Reviewer #2 (Public review):

Summary:

This paper from Sutlief et al. focuses on an apparent contradiction observed in experimental data from two related types of pursuit-based decision tasks. In "forgo" decisions, where the subject is asked to choose whether or not to accept a presented pursuit, after which they are placed into a common inter-trial interval, subjects have been shown to be nearly optimal in maximizing their overall rate of reward. However, in "choice" decisions, where the subject is asked which of two mutually-exclusive pursuits they will take, before again entering a common inter-trial interval, subjects exhibit behavior that is believed to be sub-optimal. To investigate this contradiction, the authors derive a consistent reward-maximizing strategy for both tasks using a novel and intuitive geometric approach that treats every phase of a decision (pursuit choice and inter-trial interval) as vectors. From this approach, the authors are able to show that previously-reported examples of sub-optimal behavior in choice decisions are in fact consistent with a reward-maximizing strategy. Additionally, the authors are able to use their framework to deconstruct the different ways the passage of time impacts decisions, demonstrating the time cost contains both an opportunity cost and an apportionment cost, as well as examine how a subject's misestimation of task parameters impacts behavior.

Strengths:

The main strength of the paper lies in the authors' geometric approach to studying the problem. The authors chose to simplify the decision process by removing the highly technical and often cumbersome details of evidence accumulation that is common in most of the decision-making literature. In doing so, the authors were able to utilize a highly accessible approach that is still able to provide interesting insights into decision behavior and the different components of optimal decision strategies.

Weaknesses:

The authors have made great improvements to the strength of their evidence through revision, especially concerning their treatment of apportionment cost. However, I am concerned that the story this paper tells is far from concise, and that this weakness may limit the paper's audience and overall impact. I would strongly suggest making an effort to tighten up the language and structure of the paper to improve its readability and accessibility.

Reviewer #3 (Public review):

Summary:

The goal of the paper is to examine the objective function of total reward rate in an environment to understand behavior of humans and animals in two types of decision-making tasks: 1) stay/forgo decisions and 2) simultaneous choice decisions. The main aims are to reframe the equation of optimizing this normative objective into forms that are used by other models in the literature like subjective value and temporally discounted reward. One important contribution of the paper is the use of this theoretical analysis to explain apparent behavioral inconsistencies between forgo and choice decisions observed in the literature.

Strengths:

The paper provides a nice way to mathematically derive different theories of human and animal behavior from a normative objective of global reward rate optimization. As such, this work has value in trying to provide a unifying framework for seemingly contradictory empirical observations in literature, such as differentially optimal behaviors in stay-forgo v/s choice decision tasks. The section about temporal discounting is particularly well motivated as it serves as another plank in the bridge between ecological and economic theories of decision-making. The derivation of the temporal discounting function from subjective reward rate is much appreciated as it provides further evidence for potential equivalence between reward rate optimization and hyperbolic discounting, which is known to explain a slew of decision-making behaviors in the economics literature.

Weaknesses:

(1) Readability and organization:<br /> While I appreciate the detailed analysis and authors' attempts to provide as many details as possible, the paper would have benefitted from a little selectivity on behalf of the authors so that the main contributions aren't buried by the extensive mathematical detail provided.<br /> For instance, in Figure 5, the authors could have kept the most important figures (A, B and G) to highlight the most relevant terms in the subjective value instead of providing all possible forms of the equation.

Further, in subfigure 5E, is there a reason that the outside reward r_out is shown to be zero? The text referencing 5E is also very unclear: "In so downscaling, the subjective value of a considered pursuit (green) is to the time it would take to traverse the world were the pursuit not taken, 𝑡_out, as its opportunity cost subtracted reward (cyan) is to the time to traverse the world were it to be taken (𝑡_in+ 𝑡_out) (Figure 5E)."

In the abstract, the malapportionment of time is mentioned as a possible explanation for reconciling observed empirical results between simultaneous and sequential decision-making. However, perhaps due to the density of mathematical detail presented, the discussion of the malapportionment hypothesis is pushed all the way to the end of the discussion section.

(2) Apportionment Cost definition and interpretation<br /> This additional cost arises in their analyses from redefining the opportunity cost in terms of just "outside" rewards so that the subjective value of the current pursuit and the opportunity cost are independent of each other. However, in doing so, an additional term arises in defining the subjective value of a pursuit, named here the "apportionment cost". The authors have worked hard to provide a definition to conceptualize the apportionment cost though it remains hard to intuit, especially in comparison to the opportunity cost. The additive form of apportionment cost (Equation 9) doesn't add much in way of intuition or their later analyses for the malapportionment hypothesis. It appears that the most important term is the apportionment scaling term so just focusing on this term will help the reader through the subsequent analyses.

(3) Malapportionment Hypothesis: From where does this malapportionment arise?<br /> The authors identify the range of values for t_in and t_out in Figure 18, the terms comprising the apportionment scaling term, that lead to optimal forgo behaviors despite suboptimally rejecting the larger-later (LL) choice in choice decisions. They therefore conclude that a lower apportionment scale, which arises from overestimating the time required outside the pursuit (t_out) or underestimating the time required at the current pursuit (t_in). What is not discussed though is whether and how the underestimation of t_out and overestimation of t_in can be dissociated, though it is understood that empirical demonstration of this dissociation is outside the scope of this work.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of HSPA2 during mouse preimplantation development. Knocking down HSPA2 in zygotes, the authors describe lower chances of developing into blastocysts, which show a reduced number of inner cell mass cells. They find that HSPA2 mRNA and protein levels show some heterogeneity among blastomeres at the 4-cell stage and propose that HSPA2 could contribute to skewing their relative contribution to embryonic lineages. To test this, the authors try to reduce HSPA2 expression in one of the 2-cell stage blastomere and propose that it biases their contribution to towards extra-embryonic lineages. To explain this, the authors propose that HSPA2 would interact with CARM1, which controls chromatin accessibility around genes regulating differentiation into embryonic lineage.

Strengths:

(1) The study offers simple and straightforward experiments with large sample sizes.

(2) Unlike most studies in the field, this research often relies on both mRNA and protein levels to analyse gene expression and differentiation.

Weaknesses:

(1) Image and statistical analyses are not well described.

(2) The functionality of the overexpression construct is not fully validated.

(3) Tracking of KD cells in embryos injected at the 2-cell stage with GFP is unclear.

(4) A key rationale of the study relies on measuring small differences in the levels of mRNA and proteins using semi-quantitative methods to compare blastomeres. As such, it is not possible to know whether those subtle differences are biologically meaningful. For example, the lowest HSPA2 level of the embryo with the highest level is much higher than the top cell from the embryo with the lowest level. What does this level mean then? Does this mean that some blastomeres grafted from strong embryos would systematically outcompete all other blastomeres from weaker embryos? That would be very surprising. I think the authors should be more careful and consider the lack of quantitative power of their approach before reaching firm conclusions. Although to be fair, the authors only follow a long trend of studies with the same intrinsic flaw of this approach.

(5) Some of the analyses on immunostaining do not take into account that this technique only allows for semi-quantitative measurements and comparisons.<br /> a) Some of the microscopy images are shown with an incorrect look-up table.<br /> b) Some of the schematics are incorrect and misleading.

Reviewer #2 (Public review):

Summary:

In this study, Gao et al. use RNA-seq to identify Hspa2 as one of the earliest transcripts heterogeneously distributed between blastomeres. Functional studies are performed using siRNA knockdown showing Hspa2 may bias cells toward the ICM lineage via interaction with the known methyltransferase CARM1.

Strengths:

This study tackles an important question regarding the origins of the first cell fate decision in the preimplantation embryo. It provides novelty in its identification of Hspa2 as a heterogeneous transcript in the early embryo and proposes a plausible mechanism showing interactions with Carm1. Multiple approaches are used to validate their functional studies (FISH, WB, development rates, proteomics). Given only 4 other transcripts/RNA have been identified at or before the 4-cell stage (LincGET, CARM1, PRDM14, HMGA1), this would be an important addition to our understanding of how TE vs ICM fate is established.

Weaknesses:

The RNA-seq results leading the authors to focus on Hspa2 are not included in the manuscript. This dataset would serve as an important resource but is neither included nor discussed. Nor is it mentioned whether Hspa2 was identified in prior RNA-seq embryos studies (for example Deng Science 2014).

Furthermore, the authors show that Hspa2 knockdown at the 1-cell stage lowers total Carm1 levels at the 4-cell stage. However, it is unclear how total abundance within the embryo alters lineage specification within blastomeres. The authors go on to propose a plausible mechanism involving Hspa2 and Carm1 interaction, but do not discuss how expression levels may be involved.

Reviewer #1 (Public review):

Summary:

Boldt et al test several possible relationships between trandiagnostically-defined compulsivity and cognitive offloading in a large online sample. To do so, they develop a new and useful cognitive task to jointly estimate biases in confidence and reminder-setting. In doing so, they find that over-confidence is related to less utilization of reminder-setting, which partially mediates the negative relationship between compulsivity and lower reminder-setting. The paper thus establishes that, contrary to the over-use of checking behaviors in patients with OCD, greater levels of transdiagnostically-defined compulsivity predicts less deployment of cognitive offloading. The authors offer speculative reasons as to why (perhaps it's perfectionism in less clinically-severe presentations that lowers the cost of expending memory resources), and sets an agenda to understand the divergence in cognitive between clinical and nonclinical samples. Because only a partial mediation had robust evidence, multiple effects may be at play, whereby compulsivity impacts cognitive offloading via overconfidence and also by other causal pathways.

Strengths:

The study develops an easy-to-implement task to jointly measure confidence and replicates several major findings on confidence and cognitive offloading. The study uses a useful measure of cognitive offloading - the tendency to set reminders to augment accuracy in the presence of experimentally manipulated costs. Moreover, the utilizes multiple measures of presumed biases -- overall tendency to set reminders, the empirically estimated indifference point at which people engage reminders, and a bias measure that compares optimal indifference points to engage reminders relative to the empirically observed indifference points. That the study observes convergenence along all these measures strengthens the inferences made relating compulsivity to the under-use of reminder-setting. Lastly, the study does find evidence for one of several a priori hypotheses and sets a compelling agenda to try to explain why such a finding diverges from an ostensible opposing finding in clinical OCD samples and the over-use of cognitive offloading.

Weaknesses:

Although I think this design and study are very helpful for the field, I felt that a feature of the design might reduce the tasks's sensitivity to measuring dispositional tendencies to engage cognitive offloading. In particular, the design introduces prediction errors, that could induce learning and interfere with natural tendencies to deploy reminder-setting behavior. These PEs comprise whether a given selected strategy will be or not be allowed to be engaged. We know individuals with compulsivity can learn even when instructed not to learn (e.g., Sharp, Dolan and Eldar, 2021, Psychological Medicine), and that more generally, they have trouble with structure knowledge (eg Seow et al; Fradkin et al), and thus might be sensitive to these PEs. Thus, a dispositional tendency to set reminders might be differentially impacted for those with compulsivity after an NPE, where they want to set a reminder, but aren't allowed to. After such an NPE, they may avoid moreso the tendency to set reminders. Those with compulsivity likely have superstitious beliefs about how checking behaviors lead to a resolution of catastrophes, that might in part originate from inferring structure in the presence of noise or from purely irrelevant sources of information for a given decision problem.<br /> It would be good to know if such learning effects exist, if they're modulated by PE (you can imagine PEs are higher if you are more incentivized - e.g., 9 points as opposed to only 3 points - to use reminders, and you are told you cannot use them), and if this learning effect confounds the relationship between compulsivity and reminder-setting.

A more subtle point, I think this study can be more said to be an exploration than a deductive of test of a particular model -> hypothesis -> experiment. Typically, when we test a hypothesis, we contrast it with competing models. Here, the tests were two-sided because multiple models, with mutually exclusive predictions (over-use or under-use of reminders) were tested. Moreover, it's unclear exactly how to make sense of what is called the direct mechanism, which is supported by the partial (as opposed to complete) mediation.

Comments on revisions:

I have the following final comments for your manuscript revisions:

To improve the clarity of the work, I suggest a final note to the authors to say more explicitly that objective accuracy has a finer resolution *due to the number of "special circles" per trial* in their task. This task detail got lost in my read of the manuscript, and confused me with respect to the resolution of each accuracy measure. Similarly for clarification, they could point out that their exclusion criteria removes subjects that have lower OIP than their AIP analysis allows (which is good for comparison between OIP and AIP). Thus, it removes the possibility that very poor performing subjects (OIP) are forced to have a higher than actual AIP due to the range).

Reviewer #1 (Public review):

Summary:

You, Zhang et al. comprehensively characterize the long-term fates of mouse HSCs in the unperturbed setting using transposon-based lineage tracing for up to 2 years post-labeling. Their analyses reveal a complex heterogeneity of long-term fates, dominated by two behaviors: i) long-lived differentiation-biased clones, and ii) self-renewal & platelet-biased clones. They further identify two categories of multipotent progenitor clones, with one group showing a markedly reduced differentiation activity.

Strengths:

You et al. present a very comprehensive and high-resolution characterization of mouse hematopoietic clonal dynamics, with robust replicates, and technical prowess. The manuscript is beautifully written, with in-depth and clear explanations of the logic behind experimental design choices, and very well-thought-out interpretations of results.

Some of the results integrate well with past observations in the field, whereas many of them are quite unique and novel.

This will surely be a highly impactful study in the field of hematopoiesis and stem cell biology.

Weaknesses:

The authors trace hematopoiesis in situ, in a fully unbiased way for almost 2-years. They compare this time course with the last few years of Cre-LoxP-based tracing studies and they make an assumption that most hematopoiesis will be derived from some type of HSC at that point in time. They then use this assumption to support that what is being measured in their model are the long-term fates of HSCs (or at least cells that were HSC at the point of labeling). While this is a generally valid assumption, the short-lived nature of certain populations (myeloid cells, megakaryocytes) means that these cells are being produced in the context of a relatively aged environment by the time of sampling, which might change the properties of the system. In other words, the "steady-state" is always changing. It is important to read and interpret this manuscript with this in consideration.

Reviewer #2 (Public review):

Summary:

The work from You et al. elucidates the clonal contribution of ageing stem and progenitor cells to both native and perturbed hematopoiesis. The authors use a previously published in vivo lineage tracing system (Patel et al., 2022) that relies on the random integration of a transposon element in the mouse genome. They barcode all mouse cells and then look at lineage relationships between HSPC and mature populations after ~90 weeks.

Strengths:

This work offers very interesting insights into the clonal behaviour of HSPC in the native and perturbed setting during ageing. Experiments are well-planned and well-executed. Understanding the clonal output of HSPCs in aged mice in a native setting, after 5-FU treatment, and upon transplantation are important findings for the field.

Weaknesses:

We found appraising the graphs, interpreting the findings, and understanding those findings in the main text very difficult to follow. While we have made some suggestions below, we encourage the authors to think carefully about what the core messages are, and how best to visualise those, both in terms of data viz and in a schematic to summarise the key findings, and to use plain language in the text.

Reviewer #1 (Public review):

Summary

Pyrazinamide (PZA) is a key drug in the anti-TB arsenal, yet despite over 50 years of clinical use, its precise mechanism of action remains unclear. This study offers valuable insights into the in vitro potentiating effect of PZA when used with exogenous oxidative agents. The authors suggest that oxidative stress, specifically thiol oxidation, may be a primary driver of PZA/POA's bactericidal activity. Although the work is substantial, conceptually innovative, and timely, the evidence supporting the authors' conclusions requires further investigation with additional controls and experiments to fully validate the proposed mechanism of action. Once revised, this work will undoubtedly be of significant interest to the TB drug discovery community and researchers focusing on mycobacterial diseases.

Strengths

The authors have long-standing experience in the field of PZA mode of action, with several publications that have been highly relevant to the field. They are particularly well aware of the literature, and this is clearly visible in the introduction of the manuscript which is beautifully articulated. The biological question(s) and their hypotheses are also well-formulated in the introduction section.

The understanding of PZA mode of action is a long-lasting question in the TB community, therefore studies reporting well-conducted research that aims at deciphering the underlying mechanism responsible for PZA peculiar activity is always appreciated. Since PZA/POA are poorly active in conventional 7H9 media, but very potent in cellulo or in vivo; looking at host-mediated stress that can eventually lead to an increased vulnerability is extremely relevant. In that context, most of the work has been focused on host-cell endolysosomal pH but very little information is available on other stress. Thus, investigating the contribution of oxidative stress and ROS as specific host environments that might contribute to PZA/POA activity is overall novel and conceptually very interesting.

To address this question, the authors combine multiple approaches including conventional antimicrobial susceptibility profiling, CFU-based counting, and checkerboard assays to report the potentiating effect of PZA pre-treatment on hydrogen peroxide- and diamide-mediated antibacterial action. The use of multiple reference strains including Mtb H37Ra, Mtb H37Rv, M.bovis BCG, and M.bovis BCG::pncA is a great asset of the manuscript, even though they might have been more appropriately used to get further mechanistical insights on the proposed model of action. The findings are reported in 4 major figures that are clear and in an order that appears logical for the understanding of the story.

Weaknesses

Although the manuscript is conceptually very interesting and contains intriguing results, it sometimes fails to fully convince and some additional controls/experiments might help to better back up the authors' claims and really strengthen the study. Indeed, some conclusions seem premature therefore leading to some molecular assumptions regarding a potential mode of action that is not fully supported by the presented data.<br /> The rationale behind some of the experiments is not always clearly explained which makes difficult to follow the authors ideas, the biological hypothesis/model that they test, and therefore the overall scientific story.

The authors conclude their study by proposing a mechanism by which the active form of the drug POA acts in concert with exogenous ROS to promote cellular oxidative damage. This is tested within two models of macrophage infection where they propose that IFN-γ mediated ROS production is essential for PZA activity. Unfortunately, the in cellulo part presents some weaknesses and inconsistencies that the authors need to carefully address.

Finally, the in vitro experiments performed in this manuscript mainly report that PZA pre-treatment increases H2O2-mediated killing or inhibition. There is no direct evidence that clearly shows that oxidative stress drives the potent bactericidal activity of PZA. In these settings, the oxidative stress is always applied after PZA pre-treatment and is therefore likely displaying the major lethal effect.

Reviewer #2 (Public review):

Summary:

The authors tested how ROS and PZA affected Mycobacterium survival to determine if ROS could have a role in the remarkable in vivo efficacy of PZA.

Strengths:

This is a well-written and clear manuscript convincingly demonstrating the synergy between PZA and reactive oxygen species in the inhibition of growth and survival of Mycobacterium tuberculosis.

Weaknesses:

The manuscript would benefit from a clear statement of the rationale for the protocols used to examine the synergy of PZA with ROS, the possible models their protocols could be testing, and then how their data supports or disproves the models being tested. The manuscript appears to propose, as stated in the title, that "Oxidative stress drives potent bactericidal activity of pyrazinamide...". However their experimental design more likely tests the effect of PZA on ROS sensitivity. Indeed, by the last figure, the authors begin the present their data as PZA sensitizing the bacteria to ROS. More clarity on these possible models and the different interpretations of the data should be considered.

Impact:

The data provide important insight to expand our understanding of the in vivo efficacy of PZA in the treatment of tuberculosis.

Reviewer #1 (Public review):

Summary:

The question of how central nervous system (CNS) lamination defects affect functional integrity is an interesting topic, though it remains a subject of debate. The authors focused on the retina, which is a relatively simple yet well-laminated tissue, to investigate the impact of afadin - a key component of adherens junctions on retinal structure and function. Their findings show that the loss of afadin leads to significant disruptions in outer retinal lamination, affecting the morphology and localization of photoreceptors and their synapses, as illustrated by high-quality images. Despite these severe changes, the study found that some functions of the retinal circuits, such as the ability to process light stimuli, could still be partially preserved. This research offers new insights into the relationship between retinal lamination and neural circuit function, suggesting that altered retinal morphology does not completely eliminate the capacity for visual information processing.

Strengths:

The retina serves as an excellent model for investigating lamination defects and functional integrity due to its relatively simple yet well-organized structure, along with the ease of analyzing visual function. The images depicting outer retinal lamination, as well as the morphology and localization of photoreceptors and their synapses, are clear and well-described. The paper is logically organized, progressing from structural defects to functional analysis. Additionally, the manuscript includes a comprehensive discussion of the findings and their implications.

Weaknesses:

While this work presents a wealth of descriptive data, it lacks quantification, which would help readers fully understand the findings and compare results with those from other studies. Furthermore, the molecular mechanisms underlying the defects caused by afadin deletion were not explored, leaving the role of afadin and its intracellular signaling pathways in retinal cells unclear. Finally, the study relied solely on electrophysiological recordings to demonstrate RGC function, which may not be robust enough to support the conclusions. Incorporating additional experiments, such as visual behavior tests, would strengthen the overall conclusions.

Reviewer #2 (Public review):

Summary:

Ueno et al. described substantial changes in the afadin knockout retina. These changes include decreased numbers of rods and cones, an increased number of bipolar cells, and disrupted somatic and synaptic organization of the outer limiting membrane, outer nuclear layer, and outer plexiform layer. In contrast, the number and organization of amacrine cells and retinal ganglion cells remain relatively intact. They also observed changes in ERG responses and RGC receptive fields and functions using MEA recordings.

Strengths:

The morphological characterization of retinal cell types and laminations is detailed and relatively comprehensive.

Weaknesses:

(1) The major weakness of this study, perhaps, is that its findings are predominantly descriptive and lack any mechanistic explanation. As afadin is key component of adherent junctions, its role in mediating retinal lamination has been reported previously (see PMCID: PMC6284407). Thus, a more detailed dissection of afadin's role in processes, such as progenitor generation, cell migration, or the formation of retinal lamination would provide greater insight into the defects caused by knocking out afadin.

(2) The authors observed striking changes in the numbers of rods, cones, and BCs, but not in ACs or RGCs. The causes of these distinct changes in specific cell classes remain unclear. Detailed characterizations, such as the expression of afadin in early developing retina, tracing cell numbers across various early developmental time points, and staining of apoptotic markers in developing retinal cells, could help to distinguish between defects in cell generation and survival, providing a better understand of the underlying causes of these phenotypes.

(3) Although the total number of ACs or RGCs remains unchanged, their localizations are somewhat altered (Figures 2E and 4E). Again, the cause of the altered somatic localization in ACs and RGCs is unclear.

(4) One conclusion that the authors emphasise is that the function of RGCs remains detectable despite a major disrupted outer plexiform layer. However, the organization of the inner plexiform layer remains largely intact, and the axonal innervation of BCs remains unchanged. This could explain the function integrity of RGCs. In addition, the resolution of detecting RGCs by MEA is low, as they only detected 5 clusters in heterozygous animals. This represents an incomplete clustering of RGC functional types and does not provide a full picture of how functional RGC types are altered in the afadin knockout.

Minor Comments:

(1) Line 56-67: "Overall, these findings provide the first evidence that retinal circuit function can be partially preserved even when there are significant disruptions in retinal lamination and photoreceptor synapses" There is existing evidence showing substantial adaption in retinal function when retinal lamination or photoreceptor synapses are disrupted, such as PMCID: PMC10133175.

(2) Line 114-115: "we focused on afadin, which is a scaffolding protein for nectin and has no ortholog in mice." The term "Ortholog" is misused here, as the mouse has an afadin gene. Should the intended meaning be that afadin has no other isoforms in mouse?

Reviewer #1 (Public review):

Summary:

In this paper, Derkaloustian et. al look at the important topic of what affects fine touch perception. The observations that there may be some level of correlation with instabilities are intriguing. They attempted to characterize different materials by counting the frequency (occurrence #, not of vibration) of instabilities at various speeds and forces of a PDMS slab pulled lengthwise over the material. They then had humans make the same vertical motion to discriminate between these samples. They correlated the % correct in discrimination with differences in frequency of steady sliding over the design space as well as other traditional parameters such as friction coefficient and roughness. The authors pose an interesting hypothesis and make an interesting observation about the occurrences of instability regimes in different materials while in contact with PDMS, which is interesting for the community to see in the publication. It should be noted that the finger is complex, however, and there are many factors that may be quite oversimplified with the use of the PDMS finger, and the consideration and discounting of other parameters are not fully discussed in the main text or SI. Most importantly, however, the conclusions as stated do not align with the primary summary of the data in Figure 2.

Strengths:

The strength of this paper is in its intriguing hypothesis and important observation that instabilities may contribute to what humans are detecting as differences in these apparently similar samples.

Weaknesses:

The most important weakness is that the findings do not support the statements of findings made in the abstract. Of specific note in this regard is the primary correlation in Figure 2B between SS (steady sliding) and percent correct discrimination. While the statistical test shows significance (and is interesting!), the R-squared value is 0.38, while the R-squared value for the "Friction Coefficient vs. Percent Correct" plot has an R-squared of 0.6 and a p-value of < 0.01 (including Figure 2B). This suggests that the results do not support the claim in the abstract: "We found that participant accuracy in tactile discrimination was most strongly correlated with formations of steady sliding, and response times were negatively correlated with stiction spikes. Conversely, traditional metrics like surface roughness or average friction coefficient did not predict tactile discriminability." This is the most fundamental weakness of this paper.

Along the same lines, other parameters that were considered such as the "Percent Correct vs. Difference in Sp" and "Percent Correct vs. Difference in SFW" were not plotted for consideration in the SI. It would be helpful to compare these results with the other three metrics in order to fully understand the relationships. Other parameters such as stiction magnitude and differences in friction coefficient over the test space could also be important and interesting.

Beyond this fundamental concern, there is a weakness in the representativeness of the PDMS finger, the vertical motion, and the speed of sliding to real human exploration. The real finger has multiple layers with different moduli. In fact, the stratum corneum cells, which are the outer layer at the interface and determine the friction, have a much higher modulus than PDMS. In addition, the slanted position of the finger can cause non-uniform pressures across the finger. Both can contribute to making the PDMS finger have much more stick-slip than a real finger. In fact, if you look at the regime maps, there is very little space that has steady sliding. This does not represent well human exploration of surfaces. We do not tend to use a force and velocity that will cause extensive stick-slip (frequent regions of 100% stick-slip) and, in fact, the speeds used in the study are on the slow side, which also contributes to more stick-slip. At higher speeds and lower forces, all of the materials had steady sliding regions. Further, on these very smooth surfaces, the friction and stiction are more complex and cannot dismiss considerations such as finger material property change with sweat pore occlusion and sweat capillary forces. Also, the vertical motion of both the PDMS finger and the instructed human subjects is not the motion that humans typically use to discriminate between surfaces. Finally, fingerprints may not affect the shape and size of the contact area, but they certainly do affect the dynamic response and detection of vibrations.

This all leads to the critical question, why are friction, normal force, and velocity not measured during the measured human exploration and in a systematic study using the real human finger? The authors posed an extremely interesting hypothesis that humans may alter their speed to feel the instability transition regions. This is something that could be measured with a real finger but is not likely to be correlated accurately enough to match regime boundaries with such a simplified artificial finger.

Reviewer #2 (Public review):

Summary:

In this paper, the authors want to test the hypothesis that frictional instabilities rather than friction are the main drivers for discriminating flat surfaces of different sub-nanometric roughness profiles.

They first produced flat surfaces with 6 different coatings giving them unique and various properties in terms of roughness (picometer scale), contact angles (from hydrophilic to hydrophobic), friction coefficient (as measured against a mock finger), and Hurst exponent.

Then, they used those surfaces in two different experiments. In the first experiment, they used a mock finger (PDMS of 100kPA molded into a fingertip shape) and slid it over the surfaces at different normal forces and speeds. They categorized the sliding behavior as steady sliding, sticking spikes, and slow frictional waves by visual inspection, and show that the surfaces have different behaviors depending on normal force and speed. In a second experiment, participants (10) were asked to discriminate pairs of those surfaces. It is found that each of those pairs could be reliably discriminated by most participants.

Finally, the participant's discrimination performance is correlated with differences in the physical attributes observed against the mock finger. The authors found a positive correlation between participants' performances and differences in the count of steady sliding against the mock finger and a negative correlation between participants' reaction time and differences in the count of stiction spikes against the mock finger. They interpret those correlations as evidence that participants use those differences to discriminate the surfaces.

Strengths:

The created surfaces are very interesting as they are flat at the nanometer scale, yet have different physical attributes and can be reliably discriminated.

Weaknesses:

In my opinion, the data presented in the paper do not support the conclusions. The conclusions are based on a correlation between results obtained on the mock finger and results obtained with human participants but there is no evidence that the human participants' fingertips will behave similarly to the mock finger during the experiment. Figure 3 gives a hint that the 3 sliding behaviors can be observed in a real finger, but does not prove that the human finger will behave as the mock finger, i.e., there is no evidence that the phase maps in Figure 1C are similar for human fingers and across different people that can have very different stiffness and moisture levels.

I believe that the authors collected the contact forces during the psychophysics experiments, so this shortcoming could be solved if the authors use the actual data, and show that the participant responses can be better predicted by the occurrence of frictional instabilities than by the usual metrics on a trial by trial basis, or at least on a subject by subject basis. I.e. Poor performers should show fewer signs of differences in the sliding behaviors than good performers.

The sample size (10) is very small.

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 #1 (Public review):

This manuscript by Ori and colleagues investigates the role of Lmod1 in muscle stem cell activation and differentiation. The study begins with a time-course mass spectrometry analysis of primary muscle stem cells, identifying Lmod1 as a pro-myogenic candidate (Figure 1). While the initial approach is robust, the subsequent characterization lacks depth and clarity. Although the data suggest that Lmod1 promotes myogenesis, the underlying mechanisms remain vague, and key experiments are missing. Please find my comments below.

(1) The authors mainly rely on coarse and less-established readouts such as myotube length and spherical Myh-positive cells. More comprehensive and standard analyses, such as co-staining for Pax7, MyoD, and Myogenin, would allow quantification of quiescent, activated, and differentiating stem cells in knockdown and overexpression experiments. The exact stage at which Lmod1 functions (stem cell, progenitor, or post-fusion) is unclear due to the limited depth of the analysis. Performing similar experiments on cultured single EDL fibers would add valuable insights.

(2) In supplementary Figure 2E, the distinction between Hoechst-positive cells and total cell counts is unclear. The authors should clarify why Hoechst-positive cells increase and relabel "reserve cells," as the term is confusing without reading the legend.

(3) The specificity of Lmod1 and Sirt1 immunostaining needs validation using siRNA-treated samples, especially as these data form the basis of the mechanistic conclusions.

(4) The authors must test the effect of Lmod1 siRNA on Sirt1 localization, as only overexpression experiments are shown.

(5) In Figure S3, the biotin signal in LMOD2 samples appears weak. The authors need to address whether comparing LMOD1 and LMOD2 is valid given the apparent difference in reaction efficiency. It would also help to highlight where Sirt1 falls on the volcano plot in S3B.

(6) The immunostaining data suggest that Lmod1 remains cytoplasmic throughout differentiation, whereas Sirt1 shows transient cytoplasmic localization at day 1 of differentiation. The authors should explain why Sirt1 is not constantly sequestered if Lmod1's cytoplasmic localization is consistent. It is also unclear whether day 1 is the key time point for Lmod1 function, as its precise role during myogenesis remains ambiguous.

(7) The introduction does not sufficiently establish the motivation or knowledge gap this work aims to address. Instead, it reads like a narration of disparate topics in a single paragraph. The authors should clarify the statement in line 150, "since this protein has been...,".

Overall, while the identification of Lmod1 as a pro-myogenic factor is convincing, the mechanistic insights are insufficient, and the manuscript would benefit from addressing these concerns.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors identify Leiomodin-1 (LMOD1) as a key regulator of early myogenic differentiation, demonstrating its interaction with SIRT1 to influence SIRT1's cellular localization and gene expression. The authors propose that LMOD1 translocates SIRT1 from the nucleus to the cytoplasm to permit the expression of myogenic differentiating genes such as MYOD or Myogenin.

Strengths:

A major strength of this work lies in the robust temporal resolution achieved through a time-course mass spectrometry analysis of in vitro muscle differentiation. This provides novel insights into the dynamic process of myogenic differentiation, often under-explored in terms of temporal progression. The authors provide a strong mechanistic case for how LMOD1 exerts its role in muscle differentiation which opens avenues to modulate.

Weaknesses:

One limitation of the study is the in vivo data. Although the authors do translate their findings in vivo for LMOD1 localization and expression, the cross-sectional imaging is not highly convincing. Longitudinal cuts or isolated fibers could have been more useful specimens to answer these questions. Moreover, the authors do not assess their in vitro SIRT1 findings in vivo. A few key experiments in regenerating or aged mice would strengthen the mechanistic insight of the findings.

Discussion:

Overall, the study emphasizes the importance of understanding the temporal dynamics of molecular players during myogenic differentiation and provides valuable proteomic data that will benefit the field. Future studies should explore whether LMOD1 modulates the nuclear-cytoplasmic shuttling of other transcription factors during muscle development and how these processes are mechanistically achieved. Investigating whether LMOD1 can be therapeutically targeted to enhance muscle regeneration in contexts such as exercise, aging, and disease will be critical for translational applications. Additionally, elucidating the interplay among LMOD1, LMOD2, and LMOD3 could uncover broader implications for actin cytoskeletal regulation in muscle biology.

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 #1 (Public review):

Summary:

The authors introduce a denoising-style model that incorporates both structure and primary-sequence embeddings to generate richer embeddings of peptides. My understanding is that the authors use ESM for the primary sequence embeddings, take resolved structures (or use structural predictions from AlphaFold when they're not available), then develop an architecture to combine these two with a loss that seems reminiscent of diffusion models or masked language model approaches. The embeddings can be viewed as ensemble-style embedding of the two levels of sequence information, or with AlphaFold, an ensemble of two methods (ESM+AlphaFold). The authors also gather external datasets to evaluate their approach and compare it to previous approaches. The approach seems promising and appears to out-compete previous methods at several tasks. Nonetheless, I have strong concerns about a lack of verbosity as well as exclusion of relevant methods and references.

Advances:

I appreciate the breadth of the analysis and comparisons to other methods. The authors separate tasks, models, and sizes of models in an intuitive, easy-to-read fashion that I find valuable for selecting a method for embedding peptides. Moreover, the authors gather two datasets for evaluating embeddings' utility for predicting thermostability. Overall, the work should be helpful for the field as more groups choose methods/pretraining strategies amenable to their goals, and can do so in an evidence-guided manner.

Considerations:

Primarily, a majority of the results and conclusions (e.g., Table 3) are reached using data and methods from ProteinGym, yet the best-performing methods on ProteinGym are excluded from the paper (e.g., EVE-based models and GEMME). In the ProteinGym database, these methods outperform ProtSSN models. Moreover, these models were published over a year---or even 4 years in the case of GEMME---before ProtSSN, and I do not see justification for their exclusion in the text.

Secondly, related to comparison of other models, there is no section in the methods about how other models were used, or how their scores were computed. When comparing these models, I think it's crucial that there are explicit derivations or explanations for the exact task used for scoring each method. In other words, if the pre-training is indeed the important advance of the paper, the paper needs to show this more explicitly by explaining exactly which components of the model (and previous models) are used for evaluation. Are the authors extracting the final hidden layer representations of the model, treating these as features, then using these features in a regression task to predict fitness/thermostability/DDG etc.? How are the model embeddings of other methods being used, since, for example, many of these methods output a k-dimensional embedding of a given sequence, rather than one single score that can be correlated with some fitness/functional metric. Summarily, I think the text is lacking an explicit mention of how these embeddings are being summarized or used, as well as how this compares to the model presented.

