Reviewer #3 (Public review):

Summary:

In this paper, Rethemeier et al capitalize on their previous observation that the beetle central complex develops heterochronically compared to the fly and try to identify the developmental origin of this difference. For this reason, they use a fez enhancer trap line that they generated to study the neuronal stem cells (INPs) that give rise to the central complex. Using this line and staining against Drosophila type-II neuroblast markers, they elegantly dissect the number of developmental progression of the beetle type II neuroblasts. They show that the NBs, INPs, and GMCs have a conserved marker progression by comparing to Drosophila marker genes, although the expression of some of the lineage markers (otd, six3, and six4) is slightly different. Finally, they show that the beetle type II neuroblasts lineages are likely longer than the equivalent ones in Drosophila and argue that this might be the underlying reason for the observed heterochrony.

Strengths:

- Very interesting study system that compares a conserved structure that, however, develops in a heterochronic manner.<br /> - Identification of a conserved molecular signature of type-II neuroblasts between beetles and flies. At the same time, identification of transcription factors expression differences in the neuroblasts, as well as identification of an extra neuroblast.<br /> - Nice detailed experiments to describe the expression of conserved and divergent marker genes, including some lineaging looking into co-expression of progenitor (fez) and neuronal (skh) markers.

Weaknesses:

- The link between size and number of neuroblast lineages and the earlier central complex development in beetles is not examined.

Reviewer #3 (Public review):

The authors aim to understand how gene pleiotropy affects parallel evolutionary changes among independent replicates of adaptation to a new hot environment of a set of experimental lines of Drosophila simulans using experimental evolution. The flies were RNAsequenced after more than 100 generations of lab adaptation and the changes in average gene expression were obtained relative to ancestral expression levels from reconstructed ancestral lines. Parallelism of gene expression change among lines is evaluated as variance in differential gene expression among lines relative to error variance. Similarly, the authors ask how the standing variation in gene expression estimated from a handful of flies from a reconstructed outbred line affects parallelism. The main findings are that parallelism in gene expression responses is positively associated with pleiotropy and negatively associated with expression variation. Those results are in contradiction with theoretical predictions and empirical findings. To explain those seemingly contradictory results the authors invoke the role of synergistic pleiotropy and correlated selection, although they do not attempt to measure either.

Strengths:

The study uses highly replicated outbred laboratory lines of Drosophila simulans evolved in the lab under constant hot regime for over 100 generations. This allows for robust comparisons of evolutionary responses among lines.

The manuscript is well written and the hypotheses are clearly delineated at the onset.

The authors have run a causal analysis to understand the causal dependencies between pleiotropy and expression variation on parallelism.

The use of whole-body RNA extraction to study gene expression variation is well justified.

Weaknesses:

The accuracy of the estimate of ancestral phenotypic variation in gene expression is likely low because estimated from a small sample of 20 males from a reconstructed outbred line. It might not constitute a robust estimate of the genetic variation of the evolved lines under study.

There are no estimates of the standing genetic variation of expression levels of the genes under study, only estimates of their phenotypic variation. I wished the authors had been clear about that limitation and had refrained from equating phenotypic variation in expression level with standing genetic variation.

Moreover, since the phenotype studied is gene expression, its genetic basis extends beyond expressed sequences. The phenotypic variation of a gene's expression may thus likely misrepresent the genetic variation available for its evolution. The authors do not present evidence that sequence variation correlates with expression variation.

The authors have not attempted to estimate synergistic pleiotropy among genes, nor how selection acts on gene expression modules. It makes their conclusion regarding the role of synergistic pleiotropy rather speculative.

Reviewer #3 (Public review):

The authors apply multivoxel decoding analyses from fMRI during reward feedback about the cues previously chosen that led to that feedback. They compare two versions of the task - one in which the feedback is provided about the current trial, and one in which the feedback is provided about the previous trial. Reward probability changes slowly over time, so subjects need to identify which cues are leading to reward at a given time. They find that evidence for recall of the cue in lateral orbitofrontal cortex (lOFC) and hippocampus (HC). They also find that in the second condition, where feedback is for the one-back trial, this representation is mediated by the lateral frontal pole (FPl).

Overall, the analyses are clean and elegant and seem to be complete. I have only a few comments, all of which can be public.

(1) They do find (not surprisingly) that the one-back task is harder. It would be good to ensure that the reason that they had more trouble detecting direct HC & lOFC effects on the harder task was not because the task is harder and thus that there are more learning failures on the harder one-back task. (I suspect their explanation that it is mediated by FPl is likely to be correct. But it would be nice to do some subsampling of the zero-back task [matched to the success rate of the one-back task] to ensure that they still see the direct HC and lOFC there.)

(2) The evidence that they present in the main text (Figure 3) that the HC and lOFC are mediated by FPl is a correlation. I found the evidence presented in Supplemental Figure 7 to be much more convincing. As I understand it, what they are showing in SF7 is that when FPl decodes the cue, then (and only then) HC and lOFC decode the cue. If my understanding is correct, then this is a much cleaner explanation for what is going on than the secondary correlation analysis. If my understanding here is incorrect, then they should provide a better explanation of what is going on so as to not confuse the reader.

(3) I like the idea of "credit spreading" across trials (Figure 1E). I think that credit spreading in each direction (into the past [lower left] and into the future [upper right]) is not equivalent. This can be seen in Figure 1D, where the two tasks show credit spreading differently. I think a lot more could be studied here. Does credit spreading in each of these directions decode in interesting ways in different places in the brain?

Comments on revisions:

After revision, I have no additional comments.

Reviewer #3 (Public review):

Summary:

The authors applied an innovative approach (CO-Detection by indEXing - CODEX) together with sophisticated computational analyses to image pancreas tissues from rare organ donors with type 1 diabetes. They aimed to assess key features of inflammation in both islet and extra-islet tissue areas; they report that the extra-islet space of lobules with extensive islet infiltration differs from the extra-islet space of less infiltrated areas within the same tissue section. The study also identifies four sub-states of inflamed islets characterized by the activation profiles of CD8+T cells enriched in islets relative to the surrounding tissue. Lymphoid structures are identified in the pancreas tissue away from islets, and these were enriched in CD45RA+ T cells - a population also enriched in one of the inflamed islet sub-states. Together, these data help define the coordination between islets and the extra-islet pancreas in the pathogenesis of human T1D.

Strengths:

The analysis of tissue from well-characterized organ donors, provided by the Network for the Pancreatic Organ Donor with Diabetes, adds strength to the validity of the findings.

By using their innovative imaging/computation approaches, key known features of islet autoimmunity were confirmed, providing validation of the methodology.

The detection of IDO+ vasculature in inflamed islets - but not in normal islets or islets that have lost insulin-expression links this expression to the islet inflammation, and it is a novel observation. IDO expression in the vasculature may be induced by inflammation and may lost as disease progresses, and it may provide a potential therapeutic avenue.

The high-dimensional spatial phenotyping of CD8+T cells in T1D islets confirmed that most T cells were antigen experienced. Some additional subsets were noted: a small population of T cells expressing CD45RA and CD69, possibly naive or TEMRA cells, and cells expressing Lag-3, Granzyme-B, and ICOS.

While much attention has been devoted to the study of the insulitis lesion in T1D, our current knowledge is quite limited; the description of four sub-clusters characterized by the<br /> activation profile of the islet-infiltrating CD8+T cells is novel. Their presence in all T1D donors, indicates that the disease process is asynchronous and is not at the same stage across all islets. Although this concept is not novel, this appears to be the most advanced characterization of insulitis stages.

When examining together both the exocrine and islet areas, which is rarely done, authors report that pancreatic lobules affected by insulitis are characterized by distinct tissue markers. Their data support the concept that disease progression may require crosstalk between cells in the islet and extra-islet compartments. Lobules enriched in β-cell-depleted islets were also enriched in nerves, vasculature, and Granzyme-B+/CD3- cells, which may be natural killer cells.

Lastly, authors report that immature tertiary lymphoid structures (TLS) exist both near and away from islets, where CD45RA+ CD8+T cells aggregate, and also observed an inflamed islet-subcluster characterized by an abundance of CD45RA+/CD8+ T cells. These TLS may represent a point of entry for T cells and this study further supports their role in islet autoimmunity.

Weaknesses:

As the author themselves acknowledge, the major limitation is that the number of donors examined is limited as those satisfying study criteria are rare. Thus, it is not possible to examine disease heterogeneity, and the impact of age at diagnosis. Of 8 T1D donors examined, 4 would be considered newly diagnosed (less than 3 months from onset) and 4 had longer disease durations (2, 2, 5 and 6 years). It was unclear if disease duration impacted the results in this small cohort. In the introduction, the authors discuss that most of the pancreata from nPOD donors with T1D lack insulitis. This is correct, yet it is a function of time from diagnosis. Donors with shorter duration will be more likely to have insulitis. A related point is that the proportion of islets with insulitis is low even near diagnosis, Finally, only one donor was examined that while not diagnosed with T1D, was likely in the preclinical disease stage and had autoantibodies and insulitis. This is a critically important disease stage where the methodology developed by the investigators could be applied in future efforts.

While this was not the focus of this investigation, it appears that the approach was very much immune-focused and there could be value in examining islet cells in greater depth using the methodology the authors developed.

Additional comments

Overall, the authors were able to study pancreas tissues from T1D donors and perform sophisticated imaging and computational analysis that reproduce and importantly extend our understanding of inflammation in T1D. Despite the limitations associated with the small sample size, the results appear robust, and the claims are well supported.

The study expands the conceptual framework of inflammation and islet autoimmunity, especially by the definition of different clusters (stages) of insulitis and by the characterization of immune cells in and outside the islets.

Comments on revisions:

I have not felt the need to update the initial review.

However, I note that the paragraph describing the nPOD repository (lines 154-158) can be misinterpreted that insulitis is infrequent in T1D (17 of 200 donors had it) without the clarification that insulitis is present around the time of diagnosis in most patients and it subsides over time. Thus, authors are urged to clarify that the presence of insulitis and its severity are impacted by the disease stage and disease duration.

The last sentence of this paragraph, lines 164-165, although linked to the previous sentence about the cause of death in the donors, may be misconstrued in the context of this paragraph, and it is unclear what data support this statement. Please delete this sentence.

Reviewer #3 (Public review):

Summary:

Ruan and colleagues consider a branching process model (in their terminology the "Haldane model") and the most basic Wright-Fisher model. They convincingly show that offspring distributions are usually non-Poissonian (as opposed to what's assumed in the Wright-Fisher model), and can depend on short-term ecological dynamics (e.g., variance in offspring number may be smaller during exponential growth). The authors discuss branching processes and the Wright-Fisher model in the context of 3 "paradoxes" --- 1) how Ne depends on N might depend on population dynamics; 2) how Ne is different on the X chromosome, the Y chromosome, and the autosomes, and these differences do match the expectations base on simple counts of the number of chromosomes in the populations; 3) how genetic drift interacts with selection. The authors provide some theoretical explanations for the role of variance in the offspring distribution in each of these three paradoxes. They also perform some experiments to directly measure the variance in offspring number, as well as perform some analyses of published data.

Strengths:

- The theoretical results are well-described and easy to follow.<br /> - The analyses of different variances in offspring number (both experimentally and analyzing public data) are convincing that non-Poissonian offspring distributions are the norm.<br /> - The point that this variance can change as the population size (or population dynamics) change is also very interesting and important to keep in mind.<br /> - I enjoyed the Density-Dependent Haldane model. It was a nice example of the decoupling of census size and effective size.<br /> - Equation (10) is a nice result

Comments on revisions:

I appreciate the effort that the authors have put into the revision, but I still find the framing to be a bit confusing -- these apparent paradoxes only appear in the most basic version of Wright-Fisher models, and so framing the paper as the solution to these paradoxes overlooks much previous work. Saying that existing work discussing exactly these phenomena is "beyond the scope of this study", without citing or interacting in any way with that work is unscholarly. I agree with the authors that the apparent paradoxes that they consider and interesting, and by thinking about branching processes, the apparent paradoxes appear to be less paradoxical, but without contextualizing this work in the substantial Wright-Fisher literature (e.g., Cannings Exchangeable Models and the work of Möhle) it misrepresents the state of the field and the contributions of this paper.

Reviewer #3 (Public review):

Summary:

Notch is active in HCC, but generally not mutated. The authors use a JAG1-selective blocking antibody in a large panel of liver cancer patient-derived xenograft models. They find JAG-dependent HCCs, and these are aggressive and proliferative. Notch inhibition induces cycle arrest and promotes hepatocyte differentiation, through upregulation of CEBPA expression and activation of existing HNF4A, mimicking normal developmental programs.

The authors use aJ1.b70, a potent and selective therapeutic antibody that inhibits JAG1 against PDX models. They tested over 40 PDX models and found a handful of super-responders to single-agent inhibition. In LIV78 and Li1035 cancer cells, NOTCH2 was expressed and required, in contrast to NOTCH1. RNA-seq showed that the responsive HCCs resembled the S2 transcriptional class of HCCs, which were enriched for Notch-dependent models. They conclude that these dependent tumors have transcriptomes that resemble a hybrid progenitor cell expressing FGF9 and GAS7. Inhibition was able to induce hepatocyte differentiation away from a NOTCH-driven progenitor program. scRNA-seq analysis showed a large population of NOTCH-JAG expressing cells but also showed that there are cells that did not. Not surprisingly, NOTCH2 inhibition leads to increased CEBPA and HNF4A transcriptional activity, which are standard TFs in hepatocytes.

Strengths:

The paper provides useful information about the frequency of HCCs and CCA that respond to NOTCH inhibition and could allow us to anticipate the super-responder rate if these antibodies were actually used in the clinic. The inhibitor tools are highly specific, and provide useful information about NOTCH activities in liver cancers. The large number of PDXs and the careful transcriptomic analyses were positives about the study.

Weaknesses:

The paper is mostly descriptive.

Reviewer #3 (Public review):

In this study, Cao et al. explore the neural mechanisms by which chronic heat exposure induces negative valence and hyperarousal in mice, focusing on the role of the posterior paraventricular nucleus (pPVT) neurons that receive projections from the preoptic area (POA). The authors show that chronic heat exposure leads to heightened activity of the POA projection-receiving pPVT neurons, potentially contributing to behavioral changes such as increased anxiety level and reduced sociability, along with heightened startle responses. In addition, using electrophysiological methods, the authors suggest that increased membrane excitability of pPVT neurons may underlie these behavioral changes. The use of a variety of behavioral assays enhances the robustness of their claim. Moreover, while previous research on thermoregulation has predominantly focused on physiological responses to thermal stress, this study adds a unique and valuable perspective by exploring how thermal stress impacts affective states and behaviors, thereby broadening the field of thermoregulation.

While the manuscript has been revised and some efforts have been made to address the reviewers' concerns, the majority of the issues raised remain insufficiently resolved. Therefore, the reviewer has highlighted key major points that the authors should address to strengthen the manuscript's conclusions.

Major points<br /> The manuscript highlights the increased activity in pPVT neurons receiving projections from the POA (Figure 3) and shows that these neurons are necessary for heat-induced behavioral changes (Figures 4N-W). However, it remains unclear whether the POA-to-pPVT projection itself plays a critical role. Since pPVT recipient neurons can receive inputs from various brain regions, the role of the POA input in driving these effects needs to be validated more explicitly.<br /> (1) To establish this, the authors should conduct experiments directly inhibiting the POA-to-pPVT projection and demonstrate whether the increased activity in pPVT neurons due to chronic heat exposure is abolished when the POA is blocked.<br /> (2) Alternatively, the authors could use anterograde labeling from the POA and specifically target recipient neurons in the pPVT to confirm that the observed excitatory inputs originate from the POA (related to Figure 6).<br /> (3) If these experiments are not feasible, the authors should consider toning down the emphasis on the POA's role throughout the manuscript and discussing this limitation explicitly. The term "POA recipient pPVT neurons" should be used consistently to avoid misleading implications that the POA-to-pPVT excitatory projection is definitively established as the key pathway.<br /> a) For example, in lines 368-369, the phrase "The increase in presynaptic excitability of the POA to pPVT excitatory pathway" represents a logical jump, as the data only support the "differential increase in presynaptic excitability of the excitatory pathway" (as described in lines 358-359) without specifically confirming the POA-to-pPVT pathway.<br /> b) Similarly, in lines 442-446, the statement "the role of excitatory projections from POA to pPVT in chronic heat exposure-induced emotional changes" should be revised to "the role of excitatory projection recipient pPVT in chronic heat~," as the data do not provide direct evidence that heat-responsive POA neurons projecting to pPVT mediate these effects. Such revisions would improve clarity and ensure that the conclusions remain aligned with the presented data.

Reviewer #3 (Public review):

Summary:

The authors used cTBS TMS, magnetic resonance spectroscopy (MRS), and functional magnetic resonance imaging (fMRI) as the main methods of investigation. Their data show that cTBS modulates GABA concentration and task-dependent BOLD in the ATL, whereby greater GABA increase following ATL cTBS showed greater reductions in BOLD changes in ATL. This effect was also reflected in the performance of the behavioural task response times, which did not subsume to practice effects after AL cTBS as opposed to the associated control site and control task. This is in line with their first hypothesis. The data further indicates that regional GABA concentrations in the ATL play a crucial role in semantic memory because individuals with higher (but not excessive) GABA concentrations in the ATLs performed better on the semantic task. This is in line with their second prediction. Finally, the authors conducted additional analyses to explore the mechanistic link between ATL inhibitory GABAergic action and semantic task performance. They show that this link is best captured by an inverted U-shaped function as a result of a quadratic linear regression model. Fitting this model to their data indicates that increasing GABA levels led to better task performance as long as they were not excessively low or excessively high. This was first tested as a relationship between GABA levels in the ATL and semantic task performance; then the same analyses were performed on the pre and post-cTBS TMS stimulation data, showing the same pattern. These results are in line with the conclusions of the authors.

Comments on revisions:

The authors have comprehensively addressed my comments from the first round of review, and I consider most of their answers and the steps they have taken satisfactorily. Their insights prompted me to reflect further on my own knowledge and thinking regarding the ATL function.

I do, however, have an additional and hopefully constructive comment regarding the point made about the study focusing on the left instead of bilateral ATL. I appreciate the methodological complexities and the pragmatic reasons underlying this decision. Nevertheless, briefly incorporating the justification for this decision into the manuscript would have been beneficial for clarity and completeness. The presented argument follows an interesting logic; however, despite strong previous evidence supporting it, the approach remains based on an assumption. Given that the authors now provide the group-level fMRI results captured more comprehensively in Supplementary Figure 2, where the bilateral pattern of fMRI activation can be observed in the current data, the authors could have strengthened their argument by asserting that the activation related to the given semantic association task in this data was bilateral. This would imply that the TMS effects and associated changes in GABA should be similar for both sites. Furthermore, it is worth noting the approach taken by Pobric et al. (2007, PNAS), who stimulated a site located 10 mm posterior to the tip of the left temporal pole along the middle temporal gyrus (MTG) and not the bilateral ATL.

Reviewer #3 (Public review):

A bias in how people infer the amount of control they have over their environment is widely believed to be a key component of several mental illnesses including depression, anxiety, and addiction. Accordingly, this bias has been a major focus in computational models of those disorders. However, all of these models treat control as a unidimensional property, roughly, how strongly outcomes depend on action. This paper proposes---correctly, I think---that the intuitive notion of "control" captures multiple dimensions in the relationship between action and outcome is multi-dimensional. In particular, the authors propose that the degree to which outcome depends on how much *effort* we exert, calling this dimension the "elasticity of control". They additionally propose that this dimension (rather than the more holistic notion of controllability) may be specifically impaired in certain types of psychopathology. This idea thus has the potential to change how we think about mental disorders in a substantial way, and could even help us better understand how healthy people navigate challenging decision-making problems.

Unfortunately, my view is that neither the theoretical nor empirical aspects of the paper really deliver on that promise. In particular, most (perhaps all) of the interesting claims in the paper have weak empirical support.

Starting with theory, the elasticity idea does not truly "extend" the standard control model in the way the authors suggest. The reason is that effort is simply one dimension of action. Thus, the proposed model ultimately grounds out in how strongly our outcomes depend on our actions (as in the standard model). Contrary to the authors' claims, the elasticity of control is still a fixed property of the environment. Consistent with this, the computational model proposed here is a learning model of this fixed environmental property. The idea is still valuable, however, because it identifies a key dimension of action (namely, effort) that is particularly relevant to the notion of perceived control. Expressing the elasticity idea in this way might support a more general theoretical formulation of the idea that could be applied in other contexts. See Huys & Dayan (2009), Zorowitz, Momennejad, & Daw (2018), and Gagne & Dayan (2022) for examples of generalizable formulations of perceived control.

Turning to experiment, the authors make two key claims: (1) people infer the elasticity of control, and (2) individual differences in how people make this inference are importantly related to psychopathology.

Starting with claim 1, there are three sub-claims here; implicitly, the authors make all three. (1A) People's behavior is sensitive to differences in elasticity, (1B) people actually represent/track something like elasticity, and (1C) people do so naturally as they go about their daily lives. The results clearly support 1A. However, 1B and 1C are not supported.

Starting with 1B, the experiment cannot support the claim that people represent or track elasticity because the effort is the only dimension over which participants can engage in any meaningful decision-making (the other dimension, selecting which destination to visit, simply amounts to selecting the location where you were just told the treasure lies). Thus, any adaptive behavior will necessarily come out in a sensitivity to how outcomes depend on effort. More concretely, any model that captures the fact that you are more likely to succeed in two attempts than one will produce the observed behavior. The null models do not make this basic assumption and thus do not provide a useful comparison.

For 1C, the claim that people infer elasticity outside of the experimental task cannot be supported because the authors explicitly tell people about the two notions of control as part of the training phase: "To reinforce participants' understanding of how elasticity and controllability were manifested in each planet, [participants] were informed of the planet type they had visited after every 15 trips." (line 384).

Finally, I turn to claim 2, that individual differences in how people infer elasticity are importantly related to psychopathology. There is much to say about the decision to treat psychopathology as a unidimensional construct. However, I will keep it concrete and simply note that CCA (by design) obscures the relationship between any two variables. Thus, as suggestive as Figure 6B is, we cannot conclude that there is a strong relationship between Sense of Agency and the elasticity bias---this result is consistent with any possible relationship (even a negative one). The fact that the direct relationship between these two variables is not shown or reported leads me to infer that they do not have a significant or strong relationship in the data.

There is also a feature of the task that limits our ability to draw strong conclusions about individual differences in elasticity inference. As the authors clearly acknowledge, the task was designed "to be especially sensitive to overestimation of elasticity" (line 287). A straightforward consequence of this is that the resulting *empirical* estimate of estimation bias (i.e., the gamma_elasticity parameter) is itself biased. This immediately undermines any claim that references the directionality of the elasticity bias (e.g. in the abstract). Concretely, an undirected deficit such as slower learning of elasticity would appear as a directed overestimation bias.

When we further consider that elasticity inference is the only meaningful learning/decision-making problem in the task (argued above), the situation becomes much worse. Many general deficits in learning or decision-making would be captured by the elasticity bias parameter. Thus, a conservative interpretation of the results is simply that psychopathology is associated with impaired learning and decision-making.

Minor comments:

Showing that a model parameter correlates with the data it was fit to does not provide any new information, and cannot support claims like "a prior assumption that control is likely available was reflected in a futile investment of resources in uncontrollable environments." To make that claim, one must collect independent measures of the assumption and the investment.

Did participants always make two attempts when purchasing tickets? This seems to violate the intuitive model, in which you would sometimes succeed on the first jump. If so, why was this choice made? Relatedly, it is not clear to me after a close reading how the outcome of each trial was actually determined.

It should be noted that the model is heuristically defined and does not reflect Bayesian updating. In particular, it overestimates control by not using losses with less than 3 tickets (intuitively, the inference here depends on your beliefs about elasticity). I wonder if the forced three-ticket trials in the task might be historically related to this modeling choice.

Reviewer #3 (Public review):

Summary:

This is a retrospective analysis of 53 individuals over 26 features (12 clinical phenotypes, 12 CGM features, and 2 autocorrelation features) to examine which features were most informative in predicting percent necrotic core (%NC) as a parameter for coronary plaque vulnerability. Multiple regression analysis demonstrated a better ability to predict %NC from 3 selected CGM-derived features than 3 selected clinical phenotypes. LASSO regularization and partial least squares (PLS) with VIP scores were used to identify 4 CGM features that most contribute to the precision of %NC. Using factor analysis they identify 3 components that have CGM-related features: value (relating to the value of blood glucose), variability (relating to glucose variability), and autocorrelation (composed of the two autocorrelation features). These three groupings appeared in the 3 validation cohorts and when performing hierarchical clustering. To demonstrate how these three features change, a simulation was created to allow the user to examine these features under different conditions.

Review:

The goal of this study was to identify CGM features that relate to %NC. Through multiple feature selection methods, they arrive at 3 components: value, variability, and autocorrelation. While the feature list is highly correlated, the authors take steps to ensure feature selection is robust. There is a lack of clarity of what each component (value, variability, and autocorrelation) includes as while similar CGM indices fall within each component, there appear to be some indices that appear as relevant to value in one dataset and to variability in the validation. We are sceptical about statements of significance without documentation of p-values. While hesitations remain, the ability of these authors to find groupings of these many CGM metrics in relation to %NC is of interest. The believability of the associations is impeded by an obtuse presentation of the results with core data (i.e. correlation plots between CGM metrics and %NC) buried in the supplement while main figures contain plots of numerical estimates from models which would be more usefully presented in supplementary tables. Given the small sample size in the primary analysis, there is a lot of modeling done with parameters estimated where simpler measures would serve and be more convincing as they require less data manipulation. A major example of this is that the pairwise correlation/covariance between CGM_mean, CGM_std, and AC_var is not shown and would be much more compelling in the claim that these are independent factors. Lack of methodological detail is another challenge. For example, the time period of CGM metrics or CGM placement in the primary study in relation to the IVUS-derived measurements of coronary plaques is unclear. Are they temporally distant or proximal/ concurrent with the PCI? A patient undergoing PCI for coronary intervention would be expected to have physiological and iatrogenic glycemic disturbances that do not reflect their baseline state. This is not considered or discussed. The attempts at validation in external cohorts, Japanese, American, and Chinese are very poorly detailed. We could only find even an attempt to examine cardiovascular parameters in the Chinese data set but the outcome variables are unspecified with regard to what macrovascular events are included, their temporal relation to the CGM metrics, etc. Notably macrovascular event diagnoses are very different from the coronary plaque necrosis quantification. This could be a source of strength in the findings if carefully investigated and detailed but due to the lack of detail seems like an apples-to-oranges comparison. Finally, the simulations at the end are not relevant to the main claims of the paper and we would recommend removing them for the coherence of this manuscript.

Reviewer #3 (Public review):

Summary:

Ribosomes are generally considered homogeneous complexes with no inherent role in regulating translation. However, recent studies have found heterogeneity in the composition of ribosome accessory factors, proteins, and ribosomal RNA. Moreover, there is evidence that district ribosomal isoforms are produced at different developmental stages in Xenopus, Drosophila, and zebrafish. In Drosophila, germline-derived ribosomes have a different protein composition to those produced by somatic cell types. In zebrafish, germline vs. somatic ribosomes have been shown to incorporate distinct rRNA isoforms. However, the functional significance of ribosome heterogeneity is not known.

The manuscript by Shah et al., uses the power of the zebrafish to test the hypothesis that maternal ribosome isoforms have a distinct function relative to ribosome isoforms produced by somatic cells after the maternal-to-zygotic transition (MTZ). They confirm previous findings that all maternal rRNA are derived from the maternal-specific rRNA locus on Chromosome 4. Additionally, proteomic analysis showed that maternal and somatic ribosomes also differ in protein composition. Using ribosome tagging experiments they showed that maternally derived subunits can form functional heteroduplexes (hybrids) with somatic-derived subunits. Finally, they show that maternal-derived ribosomes continue to be expressed in germ cells where they preferentially associate with the maternally derived and germline localized nanos3 mRNA. This suggests a possible role of maternal ribosomes in germ cell-specific translational regulation.

Strengths:

The authors use the experimental power of zebrafish to test the hypothesis that maternal and somatic-derived ribosomes have distinct functions. They use state-of-the art proteomics, molecular modeling, and transgenesis techniques. For the most part, the data presented is clear and supports their conclusions.

Weaknesses:

Using pulldown experiments they show that maternal ribosomes associate with the PGC-enriched nanos3 RNA, suggesting a role for the maternal isoform in germline-specific translation. However, they acknowledge that the level of enrichment is similar to the level of maternal vs. somatic isoforms that localize to PGCs. The nanos3 mRNA is unique in that it is actively degraded in somatic cells shortly after MTZ so is never present in cells that express the somatic isoforms. Therefore, the association of nanos3 with maternal ribosomes shows that these ribosomes can associate with germline-specific RNAs, but does not provide compelling evidence for a maternal isoform-specific role in translational regulation.

