that MA can enhance performance for low-performers, and that the enhancement reflects a reduction in the degree to which these low-performers dynamically up-regulate their learning rates when they encounter unexpected outcomes. The net effect is that poor performers show more volatile learning rates (e.g. jumping up when they receive misleading feedback), when the environment is actually stable, undermining their performance over trials.

      Strengths:

      The study has multiple strengths, including a large sample size, placebo control, double-blind randomized design, and rigorous computational modeling of a validated task. Additionally, the analytic methods are rigorous and offer new types of analyses for people interested in exploring learning as a function of dynamically changing volatility.

      Weaknesses:

      The limitations, which are acknowledged, include that the drug they use, methamphetamine, can influence multiple neuromodulatory systems including catecholamines and acetylcholine, all of which have been implicated in learning rate dynamics. They also do not have any independent measures of any of these systems, so it is impossible to know which is having an effect.

      Another limitation which they should acknowledge is that the fact that participants were aware of having different experiences in the drug sessions means that their blinding was effectively single-blind (to the experimenters) and not double-blind. That said, the authors do provide some evidence that subjective effects of drugs (e.g. arousal, mood, etc.) did not drive differences in performance.

      Comments on revisions:

      The authors have done an outstanding job responding to, and allaying my prior concerns about their analyses.

    1. Reviewer #2 (Public review):

      Summary:

      Bonnifet et al. sought to characterize the expression pattern of L1 ORF1p expression across the entire mouse brain, in young and aged animals and to corroborate their characterization with Western blotting for L1 ORF1p and L1 RNA expression data from human samples. They also queried L1 ORF1p interacting partners in the mouse brain by IP-MS.

      Strengths:

      A major strength of the study is the use of two approaches: a deep-learning detection method to distinguish neuronal vs. non-neuronal cells and ORF1p+ cells vs. ORF1p- cells across large-scale images encompassing multiple brain regions mapped by comparison to the Allen Brain Atlas, and confocal imaging to give higher resolution on specific brain regions. These results are also corroborated by Western blotting on six mouse brain regions. Extension of their analysis to post-mortem human samples, to the extent possible, is another strength of the paper. The identification of novel ORF1p interactors in brain is also a strength in that it provides a novel dataset for future studies.

      Weaknesses:

      The main weakness of the study is that cell type specificity of ORF1p expression was not examined beyond neuron (NeuN+) vs non-neuron (NeuN-). Indeed, a recent study (Bodea et al. 2024, Nature Neuroscience) found that ORF1p expression is characteristic of parvalbumin-positive interneurons, and it would be very interesting to query whether other neuronal subtypes in different brain regions are distinguished by ORF1p expression. The data suggesting that ORF1p expression is increased in aged mouse brains is intriguing, although it seems to be based upon modestly (up to 27%, dependent on brain region) higher intensity of ORF1p staining rather than a higher proportion of ORF1+ neurons. Indeed, the proportion of NeuN+/Orf1p+ cells actually decreased in aged animals. It is difficult to interpret the significance and validity of the increase in intensity, as Hoechst staining of DNA, rather than immunostaining for a protein known to be stably expressed in young and aged neurons, was used as a control for staining intensity. The main weakness of the IP-MS portion of the study is that none of the interactors were individually validated or subjected to follow-up analyses. The list of interactors was compared to previously published datasets, but not to ORF1p interactors in any other mouse tissue.

      The authors achieved the goals of broadly characterizing ORF1p expression across different regions of the mouse brain, and identifying putative ORF1p interactors in the mouse brain. However, findings from both parts of the study are somewhat superficial in depth.

      This provides a useful dataset to the field, which likely will be used to justify and support numerous future studies into L1 activity in the aging mammalian brain and in neurodegenerative disease. Similarly, the list of ORF1p interacting proteins in the brain will likely be taken up and studied in greater depth.

      Comments on revisions:

      The co-staining of Orf1p with Parvalbumin (PV) presented in Supplemental Figure S5 is a welcome addition exploring the cell type-specificity of Orf1p staining, and broadly corroborates the work of Bodea et al. while revealing that Orf1p also is expressed in non-PV+ cells, consistent with L1 activity across a range of neuronal subtypes. The authors also have strengthened their findings regarding the increased intensity of ORF1p staining in aged compared to young animals, and the newly presented results are indeed more convincing. The prospect of increased neuronal L1 activity with age is exciting, and the results in this paper have provided the groundwork for ongoing discoveries in this area. While it is disappointing that no Orf1p interactors were followed up, this is understandable and the data are nonetheless valuable and will likely prove useful to future studies.

    1. Reviewer #2 (Public review):

      Summary:

      This paper by Howell and colleagues focuses on describing macro patterns of anatomical connections between cortical areas and the thalamus in the human brain. This research topic poses significant challenges due to the inability to apply the gold standard of mapping anatomical connections, viral tracing, to humans. Moreover, when applied to animal models, viral tracing often has limited scope and resolution. As a result, the field has thus far lacked a comprehensive and validated description of thalamocortical anatomical connectivity in humans.

      The paper focuses on an intriguing question: whether anatomical connections from the cortex to the thalamus exhibit a diffuse pattern, targeting multiple thalamic sub-regions, or a more focal pattern, selectively targeting specific thalamic subregions. This novel and significant question holds substantial implications for our understanding of thalamocortical information processing. The authors have developed a sophisticated and innovative quantitative metric to address this question. The study revealed two main patterns: a focal pattern originating from sensorimotor cortical regions to the posterior thalamus and a more diffuse pattern from associative cortical regions to the anterior-medial thalamus. These findings are then framed within the context of thalamocortical motifs involved in feedforward versus feedback processing.

      While this paper has several strengths, including its significance and methodological sophistication, its extension to non-human primates and other forms of data for testing hierarchy, there are important limitations. These limitations, discussed in more detail below, primarily concern tracking accuracy and the known limitations of using diffusion data to track thalamocortical connections in humans. These limitations may potentially introduce systematic biases into the results, weakening their support. Addressing these limitations through better validation is crucial, though some may remain unresolved due to the fundamental constraints of diffusion imaging.

      Strengths:

      This research holds significant basic, clinical, and translational importance as it contributes to our understanding of how thalamocortical anatomical connectivity is organized. Its relevance spans across cognitive, systems, and clinical neuroscientists in various subfields.

      The central question addressed in this study, concerning whether cortico-thalamic projections are focal or diffuse, is both novel and previously unexplored to the best of my knowledge. It offers valuable insights into the potential capabilities of the thalamocortical system in terms of parallel or integrative processing.

      The development of quantitative metrics to analyze anatomical connectivity is highly innovative and suitable for addressing the research questions at hand.

      The findings are not only interesting but also robust, aligning with data from other sources that suggest a hierarchical organization in the brain.

      Using PCA to integrate results across a range of thresholds is innovative.

      The study's sophisticated integration of a diverse range of data and methods provides strong, converging support for its main findings, enhancing the overall credibility of the research.

      Weaknesses:

      Structural thalamocortical connectivity was estimated from diffusion imaging data obtained from the HCP dataset. Consequently, the robustness and accuracy of the results depend on the suitability of this data for such a purpose. Conducting tractography on the cortical-thalamic system is recognized as a challenging endeavour for several reasons. First, diffusion directions lose their clearly defined principal orientations once they reach the deep thalamic nuclei, rendering the tracking of structures on the medial side, such as the medial dorsal (MD) and pulvinar nuclei difficult. Somewhat concerning is those are regions that authors found to show diffuse connectivity patterns. Second, the thalamic radiata diverges into several directions, and routes to the lateral surface often lack the clarity necessary for successful tracking. It is unclear if all cortical regions have similar levels of accuracy, and some of the lateral associative regions might have less accurate tracking, making them appear to be more diffuse, biasing the results.

      While the methodology employed by the authors appears to be state-of-the-art, there exists uncertainty regarding its appropriateness for validation, given the well-documented issues of false positives and false negatives in probabilistic diffusion tractography, as discussed by Thomas et al. 2014 PNAS. Although replicating the results in both humans and non-human primates strengthens the study, a more compelling validation approach would involve demonstrating the method's ability to accurately trace known tracts from established tracing studies or, even better, employing phantom track data. Many of the control analyses the authors presented, such as track density, do not speak to accuracy.

      Because the authors included data from all thresholds into, it seems likely that false positives tracks were included into the results. The methodology described seems to unavoidably include anatomically implausible pathways in the spatial extent analyses.

      If tracking the medial thalamus is indeed less accurate, characterized by higher false positives and false negatives, it could potentially lead to increased variability among individual subjects. In cases where results are averaged across subjects, as the authors have apparently done, this could inadvertently contribute to the emergence of the "diffuse" motif, as described in the context of the associative cortex. This presents a critical issue that requires a more thorough control analysis and validation process to ensure that the main results are not artifacts resulting from limitations in tractography.

      The thresholding approach taken in the manuscript was aimed to control for inter-areal differences in anatomical connection strength that could confound the ED estimates. Here I am not quite clear why inter-areal differences in anatomical connection strength have to be controlled. A global threshold applied on all thalamic voxels might kill some connections that are weak but do exist. Those weak pathways are less possible to survive at high thresholds. In the meantime, the mean ED is weighted, with more conservative thresholds having higher weights. That being said, isn't it possible that more robust pathways might contribute more to the mean ED than weaker pathways?

      Comments on revised version:

      I appreciate the additional supplementary figures and responses from the authors. I think this is an important study, and the review I wrote should provide important context for readers to digest their responses.

    1. Reviewer #3 (Public review):

      Summary:

      This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. The experimental design and results are robust, the manuscript's discussion has been satisfactorily updated. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.

      Strengths:

      The manuscript asks an important question in plant-microbe interaction studies with interesting findings.

      Overall, the experiments are detailed, thorough and very well-designed. The findings appear to be robust.

      The authors provide results that are not overinterpreted and are instead measured and logical.

      Weaknesses:

      No major weaknesses.

    1. Reviewer #2 (Public review):

      Murphy et al. expressed ChrimsonR and GCaMP6s in retinal ganglion cells of a living macaque. They recorded calcium responses and stimulated individual cells, optically. Neurons targeted for stimulation were activated strongly whereas neighboring neurons were not.

      The ability to record from neuronal populations while simultaneously stimulating a subset in controlled way is a high priority for systems neuroscience, and this has been particularly challenging in primates. This study marks an important milestone in the journey towards this goal.

    1. Reviewer #2 (Public Review):

      Raymond Laboy et.al explored how transcriptional Mondo/Max-like complex (MML-1/MXL-2) is regulated by glucose metabolic signals using germ-line removal longevity model. They believed that MML-1/MXL-2 integrated multiple longevity pathways through nutrient sensing and therefore screened the glucose metabolic enzymes that regulated MML-1 nuclear localization. Hexokinase 1 and 2 were identified as the most vigorous regulators, which function through mitochondrial beta-oxidation and the pentose phosphate pathway (PPP), respectively. MML-1 localized to mitochondria associated with lipid droplets (LD), and MML-1 nuclear localization was correlated with LD size and metabolism. Their findings are interesting and may help us to further explore the mechanisms in multiple longevity models. The data support their proposed working model.

      Comments on Revised Version (from the Reviewing Editor):

      The authors have addressed the remaining concerns from both reviewers, adding textual information for reviewer 1 and testing the roles of hxk-1 and lipid oxidation in regulating lipid droplets for reviewer 2. Specifically, they find that knockdown of acs-2 and hxk-1 acs-2 double knockdown each result in a mild but significant increase in LD size. This result supports that the two hexokinases regulate MML-1 via distinct mechanisms, and is reflected in the updated model.

    1. Reviewer #2 (Public review):

      Oracová et al. present data supporting a role for SIMC1/SLF2 in silencing plasmid DNA via the SMC5/6 complex. Their findings are of interest, and they provide further mechanistic detail of how the SMC5/6 complex is recruited to disparate DNA elements. In essence, the present report builds on the author's previous paper in eLife in 2022 (PMID: 36373674, "The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers") by showing the role of SIMC1/SLF2 in localisation of the SMC5/6 complex to plasmid DNA, and the distinct requirements as compared to recruitment to DNA damage foci. Although the findings of the manuscript are of interest, we are not yet convinced that the new data presented here represents a compelling new body of work and would better fit the format of a "research advance" article. In their previous paper, Oracová et al. show that the recruitment of SMC5/6 to SV40 replication centres is dependent on SIMC1, and specifically, that it is dependent on SIMC1 residues adjacent to neighbouring SLF2.

      Other comments

      (1) The mutations chosen in Figure 1 are quite extensive - 5 amino acids per mutant. In addition, they are in many cases 'opposite' changes, e.g., positive charge to negative charge. Is the effect lost if single mutations to an alanine are made?

      (2) In Figure 2c, it isn't clear from the data shown that the 'SLF2-only' mutations in SMC6 result in a substantial reduction in SIMC1/SLF2 binding.

      (3) In the GFP reporter assays (e.g. Figure 3), median fluorescence is reported - was there any observed difference in the percentage of cells that are GFP positive?

      (4) The potential role of the large T antigen as an SMC5/6 evasion factor is intriguing. However, given the role of the large T antigen as a transcriptional activator, caution is required when interpreting enhanced GFP fluorescence. Antagonism of the SMC5/6 complex in this context might be further supported by ChIP experiments in the presence or absence of large T. Can large T functionally substitute for HBx or HIV-Vpr?

      (5) In Figure 5c, the apparent molecular weight of large T and SMC6 appears to change following transfection of GFP-SMC5 - is there a reason for this?

    1. Reviewer #2 (Public review):

      The study by Setogawa et al. aims to understand the role that different striatal subregions belonging to parallel brain circuits have in associative learning and discrimination learning (S-O-R and S-R tasks). Strengths of the study are the use of multiple methodologies to measure and manipulate brain activity in rats, from microPET imaging to excitotoxic lesions and multielectrode recordings across anterior dorsolateral (aDLS), posterior ventral lateral (pVLS)and dorsomedial (DMS) striatum.

    1. Reviewer #2 (Public review):

      Summary:

      This study investigated the impact of early HIV specific CD8 T cell responses on the viral reservoir size after 24 weeks and 3 years of follow-up in individuals who started ART during acute infection. Viral reservoir quantification showed that total and defective HIV DNA, but not intact, declined significantly between 24 weeks and 3 years post-ART. The authors also showed that functional HIV-specific CD8⁺ T-cell responses persisted over three years and that early CD8⁺ T-cell proliferative capacity was linked to reservoir decline, supporting early immune intervention in the design of curative strategies.

      Strengths:

      The paper is well written, easy to read, and the findings are clearly presented. The study is novel as it demonstrates the effect of HIV specific CD8 T cell responses on different states of the HIV reservoir, that is HIV-DNA (intact and defective), the transcriptionally active and inducible reservoir. Although small, the study cohort was relevant and well-characterized as it included individuals who initiated ART during acute infection, 12 of whom were followed longitudinally for 3 years, providing unique insights into the beneficial effects of early treatment on both immune responses and the viral reservoir. The study uses advanced methodology. I enjoyed reading the paper.

      Weaknesses:

      All participants were male (acknowledged by the authors), potentially reducing the generalizability of the findings to broader populations. A control group receiving ART during chronic infection would have been an interesting comparison.

    1. Reviewer #2 (Public review):

      In this paper, the authors examine the extent to which epigenetic variation acquired during a selection treatment (as opposed to standing epigenetic variation) can contribute to adaptation in Arabidopsis. They find weak evidence for such adaptation and few differences in DNA methylation between experimental groups, which contrasts with another recent study (reference 26) that reported extensive heritable variation in response to the environment. The authors convincingly demonstrate that the conclusions of the previous study were caused by experimental error, so that standing genetic variation was mistaken for acquired (epigenetic) variation. Given the controversy surrounding the possible role of epigenetic variation in mediating phenotypic variation and adaptation, this is an important, clarifying contribution.

      I have a few specific comments about the analysis of DNA methylation:

      (1) The authors group their methylation analysis by sequence context (CG, CHG, CHH). I feel this is insufficient, because CG methylation can appear in two distinct forms: gene body methylation (gbM), which is CG-only methylation within genes, and transposable element (TE) and TE-like methylation (teM), which typically involves all sequence contexts and generally affects TEs, but can also be found within genes. GbM and teM have distinct epigenetic dynamics, and it is hard to know how methylation patterns are changing during the experiment if gbM and teM are mixed. This can also have downstream consequences (see point below).

      (2) For GO analysis, the authors use all annotated genes as a control. However, most of the methylation differences they observe are likely gbM, and gbM genes are not representative of all genes. The authors' results might therefore be explained purely as a consequence of analyzing gbM genes, and not an enrichment of methylation changes in any particular GO group.

    1. Reviewer #2 (Public review):

      Summary:

      This manuscript by Syed et al. presents a detailed investigation of inhibitory interneurons, specifically from the 13A and 13B hemilineages, which contribute to the generation of rhythmic leg movements underlying grooming behavior in Drosophila. After performing a detailed connectomic analysis, which offers novel insights into the organization of premotor inhibitory circuits, the authors build on this anatomical framework by performing optogenetic perturbation experiments to functionally test predictions derived from the connectome. Finally, they integrate these findings into a computational model that links anatomical connectivity with behavior, offering a systems-level view of how inhibitory circuits may contribute to grooming pattern generation.

      Strengths:

      (1) Performing an extensive and detailed connectomic analysis, which offers novel insights into the organization of premotor inhibitory circuits.

      (2) Making sense of the largely uncharacterized 13A/13B nerve cord circuitry by combining connectomics and optogenetics is very impressive and will lay the foundation for future experiments in this field.

      (3) Testing the predictions from experiments using a simplified and elegant model.

      Weaknesses:

      (1) In Figure 4, while the authors report statistically significant shifts in both proximal inter-leg distance and movement frequency across conditions, the distributions largely overlap, and only in Panel K (13B silencing) is there a noticeable deviation from the expected 7-8 Hz grooming frequency. Could the authors clarify whether these changes truly reflect disruption of the grooming rhythm? More importantly, all this data would make the most sense if it were performed in undusted flies (with controls) as is done in the next figure.

      (2) In Figure 4-Figure Supplement 1, the inclusion of walking assays in dusted flies is problematic, as these flies are already strongly biased toward grooming behavior and rarely walk. To assess how 13A neuron activation influences walking, such experiments should be conducted in undusted flies under baseline locomotor conditions.

      (3) For broader lines targeting six or more 13A neurons, the authors provide specific predictions about expected behavioral effects-e.g., that activation should bias the limb toward flexion and silencing should bias toward extension based on connectivity to motor neurons. Yet, when using the more restricted line labeling only two 13A neurons (Figure 4 - Figure Supplement 2), no such prediction is made. The authors report disrupted grooming but do not specify whether the disruption is expected to bias the movement toward flexion or extension, nor do they discuss the muscle target. This is a missed opportunity to apply the same level of mechanistic reasoning that was used for broader manipulations.