I think the above issues can mainly be addressed by considering and incorporating points from Li et al. 2024[1] and potentially Tang & Koo 2024[2]. Li et al.[1] make extremely explicit the use of pretraining for downstream prediction tasks. Moreover, they benchmark pretraining strategies explicitly on thermostability (one of the main considerations in the submitted manuscript), yet there is no mention of this work nor the dataset used (FLIP (Dallago et al., 2021)) in this current work. I think a reference and discussion of [1] is critical, and I would also like to see comparisons in line with [1], as [1] is very clear about what features from pretraining are used, and how. If the comparisons with previous methods were done in this fashion, this level of detail needs to be included in the text.

To conclude, I think the manuscript would benefit substantially from a more thorough comparison of previous methods. Maybe one way of doing this is following [1] or [2], and using the final embeddings of each method for a variety of regression tasks---to really make clear where these methods are performing relative to one another. I think a more thorough methods section detailing how previous methods did their scoring is also important. Lastly, TranceptEVE (or a model comparable to it) and GEMME should also be mentioned in these results, or at the bare minimum, be given justification for their absence.

[1] Feature Reuse and Scaling: Understanding Transfer Learning with Protein Language Models, Francesca-Zhoufan Li, Ava P. Amini, Yisong Yue, Kevin K. Yang, Alex X. Lu bioRxiv 2024.02.05.578959; doi: https://doi.org/10.1101/2024.02.05.578959<br /> [2] Evaluating the representational power of pre-trained DNA language models for regulatory genomics, Ziqi Tang, Peter K Koo bioRxiv 2024.02.29.582810; doi: https://doi.org/10.1101/2024.02.29.582810

Comments on revisions:

My concerns have been addressed. What seems to remain are some semantical disagreements and I'm not sure that these will be answered here. Do MSAs and other embedding methods lead to some notable type of data leakage? Does this leakage qualify as "x-shot" learning under current definitions?

Reviewer #2 (Public review):

Summary:

To design proteins and predict disease, we want to predict the effects of mutations on the function of a protein. To make these predictions, biologists have long turned to statistical models that learn patterns that are conserved across evolution. There is potential to improve our predictions however by incorporating structure. In this paper the authors build a denoising auto-encoder model that incorporates sequence and structure to predict mutation effects. The model is trained to predict the sequence of a protein given its perturbed sequence and structure. The authors demonstrate that this model is able to predict the effects of mutations better than sequence-only models.

As well, the authors curate a set of assays measuring the effect of mutations on thermostability. They demonstrate their model also predicts the effects of these mutations better than previous models and make this benchmark available for the community.

Strengths:

The authors describe a method that makes accurate mutation effect predictions by informing its predictions with structure.

The authors curate a new dataset of assays measuring thermostability. These can be used to validate and interpret mutation effect prediction methods in the future.

Weaknesses:

In the review period, the authors included a previous method, SaProt, that similarly uses protein structure to predict the effects of mutations, in their evaluations. They see that SaProt performs similarly to their method.

ProteinGym is largely made of deep mutational scans, which measure the effect of every mutation on a protein. These new benchmarks contain on average measurements of less than a percent of all possible point mutations of their respective proteins. It is unclear what sorts of protein regions these mutations are more likely to lie in; therefore it is challenging to make conclusions about what a model has necessarily learned based on its score on this benchmark. For example, several assays in this new benchmark seem to be similar to each other, such as four assays on ubiquitin performed in pH 2.25 to pH 3.0.

Comments on revisions:

I think the rounds of review have improved the paper and I've raised my score.

Reviewer #1 (Public review):

This manuscript makes a significant contribution to the field by exploring the dichotomy between chemical synaptic and gap junctional contributions to extracellular potentials. While the study is comprehensive in its computational approach, adding experimental validation, network-level simulations, and expanded discussion on implications would elevate its impact further.

Strengths:

Novelty and Scope:<br /> The manuscript provides a detailed investigation into the contrasting extracellular field potential (EFP) signatures arising from chemical synapses and gap junctions, an underexplored area in neuroscience.<br /> It highlights the critical role of active dendritic processes in shaping EFPs, pushing forward our understanding of how electrical and chemical synapses contribute differently to extracellular signals.

Methodological Rigor:<br /> The use of morphologically and biophysically realistic computational models for CA1 pyramidal neurons ensures that the findings are grounded in physiological relevance.<br /> Systematic analysis of various factors, including the presence of sodium, leak, and HCN channels, offers a clear dissection of how transmembrane currents shape EFPs.

Biological Relevance:<br /> The findings emphasize the importance of incorporating gap junctional inputs in analyses of extracellular signals, which have traditionally focused on chemical synapses.<br /> The observed polarity differences and spectral characteristics provide novel insights into how neural computations may differ based on the mode of synaptic input.

Clarity and Depth:<br /> The manuscript is well-structured, with a logical progression from synchronous input analyses to asynchronous and rhythmic inputs, ensuring comprehensive coverage of the topic.

Weaknesses and Areas for Improvement:

Generality and Validation:<br /> The study focuses exclusively on CA1 pyramidal neurons. Expanding the analysis to other cell types, such as interneurons or glial cells, would enhance the generalizability of the findings.<br /> Experimental validation of the computational predictions is entirely absent. Empirical data correlating the modeled EFPs with actual recordings would strengthen the claims.

Role of Active Dendritic Currents:<br /> The paper emphasizes active dendritic currents, particularly the role of HCN channels in generating outward currents under certain conditions. However, further discussion of how this mechanism integrates into broader network dynamics is warranted.

Analysis of Plasticity:<br /> While the manuscript mentions plasticity in the discussion, there are no simulations that account for activity-dependent changes in synaptic or gap junctional properties. Including such analyses could significantly enhance the relevance of the findings.

Frequency-Dependent Effects:<br /> The study demonstrates that gap junctional inputs suppress high-frequency EFP power due to membrane filtering. However, it could delve deeper into the implications of this for different brain rhythms, such as gamma or ripple oscillations.

Visualization:<br /> Figures are dense and could benefit from more intuitive labeling and focused presentations. For example, isolating key differences between chemical and gap junctional inputs in distinct panels would improve clarity.

Contextual Relevance:<br /> The manuscript touches on how these findings relate to known physiological roles of gap junctions (e.g., in gamma rhythms) but does not explore this in depth. Stronger integration of the results into known neural network dynamics would enhance its impact.

Suggestions for Improvement:

Broader Application:<br /> Simulate EFPs in multi-neuron networks to assess how the findings extend to network-level interactions, particularly in regions with mixed synaptic connectivity.

Experimental Correlation:<br /> Collaborate with experimental groups to validate the computational predictions using in vivo or in vitro recordings.

Mechanistic Insights:<br /> Provide a more detailed mechanistic explanation of how specific ionic currents (e.g., HCN, sodium, leak) interact during gap junctional vs. chemical synaptic inputs.

Implications for Neural Coding:<br /> Discuss how the observed differences in EFP signatures might influence neural coding, especially in circuits with heavy gap junctional connectivity.

Reviewer #2 (Public review):

Summary:

This computational work examines whether the inputs that neurons receive through electrical synapses (gap junctions) have different signatures in the extracellular local field potential (LFP) compared to inputs via chemical synapses. The authors present the results of a series of model simulations where either electric or chemical synapses targeting a single hippocampal pyramidal neuron are activated in various spatio-temporal patterns, and the resulting LFP in the vicinity of the cell is calculated and analyzed. The authors find several notable qualitative differences between the LFP patterns evoked by gap junctions vs. chemical synapses. For some of these findings, the authors demonstrate convincingly that the observed differences are explained by the electric vs. chemical nature of the input, and these results likely generalize to other cell types. However, in other cases, it remains plausible (or even likely) that the differences are caused, at least partly, by other factors (such as different intracellular voltage responses due to, e.g., the unequal strengths of the inputs). Furthermore, it was not immediately clear to me how the results could be applied to analyze more realistic situations where neurons receive partially synchronized excitatory and inhibitory inputs via chemical and electric synapses.

Strengths:

The main strength of the paper is that it draws attention to the fact that inputs to a neuron via gap junctions are expected to give rise to a different extracellular electric field compared to inputs via chemical synapses, even if the intracellular effects of the two types of input are similar. This is because, unlike chemical synaptic inputs, inputs via gap junctions are not directly associated with transmembrane currents. This is a general result that holds independent of many details such as the cell types or neurotransmitters involved.

Another strength of the article is that the authors attempt to provide intuitive, non-technical explanations of most of their findings, which should make the paper readable also for non-expert audiences (including experimentalists).

Weaknesses:

The most problematic aspect of the paper relates to the methodology for comparing the effects of electric vs. chemical synaptic inputs on the LFP. The authors seem to suggest that the primary cause of all the differences seen in the various simulation experiments is the different nature of the input, and particularly the difference between the transmembrane current evoked by chemical synapses and the gap junctional current that does not involve the extracellular space. However, this is clearly an oversimplification: since no real attempt is made to quantitatively match the two conditions that are compared (e.g., regarding the strength and temporal profile of the inputs), the differences seen can be due to factors other than the electric vs. chemical nature of synapses. In fact, if inputs were identical in all parameters other than the transmembrane vs. directly injected nature of the current, the intracellular voltage responses and, consequently, the currents through voltage-gated and leak currents would also be the same, and the LFPs would differ exactly by the contribution of the transmembrane current evoked by the chemical synapse. This is evidently not the case for any of the simulated comparisons presented, and the differences in the membrane potential response are rather striking in several cases (e.g., in the case of random inputs, there is only one action potential with gap junctions, but multiple action potentials with chemical synapses). Consequently, it remains unclear which observed differences are fundamental in the sense that they are directly related to the electric vs. chemical nature of the input, and which differences can be attributed to other factors such as differences in the strength and pattern of the inputs (and the resulting difference in the neuronal electric response).

Some of the explanations offered for the effects of cellular manipulations on the LFP appear to be incomplete. More specifically, the authors observed that blocking leak channels significantly changed the shape of the LFP response to synchronous synaptic inputs - but only when electric inputs were used, and when sodium channels were intact. The authors seemed to attribute this phenomenon to a direct effect of leak currents on the extracellular potential - however, this appears unlikely both because it does not explain why blocking the leak conductance had no effect in the other cases, and because the leak current is several orders of magnitude smaller than the spike-generating currents that make the largest contributions to the LFP. An indirect effect mediated by interactions of the leak current with some voltage-gated currents appears to be the most likely explanation, but identifying the exact mechanism would require further simulation experiments and/or a detailed analysis of intracellular currents and the membrane potential in time and space.

In every simulation experiment in this study, inputs through electric synapses are modeled as intracellular current injections of pre-determined amplitude and time course based on the sampled dendritic voltage of potential synaptic partners. This is a major simplification that may have a significant impact on the results. First, the current through gap junctions depends on the voltage difference between the two connected cellular compartments and is thus sensitive to the membrane potential of the cell that is treated as the neuron "receiving" the input in this study (although, strictly speaking, there is no pre- or postsynaptic neuron in interactions mediated by gap junctions). This dependence on the membrane potential of the target neuron is completely missing here. A related second point is that gap junctions also change the apparent membrane resistance of the neurons they connect, effectively acting as additional shunting (or leak) conductance in the relevant compartments. This effect is completely missed by treating gap junctions as pure current sources.

One prominent claim of the article that is emphasized even in the abstract is that HCN channels mediate an outward current in certain cases. Although this statement is technically correct, there are two reasons why I do not consider this a major finding of the paper. First, as the authors acknowledge, this is a trivial consequence of the relatively slow kinetics of HCN channels: when at least some of the channels are open, any input that is sufficiently fast and strong to take the membrane potential across the reversal potential of the channel will lead to the reversal of the polarity of the current. This effect is quite generic and well-known and is by no means specific to gap junctional inputs or even HCN channels. Second, and perhaps more importantly, the functional consequence of this reversed current through HCN channels is likely to be negligible. As clearly shown in Supplementary Figure S3, the HCN current becomes outward only for an extremely short time period during the action potential, which is also a period when several other currents are also active and likely dominant due to their much higher conductances. I also note that several of these relevant facts remain hidden in Figure 3, both because of its focus on peak values, and because of the radically different units on the vertical axes of the current plots.

Finally, I missed an appropriate validation of the neuronal model used, and also the characterization of the effects of the in silico manipulations used on the basic behavior of the model. As far as I understand, the model in its current form has not been used in other studies. If this is the case, it would be important to demonstrate convincingly through (preferably quantitative) comparisons with experimental data using different protocols that the model captures the physiological behavior of at least the relevant compartments (in this case, the dendrites and the soma) of hippocampal pyramidal neurons sufficiently well that the results of the modeling study are relevant to the real biological system. In addition, the correct interpretation of various manipulations of the model would be strongly facilitated by investigating and discussing how the physiological properties of the model neuron are affected by these alterations.

Reviewer #2 (Public review):

Summary:

This manuscript reports a comparison of microbial traits and host response traits in a laboratory model of infected granuloma using Mtb strains from different lineages. The authors report increased bacillary growth and granuloma formation, inversely associated with T cell activation that is characterized by CXCL9, granzyme B and TNF expression. They therefore infer that these T cell responses are likely to be host-protective and that the greater virulence of modern Mtb lineages may be driven by their ability to avoid triggering these responses.

Strengths:

The comparison of multiple Mtb lineages in a granuloma model that enables evaluation of the potential role of multiple host cells in Mtb control, offers a valuable experimental approach to study the biological mechanisms that underpin differential virulence of Mtb lineages that has been previously reported in clinical and epidemiological studies.

Weaknesses:

The study is rather limited to descriptive observations, and lacks experiments to test causal relationships between host and pathogen traits. Some of the presentation of the data are difficult to interpret, and some conclusions are not adequately supported by the data.

Comments on revisions:

The authors have addressed my previous comments with appropriate revisions and explanations.

Reviewer #3 (Public review):

Arbués and colleagues describe the impact of mycobacterial genetic diversity on host-infection phenotypes. The authors evaluate Mtb infection and contextualize host-responses, bacterial growth and metabolic transitioning in vitro using their previously established model of blood-derived, primary-human-cells cultured within a collagen/fibronectin matrix. They seek to demonstrate the effectiveness of the model in determining mycobacterial strain specific granuloma-dependent host-pathogen interactions.

Understanding the way mycobacterial genetic diversity impacts granuloma biology in tuberculosis is an important goal. One of this works strengths is the use of primary human cells and two constituents of pulmonary extracellular matrix to model Mtb infection. The authors and others have previously shown that Mtb infected PBMC aggregates share important characteristics with early pulmonary TB granulomas. Use of multiple genetically distinct strains of Mtb defines this work and further bolsters it potential impact. However, the study is not comprehensive as lineages 6 and 7 are not tested. Experiments are primarily descriptive, and the methodologies are conventional. Correlative relationships are the manuscripts focus and effect sizes are generally small.

The main aim of this work is to extend an in vitro granuloma model to the study of a large collection of well characterized, genetically diverse representatives of the mycobacterium tuberculosis complex (MTBC). I believe that they accomplish that aim. The work does investigate MTBC infection of aggregated PBMCs using three strains each of Mtb lineages 1-5 and H37Rv, which is not a trivial undertaking. The experimental aims are to show that MTBC genetic diversity impacts growth and dormancy of granuloma bound bacteria and, the host responses of granulomatous aggregation as well as macrophage apoptosis, lymphocyte activation and soluble mediator release within granulomas. The methodologies employed are sufficient to test most of these aims. The authors conclusions regarding their results are mostly supported by the data. The conclusion that lineage impacts growth within granulomas is likely true and the data as presented reflect such a relationship. Their conclusions regarding lineage's impact on dormancy are partially supported, as their findings demonstrate that assays for dormancy identify strain-specific metabolic changes in the bacteria consistent with a dormancy-like state but also identify replicating bacteria as being dormant. The data strongly supports the impact of mycobacterial genetic diversity on a spectrum of granulomatous responses in their model system. Those findings are a highlight of the publication. The data further supports the idea that strain diversity impacts macrophage apoptosis but a relationship of apoptosis to the granulomatous response is not effectively evaluated. The association of lymphocyte activation with reduced mycobacterial growth as an aspect of granulomas is well documented in the literature and a negative correlation between T cell activation and growth is supported by the authors results. Their data also support the conclusion that soluble mediator production by PBMCs is different based on the infecting strain of mycobacteria and that IL1b modulates aggregate phenotypes in their model.

The authors contribute some valuable insights, particularly in Figure 3. Their model is higher echelon relative to others in the field, but I don't believe that it possesses all the components necessary to replicate formation of mycobacterial granulomas in vivo. That being said, their identification of donor-dependent aggregation phenotypes by mycobacterial strain has the potential to enable future investigations of human and mycobacterial genetic components that are involved in the formation of TB granulomas.

Reviewer #1 (Public review):

Summary:

Chen and Phillips describe the dynamic appearance of cytoplasmic granules during embryogenesis analogous to SIMR germ granules, and distinct from CSR-1-containing granules, in the C. elegans germline. They show that the nuclear Argonaute NRDE-3, when mutated to abrogate small RNA binding, or in specific genetic mutants, partially colocalizes to these granules along with other RNAi factors, such as SIMR-1, ENRI-2, RDE-3, and RRF-1. Furthermore, NRDE-3 RIP-seq analysis in early vs. late embryos is used to conclude that NRDE-3 binds CSR-1-dependent 22G RNAs in early embryos and ERGO-1-dependent 22G RNAs in late embryos. These data lead to their model that NRDE-3 undergoes small RNA substrate "switching" that occurs in these embryonic SIMR granules and functions to silence two distinct sets of target transcripts - maternal, CSR-1 targeted mRNAs in early embryos and duplicated genes and repeat elements in late embryos.

Strengths:

The identification and function of small RNA-related granules during embryogenesis is a poorly understood area and this study will provide the impetus for future studies on the identification and potential functional compartmentalization of small RNA pathways and machinery during embryogenesis.

Weaknesses:

(1) The authors acknowledge the following issue that loss of SIMR granules have no significant impact on NRDE-3 small RNA loading weakens the functional relevance of these structures. However, this point is clearly discussed and, as they note in their Discussion, it is entirely possible that these embryonic granules may be "incidental condensates."

Reviewer #2 (Public review):

Summary:

NRDE-3 is a nuclear WAGO-clade Argonaute that, in somatic cells, binds small RNAs amplified in response to the ERGO-class 26G RNAs that target repetitive sequences. This manuscript reports that, in the germline and early embryos, NRDE-3 interacts with a different set of small RNAs that target mRNAs. This class of small RNAs were previously shown to bind to a different WAGO-clade Argonaute called CSR-1, which is cytoplasmic unlike nuclear NRDE-3. The switch in NRDE-3 specificity parallels recent findings in Ascaris where the Ascaris NRDE homolog was shown to switch from sRNAs that target repetitive sequences to CSR-class sRNAs that target mRNAs.

The manuscript also correlates the change in NRDE-3 specificity with the appearance in embryos of cytoplasmic condensates that accumulate SIMR-1, a scaffolding protein that the authors previously implicated in sRNA loading for a different nuclear Argonaute HRDE-1. By analogy, and through a set of corelative evidence, the authors argue that SIMR foci arise in embryogenesis to facilitate the change in NRDE-3 small RNA repertoire. The paper presents lots of data that beautifully documents the appearance and composition of the embryonic SIMR-1 foci, including evidence that a mutated NRDE-3 that cannot bind sRNAs accumulate in SIMR-1 foci in SIMR-1-dependent fashion.

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 #1 (Public review):

Summary:

The works seeks to investigate the efficacy of linalool as a natural alternative for combating Saprolegnia parasitica infections, which would provide great benefit to aquaculture. This paper shows the effect of linalool in vitro using a variety of techniques including changes in S. parasitica membrane integrity following linalool exposure and alterations in cell metabolism and ribosome function. Additionally, this work goes on to show that prophylactic and concurrent treatment of linalool at the time of S. parasitica infection can improve survival and tissue damage in vivo in their grass carp infection model. The conclusions of the paper are partially supported by the data with the corrections done by the authors improving clarity such that I believe there is merit in the work.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to delineate the antimicrobial activity of linalool and tried to investigate the mode of action on linalool against S. parasitica infection. One of the main focus of this work was to identify the in vitro and in vivo mechanisms associated with the protective role of linalool against S. parasitica infection.

Strengths:

(1) Authors have used a variety of techniques to prove their hypothesis.<br /> (2) Adequate number of replicates were used in their studies.<br /> (3) Their findings showed a protective role of linalool against oomycetes and makes it an attractive future antibiotic in the aquaculture industry.

Weaknesses: The revised version of the manuscript is more thoroughly written with clearer explanations, however there are a few weaknesses in this manuscript.

(1) Although the introduction section was rewritten with rationale, it's still lengthy and not very much to the point.<br /> (2) The claim of linalool regulating the gut microbiota is based on the correlation analysis only. It's not super convincing and requires experimental validation to strengthen the claim.

Overall, the conclusions drawn by the authors are justified by the data. Importantly, this paper has discovered the novelty of the compound linalool as a potent antimicrobial agent and might open up future possibilities to use this compound in the aquaculture industry.

Reviewer #1 (Public review):

Summary:

The manuscript by Wang et al. investigates the relationship between Streptococcus Suis (S. Suis) growth phases and levels of virulence factor, capsular polysaccharide (CPS), in the bacterial cell wall. They use an understudied mouse intranasal infection model to connect growth phase related CPS abundance to the pathogenicity of the bacteria in the nose, blood, and other organs. Adoptive transfer of serum against either CPS or V5 (five other virulence factors) reinforces their discovery of CPS levels on S. Suis in different organs and stages of infection. Vaccination against bacterial infections can be difficult, and understanding how the serotype of a bacterial pathogen changes between infection sights and systemic disease is critical. Further, understanding host-pathogen interactions at early time points in the upper respiratory tract may have broad implications for vaccine development. While some of the results are interesting and compelling, others are not supported by the data and require further experimental work.

Strengths:

The model of intranasal infection is compelling to expand upon work previously done in vitro and with systemic routes of infection. The histology and fluorescent imaging of the olfactory epithelium and olfactory bulb complement work in Figure 2 about the attachment of S. suis to epithelial cells and the bacterial burden over time in different organs of Figure 3. Histology was performed at 1 hour and 9 days after intranasal infection with stationary phase S. suis and drives home that this pathogen can invade the olfactory nerve and may potentially cause bacterial meningitis seen in some infected swine.

The adoptive transfer of either anti-CPS or anti-V5 to mice before infection at both longer (12 hr), and shorter (0.5 hr) time points is useful to demonstrate that the changes in cell wall composition between the NALT/CSF and blood compartments result in different efficacy in clearing bacteria from those locations. This is fundamental for the development of vaccines for the swine industry and begs those developing other bacterial vaccines to consider what virulence factors are the most useful as neutralizing antibody targets at the sight of bacterial invasion.

Demonstrating that the amount of CPS within the cell wall of S. Suis is related to the growth phase of the bacteria is an important consideration for vaccine development. While others had previously shown that CPS levels were higher in the blood than in the CNS, and that CPS decreases the invasion of epithelial cells, the close look at the olfactory epithelium at an early time point ties together in vitro findings. The control of a CPS-negative strain was critical to understanding their findings. The location and the microbial community that bacterial pathogens live within may change the growth phase and therefore also the cell wall components.

Weaknesses:

The authors present compelling data that is relevant to the development of anti-bacterial vaccinations and show a relationship between CPS levels and pathogenicity. However, the use of a laboratory murine model requiring acetic acid pre-treatment and a high i.n. dose. Therefore, the findings presented may not represent what occurs in swine. Furthermore, several conclusions are not supported by the data and require substantial new experimental support. Thus, major concerns remain that impact the validity of the findings.

Major concerns for the manuscript:

The intranasal infections were done with S. Suis in the stationary phase which has been shown to have less CPS on the cell wall. While this mimics the literature that shows S. Suis to have less CPS in the CNS, the difference in the pathogenesis of a log phase vs. stationary phage intranasal infection would be interesting. Especially because the bacteria is a part of the natural microbial community of swine tonsils, it is curious if the change in growth phase and therefore CPS levels may be a causative reason for pathogenic invasion in some pigs. To take this line of thought a step further, the authors should consider taking the bacteria from NALT/CSF and blood and compare the lag times bacteria from different organs take to enter a log growth phase to show whether the difference in CPS is because S. Suis in each location is in a different growth phase. If log phase bacteria were intranasally delivered, would it adapt a stationary phase life strategy? How long would that take? Lastly, the authors should be cautious about claims about S. suis downregulating CPS in the NALT for increased invasion and upregulating CPS to survive phagocytosis in blood. While it is true that the data shows that there are different levels of CPS in these locations, the regulation and mechanism of the recorded and observed cell wall difference is not investigated past the correlation to the growth phase. While mechanistic work is outside the scope of the current work, readers should keep in mind that these results may be explained multiple ways. In addition, the mouse model is used rather than the usual host of a pig. The NALTs of conventional pigs and SPF mice certainly have unique microbial communities and this may affect the pathogenesis of S. suis in the mouse, therefore influencing the results. Because the authors show a higher infection rate in the mouse with acetic acid, they may want to consider investigating what the mouse NALT microenvironment is naturally doing to exclude more bacterial invasion in future studies. Is it simply a host mismatch or is there something about the microbiome or steady-state immune system in the nose of mice that is different from pigs?

Reviewer #1 (Public review):

The authors have strengthened their conclusions by providing additional information about the specificity of their antibodies, but at the same time the authors have revealed concerning information about the source of their antibodies.

It appears that many of the antibodies used in this study have been discontinued because the supplier company was involved in a scandal of animal cruelty and all their goats and rabbits Ab products were sacrificed. The authors acknowledge that this is unfortunate but they also claim that the issue is out of their hands.

The authors' statement is false; the authors ought to not use these antibodies, just as the providing company chose to discontinue them, as<br /> those antibodies are tied to animal cruelty. The issue that the authors feel OK with using them is of concern. In short, please remove any results from unethical antibodies.

Removal of such results also best serves science. That is, any of their results using the discontinued antibodies means that the authors' results are non-reproducible and we should be striving to publish good, reproducible science.

For the antibodies that do not have unethical origins the authors claim that their antibodies have been appropriately validated, by "testing in positive control tissue and/or Western blot or in situ hybridization". This is good but needs to be expanded upon. It is a strong selling point that the Abs are validated and I want to see additional information in their Supplementary Table 2 stating for each Ab specifically:

(1) What +ve control tissue was used in the validation of each Ab and which species that +ve control came from. Likewise, if competition assays to confirm validity was used, please also specify.

(2) Which assay was the Ab validated for (WB, IHC, ELISA, all etc)

(3) For Antibodies that were validated for, or using WBs please let the reader know if there were additional bands showing.

(4) Include references to the literature that supports these validations. That is, please make it easy for the reader to appreciate the hard work that went into the validation of the Antibodies.

Finally, for the Abs, when the authors write that "All antibodies used have been validated by testing in positive control tissue and/or Western blot or in situ hybridization" I fail to understand what in situ hybridisation means in this context. I am under the impression that in situ hybridisation is some nucleic acid -hybridising-to-organ or tissue slice. Not polypeptide binding.

Reviewer #3 (Public review):

In this study, the authors tested a dietary intervention focused on improving meal regularity. Participants first utilized a smartphone application to track their meal frequencies, and then they were asked to restrict their meal intake to times when they most often eat to enhance meal regularity for six weeks. This, supposedly, resulted in some weight loss, supposedly independent of changes in caloric intake.

The concept is appealing, and it is interesting to use a smartphone app in participants' typical everyday environment to regularize food intake. It asks from participants to stick to meal intake times that are supported in many cultures, and it asks them not to eat outside of what are likely unhealthy habits such as grazing a refrigerator late at night. In essence, this is a restrictive diet, not restricting caloric intake but the timing of food intake, and it has many parallel to time restricted feeding. It is important to note that there are many restrictive diets, and a common problem with restrictive diets is that while they allow one to lose a couple of pounds for a couple of months just as with this diet, the long-term success is very poor because they depend on restriction. This issue is still not discussed.

Further, why the participants lose weight, whether this is indeed due to a reduction in food intake as implied, or if the weight loss occurred without a reduction in caloric intake as first stated by the authors and now suggested remains to be determined as the method of food diary as a method to assess caloric intake lacks rigor as has been well established and has been shown again and again to be misleading even though many readers without that knowledge draw conclusions from such studies and they should best have been omitted.

The authors hypothesize that the intervention improves metabolism by improving circadian rhythmicity. That's plausible, but the study provides only a subjective questionnaire and lacks more objective measures such as actigraphy.

While the authors now state now that this as a pilot study, the study falls short of providing mechanistic insights into what underlies the weight loss and the many correlations provided do not make up for this weakness.

Overall, while this pilot study introduces an interesting approach to meal regularity, its limitations highlight the need for more rigorous studies to validate these findings.

(1) Unreliable method of caloric intake

The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake. As pointed out earlier by me and now cited in the revised manuscript, the NEJM paper (DOI: 10.1056/NEJM199212313272701) reported that some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. The question is, why include such unreliable data that is more misleading than informative at all? These data should have been omitted. More rigorous methods for assessing food intake should have been utilized. I understand this requires more effort, such as providing participants with meals, or using better methods that photograph and weigh the meals, etc., but it is certainly feasible. It has been done many times in other studies. Further, the control group was not asked to restrict their diet in any way, and hence, asking for a restriction in timing in the treatment group may be sufficient to reduce caloric intake and induce weight loss.<br /> Merely acknowledging the unreliability of self-reported caloric intake is insufficient, as it still leaves the reader with the impression that this weight loss is independent of caloric intake when, in reality, we actually have no idea if food intake contributes to it. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced a more rigorous study would unveil testing this paradigm, this intervention may merely represent another restrictive diet among countless others that show that one may lose weight by going on a diet. Seemingly, any restrictive diet works for a few months. The trouble is they do not work long-term because they depend on restriction. I agree with the authors that their intervention seems common sense and has little downside, but one also needs to be realistic about the prospects of this intervention.

(2) Lack of objective data regarding circadian rhythm

The assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, is subjective. More objective methods like actigraphy would have strengthened the study.

Actigraphy is considered better than a sleep questionnaire for assessing circadian rhythms because it provides objective data on activity patterns over time, offering a more accurate picture of sleep-wake cycles compared to subjective self-reported information from a questionnaire.

The authors' responses to my prior review are misleading.

I understand that this is a pilot study. Is it appropriate to point out weaknesses and flaws in the conclusion drawn from a pilot study? Absolutely, that is the reviewer's job.

I also understand that food intake can affect circadian rhythm, which was part of the rationale behind the study. Is it appropriate to criticize the study for not examining the effect of the intervention on circadian rhythm using objective measures provided by actigraphy? Yes, it is, as this would have provided mechanistic insights that are more rigorous. I understand that this was not the declared goal, but it should have been examined in a pilot study. To jump to the conclusion that based on prior studies, the intervention will improve circadian rhythms as the authors do is not rigorous and hence a weakness.

A less rigorous method, such as a food questionnaire, to assess caloric intake can result in inadequately supported and potentially misleading conclusions. By including it, the reader may conclude that there was no change in caloric intake when indeed we do not know. I disagree with the authors that this is a minor issue. The associations and correlations the authors provide do not solve the issue. Hence, to make it very clear, it remains to be studied if this intervention reduces weight by reducing caloric intake or other mechanisms. Including this data reduces the study's rigor as it suggests that there is no difference in food intake.

I did not suggest to only use an actimeter (which is a device); I suggested actigraphy. Actigraphy is widely recognized in the field for its utility in circadian rhythm research and provides objective data, while the questionnaire used is subjective. The authors do quote papers comparing their survey to actigraphy by correlation analysis, but the fundamental difference of the two approaches remains. Does an objective measure increase rigor compared to a subjective assessment? Yes, it does.

Similarly, I did not state "that any form of imposed diet appears to lead to weight loss over several months." I said that many forms of restrictive diets do induce weight loss of a similar magnitude to this diet.

The authors should have discussed the fundamental confounder of the study in that the treatment group is asked to restrict food intake to specific times while the control group is not asked to restrict in any way and the potential contribution of this to the weight loss observed.

Reviewer #1 (Public review):

Summary:

The authors found that IL-1b signaling is pivotal for hypoxemia development and can modulate NETs formation in LPS+HVV ALI model.

Strengths:

They used IL1R1 ko mice and proved that IL1R1 is involved in ALI model proving that IL1b signalling leads towards ARDS. In addition, hypothermia reduces this effect, suggesting a therapeutic option.

Weaknesses:

(1) IL1R1 binds IL1a and IL1b. What would be the role of IL1a in this scenario?

(2) The authors depleted neutrophils using anti-Ly6G. What about MDSCs? Do these latter cells be involved in ARDS and VILI?

(3) The authors found that TH inhibited IL-1β release from macrophages led to less NETs formation and albumin leakage in the alveolar space in their lung injury model. A graphical abstract could be included suggesting a cellular mechanism.

(4) If Macrophages are responsible for IL1b release that via IL1R1 induces NETosis, what happens if you deplete macrophages? what is the role of epithelial cells?

Reviewer #2 (Public review):

Summary:

The manuscript by Nosaka et al is a comprehensive study exploring the involvement of IL1beta signaling in a 2-hit model of lung injury + ventilation, with a focus on modulation by hypothermia.

Strengths:

The authors demonstrate quite convincingly that interleukin 1 beta plays a role in the development of ventilator-induced lung injury in this model, and that this role includes the regulation of neutrophil extracellular trap formation. The authors use a variety of in vivo animal-based and in vitro cell culture work, and interventions including global gene knockout, cell-targeted knockout and pharmacological inhibition, which greatly strengthen the ability to make clear biological interpretations.

Weaknesses:

A primary point for open discussion is the translatability of the findings to patients. The main model used, one of intratracheal LPS plus mechanical ventilation is well accepted for research exploring the pathogenesis and potential treatments for acute respiratory distress syndrome (ARDS). However, the interpretation may still be open to question - in the model here, animals were exposed to LPS to induce inflammation for only 2 hours, and seemingly displayed no signs of sickness, before the start of ventilation. This would not be typical for the majority of ARDS patients, and whether hypothermia could be effective once substantial injury is already present remains an open question. The interaction between LPS/infection and temperature is also complicated - in humans, LPS (or infection) induces a febrile, hyperthermic response, whereas in mice LPS induces hypothermia (eg. Ganeshan K, Chawla A. Nat Rev Endocrinol. 2017;13:458-465). Given this difference in physiological response, it is therefore unclear whether hypothermia in mice and hypothermia in humans are easily comparable. Finally, the use of only young, male animals such as in the current study has been typical but may be criticised as limiting translatability to people.

Therefore while the conclusions of the paper are well supported by the data, and the biological pathways have been impressively explored, questions still remain regarding the ultimate interpretations.

