Reviewer #1 (Public Review):

The authors present a detailed analysis of a set of molecular dynamics computer simulations of several variants of a T-cell receptor (TCR) in isolation and bound to a Major Histocompatibility Complex with peptide (pMHC), with the aim of improving our understanding of the mechanism T cell activation in immunity. By analyzing simulations of peptide mutants and partially truncated TCRs, the authors find that native peptide agonists lead to a so-called catch-bond response, whereby tensile force applied in the direction of separation between TCR/pMHC appears to strengthen the TCR/pMHC interface, whereas mutated peptides exhibit the more common slip-bond response, in which applied force destabilizes the binding interface.

Using various computational metrics and simulation statistics, the authors propose a model in which tensile force preferentially suppresses thermal fluctuations in the variable α domain of the TCR (vs the β domain) in a peptide-dependent manner, which orders and strengthens the binding interface by bringing together the complementarity-determining regions (CDRs) in the TCR variable chains, but only if the peptide is correctly matched to the TCR.

The study is detailed and written clearly, and conclusions appear convincing and are supported by the simulation data. However, the actual motions at the molecular or amino-acid level of how the catch-bond vs slip bond response originates remain somewhat unclear, and will probably warrant further investigations. Specific hypotheses that could be testable in experiments, such as predictions of which peptide (or TCR) mutations or which peptides could generate a catch-vs-slip response or activation, would have especially strengthened this study.

Reviewer #1 (Public Review):

Chang et al. demonstrate through their findings that COVID-19 mRNA vaccination of hemodialysis patients produces no significant difference in antibody levels achieved across the vaccination series. They correlate the antibody responses through RNA sequencing data of dialysis patients versus healthy controls throughout the vaccination series. They also compare those with prior infection versus those who are infection naive. The antibody findings are interesting because they disagree with previous publications showing that dialysis patients have a significantly lower antibody titer level achieved from vaccination than controls. The authors posit that this may be age-related, but subject numbers in the current study are not adequately powered to make that definitive determination.

However, they find that T-cell responses may be muted in hemodialysis patients as they have lower activation of T-cell genes than healthy controls. The RNA sequencing evidence is solid. However, they lack data on a clinical correlation to T-cell responses.

Reviewer #1 (Public Review):

This work introduces a novel framework for evaluating the performance of statistical methods that identify replay events. This is challenging because hippocampal replay is a latent cognitive process, where the ground truth is inaccessible, so methods cannot be evaluated against a known answer. The framework consists of two elements:<br /> 1. A replay sequence p-value, evaluated against shuffled permutations of the data, such as radon line fitting, rank-order correlation, or weighted correlation. This element determines how trajectory-like the spiking representation is. The p-value threshold for all accepted replay events is adjusted based on an empirical shuffled distribution to control for the false discovery rate.<br /> 2. A trajectory discriminability score, also evaluated against shuffled permutations of the data. In this case, there are two different possible spatial environments that can be replayed, so the method compares the log odds of track 1 vs. track 2.

The authors then use this framework (accepted number of replay events and trajectory discriminability) to study the performance of replay identification methods. They conclude that sharp wave ripple power is not a necessary criterion for identifying replay event candidates during awake run behavior if you have high multiunit activity, a higher number of permutations is better for identifying replay events, linear Bayesian decoding methods outperform rank-order correlation, and there is no evidence for pre-play.

The authors tackle a difficult and important problem for those studying hippocampal replay (and indeed all latent cognitive processes in the brain) with spiking data: how do we understand how well our methods are doing when the ground truth is inaccessible? Additionally, systematically studying how the variety of methods for identifying replay perform, is important for understanding the sometimes contradictory conclusions from replay papers. It helps consolidate the field around particular methods, leading to better reproducibility in the future. The authors' framework is also simple to implement and understand and the code has been provided, making it accessible to other neuroscientists. Testing for track discriminability, as well as the sequentiality of the replay event, is a sensible additional data point to eliminate "spurious" replay events.

However, there are some concerns with the framework as well. The novelty of the framework is questionable as it consists of a log odds measure previously used in two prior papers (Carey et al. 2019 and the authors' own Tirole & Huelin Gorriz, et al., 2022) and a multiple comparisons correction, albeit a unique empirical multiple comparisons correction based on shuffled data.

With respect to the log odds measure itself, as presented, it is reliant on having only two options to test between, limiting its general applicability. Even in the data used for the paper, there are sometimes three tracks, which could influence the conclusions of the paper about the validity of replay methods. This also highlights a weakness of the method in that it assumes that the true model (spatial track environment) is present in the set of options being tested. Furthermore, the log odds measure itself is sensitive to the defined ripple or multiunit start and end times, because it marginalizes over both position and time, so any inclusion of place cells that fire for the animal's stationary position could influence the discriminability of the track. Multiple track representations during a candidate replay event would also limit track discriminability. Finally, the authors call this measure "trajectory discriminability", which seems a misnomer as the time and position information are integrated out, so there is no notion of trajectory.

The authors also fail to make the connection with the control of the false discovery rate via false positives on empirical shuffles with existing multiple comparison corrections that control for false discovery rates (such as the Benjamini and Hochberg procedure or Storey's q-value). Additionally, the particular type of shuffle used will influence the empirically determined p-value, making the procedure dependent on the defined null distribution. Shuffling the data is also considerably more computationally intensive than the existing multiple comparison corrections.

Overall, the authors make interesting conclusions with respect to hippocampal replay methods, but the utility of the method is limited in scope because of its reliance on having exactly two comparisons and having to specify the null distribution to control for the false discovery rate. This work will be of interest to electrophysiologists studying hippocampal replay in spiking data.

Reviewer #1 (Public Review):

This work introduces a novel framework for evaluating the performance of statistical methods that identify replay events. This is challenging because hippocampal replay is a latent cognitive process, where the ground truth is inaccessible, so methods cannot be evaluated against a known answer. The framework consists of two elements:<br /> 1. A replay sequence p-value, evaluated against shuffled permutations of the data, such as radon line fitting, rank-order correlation, or weighted correlation. This element determines how trajectory-like the spiking representation is. The p-value threshold for all accepted replay events is adjusted based on an empirical shuffled distribution to control for the false discovery rate.<br /> 2. A trajectory discriminability score, also evaluated against shuffled permutations of the data. In this case, there are two different possible spatial environments that can be replayed, so the method compares the log odds of track 1 vs. track 2.

The authors then use this framework (accepted number of replay events and trajectory discriminability) to study the performance of replay identification methods. They conclude that sharp wave ripple power is not a necessary criterion for identifying replay event candidates during awake run behavior if you have high multiunit activity, a higher number of permutations is better for identifying replay events, linear Bayesian decoding methods outperform rank-order correlation, and there is no evidence for pre-play.

The authors tackle a difficult and important problem for those studying hippocampal replay (and indeed all latent cognitive processes in the brain) with spiking data: how do we understand how well our methods are doing when the ground truth is inaccessible? Additionally, systematically studying how the variety of methods for identifying replay perform, is important for understanding the sometimes contradictory conclusions from replay papers. It helps consolidate the field around particular methods, leading to better reproducibility in the future. The authors' framework is also simple to implement and understand and the code has been provided, making it accessible to other neuroscientists. Testing for track discriminability, as well as the sequentiality of the replay event, is a sensible additional data point to eliminate "spurious" replay events.

However, there are some concerns with the framework as well. The novelty of the framework is questionable as it consists of a log odds measure previously used in two prior papers (Carey et al. 2019 and the authors' own Tirole & Huelin Gorriz, et al., 2022) and a multiple comparisons correction, albeit a unique empirical multiple comparisons correction based on shuffled data.

With respect to the log odds measure itself, as presented, it is reliant on having only two options to test between, limiting its general applicability. Even in the data used for the paper, there are sometimes three tracks, which could influence the conclusions of the paper about the validity of replay methods. This also highlights a weakness of the method in that it assumes that the true model (spatial track environment) is present in the set of options being tested. Furthermore, the log odds measure itself is sensitive to the defined ripple or multiunit start and end times, because it marginalizes over both position and time, so any inclusion of place cells that fire for the animal's stationary position could influence the discriminability of the track. Multiple track representations during a candidate replay event would also limit track discriminability. Finally, the authors call this measure "trajectory discriminability", which seems a misnomer as the time and position information are integrated out, so there is no notion of trajectory.

The authors also fail to make the connection with the control of the false discovery rate via false positives on empirical shuffles with existing multiple comparison corrections that control for false discovery rates (such as the Benjamini and Hochberg procedure or Storey's q-value). Additionally, the particular type of shuffle used will influence the empirically determined p-value, making the procedure dependent on the defined null distribution. Shuffling the data is also considerably more computationally intensive than the existing multiple comparison corrections.