      (4) Regarding Figure 5: The 70ms on/off stimulation with a slow opsin seems problematic. CsChrimson off kinetics are slow and unlikely to cause actual activity changes in the desired neurons with the temporal precision the authors are suggesting they get. Regardless, it is amazing that the authors get the behavior! It would still be important for the authors to mention the optogenetics caveat, and potentially supplement the data with stimulation at different frequencies, or using faster opsins like ChrimsonR.

      Overall, I think the strengths outweigh the weaknesses, and I consider this a timely and comprehensive addition to the field.

    1. Reviewer #2 (Public review):

      Summary:

      Ray and coworkers introduce a discrete model of cellular layers aimed at investigating the role of inter-cellular adhesion in collective cell migration. The model combines aspects of particle-based models, in which cells are treated as simple point-particles with pair-interactions, and "morphological models", where interactions primarily depend on the cellular shape. In this case, cells are modeled as rings of beads connected by springs, thus allowing for exploration of the role of cell morphology while treating intercellular interactions as particle-like. Upon exploring the parameter space of this model, the authors recover physical behaviors reminiscent of reconstituted cell layers, including the onset of collective cell migration, when the forces leading to cell propulsion overweight inter-cellular adhesion, and various signatures of glassy dynamics.

      Strengths:

      The model presented in the article is simple, easy to implement, and scalable. The analysis appears solid and delivers a number of clear physical properties that could be tested in more depth in experiments and future numerical studies (e.g., distribution of displacements, etc.). The authors make an appreciable effort to make contact with other models and share their ideas for further investigations.

      Weaknesses:

      I found two main weaknesses in the original version of this manuscript, which I strongly encourage the authors to address.

      (1) The manuscript explicitly aims at resolving an apparent contradiction of tessellation-based models, such as the Vertex and the Voronoi model. Both models used the so-called shape index p0 - i.e. the ratio between the preferential perimeter and the preferential area of the cells - to drive a solid/liquid phase transition in the presence of Brownian and/or rotational noise. Specifically, for sufficiently large p0 values, these in silico cell layers undergo a transition to a state of collective migration, where a rigid junction network becomes unstable to T1 events. Because p0 is often interpreted as "adhesion strength", this leads to the paradoxical conclusion that cell intercalation is favored by intercellular adhesion. The paradox, however, only lies in this interpretation, which assigns to the shape index p0 a biophysical role that is too specific. To illustrate this concept, let us consider the energy of an individual cell of area A and perimeter P: i.e. e = (a-1)^2+c*(p-p0)^2, where a=A/A_0, with A_0 the preferred area, p=P/sqrt(A_0) and p_0 = P_0/sqrt(A_0), with P_0 the preferred perimeter. Expanding the square in the second term gives e ~ p^2 - 2p_0 p. Thus, increasing p_0, favors longer cell junctions, from which it appears reasonable to interpret p0 as a dimensionless measure of intercellular adhesion. Such an increase in the length of the junctions is, however, only a byproduct of the effect of p0 on the overall shape of the cell, which becomes progressively less rounded as p0 is increased (e.g., for a circle, p0≈3.55, for an equilateral triangle, p0≈4.56). The roundness of an individual cell, on the other hand, cannot single-handedly be ascribed to intercellular adhesion, despite intercellular adhesion being undoubtedly one of the biophysical properties affecting this geometrical feature. Moreover, the shape index p0 ​enters the energy functional at the single-cell level, implying that even in isolation, without intercellular adhesion, an increase in p0 leads to a less rounded cell morphology. These peculiarities of the Vertex/Voronoi model do raise questions about its accuracy and validity, thus justify seeking for alternative cell-resolved models such as that introduced here by Ray et al., but, on the other hand, make the interpretation of p0 as an exclusive measure of adhesion evidently dubious.

      (2) The spring-bead model by Ray and coworkers has at least two predecessors in the recent literature, none of which have been cited in the present manuscript. These are Boromand et al., Phys. Rev. Lett. 121, 248003 (2018) and Pasupalak et al. arXiv:2409.16128 (2024). The former paper investigates the packing of flexible polygons and is not specific to epithelial layers, while the latter is specifically designed to address various outstanding problems in tissue mechanics, including collective migration and wound healing. While none of these models is identical to that by Ray et al., it would be fair to present the latter as a member of the family rather than the first one of its kind and possibly comment about the differences and similarities with these previous models.

    1. Reviewer #2 (Public review):

      Summary:

      This study examines whether the localization of endocytic proteins to presynaptic periactive zones depends on synaptic activity or active zone scaffolds. Using a combination of genetic and pharmacological perturbations in Drosophila and mouse neurons, the authors show that proteins such as Dynamin, Amphiphysin, AP-180, and others are still recruited to periactive zones even when evoked release or active zone architecture is disrupted. While the results are mostly negative, the study is methodologically solid and contributes to a more nuanced understanding of synaptic vesicle recycling machinery.

      Strengths:

      (1) The experimental design is careful and systematic, covering both fly and mammalian systems.

      (2) The use of advanced genetic models (e.g., Liprin-α quadruple knockout mice) is a notable strength.

      (3) High-resolution imaging (STED, Airyscan) is well used to assess spatial localization.

      (4) The findings clarify that certain core assumptions - such as strict activity dependence of endocytic recruitment - may not hold universally.

      Weaknesses:

      (1) The study would benefit from a clearer positive control to demonstrate activity-dependent recruitment (e.g., Endophilin).

      (2) The reliance on Tetanus toxin in the Drosophila NMJ experiments in my eyes is a limitation, as it does not block all presynaptic fusion events; this should be discussed more directly.

      (3) The potential role of Dynamin in organizing other periactive zone proteins is not addressed and could be an important next step.

      (4) Some small changes in protein levels upon silencing are reported; their biological meaning (e.g., compensation vs. variability) is not fully clarified.

      (5) While alternative organizing mechanisms (actin, lipids, adhesion molecules) are mentioned, a more forward-looking discussion of how to test these models would be helpful.

      (6) The authors should consider including, or at least discussing, a well-established activity-dependent endocytic protein (e.g., Endophilin) as a positive control to help contextualize the negative findings.

    1. Reviewer #2 (Public review):

      Summary:

      The overall goal of this study by Smith et al. was to understand the mechanisms through which groups of cells form specific nuclei during development. These cell groupings may have importance for the development of nervous system connections. Smith et al. have taken advantage of the ordered structure of the nucleus laminaris of the chick, which plays an important role in sound source localization. They used a candidate gene approach to both mark cells in nL and to test for signaling pathways that regulate nucleogenesis. They found that MafB, FGF8, and cadherins were expressed in the auditory hindbrain at the critical ages. They used in ovo electroporation to test gene function effects on nL lamina formation. They found that both increasing and decreasing FGF signaling (through introduction of mouse FGF8 and expression of a dominant negative FGF receptor, respectively) reduced lamina formation in the nL. An optimal concentration of FGF needed for this process was obtained using cultured hindbrain slices. Misexpression of cadherins also perturbed the normal lamina formation. The authors showed that FGF regulates MafB expression, which in turn regulates cadherin expression, suggesting a pathway that shapes lamina development. They constructed computational models of adhesion on the development of nL cells and found that laminar formation is favored by nL cells modeled as bipolar adhesive units. Overall, the study has demonstrated the importance of these adhesion pathways for the formation of the nucleus laminaris, and the findings likely have significance for the development of other nuclei as well.

      Strengths:

      The experiments have used in situ hybridization, immunofluorescence, electroporation, and brainstem slice cultures to test their hypotheses, which were based on well-selected candidate molecules. The modeling adds to the rigor of the studies, particularly in light of the observation that cadherin expression is localized to nL dendrites.

      Weaknesses:

      (1) Some references should be considered more carefully for accuracy, and additional references may be needed (introduction and results).

      (2) Information on animal numbers and statistical tests should be added.

    1. Reviewer #2 (Public review):

      Summary:

      This manuscript examined the neural correlates of the temporal-spatial dynamics of emotional processing while participants were watching short movie clips (each 12.5 s long) from the movie "Forrest Gump". Participants not only watched each film clip, but also gave emotional responses, followed by a brief resting period. Employing fMRI to track the BOLD responses during these stages of emotional processing, the authors found four large-scale brain networks (labeled as IC0,1,2,4) were differentially involved in emotional processing. Overall, this work provides valuable information on the neurodynamics of emotional processing.

      Strengths:

      This work employs a naturalistic movie watching paradigm to elicit emotional experiences. The authors used a slice-based fMRI method to examine the temporal dynamics of BOLD responses. Compared to previous emotional research that uses static images, this work provides some new data and insights into how the brain supports emotional processing from a temporal dynamics view.

      Weaknesses:

      Some major conclusions are unwarranted and do not have relevant evidence. For example, the authors seemed to interpret some neuroimaging results to be related to emotion regulation. However, there were no explicit instructions about emotional regulation, and there was no evidence suggesting participants regulated their emotions. How to best interpret the corresponding results thus requires caution.

      Relatedly, the authors argued that "In turn, our findings underscore the utility of examining temporal metrics to capture subtle nuances of emotional processing that may remain undetectable using standard static analyses." While this sentence makes sense and is reasonable, it remains unclear how the results here support this argument. In particular, there were only three emotional categories: sad, happy, and fear. These three emotional categories are highly different from each other. Thus, how exactly the temporal metrics captured the "subtle nuances of emotional processing" shall be further elaborated.

      The writing also contained many claims about the study's clinical utility. However, the authors did not develop their reasoning nor elaborate on the clinical relevance. While examining emotional processing certainly could have clinical relevance, please unpack the argument and provide more information on how the results obtained here can be used in clinical settings.

      Importantly, how are the temporal dynamics of BOLD responses and subjective feelings related? The authors showed that "the time-to-peak differences in IC2 ("response") align closely with response latency results, with sad trials showing faster response latencies and earlier peak times". Does this mean that people typically experience sad feelings faster than happy or fear? Yet this is inconsistent with ideas such that fear detection is often rapid, while sadness can be more sustained. Understandably, the study uses movie clips, which can be very different from previous work, mostly using static images (e.g., a fearful or a sad face). But the authors shall explicitly discuss what these temporal dynamics mean for subjective feelings.

    1. Reviewer #2 (Public Review):

      Here I submit my previous review and a great deal of additional information following on from the initial review and the response by the authors.

      * Initial Review *

      Assessment:

      This manuscript is based upon the unprecedented identification of an apparently highly unusual trigeminal nuclear organization within the elephant brainstem, related to a large trigeminal nerve in these animals. The apparently highly specialized elephant trigeminal nuclear complex identified in the current study has been classified as the inferior olivary nuclear complex in four previous studies of the elephant brainstem. The entire study is predicated upon the correct identification of the trigeminal sensory nuclear complex and the inferior olivary nuclear complex in the elephant, and if this is incorrect, then the remainder of the manuscript is merely unsupported speculation. There are many reasons indicating that the trigeminal nuclear complex is misidentified in the current study, rendering the entire study, and associated speculation, inadequate at best, and damaging in terms of understanding elephant brains and behaviour at worst.

      Original Public Review:

      The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.<br /> The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

      The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, but the current study differs.

      Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others appears to be just "lateral nuclei" with no suggestion of what might be there instead.

      Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occur. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

      The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400,000 and 500,000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

      But let's look more specifically at the justification outlined in the current study to support their identification of the unusually located trigeminal sensory nuclei of the brainstem.

      (1) Intense cytochrome oxidase reactivity<br /> (2) Large size of the putative trunk module<br /> (3) Elongation of the putative trunk module<br /> (4) Arrangement of these putative modules correspond to elephant head anatomy<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds<br /> (6) Location apparently matches other mammals<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals

      Let's examine these justifications more closely.

      (1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. In order to obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. It is likely that the histochemical staining observed is background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.<br /> Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

      (4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

      (6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species, the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

      (7) The dual to quadruple repetition of rostro-caudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. But, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

      (8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

      Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

      What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to detail to support the major changes indicated by the authors.

      So what are these "bumps" in the elephant brainstem?

      Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

      The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

      Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

      What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship to the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

      What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

      * Review of Revised Manuscript *

      Assessment:

      There is a clear dichotomy between the authors and this reviewer regarding the identification of specific structures, namely the inferior olivary nuclear complex and the trigeminal nuclear complex, in the brainstem of the elephant. The authors maintain the position that in the elephant alone, irrespective of all the published data on other mammals and previously published data on the elephant brainstem, these two nuclear complexes are switched in location. The authors maintain that their interpretation is correct, but this reviewer maintains that this interpretation is erroneous. The authors expressed concern that the remainder of the paper was not addressed by the reviewer, but the reviewer maintains that these sequelae to the misidentification of nuclear complexes in the elephant brainstem render any of these speculations irrelevant as the critical structures are incorrectly identified. It is this reviewer's opinion that this paper is incorrect. I provide a lot of detail below in order to provide support to the opinion I express.

      Public Review of Current Submission:

      As indicated in my previous review of this manuscript (see above), it is my opinion that the authors have misidentified, and indeed switched, the inferior olivary nuclear complex (IO) and the trigeminal nuclear complex (Vsens). It is this specific point only that I will address in this second review, as this is the crucial aspect of this paper - if the identification of these nuclear complexes in the elephant brainstem by the authors is incorrect, the remainder of the paper does not have any scientific validity.

      The authors, in their response to my initial review, claim that I "bend" the comparative evidence against them. They further claim that as all other mammalian species exhibit a "serrated" appearance of the inferior olive, and as the elephant does not exhibit this appearance, what was previously identified as the inferior olive is actually the trigeminal nucleus and vice versa.

      For convenience, I will refer to IOM and VsensM as the identification of these structures according to Maseko et al (2013) and other authors and will use IOR and VsensR to refer to the identification forwarded in the study under review.<br /> The IOM/VsensR certainly does not have a serrated appearance in elephants. Indeed, from the plates supplied by the authors in response (Referee Fig. 2), the cytochrome oxidase image supplied and the image from Maseko et al (2013) shows a very similar appearance. There is no doubt that the authors are identifying structures that closely correspond to those provided by Maseko et al (2013). It is solely a contrast in what these nuclear complexes are called and the functional sequelae of the identification of these complexes (are they related to the trunk sensation or movement controlled by the cerebellum?) that is under debate.

      Elephants are part of the Afrotheria, thus the most relevant comparative data to resolve this issue will be the identification of these nuclei in other Afrotherian species. Below I provide images of these nuclear complexes, labelled in the standard nomenclature, across several Afrotherian species.

      (A) Lesser hedgehog tenrec (Echinops telfairi)

      Tenrecs brains are the most intensively studied of the Afrotherian brains, these extensive neuroanatomical studies were undertaken primarily by Heinz Künzle. Below I append images (coronal sections stained with cresol violet) of the IO and Vsens (labelled in the standard mammalian manner) in the lesser hedgehog tenrec. It should be clear that the inferior olive is located in the ventral midline of the rostral medulla oblongata (just like the rat) and that this nucleus is not distinctly serrated. The Vsens is located in the lateral aspect of the medulla skirted laterally by the spinal trigeminal tract (Sp5). These images and the labels indicating structures correlate precisely with that provided by Künzle (1997, 10.1016/S0168- 0102(97)00034-5), see his Figure 1K,L. Thus, in the first case of a related species, there is no serrated appearance of the inferior olive, the location of the inferior olive is confirmed through connectivity with the superior colliculus (a standard connection in mammals) by Künzle (1997), and the location of Vsens is what is considered to be typical for mammals. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

      Review image 1.

      (B) Giant otter shrew (Potomogale velox)

      The otter shrews are close relatives of the Tenrecs. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see hints of the serration of the IO as defined by the authors, but we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

      Review image 2.

      (C) Four-toed sengi (Petrodromus tetradactylus)

      The sengis are close relatives of the Tenrecs and otter shrews, these three groups being part of the Afroinsectiphilia, a distinct branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see vague hints of the serration of the IO (as defined by the authors), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

      Review image 3.

      (D) Rock hyrax (Procavia capensis)

      The hyraxes, along with the sirens and elephants form the Paenungulata branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per the standard mammalian anatomy. Here we see hints of the serration of the IO (as defined by the authors), but we also see evidence of a more "bulbous" appearance of subnuclei of the IO (particularly the principal nucleus), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

      Review image 4.

      (E) West Indian manatee (Trichechus manatus)

      The sirens are the closest extant relatives of the elephants in the Afrotheria. Below I append images of cresyl violet (top) and myelin (bottom) stained coronal sections (taken from the University of Wisconsin-Madison Brain Collection, https://brainmuseum.org, and while quite low in magnification they do reveal the structures under debate) through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see the serration of the IO (as defined by the authors). Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

      Review image 5.

      These comparisons and the structural identification, with which the authors agree as they only distinguish the elephants from the other Afrotheria, demonstrate that the appearance of the IO can be quite variable across mammalian species, including those with a close phylogenetic affinity to the elephants. Not all mammal species possess a "serrated" appearance of the IO. Thus, it is more than just theoretically possible that the IO of the elephant appears as described prior to this study.

      So what about elephants? Below I append a series of images from coronal sections through the African elephant brainstem stained for Nissl, myelin, and immunostained for calretinin. These sections are labelled according to standard mammalian nomenclature. In these complete sections of the elephant brainstem, we do not see a serrated appearance of the IOM (as described previously and in the current study by the authors). Rather the principal nucleus of the IOM appears to be bulbous in nature. In the current study, no image of myelin staining in the IOM/VsensR is provided by the authors. However, in the images I provide, we do see the reported myelin stripes in all stains - agreement between the authors and reviewer on this point. The higher magnification image to the bottom left of the plate shows one of the IOM/VsensR myelin stripes immunostained for calretinin, and within the myelin stripes axons immunopositive for calretinin are seen (labelled with an arrow). The climbing fibres of the elephant cerebellar cortex are similarly calretinin immunopositive (10.1159/000345565). In contrast, although not shown at high magnification, the fibres forming the Sp5 in the elephant (in the Maseko description, unnamed in the description of the authors) show no immunoreactivity to calretinin.

      Review image 6.

      Peripherin Immunostaining

      In their revised manuscript the authors present immunostaining of peripherin in the elephant brainstem. This is an important addition (although it does replace the only staining of myelin provided by the authors which is unusual as the word myelin is in the title of the paper) as peripherin is known to specifically label peripheral nerves. In addition, as pointed out by the authors, peripherin also immunostains climbing fibres (Errante et al., 1998). The understanding of this staining is important in determining the identification of the IO and Vsens in the elephant, although it is not ideal for this task as there is some ambiguity. Errante and colleagues (1998; Fig. 1) show that climbing fibres are peripherin-immunopositive in the rat. But what the authors do not evaluate is the extensive peripherin staining in the rat Sp5 in the same paper (Errante et al, 1998, Fig. 2). The image provided by the authors of their peripherin immunostaining (their new Figure 2) shows what I would call the Sp5 of the elephant to be strongly peripherin immunoreactive, just like the rat shown in Errant et al (1998), and moreover in the precise position of the rat Sp5! This makes sense as this is where the axons subserving the "extraordinary" tactile sensitivity of the elephant trunk would be found (in the standard model of mammalian brainstem anatomy). Interestingly, the peripherin immunostaining in the elephant is clearly lamellated...this coincides precisely with the description of the trigeminal sensory nuclei in the elephant by Maskeo et al (2013) as pointed out by the authors in their rebuttal. Errante et al (1998) also point out peripherin immunostaining in the inferior olive, but according to the authors this is only "weakly present" in the elephant IOM/VsensR. This latter point is crucial. Surely if the elephant has an extraordinary sensory innervation from the trunk, with 400,000 axons entering the brain, the VsensR/IOM should be highly peripherin-immunopositive, including the myelinated axon bundles?! In this sense, the authors argue against their own interpretation - either the elephant trunk is not a highly sensitive tactile organ, or the VsensR is not the trigeminal nuclei it is supposed to be.