Reviewer #3 (Public review):

Summary:

Dong et al. described a deep learning-based framework of antimicrobial (AMP) generator and regressor to design and rank de novo antimicrobial peptides (AMPs). For generated AMPs, they predicted their minimum inhibitory concentration (MIC) using a model that combines the Morgan fingerprint, contact map and ESM language model. For their selected AMPs based on predicted MIC, they also use a combination of antiviral peptide (AVP) prediction models to select AMPs with potential antiviral activity. They experimentally validated 3 candidates for antimicrobial activity against S. aureus, A. baumannii, E. coli, and P. aeruginosa, and their toxicity on mouse blood and three human cell lines. The authors select their most promising AMP (P076) for in vivo experiments in A. baumannii-infected mice. They finally test the antiviral activity of their 3 AMPs against viruses.

Strengths:

- The development of de novo antimicrobial peptides (AMPs) with the novelty of being bifunctional (antimicrobial and antiviral activity).

- Novel, combined approach to AMP activity prediction from their amino acid sequence.

Weaknesses:

- I missed the justification for combined antiviral and antibacterial activities. As the authors responded, less than 10% of the training data has antiviral activity. Therefore, I do not understand how the high percentage of antiviral activities was achieved. Especially reading that the antiviral filtering did not have an influence on the number of antiviral peptides obtained.

- I had difficulty in reading the story because of the use of acronyms without referring to their full name for the first time, and incomplete information annotation in figures and captions.

Reviewer #3 (Public review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The authors have provided evidences that the Cad96Ca and FGF1 RTK receptors contribute to JH signaling through CRISPR/Cas9, inducing precocious metamorphosis, although not to the same extent as absence of JH. Therefore, it still remains unclear whether these RTKs are completely required for pathway activation or only necessary for high activation levels during the last larval stage.

While the authors have included some additional data, the mechanism by which different RTKs function in transducing JH signaling in a tissue specific manner is still unclear. As the authors note in the discussion, it is possible that other RTKs may also play a role in facilitating the transduction of JH signaling.

Lastly, the study does not yet explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. Although receptor promiscuity has been suggested as a possible mechanism, future studies could explore whether activation of RTK pathways by their known ligands induces certain levels of JH transducer phosphorylation, which, in the presence of JH, could contribute to full pathway activation without the need for direct JH-RTK binding.

Reviewer #3 (Public review):

In this manuscript, Fang et al. describe a new oncogenic function of the STAMBPL1 protein in triple-negative breast cancer (TNBC). STAMBPL1 is a deubiquitinase that has been poorly studied in cancer. Previous reports identify it as a promoter of epithelial to mesenchymal transition or an inhibitor of cisplatin-induced cell death, but its participation to other cancer phenotypes has not been investigated. Fang et al. find that in cell line models of TNBC, STAMBPL1 promotes expression of the transcription factor HIF-1a and its downstream target VEGF, with the consequence of stimulating neo-angiogenesis in vitro and in vivo. Mechanistically, the authors find that this occurs via a non-enzymatic and indirect mechanism, that is by promoting the expression of GRHL3, a transcription factor that in turn binds to the HIF-1a promoter to stimulate its transcription. Interestingly, the way by which STAMPB1 promotes GRHL3 expression is by facilitating the transcriptional activity of FOXO1, a known regulator of GRHL3. Because the authors find that STAMBPL1 and FOXO1 interact, they suggest that STAMBPL1 may promote the formation of an active transcriptional complex containing FOXO1, perhaps by facilitating the recruitment of transcriptional coactivators.

In conclusion, these data position for the first time the STAMBPL1 deubiquitinase in a FOXO-GRHL3 regulatory axis for the control of VEGF expression and tumor angiogenesis.

The main weaknesses of this work are that the relevance of this molecular axis to the pathogenesis of TNBC is not clear, and it is not clearly established whether this is a regulatory pathway that occurs in hypoxic conditions or independently of oxygen levels.

Major criticisms:

(1) Both FOXO1 and GRHL3 have been previously described as tumor suppressors, with reports of FOXO1 inhibiting tumor angiogenesis. Therefore, this work describes an apparently contradictory function of these proteins in TNBC. While it is not surprising that the same genes perform divergent functions in different tumor contexts, a stronger evidence in support of the oncogenic function of these two genes should be provided to make the data more convincing.<br /> To strengthen the notion that STAMBPL1, FOXO and GRHL3 are overexpressed in TNBC, the authors have utilized the BCIP tool to analyze their expression in the Metabric database. According to this analysis, the levels of STAMBPL1and GRHL3 are not higher in breast cancer than in adjacent tissues, and the levels of FOXO1 are lower. Nonetheless, the authors observe that their expression levels are significantly (yet not dramatically) higher in TNBC compared to non-TNBC (Fig.S6A-C). However, these new data do not provide convincing evidence of the relevant tumor suppressive function of these genes in TNBC, as neither is more expressed in tumors compared to adjacent normal tissues.

(2) Because STAMBPL1 overexpression in normoxic conditions is sufficient to cause HIF-1a protein accumulation, it is not clear why the authors then use hypoxic conditions to analyze the effect of STAMBPL1 on HIF-1a transcription Avoiding HIF1-a protein degradation should not have any effect on its transcription. At the same time, it is not clear nor is being explained why different hypoxic conditions are sometimes used, resulting in different mRNA levels of HIF-1a and its downstream targets and quite significant fluctuations within the same cell line from one experimental setting to the next. In conclusion, it is not clear what is the relevance of the new HIF-1a regulatory axis described in this paper in normoxic or hypoxic conditions.

(3) Another critical point is that necessary experimental controls are sometimes missing, and this is reducing the strength of some of the conclusions enunciated by the authors. As an example, experiments where overexpression of STAMBPL1 is coupled to silencing of FOXO1 to demonstrate dependency lack FOXO1silencing the absence of STAMBPL1 overexpression. Because diminishing FOXO1 expression affects HIF-1a/VEGF transcription even in the absence of STAMBPL1 (shown in Figure 7C, D), it is not clear if the data presented in Figure 7G are significant. The difference between HIF-1a expression upon FOXO1 silencing should be compared in the presence or absence of STAMBPL1 overexpression to understand if FOXO1 impacts HIF-1a transcription dependently or independently of STAMBPL1.

In addition, some minor comments to improve the quality of this manuscript are provided.

(1) In Figures 2A and D, where endogenous versus STAMBPL1 expression is shown, it is not clear what is the molecular weight of these proteins as they both appear to be of 55 KDa, even though according to the authors the exogenous protein is bigger than the endogenous and the lower band in Figure 2D is reported to be the endogenous STAMBPL1.

(2) In Figure 2, the effect of STAMBPL1 overexpression on HIF-1a mRNA is minor. At the same time, it seems that the protein levels of HIF-1a are quite high (or at least visible by WB) in normoxic cells even in the absence of STAMBPL1 overexpression. This raises questions about the type of regulation that HIF-1a is subjected to in these cells.

In general, because only two cell lines are used in this study and the data in patients do not appear to strongly support an oncogenic function of STAMBPL1 in TNBC (via its overexpression), data should be more solid and additional experiments should be provided to substantiate the oncogenic function of this pathway in TNCB.

Reviewer #3 (Public review):

Summary:

Rapamycin is a macrolide of immunologic therapeutic importance, proposed as a ligand of mTOR. It is also employed as in essays to probe protein-protein interactions.<br /> The authors serendipitously found that the drug rapamycin and some related compounds, potently activate the cationic channel TRPM8, which is the main mediator of cold sensation in mammals. The authors show that rapamycin might bind to a novel binding site that is different from the binding site for menthol, the prototypical activator of TRPM8. These convincing results are important to a wide audience, since rapamycin is a widely used drug and is also employed in essays to probe protein-protein interactions, which could be affected by potential specific interactions of rapamycin with other membrane proteins, as illustrated herein.

Strengths:

The authors employ several experimental approaches to convincingly show that rapamycin activates directly the TRPM8 cation channel and not an accessory protein or the surrounding membrane. In general, the electrophysiological, mutational and fluorescence imaging experiments are adequately carried out and cautiously interpreted, presenting a clear picture of the direct interaction with TRPM8. In particular, the authors convincingly show that the interactions of rapamycin with TRPM8 are distinct from interactions of menthol with the same ion channel.

Weaknesses:

The main weakness of the manuscript was the NMR method employed to show that rapamycin binds to TRPM8. The authors developed and deployed a novel signal processing approach based on subtraction of several independent NMR spectra to show that rapamycin binds to the TRPM8 protein and not to the surrounding membrane or other proteins. In this revised version the authors have strengthened the evidence that the method gives solid results and have improved the clarity of the presentation.

Comments on revisions:

The authors have greatly improved the quality of the presentation of the NMR data and have answered my concerns regarding the new methodology. The manuscript is improved and represents an important contribution.

Reviewer #3 (Public review):

Summary:

In this study, the authors address the paradox of how tyrosine can act as a stronger sticker for phase separation than phenylalanine, despite phenylalanine being higher on the hydrophobicity scale and exhibiting more prominent pairwise contact statistics in folded protein structures compared to tyrosine.

Strengths:

This is a fascinating problem for the protein science community with special relevance for the biophysical condensate community. Using atomistic simulations of simple model peptides and condensates as well as quantum calculations, the authors provide an explanation that relies on the dielectric constant of the medium and the hydration level that either tyrosine or phenylalanine can achieve in highly hydrophobic vs. hydrophilic media. The authors find that as the dielectric constant decreases, phenylalanine becomes a stronger sticker than tyrosine. The conclusions of the paper seem to be solid, it is well-written and it also recognises the limitations of the study. Overall, the paper represents an important contribution to the field.

Weaknesses:

How can the authors ensure that a condensate of GSY or GSF peptides is a representative environment of a protein condensate? First, the composition in terms of amino acids is highly limited, second the effect of peptide/protein length compared to real protein sequences is also an issue, and third, the water concentration within these condensates is really low as compared to real experimental condensates. Hence, how can we rely on the extracted conclusions from these condensates to be representative for real protein sequences with a much more complex composition and structural behaviour?

Reviewer #3 (Public review):

Summary:

The authors performed snRNA-seq in the pre-optic area (POA), a heterogeneous brain region implicated in multiple innate behaviors, comparing two species of Peromyscus mice that possess strikingly different parenting behaviors. P. polionotus show high levels of parental care from both sexes of parent, and P. maniculatus show lower levels of care, predominantly displayed by dams rather than sires. The overall goal of understanding the genomic basis of behavioral variation is significant and of broad interest and comparative studies in POA in these two species is an excellent approach to tackle this question. The authors correctly point out that existing studies largely compare species that are highly divergent, such as mice and humans, which confounds the association of specific neuronal populations or gene expression patterns with distinct behaviors. They identify neuronal populations with differential abundance between species and sexes, and additionally report sex and species differences in gene expression within each transcriptomic cell type. Their cell type classification is aided by mapping their Peromyscus cells onto a previously existing POA single cell dataset generated in lab mice. The detection and validation of previously observed sex differences in the Gal/Moxd1 cell type, and species differences in Avp expression provides additional support that their data are robust. Importantly, the authors demonstrate reduced sexual dimorphism in the POA of P. polionotus, compared to P. maniculatus, and prior knowledge in rats and mice. This finding suggests a potential neural substrate for the increased parental behavior in P. polionotus.

Strengths:

This is a pioneering comparative snRNA-seq study that provides a roadmap for similar approaches in non-traditional model organisms.

The authors have identified populations that may underlie sex- and species- differences in parenting behavior in rodents.

A significant strength of the manuscript is the histological validation of their most robust marker genes.

Weaknesses:

My primary concern is that the dataset is limited: 52,121 neuronal nuclei across 24 samples, which does not provide many cells per cluster to analyze comparatively across sex and species, particularly given the heterogeneity of the large region dissected, which contains adjacent regions such as the PVN and SCN.

There is no explanation for the finding that there is a female-bias in gene expression across all cell types in P. polionotus.

Reviewer #3 (Public review):

Summary:

In this study by Kawadkar et al, the authors investigate the developmental role of Nup107, a nucleoporin, in regulating the larval-to-pupal transition in Drosophila through RNAi knockdown and CRISPR-Cas9-mediated gene editing. They demonstrate that Nup107, an essential component of the nuclear pore complex (NPC), is crucial for regulating ecdysone signaling during developmental transitions. The authors show that the depletion of Nup107 disrupts these processes, offering valuable insights into its role in development.

Specifically, they find that:

(1) Nup107 depletion impairs pupariation during the larval-to-pupal transition.<br /> (2) RNAi knockdown of Nup107 results in defects in EcR nuclear translocation, a key regulator of ecdysone signaling.<br /> (3) Exogenous 20-hydroxyecdysone (20E) rescues pupariation blocks, but rescued pupae fail to close.<br /> (4) Nup107 RNAi-induced defects can be rescued by activation of the MAP kinase pathway.

Strengths:

The manuscript provides strong evidence that Nup107, a component of the nuclear pore complex (NPC), plays a crucial role in regulating the larval-to-pupal transition in Drosophila, particularly in ecdysone signaling.

The authors employ a combination of RNAi knockdown, CRISPR-Cas9 gene editing, and rescue experiments, offering a comprehensive approach to studying Nup107's developmental function.

The study effectively connects Nup107 to ecdysone signaling, a key regulator of developmental transitions, offering novel insights into the molecular mechanisms controlling metamorphosis.

The use of exogenous 20-hydroxyecdysone (20E) and activation of the MAP kinase pathway provides a strong mechanistic perspective, suggesting that Nup107 may influence EcR signaling and ecdysone biosynthesis.

Weaknesses:

The authors do not sufficiently address the potential off-target effects of RNAi, which could impact the validity of their findings. Alternative approaches, such as heterozygous or clonal studies, could help confirm the specificity of the observed phenotypes.

NPC Complex Specificity: While the authors focus on Nup107, it remains unclear whether the observed defects are specific to this nucleoporin or if other NPC components also contribute to similar defects. Demonstrating similar results with other NPC components would strengthen their claims.

Although the authors show that Nup107 depletion disrupts EcR signaling, the precise molecular mechanism by which Nup107 influences this process is not fully explored. Further investigation into how Nup107 regulates EcR nuclear translocation or ecdysone biosynthesis would improve the clarity of the findings.

There are some typographical errors and overly strong phrases, such as "unequivocally demonstrate," which could be softened. Additionally, the presentation of redundant data in different tissues could be streamlined to enhance clarity and flow.

Reviewer #3 (Public review):

Neural activity in the visual cortex has primarily been studied in terms of responses to external visual stimuli. While the noisiness of inputs to a visual area is known to also influence visual responses, the contribution of this noisy component to overall visual responses has not been well characterized.

In this study, the authors reanalyze two previously published datasets - a Ca++ imaging study from mouse V1 and a large-scale electrophysiological study from monkey V1-V4. Using regression models, they examine how neural activity in one layer (in mice) or one cortical area (in monkeys) predicts activity in another layer or area. Their main finding is that significant predictions are possible even in the absence of visual input, highlighting the influence of non-stimulus-related downstream activity on neural responses. These findings can inform future modeling work of neural responses in the visual cortex to account for such non-visual influences.

A major weakness of the study is that the analysis includes data from only a single monkey. This makes it hard to interpret the data as the results could be due to experimental conditions specific to this monkey, such as the relative placement of electrode arrays in V1 and V4. The authors perform a thorough analysis comparing regression-based predictions for a wide variety of combinations of stimulus conditions and directions of influence. However, the comparison of stimulus types (Figure 4) raises a potential concern. It is not clear if the differences reported reflect an actual change in predictive influence across the two conditions or if they stem from fundamental differences in the responses of the predictor population, which could in turn affect the ability to measure predictive relationships. The authors do control for some potential confounds such as the number of neurons and self-consistency of the predictor population. However, the predictability seems to closely track the responsiveness of neurons to a particular stimulus. For instance, in the monkey data, the V1 neuronal population will likely be more responsive to checkerboards than to single bars. Moreover, neurons that don't have the bars in their RFs may remain largely silent. Could the difference in predictability be just due to this? Controlling for overall neuronal responsiveness across the two conditions would make this comparison more interpretable.

Reviewer #3 (Public review):

Summary:

In their report, Tsutsumi et al., use single nucleus transcriptional and chromatin accessibility analyses of mouse achilles tendon in an attempt to uncover new markers of tendon stem/progenitor cells. They propose CD55 and CD248 as novel markers of tendon stem/progenitor cells.

Strengths:

This is an interesting and important research area. The paper is overall well written.

Weaknesses:

Major problems:

(1) It is not clear what tissue exactly is being analyzed. The authors build a story on tendons, but there is little description of the dissection. The authors claim to detect MTJ and cartilage cells, but not bone or muscle cells. The tendon sheath is known to express CD55, so the population of "progenitors" may not be of tendon origin.

(2) Cluster annotations are seemingly done with a single gene. Names are given to cells without functional or spatial validation. For example, MTJ cells are annotated based on Postn, but it is never shown that Postn is only expressed at the MTJ, and not in other anatomical locations in the tendon.

(3) The authors compare their data to public data based on interrogating single genes in their dataset. It is now standard practice to integrate datasets (eg, using harmony), or at a minimum using gene signatures built into Seurat (eg AddModuleScore).

(4) Progenitor populations (SP1, SP2). The authors claim these are progenitors but show very clearly that they express macrophage genes. What are they, macrophages or fibroblasts?

(5) All omics analysis is done on single data points (from many mice pooled). The authors make many claims on n=1 per group for readouts dependent on sample number (eg frequency of clusters).

(6) The scRNAseq atlas in Figure 1 is made by analyzing 2W and 6W tendons at the same time. The snRNAseq and ATACseq atlas are built first on 2W data, after which the 6W data is compared. Why use the 2W data as a reference? Why not analyze the two-time points together as done with the scRNAseq?

(7) Figure 5: The authors should show the gating strategy for FACS. Were non-fibroblasts excluded (eg, immune cells, endothelia...etc). Was a dead cell marker used? If not, it is not surprising that fibroblasts form colonies and express fibroblast genes when compared to CD55-CD248- immune cells, dead cells, or debris. Can control genes such as Ptprc or Pecam1 be tested to rule out contamination with other cell types?

Minor problems:

(1) Report the important tissue processing details: type of collagenase used. Viability before loading into 10x machine.

Reviewer #3 (Public review):

The manuscript of Fuchsberger et al. investigates the cellular mechanisms underlying dopamine-dependent long-term potentiation (DA-LTP) in mouse hippocampal CA1 neurons. The authors conducted a series of experiments to measure the effect of dopamine on the protein synthesis rate in hippocampal neurons and its role in enabling DA-LTP. The key results indicate that protein synthesis is increased in response to dopamine and neuronal activity in the pyramidal neurons of the CA1 hippocampal area, mediated via the activation of adenylate cyclases subtypes 1 and 8 (AC1/8) and the cAMP-dependent protein kinase (PKA) pathway. Additionally, the authors show that postsynaptic DA-induced increases in protein synthesis are required to express DA-LTP, while not required for conventional t-LTP.

The increased expression of the newly synthesized GluA1 receptor subunit in response to DA supports the formation of homomeric calcium-permeable AMPA receptors (CP-AMPARs). This evidence aligns well with data showing that DA-LTP expression requires the GluA1 AMPA subunit and CP-AMPARs, as DA-LTP is absent in the hippocampus of a GluA1 genetic knock-out mouse model.

Comments on revisions:

The authors addressed adequately all my comments.

Reviewer #3 (Public review):

Strengths:

The paper describes a new perspective on friction perception, with the hypothesis that humans are sensitive to the instabilities of the surface rather than the coefficient of friction. The paper is very well written and with a comprehensive literature survey.

One of the central tools used by the author to characterize the frictional behavior is the frictional instabilities maps. With these maps, it becomes clear that two different surfaces can have both similar and different behavior depending on the normal force and the speed of exploration. It puts forward that friction is a complicated phenomenon, especially for soft materials.

The psychophysics study is centered around an odd-one-out protocol, which has the advantage of avoiding any external reference to what would mean friction or texture for example. The comparisons are made only based on the texture being similar or not.

The results show a significant relationship between the distance between frictional maps and the success rate in discriminating two kinds of surface.

Weaknesses:

The main weakness of the paper comes from the fact that the frictional maps and the extensive psychophysics study are not made at the same time, nor with the same finger. The frictional maps are produced with an artificial finger made out of PDMS which is a poor substitute for the complex tribological properties of skin.

The evidence would have been much stronger if the measurement of the interaction was done during the psychophysical experiment. In addition, because of the protocol, the correlation is based on aggregates rather than on individual interactions.

The authors compensate with a third experiment where they used a 2AFC protocol and an online force measurement. But the results of this third study, fail to convince the relation.

No map of the real finger interaction is shown, bringing doubt to the validity of the frictional map for something as variable as human fingers.

Reviewer #3 (Public review):

Summary:

In this manuscript, the investigators identified LMOD1 as one of a subset of cytoskeletal proteins whose levels increase in the early stages of myogenic differentiation. Lmod1 is understudied in striated muscle and in particular in myogenic differentiation. Thus, this is an important study. It is also a very thorough study - with perhaps even too much data presented. Importantly, the investigators observed that LMOD1 appears to be important for skeletal regeneration, and myogenic differentiation and that it interacts with SIRT1. Both primary myoblast differentiation and skeletal muscle regeneration were studied. Rescue experiments confirmed these observations: SIRT1 can rescue perturbations of myogenic differentiation as a result of LMOD1 knockdown.

Strengths:

Particular strengths include: important topic, the use of primary skeletal cultures, the use of both cell culture and in vivo approaches, careful biomarker analysis of primary mouse myoblast differentiation, the use of two methods to probe the function of the Lmod1/SIRT1 pathway via using depletion approaches and inhibitors, and generation of six independent myoblast cultures. Results support their conclusions.

Weaknesses:

(1) Figure 1. Images of cells in Figure 1A are too small to be meaningful (especially in comparison to the other data presented in this figure). Perhaps the authors could make graphs smaller?

(2) Line 148 "We found LMOD2 to be the most abundant Lmod in whole skeletal muscle." This is confusing since most if not all prior studies have shown that Lmod3 is the predominant isoform in skeletal muscle. The two papers that are cited are incorrectly cited. Clarification to resolve this discrepancy is needed.

(3) Figure 2. Immunoflorescence (IF) panels are too small to be meaningful. Perhaps the graphs could be made smaller and more space allocated for the IF panels? This issue is apparent for just about all IF panels - they are simply too small to be meaningful. Additionally, in many of the immunofluorescence figures, the colors that were used make it difficult to discern the stained cellular structures. For example in Figure S1, orange and purple are used - they do not stand out as well as other colors that are more commonly used.

(4) There is huge variability in many experiments presented - as such, more samples appear to be required to allow for meaningful data to be obtained. For example, Figure S2. Many experimental groups, only have 3 samples - this is highly problematic - I would estimate that 5-6 would be the minimum.

(5) Ponceau S staining is often used as a loading control in this manuscript for western blots. The area/molecular weight range actually used should be specified. Not clear why in some experiments GAPDH staining is used, in other experiments Ponceau S staining is used, and in some, both are used. In some experiments, the variability of total protein loaded from lane to lane is disconcerting. For example, in Figure S4C there appears to be more than normal variability. Can the protein assay be redone and samples run again?

(6) Figure S3 - Lmod3 is included in the figure but no mention of it occurs in the title of the figure and/or legend.

(7) Abstract, line 25. "overexpression accelerates and improves the formation of myotubes". This is a confusing sentence. How is it improving the formation? A little more information about how they are different than developing myotubes in normal/healthy muscles would be helpful.

(8) It is impossible from the IF figures presented to determine where Lmod1 localizes in the myocytes. Information on its subcellular localization is important. Does it localize with Lmod2 and Lmod3 at thin filament pointed ends?

Reviewer #3 (Public review):

Summary:

Chen and Phillips present intriguing work that extends our view on the C. elegans small RNA network significantly. While the precise findings are rather C. elegans specific there are also messages for the broader field, most notably the switching of small RNA populations bound to an argonaute, and RNA granules behavior depending on developmental stage. The work also starts to shed more light on the still poorly understood role of the CSR-1 argonaute protein and supports its role in the decay of maternal transcripts. Overall, the work is of excellent quality, and the messages have a significant impact.

Strengths:

Compelling evidence for major shift in activities of an argonaute protein during development, and implications for how small RNAs affect early development. Very balanced and thoughtful discussion.

Weaknesses:

The switch between maternal and zygotic NRDE-3 remains unaddressed

Reviewer #3 (Public review):

Summary:

Xiang et al. investigated the role of ubiquitin E3 ligase ITCH in SARS-CoV-2 replication. First, they described the role of ITCH on the structural proteins. Here, the ubiquitination of E and M (but not S) leads to an enhanced interaction and presumably virion assembly. In addition, E and M ubiquitination seems to be necessary for p62-guided sequestration into autophagosomes for secretion. Furthermore, ITCH regulates S proteolytic cleavage by changing furin localization and inhibiting CTSL protease maturation. In addition, SARS-CoV-2 infection upregulates ITCH phosphorylation, whereas knockout of ITCH reduces SARS-CoV-2 replication.

Strengths:

The proposed study is of interest to the virology community because it aims to elucidate the role of ubiquitination by ITCH in SARS-CoV-2 proteins. Understanding these mechanisms will address broadly applicable questions about coronavirus biology and enhance our knowledge of ubiquitination's diverse functions in cell biology.

Weakness:

The involvement of ubiquitin ligases in SARS-CoV-2 replication is not entirely new (see E3 Ubiquitin Ligase RNF5; Yuan et al., 2022; Li et al., 2023). While the data generally support the conclusions, additional work is needed to confirm the role of ITCH in SARS-CoV-2 replication in a biologically relevant context. The vast majority of data is based on transient overexpression experiments of ITCH, which ultimately leads to massive ubiquitination of several viral and host cell factors, including potentially low-affinity substrates not typically recognized under physiological conditions. In addition to that, nearly all experiments were done in cells co-overexpressing ITCH and the viral structural proteins (or cellular proteases) in HEK293T cells. Therefore, a proteomic analysis of protein ubiquitination in a) SARS-CoV-2-infected cells (ideally several cell types) and b) SARS-CoV-2-infected v2T-ITCH-KO cells would verify the ITCH-related ubiquitination of e.g., E and M and would strengthen the whole manuscript. In addition, the few key experiments using SARS-CoV-2 infected cells were performed in VeroE6 cells, which are neither human nor lung-derived. Only in one experiment were lung-derived Calu3 cells included.<br /> Moreover, the manuscript names ITCH as a central regulator of SARS-CoV-2 replication. If ITCH is beneficial for E and M interaction and thereby aids virion assembly, showing its effect on VLP production would be desirable. Clarifications regarding data acquisition and data analysis could strengthen the manuscript and its conclusions.

Reviewer #3 (Public review):

NCXs are key Ca2+ transporters located on the plasma membrane, essential for maintaining cellular Ca2+ homeostasis and signaling. The activities of NCX are tightly regulated in response to cellular conditions, ensuring precise control of intracellular Ca2+ levels, with profound physiological implications. Building upon their recent breakthrough in determining the structure of human NCX1, the authors obtained cryo-EM structures of NCX1 in complex with its modulators, including the cellular activator PIP2 and the small molecule inhibitor SEA0400. Structural analyses revealed mechanistically informative conformational changes induced by PIP2 and elucidated the molecular basis of inhibition by SEA0400. These findings underscore the critical role of the interface between the transmembrane and cytosolic domains in NCX regulation and small molecule modulation. Overall, the results provide key insights into NCX regulation, with important implications for cellular Ca2+ homeostasis.

Reviewer #3 (Public review):

Summary:

The authors develop automated methods to visually identify micronuclei (MN) and MN-containing cells. The authors then use these methods to isolate MN-containing RPE-1 cells post-photoactivation and analyze transcriptional changes in cells with and without micronuclei. The authors find that RPE-1 cells with MN have similar transcriptomic changes as aneuploid cells and that MN rupture does not lead to vast changes in the transcriptome.

Strengths:

The authors develop a method that allows for automating measurements and analysis of micronuclei. This has been something that the field has been missing for a long time. Using such a method has the potential to greatly enhance the field's ability to analyze micronuclei and understand the downstream consequences. The authors also develop a method to identify cells with micronuclei in real-time, mark them using photoconversion, and then isolate them via cell sorting, which could change the way we isolate and study MN-containing cells, and the scale at which we do it. The authors use this method to look at the transcriptome. This method is very powerful as it can allow for the separation of a heterogenous population and subsequent analysis with a much higher sample number than previously possible.

Weaknesses:

The major weakness of this paper is the transcriptomic analysis of MN. There is in general large variance between replicates in experiments looking at cells with ruptured versus intact micronuclei. This limits our ability to assess if lack of changes are due to truly not having changes between these populations or experimental limitations. More transcriptomic analysis will be necessary to fully understand the downstream consequences of MN rupture.