Overall, the authors make interesting conclusions with respect to hippocampal replay methods, but the utility of the method is limited in scope because of its reliance on having exactly two comparisons and having to specify the null distribution to control for the false discovery rate. This work will be of interest to electrophysiologists studying hippocampal replay in spiking data.

replying to Will at https://forum.zettelkasten.de/discussion/comment/18885/#Comment_18885

I've generally found that this is much easier to do when it's an area you tend to specialize in and want to delve ever deeper (or on which you have larger areas within your zettelkasten) versus those subjects which you care less about or don't tend to have as much patience for.

Perhaps it's related to the System 1/System 2 thinking of Kahneman/Tversky? There are only some things that seem worth System 2 thinking/clarifying/citing and for all the rest one relies on System 1 heuristics. I find that the general ease of use of my zettelkasten (with lots of practice) allows me to do a lot more System 2 thinking than I had previously done, even for areas which I don't care as much about.

syndication link: https://forum.zettelkasten.de/discussion/comment/18888/#Comment_18888

DOI: 10.7554/eLife.85930

Resource: ZFIN_ZDB-GENO-101203-1

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-GENO-101203-1

What is this?

DOI: 10.7554/eLife.85930

Resource: ZFIN_ZDB-ALT-080910-1

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-080910-1

What is this?

DOI: 10.7554/eLife.85930

Resource: ZFIN_ZDB-ALT-170329-1

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-170329-1

What is this?

Reviewer #1 (Public Review):

In this study, Li et al., report that FBXO24 contributes to sperm development by modulating alternative mRNA splicing and MIWI degradation during spermiogenesis. The authors demonstrated that FBXO24 deficiency impairs sperm head formation, midpiece compartmentalization, and axonemal/peri-axonemal organization in mature sperm, which causes sperm motility defects and male infertility. In addition, FBXO24 interacts with various mRNA splicing factors, which causes altered splicing events in Fbxo24-null round spermatids. Interestingly, FBXO24 also modulates MIWI levels via its polyubiquitination in round spermatids. Thus, the authors address that FBXO24 modulates global mRNA levels by regulating piRNA-mediated MIWI function and splicing events in testicular haploid germ cells.

This study is performed with various experimental approaches to explore and elucidate underlying molecular mechanisms for the FBXO24-mediated sperm defects during germ cell development. Overall, the experiments were designed properly and performed well to support the authors' observation in each part. In addition, the finding in this study is useful for understanding the physiological and developmental significance of the FBXO24 in the male germ line, which can provide insight into impaired sperm development and male infertility. However, there are several concerns to be explained more in this study. In addition, some results should be revised and updated.

Reviewer #1 (Public Review):

Summary:<br /> This paper examines patterns of diversity and divergence in two closely related sub-species of Zea mays. While the patterns are interesting, the strength of evidence in support of the conclusions drawn from these patterns is weak overall. Most of the main conclusions are not supported by convincing analyses.

Strengths:<br /> The paper presents interesting data from sets of sympatric populations of the two sub-species, maize and teosinte. This sampling offers unique insights into the diversity and divergence between the two, as well as the geographic structure of each.

Weaknesses:<br /> There were issues with many parts of the paper, especially with the strength of conclusions that can be drawn from the analyses. I list the major issues in the order in which they appear in the paper.

1. Gene flow and demography.<br /> The f4 tests of introgression (Figure 1E) are not independent of one another. So how should we interpret these: as gene flow everywhere, or just one event in an ancestral population? More importantly, almost all the significant points involve one population (Crucero Lagunitas), which suggests that the results do not simply represent gene flow between the sub-species. There was also no signal of increased migration between sympatric pairs of populations. Overall, the evidence for gene flow presented here is not convincing. Can some kind of supporting evidence be presented?

The paper also estimates demographic histories (changes in effective population sizes) for each population, and each sub-species together. The text (lines 191-194) says that "all histories estimated a bottleneck that started approximately 10 thousand generations ago" but I do not see this. Figure 2C (not 2E, as cited in the text) shows that teosinte had declines in all populations 10,000 generations ago, but some of these declines were very minimal. Maize has a similar pattern that started more recently, but the overall species history shows no change in effective size at all. There's not a lot of signal in these figures overall.

I am also curious: how does the demographic model inferred by mushi address inbreeding and homozygosity by descent (lines 197-202)? In other words, why does a change in Ne necessarily affect inbreeding, especially when all effective population sizes are above 10,000?

2. Proportion of adaptive mutations.<br /> The paper estimates alpha, the proportion of nonsynonymous substitutions fixed by positive selection, using two different sampling schemes for polymorphism. One uses range-wide polymorphism data and one uses each of the single populations. Because the estimates using these two approaches are similar, the authors conclude that there is little local adaptation. However, this conclusion is not justified.

There is little information as to how the McDonald-Kreitman test is carried out, but it appears that polymorphism within either teosinte or maize (using either sampling scheme) is compared to fixed differences with an outgroup. These species might be Z. luxurians or Z. diploperennis, as both are mentioned as outgroups. Regardless of which is used, this sampling means that almost all the fixed differences in the MK test will be along the ancestral branch leading to the ancestor of maize or teosinte, and on the branch leading to the outgroup. Therefore, it should not be surprising that alpha does not change based on the sampling scheme, as this should barely change the number of fixed differences (no numbers are reported).

The lack of differences in results has little to do with range-wide vs restricted adaptation, and much more to do with how MK tests are constructed. Should we expect an excess of fixed amino acid differences on very short internal branches of each sub-species tree? It makes sense that there is more variation in alpha in teosinte than maize, as these branches are longer, but they all seem quite short (it is hard to know precisely, as no Fst values or similar are reported).

3. Shared and private sweeps.<br /> In order to make biological inferences from the number of shared and private sweeps, there are a number of issues that must be addressed.

One issue is false negatives and false positives. If sweeps occur but are missed, then they will appear to be less shared than they really are. Table S3 reports very high false negative rates across much of the parameter space considered, but is not mentioned in the main text. How can we make strong conclusions about the scale of local adaptation given this? Conversely, while there is information about the false positive rate provided, this information doesn't tell us whether it's higher for population-specific events. It certainly seems likely that it would be. In either case, we should be cautious saying that some sweeps are "locally restricted" if they can be missed more than 85% of the time in a second population or falsely identified more than 25% of the time in a single population.

A second, opposite, issue is shared ancestral events. Maize populations are much more closely related than teosinte (Figure 2B). Because of this, a single, completed sweep in the ancestor of all populations could much more readily show a signal in multiple descendant populations. This is consistent with the data showing more shared events (and possibly more events overall). There also appear to be some very closely (phylogenetically) related teosinte populations. What if there's selection in their shared ancestor? For instance, Los Guajes and Palmar Chico are the two most closely related populations of teosinte and have the fewest unique sweeps (Figure 4B). How do these kinds of ancestrally shared selective events fit into the framework here?

These analyses of shared sweeps are followed by an analysis of sweeps shared by sympatric pairs of teosinte and maize. Because there are not more events shared by these pairs than expected, the paper concludes that geography and local environment are not important. But wouldn't it be better to test for shared sweeps according to the geographic proximity of populations of the same sub-species? A comparison of the two sub-species does not directly address the scale of adaptation of one organism to its environment, and therefore it is hard to know what to conclude from this analysis.

4. Convergent adaptation<br /> My biggest concern involves the apparent main conclusion of the paper about the sources of "convergent adaptations". I believe the authors are misapplying the method of Lee and Coop (2017), and have not seriously considered the confounding factors of this method as applied. I am unconvinced by the conclusions that are made from these analyses.

The method of Lee and Coop (referred to as rdmc) is intended to be applied to a single locus (or very tightly linked loci) that shows adaptation to the same environmental factor in different populations. From their paper: "Geographically separated populations can convergently adapt to the same selection pressure. Convergent evolution at the level of a gene may arise via three distinct modes." However, in the current paper, we are not considering such a restricted case. Instead, genome-wide scans for sweep regions have been made, without regard to similar selection pressures or to whether events are occurring in the same gene. Instead, the method is applied to large genomic regions not associated with known phenotypes or selective pressures.