      Summary:

      (1) Comparative data of species closely related to elephants (Afrotherians) demonstrates that not all mammals exhibit the "serrated" appearance of the principal nucleus of the inferior olive.

      (2) The location of the IO and Vsens as reported in the current study (IOR and VsensR) would require a significant, and unprecedented, rearrangement of the brainstem in the elephants independently. I argue that the underlying molecular and genetic changes required to achieve this would be so extreme that it would lead to lethal phenotypes. Arguing that the "switcheroo" of the IO and Vsens does occur in the elephant (and no other mammals) and thus doesn't lead to lethal phenotypes is a circular argument that cannot be substantiated.

      (3) Myelin stripes in the subnuclei of the inferior olivary nuclear complex are seen across all related mammals as shown above. Thus, the observation made in the elephant by the authors in what they call the VsensR, is similar to that seen in the IO of related mammals, especially when the IO takes on a more bulbous appearance. These myelin stripes are the origin of the olivocerebellar pathway and are indeed calretinin immunopositive in the elephant as I show.

      (4) What the authors see aligns perfectly with what has been described previously, the only difference being the names that nuclear complexes are being called. But identifying these nuclei is important, as any functional sequelae, as extensively discussed by the authors, is entirely dependent upon accurately identifying these nuclei.

      (4) The peripherin immunostaining scores an own goal - if peripherin is marking peripheral nerves (as the authors and I believe it is), then why is the VsensR/IOM only "weakly positive" for this stain? This either means that the "extraordinary" tactile sensitivity of the elephant trunk is non-existent, or that the authors have misinterpreted this staining. That there is extensive staining in the fibre pathway dorsal and lateral to the IOR (which I call the spinal trigeminal tract), supports the idea that the authors have misinterpreted their peripherin immunostaining.

      (5) Evolutionary expediency. The authors argue that what they report is an expedient way in which to modify the organisation of the brainstem in the elephant to accommodate the "extraordinary" tactile sensitivity. I disagree. As pointed out in my first review, the elephant cerebellum is very large and comprised of huge numbers of morphologically complex neurons. The inferior olivary nuclei in all mammals studied in detail to date, give rise to the climbing fibres that terminate on the Purkinje cells of the cerebellar cortex. It is more parsimonious to argue that, in alignment with the expansion of the elephant cerebellum (for motor control of the trunk), the inferior olivary nuclei (specifically the principal nucleus) have had additional neurons added to accommodate this cerebellar expansion. Such an addition of neurons to the principal nucleus of the inferior olive could readily lead to the loss of the serrated appearance of the principal nucleus of the inferior olive and would require far less modifications in the developmental genetic program that forms these nuclei. This type of quantitative change appears to be the primary way in which structures are altered in the mammalian brainstem.

    1. Reviewer #2 (Public review):

      In this manuscript, Timblin et al provide a model where exogenous CoA is taken up by macrophages and utilized to support transcriptional events associated with activation. They provide a series of important findings, and for the most part, the data are clear and convincing. However, additional clarity on a few points would be helpful.

      First of all, the contention that endogenous TLR ligands from the bone marrow cultures are driving the tonic signaling that makes exogenous CoA beneficial in unstimulated cells seems counter to the well-described anergic state of myeloid cells derived from TLR-null mice. This reviewer's understanding was that myeloid cells in MyD88 nulls or similar are developmentally anergic due to the lack of TLR stimulation in vivo. The data here (Figure 5G, etc) show these cells have much lower TLR responses, but the authors attribute it to loss of response to endogenous ligands during the cultures rather than in vivo. Testing some of the phenotypes ex vivo, etc, might make this argument more compelling and rule out that this is an effect in vivo.

      Second, the data suggesting that CoA enhances anti-microbial activity via itaconate production needs additional context and/or clarification. Interactions between itaconate and CoA have been demonstrated. Itaconate exposure can deplete the CoA pool as it is converted into Itaconyl-CoA. The Irg-/- cells should not have reduced CoA due to the lack of the need to activate itaconate for metabolism. Has this been addressed by the authors? I believe that low levels of itaconate production have been shown in "resting" bone marrow cultures. The data show a full log of more bugs in the macs that lack Irg, confirming that endogenous itaconate is at work. In addition, itaconate, which is made very quickly and is likely there in considerable amounts in 4 hrs, is known to affect transcription via action on TET2. Perhaps this explains some of the connections to CoA?

      Lastly, the idea that Acetyl-CoA phenocopies CoA suggests that CoA is the effector is interesting but could be supported more. Did the authors do the "unlabeling" experiment with Acetyl-CoA to confirm that it is readily converted to the CoA pool?

      Do the ACLY inhibitors have the expected effects on the ChIP seq data?

    1. Reviewer #2 (Public review):

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

      Strengths:

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

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

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

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

      Weaknesses:

      (1) The highly controlled stimuli are far removed from natural scenes, raising questions about generalisability. But, of course, control (almost) excludes ecological validity. The study does not address the challenges of natural vision or leverage the rich statistical structure afforded by natural scenes.

      (2) The experimental design appears primarily confirmatory rather than attempting to challenge the TWCO framework or test boundary conditions where it might fail.

      (3) Alternative explanations for the observed behavioral effects are not thoroughly explored. While the model provides a good fit to the data, this does not conclusively prove that gamma synchrony is the actual mechanism underlying the observed effects.

      (4) Direct neurophysiological evidence linking the observed behavioral effects to gamma synchrony in humans is absent, creating a gap between the model and the neural mechanism.

      Achievement of Aims and Support for Conclusions:

      The authors largely achieved their primary aim of demonstrating that human figure-ground perception follows patterns predicted by TWCO principles. Their psychophysical results reveal a behavioral "Arnold tongue" that matches the synchronization patterns predicted by their model, and their learning experiment shows that perceptual improvements correlate with predicted increases in synchrony.

      The evidence supports their conclusion that gamma synchrony could serve as a viable neural grouping mechanism for figure-ground segregation. However, the conclusion that "stimulus-dependence of gamma synchrony is adaptable to the statistics of visual experiences" is only partially supported, as the study uses highly controlled artificial stimuli rather than naturalistic visual statistics, or shows a sensitivity to the structure of experience.

      Likely Impact and Utility:

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

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

      The learning component of the study may have a particular impact, as it suggests a mechanism by which perceptual expertise develops through modified coupling between neural assemblies. This could influence thinking about perceptual learning more broadly, but also raises questions about the underlying mechanism that the paper does not address.

      Additional Context:

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

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

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

    1. Reviewer #2 (Public review):

      This work introduces a Vermouth library framework to enhance software development within the Martini community. Specifically, it presents a Vermouth-powered program, Martinize2, for generating coarse-grained structures and topologies from atomistic structures. In addition to introducing the Vermouth library and the Martinize2 program, this paper illustrates how Martinize2 identifies atoms, maps them to the Martini model, generates topology files, and identifies protonation states or post-translational modifications. Compared with the prior version, the authors provide a new figure to show that Martinize2 can be applied to various molecules, such as proteins, cofactors, and lipids. To demonstrate the general application, Martinize2 was used for converting 73% of 87,084 protein structures from the template library, with failed cases primarily blamed on missing coordinates.

      I appreciate the changes that the authors made to clarify the novelty. I have no doubt this paper will receive attention and citations.

    1. The strikes were the first operational use of the GBU-57, Mr. Hegseth said. It is a 30,000-pound guided bomb that contains the explosive power of approximately 5,500 pounds of TNT and is designed to attack deeply buried targets. It can be carried only by B-2 stealth bombers.
    2. Whiteman Air Force Base in Missouri, home to the B-2 stealth bombers
    1. Reviewer #2 (Public review):

      Summary:

      This manuscript explores the role of the C-terminal tail of EmrE in controlling uncoupled proton flux. Leakage occurs in the wild-type transporter under certain conditions but is amplified in the C-terminal truncation mutant D107. The authors use an impressive combination of growth assays, transport assays, NMR on WT and mutants with and without key substrates, classical MD, and reactive MD to address this problem. Overall, I think that the claims are well supported by the data, but I am most concerned about the reproducibility of the MD data, initial structures used for simulations, and the stochasticity of the water wire formation. These can all be addressed in a revision with more simulations as I point out below. I want to point out that the discussion was very nicely written, and I enjoyed reading the summary of the data and the connection to other studies very much.

      Strengths:

      The Henzler-Wildman lab is at the forefront of using quantitative experiments to probe the peculiarities in transporter biophysics, and the MD work from the Voth lab complements the experiments quite well. The sheer number of different types of experimental and computational approaches performed here is impressive.

      Weaknesses:

      The primary weaknesses are related to the reproducibility of the MD results with regard to the formation of water wires in the WT and truncation mutant. This could be resolved with simulations starting from structures built using very different loops and C-terminal tails.

      The water wire gates identified in the MD should be tested experimentally with site-directed mutagenesis to determine if those residues do impact leak.

      Comments on revisions:

      Having reviewed the latest version of the manuscript, I continue to believe that this is a solid paper with important results. I find the new data regarding the computational pKa estimate of E14 compelling.

    1. Reviewer #2 (Public review):

      Summary:

      The authors use a genetic screen in C. elegans to investigate the physiological roles of polyunsaturated fatty acids (PUFAs). They screen for mutations that rescue fat-2 mutants, which have strong reductions in PUFAs. As a result, either mutations in fat-2 itself or mutations in genes involved in the HIF-1 pathway were found to rescue fat-2 mutants. Mutants in the HIF-1 pathway rescue fat-2 mutants by boosting their catalytic activity (via upregulated Fe2+). Thus, the authors show that in the context of fat-2 mutation, the sole genetic means to rescue PUFA insufficiency is to restore PUFA levels.

      Strengths:

      As C. elegans can produce PUFAs de novo as essential lipids, the genetic model is well-suited to study the fundamental roles of PUFAs. The genetic screen finds mutations in convergent pathways, suggesting that it has reached near-saturation. The authors extensively validate the results of the screening and provide sufficient mechanistic insights to show how PUFA levels are restored in HIF-1 pathway mutants. As many of the mutations found to rescue fat-2 mutants are of gain-of-function, it is unlikely that similar discoveries could have been made with other approaches like genome-wide CRISPR screenings, making the current study distinctive. Consequently, the study provides important messages. First, it shows that PUFAs are essential for life. The inability to genetically rescue PUFA deficiency, except for mutations that restore PUFA levels, suggests that they have pleiotropic essential functions. In addition, the results suggest that the most essential functions of PUFAs are not in fluidity regulation, which is consistent with recent reviews proposing that the importance of unsaturation goes beyond fluidity (doi: 10.1016/j.tibs.2023.08.004 and doi: 10.1101/cshperspect.a041409). Thus, the study provides fundamental insights about how membrane lipid composition can be linked to biological functions.

      Weaknesses:

      The authors put in a lot of effort to answer the questions that arose through peer review, and now all the claims seem to be supported by experimental data. Thus, I do not see obvious weaknesses. Of course, it remains unclear what PUFAs do beyond fluidity regulation, but this is something that cannot be answered from a single study.

    1. Reviewer #2 (Public review):

      Summary:

      This manuscript examines the role of the dopamine receptor, Dop1R2, in memory formation. This receptor has complex roles in supporting different stages of memory, and the neural mechanisms for these functions is poorly understood. The authors are able to localize Dop1R2 function to the vertical lobes of the mushroom body, revealing a role in later (presumably middle-term) aversive and appetitive memory. In general the experimental design is rigorous, and statistics are appropriately applied. The manuscript provides a thorough assessment of how Dop1R2 functions within the mushroom bodies to regulate protein-synthesis dependent and independent memory, and provides a valuable new tool for the community.

      Strengths:

      (1) The FRT lines generated provide a novel tool for temporal and spatially precise manipulation of Dop1R2 function. This tool will be valuable to study the role of Dop1R2 in memory and other behaviors potentially regulated by this gene.

      (2) Given the highly conserved role of Dop1R2 in memory and other processes, these findings have high potential to translate to vertebrate species.

    1. Reviewer #2 (Public review):

      Summary:

      The study aims to probe the neural correlates of visual serial dependence - the phenomenon that estimates of a visual feature (here motion direction) are attracted towards the recent history of encoded and reported stimuli. The authors utilize an established retro-cue working memory task together with magnetoencephalography, which allows to probe neural representations of motion direction during encoding and retrieval (retro-cue) periods of each trial. The main finding is that neural representations of motion direction are not systematically biased during the encoding of motion stimuli, but are attracted towards the motion direction of the previous trial's target during the retrieval (retro-cue period), just prior to the behavioral response. By demonstrating a neural signature of attractive biases in working memory representations, which align with attractive behavioral biases, this study highlights the importance of post-encoding memory processes in visual serial dependence.

      Strengths:

      The main strength of the study is its elegant use of a retro-cue working memory task together with high temporal resolution MEG, enabling to probe neural representations related to stimulus encoding and working memory. The behavioral task elicits robust behavioral serial dependence and replicates previous behavioral findings by the same research group. The careful neural decoding analysis benefits from a large number of trials per participant, considering the slow-paced nature of the working memory paradigm. This is crucial in a paradigm with considerable trial-by-trial behavioral variability (serial dependence biases are typically small, relative to the overall variability in response errors). While the current study is broadly consistent with previous studies showing that attractive biases in neural responses are absent during stimulus encoding (prev. studies reported repulsive biases), to my knowledge, it is the first study showing attractive biases in current stimulus representations during working memory. The study also connects to previous literature showing reactivations of previous stimulus representations, although the link between reactivations and biases remains somewhat vague in the current manuscript. Together, the study reveals an interesting avenue for future studies investigating the neural basis of visual serial dependence.

      Weaknesses:

      The main weakness of the current manuscript is that the authors could have done more analyses to address the concern that their neural decoding results are driven by signals related to eye movements. The authors show that participants' gaze position systematically depended on the current stimuli's motion directions, which, together with previous studies on eye movement-related confounds in neural decoding, justifies such a concern. The authors seek to rule out this confound by showing that the consistency of stimulus-dependent gaze position does not correlate with (a) the neural reconstruction fidelity and (b) the attractive shift in reconstructed motion direction. However, the authors' approach of quantifying stimulus-dependent eye movements only considers gaze angle and not gaze amplitude, and thus potentially misses important features of eye movements that could manifest in the MEG data. Moreover, it is unclear whether the gaze consistency metric should correlate with attractive history biases in neural decoding, if there were a confound. These two concerns could be potentially addressed by (1) directly decoding stimulus motion direction from x-y gaze coordinates and relating this decoding performance to neural reconstruction fidelity, and (2) investigating whether gaze coordinates themselves are history-dependent and are attracted to the average gaze position associated with the previous trials' target stimulus. If the authors could show that (2) is not the case, I would be much more convinced that their main finding is not driven by eye movement confounds.

      The sample size (n = 10) is definitely at the lower end of sample sizes in this field. The authors collected two sessions per participant, which partly alleviates the concern. However, given that serial dependencies can be very variable across participants, I believe that future studies should aim for larger sample sizes.

      It would have been great to see an analysis in source space. As the authors mention in their introduction, different brain areas, such as PPC, mPFC and dlPFC have been implicated in serial biases. This begs the question which brain areas contribute to the serial dependencies observed in the current study? For instance, it would be interesting to see whether attractive shifts in current representations and pre-stimulus reactivations of previous stimuli are evident in the same or different brain areas.

    1. Reviewer #2 (Public review):

      Summary:

      This interesting study compared two different intensities of aerobic exercise (low-intensity, high-intensity) and their efficacy in inducing a hypoalgesic reaction (i.e. exercise-induced hypoalgesia; EIH). fMRI was used to identify signal changes in the brain, with infusion of naloxone used to identify hypoalgesia mechanisms. No differences were found in post exercise pain perception between the high-intensity and low-intensity conditions, with naloxone infusion causing increased pain perception across both conditions which was mirrored by activation in the medial frontal cortex (identified by fRMI).

      Strengths:

      • The use of fMRI and naloxone provides a strong approach by which to identify possible mechanisms of EIH.

      • The infusion of naloxone to maintain a stable concentration helps to ensure a consistent effect and that the time-course of the protocol won't affect consistency of changes in pain perception

      • The manipulation checks (differences in intensity of exercise, appropriate pain induction) are approached in a systematic way.

      • The interactions for fitness level and sex provide some interesting findings which should be explored further.

      Weaknesses:

      • Given the absence of a baseline/control condition (for exercise), the efficacy of high/low intensity exercise on EIH cannot be assessed. Providing this would have extended and strengthened the findings/conclusions.

      • Whilst the exercise test (functional threshold power) used to set the intensity of the low/high exercise bouts set participants to exercise at different intensities, this method does not ensure that they exercised above/below particular thresholds (i.e. within either heavy or severe domains). This could have created very different relative challenges between participants.

    1. Reviewer #2 (Public review):

      The work has significant implications for understanding immune evasion and nutrient uptake mechanisms in trypanosomes.

      While the experimental rigor is commendable, revisions are needed to clarify methodological limitations and to broaden the discussion of functional consequences.

      The authors argue that prior studies missed surface-localized TfR due to harsh washing/fixation (e.g., methanol). While this is plausible, additional evidence would strengthen the claim.

      It remains unclear how centrifugation steps of various lengths (as in previous publications) can equally and quantitatively redistribute TfR into the flagellar pocket. If this were the case, it should be straightforward for the authors to test this experimentally.

      If TfR is distributed over the cell surface, live-cell imaging with fluorescent transferrin should be performed as a control. Modern detection limits now reach the single-molecule level, and transient immobilization of live trypanosomes has been established, which would exclude hydrodynamic surface clearance as a confounding factor.

      In most images, TfR is not evenly distributed on the surface but rather appears punctate. Could this reflect localization to membrane domains? Immuno-EM with high-pressure frozen parasites could resolve this question and is relatively straightforward.

      The authors might consider discussing whether differences in parasite life cycle stages (procyclic versus bloodstream forms) or culture conditions (e.g., cell density) affect localization. The developmentally regulated retention of GPI-anchored procyclin in the flagellar pocket might be worth mentioning.