Reviewer #3 (Public review):

Summary:

Type VI secretion systems (T6SS) are employed by bacteria to inject competitor cells with numerous effector proteins. These effectors can kill injected cells via an array of enzymatic activities. A common class of T6SS effector are peptidoglycan (PG) lysing enzymes. In this manuscript, the authors characterize a PG-lysing effector-TseP-from the pathogen Aeromonas dhakensis. While the C-terminal domain of TseP was known to have lysozyme activity, the N-terminal domain was uncharacterized. Here, the authors functionally characterize TsePN as a zinc-dependent amidase. This discovery is somewhat novel because it is rare for PG-lysing effectors to have amidase and lysozyme activity. In the second half of the manuscript, the authors utilize a crystal structure of the lysozyme TsePC domain to inform the engineering of this domain to lyse gram-positive peptidoglycan.

Strengths:

The two halves of the manuscript considered together provide a nice characterization of a unique T6SS effector and reveal potentially general principles for lysozyme engineering.

Weaknesses:

The advantage of fusing amidase and lysozyme domains in a single effector is not discussed but would appear to be a pertinent question.

Comments on revisions:

The authors have adequately addressed my previous comments. The authors did not conduct any additional experiments to address the comments made by other reviewers. However, in most cases it seems that paring down the strength of claims made in the text or adding data to the supplement is sufficient to address these concerns.

Reviewer #3 (Public review):

Summary:

This manuscript introduces a differentiable variant of the Gillespie algorithm (DGA) that allows gradient calculation using backpropagation. The most significant contribution of this work is the development of the DGA itself, a novel approach to making stochastic simulations differentiable. This is achieved by replacing discontinuous operations in the traditional Gillespie algorithm with smooth, differentiable approximations using sigmoid and Gaussian functions. This conceptual advance opens up new avenues for applying powerful gradient-based optimization techniques, prevalent in machine learning, to studying stochastic biological systems.

The method was tested on a simple two-state promoter model of gene expression. The authors found that the DGA accurately captured the moments of the steady-state distribution and other major qualitative features. However, it was less accurate at capturing information about the distribution's tails, potentially because rare events result from frequent low-probability reaction events where the approximations made by the DGA have a greater impact. The authors also used the DGA to design a four-state promoter model of gene regulation that exhibited a desired input-output relationship. The DGA could learn parameters that produced a sharper response curve, which was achieved by consuming more energy.

The authors conclude that the DGA is a powerful tool for analyzing and designing stochastic systems. The discussion lays several open questions in the field and constructively addresses shortcomings of the proposed method as well as potential ways forward.

Strengths:

The DGA allows gradient-based optimization techniques to estimate parameters and design networks with desired properties.

The DGA efficacy in estimating kinetic parameters from both synthetic and experimental data. This capability highlights the DGA's potential to extract meaningful biophysical parameters from noisy biological data.

The DGA's ability to design a four-state promoter architecture exhibits a desired input-output relationship. This success indicates the potential of the DGA as a valuable tool for synthetic biology, enabling researchers to engineer biological circuits with predefined behaviours.

Weaknesses:

The study primarily focuses on analysing the steady-state properties of stochastic systems.

Comments on revisions:

Thank you for addressing all the points raised. I am looking forward to seeing the next steps in DGAs development and performance!

Reviewer #3 (Public review):

Ito et al. investigate the role of synaptic plasticity in the medial preoptic area (MPOA) pathway of male mice and its involvement in transitions from infanticidal aggression to parental behavior. Using optogenetics, whole-cell patch-clamp recordings, and behavioral assays, they demonstrate that inhibitory synaptic transmission from the posterior-dorsal medial amygdala (MePD) to the central MPOA (cMPOA) decreases following mating and cohabitation with pregnant females. This synaptic disinhibition is correlated with a reduction in aggressive behavior toward pups. They further show that paternal experience induces enhanced inhibitory transmission in the rhomboid nucleus of the bed nucleus of the stria terminalis (BSTrh), downstream of the MPOA, through postsynaptic mechanisms. These findings suggest a circuit-based model where social experiences and mating induce synaptic changes in the Me-cMPOA-BSTrh pathway, mediating the transition to parental behavior.

The conclusions of this paper are largely supported by the data, but several methodological and conceptual aspects require clarification or additional experiments.

(1) When evaluating the Me Cartpt-expressing neuron projection to the cMPOA, the authors compared excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs). However, the standard procedure for isolating these currents is to hold the membrane potential at the reversal potential for inhibitory or excitatory currents, respectively. The authors appear not to have followed this procedure, making it unclear how EPSCs and IPSCs were calculated. This requires clarification to ensure the validity of their reported E/I balance changes.

(2) The authors chose to assess parental behavior over four consecutive days. It is unclear why this specific timeframe was selected. A justification for this choice would strengthen the interpretation of the behavioral data.

(3) The experimental design in Figure 5, where the authors lesioned the entire cMPOA to assess its role in BSTrh inhibition, presents several limitations: First, the effects on BSTrh activity could result from indirect circuit alterations rather than direct cMPOA projections. The current lesion approach cannot disentangle these possibilities. Second, the cMPOA is a heterogeneous region containing diverse neuronal subtypes. Full lesions prevent the differentiation of the roles played by distinct populations within this region. Third, lesion specificity is questionable, as some lesions extended beyond the cMPOA boundaries (Figure S5). This overextension complicates the interpretation of the results and requires tighter control.

(4) In Figure 3, the authors show that optogenetic inhibition of Me projections to the cMPOA modifies the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs). However, the proposed mechanism that this modulation reflects inter-neuronal network activity within the cMPOA lacks sufficient experimental validation. Additional experiments assessing circuit-level interactions could substantiate these claims.

(5) While the paper highlights synaptic changes in the cMPOA, it does not establish a direct relationship between these changes and the social experience. How do mating and cohabitation with females impact this pathway and modulate synaptic strength? The discussion could benefit from integrating these factors into their proposed model.

Overall, the paper offers valuable insights into the neural circuitry underlying male parental behavior, particularly the synaptic dynamics of the Me-cMPOA-BSTrh pathway. However, addressing these methodological and conceptual limitations would significantly enhance the clarity and impact of the work.

Reviewer #3 (Public review):

Summary:

This study indicates that connections across human cortical pyramidal cells have identical latencies despite a larger mean dendritic and axonal length between somas in human cortex. A precise demonstration combining detailed electrophysiology and modeling, indicates that this property is due to faster propagation of signals in proximal human dendrites. This faster propagation is itself due to a slightly thicker dendrite, to a larger capacitive load, and to stronger hyperpolarizing currents. Hence, the biophysical properties of human pyramidal cells are adapted such that they do not compromise information transfer speed.

Strengths:

The manuscript is clear and very detailed. The authors have experimentally verified a large number of aspects that could affect propagation speed and have pinpointed the most important one. This paper provides an excellent comparision of biophysical properties between rat and human pyramidal cells. Thanks to this approach a comprehensive description of the mechanisms underlying the acceleration of propagation in human dendrite is provided.

Weaknesses:

The weaknesses I had identified have been addressed by the authors.

Reviewer #3 (Public review):

Summary:

Li et al propose to better understand the mechanisms of drug resistance in nematode parasites by studying mutants of the model roundworm C. elegans that are resistant to the deworming drug ivermectin. They provide compelling evidence that loss-of-function mutations in the E3 ubiquitin ligase encoded by the UBR-1 gene make worms resistant to the effects of ivermectin (and related compounds) on viability, body size, pharyngeal pumping rate, and locomotion and that these mutant phenotypes are rescued by a UBR-1 transgene. They propose that the mechanism is resistance is indirect, via the effects of UBR-1 on glutamate production. They show mutations (vesicular glutamate transporter eat-4, glutamate synthase got-1) and drugs (glutamate, glutamate uptake enhancer ceftriaxone) affecting glutamate metabolism/transport modulate sensitivity to ivermectin in wild type and ubr-1 mutants. The data are generally consistent with greater glutamate tone equating to ivermectin resistance. Finally, they show that manipulations that are expected to increase glutamate tone appear to reduce expression of the targets of ivermectin, the glutamate-gated chloride channels, which is known to increase resistance.

There is a need for genetic markers of ivermectin resistance in livestock parasites that can be used to better track resistance and to tailor drug treatment. The discovery of UBR-1 as a resistance gene in C. elegans will provide a candidate marker that can be followed up in parasites. The data suggest Ceftriaxone would be a candidate compound to reverse resistance.

Strengths:

The strength of the study is the thoroughness of the analysis and the quality of the data. There can be little doubt that ubr-1 mutations do indeed confer ivermectin resistance. The use of both rescue constructs and RNAi to validate mutant phenotypes is notable. Further, the variety of manipulations they use to affect glutamate metabolism/transport makes a compelling argument for some kind of role for glutamate in resistance.

Weaknesses:

The use of single ivermectin dose assays can be misleading. A response change at a single dose shows that the dose-response curve has shifted, but the response is not linear with dose, so the degree of that shift may be difficult to discern and may result from a change in slope but not EC50.

Reviewer #3 (Public review):

Summary:

The authors have done a good job at creating a "resource" paper for the study of gut regeneration in sea cucumbers. They present a single-cell RNAseq atlas for the reconstitution of Holothuria glaberrima gut following self-evisceration in response to a potassium chloride injection. The authors provide data characterizing cellular populations and precursors of the regenerating anlage at 9 days post evisceration. As a "Tools and Resources" contribution to eLife, this work, with some revisions, could be appropriate. It will be impactful in the fields of regeneration, particularly in invertebrates, but also in comparative studies in other species, including evolutionary studies. Some of these comparative studies could extend to vertebrates and could therefore impact regenerative medicine in the future.

Strengths:

• Novel and useful information for a model organism and question for which this type of data has not yet been reported<br /> • Single-cell gene expression data will be valuable for developing testable hypotheses in the future<br /> • Marker genes for cell types provided to the field<br /> • Interesting predictions about possible lineage relationships between cells during sea cucumber gut regeneration<br /> • Authors have done a good job in the revision of making sure not to overstate the lineage claims in absence of definitive lineage-tracing experiments<br /> • Authors have improved the figures and the overall readability of the figures and text

Specific questions:

- Is there any way to systematically compare these cells to evolutionarily-diverged cells in distant relatives to sea cucumbers? Or even on a case-by-case basis? For example, is there evidence for any of these transitory cell types to have correlate(s) in vertebrate gut regeneration?

• Authors acknowledged this would be interesting and important, but they say in the response document this is outside the scope of the current manuscript and more data would be needed to do this well.

- Line 808: The authors may make a more accurate conclusion by saying that the characteristics are similar to blastemas or behaves like a blastema rather than it is blastema. There is ambiguity about the meaning of this term in the field, but most researchers seem to currently have in mind that the "blastema" definitions includes a discrete spatial organization of cells, and here these cells are much more spread out. This could be a good opportunity for the authors to engage in this dialogue, perhaps parsing out the nuances of what a "blastema" is, what the term has traditionally referred to, and how we might consider updating this term or at least re-framing the terminology to be inclusive of functions that "blastemas" have traditionally had in the literature and how they may be dispersed over geographical space in an organism more so than the more rigid, geographically-restricted definition many researchers have in mind. However, if the authors choose to elaborate on these issues, those elaborations do belong in the discussion, and the more provisional terminology we mention here could be used throughout the paper until that element of the revised discussion is presented. We would welcome the authors to do this as a way to point the field in this direction as this is also how we view the matter. For example, some of the genes whose expression has been observed to be enriched following removal of brain tissue in axolotls (such as kazald2, Lust et al.), are also upregulated in traditional blastemas, for instance, in the limb, but we appreciate that the expression domain may not be as localized as in a limb blastema. Additionally, since there is now evidence that some aspects of progenitor cell activation even in limb regeneration extend far beyond the local site of amputation injury (Johnson et al., Payzin-Dogru et al.), there is an opportunity to connect the dots and make the claim that there could be more dispersion of "blastema function" than previously appreciated in the field. Diving a bit more into these nuances may also enable a better conceptual framework of how blastema function may evolve across vast evolutionary time and between different injury contexts in super-regenerative organisms.

• Authors addressed this comment and agree it is interesting, but given how much territory they had to cover and space limitations, they will save this type of discussion and comparative theoretical work for the future.

Overall, the manuscript is much improved.

Reviewer #3 (Public review):

Background:

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel whose dysfunction underlies cystic fibrosis, a life-limiting condition caused by thick, sticky mucus buildup in the lungs and other organs. Despite multiple high-resolution structures of CFTR, these snapshots have all captured the channel in a non-conducting or "closed" conformation - even when the protein was prepared under conditions that should favor channel opening. This discrepancy has posed a key challenge: how can a channel be experimentally observed as closed while physiological tests demonstrate it conducts chloride ions?

Key Findings:

(1) Stable Open Conformation

Through repeated molecular dynamics (MD) simulations of human CFTR in lipid bilayers, researchers observed a reproducible, stable open state. Unlike previous transient openings seen in single-run or short simulations, this conformation remains consistently permeable over extended timescales.

(2) Penta-Helical Arrangement

The authors highlight a "penta-helical" pore-lining arrangement in which five transmembrane helices symmetrically organize to create a clear ion-conduction pathway. This novel configuration resolves the previously puzzling hydrophobic bottleneck found in cryo-EM structures.

(3) Conductance Close to Experimental Values

By analyzing chloride ion flow under near-physiological voltages, they calculate a channel conductance aligning well with electrophysiological measurements. This alignment provides strong support that the observed structure is functionally relevant.

(4) Roles of Key Residues

Several positively charged (cationic) residues in the pore appear crucial for guiding and stabilizing chloride ions. Simultaneously, small kinks in certain helices may act as structural "hinges," allowing or blocking chloride passage.

How to Interpret These Results:

(1) Bridging a Major Gap: The study tackles the mismatch between static "closed" CFTR structures and their known open-channel function. Successfully capturing a stable open state in MD simulations is a significant step toward reconciling what cryo-EM data shows versus what physiological experiments have long told us.

(2) Strength in Multiple Replicas: Running many simulation repeats (rather than relying on a single trajectory) lends credibility. Only if a phenomenon is reproducible across multiple runs can it be considered robust.

(3) Consistency with Mutational Data: Observing that known functional hotspots (e.g., specific charged residues) play a key role in the new pore model further validates these findings.

Important Caveats and Limitations:

(1) Simulation Timescales vs. Biology<br /> Even extended MD (on the microsecond scale) is still much faster, simpler, and more controlled than real cellular processes.

(2) Physiological existence of the penta-helical pore<br /> Although the simulations and results are highly compelling, several factors leave open the possibility of a physiological open conformation differing from the observed penta-helical pore. These factors include ATP hydrolysis, interactions with physiological binding partners, the native membrane environment, and regions not modeled in the CFTR structures, such as the R domain. Most importantly, the transmembrane voltage is very high (500mV).

Bottom Line:

This work delivers a long-awaited, near-physiological view of CFTR's open conformation. It provides a foundational structure against which future experimental and computational studies can be compared. By demonstrating reliable chloride conduction and matching established biophysical data, these simulations bring us closer to understanding - and potentially targeting - CFTR's gating mechanism in health and disease. Readers should applaud the breakthroughs while recognizing that further exploration (including more complex in vitro and in vivo experiments) will still be necessary to capture the full dynamism of CFTR in the living cell environment.

Reviewer #3 (Public review):

Summary:

The paper presents an in-depth analysis of the original colour of a fossil feather from the crest of a 125-million-year-old enantiornithine bird. From its shape and location, it would be predicted that such a feather might well have shown some striking colour and pattern. The authors apply sophisticated microscopic and numerical methods to determine that the feather was iridescent and brightly coloured and possibly indicates this was a male bird that used its crest in sexual displays.

Strengths:

The 3D micro-thin-sectioning techniques and the numerical analyses of light transmission are novel and state-of-the-art. The example chosen is a good one, as a crest feather is likely to have carried complex and vivid colours as a warning or for use in sexual display. The authors correctly warn that without such 3D study feather colours might be given simply as black from regular 2D analysis, and the alignment evidence for iridescence could be missed.

Weaknesses: Trivial.

Reviewer #3 (Public review):

Summary:

Kamal L. Nahas et al. demonstrated that pUL16, pUL21, pUL34, VP16, and pUS3 are involved in the egress of the capsids from the nucleous, since mutant viruses ΔpUL16, ΔpUL21, ΔUL34, ΔVP16, and ΔUS3 HSV-1 show nuclear egress attenuation determined by measuring the nuclear:cytoplasmic ratio of the capsids, the dfParental, or the mutants. Then, they showed that gM-mCherry+ endomembrane association and capsid clustering were different in pUL11, pUL51, gE, gK, and VP16 mutants. Furthermore, the 3D view of cytoplasmic budding events suggests an envelopment mechanism where capsid budding into spherical/ellipsoidal vesicles drives the envelopment.

Strengths:

The authors employed both structured illumination microscopy and cellular ultrastructure analysis to examine the same infected cells, using cryo-soft-X-ray tomography to capture images. This combination, set here for the first time, enabled the authors to obtain holistic data regarding a biological process, as a viral assembly. Using this approach, the researchers studied various stages of HSV-1 assembly. For this, they constructed a dual-fluorescently labelled recombinant virus, consisting of eYFP-tagged capsids and mCherry-tagged envelopes, allowing for the independent identification of both unenveloped and enveloped particles. They then constructed nine mutants, each targeting a single viral protein known to be involved in nuclear egress and envelopment in the cytoplasm, using this dual-fluorescent as the parental one. The experimental setting, both the microscopic and the virological, is robust and well-controlled. The manuscript is well-written, and the data generated is robust and consistent with previous observations made in the field.

Weaknesses:

It would be helpful to find out what role the targeted proteins play in nuclear egress or envelopment acquisition in a different orthoherpesvirus, like HSV-2. This would confirm the suitability of the technical approach set and would also act as a way to validate their mechanism at least in one additional herpesvirus beyond HSV-1. So, using the current manuscript as a starting point and for future studies, it would be advisable to focus on the protein functions of other viruses and compare them.

Reviewer #3 (Public review):

Summary:

This study identifies confirmational fingerprints of amylodogenic light chains, that set them apart from the non-amylodogenic ones.

Strengths:

The research employs a comprehensive combination of structural and dynamic analysis techniques, providing evidence that conformational dynamics at VL-CL interface and structural expansion are distinguished features of amylodogenic LCs.

Weaknesses:

The sample size is limited, which may affect the generalizability of the findings. Additionally, the study could benefit from deeper analysis of specific mutations driving this unique conformation to further strengthen therapeutic relevance.

Furthermore. p-value (statistical significance) of Rg difference should be computer. Finally, significance of mutations (SHM?) at the interface, such as A40G should be compared with previous observations. (Garofalo et al., 2021)

Reviewer #3 (Public review):

Summary:

This study identifies confirmational fingerprints of amylodogenic light chains, that set them apart from the non-amylodogenic ones.

Strengths:

The research employs a comprehensive combination of structural and dynamic analysis techniques, providing evidence that conformational dynamics at VL-CL interface and structural expansion are distinguished features of amylodogenic LCs.

Weaknesses:

The sample size is limited, which may affect the generalizability of the findings. Additionally, the study could benefit from deeper analysis of specific mutations driving this unique conformation to further strengthen therapeutic relevance.

Furthermore. p-value (statistical significance) of Rg difference should be computer. Finally, significance of mutations (SHM?) at the interface, such as A40G should be compared with previous observations. (Garofalo et al., 2021)

Reviewer #3 (Public review):

Summary:

The manuscript entitled "Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development" by Wang et al., investigates the molecular mechanism used by FGFR signaling to support lens development. The lens has long been known to depend on FGFR-signaling for proper development. Previous investigations have demonstrated the FGFR signaling is required for embryonic lens cell survival and for lens fiber cell differentiation. The requirement of FGFR signaling for lens induction has remained more controversial as deletion of both Fgfr1 and Fgfr2 during lens placode formation does not prevent the induction of definitive lens markers such as FOXE3 or αA-crystallin. Here the authors have used the Le-Cre driver to delete all four FGFR genes from the developing lens placode demonstrating a definitive failure of lens induction in the absence of FGFR-signaling. The authors focused on FGFR1 and FGFR2, the two primary FGFRs present during early lens development and demonstrated that lens development could be significantly rescued in lenses lacking both FGFR1 and FGFR2 by expressing a constitutively active allele of KRAS. They also showed that the removal of pro-apoptotic genes Bax and Bak could also lead to a substantial rescue of lens development in lenses lacking both FGFR1 and FGFR2. In both cases, the lens rescue included both increased lens size and the expression of genes characteristic of lens cells.

Significantly the authors concentrated on the juxtamembrane domain, a portion of the FGFRs associated with FRS2. Previous investigations have demonstrated the importance of FRS2 activation for mediating a sustained level of ERK activation. FRS2 is known to associate both with GRB2 and SHP2 to activate RAS. The authors utilized a mutant allele of Fgfr1, lacking the entire juxtamembrane domain (Fgfr1ΔFrs) and an allele of Fgfr2 containing two-point mutations essential for Frs2 binding (Fgfr2LR). When combining three floxed alleles and leaving only one functional allele (Fgfr1ΔFrs or Fgfr2LR) the authors got strikingly different phenotypes. When only the Fgfr1ΔFrs allele was retained, the lens phenotype matched that of deleting both Fgfr1 and Fgfr2. However, when only the Fgfr2LR allele was retained the phenotype was significantly milder, primarily affecting lens fiber cell differentiation, suggesting that something other than FRS2 might be interacting with the juxtamembrane domain to support FGFR signaling in the lens. The authors also deleted Grb2 in the lens and showed that the phenotype was similar to that of the lenses only retaining the Fgfr2LR allele, resulting a failure of lens fiber cell differentiation and decreased lens cell survival. However, mutating the major tyrosine phosphorylation site of GRB2 did not affect lens development. The authors additionally investigated the role of SHP2 in lens development by either deleting SHP2 or by making mutations in the SHP2 catalytic domain. The deletion of the SHP2 phosphatase activity did not affect lens development as severely as total loss of SHP2 protein, suggesting a function for SHP2 outside of its catalytic activity. Although the loss of Shc1 alone has only a slight effect on lens size and pERK activation in the lens, the authors showed that the loss of Shc1 exacerbated the lens phenotype in lenses lacking both Frs2 and Shp2. The authors suggest that SHC1 binds to the FGFR juxtamembrane domain allowing for the recruitment of GRB2 in independently of FRS2.

Strengths:

(1) The authors used a variety of genetic tools to carefully dissect the essential signals downstream of FGFR signaling during lens development.

(2) The authors made a convincing case that something other than FRS2 binding mediates FGFR signaling in the juxtamembrane domain.

(3) The authors demonstrated that despite the requirement of both the adaptor function and phosphatase activity of SHP2 are required for embryonic survival, neither of these activities is absolutely required for lens development.

(4) The authors provide more information as to why FGFR loss has a phenotype much more severe than the loss of FRS2 alone during lens development.

(5) The authors followed up their work analyzing various signaling molecules in the context of lens development with biochemical analyses of FGF-induced phosphorylation in murine embryonic fibroblasts (MEFs).

(6) In general, this manuscript represents a Herculean effort to dissect FGFR signaling in vivo with biochemical backing with cell culture experiments in vitro.

Weaknesses:

(1) The authors demonstrate that the loss of FGFR1 and FGFR2 can be compensated by a constitutive active KRAS allele in the lens and suggest that FGFRs largely support lens development only by driving ERK activation. However, the authors also saw that lens development was substantially rescued by preventing apoptosis through the deletion of BAK and BAX. To my knowledge, the deletion of BAK and BAX should not independently activate ERK. The authors do not show whether ERK activation is restored in the BAK/BAX deficient lenses. Do the authors suggest the FGFR3 and/or FGFR4 provide sufficient RAS and ERK activation for lens development when apoptosis is suppressed? Alternatively, is it the survival function of FGFR-signaling as much as a direct effect on lens differentiation?

(2) Do the authors suggest that GRB2 is required for RAS activation and ultimately ERK activation? If so, do the authors suggest that ERK activation is not required for FGFR-signaling to mediate lens induction? This would follow considering that the GRB2 deficient lenses lack a problem with lens induction.

(3) The increase in p-Shc is only slightly higher in the Cre FGFR1f/f FGFR2r/LR than in the FGFR1f/Δfrs FGFR2f/f. Can the authors provide quantification?

(4) The authors have not shown directly that Shc1 binds to the juxtamembrane region of either Fgfr1 or Fgfr2.

Reviewer #3 (Public review):

Summary:

In this manuscript, the group of Glickman expands on their previous studies on the function of chalkophores during the growth of and infection by Mycobacterium tuberculosis. Previously, the group had shown that chalkophores, which are metallophores specific for the scavenging of copper, are induced by M. tuberculosis under copper deprivation conditions. Here, they show that chalkophores, under copper limiting conditions, are essential for the uptake of copper and maturation of a terminal oxidase, the heme-copper oxidase, cytochrome bcc:aa3. As M. tuberculosis has two redundant terminal oxidases, growth of and infection by M. tuberculosis is only moderated if both the chalkophores and the second terminal oxidase, cytochrome bd, are inhibited.

Strengths:

A strength of this work is that the lab-culture experiments are expanded upon with mice infection models, providing strong indications that host-inflicted copper deprivation is a condition that M. tuberculosis has adapted to for virulence.

Weaknesses:

Because the phenotype of M. tuberculosis lacking chalkophores is similar, if not identical, to using Q203, an inhibitor of cytochrome bcc:aa3, the authors propose that the copper-containing cytochrome bcc:aa3 is the only recipient of copper-uptake by chalkophores. A minor weakness of the work is that this latter conclusion is not verified under infection conditions and other copper-enzymes might still be functionally required during one or more stages of infection.

Reviewer #3 (Public review):

Summary:

This manuscript studies intracellular changes and immune processes during early HIV-1 infection with an additional focus on the small CD4+ T cell subsets. The authors used single-cell omics to achieve high resolution of transcriptomic and epigenomic data on the infected cells which were verified by viral RNA expression. The results add to understanding of transcriptional regulation which may allow progression or HIV latency later in infected cells. The biosamples were derived from early HIV infection cases, providing particularly valuable data for the HIV research field.

Strengths:

The authors examined the heterogeneity of infected cells within CD4 T cell populations, identified a significant and unexpected difference between naive and effector CD4 T cells, and highlighted the differences in Th2 and Th17 cells. Multiple methods were used to show the role of the increased KLF2 factor in infected cells. This is a valuable finding of a new role for the major transcription factor in further disease progression and/or persistence.

The methods employed by the authors are robust. Single-cell RNA-Seq from PBMC samples was followed by a comprehensive annotation of immune cell subsets, 16 in total. This manuscript presents to the scientific community a valuable multi-omics dataset of good quality, which could be further analyzed in the context of larger studies.

Weaknesses:

Methods and Supplementary materials<br /> Some technical aspects could be described in more detail. For example, it is unclear how the authors filtered out cells that did not pass quality control, such as doublets and cells with low transcript/UMI content. Next, in cell annotation, what is the variability in cell types between donors? This information is important to include in the supplementary materials, especially with such a small sample size. Without this, it is difficult to determine, whether the differences between subsets on transcriptomic level, viral RNA expression level, and chromatin assessment are observed due to cell type variations or individual patient-specific variations. For the DEG analysis, did the authors exclude the most variable genes?

The annotation of 16 cell types from PBMC samples is impressive and of good quality, however, not all cell types get attention for further analysis. It's natural to focus primarily on the CD4 T cells according to the research objectives. The authors also study potential interactions between CD4 and CD8 T cells by cell communication inference. It would be interesting to ask additional questions for other underexplored immune cell subsets, such as: 1) Could viral RNA be detected in monocytes or macrophages during early infection? 2) What are the inferred interactions between NK cells and infected CD4 T cells, are interactions similar to CD4-CD8 results? 3) What are the inferred interactions between monocytes or macrophages and infected CD4 T cells?

Discussion<br /> It would be interesting to see more discussion of the observation of how naïve T cells produce more viral RNA compared to effector T cells. It seems counterintuitive according to general levels of transcriptional and translational activity in subsets.<br /> Another discussion block could be added regarding the results and conclusion comparison with Ashokkumar et al. paper published earlier in 2024 (10.1093/gpbjnl/qzae003). This earlier publication used both a cell line-based HIV infection model and primary infected CD4 T cells and identified certain transcription factors correlated with viral RNA expression.

Reviewer #3 (Public review):

Summary:

This study provides significant insights into how host metabolism, specifically of lipids, influences the pathogenesis of Mycobacterium tuberculosis (Mtb). It builds on existing knowledge about Mtb's reliance on host lipids and emphasizes the potential of targeting fatty acid metabolism for therapeutic intervention.

Strengths:

To generate the data, the authors use CRISPR technology to precisely disrupt the genes involved in lipid import (CD36, FATP1), lipid droplet formation (PLIN2) and fatty acid oxidation (CPT1A, CPT2) in mouse primary macrophages. The Mtb Erdman strain is used to infect the macrophage mutants. The study, revealsspecific roles of different lipid-related genes. Importantly, results challenge previous assumptions about lipid droplet formation and show that macrophage responses to lipid metabolism impairments are complex and multifaceted. The experiments are well-controlled and the data is convincing.