I think the larger worry here is whether we are truly considering the "same gene" in these analyses. The methods applied here attempt to find shared sweep regions, not shared genes (or mutations). Even then, there are no details that I could find as to what constitutes a shared sweep. The only relevant text (lines 802-803) describes how a single region is called: "We merged outlier regions within 50,000 Kb of one another and treated as a single sweep region." (It probably doesn't mean "50,000 kb", which would be 50 million bases.) However, no information is given about how to identify overlap between populations or sub-species, nor how likely it is that the shared target of selection would be included in anything identified as a shared sweep. Is there a way to gauge whether we are truly identifying the same target of selection in two populations?

The question then is, what does rdmc conclude if we are simply looking at a region that happened to be a sweep in two populations, but was not due to shared selection or similar genes? There is little testing of this application here, especially its accuracy. Testing in Lee and Coop (2017) is all carried out assuming the location of the selected site is known, and even then there is quite a lot of difficulty distinguishing among several of the non-neutral models. This was especially true when standing variation was only polymorphic for a short time, as is estimated here for many cases, and would be confused for migration (see Lee and Coop 2017). Furthermore, the model of Lee and Coop (2017) does not seem to consider a completed ancestral sweep that has signals that persist into current populations (see point 3 above). How would rdmc interpret such a scenario?

Overall, there simply doesn't seem to be enough testing of this method, nor are many caveats raised in relation to the strange distributions of standing variation times (bimodal) or migration rates (opposite between maize and teosinte). It is not clear what inferences can be made with confidence, and certainly the Discussion (and Abstract) makes conclusions about the spread of beneficial alleles via introgression that seem to outstrip the results.

Joint Public Review:

Summary:<br /> The study "Effect of alpha-tubulin acetylation on the doublet microtubule structure" by S. Yang et al employs a multi-disciplinary approach, including cryo-electron microscopy (cryo-EM), molecular dynamics, and mass spectrometry, to investigate the impact of α-tubulin acetylation at the lysine 40 residue (αK40) on the structure and stability of doublet microtubules in cilia. The work reveals that αK40 acetylation exerts a small-scale, but significant, effect by influencing the lateral rotational angle of the microtubules, thereby affecting their stability. Additionally, the study provided an explanation of the relationship between αK40 acetylation and phosphorylation within cilia, despite that the details still remain elusive. Overall, these findings contribute to our understanding of how post-translational modifications can influence the structure, composition, stability, and functional properties of important cellular components like cilia.

Strengths:<br /> 1. Multi-Disciplinary Approach: The study employs a robust combination of cryo-electron microscopy (cryo-EM), molecular dynamics, and mass spectrometry, providing a comprehensive analysis of the subject matter.<br /> 2. Significant Findings: The paper successfully demonstrates the impact of αK40 acetylation on the lateral rotational angles between protofilaments (inter-PF angles) of doublet microtubules in cilia, thereby affecting their stability. This adds valuable insights into the role of post-translational modifications in cellular components.<br /> 3. Exploration of Acetylation-Phosphorylation Relationship: The study also delves into the relationship between αK40 acetylation and phosphorylation within cilia, contributing to a broader understanding of post-translational modifications.<br /> 4. High-quality data: The authors are cryo-EM experts in the field and the data quality presented in the manuscript is excellent.<br /> 5. Depth of analysis: The authors analyzed the effects of αK40 acetylation in excellent depth which significantly improved our understanding of this system.

Weaknesses:<br /> I have no major concerns about this paper, but would recommend that a few minor issues be addressed.

1. Lack of Statistical Details: The review points out that the paper could benefit from providing more statistical details, such as the number of particles and maps used for analysis, randomization methods, and dataset splitting for statistical analyses.<br /> 2. Questionable Conclusion Regarding MIPs: The reviewer suggests caution in the paper's conclusion that "Acetylation of αK40 does not affect tubulin and MIPs." The reviewer recommends that this conclusion be more specific or supported by additional evidence to exclude all other possibilities.<br /> 3. Need for Additional Visual Data: The reviewer recommends that an enlarged local density map along with fitted PDB models be provided in a supplementary figure, such as Figure 4.

Overall, the paper is strong in its scientific approach and findings but could benefit from additional statistical rigor and clarification of certain conclusions.

Reviewer #1 (Public Review):

Summary: The paper by McGinnis et al. uses a combination of genetic and biochemical approaches to understand how the conserved 5'-3' RNA exonuclease Xrn1 affects autophagy in response to methionine starvation in S. cerevisiae. The authors present evidence Xrn1 affects autophagy primarily via its effect on regulating TORC1 signaling. They present some evidence that Xrn1's effect on TORC1 singnaling is via its physical interaction with the SEACIT complex.

Strengths: The experiments in general for this paper are clear and have proper controls.

Weaknesses:<br /> The authors seem to try and fit the data to a simplistic model rather than embrace the complexity of the data. I will give some examples below.

1) Figure 1 clearly shows that xrn1d results in loss of tight repression of autophagy. Specifically, the 0 timepoint has increased autophagy in both the idh-GFP and ALP assays. However, it is incorrect to say that it is related in any way to methionine deprivation. The same basic pattern of regulation occurs in WT and xrn1d strains. The only difference is the "leakiness" of repression at t=0.

2) Figure 2 shows that catalytically inactive Xrn1 has the same autophagy phenotype as a deletion, indicating that Xrn1 enzymatic activity is important for function. However, it is also clear that xrn1-deletion cells expressing wt Xm1-flag do not repress autophagy as well as XRN+ cells, even though the amount of expressed protein seems similar. Does this imply the flag-tag may be a less active version of the protein? This should be discussed.

3) Figure 3 shows Xrn1-loss effects TORC signaling and that npr2-deletion inhibits autophagy. The surprising result is that a xrn1d/npr2d behaves like WT with regards to autophagy. This needs to be discussed. To me, this seems to strongly suggest that methionine repression of autophagy is occurring downstream of both xrn1 and npr2. Measuring p-S6 in the double mutant may be informative.

4) Figure 4 appears to show that even in the absence of GTR1, autophagy is repressed in rich media, active in YPL-SL, but still responds to methionine repression. This does not seem consistent with the model presented in Figure 5. Shouldn't loss of GTR1 result in repressed Torc1? The GTP and GDP-lock mutants are either all on, or all off. Why is deletion different? This needs to be explained and discussed. Also, the Figure legend does not match figures (problem after Fig4b).

5) Figure 5B shows GTR1 IP with Xrn1-FLAG. However, there are no negative controls in this experiment, so the result could be background. RNAaseA and RNA addition experiments are convincing.

6) Line 254-255. The lead sentence is simply not supported by the data. There is no evidence that Xrn1 actually affects the regulation of Gtr1/2 binding states.

7) Line 259-260. This is again overstated. Just because a mutant can be rescued by Gtr1-GTP-locked, does not say anything about RNA decay. In fact, the double mutant has extra high levels of some ATG RNA's, so I have no idea how the Gtr1 rescues.

8) Line 268-281. Your model here ignores the fact that methionine regulation takes place in the absence of both xrn1 and npr2. Therefore the model, as proposed, can't be correct.

9) Line 290-300. The slow growth rate of Xrn1 mutants may be affecting the metabolite levels. I felt that this entire paragraph was overly speculative.

Reviewer #1 (Public Review):

Summary:<br /> Cyclic Nucleotide Binding (CNB) domains are pervasive structural components involved in signaling pathways across eukaryotes and prokaryotes. Despite their similar structures, CNB domains exhibit distinct ligand-sensing capabilities. The manuscript offers a thorough and convincing investigation that clarifies numerous puzzling aspects of nucleotide binding in Trypanosoma.

Strengths:<br /> One of the strengths of this study is its multifaceted methodology, which includes a range of techniques including crystallography, ITC (Isothermal Titration Calorimetry), fluorimetry, CD (Circular Dichroism) spectroscopy, mass spectrometry, and computational analysis. This interdisciplinary approach not only enhances the depth of the investigation but also offers a robust cross-validation of the results.

Weaknesses:<br /> None noticed.

Reviewer #1 (Public Review):

Summary:<br /> This study aims to further resolve the history of speciation and introgression in Heliconius butterflies. The authors break the data into various partitions and test evolutionary hypotheses using the Bayesian software BPP, which is based on the multispecies coalescent model with introgression. By synthesizing these various analyses, the study pieces together an updated history of Heliconius, including a multitude of introgression events and the sharing of chromosomal inversions.

Strengths:

Full-likelihood methods for estimating introgression can be very computationally expensive, making them challenging to apply to datasets containing many species. This study provides a great example of how to apply these approaches by breaking the data down into a series of smaller inference problems and then piecing the results together. On the empirical side, it further resolves the history of a genus with a famously complex history of speciation and introgression, continuing its role as a great model system for studying the evolutionary consequences of introgression. This is highlighted by a nice Discussion section on the implications of the paper's findings for the evolution of pollen feeding.