    1. Reviewer #2 (Public review):

      Summary:

      Wenke et al. investigated the role of vlPAG somatostatin-expressing neurons in the mediation of analgesia during defensive states. A newly developed paradigm of cued fear-conditioned analgesia, which consists of a combination of an auditory fear retrieval session and a pain test, was used to evaluate this cell population's contribution to fear-mediated analgesia. Optogenetic manipulation of vlPAG SST+ neurons modulated the responses to a nociceptive cue (Hot Plate) presented concomitantly with an aversively conditioned tone. At the same time, alterations in the freezing levels could be observed during optogenetic activation of vlPAG SST+ neurons. In order to disentangle the impact of these cells on analgesia from their impact on the expression of defensive behaviors, the authors performed electrophysiological recordings from the dorsal horn in the spinal cord of anesthetized mice. A vlPAG-RVM-DH pathway was identified to trigger nociceptive C-fibers upon optic activation of the RVM. Finally, pathway-specific activation of SST+ vlPAG-RVM neurons could abolish CS-induced analgesia.

      Strengths:

      The study addresses a relevant topic, that is, brainstem circuits for pain-modulatory mechanisms as part of defensive states evoked by threat. This is important because the circuit mechanisms underlying pain are still not fully understood, and defining molecular markers of cellular circuit substrates may support the identification of potential pharmaceutical targets in treating pain. The authors confirm a previous study in that a somatostatin-positive cellular population presents a crucial vlPAG circuit element mediating anti-nociceptive effects. Key novelty aspects of the present study are the demonstration that these neurons seem to play a role specifically in threat-induced analgesia. This was possible by the elegant design and application of a novel fear analgesia paradigm, combined with cell- and pathway-specific optogenetics.

      Weaknesses:

      Despite the convincing and rigorous experimental approach, the study leaves some interpretational room when it comes to the proposed circuit mechanism. This could either be addressed by additional experiments or by more discussion of alternative circuit layouts.

      Major Comments:

      (1) The paper by Zhang et al. (https://pubmed.ncbi.nlm.nih.gov/36641028/), which identified a role for vlPAG SOM+ neurons in mediating anti-nociception in neuropathic pain, needs to be referenced and its results discussed, if not reconciled. While functionally, both studies find an analgetic role of vlPAG SOM+ neurons projecting to the RVM, Zhang et al., using slice physiology, characterize those neurons as glutamatergic. In Figure 4E of Zhang et al. they find general (fear-independent) analgetic effects with PAG-RVM specificity by performing chemogenetic experiments.

      It can be argued that in addition to the two functionally distinct inhibitory SOM subtypes hypothesized by Winke et al., there is another, excitatory subpopulation. Also, the different experimental conditions (chronic vs. acute pain, non-threat vs. fearful cues/contexts may recruit different vlPAG SOM+ populations. All of this is conceivable, yet I wonder whether the contrasting findings could more parsimoniously be reconciled. The author's own results presented here in Supplementary Figure 3 suggests that SOM+ vlPAG cells are co-localizing with glutamate and thus could also be excitatory. In addition to this rather complementary piece of evidence, a more extensive characterization of vlPAG neurons using IHC and slice physiology would be needed to justify the unambiguous identification of their inhibitory nature.

      In the absence of a direct identification of these cells exclusively releasing GABA, an alternative explanation should be considered. What about looking at vlPAG SOM+ neurons as a putatively mixed bag of local, inhibitory interneurons and long-range, RVM-projecting excitatory cells? This model would then open up interesting questions as to the actual function of somatostatin as a modulator of vlPAG circuit activity and associated function, and from my perspective, would nicely fit into the view of PAG circuits as integrators of complex survival responses.

      (2) "Our data indicate that the optogenetic inhibition of SST+ vlPAG cells promotes analgesia irrespective of the animal's defensive state. In contrast, the optogenetic activation of long-range SST+ vlPAG cells that project to the rostral ventromedial medulla (RVM) abolishes the analgesia mediated by fear behavior." (lines 32-35). Consider toning down these conclusions, as contrasting activation with inhibition of two different (though overlapping) populations cannot be fully conclusive. Alternatively, a pathway-specific (vlPAG-RVM) inhibitory experiment could help to fully understand the circuit mechanism and verify the necessity of these neurons.

      (3) Despite an overall very thorough reporting style, some information is missing from the manuscript:

      a) In Figures 2d and f, what are the freezing levels during optogenetic manipulation? From Figure 3d, one can expect that freezing is inhibited during the hot plate test, which could bias the NC response towards shorter latencies. b) In Figure 5, the histological experiment showing the vlPAG-to-RVM pathway is presented by a qualitative image only. Here, some quantification would strengthen the finding. c) In Figures 6 c and d "Consistently, activation of the SST+ vlPAG-RVM pathway during CFCA had no impact on CS-presentation, whereas the same manipulation performed during CS+ blocked the increase in NC response latency compared to GFP controls." (line 194-196). Is it possible that the NC response cannot be any lower than the one during CS-, thus constituting a floor effect? d) Connected to major point 1- this experiment is important for defining the circuit mode and therefore should be as convincing as possible. However, for the colocalization experiment in Supplementary Figure 3, the methodological description is missing and thus makes it hard to comprehend how this data set was generated (how many data points, etc.). The visual depiction of the results is non-standard and not easily graspable. Consider e.g., a Venn diagram.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript by Jia and Chen addresses the structural basis of voltage-activation of BK channels using computational approaches. Although a number of experimental studies using gating current and patch-clamp recording have analyzed voltage-activation in terms of observed charge movements and the apparent energetic coupling between voltage-sensor movement and channel opening, the structural changes that underlie this phenomenon have been unclear. The present studies use a reduced molecular system comprising the transmembrane portion of the BK channel (i.e. the cytosolic domain was deleted), embedded in a POPC membrane, with either 0 or 750 mV applied across the membrane. This system enabled acquisition of long simulations of 10 microseconds, to permit tracking of conformational changes of the channel. The authors principal findings were that the side chains of R210 and R213 rapidly moved toward the extracellular side of the membrane (by 8 - 10 Å), with greater displacements than any of the other charged transmembrane residues. These movements appeared tightly coupled to movement of the pore-lining helix, pore hydration, and ion permeation. The authors estimate that R210 and R213 contribute 0.25 and 0.19 elementary charges per residue to the gating current, which is roughly consistent with estimates based on electrophysiological measurements that used the full-length channel.

      Strengths:

      The methodologies used in this work are sound, and these studies certainly contribute to our understanding of voltage-gating of BK channels. An intriguing observation is the strongly coupled movement of the S4, S5, and S6 helices that appear to underlie voltage-dependent opening. Based on Fig 2a-d, the substantial movements of the R210 and R213 side chains occur nearly simultaneously to the S6 movement (between 4 - 5 usec of simulation time). This seems to provide support for a "helix-packing" mechanism of voltage gating in the so-called "non-domain-swapped" voltage-gated K channels.

      Weaknesses:

      The main limitation is that these studies used a truncated version of the BK channel, and there are likely to be differences in VSD-pore coupling in the context of the full-length channels that will not be resolved in the present work. Nonetheless, the authors provide a strong rationale for their use of the truncated channel, and the results presented will provide a good starting point for future computational studies of this channel.

    1. Reviewer #2 (Public review):

      Summary:

      This study by Sanchez-Vasquez is a very innovative approach to induce aneuploidy and then study the contribution of treated cells to different lineages, including post implantation. It connects well to the authors previous work to induce mosaic aneuploidies. The authors identify sensitivity to HIF1a loss in treated embryos with likely aneuploidy. This work is part of an important line of work with evaluates the consequences of aneuploidy in mammalian embryo.

      Weaknesses:

      Given that this is a study on the induction of aneuploidy, it would be meaningful to assess aneuploidy immediately after induction, and then again before implantation. This is also applicable to the competition experiments on page 7/8. What is shown is the competitiveness of treated cells. Because the publication centers around aneuploidy, inclusion of such data in the main figure at all relevant points would strengthen it. There is some evaluation of karyotypes only in the supplemental - why? Would be good not to rely on a single assay that the authors appear to not give much importance.

    1. Reviewer #2 (Public review):

      This study aims to elucidate the role of fibroblasts in regulating myocardium and vascular development through signaling to cardiomyocytes and endothelial cells. This focus is significant, given that fibroblasts, cardiomyocytes, and vascular endothelial cells are the three primary cell types in the heart. The authors employed a Pdgfra-CreER-controlled diphtheria toxin A (DTA) system to ablate fibroblasts at various embryonic and postnatal stages, characterizing the resulting cardiac defects, particularly in myocardium and vasculature development. Single-cell RNA sequencing (scRNA-seq) analysis of the ablated hearts identified collagen as a crucial signaling molecule from fibroblasts that influences the development of cardiomyocytes and vascular endothelial cells.

      This is an interesting manuscript; however, there are several major issues, including an over-reliance on the scRNA-seq data, which shows inconsistencies between replicates.

      Some of the major issues are described below.

      (1) The CD31 immunostaining data (Figure 3B-G) indicate a reduction in endothelial cell numbers following fibroblast deletion using PdgfraCreER+/-; RosaDTA+/- mice. However, the scRNA-seq data show no percentage change in the endothelial cell population (Figure 4D). Furthermore, while the percentage of Vas_ECs decreased in ablated samples at E16.5, the results at E18.5 were inconsistent, showing an increase in one replicate and a decrease in another, raising concerns about the reliability of the RNA-seq findings.

      (2) Similarly, while the percentage of Ven_CMs increased at E18.5, it exhibited differing trends at E16.5 (Fig. 4E), further highlighting the inconsistency of the scRNA-seq analysis with the other data.

      (3) Furthermore, the authors noted that the ablated samples had slightly higher percentages of cardiomyocytes in the G1 phase compared to controls (Fig. 4H, S11D), which aligns with the enrichment of pathways related to heart development, sarcomere organization, heart tube morphogenesis, and cell proliferation. However, it is unclear how this correlates with heart development, given that the hearts of ablated mice are significantly smaller than those of controls (Figure 3E). Additionally, the heart sections from ablated samples used for CD31/DAPI staining in Figure 3F appear much larger than those of the controls, raising further inconsistencies in the manuscript.

      (4) The manuscript relies heavily on the scRNA-seq dataset, which shows inconsistencies between the two replicates. Furthermore, the morphological and histological analyses do not align with the scRNA-seq findings.

      (5) There is a lack of mechanistic insight into how collagen, as a key signaling molecule from fibroblasts, affects the development of cardiomyocytes and vascular endothelial cells.

      (6) In Figure 1B, Col1a1 expression is observed in the epicardial cells (Figure 1A, E11.5), but this is not represented in the accompanying cartoon.

      (7) Do the PdgfraCreER+/-; RosaDTA+/- mice survive after birth when induced at E15.5, and do they exhibit any cardiac defects?

      Comments on Revised Version (from BRE):

      The manuscript has greatly improved following the revision, and I have no additional comments to offer.

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript the authors investigate how epithelia maintain intercellular barrier function despite and during cellular rearrangements upon e.g. apoptotic extrusion in simple epithelia or regenerative turnover in stratified epithelia like this epidermis. A fundamental question in epithelial biology. Previous literature has shown that Rho mediated local regulation of actomyosin is essential not only for cellular rearrangement itself but also directly controls tight junction barrier function. The molecular mechanics however remained unclear. Here the authors use extensive fluorescence imaging of fixed and live cells together with genetic and drug mediated interference to show that Rho activation is required and sufficient to form de novo tight junctional strands at intercellular contacts in epidermal keratinocytes (HaCat) and mammary epithelial cells. After having confirmed previous literature they then show that Rho activation activates the transmembrane protease matriptase which cleaves EpCAM and TROP2, two claudin binding transmembrane proteins, to release claudins and enable claudin strand formation and therefore tight junction barrier function.

      Strengths:

      The presented mechanism is shown to be relevant for epithelial barriers being conserved in simple and stratifying epithelial cells and mainly differs due to tissue specific expression of EpCAM and TROP2. The authors present carefull state of the art imaging and logical experiments that convincingly support the statements and conclusion. The manuscript is well written and easy to follow.

      Weaknesses:

      Whereas the in vitro evidence of the presented mechanism is strongly supported by the data, the in vivo confirmation is mostly based on the predicted distribution of TROP2. Whereas the causality of Rho mediated matriptase activation has been nicely demonstrated it remains unclear how Rho activates matriptase.

    1. Reviewer #2 (Public review):

      Summary:

      Here, the authors studied the molecular mechanisms by which the chemoreceptor cluster and flagella motor of Pseudomonas aeruginosa (PA) are spatially organized in the cell. They argue that FlhF is involved in localizing the receptors-motor to the cell pole, and even without FlhF, the two are colocalized. Finally, the authors argue that the functional reason for this colocalization is to insulate chemotactic signaling from other signaling pathways, such as cyclic-di-GMP signaling.

      Strength:

      The experiments and data are high quality. It is clear that the motor and receptors co-localize, and that elevated CheY levels lead to elevated c-di-GMP.

      Weakness:

      The explanation for the functional importance of receptor-motor colocalization is plausible but is still not conclusively demonstrated. Colocalization might reduce CheY levels throughout the cell in order to reduce cross-talk with c-di-GMP. This would mean that if physiologically-relevant levels of CheYp near the pole were present throughout the cell, c-di-GMP levels would be elevated to a point that is problematic for the cell. Clearly demonstrating this seems challenging.

    1. Reviewer #2 (Public review):

      Summary:

      In their study, Cooper et al. investigated the spontaneous fluctuations in extracellular 5-HT release in the CA1 region of the hippocampus using GRAB5-HT3.0. Their findings revealed the presence of ultra-low frequency (less than 0.05 Hz) oscillations in 5-HT levels during both NREM sleep and wakefulness. The phase of these 5-HT oscillations was found to be related to the timing of hippocampal ripples, microarousals, electromyogram (EMG) activity, and hippocampal-cortical coherence. In particular, ripples were observed to occur with greater frequency during the descending phase of 5-HT oscillations, and stronger ripples were noted to occur in proximity to the 5-HT peak during NREM. Microarousal and EMG peaks occurred with greater frequency during the ascending phase of 5-HT oscillations. Additionally, the strongest coherence between the hippocampus and cortex was observed during the ascending phase of 5-HT oscillations. These patterns were observed in both NREM sleep and the awake state, with a greater prevalence in NREM. The authors posit that 5-HT oscillations may temporally segregate internal processing (e.g., memory consolidation) and responsiveness to external stimuli in the brain.

      Strengths:

      The findings of this research are novel and intriguing. Slow brain oscillations lasting tens of seconds have been suggested to exist, but to my knowledge they have never been analyzed in such a clear way. Furthermore, although it is likely that ultra-slow neuromodulator oscillations exist, this is the first report of such oscillations, and the greatest strength of this study is that it has clarified this phenomenon both statistically and phenomenologically.

      Weaknesses:

      As with any paper, this one has some limitations. While there is no particular need to pursue them, I will describe ten of them below, including future directions:

      Contralateral recordings: 5-HT levels and electrophysiological recordings were obtained from opposite hemispheres due to technical limitations. Ipsilateral simultaneous recordings may show more direct relationships.

      Sample size: The number of mice used in the experiments is relatively small (n=6). Validation with a larger sample size would be desirable.

      Lack of causality: The observed associations show correlations, not direct causal relationships, between 5-HT oscillations and neural activity patterns.

      Limited behavioral states: The study focuses primarily on sleep and quiet wakefulness. Investigation of 5-HT oscillations during a wider range of behavioral states (e.g., exploratory behavior, learning tasks) may provide a more complete understanding.

      Generalizability to other brain regions: The study focuses on the CA1 region of the hippocampus. It's unclear whether similar 5-HT oscillation patterns exist in other brain regions.

      Long-term effects not assessed: Long-term effects of ultra-low 5-HT oscillations (e.g., on memory consolidation or learning) were not assessed.

      Possible species differences: It's uncertain whether the findings in mice apply to other mammals, including humans.

      Technical limitations: The temporal resolution and sensitivity of the GRAB5-HT3.0 sensor may not capture faster 5-HT dynamics.

      Interactions with other neuromodulators: The study does not explore interactions with other neuromodulators (e.g., norepinephrine, acetylcholine) or their potential ultraslow oscillations.

      Limited exploration of functional significance: While the study suggests a potential role for 5-HT oscillations in memory consolidation and arousal, direct tests of these functional implications are not included.

    1. Reviewer #2 (Public review):

      Summary:

      This study reports on the existence of subpopulations of isogenic E. coli and P. aeruginosa cells that are tolerant to the antimicrobial peptide tachyplesin and are characterized by accumulation of low levels of a fluorescent tachyplesin-NBD conjugate. The authors then set out to address the molecular mechanisms, providing interesting insights even though the mechanism remains incompletely defined: The work demonstrates that increased efflux may cause this phenotype, putatively together with other changes in membrane lipid composition. The authors further demonstrate that pharmacological manipulation can prevent generation of tolerance. The authors are cautious in their interpretation and the claims made are largely justified by the data.

      Strengths:

      Going beyond the commonly used bulk techniques for studying susceptibility to AMPs, Lee et al. used of fluorescent antibiotic conjugates in combination with flow cytometry analysis to study variability in drug accumulation at the single cell level. This powerful approach enabled the authors to expose bimodal drug accumulation pattern that were condition dependent, but conserved across a variety of E. coli clinical isolates. Using cell sorting in combination with colony-forming unit assays as well as quantitative fluorescence microscopic analysis in a microfludics-setup the authors compellingly demonstrate that low accumulators (where fluorescence signal is mostly restricted to the membrane), can survive antibiotic treatment, whereas high accumulators (with high intracellular fluorescence) were killed.

      The relevance of efflux for the ´low accumulator´ phenotype and its survival is convincingly demonstrated by the following lines of evidence: i) A time-course experiment on tachyplesin-NBD pre-loaded cells revealed that all cells initially were high accumulators, before a subpopulation of cells subsequently managed to reduce signal intensity, demonstrating that the ´low accumulator´ phenotype is an induced response and not a pre-existing property. Ii) Double-mutants deficient in the delta acrA delta tolC double-KO, which showed reduced levels of low accumulators´. Interestingly, ´low accumulator´populations were nearly abrogated in bacteria deficient in the qse quorum sensing system, suggesting its centrality for the tachyplesin response. Even though this system may control acrA, the strength of the phenotype may suggest that it may control additional as-of-yet unidenitified factors relevant in the response to tachyplesin. Iii) treatment with efflux pump inhibitor sertraline and verapamil (even though some caution needs to be taken since it is not perfectly selective, see weakness) prevents generation of low accumulators. The observation that sertraline enhances tachyplesin-based killing is an important basis for developing combination therapies.

      The study convincingly illustrates how susceptibility to tachyplesin adaptively changes in a heterogeneous way dependent on the growth phases and nutrient availability. This is highly relevant also beyond the presented example of tachyplesin and similar subpopulation-based adaptive changes to the susceptibility towards antimicrobial peptides or other drugs may occur during infections in vivo and they would likely be missed by standardized in vitro susceptibility testing.