Overall, this well-written paper makes a meaningful contribution to the field of tuberculosis research, particularly in the context of host-directed therapies (HDTs). It suggests that manipulating macrophage metabolism could be an effective strategy to limit Mtb growth.

Weaknesses:

None noted. The manuscript provides important new knowledge that will lead mpvel to host-directed therapies to control Mtb infections.

Comments on revisions: The authors have addressed the concerns of the reviewers.

Reviewer #3 (Public review):

Summary:

Bone morphogenetic protein (BMP) signaling instructs multiple processes during development including cell proliferation and differentiation. The authors set out to understand the role of PRDM16 in these various functions of BMP signaling. They find that PRDM16 and BMP co-operate to repress stem cell proliferation by regulating the genomic distribution of BMP pathway transcription factors. They additionally show that PRDM16 impacts choroid plexus epithelial cell specification. The authors provide evidence for a regulatory circuit (constituting of BMP, PRDM16, and Wnt) that influences stem cell proliferation/differentiation.

Strengths:

I find the topics studied by the authors in this study of general interest to the field, the experiments well-controlled and the analysis in the paper sound.

Weaknesses:

I have no major scientific concerns. I have some minor recommendations that will help improve the paper (regarding the discussion).

Reviewer #3 (Public review):

This manuscript examines the role of pdgfrb-positive pericytes in the establishment and maintenance of the blood-brain barrier (BBB) in the zebrafish. Previous studies in PDGFB- or PDGFRB-deficient mice have suggested that loss of pericytes results in disruption of the BBB. The authors show that zebrafish pdgfrb mutant larvae have an intact BBB and that pdgfrb mutant adult fish show large vessel defects and hemorrhage but do not exhibit substantial leakage from brain capillaries, suggesting loss of pericytes is not sufficient to "open" the BBB. The authors use beautiful and compelling images and rigorous quantification to back up most of their conclusions. The imaging of the adult brain is particularly nice. The authors rigorously document the lack of BBB leakage in pdgfrbuq30bh mutant larvae and large vessel phenotypes (eg, enlargement and rupture) in pdgfrbuq30bh mutant adults. A few points would help the authors to further strengthen their findings contradicting the current dogma from rodent models.

Major point:

The authors document pericyte loss using a single TgBAC(pdgfrb:egfp)ncv22 transgenic line driven by the promoter of the same gene mutated in their pdgfrbuq30bh mutants. Given their findings on the consequences of pericyte loss directly contradict current dogma from rodent studies, it would be useful to further validate the absence of brain pericytes in these mutants using one of several other transgenic lines marking pericytes currently available in the zebrafish. This could be done using pdgfrb crispants, which the authors show nicely phenocopy the germline mutants, at least in larvae. This would help nail down the absence of any currently identifiable pericyte population or sub-population in the loss of pdgfrb animals and substantially strengthen the authors' conclusions.

Other issues:

The authors should provide more information about the pdgfrbuq30bh mutant and how it was generated (including a diagram in a supplemental figure would be useful).

It would be helpful to show some data on whether mutants show morphological phenotypes or developmental delay at 7 and 14 dpf, to provide some context to better assess the reduced branching and vessel length vascular phenotypes (see Figures 1c-e).

If available, it would be helpful to have a positive control for the tracer leakage experiments - a genetic manipulation that does cause disruption of the BBB and leakage at 2 hours post-tracer injection (see Figures 1f and g).

Quantification of the findings in Figure 4c,d would be useful, as would the use of germline fish for these experiments if these are now available. If this is not possible, it would be helpful to document that the crispants used in these experiments lack pdgfrb:egfp pericytes at adult stages (this is only shown for 5 dpf larvae, in Extended Data Figure 4b).

Adult mutants clearly show less dye leakage in the more superficial capillary regions than WT siblings, but dextran intensity is a bit higher, although this could well be diffusion from more central brain regions where overt hemorrhage is occurring. Along similar lines though, the authors' TEM data in Extended Data Figure 4d hints that there may be more caveolae in mutant brain capillaries, although the N number was lower here than for the measurements from TEM of larger central vessels (Figure 4g). It would be useful to carry out additional measurements to increase the N number in Figure 4d to see whether the difference between wild-type sibling and mutant capillary caveolae numbers remains as not significant.

It might be helpful to include some orienting labels and/or additional descriptions in the figure legends to help readers who are not used to looking at zebrafish brain vessels have an easier time figuring out what they are looking at and where it is in the brain.

Reviewer #3 (Public review):

Summary:

This manuscript describes an extensive application of the Yeast (SATAY) transposon mutagenesis and sequencing method to explore loss- and gain-of-function mutations conferring resistance to 20 different antifungal compounds. Impressively, the authors demonstrate that SATAY can be used to identify mutations that lead to antifungal resistance, including promoter mutations that include the direct targets of antifungal compounds and drug efflux pumps. Because SATAY is not tied to a specific genetic background, the sensitivity of an S. cerevisiae strain, AD1-8, that specifically displays Chitosan susceptibility was examined in detail, and the results suggest that Chitosan acts through interactions with the fungal cell wall. Through a series of experiments that expand upon SATAY analysis, the novel antifungal ATI-2307, the authors clearly show that the transporter Hol1 concentrates this compound within yeast.

General Comments:

This is a very impressive application of SATAY, highlighting many different strategies for exploring the mechanism of action of various antifungal compounds. It's clear from the findings presented that SATAY is a powerful and potentially highly productive approach for chemical-genetic analysis.

Reviewer #3 (Public review):

Summary:

This study examines prediction errors, information gain (Kullback-Leibler [KL] divergence), and uncertainty (entropy) from an information-theory perspective using two experimental tasks and pupillometry. The authors aim to test a theoretical proposal by Zénon (2019) that the pupil response reflects information gain (KL divergence). In particular, the study defines the prediction error in terms of KL divergence and speculates that changes in pupil size associated with KL divergence depend on entropy. Moreover, the authors examine the temporal characteristics of pupil correlates of prediction errors, which differed considerably across previous studies that employed different experimental paradigms. In my opinion, the study does not achieve these aims due to several methodological and theoretical issues.

Strengths:

(1) Use of an established Bayesian model to compute KL divergence and entropy.

(2) Pupillometry data preprocessing, including deconvolution.

Weaknesses:

(1) Definition of the prediction error in terms of KL divergence:

I'm concerned about the authors' theoretical assumption that the prediction error is defined in terms of KL divergence. The authors primarily refer to a review article by Zénon (2019): "Eye pupil signals information gain". It is my understanding that Zénon argues that KL divergence quantifies the update of a belief, not the prediction error: "In short, updates of the brain's internal model, quantified formally as the Kullback-Leibler (KL) divergence between prior and posterior beliefs, would be the common denominator to all these instances of pupillary dilation to cognition." (Zénon, 2019).

From my perspective, the update differs from the prediction error. Prediction error refers to the difference between outcome and expectation, while update refers to the difference between the prior and the posterior. The prediction error can drive the update, but the update is typically smaller, for example, because the prediction error is weighted by the learning rate to compute the update. My interpretation of Zénon (2019) is that they explicitly argue that KL divergence defines the update in terms of the described difference between prior and posterior, not the prediction error.

The authors also cite a few other papers, including Friston (2010), where I also could not find a definition of the prediction error in terms of KL divergence. For example [KL divergence:] "A non-commutative measure of the non-negative difference between two probability distributions." Similarly, Friston (2010) states: Bayesian Surprise - "A measure of salience based on the Kullback-Leibler divergence between the recognition density (which encodes posterior beliefs) and the prior density. It measures the information that can be recognized in the data." Finally, also in O'Reilly (2013), KL divergence is used to define the update of the internal model, not the prediction error.

The authors seem to mix up this common definition of the model update in terms of KL divergence and their definition of prediction error along the same lines. For example, on page 4: "KL divergence is a measure of the difference between two probability distributions. In the context of predictive processing, KL divergence can be used to quantify the mismatch between the probability distributions corresponding to the brain's expectations about incoming sensory input and the actual sensory input received, in other words, the prediction error (Friston, 2010; Spratling, 2017)."

Similarly (page 23): "In the current study, we investigated whether the pupil's response to decision outcome (i.e., feedback) in the context of associative learning reflects a prediction error as defined by KL divergence."

This is problematic because the results might actually have limited implications for the authors' main perspective (i.e., that the pupil encodes prediction errors) and could be better interpreted in terms of model updating. In my opinion, there are two potential ways to deal with this issue:

a) Cite work that unambiguously supports the perspective that it is reasonable to define the prediction error in terms of KL divergence and that this has a link to pupillometry. In this case, it would be necessary to clearly explain the definition of the prediction error in terms of KL divergence and dissociate it from the definition in terms of model updating.

b) If there is no prior work supporting the authors' current perspective on the prediction error, it might be necessary to revise the entire paper substantially and focus on the definition in terms of model updating.

(2) Operationalization of prediction errors based on frequency, accuracy, and their interaction:

The authors also rely on a more model-agnostic definition of the prediction error in terms of stimulus frequency ("unsigned prediction error"), accuracy, and their interaction ("signed prediction error"). While I see the point here, I would argue that this approach offers a simple approximation to the prediction error, but it is possible that factors like difficulty and effort can influence the pupil signal at the same time, which the current approach does not take into account. I recommend computing prediction errors (defined in terms of the difference between outcome and expectation) based on a simple reinforcement-learning model and analyzing the data using a pupillometry regression model in which nuisance regressors are controlled, and results are corrected for multiple comparisons.

(3) The link between model-based (KL divergence) and model-agnostic (frequency- and accuracy-based) prediction errors:

I was expecting a validation analysis showing that KL divergence and model-agnostic prediction errors are correlated (in the behavioral data). This would be useful to validate the theoretical assumptions empirically.

(4) Model-based analyses of pupil data:

I'm concerned about the authors' model-based analyses of the pupil data. The current approach is to simply compute a correlation for each model term separately (i.e., KL divergence, surprise, entropy). While the authors do show low correlations between these terms, single correlational analyses do not allow them to control for additional variables like outcome valence, prediction error (defined in terms of the difference between outcome and expectation), and additional nuisance variables like reaction time, as well as x and y coordinates of gaze.

Moreover, including entropy and KL divergence in the same regression model could, at least within each task, provide some insights into whether the pupil response to KL divergence depends on entropy. This could be achieved by including an interaction term between KL divergence and entropy in the model.

(5) Major differences between experimental tasks:

More generally, I'm not convinced that the authors' conclusion that the pupil response to KL divergence depends on entropy is sufficiently supported by the current design. The two tasks differ on different levels (stimuli, contingencies, when learning takes place), not just in terms of entropy. In my opinion, it would be necessary to rely on a common task with two conditions that differ primarily in terms of entropy while controlling for other potentially confounding factors. I'm afraid that seemingly minor task details can dramatically change pupil responses. The positive/negative difference in the correlation with KL divergence that the authors interpret to be driven by entropy may depend on another potentially confounding factor currently not controlled.

(6) Model validation:

My impression is that the ideal learner model should work well in this case. However, the authors don't directly compare model behavior to participant behavior ("posterior predictive checks") to validate the model. Therefore, it is currently unclear if the model-derived terms like KL divergence and entropy provide reasonable estimates for the participant data.

(7) Discussion:

The authors interpret the directional effect of the pupil response w.r.t. KL divergence in terms of differences in entropy. However, I did not find a normative/computational explanation supporting this interpretation. Why should the pupil (or the central arousal system) respond differently to KL divergence depending on differences in entropy?

The current suggestion (page 24) that might go in this direction is that pupil responses are driven by uncertainty (entropy) rather than learning (quoting O'Reilly et al. (2013)). However, this might be inconsistent with the authors' overarching perspective based on Zénon (2019) stating that pupil responses reflect updating, which seems to imply learning, in my opinion. To go beyond the suggestion that the relationship between KL divergence and pupil size "needs more context" than previously assumed, I would recommend a deeper discussion of the computational underpinnings of the result.

Reviewer #3 (Public review):

Summary:

This work aims to investigate how perceptual and attentional processes affect conscious access in humans. By using multivariate decoding analysis of electroencephalography (EEG) data, the authors explored the neural temporal dynamics of visual processing across different levels of complexity (local contrast, collinearity, and illusory perception). This is achieved by comparing the decidability of an illusory percept in matched conditions of perceptual (i.e., degrading the strength of sensory input using visual masking) and attentional impairment (i.e., impairing top-down attention using attentional blink, AB). The decoding results reveal three distinct temporal responses associated with the three levels of visual processing. Interestingly, the early stage of local contrast processing remains unaffected by both masking and AB. However, the later stage of collinearity and illusory percept processing are impaired by the perceptual manipulation but remained unaffected by the attentional manipulation. These findings contribute to the understanding of the unique neural dynamics of perceptual and attentional functions and how they interact with the different stages of conscious access.

Strengths:

The study investigates perceptual and attentional impairments across multiple levels of visual processing in a single experiment. Local contrast, collinearity, and illusory perception were manipulated using different configurations of the same visual stimuli. This clever design allows for the investigation of different levels of visual processing under similar low-level conditions.

Moreover, behavioural performance was matched between perceptual and attentional manipulations. One of the main problems when comparing perceptual and attentional manipulations on conscious access is that they tend to impact performance at different levels, with perceptual manipulations like masking producing larger effects. The study utilizes a staircasing procedure to find the optimal contrast of the mask stimuli to produce a performance impairment to the illusory perception comparable to the attentional condition, both in terms of perceptual performance (i.e., indicating whether the target contained the Kanizsa illusion) and metacognition (i.e., confidence in the response).

The results show a clear dissociation between the three levels of visual processing in terms of temporal dynamics. Local contrast was represented at an early stage (~80 ms), while collinearity and illusory perception were associated with later stages (~200-250 ms). Furthermore, the results provide clear evidence in support of a dissociation between the effects of perceptual and attentional processes on conscious access: while the former affected both neuronal correlates of collinearity and illusory perception, the latter did not have any effect on the processing of the more complex visual features involved in the illusion perception.

Weaknesses:

The design of the study and the results presented are very similar to those in Fahrenfort et al. (2017), reducing its novelty. Similar to the current study, Fahrenfort et al. (2017) tested the idea that if both masking and AB impact perceptual integration, they should affect the neural markers of perceptual integration in a similar way. They found that behavioural performance (hit/false alarm rate) was affected by both masking and AB, even though only the latter was significant in the unmasked condition. In contrast, an early classification peak was exclusively affected by masking. A later classification peak mirrored the behavioural findings, with classification performance impacted by both masking and AB.

The interpretation of the results primarily relies on the recurrent processing theory of consciousness (Lamme, 2020), which lead to the assumption that local contrast and illusory perception reflect feedforward and (lateral and feedback) recurrent connections, respectively. It should be mentioned, however, that this theoretical prediction is not directly tested in the study. Moreover, the evidence for the dissociation between illusion and collinearity in terms of lateral and feedback connections seems at least limited. For instance, Kok et al. (2016) found that, whereas bottom-up stimulation activated all cortical layers, feedback activity induced by illusory figures led to a selective activation of the deep layers. Lee & Nguyen (2001), instead, found that V1 neurons respond to illusory contours of the Kanizsa figures, particularly in the superficial layers. Although both studies reference feedback connections, neither provides clear evidence for the involvement of lateral connections.

The evidence in favour of primarily lateral connections driving collinearity seems mixed as well. On one hand, Liang et al. (2017) showed that feedback and lateral connections closely interact to mediate image grouping and segmentation. On the other hand, Stettler et al. (2002) showed that, whereas the intrinsic connections link similarly oriented domains in V1, V2 to V1 feedback displays no such specificity. Additionally, the other studies cited in the manuscript focused solely on lateral connections without examining feedback pathways, making it challenging to draw definitive conclusions.

Comments on revisions:

The authors have thoroughly addressed all my comments and provided comprehensive responses to each point raised.

Reviewer #3 (Public review):

The current manuscript investigates the effect of 2-oxoglutarate (2OG) as modulator of glutamine synthetase (GS). To do this, the authors rely of mass photometry, specific activity measurements and single particle cryo-EM data.<br /> From the results, the authors conclude that the GS from Methanosarcina mazei shifts from a dimeric, non-active state under low concentrations of 2OG, to a dodecameric and fully active complex at saturating concentrations of 2OG.

GS is a crucial enzyme in all domains of life. The dodecameric fold of GS is recurrent amongst prokaryotic and archaea organisms but the enzyme activity can be regulated in distinct ways. This is a very interesting work combining protein biochemistry with structural biology.

A novel role for 2OG is presented for this mesophilic methanoarchaeon, as a crucial effector for the enzyme oligomerization and full reactivity.

The conclusions of this paper are mostly well supported by data, but some aspects of this GS regulation and interaction with known partners like Glnk1 and sp26 need to be clarified and extended.

Reviewer #3 (Public review):

Summary:

Wang et al., examined the brain activity patterns during sleep, especially when locked to those canonical sleep rhythms such as SO, spindle, and their coupling. Analyzing data from a large sample, the authors found significant coupling between spindles and SOs, particularly during the upstate of the SO. Moreover, the authors examined the patterns of whole-brain activity locked to these sleep rhythms. To understand the functional significance of these brain activities, the authors further conducted open-ended cognitive state decoding and found a variety of cognitive processing may be involved during SO-spindle coupling and during other sleep events. The authors next investigated the functional connectivity analyses and found enhanced connectivity between the hippocampus, the thalamus, and the medial PFC. These results reinforced the theoretical model of sleep-dependent memory consolidation, such that SO-spindle coupling is conducive to systems-level memory reactivation and consolidation.

Strengths:

There are obvious strengths in this work, including the large sample size, state-of-the-art neuroimaging and neural oscillation analyses, and the richness of results.

Weaknesses:

Despite these strengths and the insights gained, there are weaknesses in the design, the analyses, and inferences.

A repeating statement in the manuscript is that brain activity could indicate memory reactivation and thus consolidation. This is indeed a highly relevant question that could be informed by the current data/results. However, an inherent weakness of the design is that there is no memory task before and after sleep. Thus, it is difficult (if not impossible) to make a strong argument linking SO/spindle/coupling-locked brain activity with memory reactivation or consolidation.

Relatedly, to understand the functional implications of the sleep rhythm-locked brain activity, the authors employed the "open-ended cognitive state decoding" method. While this method is interesting, it is rather indirect given that there were no behavioral indices in the manuscript. Thus, discussions based on these analyses are speculative at best. Please either tone down the language or find additional evidence to support these claims.

Moreover, the results from this method are difficult to understand. Figure 3e showed that for all three types of sleep events (SO, spindle, SO-spindle), the same mental states (e.g., working memory, episodic memory, declarative memory) showed opposite directions of activation (left and right panels showed negative and positive activation, respectively). How to interpret these conflicting results? This ambiguity is also reflected by the term used: declarative memory and episodic memories are both indexed in the results. Yet these two processes can be largely overlapped. So which specific memory processes do these brain activity patterns reflect? The Discussion shall discuss these results and the limitations of this method.

The coupling strength is somehow inconsistent with prior results (Hahn et al., 2020, eLife, Helfrich et al., 2018, Neuron). Specifically, Helfrich et al. showed that among young adults, the spindle is coupled to the peak of the SO. Here, the authors reported that the spindles were coupled to down-to-up transitions of SO and before the SO peak. It is possible that participants' age may influence the coupling (see Helfrich et al., 2018). Please discuss the findings in the context of previous research on SO-spindle coupling.

The discussion is rather superficial with only two pages, without delving into many important arguments regarding the possible functional significance of these results. For example, the author wrote, "This internal processing contrasts with the brain patterns associated with external tasks, such as working memory." Without any references to working memory, and without delineating why WM is considered as an external task even working memory operations can be internal. Similarly, for the interesting results on SO and reduced DMN activity, the authors wrote "The DMN is typically active during wakeful rest and is associated with self-referential processes like mind-wandering, daydreaming, and task representation (Yeshurun, Nguyen, & Hasson, 2021). Its reduced activity during SOs may signal a shift towards endogenous processes such as memory consolidation." This argument is flawed. DMN is active during self-referential processing and mind-wandering, i.e., when the brain shifts from external stimuli processing to internal mental processing. During sleep, endogenous memory reactivation and consolidation are also part of the internal mental processing given the lack of external environmental stimulation. So why during SO or during memory consolidation, the DMN activity would be reduced? Were there differences in DMN activity between SO and SO-spindle coupling events?

Reviewer #3 (Public review):

Summary:

The authors used powerful and novel reagents to carefully assess the roles of the voltage gated sodium channel (NaV) isoforms in regulating the neural excitability of principal neurons of the cerebral cortex. Using this approach, they were able to confirm that two different isoforms, NaV1.2 and NaV1.6 have distinct roles in electrogenesis of neocortical pyramidal neurons.

Strengths:

Development of very powerful transgenic mice in which NaV1.2 and/or NaV1.6 were modified to be insensitive to ASCs, a particular class of NaV blocker. This allowed them to test for roles of the two isoforms in an acute setting, without concerns of genetic or functional compensation that might result from a NaV channel knockout.

Careful biophysical analysis of ASC effects on different NaV isoforms.

Extensive and rigorous analysis of electrogenesis - action potential production - under conditions of blockade of either NaV1.2 or NaV1 or both.

Weaknesses:

Some results are overstated in that the representative example records provided do not directly support the conclusions.

Results from a computational model are provided to make predictions of outcomes, but the computational approach is highly underdeveloped.

Reviewer #3 (Public review):

Summary:

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

Strengths:

The work is interesting as it describes a novel mechanism by which cancer cells might influence CD8+ T cell activation and immunological synapse formation, and the authors have used a variety of cell biology and immunology methods to study this. However, there are some aspects of the paper that should be addressed more thoroughly to substantiate the conclusions made by the authors.

Weaknesses:

In Figure 2A-B, the authors show micrographs from live TIRF movies of HeLa and LB33-MEL cells stably expressing EndoA3-GFP and transiently expressing ICAM-1-mScarlet. The ICAM-1 signal appears diffuse across the plasma membrane while the EndoA3 signal is partially punctate and partially lining the edge of membrane patches. Previous studies of EndoA3-mediated endocytosis have indicated that this can be observed as transient cargo-enriched puncta on the cell surface. In the present study, there is only one example of such an ICAM-1 and EndoA3 positive punctate event. Other examples of overlapping signals between ICAM-1 and EndoA3 are shown, but these either show retracting ICAM-1 positive membrane protrusions or large membrane patches encircled by EndoA3. While these might represent different modes of EndoA3-mediated ICAM-1 internalization, any conclusion on this would require further investigation.

Moreover, in Figure 2C-E, uptake of the previously established EndoA3 endocytic cargo ALCAM is analyzed by quantifying total internal fluorescence in LB33-MEL cells of antibody labelled ALCAM following both overexpression and siRNA-mediated knockdown of EndoA3, showing increased and decreased uptake respectively. Why has not the same quantification been done for the proposed novel EndoA3 endocytic cargo ICAM-1? Furthermore, if endocytosis of ICAM-1 and ALCAM is diminished following EndoA3 knockdown, the expression level on the cell surface would presumably increase accordingly. This has been shown for ALCAM previously and should also be quantified for ICAM-1.

In Figure 4A the authors show micrographs from a live-cell Airyscan movie (Movie S6) of a CD8+ T cell incubated with HeLa cells stably expressing HLA-A*68012 and transiently expressing ICAM1-EGFP. From the movie, it seems that some ICAM-1 positive vesicles in one of the HeLa cells are moving towards the T cell. However, it does not appear like the T cell has formed a stable immunological synapse but rather perhaps a motile kinapse. Furthermore, to conclude that the ICAM-1 positive vesicles are transported toward the T cell in a polarized manner, vesicles from multiple cells should be tracked and their overall directionality should be analyzed. It would also strengthen the paper if the authors could show additional evidence for polarization of the cancer cells in response to T-cell interaction.

Finally, in Figures 4D-G, the authors show that the contact area between CD8+ T cells and LB33-MEL cells is increased in response to siRNA-mediated knockdown of EndoA3 and VPS26A. While this could be caused by reduced polarized delivery of ICAM-1 and ALCAM to the interface between the cells, it could also be caused by other factors such as increased cell surface expression of these proteins due to diminished endocytosis, and/or morphological changes in the cancer cells resulting from disrupted membrane traffic. More experimental evidence is needed to support the working model in Figure 4H.

Reviewer #3 (Public review):

Summary:

In the manuscript by Lapao et al., the authors uncover a role for the RAB27A effector protein SYTL5 in regulating mitochondrial function and turnover. The authors find that SYTL5 localizes to mitochondria in a RAB27A-dependent way and that loss of SYTL5 (or RAB27A) impairs lysosomal turnover of an inner mitochondrial membrane mitophagy reporter but not a matrix-based one. As the authors see no co-localization of GFP/mScarlet tagged versions of SYTL5 or RAB27A with LC3 or p62, they propose that lysosomal turnover is independent of the conventional autophagy machinery. Finally, the authors go on to show that loss of SYTL5 impacts mitochondrial respiration and ECAR and as such may influence the Warburg effect and tumorigenesis. Of relevance here, the authors go on to show that SYTL5 expression is reduced in adrenocortical carcinomas and this correlates with reduced survival rates.

Strengths:

There are clearly interesting and new findings here that will be relevant to those following mitochondrial function, the endocytic pathway, and cancer metabolism.

Weaknesses:

The data feel somewhat preliminary in that the conclusions rely on exogenously expressed proteins and reporters, which do not always align.

As the authors note there are no commercially available antibodies that recognize endogenous SYTL5, hence they have had to stably express GFP-tagged versions. However, it appears that the level of expression dictates co-localization from the examples the authors give (though it is hard to tell as there is a lack of any kind of quantitation for all the fluorescent figures). Therefore, the authors may wish to generate an antibody themselves or tag the endogenous protein using CRISPR.

In relation to quantitation, the authors found that SYTL5 localizes to multiple compartments or potentially a few compartments that are positive for multiple markers. Some quantitation here would be very useful as it might inform on function.

The authors find that upon hypoxia/hypoxia-like conditions that punctate structures of SYTL5 and RAB27A form that are positive for Mitotracker, and that a very specific mitophagy assay based on pSu9-Halo system is impaired by siRNA of SYTL5/RAB27A, but another, distinct mitophagy assay (Matrix EGFP-mCherry) shows no change. I think this work would strongly benefit from some measurements with endogenous mitochondrial proteins, both via immunofluorescence and western blot-based flux assays.

A really interesting aspect is the apparent independence of this mitophagy pathway on the conventional autophagy machinery. However, this is only based on a lack of co-localization between p62 or LC3 with LAMP1 and GFP/mScarlet tagged SYTL5/RAB27A. However, I would not expect them to greatly colocalize in lysosomes as both the p62 and LC3 will become rapidly degraded, while the eGFP and mScarlet tags are relatively resistant to lysosomal hydrolysis. -/+ a lysosome inhibitor might help here and ideally, the functional mitophagy assays should be repeated in autophagy KOs.

The link to tumorigenesis and cancer survival is very interesting but it is not clear if this is due to the mitochondrially-related aspects of SYTL5 and RAB27A. For example, increased ECAR is seen in the SYTL5 KO cells but not in the RAB27A KO cells (Fig.5D), implying that mitochondrial localization of SYTL5 is not required for the ECAR effect. More work to strengthen the link between the two sections in the paper would help with future directions and impact with respect to future cancer treatment avenues to explore.

Reviewer #3 (Public review):

Summary:

The authors have studied the mechanics of bolalipid and archaeal mixed-lipid membranes via comprehensive molecular dynamics simulations. The Cooke-Deserno 3-bead-per-lipid model is extended to bolalipids with 6 beads. Phase diagrams, bending rigidity, mechanical stability of curved membranes, and cargo uptake are studied. Effects such as the formation of U-shaped bolalipids, pore formation in highly curved regions, and changes in membrane rigidity are studied and discussed. The main aim has been to show how the mixture of bolalipids and regular bilayer lipids in archaeal membrane models enhances the fluidity and stability of these membranes.

Strengths:

The authors have presented a wide range of simulation results for different membrane conditions and conformations. For the most part, the analyses and their results are presented clearly and concisely. Figures, supplementary information, and movies very well present what has been studied. The manuscript is well-written and is easy to follow.

Major issues:

The Cooke-Deserno model, while very powerful for biophysical analysis of membranes at the mesoscale, is very much void of chemical information. It is parameterized such that it is good in producing fluid membranes and predicting values for bending rigidity, compressibility, and even thermal expansion coefficient falling in the accepted range of values for bilayer membranes. But it still represents a generic membrane. Now, the authors have suggested a similar model for the archaeal bolalipids, which have chemically different lipids (the presence of cyclopentane rings for one), and there is no good justification for using the same pairwise interactions between their representative beads in the coarse-grained model. This does not necessarily diminish the worth of all the authors' analyses. What is at risk here is the confusion between "what we observe this model of bolalipid- or mixed-membranes do" and "how real bolalipid-containing archaeal membranes behave at these mechanical and thermal conditions.".