Weaknesses:

The analyses in this study make use of a single method, BPP. The analyses are quite thorough so this is okay in my view from a methodological standpoint, but given this singularity, more attention should be paid to the weaknesses of this particular approach. Additionally, little attention is paid to comparable methods such as PhyloNet and their strengths and weaknesses in the Introduction or Discussion. BPP reduces computational burden by fixing certain aspects of the parameter space, such as the species tree topology or set of proposed introgression events. While this approach is statistically powerful, it requires users to make informed choices about which models to test, and these choices can have downstream consequences for subsequent analyses. It also might not be as applicable to systems outside of Heliconius where less previous information is available about the history of speciation and introgression. In general, it is likely that most modelling decisions made in the study are justified, but more attention should be paid to how these decisions are made and what the consequences of them could be, including alternative models.

• Co-estimating histories of speciation and introgression remains computationally challenging. To circumvent this in the study, the authors first estimate the history of speciation assuming no gene flow in BPP. While this approach should be robust to incomplete lineage sorting and gene tree estimation, it is still vulnerable to gene flow. This could result in a circular problem where gene flow causes the wrong species tree to be estimated, causing the true species tree to be estimated as a gene flow event. This is a flaw that this approach shares with summary-statistic approaches like the D-statistic, which also require an a-priori species tree. Enrichment of particular topologies on the Z chromosome helps resolve the true history in this particular case, but not all datasets will have sex chromosomes or chromosome-level assemblies to test against.

• The a-priori specification of network models necessarily means that potentially better-fitting models to the data don't get explored. Models containing introgression events are proposed here based on parsimony to explain patterns in gene tree frequencies. This is a reasonable and common assumption, but parsimony is not always the best explanation for a dataset, as we often see with phylogenetic inference. In general, there are no rigorous approaches to estimating the best-fitting number of introgression events in a dataset. Likewise, the study estimates both pulse and continuous introgression models for certain partitions, though there is no rigorous way to assess which of these describes the data better.

• Some aspects of the analyses involving inversions warrant additional consideration. Fewer loci were able to be identified in inverted regions, and such regions also often have reduced rates of recombination. I wonder if this might make inferences of the history of inverted regions vulnerable to the effects of incomplete lineage sorting, even when fitting the MSC model, due to a small # of truly genealogically independent loci. Additionally, there are several models where introgression events are proposed to explain the loss of segregating inversions in certain species. It is not clear why these scenarios should be proposed over those in which the inversion is lost simply due to drift or selection.

Reviewer #1 (Public Review):

Summary:

The authors embarked on a journey to understand the mechanisms and intensity-dependency of ultrasound (US)-induced extracellular vesicle (EV) release from myotubes and the potential anti-inflammatory effects of these EVs on macrophages. This study builds on their prior work from 2021 that initially reported US-induced EV secretion.

Strengths:

1. The finding that US-treated myotube EVs can suppress macrophage inflammatory responses is particularly intriguing, hinting at potential therapeutic avenues in inflammation modulation.

Weaknesses:

1. The exploration of output parameters for US induction appears limited, with only three different output powers (intensities) tested, thus narrowing the scope of their findings.<br /> 2. Their claim of elucidating mechanisms seems to be only partially met, with a predominant focus on the correlation between calcium responses and EV release.<br /> 3. While the intracellular calcium response is a dynamic activity, the method used to measure it could risk a loss of kinetic information.<br /> 4. The inclusion of miRNA sequencing is commendable; however, the interpretation of this data fails to draw clear conclusions, diminishing the impact of this segment.

While the authors have shown the anti-inflammatory effects of US-induced EVs on macrophages, there are gaps in the comprehensive understanding of the mechanisms underlying US-induced EV release. Certain aspects, like the calcium response and the utility of miRNA sequencing, were not fully explored to their potential. Therefore, while the study establishes some findings, it leaves other aspects only partially substantiated.

Joint Public Review:

Summary: Two early Cambrian taxa of linguliform brachiopods are assigned to the family Eoobolidae. The taxa exhibit a columnar shell structure and the phylogenetic implications of this shell structure in relation to other early Cambrian families is discussed.

Strengths: Interesting idea regarding the evolution of shell structure.

Weaknesses: The early record of shell structures of linguliform brachiopods is incomplete and partly contradictory. The authors maintain silence regarding contradictory information throughout the article to an extent that information is cited wrongly. The article is written under the assumption that all eoobolids have a columnar shell structure. Thus, the previously claimed columnar structure of Eoobolus incipiens which has been re-illustrated in the paper is not convincing and could be interpreted in other ways.

The article needs a proper results section. The Discussion is mainly a review of published data. Other potential results are hidden in this "discussion". In addition, a more elaborate Methods section is needed in which it is explained how the data for shell thicknesses and numbers of laminae was obtained.

A critical revision of the family Eoobolidae and Lingulellotretidae including a revision of the type species of Eoobolus and Lingulellotreta is needed.

The potential evolutionary patterns that are discussed towards the end (summarized in Fig 6) are interesting but rather unconvincing as the way the data has been obtained has never been clarified. Shell thicknesses and numbers of laminae that built up the shell of several taxa are compared, but at no point it is stated where these measurements were taken. Shell thicknesses vary within a shell and also the presence of the never mentioned tertiary layer is modifying shell thicknesses. Hence, the presented data appears random and is not comparable. The obtained evolutionary patterns must be considered as dubious.

Reviewer #1 (Public Review):

This study aims to determine the relative importance of the immediate effects of allopolyploidization from subsequent evolution in phenotypic traits associated with the selfing syndrome and in gene expression traits in the selfing allopolyploid Capsella bursa-pastoris and its diploid progenitors Capsella grandiflora, which is outcrossing, and Capsella orientalis, which is selfing. To do this, they compared five categories of plant: the two progenitors of the allopolyploid, hybrids resynthesized from the progenitors with a whole-genome duplication either before or after the hybridization event, and the naturally occurring allopolyploid.

Two lines of evidence were used: phenotypic data from the plants grown in a common environment, and RNAseq data from a subset of the plants.

The phenotypic data indicate that the selfing syndrome of C. bursa-pastoris likely evolved after the initial allopolyploidization event, and that pollen and seed viability recovered following the allopolyploidization event. The results are compelling but would benefit from small clarifications to the methods and statistics to account for possible positional effects in the growth chamber. Using a linear mixed model rather than a simple ANOVA would solve this problem.

The RNAseq data are used to explore overall expression patterns (using multi-dimensional scaling), patterns of differential expression (additive, dominant, or transgressive), and homeolog expression bias, and to determine the relative contributions of the original allopolyploidization event and subsequent evolution. Statistical cutoffs were used to categorize gene expression patterns, but the description and categorization of these patterns appears to have been largely qualitative, and might be strengthened by including more statistical detail in questions like whether homeologous expression bias did indeed show more variation in resynthesized and evolved allopolyploids.

The study includes evidence that homeolog expression bias (overrepresentation of an allele from one species) results in part from homeologous synapsis (uneven inheritance of chromosome segments). These deviations from patterns consistent with 2:2 inheritance of genomic regions are highly variable between individuals in resynthesized allopolyploids but appeared to be mostly consistent within (but not between) populations in natural C. bursa-pastoris. This is intriguing evidence that segregation can be an important source of variation in allopolyploids. However, it was limited by the difficulty of inferring homeologous recombination breakpoints with RNAseq data because of the scale of recombination in wild populations (rather than resynthesized allopolyploids). In future identifying such breakpoints will be an interesting direction for this and other allopolyploid systems.

This research suggests many follow-up questions. In particular, it may be possible to identify evidence about the mechanism of the original hybridization event. How frequently do unreduced gametes occur in these species, and is it likely that C. bursa-pastoris evolved via a triploid bridge? Exploring the viability, fertility, and phenotypes of triploids produced in both directions could be a valuable future direction.

Future research, or the current study, could also valuably explore what kinds of genes experienced what forms of expression evolution. A brief description of GO terms frequently represented in genes which showed strong patterns of expression evolution might be suggestive of which selective pressures led to the changes in expression in the C. bursa-pastoris lineage, and to what extent they related to adaptation to polyploidization (e.g. cell-cycle regulators), compensating for the initial pollen and seed inviability or adapting to selfing (endosperm- or pollen-specific genes), or adaptation to abiotic conditions.

Overall, this is an interesting and valuable contribution to the field's understanding of how expression evolves in interaction with hybridization and polyploidy. Particularly in combination with the team's previous study on these lines, this experimental design is effective for separating the contributions of hybridization, WGD, and evolution over time.