      Weaknesses:

      Some mechanistic questions regarding tachyplesin-accumulation and survival remain. One general shortcoming of the setup of the transcriptomics experiment is that the tachyplesin-NBD probe itself has antibiotic efficacy and induces phenotypes (and eventually cell death) in the ´high accumulator´ cells. As the authors state themselves, this makes it challenging to interpret whether any differences seen between the two groups are causative for the observed accumulation pattern of if they are a consequence of differential accumulation and downstream phenotypic effects.

    1. Reviewer #2 (Public review):

      Summary:

      In this study, Yagasaki et al. describe an organoid system to study the interactions between smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). While these interactions are essential for the control of rhythmic intestinal contractility (i.e., peristalsis), they are poorly understood, largely due to the complexity of and access to the in vivo environment and the inability to co-culture these cell types in vitro for long term under physiological conditions. The "gut contractile organoids" organoids described herein are reconstituted from stromal cells of the fetal chicken hindgut that rapidly reorganize into multilayered spheroids containing an outer layer of smooth muscle cells and an inner core of interstitial cells. The authors demonstrate that they contract cyclically and additionally use calcium imagining to show that these contractions occur concomitantly with calcium transients that initiate in the interstitial cell core and are synchronized within the organoid and between ICCs and SMCs. Furthermore, they use several pharmacological inhibitors to show that these contractions are dependent upon non-muscle myosin activity and, surprisingly, independent of gap junction activity. Finally, they develop a 3D hydrogel for the culturing of multiple organoids and found that they synchronize their contractile activities through interconnecting smooth muscle cells, suggesting that this model can be used to study the emergence of pacemaking activities. Overall, this study provides a relatively easy-to-establish organoid system that will be of use in studies examining the emergence of rhythmic peristaltic smooth muscle contractions and how these are regulated by interstitial cell interactions. However, further validation and quantification will be necessary to conclusively determine show the cellular composition of the organoids and how reproducible their behaviors are.

      Strengths:

      This work establishes a new self-organizing organoid system that can easily be generated from the muscle layers of the chick fetal hindgut to study the emergence of spontaneous smooth muscle cell contractility. A key strength of this approach is that the organoids seem to contain few cell types (though more validation is needed), namely smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). These organoids are amenable to live imaging of calcium dynamics as well as pharmacological perturbations for functional assays, and since they are derived from developing tissues, the emergence of the interactions between cell types can be functionally studied. Thus, the gut contractile organoids represent a reductionist system to study the interactions between SMCs and ICCs in comparison to the more complex in vivo environment, which has made studying these interactions challenging.

      Weaknesses:

      The study lacks complementary in vivo experiments, but these will be exciting to follow up in future studies.

    1. Reviewer #2 (Public review):

      Summary:

      The authors analyzed Xenopus oocytes at different stages of meiosis using quantitative phosphoproteomics. Their advanced methods and analyses revealed changes in protein abundances and phosphorylation states to an unprecedented depth and quantitative detail. In the manuscript they provide an excellent interpretation of these findings putting them in the context of past literature in Xenopus as well as in other model systems. The clarity of these explanations improved significantly in the revised version of the manuscript, and several minor imprecisions have been corrected as well.

      Strengths:

      High-quality data, careful and detailed analysis, and outstanding interpretation in the context of the large body of literature.

      Weaknesses:

      Merely a resource, none of the findings are tested in functional experiments.

      I am very impressed by the quality of the data and the careful and detailed interpretation of the findings. In this form, the manuscript will be an excellent resource to the cell division community in general, and it presents a very large number of hypotheses that can be tested in future experiments. Xenopus has been and still is a popular and powerful model system that led to critical discoveries around countless cellular processes, including the spindle, nuclear envelope, and translational regulation, just to name a few. This also includes a huge body of literature on the cell cycle describing its phosphoregulation. It is indeed somewhat frustrating to see that these earlier studies using phospho-mutants and phospho-antibodies were just scratching the surface. The phosphoproteomics analysis presented here reveals much more extensive and much more dynamic changes in phosphorylation states. Thereby, in my opinion, this manuscript opens a completely new chapter in this line of research, setting the stage for more systematic future studies.

    1. Reviewer #2 (Public review):

      Summary:

      The investigators tested a workshop intervention to improve knowledge and decrease misconceptions about sex inclusive research. There were important findings that demonstrate the difficulty in changing opinions and knowledge about the importance of studying both males and females. While interventions can improve knowledge and decrease perceived barriers, the impact was small.

      Strengths:

      The investigators included control groups and replicated the study in a second population of scientists. The results appear to be well substantiated. These are valuable findings that have practical implications for fields where sex is included as a biological variable to improve rigor and reproducibility.

      Weaknesses:

      I found the figures difficult to understand and would have appreciated more explanation of what is depicted, as well as greater space between the bars representing different categories.

    1. Reviewer #2 (Public review):

      Summary:

      I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and inflammatory responses. This study establishes that RBMX2 is an important host factor that modulates the infection process of M. bovis.

      Strengths:

      (1) This study tested multiple types of bovine and human cells, including macrophages, epithelial cells, and clinical tissues at multiple timepoints, and firmly confirmed the induced expression of RBMX2 upon M. bovis infection.

      (2) The authors have generated the monoclonal RBMX2 knockout cell lines and comprehensively characterized the RBMX2-dependent gene expression changes using a combination of global omics approaches. The study has validated the impact of RBMX2 knockout on the tight-junction pathway and on the M. bovis infection, establishing RBMX2 as a crucial host factor.

      Weaknesses:

      (1) The RBMX2 was only moderately induced (less than 2-fold) upon M. bovis infection, arguing its contribution may be small. Its value as a therapeutic target is not justified. How RBMX2 was activated by M. bovis infection was unclear.

      (2) Although multiple time points have been included in the study, most analyses lack temporal resolution. It is difficult to appreciate the impact/consequence of M. bovis infection on the analyzed pathways and processes.

    1. Reviewer #2 (Public review):

      The authors constructed an in-frame deletion of moeA gene, which is involved in molybdopterin cofactor (MoCo) biosynthesis, and investigated its role in structural colors in Flavobacterium IR1. The deletion of moeA shifted colony color from green to blue, reduced colony spreading, and increased starch degradation, which was attributed to the upregulation of various proteins in polysaccharide utilization loci. This study lays the ground for developing new colorants by modifying genes involved in structural colors.

      Overall, this is a well-written paper in which the authors effectively address their research questions through proper experimentation. This work will help us understand the genetic basis of structural colors in Flavobacterium and open new avenues to study the roles of additional genes and proteins in structural colors.

    1. Reviewer #2 (Public review):

      Summary:

      In the article Goyal et al. investigate how protein/polymer phase transition behavior is modulated by different binding partners-specifically, DNA and polyphosphate (PolyP). The authors show that while both DNA and PolyP can induce metastable condensates, only PolyP drives unique phase transition behaviors by effectively discriminating among initial protein ensembles with varying degrees of conformational heterogeneity, compactness, and plasticity. This selectivity is attributed to PolyP's ability to unfold the enzyme during condensate formation, supported by the observation of polyproline II-rich structures in two tested variants (CytR WT and DM). Overall, this work offers valuable insights into the mechanistic factors underlying condensation assembly and advances our understanding of how molecular interactions influence phase behavior.

      Strengths:

      The authors employed a well-designed and technically sound experimental approach to investigate how the initial protein conformational ensemble influences phase transition behavior in the presence of two charged polymers. Specifically, they examined phase transitions of CytR and FruR variants in the context of either polyphosphate (PolyP) or DNA, enabling a direct comparison that effectively highlights key differences. This study provides mechanistic insights into the role of PolyP in driving condensation and may contribute to a broader understanding of assembly processes involving PolyP, particularly in the context of bacterial stress responses.

      Weaknesses:

      The primary weakness of this manuscript lies in the lack of a consistent trend linking the unique phase transitions observed in protein/PolyP systems to the initial protein conformational ensemble. The observed differences in assembly and maturation behavior do not consistently correlate with conformational heterogeneity, plasticity, or compactness of the starting ensemble. This is particularly evident in the divergent outcomes between the CytR/PolyP and FruR/PolyP systems. Consequently, the phase behavior of protein/PolyP condensates does not reliably reflect the composition of the initial conformational ensemble, limiting its effectiveness as a probe for conformational state characterization.

    1. Reviewer #2 (Public review):

      Summary:

      An initial screening of pretreatment with different stress treatments of K. pneumonia allowed the identification of heat stress as a protection factor against the infection of the lytic phage Kp11. Then experiments prove that this is mediated not by an increase of phage resistant bacteria but due to an increase in phage transient tolerant population, that the authors identified as bacteriophage persistence in analogy to antibiotic persistence. Then they proved that phage persistence mediated by heath shock enhanced the evolution of bacterial resistance against the phage. The same trait was observed using other lytic phages, their combinations and two clinical strains, as well as E. coli and two T phages, hence the phenomenon may be widespread in enterobacteria.

      Next, the elucidation of heat induced phage persistence was done, determining that phage adsorption was not affected but phage DNA internalization was impaired by the heat pretreatment, likely to alterations in the bacterial envelope, including the downregulation of envelope proteins and of LPS; furthermore, heat treated bacteria were less sensitive to polymyxins due to the decrease in LPS.

      Finally, cyclic exposure to heat stress allowed the isolation of a mutant that was both resistant to heat treatment, polymyxins and lytic phage, that mutant had alterations in PspA protein that allowed a gain of function and that promoted the reduction of capsule production and loss of its structure; nevertheless this mutant was severely impaired in immune evasion as it was easily cleared from mice blood, evidencing the trade-off's between phage/heat and antibiotic resistance and the ability to counteract the immune response.

      Strengths:

      The experimental design and the sequence in which they are presented is ideal for the understanding of their study and the conclusions are supported by the findings, also the discussion points out the relevance of their work particularly in the effectiveness of phage therapy and allow the design of strategies to improve their effectiveness.

      Weaknesses:

      In its present form it lacks the incorporation of some relevant previous work that explored the role of heat stress in phage susceptibility, antibiotic susceptibility, trade offs between phage resistance and resistance against other kinds of stress, virulence, etc. and the fact that exposure to lytic phages induces antibiotic persistence.

      Comments on revised version:

      Thanks for addressing most of my comments; however, although you replied this in the rebuttal:

      "Thank you for highlighting these important studies. We have incorporated the work by Majkowska-Skrobek et al. (2021), Gordillo Altamirano et al. (2021), and García-Cruz et al. (2024) into the discussion "

      I was not able to find the new section in the discussion of the manuscript.

    1. Reviewer #3 (Public review):

      The Mutational Hazard Hypothesis (MHH) suggests that lineages with smaller effective population sizes should accumulate slightly deleterious transposable elements leading to larger genome size. Marino and colleagues tested the MHH using a set of 807 vertebrate, mollusc and insect species. The authors mined repeats de novo and estimated dN/dS for each genome. Then, they used dN/dS and life history traits as reliable proxies for effective population size and tested for correlations between these proxies and repeat content while accounting for phylogenetic nonindependence. The results suggest that overall, lineages with lower effective population sizes do not exhibit increases in repeat content or genome size. This contrasts with expectations from the MHH. The authors speculate that changes in genome size may be driven by lineage-specific host-TE conflicts rather than effective population size.

      Strengths:

      The general conclusions of this paper are supported by a powerful dataset of phylogenetically diverse species. Furthermore, the hypothesis tested is important and has proved challenging to test in the past due to technical challenges and confounding factors. The use of C-values rather than assembly size for many species (when available) helps to mitigate the challenges associated with underrepresentation of repetitive regions in short-read based genome assemblies. Overall, both the phylogenetic breadth of species considered and the approaches employed make the results highly convincing.

      Weaknesses:

      My primary concerns were related to possible biases in the author's data due to their approach to TE annotation. The authors have sufficiently acknowledged and addressed these concerns in their revised manuscript. I note no further weaknesses.

    1. Reviewer #2 (Public review):

      This is an interesting paper that delves into the post-translational modifications of the yeast Srs2 helicase and proteins with which it interacts in coping with DNA damage. The authors use mutants in some interaction domains with RPA and Srs2 to argue for a model in which there is a balance between RPA binding to ssDNA and Srs2's removal of RPA.

      The manuscript mostly addresses previous concerns by doubling down on the model without providing additional direct evidence of interactions between Srs2 and PCNA, and that "precise sites of Srs2 actions in the genome remain to be determined." One additional Srs2 allele has been examined, showing some effect in combination with rfa1-zm2.

    1. Reviewer #2 (Public review):

      Summary:

      This study aimed to uncover the protein composition and evolutionary conservation of electrical synapses in retinal neurons. The authors employed two complementary BioID approaches: expressing a Cx35b-TurboID fusion protein in zebrafish photoreceptors and using GFP-directed TurboID in Cx36-EGFP-labeled mouse AII amacrine cells. They identified conserved ZO proteins and endocytosis components in both species, along with over 50 novel proteins related to adhesion, cytoskeleton remodeling, membrane trafficking, and chemical synapses. Through a series of validation studies¬-including immunohistochemistry, in vitro interaction assays, and immunoprecipitation-they demonstrate that novel scaffold protein SIPA1L3 interacts with both Cx36 and ZO proteins at electrical synapse. Furthermore, they identify and localize proteins ZO-1, ZO-2, CGN, SIPA1L3, Syt4, SJ2BP, and BAI1 at AII/cone bipolar cell gap junctions.

      Strengths:

      The study demonstrates several significant strengths in both experimental design and validation approaches. First, the dual-species approach provides valuable insights into the evolutionary conservation of electrical synapse components across vertebrates. Second, the authors compare two different TurboID strategies in mice and demonstrate that the HKamac promoter and GFP-directed approach can successfully target the electrical synapse proteome of mouse AII amacrine cells. Third, they employed multiple complementary validation approaches-including retinal section immunohistochemistry, in vitro interaction assays, and immunoprecipitation-providing evidence supporting the presence and interaction of these proteins at electrical synapses.

      Weaknesses:

      The major weakness of this paper is the insufficient number of replicates in the proteomics datasets. The zebrafish datasets include only two biological replicates, while the mouse dataset has only one high-quality replicate. Due to the limited number of replicates, it is not possible to determine which enriched proteins are statistically significant.

      Additionally, the Neutravidin staining in the TurboID condition is not restricted to where Cx35 is expressed but is broadly distributed throughout the INL and IPL in the zebrafish retina (Figure 1B, bottom). Therefore, it is necessary to include NeutrAvidin staining in non-labeled retinas to verify whether the biotinylated proteins are specifically associated with Cx35 expression. Although the western blot results showed increased protein enrichment in the TurboID condition compared to non-labeled retinas, this does not confirm that the streptavidin pull-down proteins are associated with Cx35.

      Similarly, it is important to include NeutrAvidin staining in both TurboID and non-labeled conditions in the mouse retina to verify that the biotinylated proteins are specifically associated with gap junctions.

    1. Reviewer #2 (Public review):

      This study presents a comprehensive investigation of remote memory deficits in the APP/PS1 mouse model of Alzheimer's disease. The authors convincingly show that these deficits emerge progressively and are paralleled by selective hyperexcitability of PV interneurons in the mPFC. Using viral-TRAP labeling and patch-clamp electrophysiology, they demonstrate that inhibitory input onto labeled engram cells is selectively increased in APP/PS1 mice, despite unaltered engram size or reactivation. These findings support the idea that alterations in inhibitory microcircuits may contribute to cognitive decline in AD.

      However, several aspects of the study merit further clarification. Most critically, the central paradox, i.e., increased inhibitory input without an apparent change in engram reactivation, remains unresolved. The authors propose possible mechanisms involving altered synchrony or impaired output of engram cells, but these hypotheses require further empirical support. Additionally, the study employs multiple crossed transgenic lines without reporting the progression of amyloid pathology in the mPFC, which is important for interpreting the relationship between circuit dysfunction and disease stage. Finally, the potential contribution of broader network dysfunction, such as spontaneous epileptiform activity reported in APP/PS1 mice, is also not addressed.

    1. Reviewer #2 (Public review):

      Summary:

      In this article, the authors investigate enhancing the therapeutic and regenerative properties of mesenchymal stem cells (MSCs) through genetic modification, specifically by overexpressing genes involved in the glycogen synthesis pathway. By creating a non-phosphorylatable mutant form of glycogen synthase (GYSmut), the authors successfully increased glycogen accumulation in MSCs, leading to significantly improved cell survival under starvation conditions. The study highlights the potential of glycogen engineering to improve MSC function, especially in inflammatory or energy-deficient environments. However, critical gaps in the study's design, including the lack of validation of key findings, limited differentiation assessments, and missing data on MSC-GYSmut resistance to reactive oxygen species (ROS), necessitate further exploration.

      Strengths:

      (1) Novel Approach: The study introduces an innovative method of enhancing MSC function by manipulating glycogen metabolism.

      (2) Increased Glycogen Storage: The genetic modification of GYS1, resulting in GYSmut, significantly increased glycogen accumulation, leading to improved MSC survival under starvation, which has strong implications for enhancing MSC therapeutic properties in energy-deficient environments.

      (3) Potential Therapeutic Impact: The findings suggest significant therapeutic potential for MSCs in conditions that require improved survival, persistence, and immunomodulation, especially in inflammatory or energy-limited settings.

      (4) In Vivo Validation: The in vivo murine model of pulmonary fibrosis demonstrated the improved survival and persistence of MSC-GYSmut, supporting the translational potential of the approach.

      Weaknesses:

      (1) Lack of Differentiation Assessments: The study did not evaluate key MSC differentiation pathways, including chondrogenic and osteogenic differentiation. The absence of analysis of classical MSC surface markers and multipotency limits the understanding of the full potential of MSC-GYSmut.

      (2) Missing Validation of RNA Sequencing Data: Although RNA sequencing data revealed promising transcriptomic changes in chondrogenesis and metabolic pathways, these findings were not experimentally validated, limiting confidence.

      (3) Lack of ROS Resistance Analysis: Resistance to reactive oxygen species (ROS), an important feature for MSCs under regenerative conditions, was not assessed, leaving out a critical aspect of MSC function.

      (4) Inconsistencies in In Vivo Data: There is a discrepancy between the number of animals shown in the figures and the graph (three individuals vs. five animals), as well as missing details on how luciferase signal intensity was quantified, requiring further clarification.

      (5) Limited Exploration of Immunosuppressive Properties: The study did not address the immunosuppressive functions of MSC-GYSmut, which are critical for MSC-based therapies in clinical settings.

      Conclusion:

      The study presents an exciting new direction for enhancing MSC function through glycogen metabolism engineering. While the results show promise, key experiments and validations are missing, and several areas, such as differentiation capacity, ROS resistance, and immunosuppressive properties, require further investigation. Addressing these gaps would solidify the conclusions and strengthen the potential clinical applications of MSC-GYSmut in regenerative medicine.