Another more specific, major issue has to do with using the Hamm-Kozlov model for fitting the power spectrum of thermal undulations. The 1/q^2 term can very well be attributed to membrane tension. While a barostat is indeed used, have the authors made absolutely sure that the deviation from 1/q^4 behavior does not correspond to lateral tension? I got more worried when I noticed in the SI that the simulations had been done with combined "fix langevin" and "fix nph" LAMMPS commands. This combination does not result in a proper isothermal-isobaric ensemble. The importance of tilt terms for bolalipids is indeed very interesting, but I believe more care is needed to establish that.

This issue is reinforced when considering Figure 3B. These results suggest that increasing the fraction of regular lipids increases the tilt modulus, with the maximum value achieved for a normal Cooke-Deserno bilayer void of bolalipids. But this is contradictory. For these bilayers, we don't need the tilt modulus in the first place.

Also, from the SI, I gathered that the authors have neglected the longest wavelength mode because it is not equilibrated. If this is indeed the case, it is a dangerous thing to do, because with a small membrane patch, this mode can very well change the general trend of the power spectrum. As a lot of other analyses in the manuscript rely on these measurements, I believe more elaboration is in order.

The authors have found that "there is a strong dependency of the bending rigidity on the membrane mean curvature of stiffer bolalipids." The effect is negative, with the membrane becoming less stiff at higher mean curvatures. Why is that? I would assume that with more flexible bolalipids, the possibility of reorganization into U-shaped chains should affect the bending rigidity more (as Figure 2E suggests). While for a stiff bolalipid, not much would change if you increase the mean curvature. This should be either a tilt effect, or have to do with asymmetry between the leaflets. But on the other hand, the tilt modulus is shown to decrease with increasing bolalipid rigidity. The authors get back to this issue only on page 10, when they consider U-shaped lipids in the inner and outer leaflets and write, "this suggested that an additional membrane-curving mechanism must be involved." But then again, in the Discussion, the authors write, "It is striking that membranes made from stiffer bolalipids showed a curvature-dependent bending modulus, which is a clear signature that bolalipid membranes exhibit plastic behavior during membrane reshaping," adding to the confusion.

This issue is repeated when the authors study nanoparticle uptake. They write: "to reconcile these seemingly conflicting observations we reason that the bending rigidity, similar to Figure 2F, is not constant but softens upon increasing membrane curvature, due to dynamic change in the ratio between bolalipids in straight and U-shaped conformation. Hence, bolalipid membranes show stroking plastic behavior as they soften during reshaping." But the softening effect that they refer to, as shown in Figure 4B, occurs for very stiff bolalipids, for which not much switching to U-shaped conformation should occur.

Another major issue is with what the authors refer to as the "effective temperature". While plotting phase diagrams for kT/eps value is absolutely valid, I'm not a fan of calling this effective temperature. It is a dimensionless quantity that scales linearly with temperature, but is not a temperature. It is usually called a "reduced temperature". Then the authors refer to their findings as studying the stability of archaeal membranes at high temperatures. I have to disagree because eps is not the only potential parameter in the simulations (there are at least space exclusion and angle-bending stiffnesses) so one cannot identify changing eps with changing the global simulation temperature. This only works when you have one potential parameter, like an LJ gas.

Minor issues:

As the authors have noted, the fact that the membrane curvature can change the ratio of U-shaped to straight bolalipids would render the curvature elasticity non-linear (though the term "plastic" should not be used, as this is still structurally reversible when the stress is removed. Technically, it is hypoelastic behavior, possibly with hysteresis.) With this in mind, when the authors use essentially linear elastic models for fluctuation analysis, they should make a comparison of maximum curvatures occurring in simulations with a range that causes significant changes in bolalipid conformational ratios.

The Introduction section of the manuscript is written with a biochemical approach, with very minor attention to the simulation works on this system. Some molecular dynamics works are only cited as existing previous work, without mentioning what has already been studied in archaeal membranes. While some information, like the binding of ESCRT proteins to archaeal membranes, though interesting, helps little to place the study within the discipline. The Introduction should be revised to show what has already been studied with simulations (as the authors mention in the Discussion) and how the presented research complements it.

The authors have been a bit loose with using the term "stability". I'd like to see the distinction in each case, as in "chemical/thermal/mechanical/conformational stability".

In the original Cooke-Deserno model, a so-called "poorman's angle-bending term" is used, which is essentially a bond-stretching term between the first and third particle. However, I notice the authors using the full harmonic angle-bending potential. This should be mentioned.

The analysis of energy of U-shaped lipids with the linear model E=c_0 + c_1 * k_bola is indeed very interesting. I am curious, can this also be corroborated with mean energy measurements? The minor issue is calling the source of the favorability of U-shaped lipids "entropic", while clearly an energetic contribution is found. The two conformations, for example, might differ in the interactions with the neighboring lipids.

The authors write in the Discussion, "In any case, our results indicate that membrane remodelling, such as membrane fission during membrane traffic, is much more difficult in bolalipid membranes [34]." Firstly, I'm not sure if studying the dependence of budding behavior on adhesion energy with nanoparticles is enough to make claims about membrane fission. Secondly, why is the 2015 paper by Markus Deserno cited here?

In the SI, where the measurement of the diffusion coefficient is discussed, the expression for D is missing the power 2 of displacement.

Where cargo uptake is discussed, the term "adsorption energy" is used. I think the more appropriate term would be "adhesion energy".

Typos:<br /> Page 1, paragraph 2: Adaption → Adaptation.<br /> Page 10, paragraph 1: Stroking → Striking.

Reviewer #3 (Public review):

Summary:

The manuscript by Squiers and colleagues uncovers a Commander-independent function for COMMD3 in endosomal recycling. The authors identified COMMD3 as a regulator of endosomal recycling for GLUT4-SPR through unbiased genetic screens. Subsequently, the authors performed COMMD3 knockout experiments to assess endosomal morphology and trafficking, demonstrating that COMMD3 regulates endosomal trafficking in a Commander-independent manner. Furthermore, the authors identified and confirmed that the N-terminal domain (NTD) of COMMD3 interacts with the GTPase Arf1. Using structure-guided mutations, they demonstrated that the COMMD3-Arf1 interaction is critical for the Commander-independent function of COMMD3.

Overall, the manuscript presents compelling evidence for a Commander-independent role of COMMD3, and I agree with the author's interpretations. The manuscript uses a combination of genetic screening, microscopy, and structural and biochemical approaches to examine and support the conclusions. This is an excellent and intriguing study and I have only a few comments and suggestions to improve the manuscript further.

Reviewer #2 (Public review):

This manuscript addresses an important question which has not yet been solved in the field, what is the contribution of different gamma oscillatory inputs to the development of "theta sequences" in the hippocampal CA1 region. Theta sequences have received much attention due to their proposed roles in encoding short-term behavioral predictions, mediating synaptic plasticity, and guiding flexible decision making. Gamma oscillations in CA1 offer a readout of different inputs to this region and have been proposed to synchronize neuronal assemblies and modulate spike timing and temporal coding. However, the interactions between these two important phenomena have not been sufficiently investigated. The authors conducted place cell and local field potential (LFP) recordings in the CA1 region of rats running on a circular track. They then analyzed the phase locking of place cell spikes to slow and fast gamma rhythms, the evolution of theta sequences during behavior and the interaction between these two phenomena. They found that place cell with the strongest modulation by fast gamma oscillations were the most important contributors to the early development of theta sequences and that they also displayed a faster form of phase precession within slow gamma cycles nested with theta. The results reported are interesting and support the main conclusions of the authors. However, the manuscript needs significant improvement in several aspects regarding data analysis, description of both experimental and analytical methods and alternative interpretations, as I detail below.

• The experimental paradigm and recordings should be explained at the beginning of the Results section. Right now, there is no description whatsoever which makes it harder to understand the design of the study.<br /> • An important issue that needs to be addressed is the very small fraction of CA1 cells phased-locked to slow gamma rhythms (3.7%). This fraction is much lower than in many previous studies, that typically report it in the range of 20-50 %. However, this discrepancy is not discussed by the authors. This needs to be explained and additional analysis considered. One analysis that I would suggest, although there are also other valid approaches, is to, instead of just analyze the phase locking in two discrete frequency bands, to compute the phase locking will all LFP frequencies from 25-100 Hz. This will offer a more comprehensive and unbiased view of the gamma modulation of place cell firing. Alternative metrics to mean vector length that are less sensitive to firing rates, such as pairwise phase consistency index (Vinck et a., Neuroimage, 2010), could be implemented. This may reveal whether the low fraction of phase locked cells could be due to a low number of spikes entering the analysis.<br /> • From the methods, it is not clear to me whether the reference LFP channel was consistently selected to be a different one that where the spikes analyzed were taken. This is the better practice to reduce the contribution of spike leakage that could substantially inflate the coupling with faster gamma frequencies. These analyses need to be described in more detail.<br /> • The initial framework of the authors of classifying cells into fast gamma and not fast gamma modulated implies a bimodality that may be artificial. The authors should discuss the nuances and limitations of this framework. For example, several previous work has shown that the same place cell can couple to different gamma oscillations (e.g., Lastoczni et al., Neuron, 2016; Fernandez-Ruiz et al., Neuron, 2017; Sharif et al., Neuron,2021).<br /> • It would be useful to provide a more through characterization of the physiological properties of FG and NFG cells, as this distinction is the basis of the paper. Only very little characterization of some place cell properties is provided in Figure 5. Important characteristics that should be very feasible to compare include average firing rate, burstiness, estimated location within the layer (i.e., deep vs superficial sublayers) and along the transverse axis (i.e., proximal vs distal), theta oscillation frequency, phase precession metrics (given their fundamental relationship with theta sequences), etc.<br /> • It is not clear to me how the analysis in Figure 6 was performed. In Fig. 6B I would think that the grey line should connect with the bottom white dot in the third panel, which would the interpretation of the results.

Comments on revisions:

The authors have conducted new analysis to address the issues I and the other reviewers raised in our original revision. As a result, the revised manuscript has been substantially improved.

Reviewer #3 (Public review):

Summary:

The authors describe a new method for measuring DNA torsion in cells using the photoactivatable intrastrand cross-linker trimethyl psoralen (TMP). However, their method differs from previous TMP-based torsion mapping methods by comparing formaldehyde cross-linked and torsionally trapped chromatin to torsion-relieved (zero-torsion) chromatin in parallel. Comparison between the two datasets reveals a very slight difference, but enough to provide extremely high resolution genome-wide maps of torsion in the yeast genome. This direct comparison of the two maps confirms that blockage of TMP binding by nucleosomes and some DNA-binding proteins from TMP intercalation is a major complication of previous methods, and analysis of the data provides a glimpse of chromatin-based processes from within the DNA gyre.

Strengths:

In addition to providing direct evidence for the twin-supercoiled domain model and for torsional effects at transcription start (TSS) and end (TES) sites, the authors' analyses reveal some novel features of yeast higher-order structure. These include the cohesin-dependent anchoring of DNA loops at sites of positive supercoiling and the insulation of torsion between closely spaced divergent genes by general transcription factors, which implies that these factors resist free rotation. The fact that method should be generalizable to complex eukaryotic cells with large genomes, and the implications for understanding how torsion impacts transcription and gene regulation will be of substantial interest to a broad community.

Weaknesses:

No serious weaknesses.

Reviewer #3 (Public review):

Summary:

This intriguing paper addresses a special case of a fundamental statistical question: how to distinguish between stochastic point processes that derive from a single "state" (or single process) and more than one state/process. In the language of the paper, a "state" (perhaps more intuitively called a strategy/process) refers to a set of rules that determine the temporal statistics of the system. The rules give rise to probability distributions (here, the probability for turning events). The difficulty arises when the sampling time is finite, and hence, the empirical data is finite, and affected by the sampling of the underlying distribution(s). The specific problem being tackled is the foraging behavior of C. elegans nematodes, removed from food. Such foraging has been studied for decades, and described by a transition over time from 'local'/'area-restricted' search'(roughly in the initial 10-30 minutes of the experiments, in which animals execute frequent turns) to 'dispersion', or 'global search' (characterized by a low frequency of turns). The authors propose an alternative to this two-state description - a potentially more parsimonious single 'state' with time-changing parameters, which they claim can account for the full-time course of these observations.

Figure 1a shows the mean rate of turning events as a function of time (averaged across the population). Here, we see a rapid transient, followed by a gradual 4-5 fold decay in the rate, and then levels off. This picture seems consistent with the two-state description. However, the authors demonstrate that individual animals exhibit different "transition" statistics (Figure 1e) and wish to explain this. They do so by fitting this mean with a single function (Equations 1-3).

Strengths:

As a qualitative exercise, the paper might have some merit. It demonstrates that apparently discrete states can sometimes be artifacts of sampling from smoothly time-changing dynamics. However, as a generic point, this is not novel, and so without the grounding in C. elegans data, is less interesting.

Weaknesses:

(1) The authors claim that only about half the animals tested exhibit discontinuity in turning rates. Can they automatically separate the empirical and model population into these two subpopulations (with the same method), and compare the results?

(2) The equations consider an exponentially decaying rate of turning events. If so, Figure 2b should be shown on a semi-logarithmic scale.

(3) The variables in Equations 1-3 and the methods for simulating them are not well defined, making the method difficult to follow. Assuming my reading is correct, Omega should be defined as the cumulative number of turning events over time (Omega(t)), not as a "turn" or "reorientation", which has no derivative. The relevant entity in Figure 1a is apparently , i.e. the mean number of events across a population which can be modelled by an expectation value. The time derivative would then give the expected rate of turning events as a function of time.

(4) Equations 1-3 are cryptic. The authors need to spell out up front that they are using a pair of coupled stochastic processes, sampling a hidden state M (to model the dynamic turning rate) and the actual turn events, Omega(t), separately, as described in Figure 2a. In this case, the model no longer appears more parsimonious than the original 2-state model. What then is its benefit or explanatory power (especially since the process involving M is not observable experimentally)?

(5) Further, as currently stated in the paper, Equations 1-3 are only for the mean rate of events. However, the expectation value is not a complete description of a stochastic system. Instead, the authors need to formulate the equations for the probability of events, from which they can extract any moment (they write something in Figure 2a, but the notation there is unclear, and this needs to be incorporated here).

(6) Equations 1-3 have three constants (alpha and gamma which were fit to the data, and M0 which was presumably set to 1000). How does the choice of M0 affect the results?

(7) M decays to near 0 over 40 minutes, abolishing omega turns by the end of the simulations. Are omega turns entirely abolished in worms after 30-40 minutes off food? How do the authors reconcile this decay with the leveling of the turning rate in Figure 1a?

(8) The fit given in Figure 2b does not look convincing. No statistical test was used to compare the two functions (empirical and fit). No error bars were given (to either). These should be added. In the discussion, the authors explain the discrepancy away as experimental limitations. This is not unreasonable, but on the flip side, makes the argument inconclusive. If the authors could model and simulate these limitations, and show that they account for the discrepancies with the data, the model would be much more compelling. To do this, I would imagine that the authors would need to take the output of their model (lists of turning times) and convert them into simulated trajectories over time. These trajectories could be used to detect boundary events (for a given size of arena), collisions between individuals, etc. in their simulations and to see their effects on the turn statistics.

(9) The other figures similarly lack any statistical tests and by eye, they do not look convincing. The exception is the 6 anecdotal examples in Figure 2e. Those anecdotal examples match remarkably closely, almost suspiciously so. I'm not sure I understood this though - the caption refers to "different" models of M decay (and at least one of the 6 examples clearly shows a much shallower exponential). If different M models are allowed for each animal, this is no longer parsimonious. Are the results in Figure 2d for a single M model? Can Figure 2e explain the data with a single (stochastic) M model?

(10) The left axes of Figure 2e should be reverted to cumulative counts (without the normalization).

(11) The authors give an alternative model of a Levy flight, but do not give the obvious alternative models:<br /> a) the 1-state model in which P(t) = alpha exp (-gamma t) dt (i.e. a single stochastic process, without a hidden M, collapsing equations 1-3 into a single equation).<br /> b) the originally proposed 2-state model (with 3 parameters, a high turn rate, a low turn rate, and the local-to-global search transition time, which can be taken from the data, or sampled from the empirical probability distributions). Why not? The former seems necessary to justify the more complicated 2-process model, and the latter seems necessary since it's the model they are trying to replace. Including these two controls would allow them to compare the number of free parameters as well as the model results. I am also surprised by the Levy model since Levy is a family of models. How were the parameters of the Levy walk chosen?

(12) One point that is entirely missing in the discussion is the individuality of worms. It is by now well known that individual animals have individual behaviors. Some are slow/fast, and similarly, their turn rates vary. This makes this problem even harder. Combined with the tiny number of events concerned (typically 20-40 per experiment), it seems daunting to determine the underlying model from behavioral statistics alone.

(13) That said, it's well-known which neurons underpin the suppression of turning events (starting already with Gray et al 2005, which, strangely, was not cited here). Some discussion of the neuronal predictions for each of the two (or more) models would be appropriate.

(14) An additional point is the reliance entirely on simulations. A rigorous formulation (of the probability distribution rather than just the mean) should be analytically tractable (at least for the first moment, and possibly higher moments). If higher moments are not obtainable analytically, then the equations should be numerically integrable. It seems strange not to do this.

In summary, while sample simulations do nicely match the examples in the data (of discontinuous vs continuous turning rates), this is not sufficient to demonstrate that the transition from ARS to dispersion in C. elegans is, in fact, likely to be a single 'state', or this (eq 1-3) single state. Of course, the model can be made more complicated to better match the data, but the approach of the authors, seeking an elegant and parsimonious model, is in principle valid, i.e. avoiding a many-parameter model-fitting exercise.

As a qualitative exercise, the paper might have some merit. It demonstrates that apparently discrete states can sometimes be artifacts of sampling from smoothly time-changing dynamics. However, as a generic point, this is not novel, and so without the grounding in C. elegans data, is less interesting.

Reviewer #3 (Public review):

Summary:

In this set of experiments, the authors describe a novel research tool for studying complex cognitive tasks in mice, the HABITS automated training apparatus, and a novel "machine teaching" approach they use to accelerate training by algorithmically providing trials to animals that provide the most information about the current rule state for a given task.

Strengths:

There is much to be celebrated in an inexpensively constructed, replicable training environment that can be used with mice, which have rapidly become the model species of choice for understanding the roles of distinct circuits and genetic factors in cognition. Lingering challenges in developing and testing cognitive tasks in mice remain, however, and these are often chalked up to cognitive limitations in the species. The authors' findings, however, suggest that instead, we may need to work creatively to meet mice where they live. In some cases, it may be that mice may require durations of training far longer than laboratories are able to invest with manual training (up to over 100k trials, over months of daily testing) but the tasks are achievable. The "machine teaching" approach further suggests that this duration could be substantially reduced by algorithmically optimizing each trial presented during training to maximize learning.

Weaknesses:

Cognitive training and testing in rodent models fill a number of roles. Sometimes, investigators are interested in within-subjects questions - querying a specific circuit, genetically defined neuron population, or molecule/drug candidate, by interrogating or manipulating its function in a highly trained animal. In this scenario, a cohort of highly trained animals that have been trained via a method that aims to make their behavior as similar as possible is a strength.

However, often investigators are interested in between-subjects questions - querying a source of individual differences that can have long-term and/or developmental impacts, such as sex differences or gene variants. This is likely to often be the case in mouse models especially, because of their genetic tractability. In scenarios where investigators have examined cognitive processes between subjects in mice who vary across these sources of individual difference, the process of learning a task has been repeatedly shown to be different. The authors do not appear to have considered individual differences except perhaps as an obstacle to be overcome.

The authors have perhaps shown that their main focus is highly-controlled within-subjects questions, as their dataset is almost exclusively made up of several hundred young adult male mice, with the exception of 6 females in a supplemental figure. It is notable that these female mice do appear to learn the two-alternative forced-choice task somewhat more rapidly than the males in their cohort.

Considering the implications for mice modeling relevant genetic variants, it is unclear to what extent the training protocols and especially the algorithmic machine teaching approach would be able to inform investigators about the differences between their groups during training. For investigators examining genetic models, it is unclear whether this extensive training experience would mitigate the ability to observe cognitive differences, or select the animals best able to overcome them - eliminating the animals of interest. Likewise, the algorithmic approach aims to mitigate features of training such as side biases, but it is worth noting that the strategic uses of side biases in mice, as in primates, can benefit learning, rather than side biases solely being a problem. However, the investigators may be able to highlight variables selected by the algorithm that are associated with individual strategies in performing their tasks, and this would be a significant contribution.

A final, intriguing finding in this manuscript is that animal self-paced training led to much slower learning than "manual" training, by having the experimenter introduce the animal to the apparatus for a few hours each day. Manual training resulted in significantly faster learning, in almost half the number of trials on average, and with significantly fewer omitted trials. This finding does not necessarily argue that manual training is universally a better choice because it leads to more limited water consumption. However, it suggests that there is a distinct contribution of experimenter interactions and/or switching contexts in cognitive training, for example by activating an "occasion setting" process to accelerate learning for a distinct period of time. Limiting experimenter interactions with mice may be a labor-saving intervention, but may not necessarily improve performance. This could be an interesting topic of future investigation, of relevance to understanding how animals of all species learn.

Reviewer #3 (Public review):

Summary

In this work, the authors asked how mating experience impacts reward perception and processing. For this, they employ fruit flies as a model, with a combination of behavioral, immunostaining, and live calcium imaging approaches.

Their study allowed them to demonstrate that courtship failure decreases the fraction of flies motivated to eat sweet compounds, revealing a link between reproductive stress and reward-related behaviors. This effect is mediated by a small group of dopaminergic neurons projecting to the SEZ. After courtship failure, these dopaminergic neurons exhibit reduced activity, leading to decreased Gr5a+ neuron activity via Dop1R1 and Dop2R signaling, and leading to reduced sweet sensitivity. The authors therefore showed how mating failure influences broader behavioral outputs through suppression of the dopamine-mediated reward system and underscores the interactions between reproductive and reward pathways.

Concern

My main concern regarding this study lies in the way the authors chose to present their results. If I understood correctly, they provided evidence that mating failure induces a decrease in the fraction of flies exhibiting PER. However, they also showed that food consumption was not affected (Fig. 1, supplement), suggesting that individuals who did eat consumed more. This raises questions about the analysis and interpretation of the results. Should we consider the group as a whole, with a reduced sensitivity to sweetness, or should we focus on individuals, with each one eating more? I am also concerned about how this could influence the results obtained using live imaging approaches, as the flies being imaged might or might not have been motivated to eat during the feeding assays. I would like the authors to clarify their choice of analysis and discuss this critical point, as the interpretation of the results could potentially be the opposite of what is presented in the manuscript.

Reviewer #3 (Public review):

Summary:

Krishnan et al. present a novel contextual fear conditioning (CFC) paradigm using a virtual reality (VR) apparatus to evaluate whether conditioned context-induced freezing can be elicited in head-fixed mice. By combining this approach with two-photon imaging, the authors aim to provide high-resolution insights into the neural mechanisms underlying learning, memory, and fear. Their experiments demonstrate that head-fixed mice can discriminate between threat and non-threat contexts, exhibit fear-related behavior in VR, and show context-dependent variability during extinction. Supplemental analyses further explore alternative behaviors and the influence of experimental parameters, while hippocampal neuron remapping is tracked throughout the experiments, showcasing the paradigm's potential for studying memory formation and extinction processes.

Strengths:

Methodological Innovation: The integration of a VR-based CFC paradigm with real-time two-photon imaging offers a powerful, high-resolution tool for investigating the neural circuits underlying fear, learning, and memory.

Versatility and Utility: The paradigm provides a controlled and reproducible environment for studying contextual fear learning, addressing challenges associated with freely moving paradigms.

Potential for Broader Applications: By demonstrating hippocampal neuron remapping during fear learning and extinction, the study highlights the paradigm's utility for exploring memory dynamics, providing a strong foundation for future studies in behavioral neuroscience.

Comprehensive Data Presentation: The inclusion of supplemental figures and behavioral analyses (e.g., licking behaviors and variability in extinction) strengthens the manuscript by addressing additional dimensions of the experimental outcomes.

Weaknesses:

Characterization of Freezing Behavior: The evidence supporting freezing behavior as the primary defensive response in VR is unclear. Supplementary videos suggest the observed behaviors may include avoidance-like actions (e.g., backing away or stopping locomotion) rather than true freezing. Additional physiological measurements, such as EMG or heart rate, are necessary to substantiate the claim that freezing is elicited in the paradigm.

Analysis of Extinction: Extinction dynamics are only analyzed through between-group comparisons within each Recall day, without addressing within-group changes in behavior across days. Statistical comparisons within groups would provide a more robust demonstration of extinction processes.

Low Sample Sizes: Paradigm 1 includes conditions with very low sample sizes (N=1-3), limiting the reliability of statistical comparisons regarding the effects of shock number and intensity. Increasing sample sizes or excluding data from mice that do not match the conditions used in Paradigms 2 and 3 would improve the rigor of the analysis.

Potential Confound of Water Reward: The authors critique the use of reward in conjunction with fear conditioning in prior studies but do not fully address the potential confound introduced by using water reward during the training phase in their own paradigm.

Reviewer #3 (Public review):

Summary:

This paper by Esmaeili and co-authors presents a connectome prediction study to predict episodic memory and relate prediction errors to other phonotypic variables.

Strengths:

(1) A primary and external validation dataset.

(2) Novel use of prediction errors (i.e., brain-cognitive gap).

(3) A wide range of data was investigated.

Weaknesses:

(1) Lack of comparisons to other methods for prediction.

(2) Several different points are being investigated that don't allow any particular one to shine through.

(3) Some choices of analysis are not well-motivated.

(4) How do the n-back connectomes perform for prediction if the authors do not regress task activations from the n-back task?

(5) I am a little concerned about overfitting with the convolutional neural net. For example, the drop-off in prediction performance in the external sample is stark. How does the deep learning approach used here compare to something simpler, like a connectome-based predictive model or ridge regression?

(6) It may be nice to try the other models in the validation dataset. This would also provide a sense of the overfitting that may be going on with overfitting.

(7) While predictive models increase the power over association studies, they still require large samples to prevent overfitting. Do the authors have a sense of the power their main and external validation sample sizes provide?

(8) I am not sure that the Mann-Whitney is the correct test for comparing the distributions of prediction performances. The distributions are dependent on each other as they are each predicting the same outcomes. Using the typical degrees of freedom formula would overestimate the degrees of freedom.

(9) The brain cognition gap is interesting. It is very similar conceptually to the brain age gap. When associating the brain age gap with other phenotypes, typically age is regressed from the brain age gap and the other phenotype. In other words, age is typically associated with a brain age gap as individuals at the tail ages often show the largest gaps. Is the brain cognition gap correlated with episodic memory and do the group differences hold if episodic memory is controlled for?

(10) I have the same question for the dopamine results. Particularly, in the correlations that are divided by brain cognition gap sign. I could see these types of patterns arise due to a correlation with a third variable.

Reviewer #3 (Public review):

Summary:

This manuscript by Guo and colleagues features the documentation and interpretation of three successions of continental to marginal marine deposits spanning the P/T transition and their respective ichnofaunas. Based on these new data inferences concerning end-Permian mass extinction and Triassic recovery in the tropical realm are discussed.

Strengths:

The manuscript is well-written and organized and includes a large amount of new lithological and ichnological data that illuminate ecosystem evolution in a time of large-scale transition. The lithological documentations, facies interpretations, and ichnotaxonomic assignments look okay (with a few exceptions).

Weaknesses:

Some interpretations in Table 1 could be questioned: For facies association FA2 the interpretation as „terrestrial facies with periodical flooding" should be put into the right column and, given the fossil content, other interpretations, such as "marine facies" or "lagoonal environment" with some plant debris and (terrestrial) animal remains washed in, could also be possible. For FA3 the statement "bioturbation is absent" is in conflict with the next statement "strata are moderately reworked". For FA5 the observation of a "monospecific ichnoassemblage" contradicts the listing of several ichnotaxa.

Concerning the structure of the manuscript, certain hypotheses related to the end-Permian mass extinction and the process of the P/T extinction and recovery, namely the existence of a long-persisting "tropic dead zone" are introduced as a foregone conclusion to which the new data seemingly shall be fit as corroborating evidence. Some of the data - e.g. the presence of a supposedly Smithian-age ichnofauna are interpreted as a fast recovery shortening the duration of the "tropic dead zone" episode - but these interpretations could also be interpreted as contradicting the idea of a "dead zone" sensu stricto in favour of a "normal" post-extinction environment with low diversity and occurrence of typical disaster taxa. Due to their large error bars the early Triassic radiometric ages did not put much of a constraint on the age determination of the earliest post-extinction ichnofaunas discussed here.