Update: the authors have thoughtfully and thoroughly updated the manuscript to address all the questions I raised. I appreciate the chance to review this valuable contribution to the scientific literature.

Reviewer #1 (Public Review):

Previous reports suggested an association between ceramide accumulation in skeletal muscle and disruption of insulin signaling and metabolic dysregulation. Mechanistically, however, how intracellular ceramide attenuates insulin action and reduces metabolism is not fully understood. It was suggested that insulin receptor (IR) signaling to PI3-K/AKT is inhibited by elevated intracellular ceramide. However, other studies failed to demonstrate an inhibitory effect of ceramide on PI3K/AKT. More recently, a study was published describing that intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle (PMID: 29415895). In the present study, Diaz-Vegas and colleagues used an in vitro system to investigate this topic further and better understand how intracellular ceramide accumulation causes cellular insulin resistance and metabolic dysregulations in cultured myocytes.

The authors applied multiple methods to achieve this goal. Among these procedures are:

1. The overexpression of enzymes involved in mitochondrial ceramide synthesis and degradation;<br /> 2. Treatments of myocytes with different pharmacological tools to validate their findings;<br /> 3. Mitochondrial proteomics and lipidomics analyses.

The effects of these experimental conditions and treatment on intracellular lipids contents, mitochondrial functions, and insulin signaling in myocytes were then evaluated.

Findings:

The author's findings indicate that incubation of myocytes with palmitate increases mitochondrial ceramide and reduces the insulin-stimulated GLUT4-HA translocation to the myocyte surface without affecting AKT activation. The elevation in mitochondrial ceramide lowers the coenzyme Q levels e depletes the electron transport chain (ETC) components, impairing mitochondrial respiration. Such mitochondrial dysfunction appears to attenuate the translocation of GLUT4-HA to the plasma membrane of the L6-myotubule. Also, mitochondrial proteomic analysis revealed an association of insulin sensitivity with mitochondrial ceramide and ETC expression levels in human muscle.

Based on these findings, the authors propose a mechanism whereby the building up of ceramide inside mitochondria depletes CoQ and compromises mitochondrial respiratory complexes, raising ROS. The resulting mitochondrial dysfunction causes insulin resistance in cultured myocytes. They postulate that CoQ depletion links ceramides with insulin resistance and define the respirasome as a critical connection between ceramides and mitochondrial dysfunction.

Relevance and critiques:

This original study provides direct evidence that mitochondrial ceramide accumulation depletes CoQ and downregulates multiple ETC components in myocytes. Consequently, elevation in the levels of reactive oxygen species (ROS) and mitochondrial dysfunctions occur. The authors proposed that such mitochondrial dysregulation attenuates insulin-stimulated GLUT4 translocation to the plasma membrane of L6-myotubules. Moreover, mitochondrial ceramide accumulation does not affect insulin action on AKT activation.

Overall, this is a well-done study, showing that in obesity, elevated mitochondrial ceramide suppresses mitochondrial function and attenuates insulin action on glucose transporter GLUT4 translocation into the myocyte surface. The main conclusion is supported by the results presented. The study also applied multiple methods and described several experiments designed to test the author's central hypothesis.

Importantly, these new findings shed light on possible cellular mechanisms whereby ectopic fat deposition in skeletal muscle drives insulin resistance and metabolism dysregulation. The results demonstrating that alterations in mitochondrial ceramide are sufficient to attenuate insulin-stimulated GLUT4 trafficking in cultured myocytes are very interesting. Well-done.

Comments for further discussion and suggestions:

Although the author's results suggest that higher mitochondrial ceramide levels suppress cellular insulin sensitivity, they rely solely on a partial inhibition (i.e., 30%) of insulin-stimulated GLUT4-HA translocation in L6 myocytes. It would be critical to examine how much the increased mitochondrial ceramide would inhibit insulin-induced glucose uptake in myocytes using radiolabel deoxy-glucose.

Another important question to be addressed is whether glycogen synthesis is affected in myocytes under these experimental conditions. Results demonstrating reductions in insulin-stimulated glucose transport and glycogen synthesis in myocytes with dysfunctional mitochondria due to ceramide accumulation would further support the author's claim.

In addition, it would be critical to assess whether the increased mitochondrial ceramide and consequent lowering of energy levels affect all exocytic pathways in L6 myoblasts or just the GLUT4 trafficking. Is the secretory pathway also disrupted under these conditions?

Additional suggestions:

• Figure 1: How does increased mitochondrial ceramide affect fatty acid oxidation (FAO) in L6-myocytes? As the accumulation of mitochondrial ceramide inhibits respirasome and mitochondrial activity in vitro, can reduced FAO in vivo, due to high mitochondrial ceramide, accounts for ectopic lipid deposition in skeletal muscle of obese subjects?

• Figure 2: Although the authors show that mtSMPD5 overexpression does not affect ceramide abundance in whole cell lysate, it would be critical to examine the abundance of this lipid in other cellular membranes and organelles, particularly plasma membrane. What is the effect of mtSMPD5 overexpression on plasma membrane lipids composition? Does that affect GLUT4-containing vesicles fusion into the plasma membrane, possibly due to depletion of v-SNARE or tSNARE?

• Figure 4: One critical piece of information missing is the effect (if any) of mitochondrial ceramide accumulation on the mRNAs encoding the ETC components affected by this lipid. Although the ETC protein's lower stability may account for the effect of increased ceramide, transcriptional inhibition can't be ruled out without checking the mRNA expression levels for these ETC components.

In the revised version of their study, the authors nicely addressed all concerns previously raised. The amount of work that went into the revisions is appreciated. All weak points have been properly addressed, and the manuscript has improved substantially.

Reviewer #1 (Public Review):

This study identified the truncating LRRC23 is associated with the asthenozoospermia in human and demonstrated that the truncated Lrrc23 specifically disorganizes RS3 and the junctional structure between RS2 and RS3 in the sperm axoneme, which might cause sperm motility defects and male infertility. Although LRRC23 has been reported as a component of the radial spoke and is necessary for sperm motility in mice, this study provided a precise pathogenic mechanism of truncating LRRC23 in asthenozoospermia. This work is of interest to researchers working on reproduction biology. The manuscript has been revised to address prior reviewers' comments.

Reviewer #1 (Public Review):

Summary:

Chen et al. describe the bacterial and fungal composition of cervical samples from women with/without Cesarean-section scar diverticulum (CSD) using whole metagenomic sequencing. Also, they report the metabolomic profile associated with CSD and built correlation networks at the taxonomical and taxonomic-metabolic levels to establish potential bacteria-fungi interactions. These interactions could be used, long-term, as therapeutic options to treat or prevent CSD.

After reviewing the manuscript, the authors have not integrated any of my previous recommendations into the new version of the work. Therefore, in my opinion, the limitations or weaknesses of the study remain the same.

I find it especially worrying that they do not consider the use of white controls necessary, arguing that "we considered that this study described a biomass-rich site, and the abundance of dominant species was much higher than that of the possible 'kitome', so we did not set a blank control" while describing among the most predominant species in the reproductive tract bacteria that do not colonize humans and that have been previously described as contaminants.

Lack of experimental controls can lead to artifactual results and compromise the evidence presented and the significance of the results.

Reviewer #1 (Public Review):

The study utilizes a variety of methods, chemical and expressed probes, caged release of IP3, as well as oocytes with mutations that alter zinc availability, that provide an elegant examination of how zinc deficiency and zinc excess modulate the transient and cyclic release of calcium during egg activation. In this manuscript, the authors sought to determine if there is any interplay between zinc and calcium, two divalent cations that have been demonstrated to have important roles during fertilization. They employ agents that disrupt normal zinc homeostasis and then monitor the resulting calcium oscillations during egg activation. If zinc was made unavailable via chelation with TPEN, then the calcium oscillations halted. This occurred regardless of the activation method, which included ICSI, PLC𝛇, Acetylcholine, strontium chloride, and thimerosal. This phenotype could be rescued by introducing zinc back into the egg via an ionophore, such as zinc pyrithione; however, too much zinc pyrithione also halted calcium oscillations. Taken together, these two results demonstrate that there is a threshold level of zinc that is required for proper calcium oscillations to occur.

Furthermore, the authors sought to understand how zinc affects the IP3 receptor, IP3R1. IP3R1 is the receptor that modulates the release of calcium from the endoplasmic reticulum. The authors cited a previous study that identified zinc binding sites on IP3R1. The authors highlight that there exist no studies regarding the regulation of IP3R1 by zinc; however, such studies were cited for a similar calcium channel, the RyRs. The authors use thapsigargin to inhibit the SERCA pump, leading to calcium leak from the IP3R1. TPEN blunted the amount of calcium leaked from the ER following treatment, suggesting that zinc occupancy is necessary for IP3R1 function.