Fusion Body Art Product Review - Prime Face Paint Colours

Reviewer #1 (Public review):

Summary:

The authors investigated the role of an E3 ubiquitin ligase ITCH in regulating the viral life cycle of SARS-CoV-2. The authors showed that ITCH mediates ubiquitination of the membrane (M) and envelope (E) proteins of SARS-CoV-2. Ubiquitination of E and M results in enhanced interactions between the structural proteins and redistribution of the structural proteins into autophagosomes. The authors claim that the enhanced interactions between structural proteins and trafficking of the structural proteins into autophagosomes contribute to SARS-CoV-2 replication and egress, prompting ITCH as a potential antiviral target. ITCH also alters the cellular distribution of host proteases important for spike cleavage which protect and stabilize spike with cleavage. The authors also demonstrated that SARS-CoV-2 replication is augmented by ITCH in which virus replication is significantly impaired in cells lacking ITCH expression.

Strengths:

The authors provided high-quality data with appropriate experimental controls to justify their claims and conclusions. The mechanistic analyses are excellent and presented in a logical manner. The investigation of the role of ubiquitination in coronavirus assembly and egress is novel as most previous studies focused on its role in mediating innate immune responses.

Weaknesses:

Although the authors showed that ITCH ubiquitinates E and M proteins, the claim that such ubiquitination promotes virion assembly and egress is circumstantial. The enhanced interaction between the structural proteins and targeting of ubiquitinated structural proteins into autophagosomes does not necessarily result in increased virion production and release as suggested by the authors. There is a disconnect between the ubiquitination of structural proteins and the role of ITCH in augmenting virus replication as shown in Fig. 6A and B. In addition, the authors showed that the catalytic activity of ITCH is important for the localization and maturation of host proteases. However, the mechanism behind is unknown. Also, it is unclear how protection of spike from cleavage conferred by ITCH explains its role in promoting replication as a lack of spike cleavage would inevitably compromise entry. The major weakness of the manuscript is the lack of experimental data that explains the molecular role of ITCH in relation to its phenotype observed during SARS-CoV-2 infection.

Reviewer #2 (Public review):

Summary:<br /> In this manuscript Qiwang Xiang et al. investigated the role of the E3 ubiquitin ligase ITCH in the life cycle of SARS-CoV-2. They claim the following:<br /> i) ITCH promotes virion assembly by interacting with E and M proteins and enhancing their K63-linked ubiquitination<br /> ii) ITCH-mediated ubiquitination promotes autophagosome-dependent secretion of viral particles.<br /> iii) ITCH stabilizes the viral spike protein by impairing its processing by furin and catepsin L proteases.<br /> The manuscript provides an interesting exploration of ITCH's role in the SARS-CoV-2 life cycle but requires additional work to strengthen key claims and address potential confounding factors.

Strengths:

The experiments are sufficiently clear in documenting that ITCH activity is critical for efficient SARS-CoV-2 replication and for M and E proteins K63-linked ubiquitination

Weaknesses:

• The manuscript does not convincingly demonstrate how ITCH-mediated ubiquitination of E and M impacts virus assembly and release. Identifying the specific lysine residues in M and E targeted by ITCH, and generating mutant VLPs or recombinant viruses, would strengthen the conclusions.<br /> • Most of the conclusions rely on ITCH overexpression data, which may have off-target effects on Golgi integrity and vesicular trafficking. For instance, figure 4F provides evidence of altered Golgi morphology and TGN46 fragmentation raising concerns that ITCH overexpression could indirectly mislocalize furin, affecting S1/S2 cleavage of the spike protein. In addition, inhibition of furin activity may also lead to off-target effects, given its role in processing numerous host proteins.<br /> • Similarly, ITCH overexpression is likely to indirectly affect cathepsin-L maturation. In addition, the manuscript does not clarify how impaired cathepsin L activity would influence virus assembly or release.<br /> • A major concern is also the lack of quantification and statistical analysis of immunofluorescence images throughout the manuscript, which undermines the reliability of these observations.

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 #1 (Public review):

This study uses structural and functional approaches to investigate the regulation of the Na/Ca exchanger NCX1 by an activator, PIP2, and an inhibitor, SEA0400.

State-of-the-art methods are employed, and the data are of high quality and presented very clearly. The manuscript combines two rather different studies (one on PIP2; and one on SEA0400) neither of which is explored in the depth one might have hoped to form robust conclusions and significantly extend knowledge in the field.

The novel aspect of this work is the study of PIP2. Unfortunately, technical limitations precluded structural data on binding of the native PIP2, so an unnatural short-chained analog, di-C8 PIP2, was used instead. This raises the question of whether these two molecules, which have similar but very distinctly different profiles of activation, actually share the same binding pocket and mode of action. In an effort to address this, the authors mutate key residues predicted to be important in forming the binding site for the phosphorylated head group of PIP2. However, none of these mutations prevent PIP2 activation. The only ones that have a significant effect also influence the Na-dependent inactivation process independently of PIP2, thus casting doubt on their role in PIP2 binding, and thus identification of the PIP2 binding site. A more extensive mutagenic study, based on the di-C8 PIP2 binding site, would have given more depth to this work and might have been more revealing mechanistically.

The SEA0400 aspect of the work does not integrate particularly well with the rest of the manuscript. This study confirms the previously reported structure and binding site for SEA0400 but provides no further information. While interesting speculation is presented regarding the connection between SEA0400 inhibition and Na-dependent inactivation, further experiments to test this idea are not included here.

Reviewer #2 (Public review):

The study by Xue et al. reports the structural basis for the regulation of the human cardiac sodium-calcium exchanger, NCX1, by the endogenous activator PIP2 and the small molecule inhibitor SEA400. This well-written study contextualizes the new data within the existing literature on NCX1 and the broader NCX family. This work builds upon the authors' previous study (Xue et al., 2023), which presented the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states. The 2023 study highlighted key structural differences between the active and inactive states and proposed a mechanism where the activity of NCX1 is regulated by the interactions between the ion-transporting transmembrane domain and the cytosolic regulatory domain. Specifically, in the inward-facing state and at low cytosolic calcium levels, the transmembrane (TM) and cytosolic domains form a stable interaction that results in the inactivation of the exchanger. In contrast, calcium binding to the cytosolic domain at high cytosolic calcium levels disrupts the interaction with the TM domain, leading to active ion exchange.

In the current study, the authors present two mechanisms explaining how both PIP2 stimulates NCX1 activity by destabilizing the protein's inactive state (i.e., by disrupting the interaction between the TM domain and the cytosolic domain) and how SEA400 stabilizes this interaction, thereby acting as a specific inhibitor of the system.

The first part of the results section addresses the effect of PIP2 and PIP2 diC8 on NCX1 activity. This is pertinent as the authors use the diC8 version of this lipid (which has a shorter acyl chain) in their subsequent cryo-EM structure due to the instability of native PIP2. I am not an electrophysiology expert; however, my main comment would be to ask whether there is sufficient data here to characterise fully the differences between PIP2 and PIP2 diC8 on NCX1 function. It appears from the text that this study is the first to report these differences, so perhaps this data needs to be more robust. The spread of the data points in Figure 1B is possibly a little unconvincing given that only six measurements were taken. Why is there one outlier in Figure 1A? Were these results taken using the same batch of oocytes? Are these technical or biological replicates? Is the convention to use statistical significance for these types of experiments?

I am also somewhat skeptical about the modelling of the PIP2 diC8 molecule. The authors state, "The density of the IP3 head group from the bound PIP2 diC8 is well-defined in the EM map. The acyl chains, however, are flexible and could not be resolved in the structure (Fig. S2)."

However, the density appears rather ambiguous to me, and the ligand does not fit well within the density. Specifically, there is a large extension in the volume near the phosphate at the 5' position, with no corresponding volume near the 4' phosphate. Additionally, there is no bifurcation of the volume near the lipid tails. I attempted to model cholesterol hemisuccinate (PDB: Y01) into this density, and it fits reasonably well - at least as well as PIP2 diC8. I am also concerned that if this site is specific for PIP2, then why are there no specific interactions with the lipid phosphates? How can the authors explain the difference between PIP2 and PIP2 diC8 if the acyl chains don't make any direct interactions with the TM domain? In short, the structures do not explain the functional differences presented in Figure 1.

The side chain densities for Arg167 and Arg220 are also quite weak. While there is some density for the side chain of Lys164, it is also very weak. I would expect that if this site were truly specific for PIP2, it should exhibit greater structural rigidity - otherwise, how is this specific?

Given this observation, have the authors considered using other PIP2 variants to determine if the specificity lies with PI4,5P2 as opposed to PI3,5P2 or PI3,4P2? A lack of specificity may explain the observed poor density.

I also noticed many lipid-like densities in the maps for this complex. Is it possible that the authors overlooked something? For instance, there is a cholesterol-like density near Val51, as well as something intriguing near Trp763, where I could model PIP2 diC8 (though this leads to a clash with Trp763). I wonder if the authors are working with mixed populations in their dataset. The accompanying description of the structural changes is well-written (assuming it is accurate).

I would recommend that the authors update the figures associated with this section, as they are currently somewhat difficult to interpret without prior knowledge of NCX architecture. My suggestions include:

- Including the density for the PIP2 diC8 in Figure 2A.

- Adding membrane boundaries (cytosolic vs. extracellular) in Figure 2B.

- Labeling the cytosolic domains in Figure 2B.

- Adding hydrogen bond distances in Figure 2A.

- Detailing the domain movements in Figure 2B (what is the significance of the grey vs. blue structures?).

The section on the mechanism of SEA400-induced inactivation is strong. The maps are of better quality than those for the PIP2 diC8 complex, and the ligand fits well. However, I noticed a density peak below F02 on SEA400 that lies within the hydrogen bonding distance of Asp825. Is this a water molecule? If so, is this significant?

Furthermore, there are many unmodeled regions that are likely cholesterol hemisuccinate or detergent molecules, which may warrant further investigation.

The authors introduce SEA400 as a selective inhibitor of NCX1; however, there is little to no comparison between the binding sites of the different NCX proteins. This section could be expanded. Perhaps Fig. 4C could include sequence conservation data.

Additionally, is the fenestration in the membrane physiological, or is it merely a hole forced open by the binding of SEA400? I was unclear as to whether the authors were suggesting a physiological role for this feature, similar to those observed in sodium channels.

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 #1 (Public review):

DiPeso et al. develop two tools to i) classify micronucleated (MN) cells, which they call VCS MN, and ii) segment micronuclei and nuclei with MNFinder. They then use these tools to identify transcriptional changes in MN cells.

The strengths of this study are:

- Developing highly specialized tools to speed up the analysis of specific cellular phenomena such as MN formation and rupture is likely valuable to the community and neglected by developers of more generalist methods.

- A lot of work and ideas have gone into this manuscript. It is clearly a valuable contribution.

- Combining automated analysis, single-cell labeling, and cell sorting is an exciting approach to enrich for phenotypes of interest, which the authors demonstrate here.

The authors addressed my original concerns related to the first version of this manuscript.

Reviewer #2 (Public review):

Summary:

Micronuclei are aberrant nuclear structures frequently seen following the missegregation of chromosomes. The authors present two image analysis methods, one robust and another rapid, to identify micronuclei (MN) bearing cells. To analyse their software efficacy, the authors study images of cells treated with MPS1 inhibitor to induce chromosome missegregation. Next, the authors use RNA-seq to assess the outcomes of their MN-identifying methods: they do not observe a transcriptomic signature specific to MN but find changes that correlate with aneuploidy status. Overall, this work offers new tools to identify MN-presenting cells, and it sets the stage with clear benchmarks for further software development.

Strengths:

Currently, there are no robust MN classifiers with a clear quantification of their efficiency across cell lines (mIoU score). The software presented here tries to address this gap. GitHub material (images, ground truth labels, tools, protocols, etc.) provided is a great asset to computational biologists. The method has been tested in more than one cell line. This method can help integrate cell biology and 'omics' data, making it suitable for multimodal studies.

Weaknesses:

Although the classifier outperforms available tools for MN segmentation by providing mIoU, it's not yet at a point where it can be reliably applied to functional genomics assays where we expect a range of phenotypic penetrance in most cell lines (e.g., misshapen, multinucleated, and lagging DNA in addition to micronucleated cells). The discussion considers the nature and proportion of MN in RPE1 cells, and how the classifier is well-suited for RPE1 that predominantly display MN structures. Whether the classifier can rigorously assign MN-presenting cells amidst drastic nuclear aberrancies following a spindle checkpoint loss needs to be tested in the future.

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 #2 (Public review):

The fledgling field of epitranscriptomics has encountered various technical roadblocks with implications as to the validity of early epitranscriptomics mapping data. As a prime example, the low specificity of (supposedly) modification-specific antibodies for the enrichment of modified RNAs, has been ignored for quite some time and is only now recognized for its dismal reproducibility (between different labs), which necessitates the development of alternative methods for modification detection. Furthermore, early attempts to map individual epitranscriptomes using sequencing-based techniques are largely characterized by the deliberate avoidance of orthogonal approaches aimed at confirming the existence of RNA modifications that have been originally identified.

Improved methodology, the inclusion of various controls, and better mapping algorithms as well as the application of robust statistics for the identification of false-positive RNA modification calls have allowed revisiting original (seminal) publications whose early mapping data allowed making hyperbolic claims about the number, localization and importance of RNA modifications, especially in mRNA. Besides the existence of m6A in mRNA, the detectable incidence of RNA modifications in mRNAs has drastically dropped.

As for m5C, the subject of the manuscript submitted by Zhou et al., its identification in mRNA goes back to Squires et al., 2012 reporting on >10.000 sites in mRNA of a human cancer cell line, followed by intermittent findings reporting on pretty much every number between 0 to > 100.000 m5C sites in different human cell-derived mRNA transcriptomes. The reason for such discrepancy is most likely of a technical nature. Importantly, all studies reporting on actual transcript numbers that were m5C-modified relied on RNA bisulfite sequencing, an NGS-based method, that can discriminate between methylated and non-methylated Cs after chemical deamination of C but not m5C. RNA bisulfite sequencing has a notoriously high background due to deamination artifacts, which occur largely due to incomplete denaturation of double-stranded regions (denaturing-resistant) of RNA molecules. Furthermore, m5C sites in mRNAs have now been mapped to regions that have not only sequence identity but also structural features of tRNAs. Various studies revealed that the highly conserved m5C RNA methyltransferases NSUN2 and NSUN6 do not only accept tRNAs but also other RNAs (including mRNAs) as methylation substrates, which in combination account for most of the RNA bisulfite-mapped m5C sites in human mRNA transcriptomes. Is m5C in mRNA only a result of the Star activity of tRNA or rRNA modification enzymes, or is their low stoichiometry biologically relevant?

In light of the short-comings of existing tools to robustly determine m5C in transcriptomes, other methods, like DRAM-seq, aiming to map m5C independently of ex situ RNA treatment with chemicals, are needed to arrive at a more solid "ground state", from which it will be possible to state and test various hypotheses as to the biological function of m5C, especially in lowly abundant RNAs such as mRNA.

Importantly, the identification of >10.000 sites containing m5C increases through DRAM-Seq, increases the number of potential m5C marks in human cancer cells from a couple of 100 (after rigorous post-hoc analysis of RNA bisulfite sequencing data) by orders of magnitude. This begs the question, whether or not the application of these editing tools results in editing artefacts overstating the number of actual m5C sites in the human cancer transcriptome.

Reviewer #1 (Public review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb are identified as significantly associated with post-diapause fecundity, which they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not arista are required for the effects of Dip-gamma and sbb. They show that removing antenna has a diapause specific lifespan extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause associated effects.

Strengths:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is.

Weaknesses:

A minor point is I cannot find how many DGRP lines are used.

Reviewer #1 (Public review):

Summary:

Gao et al. has demonstrated that the the pesticide emamectin benzoate (EB) treatment of brown plathopper (BPH) leads to increased egg laying in the insect, which is a common agricultural pest. The authors hypothesize that EB upregulates JH titer resulting in increased fecundity.

Strengths:

The finding that a class of pesticide increases fecundity of brown planthopper is interesting.

Weaknesses:

(1) EB is an allosteric modulator of GluCl. That means it EB physically interacts with GluCl initiating a structural change in the cannel protein. Yet the authors here central hypothesis is about how EB can upregulate the mRNA of GluCl. I do not know whether there is any evidence that an allosteric modulator can function as a transcriptional activator for the same receptor protein. The basic premise of the paper sounds counterintuitive. This is a structural problem and should be addressed by the authors by giving sufficient evidence about such demonstrated mechanisms before.<br /> (2) I am surprised to see a 4th instar larval application or treatment with EB results in upregulation of JH in the adult stages. Complicating the results further is the observation that a 4th instar EB application results in an immediate decrease in JH titer. There is a high possibility that this late JH titer increase is an indirect effect.<br /> (3) The writing quality of the paper needs improvement. Particularly with respect to describing processes, and abbreviations. In several instances authors have not adequately described the processes they have introduced, thus confusing the readers.<br /> (4) In the section 'EB promotes ovarian development' the authors have shown that EB treatment results in increased detention of eggs which contradicts their own results which show that EB promotes egg laying. Again, this is a serious contradiction that nullifies their hypothesis.<br /> (5) Furthermore, the results suggest that oogenesis is not affected by EB application. The authors should devote a section to discussing how they are observing increased egg numbers in EB-treated insects while not impacting Oogenesis.<br /> (6) Met is the receptor of JH and to my understanding, remains mostly constant in terms of its mRNA or protein levels throughout various developmental periods in many different insects. Therefore, the presence of JH becomes the major driving factor for physiological events and not the presence of the receptor Met. Here the authors have demonstrated an increase in Met mRNA as a result of EB treatment. Their central hypothesis is that EB increases JH titer to result in enhanced fecundity. JH action will not result in the activation of Met. Although not contradictory to the hypothesis, the increase in mRNA content of Met is contrary to the findings of the JH field thus far.<br /> (7) As pointed out before, it is hard to rationalize how a 4th instar exposure to EB can result in upregulation of key genes involved in JH synthesis at the adult stage. The authors must consider providing a plausible explanation and discussion in this regard.<br /> (8) I have strong reservations against such an irrational hypothesis that Met (the receptor for JH) and JH-Met target gene Kr-h1 regulates JH titer (Line 311, Fig 3 supplemental 2D). This would be the first report of such an event on the JH field and therefore must be analysed to depth before it may go to publication. I strongly suggest the authors remove such claims from the manuscript without substantiating it.<br /> (9) Kr-h1 is JH/Met target gene. The authors demonstrate that silencing of Kr-h1 results in inhibition of FAMeT, which is a gene involved in JH synthesis. The feedback loop in JH synthesis is unreported. Authors must go ahead with a mechanistic detail of Kr-h1 mediated JH upregulation before this can be concluded. Mere qPCR experiments are not sufficient to substantiate a claim that is completely contrary to the current understanding of JH signalling pathway.<br /> (10) Authors have performed knockdowns of JHAMT, Met and Kr-h1 to demonstrate the effect of these factors on fecundity n BPH. Additionally, they have performed rescue experiments with EB application on these knockdown insects (Figure 3K-M). This I believe is a very flawed experiment. The authors demonstrate EB works through JHAMT in upregulating JH titer. In the absence of JHAMT, EB application is not expected to rescue the phenotype. But authors have reported a complete rescue here. In the absence of Met, the receptor of JH, either EB or JH is not expected to rescue the phenotype. But a complete rescue has been reported. These two experimental results contradict their own hypothesis.<br /> (11) A significant section of the paper deals with how EB upregulates JH titer. JH is a hormone synthesized in the Corpora Allata. Yet the authors have chosen to use the whole body for all of their experiments. Changes in the whole body for mRNA of those enzymes involved in JH synthesis does may not reflect on the situation in Corpora Allata. Although working with corpora Allata is challenging, discarding the abdomen and thorax region and working with the head and neck region of the insect is easily doable. Results from such sampling is always more convincing when it comes to JH synthesis studies.<br /> (12) The phenomenon reported was specific for BPH and not found in other insects. This limits the implications of the study.<br /> (13) Overall, the molecular experiments are very poorly designed and can at best be termed superficial. There are several contradictions within the paper and no discussion or explanation has been provided for that.

Comments on revisions:

(1) The onus of making the revisions understandable to the reviewers lies with the authors. In its current form, how the authors have approached the review is hard to follow, in my opinion. Although the authors have taken a lot of effort in answering the questions posed by reviewers, parallel changes in the manuscript are not clearly mentioned. In many cases, the authors have acknowledged the criticism in response to the reviewer, but have not changed their narrative, particularly in the results section.<br /> (2) In the response to reviewers, the authors have mentioned line numbers in the main text where changes were made. But very frequently, those lines do not refer to the changes or mention just a subsection of changes done. The problem is throughout the document making it very difficult to follow the revision and contributing to the point mentioned above.<br /> (3) The authors need to infer the performed experiments rationally without over interpretation. Currently, many of the claims that the authors are making are unsubstantiated. As a result of the first review process, the authors have acknowledged the discrepancies, but they have failed to alter their interpretations accordingly.<br /> (4) I would like to point to the fact that there are significant experimental modifications added to the manuscript. The decision from the first cycle of review was given on 8th Nov 2024. The authors re-submitted the manuscript on 20th Nov 2024. It just beats my understanding, how so many experiments can be done in such a short time. The rush in resubmission is evident in the writing quality as well. Which I think is now poorer than the original version.<br /> (5) The writing quality is still extremely poor.

Reviewer #1 (Public review):

Summary:

The authors aimed to confirm the association between the human leukocyte antigen (HLA)-II region and tuberculosis (TB) susceptibility within admixed African populations. Building upon previous findings from the International Tuberculosis Host Genetics Consortium (ITHGC), this study sought to address the limitations of small sample size and the inclusion of admixed samples by employing the Local Ancestry Allelic Adjusted (LAAA) model, as well as identify TB susceptibility loci in an admixed South African cohort.

Strengths:

The major strengths of this study include the use of multiple TB case-control datasets from diverse South African populations and ADMIXTURE for global ancestry inference.

Weaknesses:

The major weakness of this study include insufficient significant novel discoveries and reliance on cross-validation. The use of existing models did not add value to this study.

Appraisal:<br /> The authors achieved their aims. However, the results still needed to be further validated in the future.

Impact:<br /> The innovative use of the LAAA model and the comprehensive dataset in this study may make contributions to the field of genetic epidemiology.

Reviewer #2 (Public review):

Summary:

This manuscript is about using different analytical approaches to allow ancestry adjustments to GWAS analyses amongst admixed populations. This work is a follow-on from the recently published ITHGC multi-population GWAS (https://doi.org/10.7554/eLife.84394), with the focus on the admixed South African populations. Ancestry adjustment models detected a peak of SNPs in the class II HLA DPB1, distinct from the class II HLA DQA1 loci signficant in the ITHGC analysis.

Strengths:

Excellent demonstration of GWAS analytical pipelines in highly admixed populations. Particularly the utility of ancestry adjustment to improve study power to detect novel associations. Further confirmation of the importance of the HLA class II locus in genetic susceptibility to TB.

Weaknesses:

Limited novelty compared to the group's previous existing publications and the body of work linking HLA class II alleles with TB susceptibility in South Africa or other African populations. This work includes only ~100 new cases and controls from what has already been published. High resolution HLA typing has detected significant signals in both the DQA1 and DPB1 regions identified by the larger ITHGC and in this GWAS analysis respectively (Chihab L et al. HLA. 2023 Feb; 101(2): 124-137).<br /> Despite the availability of strong methods for imputing HLA from GWAS data (Karnes J et Plos One 2017), the authors did not confirm with HLA typing the importance of their SNP peak in the class II region. This would have supported the importance of this ancestry adjustment versus prior ITHGC analysis.<br /> The populations consider active TB and healthy controls (from high-burden presumed exposed communities) and do not provide QFT or other data to identify latent TB infection.

Important methodological points for clarification and for readers to be aware of when reading this paper:

(1) One of the reasons cited for the lack of African ancestry-specific associations or suggestive peaks in the ITHGC study was the small African sample size. The current association test includes a larger African cohort and yields a near-genome-wide significant threshold in the HLA-DPB1 gene originating from the KhoeSan ancestry. Investigation is needed as to whether the increase in power is due to increased African samples and not necessarily the use of the LAAA model as stated on lines 295 and 296?

Authors response - The Manhattan plot in Figure 3 includes the results for all four models: the traditional GWAS model (GAO), the admixture mapping model (LAO), the ancestry plus allelic (APA) model and the LAAA model. In this figure, it is evident that only the LAAA model identified the association peak on chromosome 6, which lends support the argument that the increase in power is due to the use of the LAAA model and not solely due to the increase in sample size.<br /> Reviewer comment - This data supports the authors conclusions that increase power is related to the LAAA model application rather than simply increase sample size.

(2) In line 256, the number of SNPs included in the LAAA analysis was 784,557 autosomal markers; the number of SNPs after quality control of the imputed dataset was 7,510,051 SNPs (line 142). It is not clear how or why ~90% of the SNPs were removed. This needs clarification.

Authors response:<br /> In our manuscript (line 194), we mention that "...variants with minor allele frequency (MAF) < 1% were removed to improve the stability of the association tests." A large proportion of imputed variants fell below this MAF threshold and were subsequently excluded from this analysis.

Reviewers additional comment: The authors should specify the number of SNPs in the dataset before imputation and indicate what proportion of the 784,557 remaining SNPs were imputed. Providing this information might help the reader better understand the rationale behind the imputation process.

(3) The authors have used the significance threshold estimated by the STEAM p-value < 2.5x10-6 in the LAAA analysis. Grinde et al. (2019 implemented their significance threshold estimation approach tailored to admixture mapping (local ancestry (LA) model), where there is a reduction in testing burden. The authors should justify why this threshold would apply to the LAAA model (a joint genotype and ancestry approach).

Authors response: We describe in the methods (line 189 onwards) that the LAAA model is an extension of the APA model. Since the APA model itself simultaneously performs the null global ancestry only model and the local ancestry model (utilised in admixture mapping), we thus considered the use of a threshold tailored to admixture mapping appropriate for the LAAA model.

Reviewers additional comment: While the LAAA model is an extension of the APA model, the authors describe the LAAA test as 'models the combination of the minor allele and the ancestry of the minor allele at a specific locus, along with the effect of this interaction,' thus a joint allele and ancestry effects model. Grinde et al. (2019) proposed the significance threshold estimation approach, STEAM, specifically for the LA approach, which tests for ancestry effects alone and benefits from the reduced testing burden. However, it remains unclear why the authors found it appropriate to apply STEAM to the LAAA model, a joint test for both allele and ancestry effects, which does not benefit from the same reduction in testing burden.

(4) Batch effect screening and correction (line 174) is a quality control check. This section is discussed after global and local ancestry inferences in the methods. Was this QC step conducted after the inferencing? If so, the authors should justify how the removed SNPs due to the batch effect did not affect the global and local ancestry inferences or should order the methods section correctly to avoid confusion.

Authors response: The batch effect correction method utilised a pseudo-case-control comparison which included global ancestry proportions. Thus, batch effect correction was conducted after ancestry inference. We excluded 36 627 SNPs that were believed to have been affected by the batch effect. We have amended line 186 to include the exact number of SNPs excluded due to batch effect.<br /> The ancestry inference by RFMix utilised the entire merged dataset of 7 510 051 SNPs. Thus, the SNPs removed due to the batch effect make up a very small proportion of the SNPs used to conduct global and local ancestry inferences (less than 0.5%). As a result, we do not believe that the removed SNPs would have significantly affected the global and local ancestry inferences. However, we did conduct global ancestry inference with RFMix on each separate dataset as a sanity check. In the Author response tables 1 and 2, we show the average global ancestry proportions inferred for each separate dataset, the average global ancestry proportions across all datasets and the average global ancestry proportions inferred using the merged dataset. The SAC and Xhosa cohorts are shown in two separate tables due to the different number of contributing ancestral populations to each cohort. The differences between the combined average global ancestry proportions across the separate cohorts does not differ significantly to the global ancestry proportions inferred using the merged dataset.

This is an excellent response and should remain accessible to readers to clarify this issue.

Reviewer #1 (Public review):

Summary:

The authors of this study sought to define a role for IgM in responses to house dust mites in the lung.

Strengths:

Unexpected observation about IgM biology.<br /> Combination of experiments to elucidate function.

Weaknesses:

Would love more connection to human disease

Reviewer #2 (Public review):

Summary:

The manuscript by Hadebe and colleagues describes a striking reduction in airway hyperresponsiveness in Igm-deficient mice in response to HDM, OVA and papain across the B6 and BALB-c backgrounds. The authors suggest that the deficit is not due to improper type 2 immune responses, nor an aberrant B cell response, despite a lack of class switching in these mice. Through RNA-Seq approaches, the authors identify few differences between the lungs of WT and Igm-deficient mice, but see that two genes involved in actin regulation are greatly reduced in IgM-deficient mice. The authors target these genes by CRISPR-Cas9 in in vitro assays of smooth muscle cells to show that these may regulate cell contraction. While the study is conceptually interesting, there are a number of limitations, which stop us from drawing meaningful conclusions.

Strengths:

Fig. 1. The authors clearly show that IgMKO mice have striking reduced AHR in the HDM model, despite the presence of a good cellular B cell response.

Weaknesses:

Due to several technical and experimental limitations, it is unclear what leads to the reduction in airway hyperresponsiveness in IGM-KO mice. The limitations as outlined previously remain.

Reviewer #1 (Public review):

Summary:

The manuscript performs a comprehensive biochemical, structural, and bioinformatic analysis of TseP, a type 6 secretion system effector from Aeromonas dhakensis that includes identification of a domain required for secretion and residues conferring target organism specificity. Through targeted mutations, they have expanded the target range of a T6SS effector to include a gram-positive species, which are not typically susceptible to T6SS attack. Although this is not the first dual domain effector to be described, this is the first time anyone has been able to modify a T6SS effector to have an expanded target species range.

Strengths:

The thorough dissection of TseP activity and modulation of target specificity represent a novel contribution to the field of antibacterial research.

Weaknesses:

Although the mechanistic activity of TseP is fully dissected here, there are some unaddressed questions regarding the importance/evolution of the dual activity domain organization. For example, does the modified Gram-positive targeting TseP effector still kill Gram-negative bacteria in bacterial mixtures? And if so, what is the evolutionary benefit of having a TseP that cannot target Gram-positives? And can something be inferred about the biology of Aeromonas from this?

Comments on revisions:

The comments and critiques from the initial submission have been addressed. However, some of them have only been addressed in the author's rebuttal. Some of the discussion particularly regarding the validity of using E. coli PG, the ability for TseP_C4+ to still kill E. coli, and the advantages of having dual domain function effectors probably should be present in the actual manuscript.

Reviewer #2 (Public review):

Summary:

Wang et al. investigate the role of TseP, a Type VI secretion system (T6SS) effector molecule, revealing its dual enzymatic activities as both an amidase and a lysozyme. This discovery significantly enhances the understanding of T6SS effectors, which are known for their roles in interbacterial competition and survival in polymicrobial environments. TseP's dual function is proposed to play a crucial role in bacterial survival strategies, particularly in hostile environments where competition between bacterial species is prevalent.

Strengths:

(1) The dual enzymatic function of TseP is a significant contribution, expanding the understanding of T6SS effectors.<br /> (2) The study provides important insights into bacterial survival strategies, particularly in interbacterial competition.<br /> (3) The findings have implications for antimicrobial research and understanding bacterial interactions in complex environments.

Weaknesses:

(1) The manuscript assumes familiarity with previous work, making it difficult to follow. Mutants and strains need clearer definition and references.<br /> (2) Figures lack proper controls, quantification, and clarity in some areas, notably in Figures 1A and 1C.<br /> (3) The Materials and Methods section is poorly organized, hindering reproducibility. Biophysical validation of Zn²⁺ interaction and structural integrity of proteins need to be addressed.<br /> (4) Discrepancies in protein degradation patterns and activities across different figures raise concerns about data reliability.

Comments on revisions:

The authors have addressed most of the comments, significantly improving the manuscript. They provided clear details of mutant constructs and strains, including additional references and a revised strain. Individual data points and statistical analyses were added to key figures, ensuring transparency and reproducibility. Supplemental data, such as protein purification details and loading controls, were included to address concerns about experimental reliability. However, the authors did not perform new experiments, such as isothermal titration calorimetry (ITC) to demonstrate the interaction between Zn²⁺ and TsePN or stop-flow spectroscopy to examine enzymatic kinetics, which could have further strengthened the manuscript. I trust these aspects will be addressed in future studies.

The revised Materials and Methods section was significantly improved, providing detailed protocols for bioinformatics analyses, microscopic imaging, and enzymatic assays.

These revisions provide a clearer and more robust presentation of TseP's dual enzymatic functions and their implications in bacterial competition. The manuscript now represents a significant contribution to understanding T6SS effectors, and I recommend it for publication in its current form.

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 #2 (Public review):

Summary

In this extensive comparative study, Moreno-Borrallo and colleagues examine the relationships between plasma glucose levels, albumin glycation levels, diet and life-history traits across birds. Their results confirmed the expected positive relationship between plasma blood glucose level and albumin glycation rate but also provided findings that are somewhat surprising or contrast with findings of some previous studies (positive relationships between blood glucose and lifespan, or absent relationships between blood glucose and clutch mass or diet). This is the first extensive comparative analysis of glycation rates and their relationships to plasma glucose levels and life history traits in birds that is based on data collected in a single study, with blood glucose and glycation measured using unified analytical methods (except for blood glucose data for 13 species collected from a database).

Strengths

This is an emerging topic gaining momentum in evolutionary physiology, which makes this study a timely, novel and important contribution. The study is based on a novel data set collected by the authors from 88 bird species (67 in captivity, 21 in the wild) of 22 orders, except for 13 species, for which data were collected from a database of veterinary and animal care records of zoo animals (ZIMS). This novel data set itself greatly contributes to the pool of available data on avian glycemia, as previous comparative studies either extracted data from various studies or a ZIMS database (therefore potentially containing much more noise due to different methodologies or other unstandardised factors), or only collected data from a single order, namely Passeriformes. The data further represents the first comparative avian data set on albumin glycation obtained using a unified methodology. The authors used LC-MS to determine glycation levels, which does not have problems with specificity and sensitivity that may occur with assays used in previous studies. The data analysis is thorough, and the conclusions are substantiated. Overall, this is an important study representing a substantial contribution to the emerging field evolutionary physiology focused on ecology and evolution of blood/plasma glucose levels and resistance to glycation.

Weaknesses

Unfortunately, the authors did not record handling time (i.e., time elapsed between capture and blood sampling), which may be an important source of noise because handling-stress-induced increase in blood glucose has previously been reported. Moreover, the authors themselves demonstrate that handling stress increases variance in blood glucose levels. Both effects (elevated mean and variance) are evident in Figure ESM1.2. However, this likely makes their significant findings regarding glucose levels and their associations with lifespan or glycation rate more conservative, as highlighted by the authors.

Reviewer #1 (Public review):

Summary:

This work introduces the differentiable Gillespie algorithm, DGA, which is a differentiable variant of the celebrated (and exact) Gillespie algorithm commonly used to perform stochastic simulations across numerous fields, notably in the life sciences. The proposed DGA approximates the exact Gillespie algorithm using smooth functions yielding a suitable approximate differentiable stochastic system as a proxy for the underlying discrete stochastic system, where DGA stochastic reactions have continuous reaction index and the species abundances. To illustrate their methodology, the authors specifically consider in detail the case of a well-studied two-state promoter gene regulation system that they analyze using a machine learning approach, and by combining simulation data with analytical results. For the two-state promoter gene system, the DGA is benchmarked by accurately reproducing the results of the exact Gillespie algorithm. For this same simple system, the authors also show how the DGA can be used for estimating kinetic parameters of both simulated and real noisy experimental data. This lets them argue convincingly that the DGA can become a powerful computation tool for applications in quantitative and synthetic biology. In order to argue that the DGA can be employed to design circuits with ad-hoc input-output relations, these considerations are then extended to a more complex four-state promoter model of gene regulation. The main strength of the paper is its clarity and its pedagogical presentation of the simulation methods.