Considering the somewhat equivocal evidence and controversial ideas about the P/T transition, the introduction could be improved by describing how the idea of a "tropic dead zone" arose against the background of earlier ideas, alternative views, and conflicting data. In the discussion section, alternative interpretations of the extensive data presented here - e.g. proximal-distal shifts in lithofacies with respect to the sediment source, sea level changes, preservation bias, the local occurrence of hostile environments instead of a regional scale, etc. should be discussed, also to avoid the impression that the author's conclusion was driven by confirmation bias.

Contrary to the authors' claim, Figures S7 and S8 suggest that burrow size does not vary much within the studied sections. Size decreases and increases in the Shichuanhe and Liulin sections do not contemporaneously, are usually within the error-bar range, and might be driven by ichnotaxa composition, i.e. the presence or absence of larger ichnotaxa, rather than by size changes in the same ichnotaxon (and producer group). Here the measurement data would be needed as well to check the basis of the authors' interpretations.

Some arthropod tracks assigned here to Kouphichnium might not represent limulid traces but other (non-marine) arthropod taxa in accordance with their occurrence in terrestrial facies/non-marine units of the succession. More generally, the ichnotaxonomy of arthropod trackways is not yet well reserved - beyond Kouphichnium and Diplichnites various similar-looking types may occur that can have a variety of distinct insect, crustacean, millipede, etc. producers (including larval stages).

Reviewer #3 (Public review):

In this manuscript, the authors use HiC to study the 3D genome of CD14+ CD16+ monocytes from the blood of healthy and those from patients with Alcohol-associated Hepatitis.

Overall, the authors perform a cursory analysis of the HiC data and conclude that there are a large number of changes in 3D genome architecture between healthy and AH patient monocytes. They highlight some specific examples that are linked to changes in gene expression. The analysis is of such a preliminary nature that I would usually expect to see the data from all figures in just one or two figures.

In addition, I have a number of concerns regarding the experimental design and the depth of the analyses performed that I think must be addressed.

(1) There is a myriad of literature that describes the existence of cell type-specific 3D genome architecture. In this manuscript, there is an assumption by the authors that the CD14+ CD16+ monocytes represent the same population from both healthy and diseased patients. Therefore, the authors conclude that the differences they see in the HiC data are due to disease-related changes in the equivalent cell types. However, I am concerned that the AH patient monocytes may have differentiated due to their environment so that they are in fact akin to a different cell type and the 3D genome changes they describe reflect this. This is supported by published articles for example: Dhanda et al., Intermediate Monocytes in Acute Alcoholic Hepatitis Are Functionally Activated and Induce IL-17 Expression in CD4+ T Cells. J Immunol (2019) 203 (12): 3190-3198, in which they show an increased frequency of CD14+ CD16+ intermediate monocytes in AH patients that are functionally distinct.

I suggest that if the authors would like to study the specific effects of AH on 3D genome architecture then they should carefully FACsort the equivalent monocyte populations from the healthy and AH patients.

(2) The analysis of the HiC data is quite preliminary. In the 3D genome field, it is usual to report the different scales of genome architecture, for example, compartments, topologically associated domains (TADs), and loops. I think that reporting this information and how it changes in AH patients in the appropriate cell types would be of great interest to the field.

Reviewer #3 (Public review):

Summary:

The study highlights how the initiation, reversal, and cessation of movements are linked to changes in beta synchronization within the basal ganglia-cortex loops. It was observed that different movement phases, such as starting, stopping briefly, and stopping completely, affect beta oscillations in the motor system.

It was found that unpredictable cues lead to stronger changes in STN-cortex beta coherence. Additionally, specific patterns of beta and gamma oscillations related to different movement actions and contexts were observed. Stopping movements was associated with a lack of the expected beta rebound during brief pauses within a movement sequence.

Overall, the results underline the complex and context-dependent nature of motor control and emphasize the role of beta oscillations in managing movement according to changing external cues.

Strengths:

The paper is very well written, clear and appears methodologically sound.

Although the use of continuous movement (turning) with reversals is more naturalistic than many previous button push paradigms.

Weaknesses:

The generalizability of the findings are somewhat curtailed by the fact that this was performed peri-operatively during the period of the microlesion effect. Given the availability of sensing enabled DBS devices now and HD-EEG, does MEG offer a significant enough gain in spatial localizability to offset the fact that it has to be done shortly postoperatively with externalized leads, with attendant stun effect? Specifically, for paradigms that are not asking very spatially localized questions as a primary hypothesis?

Further investigation of the gamma signal seems warranted, even though it has a slightly lower proportional change in amplitude in beta. Given that the changes in gamma here are relatively wide band, this could represent a marker of neural firing that could be interestingly contrasted against the rhythm account presented.

Comments on revisions: I congratulate the authors on their paper and their revisions and I have no further comments. I look forward to seeing the continuous analyses in the future. Good luck!

Reviewer #3 (Public review):

The authors tested whether: 1. The number of stimulus-stimulus pairings alters whether preconditioned fear depends on online integration during formation of the stimulus-outcome memory or during the probe test/mobilization phase, when the original stimulus, which was never paired with aversive events, elicits fear via chaining of stimulus-stimulus and stimulus-outcome memories. They found that sensory preconditioning was successful with either 8 or 32 stimulus-stimulus pairings. Perirhinal cortex NMDA receptor blockade during stimulus-outcome learning impaired preconditioning following 8 but not 32 pairings during preconditioning. Therefore, perirhinal cortex NMDA activity is required for online integration or mediated learning. Perirhinal-basolateral amygdala had nearly identical effects with the same interpretation: these areas communicate during stimulus-outcome learning, and this online communication is required for later expressing preconditioned fear. Disconnection prior to the probe test, when chaining might occur, had different effects: it impaired the expression of preconditioned fear in rats that received 32, but not 8, pairings during preconditioning. The study has several strengths and provides a thoughtful discussion of future experiments. The study is highly impactful and significant; the authors were successful in describing the behavioral and neurobiological mechanisms of mediated learning versus chaining in sensory preconditioning, which is often debated in the learning field. Therefore this study will have a significant impact on the behavioral neurobiology and learning fields.

Strengths:

Careful, rigorous experimental design and statistics

The discussion leaves open questions that are very much worth exploring. For example - why did perirhinal-amygdala disconnection prior to the probe have no effect in the 8-pairing group, when bilateral perirhinal inactivation did (in Wong et al, 2019)? The authors propose that perirhinal cortex outputs bypass the amygdala during the probe test, which is an excellent hypothesis to test.

The experiments are very explicitly hypothesis-driven, and the authors provide evidence of how and why mediated learning and chaining occur during sensory-sensory learning.

Reviewer #3 (Public review):

Summary:

Ishii et al used molecular genetics, behavioral analyses, in vivo neural activity imaging, and neural activity manipulations in mice to study the functional role of a subset of medial preoptic area (MPOA) neurons in the regulation of female sexual drive. They first employed a self-paced mating assay during which a female could control the amount of interaction time with a male to assess female sexual drive after completion of mating. The authors observed that after mating completion (i.e., male ejaculation) females spend significantly less time interacting with males, indicating that their sexual drive is reduced. Next, the authors performed a brain-wide analysis of neurons activated following male ejaculation and identified the MPOA as a strong candidate region. One caveat is that the activity labeling was not exclusive to neurons activated following male ejaculation but included all neurons activated before, during, and after the mating encounter. However, in this revised version of the manuscript, the authors have included a key control group that labels all neurons activated up to but not including male ejaculation. Comparison of the number of activated neurons in these two groups revealed a significant additional set of neurons in the female MPOA following ejaculation. Importantly, the authors also provided in vivo calcium imaging data showing that a subset of MPOA neurons responds significantly and specifically to male ejaculation and not other behaviors during the social encounter. The authors performed these studies in both excitatory and inhibitory populations of the MPOA. Their analysis identified a subpopulation of inhibitory neurons that exhibit sustained increased activity for 90 sec following male ejaculation. Finally, the authors used chemogenetics to activate MPOA neurons during home cage mating, condition place preference, pup retrieval, and the self-paced mating assay. They found that activation of female MPOA neurons that were previously activated following male ejaculation significantly reduces mating behaviors and time spent interacting with a male during the self-paced mating assay. Whereas, activation of female MPOA neurons that were previously activated during consummatory behaviors but not male ejaculation does not alter mating behaviors and time spent interacting with a male. Therefore, MPOA neurons activated following ejaculation are sufficient to suppress female sexual motivation.

The authors' experimental execution is rigorous and well performed. Their data identify inhibitory neurons in the female MPOA as a neural locus that is activated following male ejaculation and whose prolonged activity plays a key role in the regulation of female sexual motivation. The addition of some key control groups to this revised version of the manuscript greatly strengthens the interpretation of the authors' findings.

Strengths:

(1) The use of the self-paced mating assay in combination with neural imaging and manipulation to assess female sexual drive is innovative. The authors correctly assert that relatively little is known about how male ejaculation affects sexual motivation in females as compared to males. Therefore, the data collected from these studies is important and valuable.

(2) The authors provide convincing histological data and analyses to verify and validate their brain-wide activity labeling, neural imaging, and chemogenetic studies.

(3) The single cell in vivo calcium imaging data are well performed and analyzed. They provide key insights into the activity profiles of both excitatory and inhibitory neurons in the female MPOA during mating encounters. The authors identification of an inhibitory subpopulation of female MPOA neurons that is selectively activated following completion of mating is fundamental for future experiments which could potentially find a molecular marker for this population and specifically manipulate these neurons to understand their role in female sexual motivation in greater detail.

(4) The authors provide convincing evidence that activation of female MPOA neurons activated following male ejaculation is sufficient to suppress female sexual motivation. Importantly, the authors addition of the consummatory-hM3Dq group demonstrates that activation of female MPOA neurons activated during mating behaviors prior to male ejaculation is not sufficient to suppress female sexual motivation.

Weaknesses:

In this revised version of the manuscript, the authors have added important controls as well as additional clarifying text that adequately address the weaknesses that were present in the original version of the manuscript.

Reviewer #3 (Public review):

This manuscript presents a number of interesting findings that have the potential to increase our understanding of the mechanism underlying homeostatic synaptic plasticity (HSP). The data broadly support that Rab3A plays a role in HSP, although the site and mechanism of action remain uncertain.

The authors clearly demonstrate that Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength is already elevated. In this context, it is unclear if the plasticity is absent, already induced by this mutation, or just occluded by a ceiling effect due to the synapses already being strengthened. Occlusion may also occur in the mixed cultures when Rab3A is missing from neurons but not astrocytes. The authors do appropriately discuss these options. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between changes in synaptic strength and AMPA receptor trafficking during HSP, and conclude that trafficking may not be solely responsible for the changes in synaptic strength during HSP.

Strengths:

This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is likely only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms, including whether Rab3A is active pre-synaptically to regulate quantal amplitude.

As Rab3A is primarily known as a pre-synaptic molecule, this possibility is intriguing. However, it is based on the partial dissociation of AMPAR trafficking and synaptic response and lacks strong support. On average, they saw a similar magnitude of change in mEPSC amplitude and GluA2 cluster area and integral, but the GluA2 data was not significant due to higher variability. It is difficult to determine if this is due to biology or methodology - the imaging method involves assessing puncta pairs (GluA2/VGlut1) clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, with usually less than 20 synapses per neuron analyzed, which would be expected to be more variable than mEPSC recordings averaged across several hundred events. However, when they reduce the mEPSC number of events to similar numbers as the imaging, the mESPC amplitudes are still less variable than the imaging data. The reason for this remains unclear. The pool of sampled synapses is still different between the methods and recent data has shown that synapses have variable responses during HSP. Further, there could be variability in the subunit composition of newly inserted AMPARs, and only assessing GluA2 could mask this (see below). It is intriguing that pre-synaptic changes might contribute to HSP, especially given the likely localization of Rab3A. But it remains difficult to distinguish if the apparent difference in imaging and electrophysiology is a methodological issue rather than a biological one. Stronger data, especially positive data on changes in release, will be necessary to conclude that pre-synaptic factors are required for HSP, beyond the established changes in post-synaptic receptor trafficking.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a strong frequency effect that is unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. But the change in frequency seems to argue (as the authors do) that some synapses only have CP-AMPARs, while the rest of the synapses have few or none. Another possibility is that there are pre-synaptic NASPM-sensitive receptors that influence release probability. Further, the amplitude data show a strong trend towards smaller amplitude following NASPM treatment (Fig 3B). The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. The decrease on average is larger in the TTX neurons, and some cells show a strong effect. It is possible there is some heterogeneity between neurons on whether GluA1/A2 heteromers or GluA1 homomers are added during HSP. This would impact the conclusions about the GluA2 imaging as compared to the mEPSC amplitude data.

To understand the role of Rab3A in HSP will require addressing two main issues:

(1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role. The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. More concrete support for the authors' suggestion of a pre-synaptic site of control would be helpful.

(2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs or a decrease in GABA release (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at those synapses.

Reviewer #3 (Public review):

Huang et al. investigated the phenotype of Bend2 mutant mice which expressed truncated isoform. Bend2 deletion in male showed fertility and this enabled them to analyze the BEND2 function in females. They showed that Bend2 deletion in females showed decreasing follicle number which may lead to loss of ovarian reserve.

Strengths:

They found the truncated isoform of Bend2 and the depletion of this isoform showed decreasing follicle number at birth.

Weaknesses:

The authors showed novel factors that impact ovarian reserve. Although the number of follicles and conception rate are reduced in mutant mice, the in vitro fertilization rate is normal and follicles remain at 40 weeks of age. It is difficult to know how critical this is when applied to the human case.

Reviewer #3 (Public review):

Summary:

The authors performed a detailed single-cell analysis of the early embryonic cranial neural plate with unprecedented temporal resolution between embryonic days 7.5 and 8.75. They employed diffusion analysis to identify genes that correspond to different temporal and spatial locations within the embryo. Finally, they also examined the global response of cranial tissue to a Smoothened agonist.

Strengths:

Overall, this is an impressive resource, well-validated against sets of genes with known temporal and spatial patterns of expression. It will be of great value to investigators examining early stages of neural plate patterning, neural progenitor diversity, and the roles of signaling molecules and gene regulatory networks controlling regionalization and diversification of the neural plate.

Weaknesses:

The manuscript should be considered a resource. Experimental manipulation is limited to analysis of neural plate cells that were cultured in vitro for 12 hours with SAG. They have identified a significant set of previously unreported genes that are differentially expressed in the cranial neural plate. Some additional analyses might help to highlight novel hypotheses arising from this remarkable resource.

Comments on revisions: I am satisfied with the responses of the authors and do not have any further concerns.

Reviewer #3 (Public review):

Summary:

This study from Oriol et al. first uses transgenic animals to examine projection targets of specific subtypes of VTA GABA neurons (expressing PV, SST, MOR, or NTS). They follow this with a set of optogenetic experiments showing that VTA projection neurons (regardless of genetic subtype) make local functional connections within the VTA itself. Both of these findings are important advances in the field. Notably, both GABAergic and glutamatergic neurons in the VTA likely exhibit these combined long/short-range projections.

Strengths:

The main strength of this study is the series of optogenetic/electrophysiological experiments that provide detailed circuit connectivity of VTA neurons. The long-range projections to the VP (but not other targets) are also verified to have functional excitatory and inhibitory components. Overall, the experiments are well executed and the results are very relevant in light of the rapidly growing knowledge about the complexity and heterogeneity of VTA circuitry.

Another strength of this study is the well-written and thoughtful discussion regarding the current findings in the context of the long-standing question of whether the VTA does or does not have true interneurons.

Comments on revisions:

The authors have addressed all of my questions admirably, and the final result is considerably improved and remains a valuable contribution to the field.

Reviewer #3 (Public review):

Summary:

Mutations that result in consistent RAS activation constitute a major driver of cancer. Therefore, RAS is a favorable target for cancer therapy. However, since normal RAS activity is essential for the function of normal cells, a mechanism that differentiates aberrant RAS activity from normal one is required to avoid severe adverse effects. To this end, the authors designed and optimized a synthetic gene circuit that is induced by active RAS-GTP. The circuit components, such as RAS-GTP sensors, dimerization domains, and linkers. To enhance the circuit selectivity and dynamic range, the authors designed a synthetic promoter comprised of MAPK-responsive elements to regulate the expression of the RAS sensors, thus generating a feed-forward loop regulating the circuit components. Circuit outputs with respect to circuit design modification were characterized in standard model cell lines using basal RAS activity, active RAS mutants, and RAS inactivation.

This approach is interesting. The design is novel and could be implemented for other RAS-mediated applications. The data support the claims, and while this circuit may require further optimization for clinical application, it is an interesting proof of concept for targeting aberrant RAS activity.

Strengths:

Novel circuit design, through optimization and characterization of the circuit components, solid data.

Weaknesses:

This manuscript could significantly benefit from testing the circuit performance in more realistic cell lines, such as patient-derived cells driven by RAS mutations, as well as in corresponding non-cancer cell lines with normal RAS activity. Furthermore, testing with therapeutic output proteins in vitro, and especially in vivo, would significantly strengthen the findings and claims.

Reviewer #3 (Public review):

Summary:

The laboratory mouse is an ideal animal to study the neural and psychological underpinnings of social dominance behavior because of its economic cost and the animals' readiness to display dominant and subordinate behaviors in simple and testable environments. Here, a new and novel method for measuring dominance and the individual social status of mice is presented using a food competition assay. Historically, food competition assays have been avoided because they occur in an open arena or the home cage, and it can be difficult to assess who gets priority access to the resource and to avoid aggressive interactions such as bite wounding. Now, the authors have designed a narrow rectangular arena separated in half by a sliding floor-to-ceiling obstacle, where the mice placed at opposite sides of the obstacle compete by pushing the obstacle to gain priority access to a food pellet resting on the arena floor under the obstacle. One can also place the food pellet within the obstacle to restrict priority access to the food and measure the time or effort spent pushing the obstacle back and forth. As hypothesized, the outcomes in the food competition test were significantly consistent with those of the more common tube test (space competition) and warm spot competition test. This suggests that these animals have a stereotypic dominance organization that exists across multiple resource domains (i.e., food, space, and temperature). Only male and female C57 mice in same-sex pairs or triads were tested.

Strengths:

The design of the apparatus and the inclusion of females are significant strengths within the study.

Weaknesses:

There are at least two major weaknesses of the study: neglecting the value of test inconsistency and not providing the mice time to recognize who they are competing with.

Several studies have demonstrated that although inbred mice in laboratory housing share similar genetics and environment, they can form diverse types of hierarchical organizations (e.g., loose, stable, despotic, linear, etc.) and there are multiple resource domains in the home cage that mice compete over (e.g., space, food, water, temperature, etc.). The advantage of using multiple dominance assays is to understand the nuances of hierarchical organizations better. For example, some groups may have clear dominant and subordinate individuals when competing for food, but the individuals may "change or switch" social status when competing for space. Indeed, social relationships are dynamic, not static. Here, the authors have provided another test to measure another dimension of dominance: food competition. Rather than highlight this advantage, the authors highlight that the test is in agreement with the standard tube test and warm spot test and that C57 mice have stereotypic dominance across multiple domains. While some may find this great, it will leave many to continue using the tube test only (which measures the dimension of space competition) and avoid measuring food competition. If the reader looks at Figures 6E, F, and G they will see examples of inconsistency across the food competition test, tube test, and warm spot test in triads of mice. These groups are quite interesting and demonstrate the diversity of social dynamics in groups of inbred mice in highly standardized environmental conditions. Scientists interested in dominance should study groups that are consistent and inconsistent across multiple dimensions of dominance (e.g., space, food, mates, etc.).

Unlike the tube test and warm spot test, the food competition test presented here provides no opportunity for the animals to identify their opponent. That is, they cannot sniff their opponent's fur or anogenital region, which would allow them an opportunity to identify them individually. Thus, as the authors state, the test only measures psychological motivation to get a food reward. Notably, the outcome in the direct and indirect testing of food competition is in agreement, leaving many to wonder whether they are measuring the social relationship or the effort an individual puts forth in attaining a food reward regardless of the social opponent. Specifically, in the direct test, an individual can retrieve the food reward by pushing the obstacle out of the way first. In the indirect test, the animals cannot retrieve the reward and can only push the obstacle back and forth, which contains the reward inside. In Figure 4E, you can see that winners spent more time pushing the block in the indirect test. Thus, whether the test measures a social relationship or just the likelihood of gaining priority access to food is unclear. To rectify this issue, the authors could provide an opportunity for the animals to interact before lowering the obstacle and raising(?) a food reward. They may also create a very long one-sided apparatus to measure the amount of effort an individual mouse puts forth in the indirect test with only one individual - or any situation with just one mouse where the moving obstacle is not pushed back, and the animal can just keep pushing until they stop. This would require another experiment. It also may not tell us much more since it remains unclear whether inbred mice can individually identify one another (see https://doi.org/10.1098/rspb.2000.1057 for more details).

A minor issue is that the write-up of the history of food competition assays and female dominance research is inaccurate. Food competition assays have a long history since at least the 1950s and many people study female dominance now.

Food competition: https://doi.org/10.1080/00223980.1950.9712776, https://psycnet.apa.org/fulltext/1953-03267-001.pdf, https://doi.org/10.1016/j.bbi.2003.11.007, https://doi.org/10.1038/s41586-022-04507-5

Female dominance: https://doi.org/10.1016/0031-9384(87)90269-1, https://doi.org/10.1016/j.cub.2023.03.020, https://doi.org/10.1016/S0031-9384(01)00494-2, https://doi.org/10.1037/0735-7036.99.4.411

Reviewer #3 (Public review):

In this study, the authors used RNAscope and immunostaining to confirm the expression of RTN4RL2 RNA and protein in hair cells and spiral ganglia. Through RTN4RL2 gene knockout mice, they demonstrated that the absence of RTN4RL2 leads to an increase in the size of presynaptic ribbons and a depolarized shift in the activation of calcium channels in inner hair cells. Additionally, they observed a reduction in GluA2/3 AMPA receptors in postsynaptic neurons and identified additional "orphan PSDs" not paired with presynaptic ribbons. These synaptic alterations ultimately resulted in an increased hearing threshold in mice, confirming that the RTN4RL2 gene is essential for normal hearing. These data are intriguing as they suggest that RTN4RL2 contributes to the proper formation and function of auditory afferent synapses and is critical for normal hearing. However, a thorough understanding of the known or postulated roles of RTN4Rl2 is lacking.

While the conclusions of this paper are generally well supported by the data, several aspects of the data analysis warrant further clarification and expansion.

(1) A quantitative assessment is necessary in Figure 1 when discussing RNA and protein expression. It would be beneficial to show that expression levels are quantitatively reduced in KO mice compared to wild-type mice. This suggestion also applies to Figure 2-supplement 3.D, which examines expression levels.

(2) In Figure 2, the authors present a morphological analysis of synapses and discuss the presence of "orphan PSDs." I agree that Homer1 not juxtaposed with Ctbp2 is increased in KO mice compared to the control group. However, in quantifying this, they opted to measure the number of Homer1 juxtaposed with Ctbp2 rather than directly quantifying the number of Homer1 not juxtaposed with Ctbp2. Quantifying the number of Homer1 not juxtaposed with Ctbp2 would more clearly represent "orphan PSDs" and provide stronger support for the discussion surrounding their presence.

(3) In Figure 2, Supplementary 3, the authors discuss GluA2/3 puncta reduction and note that Gria2 RNA expression remains unchanged. However, there is an issue with the lack of quantification for Gria2 RNA expression. Additionally, it is noted that RNA expression was measured at P4. While the timing for GluA2/3 puncta assessment is not specified, if it was assessed at 3 weeks old as in Figure 2's synaptic puncta analysis, it would be inappropriate to link Gria2 RNA expression with GluA2/3 protein expression at P4. If RNA and protein expression were assessed at P4, please indicate this timing for clarity.

(4) In Figure 3, the authors indicate that RTN4RL2 deficiency reduces the number of type 1 SGNs connected to ribbons. Given that the number of ribbons remains unchanged (Figure 2), it is important to clearly explain the implications of this finding. It is already known that each type I SGN forms a single synaptic contact with a single IHC. The fact that the number of ribbons remains constant while additional "orphan PSDs" are present suggests that the overall number of SGNs might need to increase to account for these findings. An explanation addressing this would be helpful.

(5) In Figure 4F and 5Cii, could you clarify how voltage sensitivity (k) was calculated? Additionally, please provide an explanation for the values presented in millivolts (mV).

(6) In Figure 6, the author measured the threshold of ABR at 2-4 months old. Since previous figures confirming synaptic morphology and function were all conducted on 3-week-old mice, it would be better to measure ABR at 3 weeks of age if possible.

Reviewer #3 (Public review):

Summary:

The rete ovarii (RO) has long been disregarded as a non-functional structure within the ovary. In their study, Anbarci and colleagues have delineated the markers and developmental dynamics of three distinct regions of the RO - the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). Notably focusing on the EOR, the authors presented evidence illustrating that the EOR forms a convoluted tubular structure culminating in a dilated tip. Intriguingly, microinjections into this tip revealed luminal flow towards the ovary containing potentially secreted functional proteins. Additionally, the EOR cells exhibit associations with vasculature, macrophages, and neuronal projections, proposing the notion that the RO may play a functional role in ovarian development during critical ovariogenesis stages. By identifying marker genes within the RO, the authors have also suggested that the RO could serve as a potential structure linking the ovary with the neuronal system.

Strengths:

Overall, the reviewer commends the authors for their systematic research on the RO, shedding light on this overlooked structure in developing ovaries. Furthermore, the authors have proposed a series of hypotheses that are both captivating and scientifically significant, with the potential to reshape our understanding of ovarian development through future investigations.

Weaknesses:

Although the manuscript lacks conclusive data to support many of its conclusions, the authors provide highly constructive discussions that offer valuable insights for future research on the rete ovarii in the field.

Reviewer #3 (Public review):

In this study, the authors employ a mouse ES-derived "hemogenic gastruloid" model which they generated and which they claim to be able to deconvolute YS and AGM stages of blood production in vitro. This work could represent a valuable resource for the field. However, in general, I find the conclusions in this manuscript poorly supported by the data presented. Importantly, it isn't clear what exactly are the "YS" and the "AGM"-like stages identified in the culture and where is the data that backs up this claim. In my opinion, the data in this manuscript lack convincing evidence that can enable us to identify what kind of hematopoietic progenitor cells are generated in this system. Therefore, the statement that "our study has positioned the MNX1-OE target cell within the YS-EMP stage (line 540)" is not supported by the evidence presented in this study. Overall, the system seems to be very preliminary and requires further optimization before those claims can be made.

Specific comments below:

(1) The flow cytometric analysis of gastruloids presented in Figure 1 C-D is puzzling. There is a large % of c-Kit+ cells generated, but few VE-Cad+ Kit+ double positive cells. Similarly, there are many CD41+ cells, but very few CD45+ cells, which one would expect to appear toward the end of the differentiation process if blood cells are actually generated. It would be useful to present this analysis as consecutive gating (i.e. evaluating CD41 and CD45 within VE-Cad+ Kit+ cells, especially if the authors think that the presence of VE-Cad+ Kit+ cells is suggestive of EHT). The quantification presented in D is misleading as the scale of each graph is different.

(2) The imaging presented in Figure 1E is very unconvincing. C-Kit and CD45 signals appear as speckles and not as membrane/cell surfaces as they should. This experiment should be repeated and nuclear stain (i.e. DAPI) should be included.

(3) Overall, I am not convinced that hematopoietic cells are consistently generated in these organoids. The authors should sort hematopoietic cells and perform May-Grunwald Giemsa stainings as they did in Figure 6 to confirm the nature of the blood cells generated.

(4) The scRNAseq in Figure 2 is very difficult to interpret. Specific points related to this:<br /> - Cluster annotation in Figure 2a is missing and should be included.<br /> - Why do the heatmaps show the expression of genes within sorted cells? Couldn't the authors show expression within clusters of hematopoietic cells as identified transcriptionally (which ones are they? See previous point)? Gene names are illegible.<br /> - I see no expression of Hlf or Myb in CD45+ cells (Figure 2G). Hlf is not expressed by any of the populations examined (panels E, F, G). This suggests no MPP or pre-HSC are generated in the culture, contrary to what is stated in lines 242-245. (PMID 31076455 and 34589491).<br /> Later on, it is again stated that "hGx cells... lacked detection of HSC genes like Hlf, Gfi1, or Hoxa9" (lines 281-283). To me, this is proof of the absence of AGM-like hematopoiesis generated in those gastruloids.

(5) Mapping of scRNA-Seq data onto the dataset by Thambyrajah et al. is not proof of the generation of AGM HE. The dataset they are mapping to only contains AGM cells, therefore cells do not have the option to map onto something that is not AGM. The authors should try mapping to other publicly available datasets also including YS cells.