The results of these experiments support the authors conclusions that zinc is essential for the IP3R1-mediated release of calcium in an oscillatory manner during egg activation. These results provide further insight into signals necessary for proper egg activation and the ultimate success of the resulting embryo.

Reviewer #1 (Public Review):

The study is valuable because it shows that physiologically relevant ∆9-THC concentrations have metabolic effects on early mouse embryonic cell types, which could cause developmental effects. Overall, the authors have convincing evidence showing that ∆9-THC has metabolic effects on mouse embryonic stem cells (mESCs), and that these effects persist in mESC-derived primordial germ cell-like cells even after ∆9-THC treatment has stopped. In this revised version, the authors included additional data to characterize the dose-dependence of the effects of ∆9-THC. Furthermore, they supported their finding of metabolic memory in PGCLCs by ruling out the potential alternative explanation that ∆9-THC persists in the cultured cells over the course of their experiment. This study has two significant implications: first, that ∆9-THC may alter the metabolism of early mouse embryos, and second, that mouse primordial germ cell-like cells can have a memory of previous metabolic perturbations.

The authors investigated the metabolic effects of ∆9-THC, the main psychoactive component of cannabis, on early mouse embryonic cell types. They found that ∆9-THC increases proliferation in male and female mouse embryonic stem cells (mESCs) and upregulates glycolysis. Additionally, primordial germ cell-like cells (PGCLCs) differentiated from ∆9-THC-exposed cells also show alterations to their metabolism. The study is valuable because it shows that physiologically relevant ∆9-THC concentrations have metabolic effects on cell types from the early embryo, which may cause developmental effects. Intriguingly, these effects persist in PGCLCs even after withdrawal of ∆9-THC.

The study shows that ∆9-THC increases the proliferation rate of mESCs but not mEpiLCs, without substantially affecting cell viability, except at the highest dose of 100 µM which shows toxicity (Figure 1). The dose required to cause increased proliferation was approximately 1 nM (Supplementary Figure 1), which is remarkably low. Treatment of mESCs with rimonabant (a CB1 receptor antagonist) blocks the effect of 100 nM ∆9-THC on cell proliferation, showing that the proliferative effect is mediated by CB1 receptor signaling. Similarly, treatment with 2-deoxyglucose, a glycolysis inhibitor, also blocks this proliferative effect (Figure 4G-H). Therefore, the effect of ∆9-THC depends on both CB1 signaling and glycolysis. This set of experiments strengthens the conclusions of the study by helping to elucidate the mechanism of the effects of ∆9-THC.

The study also profiles the transcriptome and metabolome of cells exposed to 100 nM ∆9-THC (Figure 4). Although the transcriptomic changes are modest overall, there is upregulation of anabolic genes, consistent with the increased proliferation rate in mESCs. Metabolomic profiling revealed a broad upregulation of metabolites in mESCs treated with 100 nM ∆9-THC. Some metabolic effects were also observed at a lower dose of 10 nM (Figure 3B).

Additionally, the study shows that ∆9-THC can influence germ cell specification. mESCs were differentiated to mEpiLCs in the presence or absence of ∆9-THC, and the mEpiLCs were subsequently differentiated to mPGCLCs. mPGCLC induction efficiency was tracked using a BV:SC dual fluorescent reporter. ∆9-THC treated cells had a moderate increase in the double positive mPGCLC population, and decrease in the double negative population. A cell tracking dye showed that mPGCLCs differentiated from ∆9-THC treated cells had undergone more divisions on average. As with the mESCs, these mPGCLCs also had altered gene expression and metabolism, consistent with an increased proliferation rate. Importantly, in the revised version, the authors supported their finding of metabolic memory in mPGCLCs by ruling out the potential alternative explanation that ∆9-THC persists in the cultured cells over the course of their experiment (Supplementary Figure 13).

Finally, the authors also observed that ∆9-THC decreases the proliferation of human ESCs (Supplementary Figure 4). These cells were in the primed pluripotent state, making them more similar to mEpiLCs than mESCs. Although this result may form the basis of follow-up experiments in a separate paper, it would be premature to conclude that the same effects will be observed in human cells as were observed in mouse cells.

Overall, this study provides good evidence for ∆9-THC having metabolic effects on mouse ESCs, and additionally shows that these effects can persist during germ cell specification. Potential effects of ∆9-THC exposure during early embryonic development are important for society to understand, and the results of this study are significant for public health.

Reviewer #1 (Public Review):

Summary:

In this paper, Steinemann et al. characterized the nature of stochastic signals underlying the trial-averaged responses observed in the lateral intraparietal cortex (LIP) of non-human primates (NHPs), while these performed the widely used random dot direction discrimination task. Ramp-up dynamics in the trial averaged LIP responses were reported in numerous papers before. However, the temporal dynamics of these signals at the single-trial level have been subject to debate. Using large-scale neuronal recordings with Neuropixels in NHPs, allows the authors to settle this debate rather compellingly. They show that drift-diffusion-like computations account well for the observed dynamics in LIP.

Strengths:

This work uses innovative technical approaches (Neuropixel recordings in behaving macaque monkeys). The authors tackle a vexing question that requires measurements of simultaneous neuronal population activity and hence leverage this advanced recording technique in a convincing way.

They use different population decoding strategies to help interpret the results.

They also compare how decoders relying on the data-driven approach using dimensionality reduction of the full neural population space compare to decoders relying on more traditional ways to categorize neurons that are based on hypotheses about their function. Intriguingly, although the functionally identified neurons are a modest fraction of the population, decoders that only rely on this fraction achieve comparable decoding performance to those relying on the full population. Moreover, decoding weights for the full population did not allow the authors to reliably identify the functionally identified subpopulation.

Weaknesses:

No major weaknesses.

Reviewer #1 (Public Review):

The study by Vengayil et al. presented a role for Ubp3 for mediating inorganic phosphate (Pi) compartmentalization in cytosol and mitochondria, which regulates metabolic flux between cytosolic glycolysis and mitochondrial processes. Although the exact function of increased Pi in mitochondria is not investigated, findings have valuable implications for understanding the metabolic interplay between glycolysis and respiration under glucose-rich conditions. They showed that UBP3 KO cells regulated decreased glycolytic flux by reducing the key Pi-dependent-glycolytic enzyme abundances, consequently increasing Pi compartmentalization to mitochondria. Increased mitochondria Pi increases oxygen consumption and mitochondrial membrane potential, indicative of increased oxidative phosphorylation. In conclusion, the authors reported that the Pi utilization by cytosolic glycolytic enzymes is a key process for mitochondrial repression under glucose conditions.

However, the main claims are only partially supported by the low number of repeats and utilizing only one strain background, which decreased the overall rigor of the study. The full-power yeast model could be utilized with testing findings in different backgrounds with increased biological repeats in many assays described in this study. In the yeast model, it has been well established that many phenotypes are genotype/strain dependent (Liti 2019, Gallone 2016, Boekout 2021, etc...). with some strains utilizing mitochondrial respiration even under high glucose conditions (Kaya 2021). It would be conclusive to test whether wild strains with increased respiration under high glucose conditions would also be characterized by increased mitochondrial Pi.

It is not described whether the drop in glycolytic flux also affects TCA cycle flux. Are there any changes in the pyruvate level? If the TCA cycle is also impaired, what drives increased mitochondrial respiration?

In addition, some of the important literature was also missed in citation and discussion. For example, in a recent study (Ouyang et al., 2022), it was reported that phosphate starvation increases mitochondrial membrane potential independent of respiration in yeast and mammalian cells, and some of the conflicting results were presented in this study.

An additional experiment with strains lacking mitochondrial DNA under phosphate-rich and restricted conditions would further strengthen the result.

Western blot control panels should include entire membrane exposure, and non-cut western blots should be submitted as supplementary.

In Figure 4, it is shown that Pi addition decreases basal OCR to the WT level. However, the Cox2 level remains significantly higher. This data is confusing as to whether mitochondrial Pi directly regulates respiration or not.

Representative images of Ubx3 KO and wild-type strains stained with CMXRos are missing.

Overall, mitochondrial copy number and mtDNA copy number should be analyzed in WT and Ubo3 KO cells as well as Pi-treated and non-treated cells, and basal OCR data should be normalized accordingly. The reported normalization against OD is not appropriate.