    1. Reviewer #3 (Public review):

      Valencia et al. aim to elucidate the biochemical and cellular mechanisms through which the human formin FHOD3 drives sarcomere assembly in cardiomyocytes. To do so, they combined rigorous in vitro biochemical assays with comprehensive in vivo characterizations, evaluating two wild type FHOD3 isoforms and two function-separating mutants. Surprisingly, they found that both wild type FHOD3 isoforms can nucleate new actin filaments, as well as elongate existing actin filaments in conjunction with profilin following barbed-end capping. This is in addition to FHOD3's proposed role as an actin bundler. Next, the authors focused on the longer isoform FHOD3L due to its essential role in sarcomere assembly in cardiomyocytes. They asked whether FHOD3L promote sarcomere assembly through its activity in actin nucleation or rather elongation. To do so, the authors designed two function-separating mutants: the K1193L mutation in the FH2 domain, known for its importance in actin nucleation, and the glycine-serine linker substitution in the FH1 domain ("GS-FH1",) known for its requirement in actin elongation. They demonstrated that while K1193L maintains its elongation activity and greatly diminishes nucleation and bundling, in GS-FH1 keeps its nucleation activity while lose its capacity to drive elongation. Armed with these tools, the authors attempted to rescue FHOD3L siRNA-treated neonatal rat ventricular myocytes (NRVM) with transgenes carrying wild type, K1193L, or GS-FH1 mutant forms of human FHOD3. In each condition, they evaluated the numbers and morphology of sarcomeres, as well as their ability to beat and generate cardiac rhythm. The authors found that while the wild type FHOD3L and the K1193L mutant can rescue sarcomere morphology and physiology, the GS-FH1 mutant fails to do so. Given that in GS-FH1 mainly elongation activity is compromised, the authors concluded that the elongation activity of FHOD3 is essential for its role in sarcomere assembly in cardiomyocytes, while its nucleator activity is dispensable. Overall, this important study provided a broadened view on the biochemical activities of FHOD3, and a pioneering view on a possible cellular mechanism of how FHOD3L drives sarcomere assembly. If further validated, this can lead to new mechanistic models of sarcomere assembly and potentially new therapeutic targets of cardiomyopathy.

      The conclusions of this paper are mostly well supported by the comprehensive biochemical analyses performed by the authors. In my original assessment, I raised the point that the extreme low level of GS-FH1 signal in transfected cells in Figure 6A may reflect a failure of actin-binding by this construct in vivo, rather than its inability of driving elongation. The authors have thoroughly addressed this concern by: 1) providing new images of the GS-FH1 rescue condition with HA-FHOD3L signal intensities matching that of the K1193L rescue condition, and 2) quantitatively demonstrating that the expression levels in the GS-FH1 rescue condition are comparable with that of wild type FHOD3L rescue condition. This is nicely complemented by the new phalloidin staining of the GS-FH1 rescue condition, which showcased additional details of actin puncta reminiscent of that present in muscle stress fibers or premyofibrils. Overall, I am now convinced that the GS-FH1 cannot rescue sarcomere formation even when expressed at comparable levels. Given that GS-FH1 demonstrates actin elongation defects in vitro, it is reasonable to conclude that the actin elongation function of FHOD3L is essential for sarcomere formation in vivo.

    1. Reviewer #2 (Public review):

      Summary:

      The authors present Altair-LSFM (Light Sheet Fluorescence Microscope), a high-resolution, open-source microscope, that is relatively easy to align and construct and achieves sub-cellular resolution. The authors developed this microscope to fill a perceived need that current open-source systems are primarily designed for large specimens and lack sub-cellular resolution or are difficult to construct and align, and are not stable. While commercial alternatives exist that offer sub-cellular resolution, they are expensive. The authors' manuscript centers around comparisons to the highly successful lattice light-sheet microscope, including the choice of detection and excitation objectives. The authors thus claim that there remains a critical need for high-resolution, economical, and easy-to-implement LSFM systems.

      Strengths:

      The authors succeed in their goals of implementing a relatively low-cost (~ USD 150K) open-source microscope that is easy to align. The ease of alignment rests on using custom-designed baseplates with dowel pins for precise positioning of optics based on computer analysis of opto-mechanical tolerances, as well as the optical path design. They simplify the excitation optics over Lattice light-sheet microscopes by using a Gaussian beam for illumination while maintaining lateral and axial resolutions of 235 and 350 nm across a 260-um field of view after deconvolution. In doing so they rest on foundational principles of optical microscopy that what matters for lateral resolution is the numerical aperture of the detection objective and proper sampling of the image field on to the detection, and the axial resolution depends on the thickness of the light-sheet when it is thinner than the depth of field of the detection objective. This concept has unfortunately not been completely clear to users of high-resolution light-sheet microscopes and is thus a valuable demonstration. The microscope is controlled by an open-source software, Navigate, developed by the authors, and it is thus foreseeable that different versions of this system could be implemented depending on experimental needs while maintaining easy alignment and low cost. They demonstrate system performance successfully by characterizing their sheet, point-spread function, and visualization of sub-cellular structures in mammalian cells, including microtubules, actin filaments, nuclei, and the Golgi apparatus.

      Weaknesses:

      There is a fixation on comparison to the first-generation lattice light-sheet microscope, which has evolved significantly since then:

      (1) The authors claim that commercial lattice light-sheet microscopes (LLSM) are "complex, expensive, and alignment intensive", I believe this sentence applies to the open-source version of LLSM, which was made available for wide dissemination. Since then, a commercial solution has been provided by 3i, which is now being used in multiple cores and labs but does require routine alignments. However, Zeiss has also released a commercial turn-key system, which, while expensive, is stable, and the complexity does not interfere with the experience of the user. Though in general, statements on ease of use and stability might be considered anecdotal and may not belong in a scientific article, unreferenced or without data.

      (2) One of the major limitations of the first generation LLSM was the use of a 5 mm coverslip, which was a hinderance for many users. However, the Zeiss system elegantly solves this problem, and so does Oblique Plane Microscopy (OPM), while the Altair-LSFM retains this feature, which may dissuade widespread adoption. This limitation and how it may be overcome in future iterations is not discussed.

      (3) Further, on the point of sample flexibility, all generations of the LLSM, and by the nature of its design, the OPM, can accommodate live-cell imaging with temperature, gas, and humidity control. It is unclear how this would be implemented with the current sample chamber. This limitation would severely limit use cases for cell biologists, for which this microscope is designed. There is no discussion on this limitation or how it may be overcome in future iterations.

      (4) The authors' comparison to LLSM is constrained to the "square" lattice, which, as they point out, is the most used optical lattice (though this also might be considered anecdotal). The LLSM original design, however, goes far beyond the square lattice, including hexagonal lattices, the ability to do structured illumination, and greater flexibility in general in terms of light-sheet tuning for different experimental needs, as well as not being limited to just sample scanning. Thus, the Alstair-LSFM cannot compare to the original LLSM in terms of versatility, even if comparisons to the resolution provided by the square lattice are fair.

      (5) There is no demonstration of the system's live-imaging capabilities or temporal resolution, which is the main advantage of existing light-sheet systems.

      While the microscope is well designed and completely open source, it will require experience with optics, electronics, and microscopy to implement and align properly. Experience with custom machining or soliciting a machine shop is also necessary. Thus, in my opinion, it is unlikely to be implemented by a lab that has zero prior experience with custom optics or can hire someone who does. Altair-LSFM may not be as easily adaptable or implementable as the authors describe or perceive in any lab that is interested, even if they can afford it. The authors indicate they will offer "workshops," but this does not necessarily remove the barrier to entry or lower it, perhaps as significantly as the authors describe.

      There is a claim that this design is easily adaptable. However, the requirement of custom-machined baseplates and in silico optimization of the optical path basically means that each new instrument is a new design, even if the Navigate software can be used. It is unclear how Altair-LSFM demonstrates a modular design that reduces times from conception to optimization compared to previous implementations.

    1. Reviewer #2 (Public review):

      In this study, the authors show that alterations in the lipid composition of the inner mitochondrial membrane, particularly changes in cardiolipin (CL) content, lead to defects in electron transport, supercomplex formation, and oxidative stress. Using liver-specific CLS knockout mice, which are characterized by dysfunctional capacity for cardiolipin synthesis, the authors highlight an underappreciated role for CL in MASH pathology. Overall, this is an interesting study highlighting the importance of functional/physiological electron transport (and in this context, electron leakage) in MASH pathophysiology. Despite that, this manuscript has several weaknesses that require attention.

      (1) For all LKO studies, it is stated that the decrease in hepatic CL is causal for the observed phenotype. However, it is evident that many other lipids are impacted by CLS KO, including a marked increase in hepatic PG. In this respect, the authors show no evidence that the observed metabolic phenotype is indeed due to the reduction in CL and not to other accompanying changes.

      (2) In the results, the authors highlight that 'MASLD has been shown to alter the total cellular lipidome in liver.' Given that this study focused on CL, it would be useful to include specific studies that pointed to changes in hepatic CL content in MASLD/MASH/fibrosis.

      (3) The initial human mitochondrial lipidomics studies show a reduction in mitochondrial CL and PG content. What was the content/expression of CL synthase and PGP synthase in these samples? If this cannot be assessed, is there any association of CLS or PGPS expression and MASLD/fibrosis (etc) in publicly available databases (e.g, GEP liver) that may explain the reduction in mitochondrial PG and CL content?

      (4) The validation of MASH in patients (Figure 1B) is not convincing (ie., no quantification/scoring provided). NAS /fibrosis scoring (according to Kleiner) would help to define if all patients have indeed MASH, and what subset has fibrosis. Could the reduction in CL/PG content be (also) associated with fibrosis? In addition, Masson's Trichrome should be added to Figure 1B.

      (5) In human lipidomics, the authors suggest that reductions are observed in tetralinoleoyl CL (Figure 1C). However, Figure 1C only shows the combined FA acyl chain length + unsaturation, therefore not allowing for FA-specific ID (unless such data are available from the LC/MS analysis).

      (6) Figures 1 J/K/I. It is obvious that the background in all murine immunoblotting analysis has been altered. The authors should provide unaltered images for these immunoblots.

      (7) For Figure 1, it is unclear what is meant by 'we performed all mitochondrial lipidomic analyses by quantifying lipids per mg of mitochondrial proteins'. Was the murine lipidomics carried out on fractionated mitochondria or whole liver? If whole liver, then how were the data corrected, particularly given that PG is not a mitochondria-specific lipid?

      (8) While total CL content seems indeed decreased across the different mouse models, this is mostly due to 1-2 CL species showing a pronounced reduction, with the remainder being unaltered. This should at least be acknowledged in the results. This is similarly the case in the LKO livers.

      (9) Figure 2. A secondary biochemical analysis of changes in lipid content should be provided, e.g., total triglyceride content, particularly given that the histology analysis does not show any major changes in hepatic lipid droplets/steatosis. In addition, the Masson's Trichrome staining shows almost no collagen deposition.

      (10) Figure 3. 'CLS deletion modestly reduced glucose handling' should be reworded. The LKO mice show improved glucose tolerance (despite the MASH phenotype), which is not evident from the above wording.

      (11) Looking at the mechanism behind the increase in hepatic steatosis, the authors state that lipid accumulation can occur due to increased lipogenesis, or dysfunctional VLDL secretion or beta oxidation, and subsequently assessed the relevant proteins/pathways. What about fatty acid uptake, which is also one of the four major pathways impacted in MASLD? This should be included in this assessment in Figure 3.

      (12) For Figure 5A, it is simply stated 'CLS deletion promotes liver fibrosis in standard chow-fed condition', and it is unclear what is highlighted within the selected EM images and what the arrows refer to. The authors should clarify this within the text.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript by Zheng et al reports the structural and biochemical study of novel effectors from the bacterial pathogen Legionella pneumophila. The authors continued from results from their earlier screening for L. pneumophila proteins that affect host F-actin dynamics to show that Llfat1 (Lpg1387) interacts with actin via a novel actin-binding domain (ABD). The authors also determined the structure of the Lfat1 ABD-F-actin complex, which allowed them to develop this ABD as a probe for F-actin. Finally, the authors demonstrated that Llfat1 is a lysine fatty acyltransferase that targets several small GTPases in host cells.

      Strengths:

      This is a very complete work that shows the structure of a novel bacterial actin-binding protein in complex with F-actin, and the biochemical activity of the protein was also revealed. Overall, this is a very exciting study and should be of great interest to scientists in both bacterial pathogenesis and the actin cytoskeleton of eukaryotic cells.

      Weaknesses:

      (1) The authors should use biochemical reactions to analyze the KFAT of Llfat1 on one or two small GTPases shown to be modified by this effector in cellulo. Such reactions may allow them to determine the role of actin binding in its biochemical activity. This notion is particularly relevant in light of recent studies that actin is a co-factor for the activity of LnaB and Ceg14 (PMID: 39009586; PMID: 38776962; PMID: 40394005). In addition, the study should be discussed in the context of these recent findings on the role of actin in the activity of L. pneumophila effectors.

      (2) The development of the ABD domain of Llfat1 as an F-actin domain is a nice extension of the biochemical and structural experiments. The authors need to compare the new probe to those currently commonly used ones, such as Lifeact, in labeling of the actin cytoskeleton structure.

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript, Monziani et al. identified long noncoding RNAs (lncRNAs) that act in cis and are coregulated with their target genes located in close genomic proximity. The authors mined the GeneHancer database, and this analysis led to the identification of four lncRNA-target pairs. The authors decided to focus on lncRNA EPB41L4A-AS1.

      They thoroughly characterised this lncRNA, demonstrating that it is located in the cytoplasm and the nuclei, and that its expression is altered in response to different stimuli. Furthermore, the authors showed that EPB41L4A-AS1 regulates EPB41L4A transcription, leading to a mild reduction in EPB41L4A protein levels. This was not recapitulated with siRNA-mediated depletion of EPB41L4AAS1. RNA-seq in EPB41L4A-AS1-depleted cells with single LNA revealed 2364 DEGs linked to pathways including the cell cycle, cell adhesion, and inflammatory response. To understand the mechanism of action of EPB41L4A-AS1, the authors mined the ENCODE eCLIP data and identified SUB1 as an lncRNA interactor. The authors also found that the loss of EPB41L4A-AS1 and SUB1 leads to the accumulation of snoRNAs, and that SUB1 localisation changes upon the loss of EPB41L4A-AS1. Finally, the authors showed that EPB41L4A-AS1 deficiency did not change the steady-state levels of SNORA13 nor RNA modification driven by this RNA. The phenotype associated with the loss of EPB41L4A-AS1 is linked to increased invasion and EMT gene signature.

      Overall, this is an interesting and nicely done study on the versatile role of EPB41L4A-AS1 and the multifaceted interplay between SUB1 and this lncRNA, but some conclusions and claims need to be supported with additional experiments. My primary concerns are using a single LNA gapmer for critical experiments, increased invasion, and nucleolar distribution of SUB1- in EPB41L4A-AS1-depleted cells. These experiments need to be validated with orthogonal methods.

      Strengths:

      The authors used complementary tools to dissect the complex role of lncRNA EPB41L4A-AS1 in regulating EPB41L4A, which is highly commendable. There are few papers in the literature on lncRNAs at this standard. They employed LNA gapmers, siRNAs, CRISPRi/a, and exogenous overexpression of EPB41L4A-AS1 to demonstrate that the transcription of EPB41L4A-AS1 acts in cis to promote the expression of EPB41L4A by ensuring spatial proximity between the TAD boundary and the EPB41L4A promoter. At the same time, this lncRNA binds to SUB1 and regulates snoRNA expression and nucleolar biology. Overall, the manuscript is easy to read, and the figures are well presented. The methods are sound, and the expected standards are met.

      Weaknesses:

      The authors should clarify how many lncRNA-target pairs were included in the initial computational screen for cis-acting lncRNAs and why MCF7 was chosen as the cell line of choice. Most of the data uses a single LNA gapmer targeting EPB41L4A-AS1 lncRNA (eg, Fig. 2c, 3B, and RNA-seq), and the critical experiments should be using at least 2 LNA gapmers. The specificity of SUB1 CUT&RUN is lacking, as well as direct binding of SUB1 to lncRNA EPB41L4A-AS1, which should be confirmed by CLIP qPCR in MCF7 cells. Finally, the role of EPB41L4A-AS1 in SUB1 distribution (Figure 5) and cell invasion (Figure 8) needs to be complemented with additional experiments, which should finally demonstrate the role of this lncRNA in nucleolus and cancer-associated pathways. The use of MCF7 as a single cancer cell line is not ideal.

    1. Reviewer #2:

      The study of Rollenhagen et al examines the ultrastructural features of Layer 1 of human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as further from the epilepsy focus, and as such considered to be non-epileptic. The analyses has included 4 patients with different age, sex, medication and onset of epilepsy. The manuscript is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:

      Layer 4 - Yakoubi et al 2019 eLife

      Layer 5 - Yakoubi et al 2019 Cerebral Cortex,

      Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex

      They find, the L1 synaptic boutons mainly have single active zone a very large pool of synaptic vesicles and are mostly devoid of astrocytic coverage.

      Strengths:

      The MS is well written easy to read. Result section gives a detailed set of figures showing many morphological parameters of synaptic boutons and surrounding glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in human brain are still very limited, the current MS has substantial relevance. The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analyses is clear and precise.

      Weaknesses:

      The authors made all the corrections required and answered all of my concerns, included additional data sets, and clarified statements where needed.

    1. Reviewer #2 (Public review):

      Summary:

      A core task of the brain is processing sensory cues from the environment. The neural mechanisms of how sensory information is transmitted from peripheral sense organs to subsequent being processing in defined brain centers remains an important topic in neuroscience. The taste system hereby assesses the palatability of food by evaluating the chemical composition and nutrient content while integrating the current need of energy by assessing the satiation level of the organism. The current manuscript provides insights into the early circuits gustatory coding using the fruit fly as model. By combining trans-tango and FACS-based bulk RNAseq to assess the target neurons of sweet sensing (using by Gr64f-Gal4) and bitter sensing (using Gr66a-Gal4) in a first set of experiments the authors investigate genes that are differentially expressed or co-expressed in normal and starved conditions. With a focus on neuropeptides and neurotransmitters differential expression in the different conditions were assessed resulting in the identification of Leucokinin as potentially interesting gene. The notion is further supported by RNAseq of Lk-Gal4>mCD8:GFP sorted cells and immunostainings. GRASP and BacTrace experiments further supports that the two Lk expressing cells in the SEZ should indeed be postsynaptic to both type of sensors. Using EM-based connectomics data (based on a previous publication by Engert et al.), the authors also look for downstream targets of the bitter versus sweet gustatory neurons to identify the Lk-neurons. Based on morphology they identify candidates and further depict the potential downstream neurons in the connectome, which appears largely in agreement with GRASP experiments. Finally silencing the Lk-neurons shows an increased PER response in starved flies (when combined with bitter compounds) as well as increased feeding in a FlyPad assay.

      Strengths:

      Overall this is an intriguing manuscript, which provides insight into the organization of 2nd order gustatory neurons. It specifically provides strong evidence for the Lk-neurons as target of sweet and bitter GRNs and provides evidence for their role in regulating sweet vs bitter based behavioral responses. Particularly the integration of different techniques and datasets in an elegant fashion is a strong side of the manuscript. Moreover to put the known LK-neurons into the context of 2nd order gustatory signalling is strengthening the knowledge about this pathway.