(6) Conclusions in Figure 3, named "hGx specify cells with preHSC characteristics" are not supported by the data presented here. Again, I am not convinced that hematopoietic cells can be efficiently generated in this system, and certainly not HSCs or pre-HSCs.<br /> - FACS analysis in 3A is again very unconvincing. I do not think the population identified as c-Kit+ CD144+ is real. Also, why not try gating the other way around, as commonly done (e.g. VE-Cad+ Kit+ and then CD41/CD45)?<br /> - The authors must have tried really hard, but the lack of short- or long-engraftment in a number of immunodeficient mouse models (lines 305-313) really suggests that no blood progenitors are generated in their system. I am not familiar with the adrenal gland transplant system, but it seems like a very non-physiological system for trying to assess the maturation of putative pre-HSCs. The data supporting the engraftment of these mice, essentially seen only by PCR and in some cases with a very low threshold for detection, are very weak, and again unconvincing. It is stated that "BFP engraftment of the Spl and BM by flow cytometry was very low level albeit consistently above control (Fig. S4E)" (lines 337-338). I do not think that two dots in a dot plot can be presented as evidence of engraftment.

(7) Given the above, I find that the foundations needed for extracting meaningful data from the system when perturbed are very shaky at best. Nevertheless, the authors proceed to overexpress MNX1 by LV transduction, a system previously shown to transform fetal liver cells, mimicking the effect of the t(7;12) AML-associated translocation. Comments on this section:<br /> - The increase in the size of the organoid when MNX1 is expressed is a very unspecific finding and not necessarily an indication of any hematopoietic effect of MNX1 OE.<br /> - The mild increase of cKit+ cells (Figure 4E) at the 144hr timepoint and the lack of any changes in CD41+ or CD45+ cells suggests that the increase in Kit+ cells % is not due to any hematopoietic effect of MNX1 OE. No hematopoietic GO categories are seen in RNA seq analysis, which supports this interpretation. Could it be that just endothelial cells are being generated?

(8) There seems to be a relatively convincing increase in replating potential upon MNX1-OE, but this experiment has been poorly characterized. What type of colonies are generated? What exactly is the "proportion of colony forming cells" in Figures 5B-D? The colony increase is accompanied by an increase in Kit+ cells; however, the flow cytometry analysis has not been quantified.

(9) Do hGx cells engraft upon MNX1-OE? This experiment, which appears not to have been performed, is essential to conclude that leukemic transformation has occurred.

Reviewer #3 (Public review):

Genetic manipulation of Leishmania has some challenges, including some limitations in the DNA repair strategies that are present in the organism and the absence of RNA interference in many species. The senior author has contributed significantly to expanding the available routes towards Leishmania genetic manipulation by developing and adapting CRISPR-Cas9 tools to allow gene manipulation via DNA double strand break repair and, more recently, base modification. This work seeks to improve on some limitations in the tools previously described for the latter approach of base modification leading to base change.

The work in the paper is meticulously described, with solid evidence for the improvements that are claimed: Fig.1 clearly describes reduced impairment in growth of parasites expressing sgRNAs via changes in promoters; Figs.2 and 3 compellingly document the usefulness of using AsCas12a for integration after transformation; Figs.1 and 4 demonstrate the capacity of the combined modifications to efficiently edit a gene in three different Leishmania species; and Fig. 5 shows that this approach can be conducted at scale, providing a means of assessing the fitness of mutant pools. There is little doubt these new tools will be adopted by the Leishmania community, adding to the growing arsenal of approaches for genetic manipulation.

Two weaknesses suggested in the initial submission have been completely addressed.

Reviewer #3 (Public review):

Summary:

The manuscript by Chen and colleagues explores the connections from cerebellar purkinje cells to various brainstem nuclei. They combine two methods - presynaptic puncta labeling as putative presynaptic markers, and optogenetics, to test the anatomical projections and functional connectivity from purkinje cells onto a variety of brainstem nuclei. Overall, their study provides an atlas of sorts of purkinje cell connectivity to the brainstem, which includes a critical analysis of some of their own data from another publication. Overall, the value of this work is to both provide neural substrates by which purkinje cells may influence the brainstem and subsequent brain regions independent of the deep cerebellar nuclei, and also, to provide a critical analysis of viral-based methods to explore neuronal connectivity.

Strengths:

The strengths lie in the simplicity of the study, the number of cells patched, and the relationship between the presence of putative presynaptic puncta and electrophysiological results. This type of study is important and should provide a foundation for future work exploring cerebellar inputs and outputs. Overall, I think that the critique of viral-based methods to define connectivity, and a more holistic assessment of what connectivity is and how it should be defined is timely and warranted, as I think this is under-appreciated by many groups and overall, there is a good deal of research being published that do not properly consider the issues that this manuscript raises about what viral-based connectivity maps do and do not tell us.

Weaknesses:

While I overall liked the manuscript, I do have a few concerns which relate to interpretation of results, and discussion of technological limitations. The main concerns I have relate to the techniques that the authors use, and an insufficient discussion of their limitations. The authors use a Cre-dependent mouse line that expresses a synaptophysin-tdtomato marker, which the authors confidently state is a marker of synapses. This is misleading. Synaptophysin is a vesicle marker, and as such, labels axons, where vesicles are present in transit, and likely cell bodies where the protein is being produced. As such, the presence of tdtomato should not be interpreted definitively as the presence of a synapse. The use of vGAT as a marker, while this helps to constrain the selection of putative pre-synaptic sites, is also a vesicle marker and will likely suffer the same limitations (though in this case the expression is endogenous and not driven by the ROSA locus). A more conservative interpretation of the data would be that the authors are assessing putative pre-synaptic sites with their analysis. This interpretation is wholly consistent with their findings showing the presence of tdtomato in some regions but only sparse connectivity - this would be expected in the event that axons are passing through. If the authors wish to strongly assert that they are specifically assessing synapses, a marker better restricted to synapses and not vesicles may be more appropriate.

Similarly, while optogenetics/slice electrophysiology remains the state of the art for assessing connectivity between cell populations, it is not without limitations. For example, connections that are not contained within the thickness of the slice (here, 200 um, which is not particularly thick for slice ephys preps) will not be detected. As such, the absence of connections are harder to interpret than the presence of connections. Slices were only made in the coronal plane, which means if that if there is a particular topology to certain connections that is orthogonal to that plane, those connections may be under-represented. As such, all connectivity analyses likely are under-representations of the actual connectivity that exists in the intact brain. Therefore, perhaps the authors should consider revising their assessments of connections, or lack thereof, of purkinje cells to e.g., LC cells. While their data do make a compelling case that the connections between purkinje cells and LC cells are not particularly strong or numerous, especially compared to other nearby brainstem nuclei, their analyses do indicate that at least some such connections do exist. Thus, rather than saying that the viral methods such as rabies virus are not accurate reflections of connectivity - perhaps a more circumspect argument would be that the quantitative connectivity maps reported by other groups using rabies virus do not always reflect connectivity defined by other means e.g., functional connections with optogenetics. In some cases the authors do suggest this (e.g., "Together, these findings indicate that reliance on anatomical tracing experiments alone is insufficient to establish the presence and important of a synaptic connection"), but in other cases they are more dismissive of viral tracing results (e.g., "it further suggests that these neurons project to the cerebellum and were not retrogradely labeled"). Furthermore, some statements are a bit misleading e.g., mentioning that rabies methods are critically dependent on starter cell identity immediately following the citation of studies mapping inputs onto LC cells. While in general this claim has merit, the studies cited (19-21) use Dbh-Cre to define LC-NE cells which does have good fidelity to the cells of interest in the LC. Therefore, rewording this section in order to raise these issues generally without proximity to the citations in the previous sentence may maintain the authors' intention without suggesting that perhaps the rabies studies from LC-NE cells that identified inputs from purkinje cells were inaccurate due to poor fidelity of the Cre line. Overall, this manuscript would certainly not be the first report indicating that rabies virus does not provide a quantitative map of input connections. In my opinion this is still under-appreciated by the broad community and should be explicitly discussed. Thus, an acknowledgement of previous literature on this topic and how their work contributes to that argument is warranted.

Comments on revisions:

The responses the authors offer in theory are good, but they still use terms such as synapses and putative presynaptic boutons relatively interchangeably - if the authors make the correction to the more conservative terminology, which I think better reflects the data, this should be more consistent throughout the manuscript.

Reviewer #3 (Public review):

Lloyd, Xia et al. utilised the existence of surface-dwelling and cave-dwelling morphs of Astyanax mexicanus to explore a proposed link between DNA damage, aging, and the evolution of sleep. Key to this exploration is the behavioural and physiological differences between cavefish and surface fish, with cavefish having been previously shown to have low levels of sleep behaviour, along with metabolic alterations (for example chronically elevated blood glucose levels) in comparison to fish from surface populations. Sleep deprivation, metabolic dysfunction and DNA damage are thought to be linked, and to all contribute to aging processes. Given that cavefish seem to show no apparent health consequences of low sleep levels, the authors suggest that they have evolved resilience to sleep loss. Furthermore, as extended wake and loss of sleep is associated with increased rates of damage to DNA (mainly double-strand breaks) and sleep is linked to repair of damaged DNA, the authors propose that changes in DNA damage and repair might underlie the reduced need for sleep in the cavefish morphs relative to their surface-dwelling conspecifics.

To fulfil their aim of exploring links between DNA damage, aging, and the evolution of sleep, the authors employ methods that are largely appropriate, and comparison of cavefish and surface fish morphs from the same species certainly provides a lens by which cellular, physiological and behavioural adaptations can be interrogated. Fluorescence and immunofluorescence are used to measure gut reactive oxygen species and markers of DNA damage and repair processes in the different fish morphs, and measurements of gene expression and protein levels are appropriately used. However, although the sleep tracking and quantification employed is quite well established, issues with the experimental design relating to attempts to link induced DNA damage to sleep regulation (outlined below). Moreover, although the methods used are appropriate for the study of the questions at hand, there are issues with the interpretation of the data and with these results being over-interpreted as evidence to support the paper's conclusions.

This study shows that a marker of DNA repair molecular machinery that is recruited to DNA double-strand breaks (γH2AX) is elevated in brain cells of the cavefish relative to the surface fish, and that reactive oxygen species are higher in most areas of the digestive tract of the cavefish than in that of the surface fish. As sleep deprivation has been previously linked to increases in both these parameters in other organisms (both vertebrates and invertebrates), their elevation in the cavefish morph is taken to indicated that the cavefish show signs of the physiological effects of chronic sleep deprivation.

It has been suggested that induction of DNA damage can directly drive sleep behaviour, with a notable study describing both the induction of DNA damage and an increase in sleep/immobility in zebrafish (Danio rerio) larvae by exposure to UV radiation (Zada et al. 2021 doi:10.1016/j.molcel.2021.10.026). In the present study, an increase in sleep/immobility is induced in surface fish larvae by exposure to UV light, but there is no effect on behaviour in cavefish larvae. This finding is interpreted as representing a loss of a sleep-promoting response to DNA damage in the cavefish morph. However, induction of DNA damage is not measured in this experiment, so it is not certain if similar levels of DNA damage are induced in each group of intact larvae, nor how the amount of damage induced compares to the pre-existing levels of DNA damage in the cavefish versus the surface fish larvae. In both this study with A. mexicanus surface morphs and the previous experiments from Zada et al. in zebrafish, observed increases in immobility following UV radiation exposure are interpreted as following from UV-induced DNA damage. However, in interpreting these experiments it is important to note that the cavefish morphs are eyeless and blind. Intense UV radiation is aversive to fish, and it has previously been shown in zebrafish larvae that (at least some) behavioural responses to UV exposure depend on the presence of an intact retina and UV-sensitive cone photoreceptors (Guggiana-Nilo and Engert, 2016, doi:10.3389/fnbeh.2016.00160). It is premature to conclude that the lack of behavioural response to UV exposure is in the cavefish is due to a difference response to DNA damage, as their lack of eyes will likely inhibit a response to the UV stimulus. Indeed, were the equivalent zebrafish experiment from Zada et al. to be repeated with mutant larvae fish lacking the retinal basis for UV detection it might be found that, in this case too, the effects of UV on behaviour are dependent on visual function. Such a finding should prompt a reappraisal of the interpretation that UV exposure's effects on fish sleep/locomotor behaviour are mediated by DNA damage. An additional note, relating to both Lloyd, Xia et al. and Zada et al., is that though increases in immobility are induced following UV exposure, in neither study have assays of sensory responsiveness been performed during this period. As a decrease in sensory responsiveness is a key behavioural criterion for defining sleep, it is therefore unclear that this post-UV behaviour is genuinely increased sleep as opposed to a stress-linked suppression of locomotion due to the intensely aversive UV stimulus. While it is true that behavioural immobility is used by many studies as a criterion to identify sleep in non-mammalian species, this is only fully appropriate when other elements of the behavioural criteria of sleep (e.g. reduced responsiveness to sensory stimuli, rapid reversibility, homeostatic regulation, circadian regulation) have been shown to be associated with these periods of behavioural quiescence. In both Lloyd, Xia et al. and Zada et al., only an increased immobility has been demonstrated, occurring at a period where the circadian clock would be promoting wake and natural homeostatic sleep drive would be expected to be at the low end of its normal range. At a minimum, testing sensory threshold would be advisable to ensure that the classification of this behaviour as sleep is accurate and to avoid the risk of being misled in the interpretation of these experiments.

The effects of UV exposure, in terms of causing damage to DNA, inducing DNA damage response and repair mechanisms, and in causing broader changes in gene expression are assessed in both surface and cavefish larvae, as well as in cell lines derived from these different morphs. Differences in the suite of DNA damage response mechanisms that are upregulated are shown to exist between surface fish and cavefish larvae, though at least some of this difference is likely to be due to differences gene expression that may exist even without UV exposure (this is discussed further below).

UV exposure induced DNA damage (as measured by levels of cyclobutene pyrimidine dimers) to a similar degree in cell lines derived from both surface fish and cave fish. However, γH2AX shows increased expression only in cells from the surface fish, suggesting an induction of an increased DNA repair response in these surface morphs, corroborated by their cells' increased ability to repair damaged DNA constructs experimentally introduced to the cells in a subsequent experiment. This "host cell reactivation assay" is a very interesting assay for measuring DNA repair in cell lines, but the power of this approach might be enhanced by introducing these DNA constructs into larval neurons in vivo (perhaps by electroporation) and by tracking DNA repair in living animals. Indeed, in such a preparation, the relationship between DNA repair and sleep/wake state could be assayed.

Comparing gene expression in tissues from young (here 1 year) and older (here 7-8 years) fish from both cavefish and surface fish morphs, the authors found that there are significant differences in the transcriptional profiles in brain and gut between young and old surface fish, but that for cavefish being 1 year old versus being 7-8 years old did not have a major effect on transcriptional profile. The authors take this as suggesting that there is a reduced transcriptional change occurring during aging and that the transcriptome of the cavefish is resistant to age-linked changes. This seems to be only one of the equally plausible interpretations of the results; it could also be the case that alterations in metabolic cellular and molecular mechanisms, and particularly in responses to DNA damage, in the cavefish mean that these fish adopt their "aged" transcriptome within the first year of life. This would mean that rather than the findings revealing that "the transcriptome of the cavefish is resilient to age-associated changes despite sleep loss, elevated ROS and elevated DNA damage", it would suggest that the cavefish transcriptome is sensitive to age-associated changes, potentially being driven by this low level of sleep, elevated reactive oxygen species, and elevated DNA damage. This alternative interpretation greatly changes the understanding of the present findings. One way in which the more correct interpretation could be determined would be by adding a further, younger group of fish to the comparison (perhaps a group in the age range of 1-3 months, relatively shortly after metamorphosis).

A major weakness of the study in its current form is the absence of sleep deprivation experiments to assay the effects of sleep loss on the cellular and molecular parameters in question. Without such experiments, the supposed link of sleep to the molecular, cellular and "aging" phenotypes remains tenuous. Although the argument might be made that the cavefish represent a naturally "sleep deprived" population, the cavefish in this study are not sleep deprived, rather they are adapted to a condition of reduced sleep relative to fish from surface populations. Comparing the effects of depriving fish from each morph on markers of DNA damage and repair, on gut reactive oxygen species, and on gene expression will be necessary to solidify any proposed link of these phenotypes to sleep.

A second important aspect that limits the interpretability and impact of this study is the absence of information about circadian variations in the parameters measured. A relationship between circadian phase, light exposure and DNA damage/repair mechanisms is known to exist in A. mexicanus and other teleosts, and for differences to exist between the cave and surface morphs in there phenomena (Beale et al. 2013, doi: 10.1038/ncomms3769). Although the present study mentions that their experiments do not align with these previous findings, they do not perform the appropriate experiments to determine if this such a misalignment is genuine. Specifically, Beale et al. 2013 showed that white light exposure drove enhanced expression of DNA repair genes (including cpdp which is prominent in the current study) in both surface fish and cavefish morphs, but that the magnitude of this change was less in the cave fish because they maintained an elevated expression of these genes in the dark, whereas darkness supressed the expression of these genes in the surface fish. If such a phenomenon is present in the setting of the current study, this would likely be a significant confound for the UV-induced gene expression experiments in intact larvae, and undermine the interpretation of the results derived from these experiments: as samples are collected 90 minutes after the dark-light transition (ZT 1.5) it would be expected that both cavefish and surface fish larvae should have a clear induction of DNA repair genes (including cpdp) regardless of 90s of UV exposure. The data in supplementary figure 3 is not sufficient to discount this potentially serious confound, as for larvae there is only gene expression data for timepoints from ZT2 to ZT 14, with all of these timepoints being in the light phase and not capturing any dynamics that would occur at the most important timepoints from ZT0-ZT1.5, in the relevant period after dark-light transition. Indeed, an appropriate control for this experiment would involve frequent sampling at least across 48 hours to assess light-linked and developmentally-related changes in gene expression that would occur in 5-6dpf larvae of each morph independently of the exposure to UV.<br /> On a broader point, given the effects of both circadian rhythm and lighting conditions that are thought to exist in A. mexicanus (e.g. Beale et al. 2013) experiments involving measurements of DNA damage and repair, gene expression, and reactive oxygen species etc. at multiple times across >1 24 hour cycle, in both light-dark and constant illumination conditions (e.g. constant dark) would be needed to substantiate the authors' interpretation that their findings indicate consistently altered levels of these parameters in the cave fish relative to the surface fish. Most of the data in this study is taken at only single timepoints.

In summary, the authors show that there are differences in gene expression, activity of DNA damage response and repair pathways, response to UV radiation, and gut reactive oxygen species between the Pachón cavefish morph and the surface morph of Astyanax mexicanus. However, the data presented does not make the precise nature of these differences very clear, and the interpretation of the results appears to be overly strong. Furthermore, the evidence of a link between these morph specific differences and sleep is unconvincing.

Comments on revisions:

I thank the authors for their engagement with the notes and recommendations I made in my original comments. I have no further recommendations to make here.

Reviewer #3 (Public review):

Summary:

In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected the other side of the brain.

Strengths:

The authors use a cutting-edge technique.

Weaknesses:

The two main messages of the manuscript indicated in the title are not supported by the data. The title gives two messages that relate to CA1 pyramidal neurons in behaving head-fixed mice: (1) synchronous ensembles are associated with theta (2) synchronous ensembles are not associated with ripples. The main problem with the work is that the theta and ripple signals were recorded using electrophysiology from the opposite hemisphere to the one in which the spiking was monitored. However, both rhythms exhibit profound differences as a function of location.

Theta phase changes with the precise location along the proximo-distal and dorso-ventral axes, and importantly, even reverses with depth. Because the LFP was recorded using a single-contact tungsten electrode, there is no way to know whether the electrode was exactly in the CA1 pyramidal cell layer, or in the CA1 oriens, CA1 radiatum, or perhaps even CA3 - which exhibits ripples and theta which are weakly correlated and in anti-phase with the CA1 rhythms, respectively. Thus, there is no way to know whether the theta phase used in the analysis is the phase of the local CA1 theta.

Although the occurrence of CA1 ripples is often correlated across parts of the hippocampus, ripples are inherently a locally-generated rhythm. Independent ripples occur within a fraction of a millimeter within the same hemisphere. Ripples are also very sensitive to the precise depth - 100 micrometers up or down, and only a positive deflection/sharp wave is evident. Thus, even if the LFP was recorded from the center of the CA1 pyramidal layer in the contralateral hemisphere, it would not suffice for the claim made in the title.

Reviewer #4 (Public review):

Summary:

Using immunostaining for the immediate early gene Fos, and employing TRAP2-mediated chemogenetic and optogenetic perturbations, the authors provide evidence that neurons in the preoptic hypothalamus, identified as 'POA-social neurons,' promote social behaviors in mice - particularly in socially isolated (or deprived) mice, who exhibit an increased motivation for social investigations.

Strengths:

The focus on female-female social interactions is a valuable contribution to the field, as these interactions are less studied and the underlying neural mechanisms are less understood. The authors should be commended for their comprehensive approach in performing and reporting multiple perturbation experiments, including optogenetics, chemogenetics, and ablation. The authors also deserve recognition for their thoughtful discussion of the nuances in the phenotypes observed across these various perturbation experiments.

Weaknesses:

A limitation of the paper, however, is the insufficient clarification of the specific functions of these POA-social neurons. In my interpretation of the results, the neurons may be crucial for motivated social behaviors in females and motivated mounting of females in males, regardless of whether the test mice are housed singly or in groups. For group-housed mice, the motivation to interact with stimulus mice was likely low in their behavioral paradigm, which may explain the reduced interactions observed in the resident-intruder assay and why these neurons were not tagged (TRAPed) in that setting. Tagging these neurons in singly housed mice following a social interaction, followed by imaging in a group setting where motivated social behaviors do occur, could elucidate whether these neurons are specifically activated during social interactions in socially deprived mice or are generally crucial for motivated social behaviors in any setting. I understand that such calcium imaging may be beyond the scope of this version of the paper, but incorporating these results in a future version would significantly enhance the paper's impact. Depending on the outcomes of such experiments, the title 'Short-term social isolation acts on hypothalamic neurons to promote social behaviors in a sex- and context-dependent manner' may need to be revised to more accurately reflect the findings.

Reviewer #3 (Public review):

In this manuscript, Masson, Scandola, et al investigate how interactions between megakaryocytes and the extracellular matrix contribute to the regulation of thrombopoiesis using primary murine bone marrow MK cultures, integrin B1/B3 knock-out mice, and high-resolution 2D and 3D imaging. They find that laminin and collagen iv create a 3D "cage" of ECM surrounding MKs and anchor them at the sinusoidal basement membrane, which contributes to MK maturation and proplatelet intravasation into circulation. Deletion of laminin a4 disrupts the localization of MKs and the endothelial basement membrane, reducing the number of MKs associated with the sinusoid while having no effect on MK-associated collagen IV. Deletion of B1/B3 integrin reduces the quantity, localization, and structural organization of multiple ECM components surrounding MKs, and reduces MK adhesion when subject to conditions of sinusoidal flow.

Further, using intravital microscopy of calvarial bone marrow and the pulmonary vasculature, they provide data suggesting that the stabilization of ECM around MKs (either in the BM or lung) prevents MKs from entering circulation as intact cells. Interestingly, deletion of B1 integrin reduces MK coverage in laminin y1, but deletion of both B1 and B3 independently results in increased MK intravasation into the sinusoidal space. Comparison of integrin KO MKs with GPVI KO MKs suggests that ECM cage formation, vessel adhesion, and intravasation are likely dependent on integrin activation/signaling rather than GPVI signals.

Further, they provide data that the balance of ECM synthesis and degradation is essential for MK maturation and also provide data showing that inhibition of ECM turnover (in vivo inhibition of MMPs) results in increased ECM cage components that correspond with reduced MK maturation, and reduced demarcation membrane development.

The conclusions of the paper are supported by the data, but there are some areas that would benefit from clarification or expansion.

(1) The data linking ECM cage formation to MK maturation raises several interesting questions. As the authors mention, MKs have been suggested to mature rapidly at the sinusoids, and both integrin KO and laminin KO MKs appear mislocalized away from the sinusoids. Additionally, average MK distances from the sinusoid may also help separate whether the maturation defects could be in part due to impaired migration towards CXCL12 at the sinusoid. Presumably, MKs could appear mislocalized away from the sinusoid given the data presented suggesting they leaving the BM and entering circulation. Additional data or commentary on intrinsic (ex-vivo) MK maturation phenotypes may help strengthen the author's conclusions and shed light on whether an essential function of the ECM cage is integrin activation at the sinusoid.

(2) The data demonstrating intact MKs inter circulation is intriguing - can the authors comment or provide evidence as to whether MKs are detectable in blood? A quantitative metric may strengthen these observations.

(3) Supplementary Figure 6 - shows no effect on in vitro MK maturation and proplt, or MK area - But Figures 6B/6C demonstrate an increase in total MK number in MMP-inhibitor treated mice compared to control. Some additional clarification in the text may substantiate the author's conclusions as to either the source of the MMPs or the in vitro environment not fully reflecting the complex and dynamic niche of the BM ECM in vivo.

(4) Similarly, one function of the ECM discussed relates to MK maturation but in the B1/3 integrin KO mice, the presence of the ECM cage is reduced but there appears to be no significant impact upon maturation (Supplementary Figure 4). By contrast, MMP inhibition in vivo (but not in vitro) reduces MK maturation. These data could be better clarified in the text, or by the addition of experiments addressing whether the composition and quantity of ECM cage components directly inhibit maturation versus whether effects of MMP-inhibitors perhaps lead to over-activation of the integrins (as with the B4galt KO in the discussion) are responsible for the differences in maturation.

Reviewer #3 (Public review):

Summary:

In this study the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste. The researchers confirmed in rats their previous work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants including: inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl; salt); citric acid (sour) and quinine hydrochloride (bitter). Robust effects of ornithine were observed in the cases of IMP, MSG, MPG and sucrose; and little or no effects were observed in the cases of sodium chloride, citric acid; quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. Inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify a role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). These alternatives are appropriately discussed and, taken together, the experimental results favor the authors' interpretation that C6A mediates the Ornithine responses. The authors provide preliminary data in Suppl. 3 for the possibility of co-expression of C6A with the CaSR.

Weaknesses:

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9).

One issue that is not addressed, and could be usefully addressed in the Discussion, relates to the potential effects of kokumi substances on the threshold concentrations of key tastants such as glutamate. Thus, an extension of taste distribution to additional areas of the mouth (previously referred to as 'mouthfulness') and persistence of taste/flavor responses (previously referred to as 'continuity') could arise from a reduction in the threshold concentrations of umami and other substances that evoke taste responses.

The status of one of the compounds used as an inhibitor of C6A, the gallate derivative EGCG, as a potential inhibitor of the CaSR or T1R1/T1R3 is unknown. It would have been helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response.

It would have been helpful to include a positive control kokumi substance in the two bottle preference experiment (e.g., one of the known gamma glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

Reviewer #3 (Public review):

Summary:

Childers et al. address a fundamental question about the complex relationship within the gut: the link between nutrient absorption, microbial presence, and intestinal physiology. They focus on the role of lysosome-rich enterocytes (LREs) and the microbiota in protein absorption within the intestinal epithelium. By using germ-free and conventional zebrafishes, they demonstrate that microbial association leads to a reduction in protein uptake by LREs. Through impressive in vivo imaging of gavaged fluorescent proteins, they detail the degradation rate within the LRE region, positioning these cells as key players in the process. Additionally, the authors map protein absorption in the gut using single-cell sequencing analysis, extensively describing LRE subpopulations in terms of clustering and transcriptomic patterns. They further explore the monoassociation of ex-germ-free animals with specific bacterial strains, revealing that the reduction in protein absorption in the LRE region is strain-specific.

Strengths:

The authors employ state-of-the-art imaging to provide clear evidence of the protein absorption rate phenotype, focusing on a specific intestinal region. This innovative method of fluorescent protein tracing expands the field of in vivo gut physiology.

Using both conventional and germ-free animals for single-cell sequencing analysis, they offer valuable epithelial datasets for researchers studying host-microbe interactions. By capitalizing on fluorescently labelled proteins in vivo, they create a new and specific atlas of cells involved in protein absorption, along with a detailed LRE single-cell transcriptomic dataset.

Weaknesses:

While the authors present tangible hypotheses, the data are primarily correlative, and the statistical methods are inadequate. They examine protein absorption in a specific, normalized intestinal region but do not address confounding factors between germ-free and conventional animals, such as size differences, transit time, and oral gavage, which may impact their in vivo observations. This oversight can lead to bold conclusions, where the data appear valuable but require more nuance.

The sections of the study describing the microbiota or attempting functional analysis are elusive, with related data being overinterpreted. The microbiome field has long used 16S sequencing to characterize the microbiota, but its variability due to experimental parameters limits the ability to draw causative conclusions about the link between LRE activity, dietary protein, and microbial composition. Additionally, the complex networks involved in dopamine synthesis and signalling cannot be fully represented by RNA levels alone. The authors' conclusions on this biological phenomenon based on single-cell data need support from functional and in vivo experiments.

Reviewer #3 (Public review):

Summary:

The authors investigate the kinase activity of IKK2, a crucial regulator of inflammatory cell signaling. They describe a novel tyrosine kinase activity of this well-studied enzyme and a highly unusual phosphotransfer from phosphorylated IKK2 onto substrate proteins in the absence of ATP as a substrate.