Reviewer #1 (Public Review):

Summary:<br /> In this paper, Ruggiero, Leite, and colleagues assess the effects of early-life seizures on a large number of anatomical, physiological, behavioral, and neurochemical measures. They find that prolonged early-life seizures do not lead to obvious cell loss, but lead to astrogliosis, working memory deficits on the radial arm maze, increased startle response, decreased paired pulse inhibition, and increased hippocampal-PFC LTP. There was a U-shape relationship between LTP and cognitive deficits. There is increased theta power during the awake state in ELS animals but reduced PFC theta-gamma coupling and reduced theta HPC-PFC coherence. Theta coherence seems to be similar in ACT and REM states in ELS animals while in decreases in active relative REM in controls.

Strengths:<br /> The main strength of the paper is the number of convergent techniques used to understand how hippocampal PFC neural dynamics and behavior change after early-life seizures. The sheer scale, breadth, and reach of the experiments are praiseworthy. It is clear that the paper is a major contribution to the field as far as understanding the impact of early-life seizures. The LTP findings are robust and provide an important avenue for future study. The experiments are performed carefully and the analysis is appropriate. The paper is well-written and the figures are clear.

Weaknesses:<br /> The main weakness of the paper is the lack of causal manipulations to determine whether prevention or augmentation of any of the findings has any impact on behavior or cognition. Alternatively, if other manipulations would enhance working memory in ELS animals, it would be interesting to see the effects on any of these parameters measured in the paper. Also, I find the sections where correlations and dimensionality reduction techniques are used to compare all possible variables to each other less compelling than the rest of the paper (with the exception of the findings of U U-shaped relationship of cognition to LTP). In fact, I think these sections take away from the impact of the actual findings. Finally, the apomorphine section seemed to hang separately from the rest of the paper and did not seem to fit well.

Reviewer #1 (Public Review):

Summary:

Protein conformational changes are often critical to protein function, but obtaining structural information about conformational ensembles is a challenge. Over a number of years, the authors of the current manuscript have developed and improved an algorithm, qFit protein, that models multiple conformations into high resolution electron density maps in an automated way. The current manuscript describes the latest improvements to the program, and analyzes the performance of qFit protein in a number of test cases, including classical statistical metrics of data fit like Rfree and the gap between Rwork and Rfree, model geometry, and global and case-by-case assessment of qFit performance at different data resolution cutoffs. The authors have also updated qFit to handle cryo-EM datasets, although the analysis of its performance is more limited due to a limited number of high-resolution test cases and less standardization of deposited/processed data.

Strengths:

The strengths of the manuscript are the careful and extensive analysis of qFit's performance over a variety of metrics and a diversity of test cases, as well as the careful discussion of the limitations of qFit. This manuscript also serves as a very useful guide for users in evaluating if and when qFit should be applied during structural refinement.

Reviewer #1 (Public Review):

The manuscript considers a hierarchical network of neurons, of the type that can be found in the sensory cortex, and assumes that they aim to constantly predict sensory inputs that may change in time. The paper describes the dynamics of neurons and rules of synaptic plasticity that minimize the integral of prediction errors over time.

The manuscript describes and analyses the model in great detail, and presents multiple and diverse simulations illustrating the model's functioning. However, the manuscript could be made more accessible and easier to read. The paper may help to understand the organization of cortical neurons, their properties, as well as the function of their particular components (such as apical dendrites).

Reviewer #1 (Public Review):

The authors bring together multiple study methods (brain recordings with EEG and behavioral coding of infant and caregiver looking, and caregiver vocal changes) to understand social processes involved in infant attention. They test different hypotheses on whether caregivers scaffold attention by structuring a child's behavior, versus whether the child's attention is guided by internal factors and caregivers then respond to infants' attentional shifts. They conclude that internal processes (as measured by brain activation preceding looking) control infants' attention, and that caregivers rapidly modify their behaviors in response to changes in infant attention.

The study is meticulously documented, with cutting-edge analytic approaches to testing alternative models; this type of work provides a careful and well-documented guide for how to conduct studies and process and analyze data for researchers in the relatively new area of neural response in infants in social contexts.

Some concerns arise around the use of terms (for example, an infant may "look" at an object, but that does not mean the infant is actually "attending); collapsing of different types of looks (to people and objects), and the averaging of data across infants that may mask some of the individual patterns.

Reviewer #1 (Public Review):

Many studies reported findings implying that rhizobial infection is associated with cell cycle re-entry and progression, however, our understanding has been fragmented. This study provides exciting new insights as it represents a comprehensive description of the cell cycle progression during early stages of nodulation using fluorescence markers.

To briefly summarize, the authors first monitor H3.1 / H3.3 replacement to distinguish between replicating (S phase) and non-replicating cells to show that M. truncatula cortex cells along the bacterial infection thread are non-replicating (while neighbors enter the S phase). Nuclear size measurements revealed that these non-replicative cells are in the post-replicative stage (G2) rather than in the pre-replicative G1 phase, which the authors confirm with the Plant Cell Cycle Indicator (PlaCCI) fluorescent marker to track cell cycle progression in more detail. Cortex cells in the trajectory of the infection thread did not accumulate the late G2 marker of the PlaCCI nor the G2/M marker KNOLLE, indicating that these cells indeed remain in G2. Because nuclear size measurements indicated that infected cells are polyploid, the authors used the centromere histone marker CENH3 to determine chromosome number. They find that cortex cells giving rise to the nodule primordium are endomitotic and tetraploid, probably because their cell cycle is halted at centromere separation. Although not a focus of this manuscript, the authors also use their fluorescent tools to track cell cycle progression during arbuscular mycorrhiza symbiosis. They confirm that infected cells transition from a replicating to a non-replicating state (H3.1 to H3.3) with progressing development of the arbuscules. In addition, the CENH3 marker confirms previous findings that cortex cells infected by fungi are endocycling (i.e., DNA synthesis without segregation of replicated parts). This represents an important confirmation of previous findings and contrasts with the situation during nodulation symbiosis, where chromosomes separate after replication.

In my view, the part about NF-YA1 is less strong - although I realize this is a compelling candidate to be a regulator of cell cycle progression, the experimental approaches used to address this question falls a bit short, in particular, compared to the very detailed approaches shown in the rest of the manuscript. The authors show that the transcription factor NF-YA1 regulates cell division in tobacco leaves; however, there is no experimental validation in the experimental system (nodules). All conclusions are based on a heterologous cell division system in tobacco leaves. The authors state that NF-YA1 has a nodule-specific role as a regulator of cell differentiation. I am concerned the tobacco system may not allow for adequate testing of this hypothesis. With the fluorescent tools the authors have at hand (in particular tools to detect G2/M transition, which the authors suggest is regulated by NF-YA1), it would be interesting to test what happens to cell division if NF-YA1 is over-expressed in Medicago roots?

Based on NF-YA1 expression data published previously and their results in tobacco epidermal cells, the authors hypothesize that NF-YA regulates the mitotic entry of nodule primordial cells. Given that much of the manuscript deals with earlier stages of the infection, I wonder if NF-YA1 could also have a role in regulating mitotic entry in cells adjacent to the infection thread?

In general, all microscopy images are of very high quality and support the authors' conclusions. While individually each set of fluorescent markers has its limitations, combined they constitute a powerful tool to track various stages of cell cycle progression in individual root cells during symbiosis. Overall, this is a very strong manuscript that comprehensively elucidates root cell cycle changes during microbial infection.

Reviewer #1 (Public Review):

Calcium channels are key regulators of synaptic strength and plasticity, yet how these channels are differentially utilized to enable synaptic diversity is not clear. In this manuscript, the authors use new endogenous tagging of the Drosophila CaV2 channel Cac and three auxiliary subunits to investigate distinct calcium channel functions at two motor neuron subtypes at the fly NMJ, Is and Ib. Although it is clear from previous studies that Pr is higher at Is over Ib, it is not clear why. The authors confirm these differences using postsynaptic calcium imaging combined with post-hoc Cac-TdTomato imaging. Then, through a series of confocal and super resolution imaging studies, the authors describe differences in calcium channel and active zone structure between Is and Ib motor neuron terminals, and the role of Brp and homeostatic plasticity in regulating channel abundance. Finally, the authors show that while the CaBeta subunit is present at similar levels at Is and Ib active zones, there is an interesting reduction in Stj at Is active zones. The authors conclude that these differences in active zone structure and architecture contribute to the generation of the observed heterogeneity in synaptic strength.