Strengths:

The main strength of the paper is its clarity and its pedagogical presentation of the simulation methods.

Weaknesses:

It would have been useful to have a brief discussion, based on a concrete example, of what can be achieved with the DGA and is totally beyond the reach of the Gillespie algorithm and the numerous existing stochastic simulation methods. A more comprehensive and quantitative analysis of the limitations of the DGA, e.g. for rare events, and how it might be used for stochastic spatial systems would have also been helpful. However, this is arguably beyond the scope of this study whose primary goal is to introduce the DGA and demonstrate that it can achieve tasks like parameter estimation and network design.

Comments on revisions:

The authors have made a sound effort to address many of the comments raised in the previous reports. This has helped improve the clarity of the discussion.

Reviewer #2 (Public review):

Summary:

In this work, the authors present a differentiable version of the widely-used Gillespie Algorithm. The Gillespie Algorithm has been used for decades to simulate the behavior of stochastic biochemical reaction networks. But while the Gillespie Algorithm is a powerful tool for the forward simulation of biochemical systems given some set of known reaction parameters, it cannot be used for reverse process, i.e. inferring reaction parameters given a set of measured system characteristics. The Differentiable Gillespie Algorithm ("DGA") overcomes this limitation by approximating two discontinuous steps in the Gillespie Algorithm with continuous functions. This makes it possible to calculate of gradients for each step in the simulation process which, in turn, allows the reaction parameters to be optimized via powerful backpropagation techniques. In addition to describing the theoretical underpinnings of DGA, the authors demonstrate different potential use-cases for the algorithm in the context of simple models of stochastic gene expression.

Overall, the DGA represents an important conceptual step forward for the field and should lay the groundwork for exciting innovations in the analysis and design of stochastic reaction networks. At the same time, significantly more work is needed to establish when the approximations made by DGA are valid and to demonstrate the viability of the algorithm in the context of complicated reaction networks.

Strengths:

This work makes an important conceptual leap by introducing a version of the Gillespie Algorithm that is end-to-end differentiable. This idea alone has the potential to drive a number of exciting innovations in the analysis, inference, and design of biochemical reaction networks. Beyond the theoretical adjustments, the authors also implement their algorithm in a Python-based codebase that combines DGA powerful optimization libraries like PyTorch. This codebase has the potential to be of interest to a wide range of researchers, even if the true scope of the method's applicability remains to be fully determined.

The authors also demonstrate how DGA can be used in practice both to infer reaction parameters from real experimental data (Figure 7) and to design networks with user-specified input-output characteristics (Figure 8). These illustrations should provide a nice roadmap for researchers interested in applying DGA to their own projects/systems.

Finally, although it does not stem directly from DGA, the exploration of pairwise parameter dependencies in different network architectures provides an interesting window into the design constraints (or lack thereof) that shape the architecture of biochemical reaction networks.

Weaknesses:

While it is clear that the DGA represents an important conceptual advancement, the authors do not do enough in the present manuscript to (i) validate the robustness of DGA inference and (ii) demonstrate that DGA inference works in the kinds of complex biochemical networks where it would actually be of legitimate use.

It is to the authors' credit that they are open and explicit about the potential limitations of DGA due to breakdowns in its continuous approximations. However they do not provide the reader with nearly enough empirical (i.e. simulation-based) or theoretical context to assess when, why, and to what extent DGA will fail in different situations. In Figure 2, they compare DGA to GA (i.e. ground-truth) in the context of a simple two state model of a stochastic transcription. Even in this minimal system, we see that DGA deviates notably from ground-truth both in the simulated mRNA distributions (Figure 2A) and in the ON/OFF state occupancy (Figure 2C). This begs the question of how DGA will scale to more complicated systems, or systems with non-steady state dynamics. Will the deviations become more severe? This is important because, in practice, there is really not much need for using DGA with a simple 2 state system-we have analytic solutions for this case. It is the more complex systems where DGA has the potential to move the needle.

A second concern is that the authors' present approach for parameter inference and error calculation does not seem to be reliable. For example, in Figure 5A, they show DGA inference results for the ON rate of a two-state system. We see substantial inference errors in this case, even though the inference problem should be non-degenerate in this case. One reason for this seems to be that the inference algorithm does not reliably find the global minimum of the loss function (Figure 2B). To turn DGA into a viable approach, it is paramount that the authors find some way to improve this behavior, perhaps by using multiple random initializations to better search the loss space.

Finally, the authors do a good job of illustrating how DGA might be used to infer biological parameters (Figure 7) and design reaction networks with desired input-output characteristics (Figure 8). However, analytic solutions exist for both of the systems they select for examples. This means that, in practice, there would be no need for DGA in these contexts, since one could directly optimize, e.g., the expressions for the mean and Fano Factor of the system in Figure 7A. I still believe that it is useful to have these examples, but it seems critical to add a use-case where DGA is the only option.

Comments on revisions:

I am concerned that the results in Figure 8D may not be correct, or that the authors may be mis-interpreting them. From my reading of the paper they cite (Lammers & Flamholz 2023), the equilibrium sharpness limit for the network they consider in Figure 8 should be 0.25. But both solutions shown in Figure 8D fall below this limit, which means that they have sharpness levels that could have been achieved with no energy expenditure. If this is the case, then it would imply that while both systems do dissipate energy, they are not doing so productively; meaning that the same results could be achieved while holding Phi=0.

I acknowledge that this could be due to a difference in how they measure sharpness, but wanted to raise it here in case it is, in fact, a genuine issue with the analysis.

There should be an easy fix for this: just set the sharper "desired response" curve in 8b to be such that it demands non-equilibrium sharpness levels (0.25)

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 #1 (Public review):

Summary:

The manuscript puts forward a statistical method to more accurately report the significance of correlations within data. The motivation for this study is two-fold. First, the publication of biological studies demands the report of p-values, and it is widely accepted that p-values below the arbitrary threshold of 0.05 give the authors of such studies justification to draw conclusions about their data. Second, many biological studies are limited by the number of replicate samples that are feasible, with replicates of less than 5 typical. The authors report a statistical tool that uses a permute-match approach to calculate p-values. Notably, the proposed method reduces p-values from around 0.2 to 0.04 as compared to a standard permutation test with a small sample size. The approach is clearly explained, including detailed mathematical explanations and derivations. The advantage of the approach is also demonstrated through analysis of computer-generated synthetic data with specified correlation and analysis of previously published data related to fish schooling. The authors make a clear case that this method is an improvement over the more standard approach currently used, and also demonstrate the impact of this methodology on the ability to obtain p-values that are the standard for biological research. Overall, this paper is very strong. While the subject matter seems somewhat specialized, I would make the case that this will be an important study that has broad general interest to readers. The findings are very general and applicable to many research contexts. Experimentalists also want to report accurate p-values in their work and better understand how these values are calculated. Although I believe the previous statement is true, I am not sure that many research groups doing biological work are reading specialized statistics journals regularly. Therefore a useful and broadly applicable statistical tool is well placed in this journal.<br /> Strengths:

The proposed method is broadly applicable to many realistic datasets in many experimental contexts.

The power of this method was demonstrated with both real experimental data and "synthetic" data. The advantages of the tool are clearly reported. The zebrafish data is a great example dataset.

The method solves a real-life problem that is frequently encountered by many experimental groups in the biological sciences.

The writing of the paper is surprisingly clear, given the technical nature of the subject matter. I would not at all consider myself a statistician or mathematician, but I found the text easy to follow. The authors did an impressive job guiding the reader through material that would often be difficult to grasp. The introduction was also well-written and clearly motivated the goals of the study.

Weaknesses:

A few changes could be made if the manuscript is revised. I would consider all of these points minor, but the paper could be improved if these points were addressed.

(1) The caption of Figure 2 doesn't seem to mention panel D. Figure A-2 also does not mention C in the caption.

(2) Figure 2D is a little hard to follow. First, the definition of "Power" is not clear, and I couldn't find the precise definition in the text. Second, the legend for the different lines in 2D is only given in Figure A-2. Perhaps a portion of the caption for Figure 2 is missing?

(3) The concept of circular variance for the fish data was heard to understand/visualize. The equation on line 326 did not help much. If there is a very simple picture that could be added near line 326 that helps to explain Ct and theta, that could be a big help for some readers who do not work on related systems. The analysis performed is understandable, the reader just has to accept that circular variance captions the degree of alignment of the fish.

(4) For the data discussed in Figure 3, I wasn't 100% sure how the time windows were selected. In the caption, it says "time series to different lengths starting from the first frame". So the 20 s time window was from t=0 to t= 20 s. Would a different result be obtained if a different 20 s window was chosen (from t = 4 min to t = 4 min 20 s just to give a specific example). I suppose by chance one of the time windows would give a p-value less than the target 0.05, that wouldn't be surprising. Maybe a random time window should be selected (although I am not indicating what was reported was incorrect)? A little more discussion on this aspect of the study may be helpful.

Reviewer #2 (Public review):

Summary:

This paper presented a hypothesis testing procedure for the independence of two time-series that was potentially suitable for nonlinear dependence and for small-sample cases. This should bring potential benefits for biology data.

Strengths:

The test offers good flexibility for different kinds of dependence (through adjusting \rho), and seems to have good finite sample performance compared to the literature. The justification regarding the validity of the test procedure is clear.

Weaknesses:

(1) The size of the test is not guaranteed to (asymptotically) equal \alpha, which may damage the power.

(2) The computational time can be an issue for a moderately large sample size when calculating the X / Y-perfect match. It will be beneficial to include discussions on the implementations of the test.

Reviewer #1 (Public review):

Summary:

Meteorin proteins were initially described as secreted neurotrophic factors. In this manuscript, Eggeler et al. demonstrate a novel role for Meteorins in establish left-right axis formation in the zebrafish embryo. The authors generated null mutations in each of the three zebrafish meteorin genes - metrn, metrnla, and metrnlab. Triple mutant embryos displayed phenotypes strongly associated with left-right defects such as heart looping and visceral organ placement, and disrupted expression of Nodal-responsive genes, as did single mutants for metrn and metrnla. The authors then go on to demonstrate that these defects in left-right asymmetry are likely to due to defects in Kupffer's Vesicle and the progenitor dorseal forerunner cells including impaired lumen formation and reduced fluid flow, reduced clustering among DFCs, impaired DFC migration, mislocalization of apical proteins ZO-1 and aPKC, and detachment of DFCs from the EVL. Notably, the authors found that expression of marker genes sox32 and sox17 were not affected, suggesting Meteorins are required for DFC/KV morphogenesis but not necessarily fate specification. Finally, the authors show genetic interaction between Meteorins and integrin receptors, which were previously implicated in left-right patterning. In a supplemental figure, the manuscript also presents data showing expression of meteorin genes around the chick Hensen's node, suggesting that the left-right patterning functions may be conserved among vertebrates.

Strengths:

Strengths of this study include the generation of a triple mutant line that targets all known zebrafish meteorin family members. The experiments presented in this study were rigorous, especially with respect to quantification and statistical analysis.

Weaknesses:

Although the authors convincingly demonstrate a role for Meteorins in zebrafish left-right patterning, data supporting a conserved role in other vertebrates is compelling but limited to one supplemental figure.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors describe their study on the role of meteorins in establishing the left-right organizer. The left-right organizer is a transient organ in vertebrate embryos in which rotating cilia cause a fluid flow that breaks the left-right symmetry and coordinates lateralization of internal organs such as gut and heart. In zebrafish, the left-right organizer (also named Kupffer's vesicle) is formed by dorsal forerunner cells, but very little is known about how dorsal forerunner cells coalles and form this ciliated vesicle in the embryo. The authors mutated the three meteorin-coding genes in zebrafish and observed that mutations in each one of these causes laterality defects with the strongest defects observed in the triple mutant. Loss of meteorins affects nodal gene expression, which play essential roles in establishing organ laterality. Meteorins are widely expressed in developing embryos and expression in lateral plate mesoderm and dorsal forerunner cells was observed. The meteorin triple mutant embryos display defects in the migration and clustering of the dorsal forerunner cells impairing kupffer's vesicle formation and cilia rotation. Finally, the authors show that meteorins genetically interact with integrins.

Strengths:

- These authors went through the lengthy process of generating triple mutants affecting all three meteorin genes. This provides robust genetic evidence on the role of meteorins in establishing organ laterality and circumvented that interpretation of the results would be hard due to redundant functions of meteorins.<br /> - The use of life imaging on triple mutants is appreciated<br /> - High-quality imaging of dorsal forerunner to quantify cell migrations and its relation to Kupffer's vesicle formation.

Weaknesses:

- Lack of a model how meteorins regulate dorsal forerunner cell migration.<br /> - Only genetic data to suggest a link between meteorins and integrins<br /> - Besides its role in DFC migration, meteorins may also play a more direct role in regulating Nodal signaling, which is not addressed here.

Reviewer #1 (Public review):

Summary:

For each of the three key transcription factor (TF) proteins in E. coli, the authors generate a large library of TF binding site (TFBS) sequences on plasmids, such that each TFBS is coupled to the expression of a fluorescence reporter. By sorting the fluorescence of individual cells and sequencing their plasmids to identify each cell's TFBS sequence (sort-seq), they are able to map the landscape of these TFBSs to the gene expression level they regulate. The authors then study the topographical features of these landscapes, especially the number and distribution of local maxima, as well as the statistical properties of evolutionary paths on these landscapes. They find the landscapes to be highly rugged, with about as many local peaks as a random landscape would have, and with those peaks distributed approximately randomly in sequence space. The authors find that there are a number of peaks that produce regulation stronger than that of the wild-type sequence for each TF and that it is not too unlikely to reach one of those "high peaks" from a random starting sequence. Nevertheless, the basins of attractions for different peaks have significant overlap, which means that chance plays a major role in determining which peak a population will evolve to.

Strengths:

(1) The experiments and analysis of this paper are very well-executed and, by and large, very thorough (with an important exception identified below). I appreciated the systematic nature of the project, both the large-scale experiments done on three TFs with replicates and the systematic analysis of the resulting landscapes. This not only makes the paper easy to follow but also inspires confidence in their results since there is so much data and so many different ways of analyzing it. It's a great recipe for other studies of genotype-phenotype landscapes to follow.

(2) Considering how technical the project was, I am really impressed at how easy to read I found the paper, and the authors deserve a lot of credit for making it so. They do a great job of building up the experiments and analyses step-by-step and explaining enough of the basics of the experimental design and the essence of each analysis in the main text without getting too complicated with details that can be left to the Methods or SI. Compared to other big data papers, this one was refreshingly not overwhelming.

Weaknesses:

(1) The main weakness of this paper, in my view, is that it felt disconnected from the larger body of work on fitness and genotype-phenotype landscapes, including previous data on TFBSs in E. coli, genotype-phenotype maps of TFBSs in other systems, protein sequence landscapes (e.g., from mutational scans or combinatorially-complete libraries), and fitness landscapes of genomic mutations (e.g., combinatorially-complete landscapes of antibiotic resistance alleles). I have no doubt the authors are experts in this literature, and they probably cite most of it already given the enormous number of references. But they don't systematically introduce and summarize what was already known from all that work, and how their present study builds on it, in the Abstract and Introduction, which left me wondering for most of the paper why this project was necessary. Eventually, the authors do address most of these points, but not until the end, in the Discussion. Readers who have no familiarity with this literature might read this paper thinking that it's the first paper ever to study topography and evolutionary paths on genotype-phenotype landscapes, which is not true.

There were two points that made this especially confusing for me. First, in order to choose which nucleotides in the binding sites to vary, the authors invoke existing data on the diversity of these sequences (position-weight matrices from RegulonDB). But since those PWMs can imply a genotype-phenotype map themselves, an obvious question I think the authors needed to have answered right away in the Introduction is why it is insufficient for their question. They only make a brief remark much later in the Results that the PWM data is just observed sequence diversity and doesn't directly reflect the regulation strength of every possible TFBS sequence. But that is too subtle in my opinion, and such a critical motivation for their study that it should be a major point in the Introduction.

The second point where the lack of motivation in the Introduction created confusion for me was that they report enormous levels of sign epistasis in their data, to the point where these landscapes look like random uncorrelated landscapes. That was really surprising to me since it contrasts with other empirical landscape data I'm familiar with. It was only in the Discussion that I found some significant explanation of this - namely that this could be a difference between prokaryotic TFBSs, as this paper studies, and the eukaryotic TFBSs that have been the focus of many (almost all?) previous work. If that is in fact the case - that almost all previous studies have focused on eukaryotic TFBSs or other kinds of landscapes, and this is the first to do a systematic test of prokaryotic TFBS, then that should be a clear point made in the Abstract and Introduction. (I find a comparable statement only in the very last paragraph of the Discussion.) If that's the case, then I would also find that point to be a much stronger, more specific conclusion of this paper to emphasize than the more general result of observing epistasis and contingency (as is currently emphasized in the Abstract), which has been discussed in tons of other papers. This raises all sorts of exciting questions for future studies - why do the landscapes of prokaryotic TFBSs differ so dramatically from almost all the other landscapes we've observed in biology? What does that mean for the evolutionary dynamics of these different systems?

(2) I am a bit concerned about the lack of uncertainties incorporated into the results. The authors acknowledge several key limitations of their approach, including the discreteness of the sort-seq bins in determining possible values of regulation strength, the existence of a large number of unsampled sequences in their genotype space, as well as measurement noise in the fluorescence readouts and sequencing. While the authors acknowledge the existence of these factors, I do not see much attempt to actually incorporate the effect of these uncertainties into their conclusions, which I suspect may be important. For example, given the bin size for the fluorescence in sort-seq, how confident are they that every sequence that appears to be a peak is actually a peak? Is it possible that many of the peak sequences have regulation strengths above all their neighbors but within the uncertainty of the fluorescence, making it possible that it's not really a peak? Perhaps such issues would average out and not change the statistical nature of their results, which are not about claiming that specific sequences are peaks, just how many peaks there are. Nevertheless, I think the lack of this robustness analysis makes the results less convincing than they otherwise would be.

Reviewer #2 (Public review):

The authors aim to investigate the ability of evolution to create strong transcription factor binding sites (TFBSs) de novo in E. coli. They focus on three global transcriptional regulators: CRP, Fis, and IHF, using a massively parallel reporter assay to evaluate the regulatory effects of over 30,000 TFBS variants. By analyzing the resulting genotype-phenotype landscapes, they explore the ruggedness, accessibility, and evolutionary dynamics of regulatory landscapes, providing insights into the evolutionary feasibility of strong gene regulation. Their experiments show that de novo adaptive evolution of new gene regulation is feasible. It is also subject to a blend of chance, historical contingency, and evolutionary biases that favor some peaks and evolutionary paths.

(1) Strengths of the methods and results:

The authors successfully employed a well-designed sort-seq assay combined with high-throughput sequencing to map regulatory landscapes. The experimental design ensures reliable measurement of regulation strengths. Their system accounts for gene expression noise and normalizes measurements using appropriate controls.

Comprehensive Landscape Mapping:<br /> The study examines ~30,000 TFBS variants per transcription factor, providing statistically robust and thorough maps of the regulatory landscapes for CRP, Fis, and IHF. The landscapes are rigorously analyzed for ruggedness (e.g., number of peaks) and epistasis, revealing parallels with theoretical uncorrelated random landscapes.

Evolutionary Dynamics Simulations:<br /> Through simulations of adaptive walks under varying population dynamics, the authors demonstrate that high peaks in regulatory landscapes are accessible despite ruggedness. They identify key evolutionary phenomena, such as contingency (multiple paths to peaks) and biases toward specific evolutionary outcomes.

Biological Relevance and Novelty:<br /> The author's work is novel in focusing on global regulators, which differ from previously studied local regulators (e.g., TetR). They provide compelling evidence that rugged landscapes are navigable, facilitating de novo evolution of regulatory interactions. The comparison of landscapes for CRP, Fis, and IHF underscores shared topographical features, suggesting general principles of global transcriptional regulation in bacteria.

(2) Weaknesses of the methods and results:

Undersampling of Genotype Space:<br /> While the quality filtering of the data ensures robustness, ~40% of the TFBS space remains uncharacterized. The authors acknowledge this limitation but could improve the analysis by employing subsampling or predictive modeling.

Simplified Regulatory Architecture:<br /> The study considers a minimal system of a single TFBS upstream of a reporter gene. While this may have been necessary for clarity, this simplification may not reflect the combinatorial complexity of transcriptional regulation in vivo.

Lack of Experimental Validation of Simulations:<br /> The adaptive walks are based on simulated dynamics rather than experimental evolution. Incorporating in vivo experimental evolution studies would strengthen the conclusions. Although this is a large request for the paper, that would not prevent publication.

Impact on the Field:<br /> This study advances our understanding of adaptive landscapes in gene regulation and offers a critical step toward deciphering how global regulators evolve de novo binding sites. The findings provide foundational insights for synthetic biology, evolutionary genetics, and systems biology by highlighting the evolutionary accessibility of strong regulation in bacteria.

Utility of Methods and Data:<br /> The sort-seq approach, combined with landscape analysis, provides a robust framework that can be extended to other transcription factors and systems. If made publicly available, the study's data and code would be valuable for researchers modeling transcriptional regulation or studying evolutionary dynamics.

Additional Context:<br /> The study builds on a growing body of work exploring regulatory evolution. For instance, recent studies on local regulators like TetR and AraC have revealed high ruggedness and epistasis in TFBS landscapes. This study distinguishes itself by focusing on global regulators, which are more biologically complex and influential in bacterial gene networks. The observed evolutionary contingency aligns with findings in other biological systems, such as protein evolution and RNA folding landscapes, underscoring the generality of these evolutionary principles.

Conclusion:<br /> The authors successfully mapped the genotype-phenotype landscapes for three global regulators and simulated evolutionary dynamics to assess the feasibility of strong TFBS evolution. They convincingly demonstrate that ruggedness and epistasis, while prominent, do not preclude the evolution of strong regulation. Their results support the notion that gene regulation evolves through a blend of chance, contingency, and evolutionary biases.

This paper makes a significant contribution to the understanding of regulatory evolution in bacteria. While minor limitations exist, the authors' methods are robust, and their findings are well-supported. The work will likely be of broad interest to researchers in molecular evolution, synthetic biology, and gene regulation.

Reviewer #1 (Public review):

Summary:

The manuscript by Cupollilo et al describes the development, characterization, and application of a novel activity labeling system; fast labelling of engram neurons (FLEN). Several such systems already exist but this study adds additional capability by leveraging an activity marker that is destabilized (and thus temporally active) as well as being driven by the full-length promoter of cFos. The authors demonstrate the activity-dependent induction and time course of expression, first in cultured neurons and then in vivo in hippocampal CA3 neurons after one trial of contextual fear conditioning. In a series of ex vivo experiments, the authors perform patch clamp analysis of labeled neurons to determine if these putative engram neurons differ from non-labelled neurons using both the FLEN system as well as the previously characterized RAM system. Interestingly the early labelled neurons at 3 h post CFC (FLEN+) demonstrated no differences in excitability whereas the RAM-labelled neurons at 24h after CFC had increased excitability. Examination of synaptic properties demonstrated an increase in sEPCS and mEPSC frequencies as well as those for sIPSCs and mIPSCs which was not due to a change in the mossy fiber input to these neurons.

Strengths:

Overall the data is of high quality and the study introduces a new tool while also reassessing some principles of circuit plasticity in the CA3 that have been the focus of prior studies.

Weaknesses:

No major weaknesses were noted.

Reviewer #2 (Public review):

Summary:

Cupollilo et al. investigate the properties of hippocampal CA3 neurons that express the immediate early gene cFos in response to a single foot shock. They compare ex-vivo the electrophysiological properties of these "engram neurons" labeled with two different cFos promoter-driven green markers: Their new tool FLEN labels neurons 2-6 h after activity, while RAM contains additional enhancers and peaks considerably later (>24 h). Since the fraction of labeled CA3 cells is comparable with both constructs, it is assumed (but not tested) that they label the same population of activated neurons at different time points. Both FLEN+ and RAM+ neurons in CA3 receive more synaptic inputs compared to non-expressing control neurons, which could be a causal factor for cFos activation, or a very early consequence thereof. Frequency facilitation and E/I ratio of mossy fiber inputs were also tested, but are not different in both cFos+ groups of neurons. One day after foot shock, RAM+ neurons are more excitable than RAM- neurons, suggesting a slow increase in excitability as a major consequence of cFos activation.

Strengths:

The study is conducted to high standards and contributes significantly to our understanding of memory formation and consolidation in the hippocampus. Modifications of intrinsic neuronal properties seem to be more salient than overall changes in the total number of (excitatory and inhibitory) inputs, although a switch in the source of the synaptic inputs would not have been detected by the methods employed in this study

Weaknesses:

With regard to the new viral tool, a direct comparison between the new tool FLEN and existing cFos reporters is missing.

Reviewer #1 (Public review):

Summary:

After mating, male mice undergo a behavioral switch from infanticide to parental behavior (postmating switch). The neural mechanisms underlying this switch are still largely unknown. Studies performed in different mouse strains have also resulted in mixed evidence for whether mating (specifically: ejaculation) itself is sufficient for this switch, or whether subsequent cohabitation with the pregnant female, and parental experience with pups is required. Recent work found that while lesions to the central part of the medial preoptic area (cMPOA) promote infanticidal behavior, lesions to the rhomboid nucleus of the bed nucleus of the stria terminalis (BSTrh) inhibit infanticide. The current work convincingly adds to this evidence by showing that mating and cohabitation lead to reduced inhibition from Cart-positive medial amygdala neurons onto cMPOA neurons, and that this synaptic change is in fact critical for the postmating switch. Further, the authors demonstrate that parental experience increases inhibitory synaptic transmission onto BSTrh neurons. The male postmating switch thus appears to rely on two sequential stages of synaptic plasticity.

Strengths:

(1) The behavioral characterization is thorough and the authors nicely manage to disentangle the relative contributions of mating, cohabitation, and parental experience to the postmating switch. Their finding of dissociable plasticity mechanisms underlying mating/cohabitation vs pup experience is intriguing.

(2) Most conclusions are based on complementary evidence from different experimental approaches and are compelling.

Weaknesses:

(1) The authors do not provide an explicit synthesis/model of the circuit-level changes underlying this switch. For instance, how does cMPOA-to-BSTrh connectivity change in fathers, and how does the necessity of the cMPOA for the exposure/sensitisation effect square with the effect being postsynaptic in the BSTrh?

(2) The presentation of the manuscript (clarity of language, grammar, reporting of stats in figures etc.) needs to be improved.

Reviewer #2 (Public review):

Summary:

The present study identifies how mating and pup experience are correlated with differences in inhibitory neurotransmission underlying the promotion of paternal behavior toward pups. The study builds on existing knowledge about the circuit between the medial amygdala, medial preoptic area, and the bed nucleus of stria terminalis to uncover synaptic changes correlated with behavior. The authors find that inhibition from the medial amygdala is decreased in the medial preoptic area and increased in the bed nucleus of stria terminalis to promote paternal behavior in mated males.

Strengths:

The authors use a combination of in vivo activity manipulation and slice electrophysiology to study the role of inhibition in this circuit in dynamic infant-directed behavior induced by mating.

Weaknesses:

(1) Some technical and methodological details are incomplete or missing for interpretation of the significance of the findings. Statistical details are also left out.

(2) The rationale for using Cartpt as a marker is not fully explained. This marker has activity-dependent expression and this possibility is not explored experimentally--for example, could exposure to objects or pups change expression (or the number of cells expressing) cartpt alone?

(3) The cfos experiment is quantified by exposing a male to a pup inside a tea ball. Therefore, it is unclear how the male was classified as infanticidal or parental based on the available criteria provided in the methods section.

(4) There is no information about inclusion/exclusion criteria for chemical and viral experiments. Specifically, there is no information provided about the validation of the lesion experiment--how large were the lesions? Is there concern about leakage of the chemical into the recorded region (MPOA and BNST are adjacent).

(5) The authors do not provide information about how long rAAV is allowed to express before quantifying retrograde transport.

(6) For statistics, the authors do not provide information distinguishing the main effects from multiple comparisons post hoc testing for the ANOVA analyses.

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 #1 (Public review):

The propagation of electrical signals within neuronal circuits is tightly regulated by the physical and molecular properties of neurons. Since neurons vary in size across species, the question arises whether propagation speed also varies to compensate for it. The present article compares numerous speed-related properties in human and rat neurons. They found that the larger size of human neurons seems to be compensated by a faster propagation within dendrites but not axons of these neurons. The faster dendritic signal propagation was found to arise from wider dendritic diameters and greater conductance load in human neurons. In addition, the article provides a careful characterization of human dendrites and axons, as the field has only recently begun to characterize post-operative human cells. There are only a few studies reporting dendritic properties and these are not all consistent, hence there is added value of reporting these findings, particularly given that the characterization is condensed in a compartmental model.

Strengths

The study was performed with great care using standard techniques in slice electrophysiology (pharmacological manipulation with somatic patch-clamp) as well as some challenging ones (axonal and dendritic patch-clamp). Modeling was used to parse out the role of different features in regulating dendritic propagation speed. The finding that propagation speed varies across species is novel as previous studies did not find a large change in membrane time constant nor axonal diameters (a significant parameter affecting speed). A number of possible, yet less likely factors were carefully tested (Ih, membrane capacitance). The main features outlined here are well known to regulate speed in neuronal processes. The modeling was also carefully done to verify that the magnitude of the effects is consistent with the difference in biophysical properties. Hence, the findings appear very solid to me.

Weaknesses

The role of diameter in regulating propagation speed is well known in the axon literature.

Comment on the revised version: the authors have now made clearer that the role of diameter was well known in the manuscript.

Reviewer #2 (Public review):

Summary:

In this paper, Oláh and colleagues introduce new research data on the cellular and biophysical elements involved in transmission within the pyramidal circuits of the human neocortex. They gathered a comprehensive set of patch-clamp recordings from human and rat pyramidal neurons to compare how the temporal aspect of neuronal processing is maintained in the larger human neocortex. A range of experimental techniques have been used, including two-photon guided dual whole-cell recordings, electron microscopy, complemented by theoretical and computational methods.

The authors find that synaptically connected pyramidal neurons within the human neocortex have longer intercellular path lengths. They go on to show that the short soma to soma latencies is not due to propagation velocity along the axon but instead reflects a higher propagation speed of synaptic potentials from dendrite to soma. Next, in a series of extensive computational modeling studies focusing on the synaptic potentials, the authors show that the shorter latency may be explained by larger diameters, affecting the cable properties and resulting is relatively faster propagation of EPSPs in the human neuron. The manuscript is well-written, and the physiological experiments and in-depth theoretical steps for the simulations are clear. Whether passive cable properties of the dendrites alone are responsible for higher velocities remains to be further investigated. Based on the present data the contribution of active membrane properties cannot be excluded.

Strengths:

The authors used complex 2P-guided dual whole-cell recordings in human neurons. In combination with detailed reconstructions, these approaches represent the next steps in unravelling the information processing in human circuits.

The computational modelling and cable theory application to the experimentally constrained simulations provides an integrated view of the passive membrane properties of human neurons.

Weaknesses:

Whether the cable properties alone are the main explanation for speeding the electrical signaling in human pyramidal neurons deserves further studies.

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:

Davenport et al have investigated how a masculinizing dose of estrogen changes the transcriptomes of several key song nuclei song and adjacent brain areas in juvenile zebra finches of both sexes. Only male zebra finches sing, learn song, and normally have a fully developed song control circuitry, so the study was aimed at further understanding how genetic and hormonal factors contribute to the dimorphism in song behavior and related brain circuitry in this species. Using WGCNA and follow-up correlations to re-analyze published transcriptome datasets, the authors provide evidence that the main variance of several identified gene co-expression modules significantly correlates with one or some of the factors examined, including sex, estrogen treatment, regional neuroanatomy, chromosomal placement, or vocal learning, noting that the latter is largely based on inference due to expression in song control nuclei.

Strengths:

Among the main strengths are the thorough gene co-expression module and correlation analyses, and the inclusion of both song nuclei and adjacent areas, the latter serving as sort of controls for areas that are not dimorphic and likely broadly present in birds in general. In situ hybridization data discussed in a previous publication (Choe et al., Hormones and Behavior, 2021) provides some support for the neuroanatomical specializations of gene expression. It is also significant that the transcriptome re-analysis was performed with an improved genome assembly that also includes the sex chromosomes, thus expanding the Z/W chromosome gene analyses in Friedrich et al, Cell Reports, 2022. The most relevant finding is arguably the identification of some modules where gene expression variation within song nuclei correlates with hormonal effects and/or gene location on sex chromosomes, which are present at different dosages between sexes. Sex differences in gene expression in areas that are not song nuclei may also bring insights into functions other than song behavior or vocal learning. The study also shows how a published RNA-seq dataset can be reanalyzed in novel and informative ways.

Weaknesses:

The validation of the inferred direction of regulation in the identified co-expression modules is limited to the in situ data mentioned above. Further evidence that representative genes in the main modules differ in expression when comparing sexes or E2- vs VEH-treated tissues using independent samples and/or methods would provide further validation and enhance rigor. Most importantly, E2 is known to exert various actions on brain physiology and neuronal function. Because there was no manipulation of candidate genes, nor assessment/manipulation of vocal behavior or vocal learning, an involvement of the identified candidate genes in setting up the sexual dimorphism of the song system or song behavior was not directly tested in this study. For the latter reason, the implication of the Title (..."gene expression associated with vocal learning...") is not well supported. While novel insights were gained into brain expression of Z chromosome genes, it cannot be excluded that the higher male expression of some Z genes may not affect brain cell function and thus may not require active compensation (as discussed for nucleus RA in Friedrich et al, Cell Reports, 2022).

Reviewer #2 (Public review):

Summary:

The authors provide an open-source graphic user interface (GUI) called Heron, implemented in Python, that is designed to help experimentalists to:

(1) Design experimental pipelines and implement them in a way that is closely aligned with their mental schemata of the experiments<br /> (2) Execute and control the experimental pipelines with numerous interconnected hardware and software on a network.

The former is achieved by representing an experimental pipeline using a Knowledge Graph and visually representing this graph in the GUI. The latter is accomplished by using an actor model to govern the interaction among interconnected nodes through messaging, implemented using ZeroMQ. The nodes themselves execute user-supplied code in, but not limited to, Python.

Using three showcases of behavioral experiments on rats, the authors highlighted four benefits of their software design:

(1) The knowledge graph serves as a self-documentation of the logic of the experiment, enhancing the readability and reproducibility of the experiment,<br /> (2) The experiment can be executed in a distributed fashion across multiple machines that each has different operating system or computing environment, such that the experiment can take advantage of hardware that sometimes can only work on a specific computer/OS, a commonly seen issue nowadays,<br /> (3) The users supply their own Python code for node execution that is supposed to be more friendly to those who do not have a strong programming background,<br /> (4) The GUI can also be used as an experiment control panel for users to control/update parameters on the fly.