      Weaknesses:

      I do not see any major weakness in the current manuscript. Novelty is to some degree lessened by the fact, that the RNAseq approach did not identify new neurons but rather put the known LK-neurons as major finding. Similarly the final behavioral section is not very deep and to some degree corroborates the previous publication by the Keene and Nässel labs- that said, the model they propose is indeed novel (but lacks depth in analyses, e.g. there is no physiology that would support the modulation of Lk neurons by either type of GRN). The connectomic section appears a bit out of place and after reading it it's not really clear what one should make of the potential downstream neurons (particularly since the Lk-receptor expression has been previously analyzed); here it might have been interesting to address if/how Lk-neurons may signal directly via a classical neurotransmitter (an information that might be found easily in the adult brain single-cell data).

      Comments on the latest version:

      I feel all points have been included to a satisfactory degree.

    1. Reviewer #2 (Public review):

      In this manuscript, Ridout et al. present an intriguing extension of beta cell mass-focused models for diabetes. Their model incorporates reversible glucose-dependent inactivation of beta cell mass, which can trigger sudden-onset hyperglycemia due to bistability in beta cell mass dynamics. Notably, this hyperglycemia can be reversed with insulin treatment. The model is simple, elegant, and thought-provoking.

    1. Reviewer #2 (Public review):

      Summary:

      Argunsah and colleagues demonstrate that SST-expressing interneurons are concentrated in the mouse septa and differentially respond to repetitive multi-whisker inputs. Identifying how a specific neuronal phenotype impacts responses is an advance.

      Strengths:

      (1) Careful physiological and imaging studies.

      (2) Novel result showing the role of SST+ neurons in shaping responses.

      (3) Good use of a knockout animal to further the main hypothesis.

      (4) Clear analytical techniques.

      Weaknesses:

      No major weaknesses were identified by this reviewer. Overall I appreciated the paper but feel it overlooked a few issues and had some recommendations on how additional clarifications could strengthen the paper. These include:

      (1) Significant work from Jerry Chen on how S1 neurons that project to M1 versus S2 respond in a variety of behavioral tasks should be included (e.g. PMID: 26098757). Similarly, work from Barry Connor's lab on intracortical versus thalamocortical inputs to SST neurons, as well as excitatory inputs onto these neurons (e.g. PMID: 12815025) should be included.

      (2) Using Layer 2/3 as a proxy to what is happening in layer 4 (~line 234). Given that layer 2/3 cells integrate information from multiple barrels, as well as receiving direct VPm thalamocortical input, and given the time window that is being looked at can receive input from other cortical locations, it is not clear that layer 2/3 is a proxy for what is happening in layer 4.

      (3) Line 267, when discussing distinct temporal response, it is not well defined what this is referring to. Are the neurons no longer showing peaks to whisker stimulation, or are the responses lasting a longer time? It is unclear why PV+ interneurons which may not be impacted by the Elfn1 KO and receive strong thalamocortical inputs, are not constraining activity.

      (4) Line 585 "the earliest CSD sink was identified as layer 4..." were post-hoc measurements made to determine where the different shank leads were based on the post-hoc histology?

      (5) For the retrograde tracing studies, how were the M1 and S2 injections targeted (stereotaxically or physiologically)? How was it determined that the injections were in the whisker region (or not)?

      (6) Were there any baseline differences in spontaneous activity in the speta versus barrel regions, and did this change in the KO animals?

    1. Reviewer #2 (Public review):

      Summary

      This study investigates the role of the human posterior inferotemporal cortex (hPIT) in attentional control, proposing that hPIT serves as an attentional priority map that integrates both top-down (endogenous) and bottom-up (exogenous) attentional processes. The authors conducted three types of fMRI experiments and collected resting-state data from 15 participants. In Experiment 1, using three different spatial attention tasks, they identified the hPIT region and demonstrated that this area is modulated by attention across tasks. In Experiment 2, by manipulating the presence or absence of visual stimuli, they showed that hPIT exhibits strong attentional modulation in both conditions, suggesting its involvement in both bottom-up and top-down attention. Experiment 3 examined the sensitivity of hPIT to stimulus features and attentional load, revealing that hPIT is insensitive to stimulus category but responsive to task load - further supporting its role as an attentional priority map. Finally, resting-state functional connectivity analyses showed that hPIT is connected to both dorsal and ventral attention networks, suggesting its potential role as a bridge between the two systems. These findings extend prior work on monkey PITd and provide new insights into the integration of endogenous and exogenous attention.

      Strengths

      (1) The study is innovative in its use of specially designed spatial attention tasks to localize and validate hPIT, and in exploring the region's role in integrating both endogenous and exogenous attention, as prior works focus primarily on its involvement in endogenous attention.

      (2) The authors provided very comprehensive experiment designs with clear figures and detailed descriptions.

      (3) A broad range of analyses was conducted to support the hypothesis that hPIT functions as an attentional priority map -- including experiments of attentional modulation under both top-down and bottom-up conditions, sensitivity to stimulus features and task load, and resting-state functional connectivity. These analyses showed consistent results.

      (4) Multiple appropriate statistical analyses - including t-tests, ANOVAs, and post-hoc tests - were conducted, and the results are clearly reported.

      Weaknesses

      (1) The sample size is relatively small (n = 15), and inter-subject variability is big in Figures 5 and 6, as seen in the spread of individual data points and error bars. The analysis of attention-modulated voxel map intersections appears to be influenced by multiple outliers.

      (2) The authors acknowledge important limitations, including the lack of exploration of feature-based attention and the temporal constraints inherent to fMRI.

      (3) Prior research has established that regions such as the prefrontal cortex (PFC) and posterior parietal cortex (PPC) are involved in both endogenous and exogenous attention and have been proposed as attentional priority maps. It remains unclear what is uniquely contributed by hPIT, how it functionally interacts with these classical attentional hubs, and whether its role is complementary or redundant. The study would benefit from more direct comparisons with these regions.

      (4) The functional connectivity analysis is only performed on resting-state data, and this approach does not capture context-dependent interactions. Task-based data analysis can provide stronger evidence.

      (5) The study does not report whether attentional modulation in hPIT is consistent across the two hemispheres. A comparison of hemispheric effects could provide important insight into lateralization and inter-individual variability, especially given the bilateral localization of hPIT.

    1. Reviewer #2 (Public review):

      This is an interesting study in Drosophila comparing potentially differential requirements for subsets of Kenyon Cells (KCs) and Dopaminergic neurons (DANS) in olfactory dishabituation driven by either a novel odor ("homosensory") or footshock ("heterosensory). The authors measure olfactory aversion to Octanol (OCT) in a T-maze, induce olfactory habituation with a 4-minute prior exposure to OCT, and use either brief yeast odor (YO) or footshock (FS) to achieve dishabituation. The major observation that YO-mediated dishabituation is mediated by reward-activated DANs (PAM cluster), while FS-mediated dishabituation is mediated by punishment-activated PPL-DANs is generally solid and convincing. Also convincing are experiments showing the involvement of KCs in the pathway for YO and FS-induced dishabituation, and the argument that KCs drive DAN activation that causes dishabituation, though not experimentally shown, is more than reasonable. The work is significant because, as the authors take pains to point out, circuits and pathways for dishabituation have been very lightly studied, and clear identification of dopaminergic neuron subsets in dishabituation achieved by different means represents unique and interesting progress.

      However, the claim that this represents a fundamental difference between homosensory and heterosensory pathways for dishabituation is overstated. The introductory section does not adequately present current broad models for habituation and dishabituation. There are many different time scales, even for Drosophila olfactory habituation. These, as well as potential underlying mechanistic differences, need to be acknowledged; any claim should be specifically qualified for the time scales being studied here. Additionally, there are several unclear, vague, and inaccurate sections and statements. A more careful, precise, and considered presentation of current views, as well as more measured claims of the impact of the findings, would substantially enhance my enthusiasm.

    1. Reviewer #2 (Public review):

      Summary:

      In this paper, entitled "SpikeMAP: An unsupervised spike sorting pipeline for cortical excitatory and inhibitory 2 neurons in high-density multielectrode arrays with ground-truth validation", the authors present spikeMAP, a pipeline for the analysis of large-scale recordings of in vitro cortical activity. According to the authors, spikeMAP not only allows for the detection of spikes produced by single neurons (spike sorting), but also allows for the reliable distinction between genetically determined cell types by utilizing viral and optogenetic strategies as ground-truth validation. While I find that the paper is nicely written and easy to follow, I find that the algorithmic part of the paper is not really new and should have been more carefully compared to existing solutions. While the GT recordings to assess the possibilities of a spike sorting tool to distinguish properly between excitatory and inhibitory neurons are interesting, spikeMAP does not seem to bring anything new to state-of-the-art solutions, and/or, at least, it would deserve to be properly benchmarked. I would suggest that the authors perform a more intensive comparison with existing spike sorters.

      Strengths:

      The GT recordings with optogenetic activation of the cells, based on the opsins, is interesting and might provide useful data to quantify how good spike sorting pipelines are, in vitro, to discriminate between excitatory and inhibitory neurons. Such an approach can be quite complementary to artificially generated ground truth.

      Weaknesses:

      (1) The global workflow of spikeMAP, described in Figure 1, seems to be very similar to that of Hilgen et al. 2020 (10.1016/j.celrep.2017.02.038). Therefore, the first question is what is the rationale of reinventing the wheel, and not using tools that are doing something very similar (as mentioned by the authors themselves). I have a hard time, in general, believing that spikeMAP has something particularly special, given its Methods, compared to state-of-the-art spike sorters. This is why, at the very least, the title of the paper is misleading, because it lets the reader think that the core of the paper will be about a new spike sorting pipeline. If this is the main message the authors want to convey, then I think that numerous validations/benchmarks are missing to assess first how good spikeMAP is, with reference to spike sorting in general, before deciding if this is indeed the right tool to discriminate excitatory vs inhibitory cells. The GT validation, while interesting, is not enough to entirely validate the paper. The details are a bit too scarce for me, or would deserve to be better explained (see other comments after).

      (2) Regarding the putative location of the spikes, it has been shown that the center of mass, while easy to compute, is not the most accurate solution [Scopin et al, 2024, 10.1016/j.jneumeth.2024.110297]. For example, it has an intrinsic bias for finding positions within the boundaries of the electrodes, while some other methods, such as monopolar triangulation or grid-based convolution,n might have better performances. Can the authors comment on the choice of the Center of Mass as a unique way to triangulate the sources?

      (3) Still in Figure 1, I am not sure I really see the point of Spline Interpolation. I see the point of such a smoothing, but the authors should demonstrate that it has a key impact on the distinction of Excitatory vs. Inhibitory cells. What is special about the value of 90kHz for a signal recorded at 18kHz? What is the gain with spline enhancement compared to without? Does such a value depend on the sampling rate, or is it a global optimum found by the authors?

      (4) Figure 2 is not really clear, especially panel B. The choice of the time scale for the B panel might not be the most appropriate, and the legend filtered/unfiltered with a dot is not clear to me in Bii. In panel E, the authors are making two clusters with PCA projections on single waveforms. Does this mean that the PCA is only applied to the main waveforms, i.e. the ones obtained where the amplitudes are peaking the most? This is not really clear from the methods, but if this is the case, then this approach is a bit simplistic and does not really match state-of-the-art solutions. Spike waveforms are quite often, especially with such high-density arrays, covering multiple channels at once, and thus the extracellular patterns triggered by the single units on the MEA are spatio-temporal motifs occurring on several channels. This is why, in modern spike sorters, the information in a local neighbourhood is often kept to be projected, via PCA, on the lower-dimensional space before clustering. Information on a single channel only might not be informative enough to disambiguate sources. Can the authors comment on that, and what is the exact spatial resolution of the 3Brain device? The way the authors are performing the SVD should be clarified in the methods section. Is it on a single channel, and/or on multiple channels in a local neighbourhood?

      (5) About the isolation of the single units, here again, I think the manuscript lacks some technical details. The authors are saying that they are using a k-means cluster analysis with k=2. This means that the authors are explicitly looking for 2 clusters per electrode? If so, this is a really strong assumption that should not be held in the context of spike sorting, because, since it is a blind source separation technique, one can not pre-determine in advance how many sources are present in the vicinity of a given electrode. While the illustration in Figure 2E is ok, there is no guarantee that one can not find more clusters, so why this choice of k=2? Again, this is why most modern spike sorting pipelines do not rely on k-means, to avoid any hard-coded number of clusters. Can the authors comment on that?

      (6) I'm surprised by the linear decay of the maximal amplitude as a function of the distance from the soma, as shown in Figure 2H. Is it really what should be expected? Based on the properties of the extracellular media, shouldn't we expect a power law for the decay of the amplitude? This is strange that up to 100um away from the soma, the max amplitude only dropped from 260 to 240 uV. Can the authors comment on that? It would be interesting to plot that for all neurons recorded, in a normed manner V/max(V) as function of distances, to see what the curve looks like.

      (7) In Figure 3A, it seems that the total number of cells is rather low for such a large number of electrodes. What are the quality criteria that are used to keep these cells? Did the authors exclude some cells from the analysis, and if yes, what are the quality criteria that are used to keep cells? If no criteria are used (because none are mentioned in the Methods), then how come so few cells are detected, and can the authors convince us that these neurons are indeed "clean" units (RPVs, SNRs, ...)?

      (8) Still in Figure 3A, it looks like there is a bias to find inhibitory cells at the borders, since they do not appear to be uniformly distributed over the MEA. Can the authors comment on that? What would be the explanation for such a behaviour? It would be interesting to see some macroscopic quantities on Excitatory/Inhibitory cells, such as mean firing rates, averaged SNRs... Because again, in Figure 3C, it is not clear to me that the firing rates of inhibitory cells are higher than Excitatory ones, whilst they should be in theory.

      (9) For Figure 3 in general, I would have performed an exhaustive comparison of putative cells found by spikeMAP and other sorters. More precisely, I think that to prove the point that spikeMAP is indeed bringing something new to the field of spike sorting, the authors should have compared the performances of various spike sorters to discriminate Exc vs Inh cells based on their ground truth recordings. For example, either using Kilosort [Pachitariu et al, 2024, 10.1038/s41592-024-02232-7], or some other sorters that might be working with such large high-density data [Yger et al, 2018, 10.7554/eLife.34518].

      (10) Figure 4 has a big issue, and I guess the panels A and B should be redrawn. I don't understand what the red rectangle is displaying.

      (11) I understand that Figure 4 is only one example, but I have a hard time understanding from the manuscript how many slices/mices were used to obtain the GT data? I guess the manuscript could be enhanced by turning the data into an open-access dataset, but then some clarification is needed. How many flashes/animals/slices are we talking about? Maybe this should be illustrated in Figure 4, if this figure is devoted to the introduction of the GT data.

      (12) While there is no doubt that GT data as the ones recorded here by the authors are the most interesting data from a validation point of view, the pretty low yield of such experiments should not discourage the use of artificially generated recordings such as the ones made in [Buccino et al, 2020, 10.1007/s12021-020-09467-7] or even recently in [Laquitaine et al, 2024, 10.1101/2024.12.04.626805v1]. In these papers, the authors have putative waveforms/firing rate patterns for excitatory and inhibitory cells, and thus, the authors could test how good they are in discriminating the two subtypes.

    1. Reviewer #2 (Public review):

      Summary:

      This work seeks to establish the Open Field Test (OFT) as a paradigm to measure emotion-like states in the fruit fly Drosophila. To do this, the authors first applied various stressors and aversive stimuli to wild-type flies and tracked their locomotion. By measuring wall-following (WAFO) and total walking (TOWA), they showed that these behaviors are generally increased by stressors, but return to baseline levels after their removal. Then, they used the same approach to analyze the effects of pharmacological, genetic, and neuronal activity manipulations, showing that diazepam, serotonin, dopamine, and neuropeptide F affect locomotion in the OFT in largely expected ways that are consistent with their functions in rodents. Finally, the authors demonstrate that wild-type fly strains from the laboratory or caught in the wild differ significantly in their OFT behavior, with wild-caught flies generally behaving as if more 'stressed'. Given the numerous advantages of Drosophila, this study can form the foundation for using the OFT in conjunction with this animal model to elucidate the molecular and neuronal mechanisms that underlie emotion primitives.

      Strengths:

      The main strength of the paper is the rigorous use of several stressful or aversive treatments and their subsequent removal to show that WAFO is a robust proxy for stress-like emotional primitives across multiple stimuli. The pharmacological, molecular, and neuronal activity manipulations, although more limited in scope, lend further credence to the authors' central claim.

      Weaknesses:

      The conceptual advance of this research is unclear, as previous work (Mohammad et al., 2016, Curr Biol.) carried out similar treatments and manipulations and reached largely similar conclusions. Moreover, while WAFO is a good proxy for 'stress', I am not convinced that TOWA necessarily represents an emotional state in all cases. Indeed, as the authors themselves acknowledge, changes in total walking may be associated with other factors, such as starvation-induced hyperactivity, physical exhaustion after sleep deprivation, increased sex drive after mating, alcohol sedation, etc. Another unclear point is the interpretation of some unexpected results, such as the finding that both serotonin transporter overexpression and its knockdown give the same phenotype. Finally, there are some issues with the use of the OFT in rodent research (e.g., inconsistent effects of anxiolytic drugs; see Rosso et al., 2022, Neurosci Biobehav Rev., for a meta-analysis). These should be explained to place the Drosophila findings in their appropriate context.

    1. Reviewer #2 (Public review):

      Summary

      Roiuk et al describe a work in which they have investigated the role of eIF2A in translation initiation in mammals without much success. Thus, the manuscript focuses on negative results. Further, the results, while original, are generally not novel, but confirmatory, since related claims have been made before independently in different systems with Haikwad et al study recently published in eLife being the most relevant.

      Despite this, we find this work highly important. This is because of a massive wealth of unreliable information and speculations regarding eIF2A role in translation arising from series of artifacts that began at the moment of eIF2A discovery. This, in combination with its misfortunate naming (eIF2A is often mixed up with alpha subunit of eIF2, eIF2S1) has generated a widespread confusion among researchers who are not experts in eukaryotic translation initiation. Given this, it is not only justifiable but critical to make independent efforts to clear up this confusion and I very much appreciate the authors' efforts in this regard.

      Strengths

      The experimental investigation described in this manuscript is thorough, appropriate and convincing.

      Weaknesses

      No major weaknesses as the authors have improved their presentation.

    1. Reviewer #2 (Public review):

      Sourmpis et al. present a study in which the importance of including certain inductive biases in the fitting of recurrent networks is evaluated with respect to the generalization ability of the networks when exposed to untrained perturbations.