Strengths:

The authors provide an extensive biochemical characterization of the processes with recombinant protein, western blot, autoradiography, protein engineering and provide MS data now.

Weaknesses:

The identity and purity of the used proteins has improved in the revised work. Since the findings are so unexpected and potentially of wide-reaching interest - this is important. Similar specific detection of phospho-Ser/Thr vs phospho-Tyr relies largely on antibodies which can have varying degrees of specificity. Using multiple antibodies and MS improves the quality of the data.

Reviewer #3 (Public review):

Summary:

The obligate intracellular bacterium Chlamydia trachomatis (Ct) divides by binary fission. It lacks FtsZ, but still has many other proteins that regulate synthesis of septal peptidoglycan, including FtsW and FtsI (PBP3) as well as divisome proteins that recruit and activate them, such as FtsK and FtsQLB. Interestingly, MreB is also required for division of Ct cells, perhaps by polymerizing to form an FtsZ-like scaffold. Here, Harpring et al. show that MreB does not act early in division and instead is recruited to a protein complex that includes FtsK and PBP2/PBP3. This indicates that Ct cell division is organized by a chimera between conserved divisome and elongasome proteins. Their work also shows convincingly that FtsK is the earliest known step of divisome activity, potentially nucleating the divisome as a single protein complex at the future division site. This is reminiscent of the activity of FtsZ, yet fundamentally different.

Strengths:

The study is very well written and presented, and the data are convincing and rigorous. The data underlying the proposed localization dependency order of the various proteins for cell division is well justified by several different approaches using small molecule inhibitors, knockdowns, and fluorescent protein fusions. The proposed dependency pathway of divisome assembly is consistent with the data and with a novel mechanism for MreB in septum synthesis in Ct.

Weaknesses:

The authors have addressed the weaknesses brought up in my previous review.

Reviewer #3 (Public review):

Childers et al. address a fundamental question about the complex relationship within the gut: the link between nutrient absorption, microbial presence, and intestinal physiology. They focus on the role of lysosome-rich enterocytes (LREs) and the microbiota in protein absorption within the intestinal epithelium. By using germ-free and conventional zebrafishes, they demonstrate that microbial association leads to a reduction in protein uptake by LREs. Through impressive in vivo imaging of gavaged fluorescent proteins, they detail the degradation rate within the LRE region, positioning these cells as key players in the process. Additionally, the authors map protein absorption in the gut using single-cell sequencing analysis, extensively describing LRE subpopulations in terms of clustering and transcriptomic patterns. They further explore the monoassociation of ex-germ-free animals with specific bacterial strains, revealing that the reduction in protein absorption in the LRE region is strain-specific.

Strengths:

- The authors employ state-of-the-art imaging to provide clear evidence of the protein absorption rate phenotype, focusing on a specific intestinal region. This innovative method of fluorescent protein tracing expands the field of in vivo gut physiology.<br /> - Using both conventional and germ-free animals for single-cell sequencing analysis, they offer valuable epithelial datasets for researchers studying host-microbe interactions. By capitalizing on fluorescently labelled proteins in vivo, they create a new and specific atlas of cells involved in protein absorption, along with a detailed LRE single-cell transcriptomic dataset.<br /> - Their robust and convincing microbiota analysis puts forward a diet-dependent mechanism of community change upon low-protein diet, intricately linked with the host.

Comments on revisions:

The authors have improved the manuscript following the revision work. No further recommendations.

Reviewer #3 (Public review):

Summary:

This important paper describes improvements to the measurement of enkephalins in vivo using microdialysis and LC-MS. The key improvement is oxidation of met- to prevent having a mix of reduced and oxidized methionine the sample which make quantification more difficult. It then shows measurements of enkephalins in the nucleus accumbens in two different stress situations-handling and exposure to predator odor. It also reports the ratio of released met- and leu-enkephalin matching that expected from digestion of proenkephalin. Measurements are also made by photometry of Ca2+ changes for the fox odor stressor. Some key takeaways are: 1) reliable measurement of met-enkephalin, significance of directly measuring peptides as opposed to proxy measurements, and the opening of a new avenue into research of enkephalins due to stress based on these direct measurements.

Strengths:

- Improved methods for measurement of enkephalins in vivo<br /> - Compelling examples of using this method<br /> - Opening a new area of looking at stress responses through the lens of enkephalin concentrations

Comments on revisions:

This revision has been improved upon in most ways. As I mentioned in the original review, there is a great deal of work here on showing the capability of measuring met- and leu-enk in different contexts. There is a technical improvement in the control of met oxidation which likely improves the detection of met-enk.

Reviewer #3 (Public review):

Summary:

This study indicates that connections across human cortical pyramidal cells have identical latencies despite a larger mean dendritic and axonal length between somas in human cortex. A precise demonstration combining detailed electrophysiology and modeling, indicates that this property is due to faster propagation of signals in proximal human dendrites. This faster propagation is itself due to a slightly thicker dendrite, to a larger capacitive load, and to stronger hyperpolarizing currents. Hence, the biophysical properties of human pyramidal cells are adapted such that they do not compromise information transfer speed.

Strengths:

The manuscript is clear and very detailed. The authors have experimentally verified a large number of aspects that could affect propagation speed and have pinpointed the most important one. This paper provides an excellent comparision of biophysical properties between rat and human pyramidal cells. Thanks to this approach a comprehensive description of the mechanisms underlying the acceleration of propagation in human dendrite is provided.

Weaknesses:

The weaknesses I had identified have been addressed by the authors.

Reviewer #3 (Public review):

Summary:

The authors used a novel technique to treat male infertility. In a proof-of-concept study, the authors were able to rescue the phenotype of a knockout mouse model with immotile sperm using this technique. This could also be a promising treatment option for infertile men.

Strengths:

In their proof-of-concept study, the authors were able to show that the novel technique rescues the infertility phenotype of Armc2 knockout spermatozoa. In the revised version of the manuscript, the authors have added data on in vitro fertilisation experiments with Armc2 mRNA-rescued sperm. The authors show that Armc2 mRNA-rescued sperm can successfully fertilise oocytes that develop to the blastocyst stage. This adds another level of reliability to the data.

Weaknesses:

Some minor weaknesses identified in my previous report have already been fixed. The technique is new and may not yet be fully established for all issues. Nevertheless, the data presented in this manuscript opens the way for several approaches to immotile spermatozoa to ensure successful fertilisation of oocytes and subsequent appropriate embryo development.

[Editors' note: The images in Figure 12 do not support the authors' interpretation that 2-cell embryos resulted from in vitro fertilization. Instead, the cells shown appear to be fragmented, unfertilized eggs. Combined with the lack of further development, it seems highly unlikely that fertilization was successful.]

Reviewer #3 (Public review):

Summary:

Protein-DNA interactions and sequence readout represent a challenging and rapidly evolving field of study. Recognizing the complexity of this task, the authors have developed a compact and elegant model. They have applied well-established approaches to address a difficult problem, effectively enhancing the information extracted from sparse contact maps by integrating artificial sequences decoy set and available experimental data. This has resulted in the creation of a practical tool that can be adapted for use with other proteins.

Strengths:

(1) The authors integrate sparse information with available experimental data to construct a model whose utility extends beyond the limited set of structures used for training.

(2) A comprehensive methods section is included, ensuring that the work can be reproduced. Additionally, the authors have shared their model as a GitHub project, reflecting their commitment to transparency of research.

Weaknesses:

(1) The coarse-graining procedure appears artificial, if not confusing, given that full-atom crystal structures provide more detailed information about residue-residue contacts. While the selection procedure for distance threshold values is explained, the overall motivation for adopting this approach remains unclear. Furthermore, since this model is later employed as an empirical potential for molecular modeling, the use of P and C5 atoms raises concerns, as the interactions in 3SPN are modeled between Cα and the nucleic base, represented by its center of mass rather than P or C5 atoms.

(2) Although the authors use a standard set of metrics to assess model quality and predictive power, some ΔΔG predictions compared to MITOMI-derived ΔΔG values appear nonlinear, which casts doubt on the interpretation of the correlation coefficient.

(3) The discussion section lacks information about the model's limitations and a comprehensive comparison with other models. Additionally, differences in model performance across various proteins and their respective predictive powers are not addressed.

Reviewer #3 (Public review):

Summary:

This work from Hira et al leverages mesoscopic 2-photon imaging to study large neural populations in different higher visual areas, in particular areas A and AM of the parietal cortex. The focus of the study is to obtain a better understanding of the representation of different task-related parameters, such as choice formation and short-term history, as well as visual responses in large neural populations across different cortical regions to obtain a better understanding of the functional specialization of neural populations in each region as well as the interaction of neural populations across regions. The authors image a large number of neurons in animals that either perform visual discrimination or a history-dependent task to test how task demands affect neural responses and population dynamics. Furthermore, by including a behavioral perturbation of animal posture they aim to dissociate the neural representation of history signals from body posture. Lastly, they relate their functional findings to anatomical data from the Allen connectivity atlas and show a strong relation between functional correlations on anatomical connectivity patterns.

Strengths:

Overall, the study is very well done and tackles a problem that should be of high interest to the field by aiming to obtain a better understanding of the function and spatial structure of different regions in the parietal cortex. The experimental approach and analyses are sound and of high quality and the main conclusions are well supported by the results. Aside from the detailed analyses, a particular strength is the additional experimental perturbation of posture to isolate history-related activity which supports the conclusion that both posture and history signals are represented in different neurons within the same region.

Weaknesses:

The main point that I found hard to understand was the fairly strong language on functional clusters of neurons while also stating that neurons encoded combinations of different types of information and leveraging the encoding model to dissociate these contributions. Do the authors find mixed selectivity or rather functional segregation of neural tuning in their data? More details on this and some other points are below.

Reviewer #3 (Public review):

Summary

The study presents an investigation into how hypothalamic orexin neurons (HONs) track body movement with high precision. Using techniques including fiber photometry, video-based movement metrics, and empirical mode decomposition (EMD), the authors demonstrate that HONs encode net body movement consistently across a range of behaviors and metabolic states. They test the ability of HONs to track body movement to that of other subcortical neural populations, from which they distinguish HONs activity from other subcortical neural populations.

Strengths:

The study characterizes HONs activity as key indicators of movement and arousal, and this method may have potential implications for understanding sleep disorders, energy regulation, and brain-body coordination. Overall, I think this is a very interesting story, with novel findings and implications about sensorimotor systems in animals. The manuscript is clearly written and the evidence presented is rigorous. The conclusions are well supported by experimental data with clear statistical analyses.

Weaknesses/suggestions:

There are a couple of issues I think the authors could address to make the paper better and more complete:

(1) The study primarily focuses on steady-state behaviors. It would be interesting if the authors' current dataset allows analyses of HON dynamics during transitions between behavioral states (e.g., resting to running or grooming to sniffing). This could provide additional insights into how HONs adapt to rapid changes in body movement.

(2) Given the established role of HONs in arousal and wakefulness, the study could further investigate how movement-related HON dynamics interact with arousal states. For example, does HON encoding of movement differ during sleep versus wakefulness?

(3) Although HON ablation experiments suggest that HONs do not shape movement frequency profiles. It would be more compelling if the authors could investigate whether HONs contribute to specific types of movements (e.g., fine motor vs. gross motor movements) or modulate movement initiation thresholds.

(4) The heterogeneous movement-related orexin dynamics observed in the LC and SNc raise intriguing questions about the circuit-level mechanisms underlying these differences. Optogenetic or chemogenetic manipulation of these projections could validate the functional implications of these dynamics.

Reviewer #4 (Public review):

The manuscript examines how patterns of selection on gene expression differ between a normal field environment and a field environment with elevated salinity based upon transcript abundances obtained from leaves of a diverse panel of rice germplasm. In addition, the manuscript also maps expression QTL (eQTL) that explains variation in each environment. One highlight from the mapping is that a small group of trans-mapping regulators explains some gene expression variation for large sets of transcripts in each environment.

The overall scope of the datasets is impressive, combining large field studies that capture information about fecundity, gene expression, and trait variation at multiple sites. The finding related to patterns indicating increased LD among eQTLs that have cis-trans compensatory or reinforcing effects in interesting in the context of other recent work finding patterns of epistatic selection. The authors have made some changes that address previous comments. However, some analyses in the manuscript remain less compelling or do not make the most from the value of collected data. Although the authors have made several improvements to the precision with which field-specific terminology is applied and to the language chosen when interpreting analytical findings, additional changes to improve these aspects of the manuscript remain necessary.

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

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

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

Selection of traits: Having paired organismal and molecular trait data is a strength of the manuscript, but the organismal trait data are underutilized. The manuscript as written only makes weak indirect inferences based on GO categories or assumed gene functions to connect selection at the organismal and molecular levels. After prompted by the initial reviews to test for correspondence between SNPs that explain organismal and gene expression trait variation or co-variance of co-expression module variation and trait variation, the response to reviewers indicates finding negative results. These findings should be included in the manuscript text and discussed.

Genetic architecture of gene expression variation: More descriptive statistics of the eQTL analysis have been included, although additional information about the variation in these measures within environments would be useful. The motivation for featuring patterns of cis-trans compensation specifically for the results obtained under high salinity conditions remains unclear to me. If the lines sampled have predominantly evolved under low salinity conditions, and the hypothesis being evaluated relates to historical experience of stabilizing selection, then evaluating the eQTL patterns under normal conditions provides the more relevant test of the hypothesis.

Lines 280-282: The revised sentence continues to read as an overstatement and merits additional revision with citations.

Lines 379-381: Following revision, it still remains unclear how the interpretation follows from the above analysis; the inference as written goes significantly beyond what may be specifically inferable from the result.

Reviewer #3 (Public review):

Summary:

This manuscript describes an in-depth analysis of the effect of the AAA+ ATPase PCH-2 on meiotic crossover formation in C. elegant. The authors reach several conclusions and attempt to synthesize a 'universal' framework for the role of this factor in eukaryotic meiosis.

Strengths:

The manuscript makes use of the advantages of the 'conveyor' belt system within the c.elegans reproductive tract, to enable a series of elegant genetic experiments

Weaknesses:

A weakness of this manuscript is that it heavily relies on certain genetic/cell biological assays that can report on distinct crossover outcomes, without clear and directed control over other aspects and variables that might also impact the final repair outcome. Such assays are currently out of reach in this model system.

Reviewer #3 (Public review):

Summary:

The authors submitted a revised manuscript that reports findings from a series of experiments suggesting that bovine oviductal fluid and species-specific oviductal glycoprotein (OVGP1 or oviductin) from bovine, murine, or human sources modulate the species specificity of bovine and murine oocytes.

Strengths:

The study reported in the manuscript deals with an important topic of interest in reproductive biology.

Weaknesses:

The authors have submitted a revised manuscript with much improvement and have answered many of this reviewer's questions. However, some of the previous questions have been dealt with inadequately. There are still several issues that need to be dealt with. In particular, there are questions regarding the specificity and/or purity of the recombinant human and mouse OVGP1 which could be detrimental to the reliability of the recombinant human and mouse OVGP1s used in the study and the validity of the results presented. This Discussion should cover more broadly what has already been published in literature.

Reviewer #3 (Public Review):

Summary:

Out of the 20 Neglected Tropical Diseases (NTD) highlighted by the WHO, three are caused by members of the trypanosomatids, namely Leishmanaisis, Trypanosomiasis, and Chagas disease. Trypanosomal glycolytic enzymes including pyruvate kinase (PyK) have long been recognised as potential targets. In this important study, single-chain camelid antibodies have been developed as novel and potent inhibitors of PyK from the T, congolense. To gain structural insight into the mode of action, binding was further characterised by biophysical and structural methods, including crystal structure determination of the enzyme-nanobody complex. The results revealed a novel allosteric mechanism/pathway with significant potential for the future development of novel drugs targeting allosteric and/or cryptic binding sites.

Strengths:

This paper covers an important area of science towards the development of novel therapies for three of the Neglected Tropical Diseases. The manuscript is very clearly written with excellent graphics making it accessible to a wide readership beyond experts. Particular strengths are the wide range of experimental and computational techniques applied to an important biological problem. The use of nanobodies in all areas from biophysical binding experiments and X-ray crystallography to in-vivo studies is particularly impressive. This is likely to inspire researchers from many areas to consider the use of nanobodies in their fields.

Weaknesses:

There is no particular weakness, but I think the computational analysis of allostery, which basically relies on a single server could have been more detailed.

Reviewer #3 (Public review):

Summary:

The authors established an experimental system that reproduced three-dimensional triphasic epithelia, i.e., the original epithelium, its EMT, and MET. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-um or larger micropores. The 3.0-um-pored membrane induced an epithelial structure with three states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain this triphasic epithelium. Live imaging revealed that KCs moved in a reciprocating manner, with actin-rich filopodia-like KC structures extending into and out of the 3.0-um micropores, while the cells migrated unidirectionally into larger micropores. KO of Piezo1 and keratin 6 increased KC entry to and exit from the 3.0-um micropores. Their results demonstrate that benign keratinocytes migrate through confined spaces in a reciprocating manner, which might help form triphasic epithelia, recapitulating wound healing processes.

Strengths:

Careful observation of the behaviour of keratinocytes on the different-sized pores. CrispR-Cas9 gene editing to KO Piezo 1 and keratin 6 isoforms in HaCaT keratinocytes.

Weaknesses:

There is no analysis of the matrix produced by the keratinocytes on the different pore sizes as this may influence migration.

HaCaT cells are quite different from normal keratinocytes in terms of migration. Pilcher et al. PMID: 9182674

Reviewer #3 (Public review):

Strengths:

This work focuses on a problem of deep significance: quantifying the structure-tension relationship and underlying mechanism for the mechanosensitive Piezo 1 and 2 channels. This objective presents a few technical challenges for molecular dynamics simulations, due to the relatively large size of each membrane-protein system. Nonetheless, the technical approach chosen is based on the methodology that is, in principle, established and widely accessible. Therefore, another group of practitioners would likely be able to reproduce these findings with reasonable effort.

Weaknesses:

The two main results of this paper are (1) that both channels exhibit a flatter structure compared to cryo-EM measurements, and (2) their estimated force vs. displacement relationship. Although the former correlates at least quantitatively with prior experimental work, the latter relies exclusively on simulation results and model parameters.

Reviewer #3 (Public review):

Summary:

The manuscript by Wu D. et al. explores an innovative approach in immunometabolism and obesity by investigating the potential of targeting macrophage Inositol-requiring enzyme 1α (IRE1α) in cases of overnutrition. Their findings suggest that pharmacological inhibition of IRE1α could influence key aspects such as adipose tissue inflammation, insulin resistance, and thermogenesis. Notable discoveries include the identification of High-Fat Diet (HFD)-induced CD9+ Trem2+ macrophages and the reversal of metabolically active macrophages' activity with IRE1α inhibition using STF. These insights could significantly impact future obesity treatments.

Strengths:

The study's key strengths lie in its identification of specific macrophage subsets and the demonstration that inhibiting IRE1α can reverse the activity of these macrophages. This provides a potential new avenue for developing obesity treatments and contributes valuable knowledge to the field.

Weaknesses:

The research lacks an in-depth exploration of the broader metabolic mechanisms involved in controlling diet-induced obesity (DIO). Addressing this gap would strengthen the understanding of how targeting IRE1α might fit into the larger metabolic landscape.

Impact and Utility:

The findings have the potential to advance the field of obesity treatment by offering a novel target for intervention. However, further research is needed to fully elucidate the metabolic pathways involved and to confirm the long-term efficacy and safety of this approach. The methods and data presented are useful, but additional context and exploration are required for broader application and understanding.

Comments on revisions:

The author has revised the manuscript and addressed the most relevant comments raised by the reviewers. The paper is now significantly improved, though two minor issues remain.

(1) Studies were limited to male mice; this should be mentioned in the paper's Title.<br /> (2) Please include the sample size (n=) in all provided tables in the main manuscript and supplementary tables.

Reviewer #3 (Public review):

Summary:

In this study, Piersma et al. successfully generated a mouse model with all Ly49 genes knocked out, resulting in the complete absence of Ly49 receptor expression on the cell surface. The absence of Ly49 expression led to the loss of NK cell education/licensing and consequently, a failure in responsiveness against missing-self target cells. The authors demonstrate the restoration of NK cell licensing by knocking in a single Ly49 gene, Ly49A, in a mouse expressing the H-2Dd ligand for this receptor, which is a novel and important finding.

Strengths:

The authors established a novel mouse model enabling them to have a clean and thorough study on the function of Ly49 on NK cell licensing. Also, by knock in a single Ly49, they were able to investigate the function of a given Ly49 receptor excluding the "contamination" of co-expression any other Ly49 genes. The experiment designing and data interpretation were logically clear and the evidence was solid.

Weaknesses:

The mouse model was somehow genetically similar to a previous study. The experimental work and findings are partially overlapping with the previous work by Zhang et al. (2019), who also performed knockout of the entire Ly49 locus in mice and demonstrated that loss of NK responsiveness was due to the removal of inhibitory, and not activating Ly49 genes.

Potential achievements and discussions: The mouse model developed by the authors holds great potential for advancing NK cell functional studies, particularly regarding the regulation of NK cell functions through receptor-ligand interactions. Moreover, it provides a valuable tool for investigating NK cell education and the development of checkpoint inhibitors. These applications could significantly contribute to the broader research efforts in cancer therapy utilizing NK cells.

Comments on revisions: The authors have successfully addressed all the concerns raised in my previous feedback. They have significantly improved the logical structure, making it clearer and more coherent. Additionally, they have ensured consistency in the use of specific terminology throughout the manuscript. The substantial revisions and re-writing efforts are commendable and have greatly enhanced the overall quality of the manuscript.

Reviewer #3 (Public Review):

Summary:

The paper aims at providing a neurocomputational account on how social perception translates in prosocial behaviors. Participants first completed a novel social perception task during fMRI scanning, in which were asked to judge the merit or need of people depicted in different situations. Second , a separate altruistic choice task was used to examine how the perception of merit and need influences the weights people place on themselves, others and fairness when deciding to provide help. Finally, a link between perception and action was drawn in those participants who completed both tasks.

Strengths:

The paper is overall very well written and presented, leaving the reader at ease when describing complex methods and results. The approach used by the author is very compelling, as it combines computational modeling of behavior and neuroimaging data analyses. Despite not being able to comment on the computational model, I find the approach used (to disentangle sensitivity and biases, for merit and need) very well described and derived from previous theoretical work. Results are also clearly described and interpreted.

Weaknesses:

In the social perception task, merit and need are evaluated by means of very different cues that rely on different cognitive processes (more abstract thinking for merit than need). Despite this limitation of the task, the authors were able to argue convincingly in the revised version about the solidity of their findings. Sample size is quite small for study 2, nevertheless the results provide convincing evidence.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The study relies on the exogenous application of GPR55 agonists. It remains unclear whether endogenous ligands released due to physiological or pathological activities would have similar effects. There is no information regarding the time course of the agonist-induced suppression. There is also little evidence that GPR55 is expressed in Purkinje cells. This study would benefit from using GPR55 knockout (KO) mice. The downstream mechanism by which GPR55 mediates the suppression of GABA release remains unknown.

Reviewer #3 (Public review):

Summary:

In this manuscript, Edwards et al. describe hamFISH, a customizable and cost-efficient method for performing targeted spatial transcriptomics. hamFISH utilizes highly amplified multiplexed branched DNA amplification, and the authors extensively describe hamFISH development and its advantages over prior variants of this approach.

The authors then used hamFISH to investigate an important circuit in the mouse brain for social behavior, the medial amygdala (MeA). To develop a hamFISH probe set capable of distinguishing MeA neurons, the authors mined published single-cell RNA-sequencing datasets of the MeA, ultimately creating a panel of 32 hamFISH probes that mostly cover the identified MeA cell types. They evaluated over 600,000 MeA cells and classified neurons into 16 inhibitory and 10 excitatory types, many of which are spatially clustered. The authors combined hamFISH with viral and other circuit tracer injections to determine whether the identified MeA cell populations sent and/or received unique inputs from connected brain regions, finding evidence that several cell types had unique patterns of input and output. Finally, the authors performed hamFISH on the brains of male mice that were placed in behavioral conditions that elicit aggressive, infanticidal, or mating behaviors, finding that some cell populations are selectively activated (as assessed by c-fos mRNA expression) in specific social contexts.

Strengths:

(1) The authors developed an optimized tissue preparation protocol for hamFISH and implemented oligopools instead of individually synthesized oligonucleotides to reduce costs. The branched DNA amplification scheme improved smFISH signal compared to previous methods, and multiple variants provide additional improvements in signal intensity and specificity. Compared to other spatial transcriptomics methods, the pipeline for imaging and analysis is streamlined and is compatible with other techniques like fluorescence-based circuit tracing. This approach is cost-effective and has several advantages that make it a valuable addition to the list of spatial transcriptomics toolkits.

(2) Using 31 probes, hamFISH was able to detect 16 inhibitory and 10 excitatory neuron types in the MeA subregions, including the vast majority of cell types identified by other transcriptomics approaches. The authors quantified the distributions of these cell types along the anterior-posterior, dorsal-ventral, and medial-lateral axes, finding spatial segregation among some, but not all, MeA excitatory and inhibitory cell types. The authors additionally identified a class of inhibitory neurons expressing Ndnf (and a subset of these that express Chrna7) that project multiple social chemosensory circuits.

(3) The authors combined hamFISH with MeA input and output mapping, finding cell-type biases in the projections to the MPOA, BNST, and VMHvl, and inputs from multiple regions.

(4) The authors identified excitatory and inhibitory cell types, and patterns of activity across cell types, that were selectively activated during various social behaviors, including aggression, mating, and infanticide, providing new insights and avenues for future research into MeA circuit function.

Weaknesses:

(1) Gene selection for hamFISH is likely to still be a limiting factor, even with the expanded (32-probe) capacity. This may have contributed to the lack of ability to identify sexually dimorphic cell types (Figure S2B). This is an expected tradeoff for a method that has major advantages in terms of cost and adaptability.

(2) Adaptation of hamFISH, for example, to adapt it to other brain regions or tissues, may require extensive optimization.

(3) Pairing this method with behavioral experiments is likely to require further optimization, as c-fos mRNA expression is an indirect and incomplete survey of neuronal activity (e.g. not all cell types upregulate c-fos when electrically active). As such, there is a risk of false negative results that limit its utility for understanding circuit function.

(4) The limited compatibility of hamFISH with thicker tissue samples and lack of optical sectioning introduce additional technical limitations. For example, it would be difficult to densely sample larger neural circuits using serial 20 micron sections. Also, because the imaging modality is not clear from the methods, it is difficult to know whether the analysis methods introduce the risk of misattributing gene expression to overlapping cells.

Reviewer #3 (Public review):

In this study, Bison et al. analyzed the role of the GATA6 transcription factor in patterning the early mesoderm and generating cardiomyocytes, using human embryonic stem cell differentiation assays and patient-derived hiPSCs with heart defects associated with mutations in the GATA6 gene. They identified a novel role for GATA6 in regulating genes involved in the WNT and BMP pathways. Modulation of the WNT and BMP pathways partially rescue early cardiac mesoderm defects in GATA6 mutant hESCs. These results provide significant insights into how GATA6 loss-of-function and heterozygous mutations contribute to heart defects.

Comments on revised version:

The authors have addressed all the concerns, using new data and modifications to the text to further strengthen the manuscript.

Reviewer #3 (Public review):

Summary:

Human cells deficient in delta-tubulin or epsilon-tubulin form unstable centrioles, which lack triplet microtubules and undergo a futile formation and disintegration cycle. In this study, the authors show that human cells lacking the associated proteins TEDC1 or TEDC2 have these identical phenotypes. They use genetics to knockout TEDC1 or TEDC2 in p53-negative RPE-1 cells and expansion microscopy to structurally characterize mutant centrioles. Biochemical methods and AlphaFold-multimer prediction software are used to investigate interactions between tubulins and TEDC1 and TEDC2.

The study shows that mutant centrioles are built only of A tubules, which elongate and extend their proximal region, fail to incorporate structural components, and finally disintegrate in mitosis. In addition, they demonstrate that delta-tubulin or epsilon-tubulin and TEDC1 and TEDC2 form one complex and that TEDC1 TEDC2 can interact independently of tubulins. Finally, they show that localization of four proteins is mutually dependent.

Strengths:

The results presented here are convincing, exciting, and important, and the manuscript is well-written. The study shows that delta-tubulin, epsilon-tubulin, TEDC1, and TEDC2 function together to build a stable and functional centriole, significantly contributing to the field and our understanding of the centriole assembly process.

Weaknesses:

The ultrastructural characterization of TEDC1 and TEDC2 in centrosomes remains challenging. Nevertheless, it is evident that these proteins occupy growing centrioles and the proximal parts of mother centrioles.

Comments on revisions:

The authors have done a great job extending the original experiments and measurements and answering outstanding questions.