Strengths:

(1) The software is light-weight and open-source, provides a clean and easy-to-use GUI,<br /> (2) The software answers the need of experimentalists, particularly in the field of behavioral science, to deal with the diversity of hardware that becomes restricted to run on dedicated systems. It can also be widely adopted in many other experimental settings.<br /> (3) The software has a solid design that seems to be functionally reliable and useful under many conditions, demonstrated by a number of sophisticated experimental setups.<br /> (4) The software is well documented. The authors pay special attention to documenting the usage of the software and setting up experiments using this software.

Comments on revisions: The authors have addressed my concerns from the initial review.

Reviewer #2 (Public review):

Summary:

Using in vivo fiber-photometry the authors first establish that DA release when contacting their partner mouse increases with days of cohabitation while this increase is not observed when contacting a stranger mouse. Similar effects are found in D1-MSNs and D2-MSNs with the D1-MSN responses increasing and D2-MSN responses decreasing with days of cohabitation. They then use slice physiology to identify underlying plasticity/adaptation mechanisms that could contribute to the changes in D1/D2-MSN responses. Last, to address causality the authors use chemogenetic tools to selectively inhibit or activate NAc shell D1 or D2 neurons that project to the ventral pallidum. They found that D2 inhibition facilitates bond formation while D2 excitation inhibits bond formation. In contrast, both D1-MSN activation and inhibition inhibits bond formation.

Strengths:

The strength of the manuscript lies in combining in vivo physiology to demonstrate circuit engagement and chemogenetic manipulation studies to address circuit involvement in pair bond formation in a monogamous vole.

Weaknesses:

Weaknesses include that a large set of experiments within the manuscript are dependent on using short promoters for D1 and D2 receptors in viral vectors. As the authors acknowledge this approach can lead to ectopic expression and the presented immunohistochemistry supports this notion. It seems to me that the presented quantification underestimates the degree of ectopic expression that is observed by eye when looking at the presented immunohistochemistry. However, given that Cre transgenic animals are not available for Microtus mandarinus and given the distinct physiological and behavioral outcomes when imaging and manipulating both viral-targeted populations this concern is minor.

The slice physiology experiments provide some interesting outcomes but it is unclear how they can be linked to the in vivo physiological outcomes and some of the outcomes don't match intuitively (e.g. cohabitation enhances excitatory/inhibitory balance in D2-MSNs but the degree of contact-induced inhibition is enhanced in D2-MSN).

One interesting finding is that the relationship between D2-MSN and pair bond formation is quite clear (inhibition facilitates while excitation inhibits pair bond formation). In contrast, the role of D1-MSNs is more complicated since both excitation and inhibition disrupts pair bond formation. This is not convincingly discussed.

It seemed a missed opportunity that physiological read out is limited to males. I understand though that adding females may be beyond the scope of this manuscript.

Comments on revised version:

The authors addressed most of my comments, some would still need to be addressed.

(1) Previous comment: "The authors do not use an isosbestic control wavelength in photometry experiments, although they do use EGFP control mice which show no effects of these interventions, a within-subject control such as an isosbestic excitation wavelength could give more confidence in these data and rule out motion artefacts within subjects."

The authors should include a paragraph in the discussion addressing the limitations of not using an internal control for the fiberphotometric measurements.

(2) Previous Comment: The slice physiology experiments provide some interesting outcomes but it is unclear how they can be linked to the in vivo physiological outcomes and some of the outcomes don't match intuitively (e.g. cohabitation enhances excitatory/inhibitory balance in D2-MSNs but the degree of contact-induced inhibition is enhanced in D2-MSN).

My comment may not have been clear and the response didn't address my comment. What is missing in the discussion is an explanation of why a relative increase in excitation of D2-MSNs in the slice (Fig. 4J) is associated with an increased inhibition in vivo (Fig. 2H)?

(3) Previous Comment: One interesting finding is that the relationship between D2-MSN and pair bond formation is quite clear (inhibition facilitates while excitation inhibits pair bond formation). In contrast, the role of D1-MSNs is more complicated since both excitation and inhibition disrupt pair bond formation. This is not convincingly discussed.

Similarly, here the response provided does not address my question. Please focus on discussing why both excitation and inhibition of D1-MSNs can disrupt pair bond formation (Figure 7).

Reviewer #3 (Public review):

Summary:

The manuscript is evaluating changes in dopamine signaling in the nucleus accumbens following pair bonding and exposure to various stimuli in mandarin voles. In addition, the authors present chemogenetic data which demonstrates excitation and inhibition of D1 and D2 MSN affect pair bond formation.

Strengths:

The experimental designs are strong. The approaches are innovative and use cutting-edge methods. The manuscript is well written.

Comments on revised version:

I appreciate the efforts by the authors to address many of my previous comments. The issues that remain are those associated with the statistics. It seems that not all statistical analyses were performed with the correct test. For example, the photometry data comparing emissions during partner vs stranger investigation over time would be best performed as a two-way ANOVA with odor type and time being separate variables. Also, there are paired t-tests being performed by calculating an average deltaF/F during the 4 second window following the being of a behavioral event. I think an area-under-the-curve calculation of these events would better capture the fluorescent emissions of these events as an index. Details in the Result describing the data being analyzed via ANOVA vs t-tests when reporting the results would be useful for the reviewer to understand each analysis.

Reviewer #1 (Public review):

The authors demonstrated that NINJ1 promotes TF-positive MV release during pyroptosis and thereby triggers coagulation. Coagulation is one of the risk factors that can cause secondary complications in various inflammatory diseases, making it a highly important therapeutic target in clinical treatment. This paper effectively explains the connection between pyroptosis and MV release with Ninj1, which is a significant strength. It provides valuable insight into the potential of targeting Ninj1 as a therapeutic strategy.

Although the advances in this paper are valuable, several aspects need to be clarified. Some comments are discussed below.

(1) Since it is not Ninj1 directly regulating coagulation but rather the MV released by Ninj1 playing a role, the title should include that. The current title makes it seem like Ninj1 directly regulates inflammation and coagulation. It would be better to revise the title.

(2) Ninj1 is known to be an induced protein that is barely expressed in normal conditions. As you showed in "Fig1G" data, control samples showed no detection of Ninj1. However, in "Figure S1", all tissues (liver, lung, kidney and spleen) expressed Ninj1 protein. If the authors stimulated the mice with fla injection, it should be mentioned in the figure legend.

(3) In "Fig3A", the Ninj1 protein expression was increased in the control of BMDM +/- cell lysate rather than fla stimulation. However, in MV, Ninj1 was not detected at all in +/- control but was only observed with Fla injection. The authors need to provide an explanation for this observation. Additionally, looking at the MV β-actin lane, the band thicknesses appear to be very different between groups. It seems necessary to equalize the protein amounts. If that is difficult, at least between the +/+ and +/- controls.

(4) Since the authors focused Ninj1-dependent microvesicle (MV) release, they need to show MV characterizations (EM, NTA, Western for MV markers, etc...).

(5) To clarify whether Ninj1-dependent MV induces coagulation, the authors need to determine whether platelet aggregation is reduced with isolated +/- MVs compared to +/+ MVs.

(6) Even with the authors well established experiments with haploid mice, it is a critical limitation of this paper. To improve the quality of this paper, the authors should consider confirming the findings using mouse macrophage cell lines, such as generating Ninj1-/- Raw264.7 cell lines, to examine the homozygous effect.

(7) There was a paper reported in 2023 (Zhou, X. et al., NINJ1 Regulates Platelet Activation and PANoptosis in Septic Disseminated Intravascular Coagulation. Int. J. Mol. Sci. 2023) that revealed the relationship between Ninj1 and coagulation. According to this paper, inhibition of Ninj1 in platelets prevents pyroptosis, leading to reduced platelet activation and, consequently, the suppression of thrombosis. How about the activation of platelets in Ninj1 +/- mice? The author should add this paper in the reference section and discuss the platelet functions in their mice.

Reviewer #2 (Public review):

Summary:

The authors main goal is to understand the mechanism by which pyroptosis (through the formation of Gasdermin D (GSDMD) pores in the plasma membrane) contributes to increased release of procoagulant Tissue Factor-containing microvesicles (MV). Their previous data demonstrate that GSDMD is critical for the release of MV that contains Tissue Factor (TF), thus making a link between pyroptosis and hypercoagulation. Given the recent identification of NINJ1 being responsible for plasma membrane rupture (Kayagaki et al. Nature 2011), the authors wanted to determine if NINJ1 is responsible for TF-containing MV release. Given the constitutive ninj1 KO mouse leads to partial embryonic lethality, the authors decide to use a heterozygous ninj1 KO mouse (ninj1+/-), and demonstrate that Ninj1 plays a role in release of TF-containing MV.

Reviewer #2 (Public review):

Summary:

Golamalamdari, van Schaik, Wang, Kumar Zhang, Zhang and colleagues study interactions between the speckle, nucleolus and lamina in multiple cell types (K562, H1, HCT116 and HFF). Their datasets define how interactions between the genome and the different nuclear landmarks relate to each other and change across cell types. They also identify how these relationships change in K562 cells in which LBR and LMNA are knocked out.

Strengths:

Overall, there are a number of datasets that are provided, and several "integrative" analyses performed. This is a major strength of the paper, and I imagine the datasets will be of use to the community to further probed and the relationships elucidated here further studied. An especially interesting result was that specific genomic regions (relative to their association with the speckle, lamina, and other molecular characteristics) segregate relative to the equatorial plane of the cell.

Weaknesses:

The experiments are primarily descriptive, and the cause-and-effect relationships are limited (though the authors do study the role of LMNA/LBR knockdown with their technologies).

Reviewer #1 (Public review):

Summary:

The drug Ivermectin is used to effectively treat a variety of worm parasites in the world, however resistance to Ivermectin poses a rising challenge for this treatment strategy. In this study, the authors found that loss of the E3 ubiquitin ligase UBR-1 in the worm C. elegans results in resistance to Ivermectin. In particular, the authors found that ubr-1 mutants are resistant to the effects of Ivermectin on worm viability, body size, pharyngeal pumping and locomotion. The authors previously showed that loss of UBR-1 disrupts homeostasis of the amino acid and neurotransmitter glutamate resulting in increased levels of glutamate in C. elegans. Here, the authors found that the sensitivity of ubr-1 mutants to Ivermectin can be restored if glutamate levels are reduced using a variety of different methods. Conversely, treating worms with exogenous glutamate to increase glutamate levels also results in resistance to Ivermectin supporting the idea that increased glutamate promotes resistance to Ivermectin. The authors found that the primary known targets of Ivermectin, glutamate-gated chloride channels (GluCls), are downregulated in ubr-1 mutants providing a plausible mechanism for why ubr-1 mutants are resistant to Ivermectin. Although it is clear that loss of GluCls can lead to resistance to Ivermectin, this study suggests that one potential mechanism to decrease GluCl expression is via disruption of glutamate homeostasis that leads to increased glutamate. This study suggests that if parasitic worms become resistant to Ivermectin due to increased glutamate, their sensitivity to Ivermectin could be restored by reducing glutamate levels using drugs such as Ceftriaxone in a combination drug treatment strategy.

Strengths:

- The use of multiple independent assays (i.e., viability, body size, pharyngeal pumping, locomotion and serotonin-stimulated pharyngeal muscle activity) to monitor the effects of Ivermectin<br /> - The use of multiple independent approaches (got-1, eat-4, ceftriaxone drug, exogenous glutamate treatment) to alter glutamate levels to support the conclusion that increased glutamate in ubr-1 mutants contributes to Ivermectin resistance

Weaknesses:

- The primary target of Ivermectin is GluCls so it is not surprising that alteration of GluCl expression or function would lead to Ivermectin resistance<br /> - It remains to be seen what percent of Ivermectin resistant parasites in the wild have disrupted glutamate homeostasis as opposed to mutations that more directly decrease GluCl expression or function.

Comments on revisions: All my concerns have been addressed by the authors.

Reviewer #2 (Public review):

Summary:

The authors provide a very thorough investigation on the role of UBR-1 in anthelmintic resistance using the non-parasitic nematode, C. elegans. Anthelmintic resistance to macrocyclic lactones is a major problem in veterinary medicine and likely just a matter of time until resistance emerges in human parasites too. Therefore, this study providing novel insight into the mechanisms of ivermectin resistance is particularly important and significant.

Strengths:

The authors use very diverse technologies (behavior, genetics, pharmacology, genetically encoded reporters) to dissect the role of UBR-1 in ivermectin resistance. Deploying such a comprehensive suite of tools and approaches provides exceptional insight into the mechanism of how UBR-1 functions in terms of ivermectin resistance.

Weaknesses:

I do not see any major weaknesses in this study. My only concern is whether the observations made by the authors would translate to any of the important parasitic helminths in which resistance has naturally emerged in the field. This is always a concern when leveraging a non-parasitic nematode to shed light on a potential mechanism of resistance of parasitic nematodes, and I understand that it is likely beyond the scope of this paper to test some of their results in parasitic nematodes.

Comments on revisions: The authors have now addressed all my concerns.

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 #1 (Public review):

Summary:

Joshua G. Medina-Feliciano et al. investigated the single-cell transcriptomic profile of holoturian regenerating intestine following evisceration, a process used to expel their viscera in response to predation. Using single-cell RNA-Sequencing and standard analysis such as "Find cluster markers", "Enrichment analysis of Gene Ontology" and "RNA velocity", they identify 13 cell clusters and their potential cell identity. Based on bioinformatic analysis they identified potentially proliferating clusters and potential trajectories of cell differentiation. This manuscript represents a useful dataset that can provide candidate cell types and cell markers for more in-depth functional analysis of the holoturian intestine regeneration.

The conclusions of this paper are supported only by bioinformatic analyses since the in vivo validation through HCR is not sufficient to support them.

Strengths:

- The Authors are providing a single-cell dataset obtained from sea cucumbers regenerating their intestines. This represents the first fundamental step to an unbiased approach to better understand this regeneration process and the cellular dynamics taking part in it.

- The Authors run all the standard analyses providing the reader with a well digested set of information about cell clusters, potential cell types, potential functions and potential cell differentiation trajectories.

Weaknesses:

- The Authors frequently report the percentage of cells with a specific feature (either labelled or expressing a certain gene or belonging to a certain cluster). This number can be misleading since that is calculated after cell dissociation and additional procedures (such as staining or sequencing and dataset cleanup) that can heavily bias the ratio between cell types. Similarly, the Authors cannot compare cell percentage between anlage and mesentery samples since that can be affected by technical aspects related to cell dissociation, tissue composition and sequencing depth.

- The Authors did not validate all the clusters.

- There is no validation of the trajectory analysis and there is no validation of the proliferating cluster with H3P or EdU co-labeling.

Reviewer #2 (Public review):

Summary:

This research offers a comprehensive analysis of the regenerative process in sea cucumbers and builds upon decades of previous research. The approach involves a detailed examination using single-cell sequencing, making it a crucial reference paper while shedding new light on regeneration in this organism.

Strengths:

Detailed analysis of single-cell sequencing data and high-quality RNA localization images provide significant new insights into regeneration in sea cucumbers and, more broadly, in animals. Identifying a proliferating cluster of cells is very interesting and may open avenues to identify the cell lineage history and deeper molecular properties of the cells that regenerate the intestine.

Weaknesses:

The spatial context of the RNA localization images is challenging to interpret in this spatially complex tissue organization. Although the authors have taken care to perform RNA localization staining, it is still challenging to relate these data to their schematic model. This is only a minor weakness that will almost certainly be clarified by future work from the authors as they follow up on findings.

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.

Joint Public Review:

Páramo et al. used 3D geometric morphometric analyses of the articulated femur, tibia, and fibula of 17 macronarian taxa (known to preserve these three skeletal elements) to investigate morphological changes that occurred in the hind limb through the evolutionary history of this sauropod clade. A principal components analysis was completed to understand the distribution of the morphological variation. A supertree was constructed to place evolutionary trends in morphological variation into phylogenetic context, and hind limb centroid size was used to investigate potential relationships between skeletal anatomy and gigantism. The majority of the results did not yield statistically significant differences, but they did identify interesting shape-change trends, especially within subclades of Titanosauria. Many previous studies have attempted to elucidate a link between wide-gauge posture and gigantism, which in this study Páramo et al. investigate among several titanosaurian subclades. They propose that morphologies associated with wide-gauge posture arose in parallel with increasing body size among basal members of Macronaria and that this connection became less significant once wide-gauge posture was acquired within Titanosauria. The authors also suggest that other biomechanical factors influenced the independent evolution of subclades within Titanosauria and that these influences resulted in instances of convergent evolution. Therefore, they infer that, overall, wide-gauge posture was not significantly correlated with gigantism, though some morphological aspects of hind limb skeletal anatomy appear to have been associated with gigantism. Their work also supports previous findings of a decrease in body size within Titanosauriformes (which they found to be not significant with shape variables but significant with Pagel's lambda). Collectively, their results support and build on previous work to elucidate more specifics on the evolution of this enigmatic clade. Further study will show if their hypotheses stand or if the inclusion of additional specimens and taxa yields alternative results.

[Editors' note: One of the original reviewers, Reviewer 2, reviewed this revised version of the manuscript; they reported satisfaction with the changes made by the authors in response to the original reviewer comments.]

Reviewer #1 (Public review):

In this paper, the authors had 2 aims:

(1) Measure macaques' aversion to sand and see if its' removal is intentional, as it likely in an unpleasurable sensation that causes tooth damage.

(2) Show that or see if monkeys engage in suboptimal behavior by cleaning foods beyond the point of diminishing returns, and see if this was related to individual traits such as sex and rank, and behavioral technique.

They attempted to achieve these aims through a combination of geochemical analysis of sand, field experiments, and comparing predictions to an analytical model.

The authors' conclusions were that they verified a long-standing assumption that monkeys have an aversion to sand as it contains many potentially damaging fine grained silicates, and that removing it via brushing or washing is intentional.

They also concluded that monkeys will clean food for longer than is necessary, i.e. beyond the point of diminishing returns, and that this is rank-dependent.

High and low-ranking monkeys tended not to wash their food, but instead over-brushed it, potentially to minimize handling time and maximize caloric intake, despite the long-term cumulative costs of sand.

This was interpreted through the *disposable soma hypothesis*, where dominants maximize immediate needs to maintain rank and increase reproductive success at the potential expense of long-term health and survival.

# Strengths

The field experiment seemed well designed, and their quantification of the physical and mineral properties of quartz particles (relative to human detection thresholds) seemed good relative to their feret diameter and particle circularity (to a reviewer that is not an expert in sand). The *Rank Determination* and *Measuring Sand* sections were clear.

In achieving Aim 1, the authors validated a commonly interpreted, but unmeasured function, of macaque and primate behavior-- a key study/finding in primate food processing and cultural transmission research.

I commend their approach in trying to develop a quantitative model to generate predictions to compare to empirical data for their second aim.<br /> This is something others should strive for.

I really appreciated the historical context of this paper in the introduction and found it very enjoyable and easy to read.

I do think that interpreting these results in the context of the *disposable soma hypothesis* and the potential implications in the *paleolithic matters* section about interpreting dental wear in the fossil record are worthwhile.

# Weaknesses

Several of my concerns in an earlier review were addressed in revision, which I appreciate. One thing I think could strengthen this paper is a clearer link to social foraging theory to explore heterogeneity in handling times (as the currency they are trying to maximize).

I am satisfied with the improvements in statistics and that I can access the code and data.

I am still struck that there was an analysis of only trials where <3 individuals are present. If rank was important, I would imagine that behavior might be different in social contexts when theft, scrounging, policing, aggression, or other distractions might occur-- where rank would have effects on foraging behavior. Maybe lower rankers prioritize rapid food intake then. If rank should be related to investment in this behavior, we might expect this to be magnified (or different) in social contexts where it would affect foraging. It might just be that the data was too hard to score or process in those settings, or the analysis was limited. Additionally, I think that more robust metrics of rank from more densely sampled focal follow data would be a better measure, but I acknowledge the limitations in getting the ideal . Since rank is central to the interpretation of these results, I think that reduced social contexts in which rank was analyzed and the robustness of the data from which rank was calculated and analyzed are the main weaknesses of the evidence presented in this paper.

While some of the boxes about raccoons and Concorde Fallacy were interesting, they did feel like a bit of a distraction from the main message in the paper.

Reviewer #3 (Public review):

This revised paper provides evidence that food washing and brushing in wild long-tailed macaques are deliberate behaviors to remove sand that can damage tooth enamel. The demonstration of the immediate functional importance of these behaviors is nicely done, and there is some interesting initial evidence that macaques differ systematically in their investment in food cleaning based on dominance rank.

The authors interpret this evidence as support for "disposable soma" effects: that reduced time and effort invested food washing in high-ranking individuals is attributable to prioritizing reproductive effort. Given that the analysis is on a single group with no longitudinal data, there are no fitness measures or fitness proxies, the energetic constraints faced by this population are not clear, and both sexes are combined into a single dominance hierarchy (trade-offs between different forms of investment are typically thought to differ between sexes), this conclusion is premature, although an interesting foundation for future studies.

More generally, the results directly supported by the data collection and analysis (grit on Koshima likely damages macaque teeth; processing food helps mitigate the damage; there is some interesting interindividual variation in food processing time, and that time is not always in line with what appears to be optimal) tend to be combined with interpretation that is much more speculative (e.g., the effect sizes observed are consequential for fitness; high-ranking animals are making choices that optimize their long-term fitness at the expense of their soma). This is in part a stylistic choice but can have the effect of drawing attention away from the stronger empirical findings and/or be misleading. Similarly, although I appreciate that the authors were trying to interpret and respond to previous feedback from reviewers, I found the addition of the box text on the raccoon nomenclature and on irrational behavior and the Concorde effect distracting (more intro-textbook style than journal article style).

Reviewer #1 (Public review):

Summary:

In their manuscript, Gomez-Frittelli and colleagues characterize the expression of cadherin6 (and -8) in colonic IPANs of mice. Moreover, they found that these cdh6-expressing IPANs are capable of initiating colonic motor complexes in the distal colon, but not proximal and midcolon. They support their claim by morphological, electrophysiological and optogenetic, and pharmacological experiments.

Strengths:

The work is very impressive and involves several genetic models and state-of-the-art physiological setups including respective controls. It is a very well-written manuscript that truly contributes to our understanding of GI-motility and its anatomical and physiological basis. The authors were able to convincingly answer their research questions with a wide range of methods without overselling their results.

Weaknesses:

The authors put quite some emphasis on stating that cdh6 is a synaptic protein (in the title and throughout the text), which interacts in a homophilic fashion. They deduct that cdh6 might be involved in IPAN-IPAN synapses (line 247ff.). However, Cdh6 does not only interact in synapses and is expressed by non-neuronal cells as well (see e.g., expression in the proximal tubuli of the kidney). Moreover, cdh6 does not only build homodimers, but also heterodimers with Chd9 as well as Cdh7, -10, and -14 (see e.g., Shimoyama et al. 2000, DOI: 10.1042/0264-6021:3490159). It would therefore be interesting to assess the expression pattern of cdh6-proteins using immunostainings in combination with synaptic markers to substantiate the authors' claim or at least add the possibility of cell-cell-interactions other than synapses to the discussion. Additionally, an immunostaining of cdh6 would confirm if the expression of tdTomato in smooth muscle cells of the cdh6-creERT model is valid or a leaky expression (false positive).

Comments on revisions:

The authors have updated their manuscript and have provided insights and discussions to my remarks.

Reviewer #2 (Public review):

Summary:

Intrinsic primary afferent neurons are an interesting population of enteric neurons that transduce stimuli from the mucosa, initiate reflexive neurocircuitry involved in motor and secretory functions, and modulate gut immune responses. The morphology, neurochemical coding, and electrophysiological properties of these cells have been relatively well described in a long literature dating back to the late 1800's but questions remain regarding their roles in enteric neurocircuitry, potential subsets with unique functions, and contributions to disease. Here, the authors provide RNAscope, immunolabeling, electrophysiological, and organ function data characterizing IPANs in mice and suggest that Cdh6 is an additional marker of these cells.

Strengths:

This paper would likely be of interest to the enteric neuroscience community and increases information regarding the properties of IPANs in mice. These data are useful and suggest that prior data from studies of IPANs in other species are likely translatable to mice.

Weaknesses:

Major weaknesses:<br /> (1) The novelty of this study is relatively limited. The main point of novelty suggests an additional marker of IPANs (Cdh6) that would add to the known list of markers for these cells. How useful this would be is unclear. Other main findings basically confirm that IPANs in mice display the same classical characteristics that have been known for many years from studies in guinea pigs, rats, mice and humans.

(2) Critical controls are needed to support the optogenetic experiments. Control experiments are needed to show that ChR2 expression 1) does not change the baseline properties of the neurons, 2) that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons, and 3) that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions focused on here. These essential controls remain absent in the study and limit confidence in the data derived from this model.

(3) The motor effects observed in optogenetic experiments are difficult to understand in the absence of good controls for optogenetic control of the proposed neuron population (discussed above). It remains unclear how stimulating IPANs in the distal colon would generate retrograde CMCs while stimulating IPANs in the proximal colon did nothing. Key controls confirming that the optogentic stimulus was adequate, specific, and relevant are needed. In addition, better characterization of the Cdh6+ population of cells in both regions would be needed to understand the mechanisms underlying these effects.

(4) From the data shown, it is clear that expression driven by the Cdh6CreERT2 driver is not confined to IPANs. There is obviously expression of GFP and ChR2 in smooth muscle cells. This is a major limitation for the physiological experiments that attempt to use this model to specifically stimulate IPANs and assess changes in gut motor function. Better characterization of this model is needed and control experiments are necessary to assess whether functional ChR2 is expressed in cells beyond the proposed subtype of enteric IPANs.

(5) Some of the main conclusions of this study are overstated and claims of priority are made that are not true. For example, the authors state on lines 27-28 of the abstract that their findings provide the "first demonstration of selective activation of a single neurochemical and functional class of enteric neurons". This is certainly not true since Gould et al (AJP-GIL 2019) expressed ChR2 in nitrergic enteric neurons and showed that activating those cells disrupted CMC activity. In fact, prior work by the authors themselves (Hibberd et al Gastro 2018) showed that activating calretinin neurons with ChR2 evoked motor responses. Work by other groups has used chemogenetics and optogenetics to show effects of activating multiple other classes of neurons in the gut.

(6) The electrophysiological characterization of mouse IPANs is useful but is limited to a small subset of Cdh6+ neurons in the distal colon myenteric plexus. Therefore, it remains unclear how well the properties reported here might reflect those of other Cdh6+ IPANs in the same or different regions. Similarly, blocking IH with ZD7288 affects all IPANs and does not add specific information regarding the role of the proposed Cdh6+ subtype.

(7) The submucosal plexus (SMP) also contains enteric IPANs and these were not included in the analysis of Cdh6 expression. Whether or not the proposed IPAN marker Cdh6 would be useful for identifying or targeting those cells remains unclear.

[Editor's note: The Reviewing Editor considers that further controls requested from the reviewers have largely been provided already in prior publications by other groups, as they concern specifically tools published years ago but in a different tissue context. Hence the methodology used to deliver the results reported here fall within the standard practices in the field. The comprehensive, multi-technique approach to the results is compelling in and of itself, and ought to suffice, rendering this work reproducible and therefore a basis for further research.]

Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal any structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are thoughtfully designed and well executed.

Weaknesses:

The cryo-FIB and cryo-ET analyses require further investigation, as detailed below. The molecular mechanism by which the loss of ANKRD5 affects sperm flagellar motility remains unclear. The current conclusion that Ankrd5 knockout reduces axoneme stability is not well-supported. Specifically, are other axonemal proteins diminished in Ankrd5 knockout sperm? Conducting immunofluorescence analyses and revisiting the quantitative proteomics data may help address these questions.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of ANKRD5 (ANKEF1) as a component of the N-DRC complex in sperm motility and male fertility. Using Ankrd5 knockout mice, the study demonstrates that ANKRD5 is essential for sperm motility and identifies its interaction with N-DRC components through IP-mass spectrometry and cryo-ET. The results provide insights into ANKRD5's function, highlighting its potential involvement in axoneme stability and sperm energy metabolism.

Strengths:

The authors employ a wide range of techniques, including gene knockout models, proteomics, cryo-ET, and immunoprecipitation, to explore ANKRD5's role in sperm biology.

Weaknesses:

(1) Limited Citations in Introduction: Key references on the role of N-DRC components (e.g., DRC1, DRC2, DRC3, DRC5) in male infertility are missing, which weakens the contextual background.

(2) Lack of Functional Insights: While interacting proteins outside the N-DRC complex were identified, their potential roles and interactions with ANKRD5 are not adequately explored or discussed.

(3) Mitochondrial Function Uncertainty: Immunofluorescence suggests possible mitochondrial localization for ANKRD5, but experiments on its role in energy metabolism (e.g., ATP production, ROS) are insufficient, especially given the observed sperm motility defects.

(4) Glycolysis Pathway Impact: Proteomic analysis indicates glycolysis pathway disruptions in Ankrd5-deficient sperm, but the link between these changes and impaired motility is not well explained.

(5) Cryo-ET Data Limitations: The structural analysis of the DMT lacks clarity on how ANKRD5 influences N-DRC or RS3. The low quality of RS3 data hinders the interpretation of ANKRD5's impact on axoneme structure.

(6) Discussion of Findings: The manuscript could benefit from a deeper discussion on the broader implications of ANKRD5's interactions and its role in sperm energy metabolism and motility mechanisms.

Reviewer #1 (Public review):

Summary:

In this study, the authors examined the role of Afadin, a key adaptor protein associated with cell-adhesion molecules, in retinal development. Using a conditional knockout mouse line (Six3-Cre; AfadinF/F), the authors successfully characterized a disorganized pattern of various neuron types in the mutant retinae. Despite these altered distributions, the retinal neurons maintained normal cell numbers and seemingly preserved some synaptic connections. Notably, tracing results indicated mistargeting of retinal ganglion cell (RGC) axon projections to the superior colliculus, and electroretinography (ERG) analyses suggested deficits in visual functions.

Strengths:

This compelling study provides solid evidence addressing the important question of how cell-adhesion molecules influence neuronal development. Compared to previous research conducted in other parts of the central nervous system (CNS), the clearly defined lamination of cell types in the retina serves as a unique model for studying the aberrant neuronal localizations caused by Afadin knockout. The data suggest that cell-cell interactions are critical for retinal cellular organization and proper axon pathfinding, while aspects of cell fate determination and synaptogenesis remain less understood. This work has broad implications not only for retinal studies but also for developmental biology and regenerative medicine.

Weaknesses:

While the phenotypes observed in the Afadin knockout (cKO) mice are intriguing, I would expect to see evidence confirming that Afadin is indeed knocked out in the retina through immunostaining. Specifically, is Afadin knocked out only in certain retinal regions and not others, as suggested by Figures 4A-B? Are Afadin levels different among distinct neuron types, which could mean that its knockout may have a more pronounced impact on certain cell types, such as rods compared to others?

The authors suggest that synapses may form between canonical synaptic partners, based on the proximity of their processes (Figure 2). However, more solid evidence is needed to verify these synapses through the use of synaptic marker staining or transsynaptic labeling before drawing further conclusions.

Although the Afadin cKO mice displayed dramatic phenotypes, additional experiments are necessary to clarify the details of this process. By manipulating Afadin levels in specific cell types or at different developmental time points, we could gain a better understanding of how Afadin regulates accurate retinal lamination and axonal projection.

Reviewer #2 (Public review):

Summary:

This study by Lum and colleagues reports on the role of Afadin, a cytosolic adapter protein that organizes multiple cell adhesion molecule families, in the generation and maintenance of complex cellular layers in the mouse retina. They used a conditional deletion approach, removing Afadin in retinal progenitors, and allowing them to analyze broad effects on retinal neuron development.

The study presents high-quality and extensive characterization of the cellular phenotypes, supporting the main conclusions of the paper. They show that Afadin loss results in significant disorganization of the retinal cellular layers and the neuropil, producing rosettes and displacement of cells away from their resident layers. The major classes of neurons in the inner retina are affected, and some neurons are, remarkably, displaced to the other side of the inner plexiform layer. Nevertheless, they mostly target their synaptic partners, including the RGCs to distant retinorecipient targets in the brain. The main conclusions are as follows. Afadin is necessary for establishing and maintaining the retinal architecture. It is not necessary for the generation of the correct numbers/densities of retinal neuron subtypes. Moreover, Afadin loss preserves associations between known synaptic partners and preserves axonal targeting to retinorecipient layers. The consequences on photoreceptor viability and visual processing are also interesting, underscoring the essential function for maintaining retinal structure and function. Overall the main conclusions describing the consequences are supported by the results.

Strengths:

The study provides new knowledge on the requirement of Afadin in retinal development. The introduction and discussion effectively set up the rationale for this work, and place it in the context of previous studies of Afadin in other regions of the CNS.

The study presents high-quality and extensive characterizations of the cellular phenotypes resulting from Afadin loss. By analyzing various aspects of retinal organization - from cellular densities to axon targeting to brain - the study narrows down the role of a structure for promoting the establishment of the layers, or maintenance. The data are straightforward and convincing, and the interpretations are bounded by the data shown (though minor weakness re. survival). Another important finding is that the targeting of retinal neuron processes to synaptic partners, including retinorecipient targets in the brain, are intact.

The study is important as it establishes a focused requirement for Afadin to set up and preserve the overall cellular organizations within the retinal tissue. The demonstration that Afadin is needed for photoreceptor viability and overall visual function enhances impact by establishing its functional importance.

The manuscript is well well-written and presented. The images are attractive and compelling, and the figures are well organized.

Weaknesses:

(1) Expanding on the developmental mechanism is beyond the scope of the study, and would not add to the main conclusions. However, the manuscript would be improved by providing more clarity on the developmental emergence of the defects. The study left me questioning whether the rosettes and cell displacements occur during earlier stages of retina development, or are progressive. For instance, do the RGCs migrate and establish within the GCL correctly at first, and then are displaced with the progressive disorganization? Or are they disorganized and delaminate en route? Images of RGC staining at P0, or earlier during their migration, would be informative. Data in Figure 1 is limited to DAPI staining at P7. Figure 4 shows an image of rod photoreceptors at P7, with their displacement in the GCL layer (and not contained within a rosette). Are the progenitors mislocalized due to delamination?

A few additional thoughts on how these defects compare to other mutants with rosettes might give us more context for understanding the results.

(2) The manuscript reports that the densities of major inner retinal classes are unaffected. There are a few details missing for this point. How were the cell densities quantified (in terms of ROI size), and normalized? This information is lacking in the methods. There is a striking thickening of the GCL in the DAPI-labeled images shown in Figure 1. What are these cells?

Reviewer #1 (Public review):

Summary:

The authors address the role of the centromere histone core in force transduction by the kinetochore.

Strengths:

They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).

Weaknesses:

The main results are shown in one figure (Figure 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that, the impact is fairly limited in scope.

Reviewer #2 (Public review):

Summary:

This paper provides a valuable addendum to the findings described in Hamilton et al. 2020 (https://doi.org/ 10.7554/eLife.56582). In the earlier paper, the authors reconstituted the budding yeast centromeric nucleosome together with parts of the budding yeast kinetochore and tested which elements are required and sufficient for force transmission from microtubules to the nucleosome. Although budding yeast centromeres are defined by specific DNA sequences, this earlier paper did not use centromeric DNA but instead the generic Widom 601 DNA. The reason is that it has so far been impossible to stably reconstitute a budding yeast centromeric nucleosome using centromeric DNA.