      The work proceeds in three stages:<br /> (1) a simple illustration of the problem is made. Two reference (ground-truth) networks with qualitatively different connectivity, but similar observable network dynamics, are constructed, and recurrent networks with varying aspects of design similarity to the reference networks are trained to reproduce the reference dynamics. The activity of these trained networks during untrained perturbations is then compared to the activity of the perturbed reference networks. It is shown that, of the design characteristics that were varied, the enforced sign (Dale's law) and locality (spatial extent) of efference were especially important.<br /> (2) The intuition from the constructed example is then extended to networks that have been trained to reproduce certain aspects of multi-region neural activity recorded from mice during a detection task with a working-memory component. A similar pattern is demonstrated, in which enforcing the sign and locality of efference in the fitted networks has an influence on the ability of the trained networks to predict aspects of neural activity during unseen (untrained) perturbations.<br /> (3) The authors then illustrate the relationship between the gradient of the motor readout of trained networks with respect to the net inputs to the network units, and the sensitivity of the motor readout to small perturbations of the input currents to the units, which (in vivo) could be controlled optogenetically. The paper is concluded with a proposed use for trained networks, in which the models could be analyzed to determine the most sensitive directions of the network and, during online monitoring, inform a targeted optogenetic perturbation to bias behavior.

      The authors do not overstate their claims, and in general, I find that I agree with their conclusions. A couple of points to be made:

      (1) Some aspects of the methods are unclear. For comparisons between recurrent networks trained from randomly initialized weights, I would expect that many initializations were made for each model variant to be compared, and that the performance characteristics are constructed by aggregating over networks trained from multiple random initializations. I could not tell from the methods whether this was done or how many models were aggregated.

      2) It is possible that including perturbation trials in the training sets would improve model performance across conditions, including held-out (untrained) perturbations (for instance, to units that had not been perturbed during training). It could be noted that if perturbations are available, their use may alleviate some of the design decisions that are evaluated here.

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript presents valuable new datasets from two ancient maize seeds that contribute to our growing understanding of the maize evolution and biodiversity landscape in pre-colonial South America. Some of the analyses are robust, but the selection elements are not supported.

      Strengths:

      The data collection is robust, and the data appear to beof sufficiently high quality to carry out some interesting analytical procedures. The central finding that aBM maize is closely related to maize from the core Inca region is well supported, although the directionality of dispersal is not supported.

      Weaknesses:

      The selection results are not justified, see examples in the detailed comments below.

      (1) The manuscript mentions cultural and natural selection (line 76), but then only gives a couple of examples of selecting for culinary/use traits. There are many examples of selection to tolerate diverse environments that could be relevant for this discussion, if desired.

      (2) I would be extremely cautious about interpreting the observations of a Spanish colonizer (lines 95-99) without very significant caveats. Indigenous agriculture and foodways would have been far more nuanced than what could be captured in this context, and the genocidal activities of the Europeans would have impacted food production activities to a degree, and any contemporaneous accounts need to be understood through that lens.

      (3) The f3 stats presented in Figure 2 are not set up to test any specific admixture scenarios, so it is unsupported to conclude that the aBM maize is not admixed on this basis (lines 201-202). The original f3 publication (Patterson et al, 2012) describes some scenarios where f3 characteristics associate with admixture, but in general, there are many caveats to this approach, and it's not the ideal tool for admixture testing, compared with e.g., f4 and D (abba-baba) statistics.

      (4) I'm a little bit skeptical that the Locator method adds value here, given the small training sample size and the wide geographic spread and genetic diversity of the ancient samples that include Central America. The paper describing that method (Battey et al 2020 eLife) uses much larger datasets, and while the authors do not specifically advise on sample sizes, they caution about small sample size issues. We have already seen that the ancient Peruvian maize has the most shared drift with aBM maize on the basis of the f3 stats, and the Locator analysis seems to just be reiterating that. I would advise against putting any additional weight on the Locator results as far as geographic origins, and personally I would skip this analysis in this case.

      (5) The overlap in PCA should not be used to confirm that aBM is authentically ancient, because with proper data handling, PCA placement should be agnostic to modern/ancient status (see lines 224-226). It is somewhat unexpected that the ancient Tehuacan maize (with a major teosinte genomic component) falls near the ancient South American maize, but this could be an artifact of sampling throughout the PCA and the lack of teosinte samples that might attract that individual.

      (6) What has been established (lines 250-251) is genetic similarity to the Inca core area, not necessarily the directionality. Might aBM have been part of a cultural region supplying maize to the Inca core region, for example? Without a specific test of dispersal directionality, which I don't think is possible with the data at hand, this is somewhat speculative.

      (7) Singleton SNPs are not a typical criterion for identifying selection; this method needs some citations supporting the exact approach and validation against neutral expectations (line 278). Without Datasets S2 and S3, which are not included with this submission, it is difficult to assess this result further. However, it is very unexpected that ~18,000 out of ~49,000 SNPs would be unique to the aBM lineage. This most likely reflects some data artifact (unaccounted damage, paralogs not treated for high coverage, which are extremely prevalent in maize, etc). I'm confused about unique SNPs in this context. How can they be unique to the aBM lineage if the SNPs used overlap the Grzybowski set? The GO results do not include any details of the exact method used or a statistical assessment of the results. It is not clear if the GO terms noted are statistically enriched.

      (8) The use of XP-EHH with pseudohaplotype variant calls is not viable (line 293). It is not clear what exact implementation of XP-EHH was used, but this method generally relies on phased or sometimes unphased diploid genotype calls to observe shared haplotypes, and some minimum population size to derive statistical power. No implementation of XP-EHH to my knowledge is appropriate for application to this kind of dataset.

    1. Reviewer #2 (Public review):

      Summary:

      In this article, Assimopoulos et al. expand the FSL-XTRACT software to include new protocols for identifying cortical-subcortical tracts with diffusion MRI, with a focus on tracts connecting to the amygdala and striatum. They show that the amygdalofugal pathway and divisions of the striatal bundle/external capsule can be successfully reconstructed in both macaques and humans while preserving large-scale topographic features previously defined in tract tracing studies. The authors set out to create an automated subcortical tractography protocol, and they accomplished this for a subset of specific subcortical connections for users of the FSL ecosystem.

      Strengths:

      A main strength of the current study is the translation of established anatomical knowledge to a tractography protocol for delineating cortical-subcortical tracts that are difficult to reconstruct. Diffusion MRI-based tractography is highly prone to false positives; thus, constraining tractography outputs by known anatomical priors is important. Key additional strengths include 1) the creation of a protocol that can be applied to both macaque and human data; 2) demonstration that the protocol can be applied to be high quality data (3 shells, > 250 directions, 1.25 mm isotropic, 55 minutes) and lower quality data (2 shells, 100 directions, 2 mm isotropic, 6.5 minutes); and 3) validation that the anatomy of cortical-subcortical tracts derived from the new method are more similar in monozygotic twins than in siblings and unrelated individuals.

      Weaknesses:

      Although this work validates the general organizational location and topographic organization of tractography-derived cortical-subcortical tracts against prior tract tracing studies (a clear strength), the validation is purely visual and thus only qualitative. Furthermore, it is difficult to assess how the current XTRACT method may compare to currently available tractography approaches to delineating similar cortical-subcortical connections. Finally, it appears that the cortical-subcortical tractography protocols developed here can only be used via FSL-XTRACT (yet not with other dMRI software), somewhat limiting the overall accessibility of the method.

      Overall Appraisal:

      This new method will accelerate research on anatomically validated cortical-subcortical white matter pathways. The work has utility for diffusion MRI researchers across fields.

  4. drive.google.com drive.google.com
    1. l _ou mu~ tell_fil1yone/' my mother said, "what Iam about to tell you. In China your father had a sister whokilled herself. She jumped into the family well. We say thatyour father has all brothers because it is as if she had neverbeen born.

      This sounds like a story, because it is too fake in real life.

    1. Reviewer #2 (Public review):

      The authors have revised their manuscript in response to reviewer feedback, incorporating several modifications to improve clarity and provide additional supporting information. To address concerns about confusing terminology, they have standardized the reference to PRDM16 overexpressing cells as Prdm16_OE, clarifying its expression from a constitutive promoter. They also revised the text to resolve seemingly contradictory statements about ChP development in the mutant. New bioinformatic analysis comparing PRDM16 binding in E12.5 ChP cells to co-repressed versus BMP-only-repressed genes has been performed and included in Supplementary Figure 5C, providing a statistical assessment of PRDM16's regulatory role on co-repressed genes. Several figures were updated, including adding an illustration of the Prdm16 cGT allele to Figure 1B, providing a zoomed-in inset for Figure 1E, and including individual channels for Wnt2b and marking boundaries in Figure 7A. Full-view images and examples of spot segmentation for SCRINSHOT analysis are now available in a new supplementary figure, and the presentation of RT-qPCR data in Supplementary Figure 2B was improved by using separate graphs for overexpression samples to avoid a broken Y-axis. Furthermore, the authors have added more references to introductory statements, annotated structures like the ChP, CH, and fourth ventricle in figures, and clarified that the beta-Gal signal was used as a marker for mutant ChP cells in Figure 1D. Finally, the manuscript now includes a discussion of the recently published, related study by Hurwitz et al. (2023) in the discussion section, highlighting similarities and differences. Overall, the authors have satisfactorily addressed the reviewers' comments.

    1. Reviewer #2 (Public review):

      The revised manuscript by Genzoni et al. reports the striking discovery of a regulatory role for trophic eggs. Prior to this study, trophic eggs were widely assumed to play a nutritional role in the colony, but this study shows that trophic eggs can suppress queen development, and therefore, can play a role in regulating caste determination in specific social contexts. In this revised version of the manuscript, the authors have addressed many of the concerns raised in the first version regarding the lack of sufficient information and context in the Introduction and Discussion.

    1. Reviewer #3 (Public review):

      Zhao et al. provide new insights into the mechanism by which a high-fat diet (HFD) induces cardiac arrhythmia employing Drosophila as a model. HFD induces cardiac arrhythmia in both mammals and Drosophila. Both glucagon and its functional equivalent in Drosophila Akh are known to induce arrhythmia. The study demonstrates that Akh mRNA levels are increased by HFD and both Akh and its receptor are necessary for high-fat diet-induced cardiac arrhythmia, elucidating a novel link. Notably, Zhao et al. identify a pair of AKH receptor-expressing neurons located at the posterior of the heart tube. Interestingly, these neurons innervate the heart muscle and form synaptic connections, implying their roles in controlling the heart muscle. The study presented by Zhao et al. is intriguing, and the rigorous characterization of the AKH receptor-expressing neurons would significantly enhance our understanding of the molecular mechanism underlying HFD-induced cardiac arrhythmia.

      Many experiments presented in the manuscript are appropriate for supporting the conclusions while additional controls and precise quantifications should help strengthen the authors' arguments. The key results obtained by loss of Akh (or AkhR) and genetic elimination of the identified AkhR-expressing cardiac neurons do not reconcile, complicating the overall interpretation.

      The most exciting result is the identification of AkhR-expressing neurons located at the posterior part of the heart tube (ACNs). The authors attempted to determine the function of ACNs by expressing rpr with AkhR-GAL4, which would induce cell death in all AkhR-expressing cells, including ACNs. The experiments presented in Figure 6 are not straightforward to interpret. Moreover, the conclusion contradicts the main hypothesis that elevated Akh is the basis of HFD-induced arrhythmia. The results suggest the importance of AkhR-expressing cells for normal heartbeat. However, elimination of Akh or AkhR restores normal rhythm in HFD-fed animals, suggesting that Akh and AkhR are not important for maintaining normal rhythms. If Akh signaling in ACNs is key for HFD-induced arrhythmia, genetic elimination of ACNs should unalter rhythm and rescue the HFD-induced arrhythmia. An important caveat is that the experiments do not test the specific role of ACNs. ACNs should be just a small part of the cells expressing AkhR. Specific manipulation of ACNs will significantly improve the study. Moreover, the main hypothesis suggests that HFD may alter the activity of ACNs in a manner dependent on Akh and AkhR. Testing how HFD changes calcium, possibly by CaLexA (Figure 2) and/or GCaMP, in wild-type and AkhR mutant could be a way to connect ACNs to HFD-induced arrhythmia. Moreover, optogenetic manipulation of ACNs may allow for specific manipulation of ACNs.

      Interestingly, expressing rpr with AkhR-GAL4 was insufficient to eliminate both ACNs. It is not clear why it didn't eliminate both ACNs. Given the incomplete penetrance, appropriate quantifications should be helpful. Additionally, the impact on other AhkR-expressing cells should be assessed. Adding more copies of UAS-rpr, AkhR-GAL4, or both may eliminate all ACNs and other AkhR-expressing cells. The authors could also try UAS-hid instead of UAS-rpr.

    1. Reviewer #2 (Public review):

      This study investigates the role of arginase-II (Arg-II) in cardiac aging. The authors challenge previous assumptions by demonstrating that Arg-II is not expressed in aged cardiomyocytes, but is upregulated in non-myocyte cells, specifically macrophages, fibroblasts, and endothelial cells. Using Arg-II knockout mice, they show protection against age-associated cardiac inflammation, fibrosis, apoptosis, endothelial-to-mesenchymal transition (EndMT), and ischemic injury. Mechanistically, Arg-II promotes IL-1β release from macrophages and increases mitochondrial ROS in fibroblasts, contributing to cardiac aging through both cell-autonomous and non-cell-autonomous mechanisms.

      The study is well-structured and combines genetic models, molecular assays, and histological analyses to support its conclusions. Including both human and mouse samples strengthens the translational relevance of the findings. The authors have addressed most of the reviewers' comments and have made efforts to improve the manuscript by adding experimental data, explanations, and further discussion.

      The data convincingly support their conclusions. This work provides valuable insights into the mechanisms of cardiac aging, aligns with growing evidence of non-cell-autonomous contributions to aging-related pathologies, and highlights the importance of intercellular signaling in maintaining cardiac health during aging.

      Although the use of cell-specific knockout mouse models would enhance the depth and translational potential of the findings, it is understandable that such an approach would be beyond the scope of a single study. This work lays the groundwork for future investigations into conditional Arg-II knockouts in specific cell types to elucidate the cell-specific roles of Arg-II in cardiac aging.

      Overall, this is a solid and impactful study with strong experimental support

    1. Reviewer #2 (Public review):

      In this manuscript by Wolfson et al., various adeno-associated viruses (AAVs) were delivered to mice to assess the cardiac-specificity, injury border-zone cardiomyocyte transduction rate, and temporal dynamics, with the goal of finding better AAVs for gene therapies targeting the heart. The authors delivered tissue regeneration enhancer elements (TREEs) controlling luciferase expression and used IVIS imaging to examine transduction in the heart and other organs. They found that luciferase expression increased in the first week after injury when using AAV9-TREE-Hsp68 promoter, waning to baseline levels by 7 weeks. However, AAV9 vectors transduced the liver, which was significantly reduced by using an AAV.cc84 liver de-targeting capsid. The authors then performed in vivo screening of AAV9 capsids and found AAV-IR41 to preferentially transduce injured myocardium when compared to AAV9. Finally, the authors combined TREEs with AAV-IR41 to show improved luciferase expression compared to AAV9-TREE at 7, 14, and 21 days after injury.

      Overall, this manuscript provides insights into TREE expression dynamics when paired with various heart-targeting capsids, which can be useful for researchers studying ischemic injury of murine hearts. While the authors have shown the success of using AAV9-TREEs in porcine hearts, it is unknown whether the expression dynamics would be similar in pigs or humans, as mentioned in the limitations.

      The following questions and concerns can be addressed to improve the manuscript:

      (1) From the IVIS data, it seems that the Hsp68 promoter might not be "normally silent in mouse tissues," specifically in the liver (Figure S1B). Are there any other promoters that can be combined with TREEs to induce cardiac-injury specific expression while minimizing liver expression? This could simplify capsid design to focus on delivery to injured areas.

      (2) Why is it that AAV9-TREE-Hsp68-Luc wane in expression (Figure 1C and 1D), whereas AAV.cc84-TREE-Hsp68-Luc expresses stably for over 2 months (3E)? This has important implications for the goal of transience in gene delivery.

      (3) AAV-IR41 was found to transduce cardiomyocytes in the injured zone. However, this capsid also shows a very strong off-target liver expression. From a capsid design perspective, is it possible to combine AAV-cc84 and AAV-IR41?

      (4) It would be helpful to see immunostaining for the various time points in Figure 5. Is it possible to use an anti-luciferase antibody (or AAV-TREE-Hsp68-eGFP) to compare the two TREE capsids?

    1. Reviewer #2 (Public review):

      Summary:

      The manuscript "Independent validation of transgenerational inheritance of learned pathogen avoidance in C. elegans" by Akinosho and Vidal-Gadea offers evidence that learned avoidance of the pathogen PA14 can be inherited for at least two generations. In spite of initial preference for the pathogen when exposed in a 'training session', 24 hours of feeding on this pathogen evoked avoidance. The data are robust, replicated in 4 trials, and the authors note that diminished avoidance is inherited in generations F1 and F2.

      Strengths:

      These results contrast with those reported by Gainey et al, who only observed intergenerational inheritance for a single generation. Although the authors' study does not explain why Gainey et el fail to reproduce the Murphy lab results, one possibility is that a difference in a media ingredient could be responsible.

      Weaknesses:

      The authors do not list the sources of their media ingredients, which might be important with regard to reproducibility.

    1. Reviewer #2 (Public review):

      Summary:

      This study presents a useful finding that the high susceptibility to CLP sepsis of Kit-mutant mice is not due to mast cell deficiency, but to dysbiosis.

      However, the present data are insufficient and incomplete to support the conclusion, and would benefit from more rigorous approaches. With the mechanism part strengthened, this paper would be of interest to researchers on mast cell biology and mucosal immunology.

      Recommendations:

      (1) The authors showed that E. coli increases in the cecum of Kit-mutant mice, which causes high CLP susceptibility. However, they did not provide any evidence E. coli is responsible for the high susceptibility. In the Figure 3 experiments, the authors administered the same number of cecal bacteria and did not show the number of E. coli after the administration. The authors should provide evidence showing that depletion of E. coli decreases susceptibility.

      (2) The author should provide direct evidence of dysbiosis by, for example, shotgun sequencing of cecal and fecal contents.

      (3) In case the authors find dysbiosis, they should analyze the mechanisms by which Kit mutation causes dysbiosis.

    1. Reviewer #2 (Public review):

      The major conclusion of the manuscript is expressed in the title: "NR2F2 is required in the embryonic testis for Fetal Leydig Cell development" and also at the end of the introduction and all along the result part. All the authors' assertions are supported by very clear and statistically validated results from ISH, IHC, precise cell counting and gene expression levels by qPCR. The authors used two different conditional Nr2f2 gene ablation systems that demonstrate the same effects at the FLC level. They also showed that the haplo-insufficiency of Wt1 in the first system (knock-in Wt1-cre-ERT2) aggravated the situation in FLC differentiation by disturbing the differentiation of Sertoli cells and their secretion of pro-FLC factors, which had a confounding effect and encouraged them to use the second system. This demonstrates the great rigor with which the authors interpreted the results. In conclusion, all authors' claims and conclusions are justified by their high-quality results.

      Comments on revised version:

      In their revised version, the authors have taken full account of all my suggestions, and I congratulate them on this. I have no further comments to make on this new version.