In this new study, the authors now report that they were able to replace part of the Widom 601 DNA with centromeric DNA from chromosome 3. This makes the assay more closely resemble the in vivo situation. Interestingly, the presence of the centromeric DNA fragment makes one type of minimal kinetochore assembly, but not the other, withstand stronger forces.

Which kinetochore assembly turned out to be affected was somewhat unexpected, and can currently not be reconciled with structural knowledge of the budding yeast centromere/kinetochore. This highlights that, despite recent advances (e.g. Guan et al., 2021; Dendooven et al., 2023), aspects of budding yeast kinetochore architecture and function remain to be understood and that it will be important to dissect the contributions of the centromeric DNA sequence.

Given the unexpected result, the study would become yet more informative if the authors were able to pinpoint which interactions contribute to the enhanced force resistance in the presence of centromeric DNA.

Strength:

The paper demonstrates that centromeric DNA can increase the attachment strength between budding yeast microtubules and centromeric nucleosomes.

Weakness:

How centromeric DNA exerts this effect remains unclear.

Reviewer #2 (Public review):

Summary:

Casas-Tinto et al., provide new insight into glial plasticity using a crush injury paradigm in the ventral nerve cord (VNC) of adult Drosophila. The authors find that both astrocyte-like glia (ALG) and ensheating glia (EG) divide under homeostatic conditions in the adult VNC and identify ALG as the glial population that specifically ramps up proliferation in response to injury, whereas the number of EGs decreases following the insult. Using lineage-tracing tools, the authors interestingly observe interconversion of glial subtypes, especially of EGs into ALGs, which occurs independent of injury and is dependent on the availability of the transcription factor Prospero in EGs, adding to the plasticity observed in the system. Finally, when tracing the progeny of glia, Casas-Tinto and colleagues detect cells of neuronal identity and provide evidence that such glia-derived neurogenesis is favored following ventral nerve cord injury, which puts forward a remarkable way in which glia can respond to neuronal damage.

Strengths:

This study highlights a new facet of adult nervous system plasticity at the level of the ventral nerve cord, supporting the view that proliferative capacity is maintained in the mature CNS and stimulated upon injury.

The injury paradigm is well chosen, as the organization of the neuromeres allows specific targeting of one segment, compared to the remaining intact and with the potential to later link observed plasticity to behavior such as locomotion.

Numerous experiments have been carried out in 7-day old flies, showing that the observed plasticity is not due to residual developmental remodeling or a still immature VNC.

Different techniques are used to observe proliferation in the VNC.

By elegantly combining different methods, the authors show glial divisions including with mitotic-dependent tracing and find that the number of generated glia is refined by apoptosis later on.

The work identifies prospero in glia as important coordinator of glial cell fate, from development to the adult context, which draws further attention to the upstream regulatory mechanisms.

Weaknesses:

The authors do not discuss their results on gliogenesis or neurogenesis in the adult VNC to previous findings made in the context of the injured adult brain.

The authors speculate about the role of glial inter-conversion for tissue homeostasis or regeneration, but no supportive evidence is cited or provided. Further experiments will be required to test the function of the described glial plasticity.

Elav+ cells originating from glia do not express markers for mature neurons at the analysed time-point. If they will eventually differentiate<br /> or what type of structure is formed by them will have to be followed up in future studies.

Context/Discussion

Highlighting some differences in the reactiveness of glia in the VNC compared to the brain could reveal important differences in repair strategies in different areas of the CNS.

Reviewer #3 (Public review):

In this manuscript, Casas-Tintó et al. explore the role of glial cell in the response to a neurodegenerative injury in the adult brain. They used Drosophila melanogaster as a model organism, and found that glial cells are able to generate new neurons through the mechanism of transdifferentiation in response to injury. This paper provides a new mechanism in regeneration, and gives an understanding to the role of glial cells in the process.

The authors have now addressed all my concerns.

Reviewer #1 (Public review):

Summary:

The authors investigated sleep and circadian rhythm disturbances in Fmr1 KO mice. Initially, they monitored daily home cage behaviors to assess sleep and circadian disruptions. Next, they examined the adaptability of circadian rhythms in response to photic suppression and skeleton photic periods. To explore the underlying mechanisms, they traced retino-suprachiasmatic connectivity. The authors further analyzed the social behaviors of Fmr1 KO mice and tested whether a scheduled feeding strategy could mitigate sleep, circadian, and social behavior deficits. Finally, they demonstrated that scheduled feeding corrected cytokine levels in the plasma of mutant mice.

Strengths:

(1) The manuscript addresses an important topic-investigating sleep deficits in an FXS mouse model and proposing a potential therapeutic strategy.

(2) The study includes a comprehensive experimental design with multiple methodologies, which adds depth to the investigation.

Weaknesses:

(1) The first serious issue in the manuscript is the lack of a clear description of how they performed the experiments and the missing definitions of various parameters in the results. Given that monitoring and analyzing sleep behaviors are the key experiments of this manuscript, I use the "Immobility-Based Sleep Behavior" section of Methods as an example to elaborate:

Incomplete or Incorrect Description of Tracking Threshold:<br /> o The phrase "tracked the (40 sec or greater as previously described" is incomplete and does not clarify what is being tracked. This appears to be an error in writing or editing.<br /> Unclear Relationship Between Threshold and EEG Validation:<br /> o The threshold "40 sec or greater" is mentioned without context or explanation of what it represents (e.g., sleep bout duration, inactivity, or another parameter). The reference to Fisher et al. (2016) and "99% correlation with EEG-defined sleep" seems misaligned with the paragraph's content.

Confusing Definition of Sleep Bout:<br /> o The definition of a sleep bout is unclear. Sleep bouts should logically be based on periods of inactivity, not activity. The sentence suggesting sleep is measured by "activity staying above the threshold" is confusing. The phrase "3 counts of sleep per minute for longer than one minute" requires clarification.

Unclear Data Selection for Analysis:<br /> o The phrase "2 days with the best recording quality" is vague and does not specify how "best" was determined or why only two days out of five were analyzed.

Awkward Grammar and Structure:<br /> o Phrases like "Acquiring data were exported in 1-min bins" are grammatically awkward. "Acquiring" should be "Acquired." Some sentences are overly long and lack clarity, making the text harder to follow.<br /> In addition to this section, the authors should review all paragraphs in the Methods section to improve readability.

(2) Although the manuscript has a relatively long Methods section, some essential information is missing. For instance, the definition of sleep bout, as described above, is unclear. Additional missing information includes:

Figure 2: "Rhythmic strength (%)" and "Cycle-to-cycle variability (min)."<br /> Figure 3: "Activity suppression."<br /> Figure 4: "Rhythmic power (V%)" (is this different from rhythmic strength (%)?) and "Subjective day activity (%)."<br /> Figure 5: Clear labeling of the SCN's anatomical features and an explanation for quantifying only the ventral part instead of the entire SCN. Alternatively, the authors should consider quantifying the whole SCN.<br /> Figure 6: Inconsistencies in terms like "Sleep frag. (bout #)" and "Sleep bouts (#)." Consistent terminology throughout the manuscript is essential.

(3) Figure 1A shows higher mouse activity during ZT13-16. It is unclear why the authors scheduled feeding during ZT15-21, as this seems to disturb the rhythm. Consistent with this, the body weights of WT and Fmr1 KO mice decreased after scheduled feeding. The authors should explain the rationale for this design clearly.

(4) The interpretation of social behavior results in Figure 6 is questionable. The authors claim that Fmr1 KO mice cannot remember the first stranger in a three-chamber test, writing, "The reduced time in exploring and staying in the novel-mouse chamber suggested that the Fmr1 KO mutants were not able to distinguish the second novel mouse from the first now-familiar mouse." However, an alternative explanation is that Fmr1 KO mice do remember the first stranger but prefer to interact with it due to autistic-like tendencies. Data in Table 5 show that Fmr1 KO mice spent more time interacting with the first stranger in the 3-chamber social recognition test, which support this possibility. Similarly, in the five-trial social test, Fmr1 KO mice's preference for familiar mice might explain the reduced interaction with the second stranger.

In Figure 6C (five-trial social test results), only the fifth trial results are shown. Data for trials 1-4 should be provided and compared with the fifth trial. The behavioral features of mice in the 5-trial test can then be shown completely. In addition, the total interaction times for trials 1-4 (154 {plus minus} 15.3 for WT and 150 {plus minus} 20.9 for Fmr1 KO) suggest normal sociability in Fmr1 KO mice (it is different from the results of 3-chamber). Thus, individual data for trials 1-4 are required to draw reliable conclusions.

In Table 6 and Figure 6G-6J, the authors claim that "Sleep duration (Figures 6G, H) and fragmentation (Figures 6I, J) exhibited a moderate-strong correlation with both social recognition and grooming." However, Figure 6I shows a p-value of 0.077, which is not significant. Moreover, Table 6 shows no significant correlation between SNPI of the three-chamber social test and any sleep parameters. These data do not support the authors' conclusions.

(5) Figure 7 demonstrates the effect of scheduled feeding on circadian activity and sleep behaviors, representing another critical set of results in the manuscript. Notably, the WT+ALF and Fmr1 KO+ALF groups in Figure 7 underwent the same handling as the WT and Fmr1 KO groups in Figures 1 and 2, as no special treatments were applied to these mice. However, the daily patterns observed in Figures 7A, 7B, 7F, and 7G differ substantially from those shown in Figures 2B and 1A, respectively. Additionally, it is unclear why the WT+ALF and Fmr1 KO+ALF groups did not exhibit differences in Figures 7I and 7J, especially considering that Fmr1 KO mice displayed more sleep bouts but shorter bout lengths in Figures 1C and 1D.

Furthermore, it is not specified whether the results in Figure 7 were collected after two weeks of scheduled feeding (for how many days?) or if they represent the average data from the two-week treatment period.

The rationale behind analyzing "ZT 0-3 activity" in Figure 7D instead of the parameters shown in Figures 2C and 2D is also unclear.

In Figure 7F, some data points appear to be incorrectly plotted. For instance, the dark blue circle at ZT13 connects to the light blue circle at ZT14 and the dark blue circle at ZT17. This is inconsistent, as the dark blue circle at ZT13 should link to the dark blue circle at ZT14. Similarly, it is perplexing that the dark blue circle at ZT16 connects to both the light blue and dark blue circles at ZT17. Such errors undermine confidence in the data. The authors need to provide a clear explanation of how these data were processed.

Lastly, in the Figure 7 legend, Table 6 is cited; however, this appears to be incorrect. It seems the authors intended to refer to Table 7.

(6) Similar to the issue in Figure 7F, the data for day 12 in Supplemental Figure 2 includes two yellow triangles but lacks a green triangle. It is unclear how the authors constructed this chart, and clarification is needed.

(7) In Figure 8, a 5-trial test was used to assess the effect of scheduled feeding on social behaviors. It is essential to present the results for all trials (1 to 4). Additionally, it is unclear whether the results for familial mice in Figure 8A correspond to trials 1, 2, 3, or 4.<br /> The legend for Figure 8 also appears to be incorrect: "The left panels show the time spent in social interactions when the second novel stranger mouse was introduced to the testing mouse in the 5-trial social interaction test. The significant differences were analyzed by two-way ANOVA followed by Holm-Sidak's multiple comparisons test with feeding treatment and genotype as factors." This description does not align with the content of the left panels. Moreover, two-way ANOVA is not the appropriate statistical analysis for Figure 8A. The authors need to provide accurate details about the analysis and revise the figure legend accordingly.

(8) The circadian activity and sleep behaviors of Fmr1 KO mice have been reported previously, with some findings consistent with the current manuscript, while others contradict it. Although the authors acknowledge this discrepancy, it seems insufficiently thorough to simply state that the reasons for the conflicts are unknown. Did the studies use the same equipment for behavior recording? Were the same parameters used to define locomotor activity and sleep behaviors? The authors are encouraged to investigate these details further, as doing so may uncover something interesting or significant.

(9) Some subtitles in the Results section and the figure legends do not align well with the presented data. For example, in the section titled "Reduced rhythmic strength and nocturnality in the Fmr1 KOs," it is unclear how the authors justify the claim of altered nocturnality in Fmr1 KO mice. How do the authors define changes in nocturnality? Additionally, the tense used in the subtitles and figure legends is incorrect. The authors are encouraged to carefully review all subtitles and figure legends to correct these errors and enhance readability.

Reviewer #2 (Public review):

Summary:

In the present study, the authors, using a mouse model of Fragile X syndrome, explore the very interesting hypothesis that restricting food access over a daily schedule will improve sleep patterns and, subsequently, behavioral capacities. By restricting food access from 12h to 6h over the nocturnal period (active period for mice), they show, in these KO mice, an improvement of the sleep pattern accompanied by reduced systemic levels of inflammatory markers and improved behavior. Using a classical mouse model of neurodevelopmental disorder (NDD), these data suggest that eating patterns might improve sleep quality, reduce inflammation and improve cognitive/behavioral capacities in children with NDD.

Strengths:

Overall, the paper is very well-written and easy to follow. The rationale of the study is generally well-introduced. The data are globally sound. The provided data support the interpretation overall.

Weaknesses:

(1) The introduction part is quite long in the Abstract, leaving limited space for the data provided by the present study.

(2) A couple of points are not totally clear for a non-expert reader:<br /> - The Fmr1/Fxr2 double KO mice are not well described.<br /> - What is the rationale for performing both LD and DD measures?

(3) The data on cytokines and chemokines are interesting. However, the rationale for the selection of these molecules is not given. In addition, these measures have been performed in the systemic blood. Measures in the brain could be very informative.

(4) An important question is the potential impact of fasting vs the impact of the food availability restriction. Indeed fasting has several effects on brain functioning including cognitive functions.

(5) How do the authors envision the potential translation of the present study to human patients? How to translate the 12 to 6 hours of food access in mice to children with Fragile X syndrome?

Reviewer #1 (Public review):

Summary:

This manuscript presents a comprehensive structure-guided secretome analysis of gall-forming microbes, providing valuable insights into effector diversity and evolution. The authors have employed AlphaFold2 to predict the 3D structures of the secretome from selected pathogens and conducted a thorough comparative analysis to elucidate commonalities and unique features of effectors among these phytopathogens.

Strengths:

The discovery of conserved motifs such as 'CCG' and 'RAYH' and their central role in maintaining the overall fold is an insightful finding. Additionally, the discovery of a nucleoside hydrolase-like fold conserved among various gall-forming microbes is interesting.

Weaknesses:

Important conclusions are not verified by experiments.

Reviewer #2 (Public review):

Summary:

Soham Mukhopadhyay et al. investigated the protein folding of the secretome from gall-forming microbes using the AI-based structure modeling tool AlphaFold2. Their study analyzed six gall-forming species, including two Plasmodiophorid species and four others spanning different kingdoms, along with one non-gall-forming Plasmodiophorid species, Polymyxa betae. The authors found no effector fold specifically conserved among gall-forming pathogens, leading to the conclusion that their virulence strategies are likely achieved through diverse mechanisms. However, they identified an expansion of the Ankyrin repeat family in two gall-forming Plasmodiophorid species, with a less pronounced presence in the non-gall-forming Polymyxa betae. Additionally, the study revealed that known effectors such as CCG and AvrSen1 belong to sequence-unrelated but structurally similar (SUSS) effector clusters.

Strengths:

(1) The bioinformatics analyses presented in this study are robust, and the AlphaFold2-derived resources deposited in Zenodo provide valuable resources for researchers studying plant-microbe interactions. The manuscript is also logically organized and easy to follow.

(2) The inclusion of the non-gall-forming Polymyxa betae strengthens the conclusion that no effector fold is specifically conserved in gall-forming pathogens and highlights the specific expansion of the Ankyrin repeat family in gall-forming Plasmodiophorids.

(3) Figure 4a and 4b effectively illustrate the SUSS effector clusters, providing a clear visual representation of this finding.

(4) Figure 1 is a well-designed, comprehensive summary of the number and functional annotations of putative secretomes in gall-forming pathogens. Notably, it reveals that more than half of the analyzed effectors lack known protein domains in some pathogens, yet some were annotated based on their predicted structures, despite the absence of domain annotations.

Weaknesses:

(1) The effector families discussed in this paper remain hypothetical in terms of their functional roles, which is understandable given the challenges of demonstrating their functions experimentally. However, this highlights the need for experimental validation as a next step.

(2) Some analyses, such as those in Figure 4e, emphasize motifs derived from sequence alignments of SUSS effector clusters. Since these effectors are sequence-unrelated, sequence alignments might be unreliable. It would be more rigorous to perform structure-based alignments in addition to sequence-based ones for motif confirmation. For instance, methods described in Figure 3E of de Guillen et al. (2015, https://doi.org/10.1371/journal.ppat.1005228) or tools like Foldseek (https://search.foldseek.com/foldmason) could be useful for aligning structures of multiple sequences.

(3) When presenting AlphaFold-generated structures, it is essential to include confidence scores such as pLDDT and PAE. For example, in Figure 1D of Derbyshire and Raffaele (2023, https://doi.org/10.1038/s41467-023-40949-9), the structural representations were colored red due to their high pLDDT scores, emphasizing their reliability.

Reviewer #1 (Public review):

Summary:

The goal of this study was to overcome the apparent difficulty in constructing structural models of the open state of the CFTR chloride channel. While several CFTR structural models at near-atomic resolution have been published under a variety of conditions, none of them have demonstrated a pore open across the full dimension of the plasma membrane. Instead, these have routinely been referred to as "near-open" models. In the present study, the authors extended their findings from a prior paper from their group that investigated a series of brief MD simulations, a small number of which exhibited permeation events where chloride ions permeated the pore. This study included massively repeated simulations initiated from these aforementioned Cl permeable conformations. Extensive analysis of the data identified a novel penta-helical structure that comprises the channel pore. This comprehensive study attempted to explain several features of conducting CFTR channels, including single-channel conductance, selectivity, and the mechanisms linking the ATP-induced dimerization of the cytosolic nucleotide-binding domains (NBDs) to the opening of the channel pore (a.k.a., "pore-gating".

Strengths:

The major strength of this study is its comprehensive nature. The approaches applied are cutting-edge and beyond, and are used to explain many different aspects of channel function in CFTR. The strength of evidence is very strong. The paper is extremely well-written, and the arguments are well-supported.

Weaknesses:

The major weakness is that none of the novel conclusions (i.e., those arising solely from this study and not previously published (have been supported by experimental confirmation. That is typical of computational studies such as this.

Reviewer #2 (Public review):

Although recent cryo-EM structures of the CFTR ion channel were reported in a putative open state (ATP-bound, NBD-dimerized), it remains unclear whether these structures explain the conductive properties of the open channel observed in functional experiments. To investigate this, the authors conducted extensive molecular dynamics simulations at different voltages. The simulations are started from snapshots of their prior work, based on the experimental putative open state and including conditions with high negative voltage. Their analysis reveals that the cryo-EM structure represents a near-open metastable state, with most trajectories transitioning to either more closed or more open conformations, leading to the identification of a potential new open state. Permeation rate analysis shows that, unlike the other states, the proposed open state exhibits functional conductive properties of the open channel, although a strong inward rectification, inconsistent with experimental data, is also noted. Further structural analysis and simulations of ATP-unbound closed states offer additional mechanistic insights.

Overall, this work tackles key questions about CFTR: What is the true open conductive state? Does the ATP-bound cryo-EM structure reflect an actual open state? What is the ion permeation mechanism, and what structural changes occur during the closed-to-open transition? Which residues are critical, particularly those linked to diseases like CF? The study, based on a comprehensive set of all-atom molecular dynamics simulations, including a range of physiologically relevant voltages, provides important insights in this regard. It identifies key structural states, permeation pathways, critical residues, and conductance properties that can be directly compared to functional data. Notably, the analysis identifies a new open state of the channel, which, systematic analysis convincingly demonstrates is a conductive conformation of the channel, in line with experimental data at negative voltages. The authors carefully address some of the limitations of their results, exploring and discussing discrepancies with functional experiments, such as inward rectification. The work is also very well written, with a clear and logical presentation of key findings.

The main weakness of this study is that the simulation data rely on the conventional CHARMM36 force field for Cl− ions, which has been shown to significantly underestimate the interaction between Cl− and proteins (J. Chem. Theory Comput. 2021, 17, 6240-6261). For example, the conventional CHARMM36 force field destabilizes the Cl-binding site in CLC-ec1. The latter ion unbinds irreversibly during microseconds-long simulations which is at odds with the experimental binding affinity.

This imbalance in Cl−/protein/water interactions could significantly impact the CFTR simulations, potentially altering state populations and Cl− permeability. Notably, recent work by Levring and Chen (Proc Natl Acad Sci U S A. 2024) identifies a likely Cl− binding site in the bottleneck region of the channel, which contradicts the simulation results showing low occupancy Cl− ions in this region (Fig. 1B and Fig. 6A). This discrepancy may be due to the underestimation of Cl−/protein interactions. Indeed, Orabi et al. have proposed corrections that specifically tune these interactions, including those with aromatic residues, in line with the binding site geometry suggested by Levring and Chen. This imbalance in interactions may also lead to an underestimation of the conductance in the experimental near-open state.<br /> Balanced Cl−/protein interactions could also influence voltage/current relationships, potentially affecting the degree of inward rectification. For example, higher Cl− occupancy in the bottleneck region may stabilize the down state of R334, along with other measured interactions, thereby increasing conductance as the authors have shown.

The experimental evidence reported and discussed by the authors in support of the proposed open state is largely qualitative. For instance, in Figure 4 Supplement 2 there is a significant overlap in the distances and SASA distributions of open and near-open states for the reported residues (are those residues water accessible in the simulations?).

Given the known limitations of the standard CHARMM36 Cl− force field and in the absence of robust experimental validation of the proposed open state, I recommend validating at least part of the results using an independent set of simulations (not started from the previous ones) with an updated Cl− force field. It would be especially important to reassess whether the experimental near-open state is truly metastable and less probable than the new open state, and confirm that the near-open state exhibits negligible conductance.

A minor point worth discussing is whether the observed inward rectification may be influenced by hysteresis or incomplete equilibration, as many simulations were started from prior trajectories at large negative voltages and may not have fully relaxed. For instance, is not uncommon that small structural changes in backbone and sidechains occur in several microseconds (Shaw et al., Science, 2010). That said, discrepancies in current-voltage relationships are not unexpected due to challenges in simulation sampling and force field accuracy (J Gen Physiol 2013 May;141(5):619-32) as the authors stated.

Another minor point to address is the preparation of the simulation setup for the ATP-free structure of the protein. It would be helpful to specify whether any particular controls or steps were taken, given that the structure is based on a relatively low resolution (3.87 Å) model.

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 #4 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

This paper investigated the dynamic self-assembly of branched actin networks and the relation between the nonequilibrium features of the dynamics with the thermodynamic cost. The authors constructed a chain model to describe the self-assembly process of a branched actin network, including events like nucleation, polymerization, and capping. The forward and backward transition rates associated with the events allowed them to investigate the entropy production rate of the dynamics. They then used the fact that the entropy production rate has to be greater than zero to derive inequalities that set bounds for the maximum force produced by the branched actin network. The idea is similar to estimating the polymerization force of actin filament via the equation F_{max} = dG/delta, which sets a bound on the maximum force by the thermodynamic potential dG which is the chemical energy associated with ATP hydrolysis and delta is the length increment upon monomer insertion. Furthermore, they speculated the dissipative cost beyond what is necessary to move the load may be necessary to maintain an adaptive steady state.

Strengths:

The authors developed a simple model that is capable of qualitatively reproducing some mechanical phenomena for a branched actin network. The model has captured the essential dynamic elements in the branched actin network and built connections between the maximum load and the adaptation behavior with the energetic cost. It is an interesting study that provides a new perspective to look at the mechanical response of the branched actin network.

Weaknesses:

The text needs to be improved, particularly in the model introduction part. It is unclear to me what happens to the state when the reverse reaction in Figure 2 occurs.

Furthermore, what the authors have done is similar to estimate the polymerization force of actin filaments but in a more complicated scenario. Their conclusion that "dissipative cost in the system beyond what is necessary to move the load may be necessary to maintain an adaptive steady state" is skeptical. The branched actin network is a nonequilibrium system driven by active processes like ATP hydrolysis that converts chemical energy into mechanical work. There has to be a gap between the actual E-C_f curve and that when dissipation rate dot{S} = 0. If the authors want to make the claim, they have to decompose the dissipation into different parts and show that a particular part is associated with adaption. Otherwise, the conclusion about the gap is baseless.

Reviewer #2 (Public review):

Summary:

Rennert et al. developed a thermodynamic framework for the assembly of branched networks to calculate the entropy dissipation associated with this process. They base their model on the simplest possible experimental system consisting of four proteins: actin, Arp2/3, capping protein, and NPF. They decompose the network assembly into a linear model where the order of events (polymerization, capping, and nucleation) is recorded sequentially. Polymerization and capping are sensitive to load and affected by Brownian ratchet effects, while nucleation is not. This simplified model provides an analytical solution that describes the load sensitivity of actin networks and agrees well with experimental data for a given set of transition rates.

Strengths:

(1) These thermodynamic approaches are original and fundamental to our understanding of these non-equilibrium systems.

(2) The fact that the model fits experimental data is encouraging.

Weaknesses:

(1) The possibility of describing branched actin assembly as a Markov process is not well justified.

(2) The choice of parameters controlling the system is open to question. Some parameters are probably completely negligible, while other ignored effects are potentially significant.

(3) The main conclusion of the manuscript, linked to the existence of a dissipation gap, is quite expected. The manuscript would have been more valuable if the authors had been able to decompose dissipation into different components in order to prove that a particular fraction is associated with adaptation.

Reviewer #1 (Public review):

Summary:

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

Strengths:

Novel set of methods applied to the study of fossil melanosomes.

Weaknesses:

(1) Firstly, several studies have argued that these structures are in fact not a crest, but rather the result of compression. Otherwise, it would seem that a large number of Jehol birds have crests that extend not only along the head but the neck and hindlimb. It is more parsimonious to interpret this as compression as has been demonstrated using actuopaleontology (Foth 2011).<br /> (2) The primitive morphology of the feather with their long and possibly not interlocking barbs also questions the ability of such feathers to be erected without geologic compression.<br /> (3) The feather is not in situ and therefore there is no way to demonstrate unequivocally that it is indeed from the head (it could just as easily be a neck feather)<br /> (4) Melanosome density may be taphonomic; in fact, in an important paper that is notably not cited here (Pan et al. 2019) the authors note dense melanosome packing and attribute it to taphonomy. This paper describes densely packed (taphonomic) melanosomes in non-avian avialans, specifically stating, "Notably, we propose that the very dense arrangement of melanosomes in the fossil feathers (Fig. 2 B, C, and G-I, yellow arrows) does not reflect in-life distribution, but is, rather, a taphonomic response to postmortem or postburial compression" and if this paper was taken into account it seems the conclusions would have to change drastically. If in this case the density is not taphonomic, this needs to be justified explicitly (although clearly these Jehol and Yanliao fossils are heavily compressed).<br /> (5) Color in modern birds is affected by the outer keratin cortex thickness which is not preserved but the authors note the barbs are much thicker (10um) than extant birds; this surely would have affected color so how can the authors be sure about the color in this feather?<br /> (6) Authors describe very strange shapes that are not present in extant birds: "...different from all other known feather melanosomes from both extant and extinct taxa in having some extra hooks and an oblique ellipse shape in cross and longitudinal sections of individual melanosome" but again, how can it be determined that this is not the result of taphonomic distortion?<br /> (7) The authors describe the melanosomes as hexagonally packed but this does not appear to be in fact the case, rather appearing quasi-periodic at best, or random. If the authors could provide some figures to justify this hexagonal interpretation?<br /> (8) One way to address these concerns would be to sample some additional fossil feathers to see if this is unique or rather due to taphonomy<br /> (9) On a side, why are the feet absent in the CT scan image?

Reviewer #2 (Public review):

Summary:

The authors reconstructed the three-dimensional organization of melanosomes in fossilized feathers belonging to a spectacular specimen of a stem avialan from China. The authors then proceed to infer the original coloration and related ecological implications.

Strengths:

I believe the study is well executed and well explained. The methods are appropriate to support the main conclusions. I particularly appreciate how the authors went beyond the simple morphological inference and interrogated the structural implications of melanosome organization in three dimensions. I also appreciate how the authors were upfront with the reliability of their methods, results, and limitations of their study. I believe this will be a landmark study for the inference of coloration in extinct species and how to interrogate its significance in the future.

Weaknesses:

I have a few minor comments.<br /> Introduction: I would suggest the authors move the paragraph on coloration in modern birds (lines 75-97) before line 64, as this is part of the reasoning behind the study. I believe this change would improve the flow of the introduction for the general reader.<br /> Melanosome organization: I was surprised to find little information in the main text regarding this topic. As this is one of the major findings of the study, I would suggest the authors include more information regarding the general geometry/morphology of the single melanosomes and their arrangement in three dimensions.<br /> Keratin: the authors use such a term pretty often in the text, but how is this inference justified in the fossil? Can the authors extend on this? Previous studies suggested the presence of degradation products deriving from keratin, rather than immaculated keratin per se.<br /> Ontogenetic assessment: the authors infer a sub-adult stage for the specimen, but no evidence or discussion is reported in the SI. Can the authors describe and discuss their interpretations?<br /> CT scan data: these data should be made freely available upon publication of the study.

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 #1 (Public review):

Summary:

Nahas et al. investigated the roles of herpes simplex virus 1 (HSV-1) structural proteins using correlative cryo-light microscopy and soft X-ray tomography. The authors generated nine viral variants with deletions or mutations in genes encoding structural proteins. They employed a chemical fixation-free approach to study native-like events during viral assembly, enabling observation of a wider field of view compared to cryo-ET. The study effectively combined virology, cell biology, and structural biology to investigate the roles of viral proteins in virus assembly and budding.

Strengths:

(1) The study presented a novel approach to studying viral assembly in cellulo.

(2) The authors generated nine mutant viruses to investigate the roles of essential proteins in nuclear egress and cytoplasmic envelopment.

(3) The use of correlative imaging with cryoSIM and cryoSXT allowed for the study of viral assembly in a near-native state and in 3D.

(4) The study identified the roles of VP16, pUL16, pUL21, pUL34, and pUS3 in nuclear egress.

(5) The authors demonstrated that deletion of VP16, pUL11, gE, pUL51, or gK inhibits cytoplasmic envelopment.

(6) The manuscript is well-written, clearly describing findings, methods, and experimental design.

(7) The figures and data presentation are of good quality.

(8) The study effectively correlated light microscopy and X-ray tomography to follow virus assembly, providing a valuable approach for studying other viruses and cellular events.

(9) The research is a valuable starting point for investigating viral assembly using more sophisticated methods like cryo-ET with FIB-milling.

(10) The study proposes a detailed assembly mechanism and tracks the contributions of studied proteins to the assembly process.

(11) The study includes all necessary controls and tests for the influence of fluorescent proteins.

Weaknesses:

Overall, the manuscript does not have any major weaknesses, just a few minor comments:

(1) The gel quality in Figure 1 is inconsistent for different samples, with some bands not well resolved (e.g., for pUL11, GAPDH, or pUL20).

(2) The manuscript would benefit from a summary figure or table to concisely present the findings for each protein. It is a large body of manuscript, and a summary figure showing the discovered function would be great.

(3) Figure 2 lacks clarity on the type of error bars used (range, standard error, or standard deviation). It says, however, range, and just checking if this is what the authors meant.

(4) The manuscript could be improved by including details on how the plasma membrane boundary was estimated from the saturated gM-mCherry signal. An additional supplementary figure with the data showing the saturation used for the boundary definition would be helpful.

(5) Additional information or supplementary figures on the mask used to filter the YFP signal for Figure 4 would be helpful.

(6) The figure legends could include information about which samples are used for comparison for significance calculations. As the color of the brackets is different from the compared values (dUL34), it would be great to have this information in the figure legend.

(7) In Figure 5B, the association between YFP and mCherry signals is difficult to assess due to the abundance of mCherry signal; single-channel and combined images might improve visualization.

(8) In Figure 6D, staining for tubulin could help identify the cytoskeleton structures involved in the observed virus arrays.

(9) It is unclear in Figure 6D if the microtubule-associated capsids are with the gM envelope or not, as the signal from mCherry is quite weak. It could be made clearer with the split signals to assess the presence of both viral components.

(10) The representation of voxel intensity in Figure 8 is somewhat confusing. Reversion of the voxel intensity representation to align brighter values with higher absorption, which would simplify interpretation.

(11) The visualization in panel I of Figure 8 might benefit from a more divergent colormap to better show the variation in X-ray absorbance.

(12) Figure 9 would be enhanced by images showing the different virus sizes measured for the comparative study, which would help assess the size differences between different assembly stages.

Overall, this is an excellent manuscript and an enjoyable read. It would be interesting to see this approach applied to the study of other viruses, providing valuable insights before progressing to high-resolution methods.

Reviewer #2 (Public review):

Summary:

For centuries, humans have been developing methods to see ever smaller objects, such as cells and their contents. This has included studies of viruses and their interactions with host cells during processes extending from virion structure to the complex interactions between viruses and their host cells: virion entry, virus replication and virion assembly, and release of newly constructed virions. Recent developments have enabled simultaneous application of fluorescence-based detection and intracellular localization of molecules of interest in the context of sub-micron resolution imaging of cellular structures by electron microscopy.

The submission by Nahas et al., extends the state-of-the-art for visualization of important aspects of herpesvirus (HSV-1 in this instance) virion morphogenesis, a complex process that involves virus genome replication, and capsid assembly and filling in the nucleus, transport of the nascent nucleocapsid and some associated tegument proteins through the inner and outer nuclear membranes to the cytoplasm, orderly association of several thousand mostly viral proteins with the capsid to form the virion's tegument, envelopment of the tegumented capsid at a virus-tweaked secretory vesicle or at the plasma membrane, and release of mature virions at the plasma membrane.

In this groundbreaking study, cells infected with HSV-1 mutants that express fluorescently tagged versions of capsid (eYFP-VP26) and tegument (gM-mCherry) proteins were visualized with 3D correlative structured illumination microscopy and X-ray tomography. The maturation and egress pathways thus illuminated were studied further in infections with fluorescently tagged viruses lacking one of nine viral proteins.

Strengths:

This outstanding paper meets the journal's definitions of Landmark, Fundamental, Important, Valuable, and Useful. The work is also Exceptional, Compelling, Convincing, and Solid. The work is a tour de force of classical and state-of-the-art molecular and cellular virology. Beautiful images accompanied by appropriate statistical analyses and excellent figures. The numerous complex issues addressed are explained in a clear and coordinated manner; the sum of what was learned is greater than the sum of the parts. Impacts go well beyond cytomegalovirus and the rest of the herpesviruses, to other viruses and cell biology in general.

Weaknesses:

I have a few suggestions for minor adjustments in the text.

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 #2 (Public review):

Summary:

The authors identified ORMDL3 as a negative regulator of the RLR pathway and anti-tumor immunity. Mechanistically, ORMDL3 interacts with MAVS and further promotes RIG-I for proteasome degradation. In addition, the deubiquitinating enzyme USP10 stabilizes RIG-I and ORMDL3 disturbs this process. Moreover, in subcutaneous syngeneic tumor models in C57BL/6 mice, they showed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8-positive T cells and IFN production in the tumor microenvironment (TME).

Strengths:

The paper has a clearly arranged structure and the English is easy to understand. It is well written. The results clearly support the conclusion.

Comments on revisions:

All questions have been answered.

Reviewer #1 (Public review):

The study investigates light chains (LCs) using three distinct approaches, with a focus on identifying a conformational fingerprint to differentiate amyloidogenic light chains from multiple myeloma light chains. The study's major contribution is the identification of a low-populated "H state," which the authors propose as a unique marker for AL-LCs. While this finding is promising, the review highlights several strengths and weaknesses. Strengths include the valuable contribution of identifying the H state and the use of multiple approaches, which provide a comprehensive understanding of LC structural dynamics. Weaknesses include a lack of physical insights explaining the changes.

Reviewer #2 (Public review):

Summary:

This well-written manuscript addresses an important but recalcitrant problem - molecular mechanism of protein misfolding in Ig light chain (LC) amyloidosis (AL), a major life-threatening form of systemic human amyloidosis. The authors use expertly recorded and analyzed small-angle X-ray scattering (SAXS) data as a restraint for molecular dynamics simulations (called M&M). Six patient-based LC proteins are explored, including four AL and two non-AL. The authors report a partially populated "H-state" determined computationally, wherein the two domains in an LC molecule acquire a straight rather than bent conformation, with an extended interdomain linker; this H-state distinguishes AL from non-AL LCs. H-D exchange mass spectrometry is used to support this conclusion. This is a novel and interesting finding with potentially important translational implications.

Strengths:

Expertly recorded and analyzed SAXS data combined with clever M&M simulations lead to a novel and interesting conclusion, which is supported by limited H-D exchange data.<br /> Stabilization of the CL-CL interface is a good idea that may help protect a subset of AL LCs from misfolding in amyloid.

Computational M&M evidence is convincing and is supported by SAXS data, which are used as restraints for simulations. Although Kratky plots reported in the main MS Fig. 1 show significant differences between the data and the structural model for only one AL protein, AL-55, H-state is also inferred for other AL proteins.

Apparent limitations:

HDX MS results show that residues 35-50 from VL-VL and VL-CL dimerization interface are less protected in AL vs. non-AL proteins, which is consistent with the H-state. However, the small number of proteins yielding useful HDX data (three AL and one non-AL) suggests that this conclusion should be treated with caution. It is unclear whether the conformational heterogeneity depicted in M&M simulations is consistent with HDX results, and whether prior HDX studies of AL and MM LCs are consistent with the conclusions that a particular domain-domain interface is weakened in AL vs. non-AL LCs. The butterfly plots in Fig. 5 could benefit from the X-axis labeling with the peptide fragments.

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 #2 (Public review):

Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.

Strengths:

The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.

Comments on revisions:

The authors did an excellent job addressing all or most of the concerns I raised in my previous review and have incorporated the necessary changes into the manuscript.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors reveal that GIF/MT-3 regulates the zinc homeostasis depending on the cellular redox status. The manuscript technically sounds, and their data concretely suggest that the recombinant MTs, not only GIF/MT-3 but also canonical MTs such as MT-1 and MT-2, contain sulfane sulfur atoms for the Zn-binding. The scenario proposed by the authors seems to be reasonable to explain the Zn homeostasis by the cellular redox balance.

Strengths:

The data presented in the manuscript solidly reveal that recombinant GIF/MT-3 contains sulfane sulfur.

Weaknesses:

It remains unclear whether native MTs, in particular induced MTs in vivo contain sulfane sulfur or not.

Comments on revisions:

Although the authors have revealed the sulfane sulfur content in native MT-3, my question, namely, whether canonical MT-1 and MT-2 contained sulfane sulfur after the induction has been left.<br /> The authors argue that the biological significance of sulfane sulfur in MTs lies in its ability to contribute to metal binding affinity, provide a sensing mechanism against oxidative stress, and aid in the regulation of the protein. Due to their biological roles, induced MT-1 and MT-2 could contain sulfane sulfur in their molecules. Thus, I expect the authors to evaluate or explain the sulfane sulfur content in induced MT-1 and MT-2.

Reviewer #3 (Public review):

Summary:

The authors were trying to show that a novel neuronal metallothionein of poorly defined function, GIF/MT3, is actually heavily persulfidated in both the Zn-bound and apo (metal-free) forms of the molecule as purified from a heterologous (bacterial) or native host. Evidence in support of this conclusion is strong, with both spectroscopic and mss spectrometry evidence strongly consistent with this general conclusion. The authors would appear to have achieved their aims.

Strengths:

The analytical data in support of the author's primary conclusions are strong. The authors also provide some modeling evidence that supports the contention that MT3 (and other MTs) can readily accommodate a sulfane sulfur on each of the 20 cysteines in the Zn-bound structure, with little perturbation of the overall structure. This is not the case with Cys trisulfides, which suggests that the persulfide-metallated state is clearly positioned at lower energy relative to the immediately adjacent thiolate- or trisulfidated metal coordination complexes.

Weaknesses:

The biological significance of the findings is not entirely clear. On the one hand, the analytical data are solid (albeit using a protein derived from a bacterial over-expression experiment), and yes, it's true that sulfane S can protect Cys from overoxidation, but everything shown in the summary figure (Fig. 9D) can be done with Zn release from a thiol by ROS, and subsequent reduction by the Trx/TR system. In addition, it's long been known that Zn itself can protect Cys from oxidation. I view this as a minor shortcoming that will motivate follow-up studies.

Impact:

The impact will be high since the finding is potentially disruptive to the MT field for sure. The sulfane sulfur counting experiment (the HPE-IAM electrophile trapping experiment) may well be widely adopted by the field. Those in the metals field always knew that this was a possibility, and it will interesting to see the extent to which metal binding thiolates broadly incorporate sulfane sulfur into their first coordination shells.

Comments on revisions:

The revised manuscript is only slightly changed from the original, with the inclusion of a supplementary figure (Fig. S2) and minor changes in the text. The authors did not choose to carry out the quantitative Zn binding experiment (which I really wanted to see), but given the complexities of the experiment, I'll let it go.

Fig. 9: the authors imply in the mechanistic "redox-switch" figure that Trx/TR can not reduce persulfide linkages. A number of groups have shown this to be the case. I recommend modifying the figure legend or text to make this clear to the reader,

Reviewer #1 (Public review):

Summary:

This manuscript uses the eye lens as a model to investigate basic mechanisms in the Fgf signaling pathway. Understanding Fgf signaling is of broad importance to biologists as it is involved in the regulation of various developmental processes in different tissues/organs and is often misregulated in disease states. The Fgf pathway has been studied in embryonic lens development, namely with regards to its involvement in controlling events such as tissue invagination, vesicle formation, epithelium proliferation and cellular differentiation, thus making the lens a good system to uncover the mechanistic basis of how the modulation of this pathway drives specific outcomes. Previous work has suggested that proteins, other than the ones currently known (e.g., the adaptor protein Frs2), are likely involved in Fgfr signaling. The present study focuses on the role of Shp2 and Shc1 proteins in the recruitment of Grb2 in the events downstream of Fgfr activation.

Strengths:

The findings reveal that the juxtamembrane region of the Fgf receptor is necessary for proper control of downstream events such as facilitating key changes in transcription and cytoskeleton during tissue morphogenesis. The authors conditionally deleted all four Fgfrs in the mouse lens that resulted in molecular and morphological lens defects, most importantly, preventing the upregulation of the lens induction markers Sox2 and Foxe3 and the apical localization of F-actin, thus demonstrating the importance of Fgfrs in early lens development, i.e. during lens induction. They also examined the impact of deleting Fgfr1 and 2, on the following stage, i.e. lens vesicle development, which could be rescued by expressing constitutively active KrasG12D. By using specific mutations (e.g. Fgfr1ΔFrs lacking the Frs2 binding domain and Fgfr2LR harboring mutations that prevent binding of Frs2), it is demonstrated that the Frs2 binding site on Fgfr is necessary for specific events such as morphogenesis of lens vesicle. Further, by studying Shp2 mutations and deletions, the authors present a case for Shp2 protein to function in a context-specific manner in the role of an adaptor protein and a phosphatase enzyme. Finally, the key surprising finding from this study is that downstream of Fgfr signaling, Shc1 is an important alternative pathway - in addition to Shp2 - involved in the recruitment of Grb2 and in the subsequent activation of Ras. The methodologies, namely, mouse genetics and state-of-the-art cell/molecular/biochemical assays are appropriately used to collect the data, which are soundly interpreted to reach these important conclusions. Overall, these findings reveal the flexibility of the Fgf signaling pathway and it downstream mediators in regulating cellular events. This work is expected to be of broad interest to molecular and developmental biologists.

Weaknesses:

A weakness that needs to be discussed is that Le-Cre depends on Pax6 activation, and hence its use in specific gene deletion will not allow evaluation of the requirement of Fgfrs in the expression of Pax6 itself. But since this is the earliest Cre available for deletion in the lens, mentioning this in the discussion would make the readers aware of this issue.

Reviewer #2 (Public review):

Summary

I have reviewed the revised manuscript submitted by Wang et al., which is entitled "Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development". In this paper, the authors first examined lens phenotypes in mice with Le-Cre-mediated knockdown (KD) of all four FGFR (FGFR1-4), and found that pERK signals, Jag1 and foxe3 expression are absent or drastically reduced, indicating that FGF signaling is essential for lens induction. Next, the authors examined lens phenotypes of FGFR1/2-KD mice and found that lens fiber differentiation is compromised and that proliferative activity and cell survival are also compromised in lens epithelium. Interestingly, Kras activation rescues defects in lens growth and lens fiber differentiation in FGFR1/2-KD mice, indicating that Ras activation is a key step for lens development, downstream of FGF signaling. Next, the authors examined the role of Frs2, Shp2 and Grb2 in FGF signaling for lens development. They confirmed that lens fiber differentiation is compromised in FGFR1/3-KD mice combined with Frs2-dysfunctional FGFR2 mutants, which is similar to lens phenotypes of Grb2-KD mice. However, lens defects are milder in mice with Shp2YF/YF and Shp2CS mutant alleles, indicating that involvement of Shp2 is limited for the Grb2 recruitment for lens fiber differentiation. Lastly, the authors showed new evidence on the possibility that another adapter protein, Shc1, promotes Grb2 recruitment independent of Frs2/Shp2-mediated Grb2 recruitment.

Strength

Overall, the manuscript provides valuable data on how FGFR activation leads to Ras activation through the adapter platform of Frs2/Shp2/Grb2, which advances our understanding on complex modification of FGF signaling pathway. The authors applied a genetic approach using mice, whose methods and results are valid to support the conclusion. The discussion also well summarizes the significance of their findings.

Weakness

The authors found that the new adaptor protein Shc1 is involved in Grb2 recruitments in response to FGF receptor activation. However, the main data on Shc1 are only histological sections and statistical evaluation of lens size. In the revised manuscript, the authors did not answer my major concern that cellular-level data are missing, which is not fully enough to support their main conclusion on the involvement of Shc1 in Grb2 recruitment of FGF signaling for lens development. Since the title of this manuscript is that Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development, it is important to provide the cellular-level evidence on Shc1.

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 #1 (Public review):

Summary:

In this study, the authors investigate the effect of mitochondrial transplantation on post-cardiac arrest myocardial dysfunction (PAMD), which is associated with mitochondrial dysfunction. The authors demonstrate that mitochondrial transplantation enhances cardiac function and increases survival rates after the return of spontaneous circulation (ROSC). Mechanistically, they found that myocardial tissues with transplanted mitochondria exhibit increased mitochondrial complex activity, higher ATP levels, reduced cardiomyocyte apoptosis, and lower myocardial oxidative stress post-ROSC.

Strengths:

Previous studies have reported that mitochondrial transplantation can improve myocardial recovery after regional ischemia, but its potential for treating myocardial injury following cardiac arrest has not been tested yet. Therefore, the findings are somewhat novel. Remarkably, the increased survival in mitochondria treated group post ROSC is very promising and highlights its translational potential.

Comments on revisions:

My concerns are adequately addressed.

Reviewer #3 (Public review):

In this manuscript titled "Transplantation of exogenous mitochondria mitigates myocardial dysfunction after cardiac arrest", Zhen Wang et al. report that exogenous mitochondrial transplantation can enhance myocardial function and survival rates. It limits mitochondrial morphology impairment, boosts complexes II and IV activity, and increases ATP levels. Additionally, mitochondrial therapy reduces oxidative stress, lessens myocardial injury, and improves PAMD after cardiopulmonary resuscitation. The results of this manuscript clearly demonstrate that mitochondrial transplantation can effectively improve PAMD after cardiopulmonary resuscitation, highlighting its significant scientific and clinical value. The findings shown in this manuscript are interesting to the readers. However, further experiments are needed to confirm this conclusion. In addition, the results should be rewritten to describe and discuss the relevant data in detail.

Major comments from the original round of review:

(1) Can isolated mitochondria be transported to cultured cardiomyocytes, such as H9C2 cells, in vitro?

(2) The description of results in the manuscript is too simple. It lacks detail on the rationale behind the experiments and the significance of the data.

(3) The authors demonstrate that mitochondrial transplantation reduces cardiomyocyte apoptosis. Therefore, Western blot analysis of apoptosis-related caspases could be provided for further confirmation.

(4) Do donor mitochondria fuse with recipient mitochondria? Relevant experiments and data should be provided to address this question.

(5) In Figure 5A, the histograms are not labeled with the specific experimental groups.

Comments on revisions:

The revised manuscript quality has been improved, and most of my concerns were addressed and resolved.

Reviewer #1 (Public review):

Summary:

This manuscript investigates a mechanism between the histone reader protein YEATS2 and the metabolic enzyme GCDH, particularly in regulating epithelial-to-mesenchymal transition (EMT) in head and neck cancer (HNC).

Strengths:

Great detailing of the mechanistic aspect of the above axis is the primary strength of the manuscript.

Weaknesses:

Several critical points require clarification, including the rationale behind EMT marker selection, the inclusion of metastasis data, the role of key metabolic enzymes like ECHS1, and the molecular mechanisms governing p300 and YEATS2 interactions.

Major Comments:

(1) The title, "Interplay of YEATS2 and GCDH mediates histone crotonylation and drives EMT in head and neck cancer," appears somewhat misleading, as it implies that YEATS2 directly drives histone crotonylation. However, YEATS2 functions as a reader of histone crotonylation rather than a writer or mediator of this modification. It cannot itself mediate the addition of crotonyl groups onto histones. Instead, the enzyme GCDH is the one responsible for generating crotonyl-CoA, which enables histone crotonylation. Therefore, while YEATS2 plays a role in recognizing crotonylation marks and may regulate gene expression through this mechanism, it does not directly catalyse or promote the crotonylation process.

(2) The study suggests a link between YEATS2 and metastasis due to its role in EMT, but the lack of clinical or pre-clinical evidence of metastasis is concerning. Only primary tumor (PT) data is shown, but if the hypothesis is that YEATS2 promotes metastasis via EMT, then evidence from metastatic samples or in vivo models should be included to solidify this claim.

(3) There seems to be some discrepancy in the invasion data with BICR10 control cells (Figure 2C). BICR10 control cells with mock plasmids, specifically shControl and pEGFP-C3 show an unclear distinction between invasion capacities. Normally, we would expect the control cells to invade somewhat similarly, in terms of area covered, within the same time interval (24 hours here). But we clearly see more control cells invading when the invasion is done with KD and fewer control cells invading when the invasion is done with OE. Are these just plasmid-specific significant effects on normal cell invasion? This needs to be addressed.

(4) In Figure 3G, the Western blot shows an unclear band for YEATS2 in shSP1 cells with YEATS2 overexpression condition. The authors need to clearly identify which band corresponds to YEATS2 in this case.

(5) In ChIP assays with SP1, YEATS2 and p300 which promoter regions were selected for the respective genes? Please provide data for all the different promoter regions that must have been analysed, highlighting the region where enrichment/depletion was observed. Including data from negative control regions would improve the validity of the results.

(6) The authors establish a link between H3K27Cr marks and GCDH expression, and this is an already well-known pathway. A critical missing piece is the level of ECSH1 in patient samples. This will clearly delineate if the balance shifted towards crotonylation.

(7) The p300 ChIP data on the SPARC promoter is confusing. The authors report reduced p300 occupancy in YEATS2-silenced cells, on SPARC promoter. However, this is paradoxical, as p300 is a writer, a histone acetyltransferase (HAT). The absence of a reader (YEATS2) shouldn't affect the writer (p300) unless a complex relationship between p300 and YEATS2 is present. The role of p300 should be further clarified in this case. Additionally, transcriptional regulation of SPARC expression in YEATS2 silenced cells could be analysed via downstream events, like Pol-II recruitment. Assays such as Pol-II ChIP-qPCR could help explain this.

(8) The role of GCDH in producing crotonyl-CoA is already well-established in the literature. The authors' hypothesis that GCDH is essential for crotonyl-CoA production has been proven, and it's unclear why this is presented as a novel finding. It has been shown that YEATS2 KD leads to reduced H3K27cr, however, it remains unclear how the reader is affecting crotonylation levels. Are GCDH levels also reduced in the YEATS2 KD condition? Are YEATS2 levels regulating GCDH expression? One possible mechanism is YEATS2 occupancy on GCDH promoter and therefore reduced GCDH levels upon YEATS2 KD. This aspect is crucial to the study's proposed mechanism but is not addressed thoroughly.

(9) The authors should provide IHC analysis of YEATS2, SPARC alongside H3K27cr and GCDH staining in normal vs. tumor tissues from HNC patients.

Reviewer #2 (Public review):

Summary:

The manuscript emphasises the increased invasive potential of histone reader YEATS2 in an SP1-dependent manner. They report that YEATS2 maintains high H3K27cr levels at the promoter of EMT-promoting gene SPARC. These findings assigned a novel functional implication of histone acylation, crotonylation.

Concerns:

(1) The patient cohort is very small with just 10 patients. To establish a significant result the cohort size should be increased.

(2) Figure 4D compares H3K27Cr levels in tumor and normal tissue samples. Figure 1G shows overexpression of YEATS2 in a tumor as compared to normal samples. The loading control is missing in both. Loading control is essential to eliminate any disparity in protein concentration that is loaded.

(3) Figure 4D only mentions 5 patient samples checked for the increased levels of crotonylation and hence forms the basis of their hypothesis (increased crotonylation in a tumor as compared to normal). The sample size should be more and patient details should be mentioned.

(4) YEATS2 maintains H3K27Cr levels at the SPARC promoter. The p300 is reported to be hyper-activated (hyperautoacetylated) in oral cancer. Probably, the activated p300 causes hyper-crotonylation, and other protein factors cause the functional translation of this modification. The authors need to clarify this with a suitable experiment.

(5) I do not entirely agree with using GAPDH as a control in the western blot experiment since GAPDH has been reported to be overexpressed in oral cancer.

(6) The expression of EMT markers has been checked in shControl and shYEATS2 transfected cell lines (Figure 2A). However, their expression should first be checked directly in the patients' normal vs. tumor samples.

(7) In Figure 3G, knockdown of SP1 led to the reduced expression of YEATS2 controlled gene Twist1. Ectopic expression of YEATS2 was able to rescue Twist1 partially. In order to establish that SP1 directly regulates YEATS2, SP1 should also be re-introduced upon the knockdown background along with YEATS2 for complete rescue of Twist1 expression.

(8) In Figure 7G, the expression of EMT genes should also be checked upon rescue of SPARC expression.

Reviewer #1 (Public review):

Summary:

It is essential for Mycobacterium tuberculosis (Mtb) to scavenge trace metals from its host to survive. In this study, the authors explore the effects of copper limitation on Mtb. Mtb synthesizes small molecular diisonitrile lipopeptides termed chalkophores, that chelate host copper for import, whereby the copper is incorporated into Mtb metalloproteins. However, the role of chalkophores in Mtb biology and their targeted metalloproteins are unknown. This study investigates Mtb proteins that require chalkophores for copper incorporation and their effect on Mtb virulence. It is known that the nrp operon is induced by copper deprivation and encodes the synthesis of chalkophores. A genetic analysis revealed transcriptional differences for WT and Mtb∆nrp when exposed to the copper chelator tetrathiomolybdate (TTM). The authors found that copper chelation results in upregulation of genes in the chalkophore cluster as well as genes involved in the respiratory chain: specifically, components of the heme-dependent oxidase CytBD and subunits of the bcc:aa3 heme-copper oxidase. Interestingly, treatment of Mtb∆nrp with an inhibitor of the QcrB subunit of the bcc:aa3 oxidase (Q203) resulted in similar transcriptional changes. The bcc:aa3 oxidase and CytBD are functionally redundant, and while both utilize heme as a cofactor, only the first utilizes heme and copper. Utilizing Mtb∆nrp, Mtb∆cydAB and MtbΔnrpΔcydAB along with single gene complementation, the authors showed that copper starvation survival requires diisonitrile chalkophore synthesis and that copper starvation results in dysfunctional bcc:aa3 oxidase. Further genetic analysis combined with inhibitor studies indicate that bcc:aa3 oxidase is the only target impacted by copper starvation. By monitoring oxygen consumption for mutants in combination with inhibitors, the authors show that copper deprivation inhibits respiration through the bcc:aa3 oxidase. Similarly, they show that TTM or Q203 treatment inhibits ATP production in MtbΔnrpΔcydAB, but not in WT, showing that chalkophores maintain oxidative phosphorylation. Lastly, the authors compare the virulence of WT Mtb, Mtb∆nrp and MtbΔnrpΔcydAB strains in mice spleen and lung. The Mtb∆nrp strain showed mild attenuation, but virulence in MtbΔnrpΔcydAB was severely attenuated, and complementation with the chalkophore biosynthetic pathway restored Mtb virulence. These results suggest that chalkophore mediated protection of the respiratory chain is critical to Mtb virulence, and the that redundant respiratory oxidases within Mtb provides respiratory chain flexibility that may promote host adaptation.

Strengths:

Overall, the paper is very clear and well-written, with thorough and well-thought-out experimentation.

The methods are all quite standard, so there are no weaknesses identified with regard to methodology.

Reviewer #2 (Public review):

Summary:

This is a well-written manuscript that clearly demonstrates that the nrp encoded diisonitrile chalkophore is necessary for the function of the bcc-aa3 oxidase supercomplex under low copper conditions. In addition, the study demonstrates that the chlakophore is important early during infection when copper sequestration is employed by the host as a method of nutritional immunity.

Strengths:

The authors use genetic approaches including single and double mutants of chalkophore biosynthesis, and both the Mtb oxidases. They use copper chelators to restrict copper in vitro. A strength of the work was the use of a synthesized a Mtb chalkophore analogue to show chemical complementation of the mutant nrp locus. Oxphos metabolic activity was measuered by oxygen consumption and ATP levels. Importantly, the study demonstrated that chalkophore, especially in a strain lacking the secondary oxidase, was necessary for early infection and ruled out a role for adaptive immunity in the chalkophore lacking Mtb by use of SCID mice. It is interesting that after two weeks of infection and onset of adaptive immunity, the chalkophore is not required, which is consistent with the host environment switching from a copper-restricted to copper overload in phagosomes.

Weaknesses:

Most claims in the manuscript are soundly justified. The one exception is the claim that "maintenance of respiration is the only cellular target of chalkophore mediated copper acquisition." While under the in vitro conditions tested this does appear to be the case; however, it can't be ruled out that the chalkophore is important in other situations. In particular, for maintenance of the periplasmic superoxide dismustase, SodC, which is the other M. tuberculosis enzyme known to require copper.

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 #2 (Public review):

In this revised manuscript, Klemm et al., build on top of past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors investigate how tissues regenerate following necrotic cell death. They find that:

(1) the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),

(2) two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may partially explain the region specificity,

(3) the presence of NiA (and/or NiCP) cells promotes regenerative proliferation in the late stages of regeneration,

(4) not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells,

(5) the initiator caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage. In the revised manuscript, the authors provide improvements through additional data quantifications and text changes to better explain NiA/NiCP lineage tracing methods.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration. The paper raises many interesting questions for future investigation, including what is the nature of the signaling between the damaged tissue and the NiA/NiCP responsive areas (such as the identity of the DAMPs)? What determines if these cells at a distance undergo apoptosis or remain viable in the tissue as caspase-positive cells? And since the authors have data that indicates that the phenomenon is distinct from 'undead cells', what are the mechanisms by which these cells promote local proliferation?

Reviewer #3 (Public review):

The manuscript "Regeneration following tissue necrosis is mediated by non-apoptotic caspase activity" by Klemm et al. is an exploration of what happens to a group of cells that experience caspase activation after necrosis occurs some distance away from the cells of interest. These experiments have been conducted in the Drosophila wing imaginal disc, which has been used extensively to study the response of a developing epithelium to damage and stress. The authors revise and refine their earlier discovery of apoptosis initiated by necrosis, here showing that many of those presumed apoptotic cells do not complete apoptosis. Thus, the most interesting aspect of the paper is the characterization of a group of cells that experience mild caspase activation in response to an unknown signal, followed by some effector caspase activation and DNA damage, but that then recover from the DNA damage, avoid apoptosis, and proliferate instead.

The authors have addressed the concerns raised, including those about drawing conclusions from RNAi knockdown without evaluating the efficacy of the knockdown, and in doing so they revised their conclusions after ascertaining that the Zfh2 RNAi was not effective.

The authors have added quantification of the imaging data throughout, which strengthens their conclusions.

In addition, the authors have revised some of the text describing the changes in EdU signal and added explanations of reagents such as the caspase sensors to clarify the experimental approaches, results, and interpretation of those results.

The authors have also addressed the minor concerns and questions about the figures and text.

A few questions remain, which the authors may choose to address.

(1) The hh>Stat92ERNAi was assessed by the 10xSTAT-GFP reporter, as shown in Fig 2 Supp1 F. The authors point out the marked reduction in GFP in the ventral part of the hinge but do not comment on the lack of change in GFP in the dorsal part of the hinge. However, the open arrowhead in Figure 2H indicating the lack of cDcp-1 signal in the hinge in the same experiment points to the dorsal hinge, where the reporter suggests no difference in JAK-STAT signaling.

(2) The data used to conclude that DRONC-DN and UAS-DIAP1 do not affect regenerative proliferation were normalized EdU intensities. As discussed in the prior review round, normalized EdU may not be a good comparison across experimental conditions given that the remainder of the disc may also have altered EdU incorporation, so this measurement may not be enough by itself to draw conclusions about regenerative proliferation. To strengthen the conclusion that regenerative proliferation is unaffected under these conditions, the authors may want to consider using a second measure such as adult wing size, PCNA, or quantitate mitoses via anti-phospho histone H3 staining.

Reviewer #1 (Public review):

Summary:

This is a very creative study using modeling and measurement of neoblast dynamics to gain insight into the mechanism that allows these highly potent cells to undergo fate-switching as part of their differentiation and self-renewal process. The authors estimate growth equation parameters for expanding neoblast clones based on new and prior experimental observations. These results indicate neoblast likely undergo much more symmetric self-amplifying division than loss of the population through symmetric differentiation, in the case of clone expansion assays after sublethal irradiation. Neoblasts take on multiple distinct transcriptional fates related to their terminally differentiated cell types, and prior work indicated neoblasts have a high plasticity to switch fates in a way linked to cell cycle progression and possibly through a random process. Here, the authors explore the impact of inhibition of key transcription factors defining such states (ie "fate specifying transcription factors", FSTFs) plus measurement and modeling in the clone expansion assay, to find that inhibition of factors like zfp1 likely cause otherwise zfp1-fated neoblasts to fail to proliferate and differentiation without causing compensatory gains in other lineages. A mathematical model of this process assuming that neoblasts do not retain a memory of prior states while they proliferate, and transition across specified states can mimic the experimentally determined decreased sizes of clones following inhibition of zfp1. Complementary approaches to inhibit more than one lineage (muscle plus intestine) supports the idea that this is a more general process in planarian stem cells. These results provide an important advance for understanding the fate-switching process and its relationship to neoblast growth.

Overall I find the evidence very well presented and the study compelling. It offers an important new perspective on the key properties of neoblasts. I do have some comments to clarify the presentation and significance of the work.

Reviewer #2 (Public review):

Summary:

Cell cycle duration and cell fate choice are critical to understanding the cellular plasticity of neoblasts in planarians. In this study, Tamar et al. integrated experimental and computational approaches to simulate a model for neoblast behaviors during colony expansion.

Strengths:

The finding that "arresting differentiation into specific lineages disrupts neoblast proliferative capacities without inducing compensatory expression of other lineages" is particularly intriguing. This concept could inspire further studies on pluripotent stem cells and their application for regenerative biology.

Weaknesses:

However, the absence of a cell-cell feedback mechanism during colony growth and the likelihood of the difference needs to be clarified. Is there any difference in interpreting the results if this mechanism is considered? More explanation and discussion should be included to distinguish the stages controlled by the one-step model from those discussed in this study. Although hnf-4 and foxF have been silenced together to validate the model, a deeper understanding of the tgs-1+ cell type and the non-significant reduction of tgs-1+ neoblasts in zfp-1 RNAi colonies is necessary, considering a high neural lineage frequency.

Reviewer #1 (Public review):

Summary:

The authors aimed to elucidate the molecular mechanisms underlying HIV-1 persistence and host immune dysfunction in CD4+ T cells during early infection (<6 months). Using single-cell multi-omics technologies-including scRNA-seq, scATAC-seq, and single-cell multiome analyses-they characterized the transcriptional and epigenomic landscapes of HIV-1-infected CD4+ T cells. They identified key transcription factors (TFs), signaling pathways, and T cell subtypes involved in HIV-1 persistence, particularly highlighting KLF2 and Th17 cells as critical regulators of immune suppression. The study provides new insights into immune dysregulation during early HIV-1 infection and reveals potential epigenetic regulatory mechanisms in HIV-1-infected T cells.

Strengths:

The study excels through its innovative integration of single-cell multi-omics technologies, enabling detailed analysis of gene regulatory networks in HIV-1-infected cells. Focusing on early infection stages, it fills a crucial knowledge gap in understanding initial immune responses and viral reservoir establishment. The identification of KLF2 as a key transcription factor and Th17 cells as major viral reservoirs, supported by comprehensive bioinformatics analyses, provides robust evidence for the study's conclusions. These findings have immediate clinical relevance by identifying potential therapeutic targets for HIV-1 reservoir eradication.

Weaknesses:

Despite its strengths, the study has several limitations. By focusing exclusively on CD4+ T cells, the study overlooks other relevant immune cells such as CD14+ monocytes, NK cells, and B cells. Additionally, while the authors generated their own single-cell datasets, they need to validate their findings using other publicly available single-cell data from HIV-1-infected PBMCs.

Reviewer #2 (Public review):

Summary:

The authors observed gene ontologies associated with upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells using scRNA-seq and scATAC-seq datasets from the PBMCs of early HIV-1-infected patients, showing immune responses contributing to HIV pathogenesis and novel targets for viral elimination.

Strengths:<br /> The authors carried out detailed transcriptomics profiling with scRNA-seq and scATAC-seq datasets to conclude upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells.

Weaknesses:

This key observation of up-regulation KLF2 associated genes family might be important in the HIV field for early diagnosis and viral clearance. However, with the limited sample size and in-vivo study model, it will be hard to conclude. I highly recommend increasing the sample size of early HIV-1-infected patients.

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 #2 (Public review):

Summary:

In the article, the authors describe their software package in R for visualizing metabolite ratio pairs. I think the work would be of interest to the mass spectrometry community.

Strengths:

The authors describe a software that would be of use to those performing MALDI MSI. This software would certainly add to the understanding metabolomics data and enhance the identification of critical metabolites.

Weaknesses:

The figures are difficult to interpret/ analyze in their current state but are significantly better in the revision.

Joint Public Review:

This study comprehensively presents data from single nuclei sequencing of Heigai pig skeletal muscle in response to conjugated linoleic acid supplementation. The authors identify changes in myofiber type and adipocyte subpopulations induced by linoleic acid at depth previously unobserved. The authors show that linoleic acid supplementation decreased the total myofiber count, specifically reducing type II muscle fiber types (IIB), myotendinous junctions, and neuromuscular junctions, whereas type I muscle fibers are increased. Moreover, the authors identify changes in adipocyte pools, specifically in a population marked by SCD1/DGAT2. To validate the skeletal muscle remodeling in response to linoleic acid supplementation, the authors compare transcriptomics data from Laiwu pigs, a model of high intramuscular fat, to Heigai pigs. The results verify changes in adipocyte subpopulations when pigs have higher intramuscular fat, either genetically or diet-induced. Targeted examination using cell-cell communication network analysis revealed associations with high intramuscular fat with fibro-adipogenic progenitors (FAPs). The authors then conclude that conjugated linoleic acid induces FAPs towards adipogenic commitment. Specifically, they show that linoleic acid stimulates FAPs to become SCD1/DGAT2+ adipocytes via JNK signaling. The authors conclude that their findings demonstrate the effects of conjugated linoleic acid on skeletal muscle fat formation in pigs, which could serve as a model for studying human skeletal muscle diseases.

[Editors' note: the authors have responded to the previous rounds of review: https://doi.org/10.7554/eLife.99790.1.sa1 and https://doi.org/10.7554/eLife.99790.2.sa1]

Reviewer #1 (Public review):

Summary:

This study investigates the role of macrophage lipid metabolism in the intracellular growth of Mycobacterium tuberculosis. By using a CRISPR-Cas9 gene-editing approach, the authors knocked out key genes involved in fatty acid import, lipid droplet formation, and fatty acid oxidation in macrophages. Their results show that disrupting various stages of fatty acid metabolism significantly impairs the ability of Mtb to replicate inside macrophages. The mechanisms of growth restriction included increased glycolysis, oxidative stress, pro-inflammatory cytokine production, enhanced autophagy, and nutrient limitation. The study demonstrates that targeting fatty acid homeostasis at different stages of the lipid metabolic process could offer new strategies for host-directed therapies against tuberculosis.

The work is convincing and methodologically strong, combining genetic, metabolic, and transcriptomic analyses to provide deep insights into how host lipid metabolism affects bacterial survival.

Strengths:

The study uses a multifaceted approach, including CRISPR-Cas9 gene knockouts, metabolic assays, and dual RNA sequencing, to assess how various stages of macrophage lipid metabolism affect Mtb growth. The use of CRISPR-Cas9 to selectively knock out key genes involved in fatty acid metabolism enables precise investigation of how each step-lipid import, lipid droplet formation, and fatty acid oxidation-affects Mtb survival. The study offers mechanistic insights into how different impairments in lipid metabolism lead to diverse antimicrobial responses, including glycolysis, oxidative stress, and autophagy. This deepens the understanding of macrophage function in immune defense.<br /> The use of functional assays to validate findings (e.g., metabolic flux analyses, lipid droplet formation assays, and rescue experiments with fatty acid supplementation) strengthens the reliability and applicability of the results.<br /> By highlighting potential targets for HDT that exploit macrophage lipid metabolism to restrict Mtb growth, the work has significant implications for developing new tuberculosis treatments.

Weaknesses:

The experiments were primarily conducted in vitro using CRISPR-modified macrophages. While these provide valuable insights, they may not fully replicate the complexity of the in vivo environment where multiple cell types and factors influence Mtb infection and immune responses. Yet, I agree that the Hoxb8 in vitro model provides a powerful genetic tool to interrogate host-Mtb interactions using primary macrophages that represent the bone marrow-derived macrophage lineage, instead of using cell lines.

Comments on revisions: The authors have addressed my comment satisfactorily.

Reviewer #2 (Public review):

Summary:

Host-derived lipids are an important factor during Mtb infection. In this study, using CRISPR knockouts of genes involved in fatty acid uptake and metabolism, the authors claim that a compromised uptake, storage or metabolism of fatty acid in the hosts restricts Mtb growth upon infection. The mechanism involves increased glycolysis, autophagy, oxidative stress, pro-inflammatory cytokines and nutrient limitation. The study may be useful for developing novel host-directed approaches against TB.

Strengths:

The study's strength is the use of clean HOXB8-derived primary mouse macrophage lines for generating CRISPR knockouts.

Weaknesses:

The strength of evidence on autophagy and redox stress remains incomplete.

Comments on revisions:

The authors have revised the manuscript and addressed some of the earlier concerns. However, some of the interpretations and responses are incorrect.

Overall, the level of evidence to state the following in the abstract- "Our analyzes demonstrate that macrophages which cannot either import, store or catabolize fatty acids restrict Mtb growth by both common and divergent anti-microbial mechanisms, including increased glycolysis, increased oxidative stress, production of pro-inflammatory cytokines, enhanced autophagy and nutrient limitation" is incomplete.

There is an increase in glycolysis and pro-inflammatory cytokines and, to some extent, oxidative stress. The same can not be said about autophagy. Unfortunately, the authors did not try to establish a direct role of any of these pathways in restricting bacterial growth in the absence of any of the three genes studied.

Major concern:

Autophagy: The LC3 WB does not, by any stretch of the imagination, convince that there is an increase in autophagy flux, as inferred by the authors. Authors correctly cite the "Guidelines to autophagy" paper. Unfortunately, they cite it only selectively to justify their assessment. The LC3II/LC3I ratio indicates the number of autophagosomes present. This ratio can also increase if there is an active block of autophagosome maturation. That's why having BafA1 or CQ controls is important to assess the active autophagosome maturation. However, the authors sidestep this serious consideration by claiming some "pleiotropic impact on Mtb". With BafA1 and CQ, the only assay one needs is to measure the impact on LC3II levels. In the absence of this assay, the evidence supporting the role of autophagy is incomplete.

The main concern regarding autophagy results is that autophagy induction can typically bring down oxidative stress and classically has anti-inflammatory outlay. Thus, increased glycolysis, inflammatory cytokine production and redox stress indicate more towards a potential block in autophagy at the maturation step. This necessitates validation using autophagy flux assays.

Oxidative stress: Showing a representative image for the corresponding representative groups would be more convincing. For example, there is no clarity on whether, in the infected group, there was any staining for Mtb to analyse only the infected cells.

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 #1 (Public review):

Summary:

This is a very creative study using modeling and measurement of neoblast dynamics to gain insight into the mechanism that allows these highly potent cells to undergo fate-switching as part of their differentiation and self-renewal process. The authors estimate growth equation parameters for expanding neoblast clones based on new and prior experimental observations. These results indicate neoblast likely undergo much more symmetric self-amplifying division than loss of the population through symmetric differentiation, in the case of clone expansion assays after sublethal irradiation. Neoblasts take on multiple distinct transcriptional fates related to their terminally differentiated cell types, and prior work indicated neoblasts have a high plasticity to switch fates in way linked to cell cycle progression and possibly through a random process. Here, the authors explore the impact of inhibition of key transcription factors defining such states (ie "fate specifying transcription factors", FSTFs) plus measurement and modeling in the clone expansion assay, to find that inhibition of factors like zfp1 likely cause otherwise zfp1-fated neoblasts to fail to proliferate and differentiation, without causing compensatory gains in other lineages. A mathematical model of this process assuming that neoblasts do not retain a memory of prior states while they proliferate and transition across specified states can mimic the experimentally determined decreased sizes of clones following inhibition of zfp1. Complementary approaches to inhibit more than one lineage (muscle plus intestine) supports the idea that this is a more general process in planarian stem cells. These results provide an important advance for understanding the fate-switching process and its relationship to neoblast growth.

Overall I find the evidence very well presented and the study compelling, and offers an important new perspective on the key properties of neoblasts. I have some comments to clarify the presentation and significance of the work.

Comments on revisions:

In this revised version, the authors nicely address all of my comments and I find the work makes a strong case for its main conclusions.

Reviewer #2 (Public review):

Summary:

Cell cycle duration and cell fate choice are critical to understanding the cellular plasticity of neoblasts in planarians. In this study, Tamar et al. integrated experimental and computational approaches to simulate a model for neoblast behaviors during colony expansion.

Strengths:

The finding that "arresting differentiation into specific lineages disrupts neoblast proliferative capacities without inducing compensatory expression of other lineages" is particularly intriguing. This concept could inspire further studies on pluripotent stem cells and their application for regenerative biology.

Comments on revisions:

The authors have addressed all of my comments and concerns.

Reviewer #1 (Public review):

Summary:

The authors investigated if/how distractor suppression derived from statistical learning may be implemented in early visual cortex. While in a scanner, participants conducted a standard additional singleton task in which one location more frequently contained a salient distractor. The results showed that activity in EVC was suppressed for the location of the salient distractor as well as for neighbouring neutral locations. This suppression was not stimulus specific - meaning it occurred equally for distractors, targets and neutral items - and it was even present in trials in which the search display was omitted. Generally, the paper was clear, the experiment was well-designed, and the data are interesting.

The authors addressed all of my concerns and the revised manuscript will make a beautiful addition to the literature.

Reviewer #2 (Public review):

The authors of this work set out to test ideas about how observers learn to ignore irrelevant visual information. Specifically, they used fMRI to scan participants who performed a visual search task. The task was designed in such a way that highly salient but irrelevant search items were more likely to appear at a given spatial location. With a region-of-interest approach, the authors found that activity in visual cortex that selectively responds to that location was generally suppressed, in response to all stimuli (search targets, salient distractors, or neutral items), as well as in the absence of an anticipated stimulus.

Strengths of the study include: A well-written and well-argued manuscript; clever application of a region of interest approach to fMRI design, which allows articulating clear tests of different hypotheses; careful application of follow-up analyses to rule out alternative, strategy-based accounts of the findings; tests of the robustness of the findings to detailed analysis parameters such as ROI size; and exclusion of the role of regional baseline differences in BOLD responses. The main findings are enhanced by supplementary analyses that distinguish between the responses of early visual areas.

The study provides an advance over previous studies, which identified enhancement or suppression in visual cortex as a function of search target/distractor predictability, but in less spatially-specific way. It also speaks to open questions about whether such suppression/enhancement is observed only in response to the arrival of visual information, or instead is preparatory, favouring the latter view. These questions have been at the heart of theoretical debates in this literature on how distractor suppression unfolds in the context of visual search.

Reviewer #1 (Public review):

Summary:

This manuscript describes the role of PRDM16 in modulating BMP response during choroid plexus (ChP) development. The authors combine PRDM16 knockout mice and cultured PRDM16 KO primary neural stem cells (NSCs) to determine the interactions between BMP signaling and PRDM16 in ChP differentiation.

They show PRDM16 KO affects ChP development in vivo and BMP4 response in vitro. They determine genes regulated by BMP and PRDM16 by ChIP-seq or CUT&TAG for PRDM16, pSMAD1/5/8, and SMAD4. They then measure gene activity in primary NSCs through H3K4me3 and find more genes are co-repressed than co-activated by BMP signaling and PRDM16. They focus on the 31 genes found to be co-repressed by BMP and PRDM16. Wnt7b is in this set and the authors then provide evidence that PRDM16 and BMP signaling together repress Wnt activity in the developing choroid plexus.

Strengths:

Understanding context-dependent responses to cell signals during development is an important problem. The authors use a powerful combination of in vivo and in vitro systems to dissect how PRDM16 may modulate BMP response in early brain development.

Main weaknesses of the experimental setup:

(1) Because the authors state that primary NSCs cultured in vitro lose endogenous Prdm16 expression, they drive expression by a constitutive promoter. However, this means the expression levels are very different from endogenous levels (as explicitly shown in Supplementary Figure 2B) and the effect of many transcription factors is strongly dose-dependent, likely creating differences between the PRDM16-dependent transcriptional response in the in vitro system and in vivo.

(2) It seems that the authors compare Prdm16_KO cells to Prdm16 WT cells overexpressing flag_Prdm16. Aside from the possible expression of endogenous Prdm16, other cell differences may have arisen between these cell lines. A properly controlled experiment would compare Prdm16_KO ctrl (possibly infected with a control vector without Prdm16) to Prdm16_KO_E (i.e. the Prdm16_KO cells with and without Prdm16 overexpression.)

Other experimental weaknesses that make the evidence less convincing:

(1) The authors show in Figure 2E that Ttr is not upregulated by BMP4 in PRDM16_KO NSCs. Does this appear inconsistent with the presence of Ttr expression in the PRDM16_KO brain in Figure1C?

(2) Figure 3: The authors use H3K4me3 to measure gene activity. This is however, very indirect, with bulk RNA-seq providing the most direct readout and polymerase binding (ChIP-seq) another more direct readout. Transcription can be regulated without expected changes in histone methylation, see e.g. papers from Josh Brickman. They verify their H3K4me3 predictions with qPCR for a select number of genes, all related to the kinetochore, but it is not clear why these genes were picked, and one could worry whether these are representative.

(3) Line 256: The overlap of 31 genes between 184 BMP-repressed genes and 240 PRDM16-repressed genes seems quite small.

(4) The Wnt7b H3K4me3 track in Fig. 3G is not discussed in the text but it shows H3K4me3 high in _KO and low in _E regardless of BMP4. This seems to contradict the heatmap of H3K4me3 in Figure 3E which shows H3K4me3 high in _E no BMP4 and low in _E BMP4 while omitting _KO no BMP4. Meanwhile CDKN1A, the other gene shown in 3G, is missing from 3E.

(5) The authors use PRDM16 CUT&TAG on dissected dorsal midline tissues to determine if their 31 identified PRDM16-BMP4 co-repressed genes are regulated directly by PRDM16 in vivo. By manual inspection, they find that "most" of these show a PRDM16 peak. How many is most? If using the same parameters for determining peaks, how many genes in an appropriately chosen negative control set of genes would show peaks? Can the authors rigorously establish the statistical significance of this observation? And why wasn't the same experiment performed on the NSCs in which the other experiments are done so one can directly compare the results? Instead, as far as I could tell, there is only ChIP-qPCR for two genes in NSCs in Supplementary Figure 4D.

(6) In comparing RNA in situ between WT and PRDM16 KO in Figure 7, the authors state they use the Wnt2b signal to identify the border between CH and neocortex. However, the Wnt2b signal is shown in grey and it is impossible for this reviewer to see clear Wnt2b expression or where the boundaries are in Figure 7A. The authors also do not show where they placed the boundaries in their analysis. Furthermore, Figure 7B only shows insets for one of the regions being compared making it difficult to see differences from the other region. Finally, the authors do not show an example of their spot segmentation to judge whether their spot counting is reliable. Overall, this makes it difficult to judge whether the quantification in Figure 7C can be trusted.

(7) The correlation between mKi67 and Axin2 in Figure 7 is interesting but does not convincingly show that Wnt downstream of PRDM16 and BMP is responsible for the increased proliferation in PRDM16 mutants.

Weaknesses of the presentation:

Overall, the manuscript is not easy to read. This can cause confusion.

Reviewer #2 (Public review):

Summary:

This article investigates the role of PRDM16 in regulating cell proliferation and differentiation during choroid plexus (ChP) development in mice. The study finds that PRDM16 acts as a corepressor in the BMP signaling pathway, which is crucial for ChP formation.

The key findings of the study are:<br /> (1) PRDM16 promotes cell cycle exit in neural epithelial cells at the ChP primordium.<br /> (2) PRDM16 and BMP signaling work together to induce neural stem cell (NSC) quiescence in vitro.<br /> (3) BMP signaling and PRDM16 cooperatively repress proliferation genes.<br /> (4) PRDM16 assists genomic binding of SMAD4 and pSMAD1/5/8.<br /> (5) Genes co-regulated by SMADs and PRDM16 in NSCs are repressed in the developing ChP.<br /> (6) PRDM16 represses Wnt7b and Wnt activity in the developing ChP.<br /> (7) Levels of Wnt activity correlate with cell proliferation in the developing ChP and CH.

In summary, this study identifies PRDM16 as a key regulator of the balance between BMP and Wnt signaling during ChP development. PRDM16 facilitates the repressive function of BMP signaling on cell proliferation while simultaneously suppressing Wnt signaling. This interplay between signaling pathways and PRDM16 is essential for the proper specification and differentiation of ChP epithelial cells. This study provides new insights into the molecular mechanisms governing ChP development and may have implications for understanding the pathogenesis of ChP tumors and other related diseases.

Strengths:

(1) Combining in vitro and in vivo experiments to provide a comprehensive understanding of PRDM16 function in ChP development.

(2) Uses of a variety of techniques, including immunostaining, RNA in situ hybridization, RT-qPCR, CUT&Tag, ChIP-seq, and SCRINSHOT.

(3) Identifying a novel role for PRDM16 in regulating the balance between BMP and Wnt signaling.

(4) Providing a mechanistic explanation for how PRDM16 enhances the repressive function of BMP signaling. The identification of SMAD palindromic motifs as preferred binding sites for the SMAD/PRDM16 complex suggests a specific mechanism for PRDM16-mediated gene repression.

(5) Highlighting the potential clinical relevance of PRDM16 in the context of ChP tumors and other related diseases. By demonstrating the crucial role of PRDM16 in controlling ChP development, the study suggests that dysregulation of PRDM16 may contribute to the pathogenesis of these conditions.

Weaknesses:

(1) Limited investigation of the mechanism controlling PRDM16 protein stability and nuclear localization in vivo. The study observed that PRDM16 protein became nearly undetectable in NSCs cultured in vitro, despite high mRNA levels. While the authors speculate that post-translational modifications might regulate PRDM16 in NSCs similar to brown adipocytes, further investigation is needed to confirm this and understand the precise mechanism controlling PRDM16 protein levels in vivo.

(2) Reliance on overexpression of PRDM16 in NSC cultures. To study PRDM16 function in vitro, the authors used a lentiviral construct to constitutively express PRDM16 in NSCs. While this approach allowed them to overcome the issue of low PRDM16 protein levels in vitro, it is important to consider that overexpressing PRDM16 may not fully recapitulate its physiological role in regulating gene expression and cell behavior.

(3) Lack of direct evidence for AP1 as the co-factor responsible for SMAD relocation in the absence of PRDM16. While the study identified the AP1 motif as enriched in SMAD binding sites in Prdm16 knockout cells, they only provided ChIP-qPCR validation for c-FOS binding at two specific loci (Wnt7b and Id3). Further investigation is needed to confirm the direct interaction between AP1 and SMAD proteins in the absence of PRDM16 and to rule out other potential co-factors.

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 #1 (Public review):

Summary:

This research focuses on C. elegans klinotaxis, a chemotactic behavior characterized by gradual turning, aiming to uncover the neural circuit mechanism responsible for the context-dependent reversal of salt concentration preference. The phenomenon observed is that the preferred salt concentration depends on the difference between the pre-assay cultivation conditions and the current environmental salt levels.

The authors propose that a synaptic-reversal plasticity mechanism at the primary sensory neuron, ASER, is critical for this memory- and context-dependent switching of preference. They build on prior findings regarding synaptic reversal between ASER and AIB, as well as the receptor composition of AIY neurons, to hypothesize that similar "plasticity" between ASER and AIY underpins salt preference behavior in klinotaxis. This plasticity differs conceptually from the classical one as it does not rely on any structural changes but rather synaptic transmission is modulated by the basal level of glutamate, and can switch from inhibitory to excitatory.

To test this hypothesis, the study employs a previously established neuroanatomically grounded model [4] and demonstrates that reversing the ASER-AIY synapse sign in the model agent reproduces the observed reversal in salt preference. The model is parameterized using a computational search technique (evolutionary algorithm) to optimize unknown electrophysiological parameters for chemotaxis performance. Experimental validity is ensured by incorporating constraints derived from published findings, confirming the plausibility of the proposed mechanism.

Finally. the circuit mechanism allowing C. elegans to switch behaviour to an exploration run when starved is also investigated. This extension highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.

Strengths and weaknesses:

The authors' approach of integrating prior knowledge of receptor composition and synaptic reversal with the repurposing of a published neuroanatomical model [4] is a significant strength. This methodology not only ensures biological plausibility but also leverages a solid, reproducible modeling foundation to explore and test novel hypotheses effectively.

The evidence produced that the original model has been successfully reproduced is convincing.

The writing of the manuscript needs revision as it makes comprehension difficult.

One major weakness is that the model does not incorporate key findings that have emerged since the original model's publication in 2013, limiting the support for the proposed mechanism. In particular, ablation studies indicate that AIY is not critical for chemotaxis, and other interneurons may play partially overlapping roles in positive versus negative chemotaxis. These findings challenge the centrality of AIY and suggest the model oversimplifies the circuit involved in klinotaxis.

Reference [1] also shows that ASER neurons exhibit complex, memory- and context-dependent responses, which are not accounted for in the model and may have a significant impact on chemotactic model behaviour.

The hypothesis of synaptic reversal between ASER and AIY is not explicitly modeled in terms of receptor-specific dynamics or glutamate basal levels. Instead, the ASER-to-AIY connection is predefined as inhibitory or excitatory in separate models. This approach limits the model's ability to test the full range of mechanisms hypothesized to drive behavioral switching.

While the main results - such as response dependence on step inputs at different phases of the oscillator - are consistent with those observed in chemotaxis models with explicit neural dynamics (e.g., Reference [2]), the lack of richer neural dynamics could overlook critical effects. For example, the authors highlight the influence of gap junctions on turning sensitivity but do not sufficiently analyze the underlying mechanisms driving these effects. The role of gap junctions in the model may be oversimplified because, as in the original model [4], the oscillator dynamics are not intrinsically generated by an oscillator circuit but are instead externally imposed via $z_\text{osc}$. This simplification should be carefully considered when interpreting the contributions of specific connections to network dynamics. Lastly, the complex and context-dependent responses of ASER [1] might interact with circuit dynamics in ways that are not captured by the current simplified implementation. These simplifications could limit the model's ability to account for the interplay between sensory encoding and motor responses in C. elegans chemotaxis.

Appraisal:

The authors show that their model can reproduce memory-dependent reversal of preference in klinotaxis, demonstrating that the ASER-to-AIY synapse plays a key role in switching chemotactic preferences. By switching the ASER-AIY connection from excitatory to inhibitory they indeed show that salt preference reverses. They also show that the curving/turn rate underlying the preference change is gradual and depends on the weight between ASER-AIY. They further support their claim by showing that curving rates also depend on cultivated (set-point).

Thus within the constraints of the hypothesis and the framework, the model operates as expected and aligns with some experimental findings. However, significant omissions of key experimental evidence raise questions on whether the proposed neural mechanisms are sufficient for reversal in salt-preference chemotaxis.

Previous work [1] has shown that individually ablating the AIZ or AIY interneurons has essentially no effect on the Chemotactic Index (CI) toward the set point ([1] Figure 6). Furthermore, in [1] the authors report that different postsynaptic neurons are required for movement above or below the set point. The manuscript should address how this evidence fits with their model by attempting similar ablations. It is possible that the CI is rescued by klinokinesis but this needs to be tested on an extension of this model to provide a more compelling argument.

The investigation of dispersal behaviour in starved individuals is rather limited to testing by imposing inhibition of the SMB neurons. Although a circuit is proposed for how hunger states modulate taxis in the absence of food, this circuit hypothesis is not explicitly modelled to test the theory or provide novel insights.

Impact :

This research underscores the value of an embodied approach to understanding chemotaxis, addressing an important memory mechanism that enables adaptive behavior in the sensorimotor circuits supporting C. elegans chemotaxis. The principle of operation - the dependence of motor responses to sensory inputs on the phase of oscillation - appears to be a convergent solution to taxis. Similar mechanisms have been proposed in Drosophila larvae chemotaxis [2], zebrafish phototaxis [3], and other systems. Consequently, the proposed mechanism has broader implications for understanding how adaptive behaviors are embedded within sensorimotor systems and how experience shapes these circuits across species.

Although the reported reversal of synaptic connection from excitatory to inhibitory is an exciting phenomenon of broad interest, it is not entirely new, as the authors acknowledge similar reversals have been reported in ASER-to-AIB signaling for klinokinesis ( Hiroki et al., 2022). The proposed reversal of the ASER-to-AIY synaptic connection from inhibitory to excitatory is a novel contribution in the specific context of klinotaxis. While the ASER's role in gradient sensing and memory encoding has been previously identified, the current paper mechanistically models these processes, introducing a hypothesis for synaptic plasticity as the basis for bidirectional salt preference in klinotaxis.

The research also highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.

The methodology of parameter search on a neural model of a connectome used here yielded the valuable insight that connectome information alone does not provide enough constraints to reproduce the neural circuits for behaviour. It demonstrates that additional neurophysiological constraints are required.

Additional Context

Oscillators with stimulus-driven perturbations appear to be a convergent solution for taxis and navigation across species. Similar mechanisms have been studied in zebrafish phototaxis [3], Drosophila larvae chemotaxis [2], and have even been proposed to underlie search runs in ants. The modulation of taxis by context and memory is a ubiquitous requirement, with parallels across species. For example, Drosophila larvae modulate taxis based on current food availability and predicted rewards associated with odors, though the underlying mechanism remains elusive. The synaptic reversal mechanism highlighted in this study offers a compelling framework for understanding how taxis circuits integrate context-related memory retrieval more broadly.

As a side note, an interesting difference emerges when comparing C. elegans and Drosophila larvae chemotaxis. In Drosophila larvae, oscillatory mechanisms are hypothesized to underlie all chemotactic reorientations, ranging from large turns to smaller directional biases (weathervaning). By contrast, in C. elegans, weathervaning and pirouettes are treated as distinct strategies, often attributed to separate neural mechanisms. This raises the possibility that their motor execution could share a common oscillator-based framework. Re-examining their overlap might reveal deeper insights into the neural principles underlying these maneuvers.

(1) Luo, L., Wen, Q., Ren, J., Hendricks, M., Gershow, M., Qin, Y., Greenwood, J., Soucy, E.R., Klein, M., Smith-Parker, H.K., & Calvo, A.C. (2014). Dynamic encoding of perception, memory, and movement in a C. elegans chemotaxis circuit. Neuron, 82(5), 1115-1128.

(2) Antoine Wystrach, Konstantinos Lagogiannis, Barbara Webb (2016) Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae eLife 5:e15504.

(3) Wolf, S., Dubreuil, A.M., Bertoni, T. et al. Sensorimotor computation underlying phototaxis in zebrafish. Nat Commun 8, 651 (2017).

(4) Izquierdo, E.J. and Beer, R.D., 2013. Connecting a connectome to behavior: an ensemble of neuroanatomical models of C. elegans klinotaxis. PLoS computational biology, 9(2), p.e1002890.

Reviewer #2 (Public review):

Summary:

This study explores how a simple sensorimotor circuit in the nematode C. elegans enables it to navigate salt gradients based on past experiences. Using computational simulations and previously described neural connections, the study demonstrates how a single neuron, ASER, can change its signaling behavior in response to different salt conditions, with which the worm is able to "remember" prior environments and adjust its navigation toward "preferred" salinity accordingly.

Strengths:

The key novelty and strength of this paper is the explicit demonstration of computational neurobehavioral modeling and evolutionary algorithms to elucidate the synaptic plasticity in a minimal neural circuit that is sufficient to replicate memory-based chemotaxis. In particular, with changes in ASER's glutamate release and sensitivity of downstream neurons, the ASER neuron adjusts its output to be either excitatory or inhibitory depending on ambient salt concentration, enabling the worm to navigate toward or away from salt gradients based on prior exposure to salt concentration.

Weaknesses:

While the model successfully replicates some behaviors observed in previous experiments, many key assumptions lack direct biological validation. As to the model output readouts, the model considers only endpoint behaviors (chemotaxis index) rather than the full dynamics of navigation, which limits its predictive power. Moreover, some results presented in the paper lack interpretation, and many descriptions in the main text are overly technical and require clearer definitions.

Reviewer #1 (Public review):

Summary:

The study investigates the role of vascular mural cells, specifically pericytes and vascular smooth muscle cells (vSMCs), in maintaining blood-brain barrier (BBB) integrity and regulating vascular patterning. Analyzing zebrafish pdgfrb mutants that lack brain pericytes and vSMCs, they show that mural cell deficiency does not impair BBB establishment or maintenance during larval and early juvenile stages. However, mural cells seem to be crucial for preventing vascular aneurysms and hemorrhage in adulthood as focal leakage, basement membrane disruption, and increased caveolae formation are observed in adult zebrafish at aneurysm hotspots. The authors challenge the paradigm that mural cells are essential for BBB regulation in early development while highlighting their importance for long-term vascular stability.

Strengths:

Previous studies have established that the zebrafish BBB shares molecular and morphological homology with e.g. the mammalian BBB and therefore represents a suitable model. By examining mural cell roles across different life stages - from larval to adult zebrafish - the study provides an unprecedented comprehensive developmental analysis of brain vascular development and of how mural cells influence BBB integrity and vascular stability over time. The use of live imaging, whole-brain clearing, and electron microscopy offers high-resolution insights into cerebrovascular patterning, aneurysm development, and structural changes in endothelial cells and basement membranes. By analyzing "leakage hotspots" and their association with structural endothelial defects in adults the presented findings add novel insights into how mural cell loss may lead to vascular instability.

Weaknesses:

The study uses quantitative tracer assays with multiple molecular weight dyes to evaluate blood-brain barrier (BBB) permeability. The study normalizes the intensity of tracer signals (e.g., 10 kDa, 70 kDa dextrans) in the brain parenchyma to the vascular signal of a 2000 kDa dextran tracer (assumed to remain within vessels). Intensity normalization is used to control for variations in tracer injection efficiency or vascular density. This method doesn't directly assess the absolute amount of tracer present in the parenchyma, potentially underestimating leakage severity. As the lack of BBB impairment is a "negative" finding, more rigorous controls or other methods might be needed to corroborate it.

Reviewer #2 (Public review):

Summary:

The authors generated a zebrafish mutant of the pdgfrb gene. The presented analyses and data confirm previous studies demonstrating that Pdgfrb signaling is necessary for mural cell development in zebrafish. In addition, the data support previously published studies in zebrafish showing that mural cell deficiency leads to hemorrhages later in life. The authors presented quantified data on vessel density and branching, assessed tracer extravasation, and investigated the vasculature of adult mice using electron microscopy.

Strengths:

The strength of this article is that it provides independent confirmation of the important role of Pdgfrb signaling for the development of mural cells in the zebrafish brain. In addition, it confirms previous literature on zebrafish that provides evidence that, in the absence of pericytes/VSMC, hemorrhages appear (Wang et al, 2014, PMID: 24306108 and Ando et al 2021, PMID: 3431092). The study by Ando et al, 2021 did not report experiments assessing BBB leakage in pdgfrb mutants but in the review article by Ando et al (PMID: 34685412) it is stated that "indicating that endothelial cells can produce basic barrier integrity without pericytes in zebrafish".

Weaknesses:

(1) The authors should avoid using violin plots, which show distribution. Instead, they should replace all violin plots in the figures with graphs showing individual data points and standard deviation. For Figure 2f specifically, the standard deviation in the analyzed cohort should be shown.

(2) The authors have not shown the reduced PDGFRB protein or the effect of mutation on mRNA level in their zebrafish mutant.

(3) Statistical data analysis: Did the authors perform analyses to investigate whether the data has a normal distribution (e.g., Figures 1d, e)?

(4) Analysis of tracer extravasation. The use of 2000 kDa dextran intensity as an internal reference is problematic because the authors have not provided data demonstrating that the 2000 kDa dextran signal remains consistent across the entire vasculature. The authors have not provided data demonstrating that the 2000 kDa dextran signal in vessels exhibits acceptable variance across the vasculature to serve as a reliable internal reference. The variability of this signal within a single animal remains unknown. The presented data do not address this aspect.

Additionally, it's intriguing that the signal intensity in the parenchyma of the tested tracers presents a substantial range, varying by 20-30% in the analysed cohort (Figure 1g, Extended Figure 1e). Such large variability raises the question of its origin. Could it be a consequence of the normalization to 2000 kDa dextran intensity which differs between different fish? Or is it due to the differences in the parenchymal signal intensity while the baseline 2000 kDa intensity is stable? Or is the situation mixed?

An alternative and potentially more effective approach would be to cross the pdgfrb mutant line with a line where endothelial cells are genetically labeled to define vessels (e.g. the line kdrl used in acquiring data presented in Figure 2a). Non-injected controls could then be used as a baseline to assess tracer extravasation into the parenchyma.

How is the data presented in Figure 3e generated? How was the dextran intensity calculated? It looks like the authors have used the kdrl line to define vessels. Was the 2000 kDa still used as in previous figures? If not, please describe this in the Materials and Methods section.

(5) The authors state that both controls and mutants show extravasation of 1 kDa NHS-ester into the parenchyma. However, the presented images do not illustrate this; it is not obvious from these images (Extended Data Figure 1c). Additionally, the presented quantification data (Extended Data Figure 1e) do not show that, at 7 dpf, the vasculature is permeable to this tracer. Note that the range of signal intensity of the 1 kDa NHS-ester is similar to the 70 kDa dextran (Figure 1g and Extended Figure 1e). Would one expect an increase in the ratio in case of extravasation, considering that the 2000 kDa dextran has the same intensity in all experiments? Please explain.

(6) The study would be strengthened by a more detailed temporal analysis of the phenotype. When do the aneurysms appear? Is there an additional loss of VSMC?

(7) The authors intended to analyze the BBB at later stages (line 128), but there is not a significant time difference between 2 months (Figure 2) and 3 months (Figure 3) considering that zebrafish live on average 3 years. Therefore, the selection of only two time-points, 2 and 3 months, to analyze BBB changes does not provide a comprehensive overview of temporal changes throughout the zebrafish's lifespan. How long do the pdgfb mutants live?

(8) Why is there a difference in tracer permeability between 2 and 3 months (Figures 2 and 3)? Are hemorrhages not detected in 2-month-old zebrafish?

(9) Figure 3: The capillary bed should be presented in magnified images as it is not clearly visible. Figure 3e shows that in the pdgfb mutant the dextran intensity is higher also in regions 6-10. How do the authors explain this?

(10) In general, the manuscript would benefit from a more detailed description of the performed experiments. How long did the tracer circulate in the experiments presented in Figures 2, 3, and 4?

(11) How do the authors explain the poor signal of the 70 kDa dextran from the vasculature of 5-month-old zebrafish presented in Extended Data Figure 3?

(12) The study would benefit from a clear separation of the phenotypes caused by the loss of VSMC. The title eludes that also capillaries present hemorrhages which is not the case. How do vascular mural cells differ from mural cells? Are there any other mural cells?

(13) I have a few comments about how the authors have interpreted the literature and why, in my opinion, they should revise their strong statements (e.g., the last sentence in the abstract).

Scientists have their own insights and interpretations of data. However, when citing published data, it should be clearly indicated whether the statement is a direct quote from the original publication or an interpretation. In the current manuscript, the authors have not correctly cited the data presented in the two published papers (references 5 and 6). These papers do not propose a model where pericytes suppress "adsorptive transcytosis" (lines 73-76). While increased transcytosis is observed in pericyte-deficient mice, the specific type of vesicular transport that is increased or induced remains unknown.

Similarly, lines 151-152 refer to references 5 and 6 and use the term "adsorptive transcytosis," but the authors of both papers did not use this term. Attributing this term to the original authors is inaccurate. Additionally, lines 152-153 do not accurately represent the findings of references 5 and 6. These papers do not state that there is an induction of "caveolae" in endothelial cells in pericyte-deficient mice. In the absence of pericytes, many vesicles can be observed in endothelial cells, but these vesicles are relatively large. It is more likely that there is some form of uncontrolled transcytosis, perhaps micropinocytosis. Please refer to the original papers accurately.

Also, the authors have missed the fact that in mice, the extent of pericyte loss correlates with the extent of BBB leakage. To a certain extent, the remaining pericytes, can compensate for the loss by making longer processes and so ensure the full longitudinal coverage of the endothelium. This was shown in the initial work of Armulik et al (reference 5) and later in other studies.

The bold assertion on lines 183 -187 that a lack of specific BBB phenotype in pdgfrb zebrafish mutant invalidates mouse model findings is unfounded. Despite the notion that zebrafish endothelium possesses a BBB, I present a few examples highlighting the differences in brain vascular development and why the authors' expectation of a straightforward extrapolation of mouse BBB phenotypes to zebrafish is untenable.

In mice Pdgfrb knockout is lethal, but in zebrafish, this is not the case. In marked contrast to mice, however, zebrafish pdgfrb null mutants reach adulthood despite extensive cerebral vascular anomalies and hemorrhage. Following the authors' argumentation about the unlikely divergence of zebrafish and mice evolution, does it mean that the described mouse phenotype warrants a revisit and that the Pdgfrb knockout in mice perhaps is not lethal? Another example where the role of a gene product is not one-to-one, which relates to pericyte development, is Notch3. Notch3-null mice do not show significant changes in pericyte numbers or distribution, suggesting a less prominent role in pericyte development compared to zebrafish.

Although many aspects of development are conserved between species, there are significant differences during brain vascular development between zebrafish and mice. These differences could reveal why the BBB is not impaired in zebrafish pdgfrb mutants. There is a difference in the temporal aspect when various cellular players emerge. The timing of microglia colonization in the brain differs. In mice, microglia colonization starts before the first vessel sprouts enter the brain, while in zebrafish, microglia enter after. Additionally, microglia in zebrafish and mice have a different ontogeny. In mice, astrocytes specialize postnatally and form astrocyte endfeet postnatally. In zebrafish, radial glia/astrocytes form at 48 hpf, and as early as 3 dpf, gfap+ cells have a close relationship with blood vessels. Thus, these radial glia/astrocyte-like cells could play an important role in BBB induction in zebrafish. It's worth noting that in Drosophila, the blood-brain barrier is located in glial cells. While speculative, these cells might still play a role in zebrafish, while the role of pericytes does not seem to be crucial. Pericytes enter the brain and contact with developing vasculature (endothelium) relatively late in zebrafish (60 hpf). In mice, the situation is different, as there is no such lag between endothelium and pericyte entry into the brain. I suggest that the authors approach the observed data with curiosity and ask: Why are these differences present? Are all aspects of the BBB induced by neural tissue in zebrafish? What is the contribution of microglia and astrocytes?"

Another interesting aspect to consider is the endothelial-pericyte ratio and longitudinal coverage of pericytes in the zebrafish brain, and how this relates to what is observed in mice. How similar is the zebrafish vasculature to the mouse vasculature when it comes to the average length of pericytes in the zebrafish brain? Does the longitudinal coverage of pericytes in the zebrafish brain reach nearly 100%, as it does in mice?

Based on the preceding arguments, it is recommended that the authors present a balanced discussion that provides insightful discussion and situates their work within a broader framework.