Overall the manuscript is well written, and the successful generation of the new endogenous Cac tags (Td-Tomato, Halo) and CaBeta, stj, and stolid genes with V5 tags will be powerful reagents for the field to enable new studies on calcium channels in synaptic structure, function, and plasticity. There are also some interesting, though not entirely unexpected, findings regarding how Brp and homeostatic plasticity modulate calcium channel abundance. However, a major concern is that the conclusions about how "molecular and organization diversity generate functional synaptic heterogeneity" are not really supported by the data presented in this study. In particular, the key fact that frames this study is that Cac levels are similar at Ib and Is active zones, but that Pr is higher at Is over Ib (which was previously known). While Pr can be influenced by myriad processes, the authors should have first assessed presynaptic calcium influx - if they had, they would have better framed the key questions in this study. As the authors reference from previous studies, calcium influx is at least two-fold higher per active zone at Is over Ib, and the authors likely know that this difference is more than sufficient to explain the difference in Pr at Is over Ib. Hence, there is no reason to invoke differences in "molecular and organization diversity" to explain the difference in Pr, and the authors offer no data to support that the differences in active zone structure at Is vs Ib are necessary for the differences in Pr. Indeed, the real question the authors should have investigated is why there are such differences in presynaptic calcium influx at Is over Ib despite having similar levels/abundance of Cac. This seems the real question, and is all that is needed to explain the Pr differences shown in Fig. 1. The other changes in active zone structure and organization at Is vs Ib may very well contribute to additional differences in Pr, but the authors have not shown this in the present study, and rely on other studies (such as calcium-SV coupling at Is vs Ib) to support an argument that is not necessitated by their data. At the end of this manuscript, the authors have found an interesting possibility that Stj levels are reduced at Is vs Ib, that might perhaps contribute to the difference in calcium influx. However, at present this remains speculative.

Overall, the authors have generated powerful reagents for the field to study calcium channels and how they are regulated, but draw conclusions about active zone structure and organization contributing to functional heterogeneity that are not strongly supported by the data presented.

Reviewer #1 (Public Review):

This manuscript investigates how homeostatic structural plasticity and synaptic scaling act under different levels of activity suppression and how this influences the network dynamics during growth and temporary or persistent silencing. To this end, the authors first use electrophysiology and chronic imaging to investigate the influence of different levels of AMPA-receptor blockade. A smaller level leads to reduced activity and up-regulation of synapse size and number, whereas a complete block abolished activity and decreases spine numbers. Along this line, the choice to block AMPAR is unconventional and needs to be better justified as both investigated homeostatic mechanisms are known to be AMPAR dependent.

Second, this finding is transferred into a mathematical rewiring rule, where spine number shrinks, grows, and shrinks again with increasing activity. It is shown that this rule, in contrast to other, simpler rules (grow, shrink), can grow healthy networks from scratch only if additional stimulation is provided. Continuing with these stable networks, the activity of a sub-network is increased, decreased, or silenced by modulating an external stimulation to the neurons. Whereas both activity and connectivity return to a stable state for small alteration, complete silencing leads to disconnection of the silenced network parts. Recovery from this can be achieved by restoring stimulation before the connectivity has completely decayed or by adding sufficiently fast synaptic scaling, although both cases can lead to unhealthy activity. A more systematic assessment of this interaction between scaling and homeostatic rewiring revealed a minimal timescale ratio that is needed for recovery. This is an important step towards disentangling the necessity of multiple, seemingly redundant mechanisms. Yet, in the simulations, the role of recurrent connectivity versus external inputs should be investigated in more detail in order to ensure the generality of the finding that a recovery of the activity is impossible for the presented rewiring rule without synaptic scaling.

Overall, the combination of experiments and simulations is a promising approach to investigating network self-organization. The gradual blocking of activity is especially valuable to inform mathematical models and distinguish them from alternatives. Here, the simulation results clearly demonstrate that the experimentally informed rule exhibits qualitatively different dynamics including the need for another homeostatic mechanism. However, a better connection between the simulations and experiment two would be desirable. In particular, it is unclear whether the model would actually reproduce the experiment, to which other experiments the model results relate, and which experimentally testable predictions the model makes.

In summary, this manuscript makes a valuable contribution to discerning the mathematical shape of a homeostatic structural plasticity model and understanding the necessity of synaptic scaling in the same network. Both experimental and computational methods are solid and well-described. Yet, both parts could be linked better in order to obtain conclusions with more impact and generality.

Reviewer #1 (Public Review):

In the manuscript, the authors explore the mechanism by which Taenia solium larvae may contribute to human epilepsy. This is extremely important question to address because T. solium is a significant cause of epilepsy and is extremely understudied. Advances in determining how T. solium may contribute to epilepsy could have significant impact on this form of epilepsy. Excitingly, the authors convincingly show that Taenia larvae contain and release glutamate sufficient to depolarize neurons and induce recurrent excitation reminiscent of seizures. They use a combination of cutting-edge tools including electrophysiology, calcium and glutamate imaging, and biochemical approaches to demonstrate this important advance. They also show that this occurs in neurons from both mice and humans. This is relevant for pathophysiology of chronic epilepsy development. This study does not rule out other aspects of T. solium that may also contribute to epilepsy, including immunological aspects, but demonstrates a clear potential role for glutamate.

Strengths:

- The authors examine not only T. solium homogenate, but also excretory/secretory products which suggests glutamate may play a role in multiple aspects of disease progression.<br /> - The authors confirm that the human relevant pathogen also causes neuronal depolarization in human brain tissue<br /> - There is very high clinical relevance. Preventing epileptogenesis/seizures possibly with Glu-R antagonists or by more actively removing glutamate as a second possible treatment approach in addition to/replacing post-infection immune response.<br /> - Effects are consistent across multiple species (rat, mouse, human) and methodological assays (GluSnFR AND current clamp recordings AND Ca imaging)<br /> - High K content (comparable levels to high-K seizure models) of larvae could have also caused depolarization. Adequate experiments to exclude K and other suspected larvae contents (i.e. Substance P).

Weaknesses:

- Acute study is limited to studying depolarization in slices and it is unclear what is necessary/sufficient for in vivo seizure generation or epileptogenesis for chronic epilepsy.<br /> - There is likely a significant role of the immune system that is not explored here. This issue is adequately addressed in the discussion, however, and the glutamate data is considered in this context.

Discuss impact:

- Interfering with peri-larval glutamate signaling may hold promise to prevent ictogenesis and chronic epileptogenesis as this is a very understudied cause of epilepsy with unknown mechanistic etiology.<br /> Additional context for interpreting significance:<br /> - High medical need as most common adult onset epilepsy in many parts of the world.

Reviewer #1 (Public Review):

The manuscript describes an interesting experiment in which an animal had to judge a duration of an interval and press one of two levers depending on the duration. The Authors recorded activity of neurons in key areas of the basal ganglia (SNr and striatum), and noticed that they can be divided into 4 types.

I would like to thank the Authors for performing the analyses I suggested in my previous review - I found their results very interesting and surprising. This is a very interesting and impressive paper.

Reviewer #1 (Public Review):

Summary: The manuscript offers a commendable exploration into the relationship between plasma omega-6/omega-3 fatty acid ratios and mortality outcomes.

Strengths: The chosen study design and analytical techniques align well with the research objectives, and the results resonate with existing literature.

Weaknesses: Lack of information on the selection criteria for participants; 5. The analysis of individual PUFAs is not appropriate; The definition of comorbidities is vague; The rationale of conducting the mediation analysis of blood biomarkers is not given.

Reviewer #1 (Public Review):

Summary:

Zanzibar archipelago is close to achieving malaria elimination, but despite the implementation of effective control measures, there is still a low-level seasonal malaria transmission. This could be due to the frequent importation of malaria from mainland Tanzania and Kenya, reservoirs of asymptomatic infections, and competent vectors. To investigate population structure and gene flow of P. falciparum in Zanzibar and mainland Tanzania, they used 178 samples from mainland Tanzania and 213 from Zanzibar that were previously sequenced using molecular inversion probes (MIPs) panels targeting single nucleotide polymorphisms (SNPs). They performed Principal Component Analysis (PCA) and identity by descent (IBD) analysis to assess genetic relatedness between isolates. Parasites from coastal mainland Tanzania contribute to the genetic diversity in the parasite population in Zanzibar. Despite this, there is a pattern of isolation by distance and microstructure within the archipelago, and evidence of local sharing of highly related strains sustaining malaria transmission in Zanzibar that are important targets for interventions such as mass drug administration and vector control, in addition to measures against imported malaria.

Strengths:

This study presents important samples to understand population structure and gene flow between mainland Tanzania and Zanzibar, especially from the rural Bagamoyo District, where malaria transmission persists and there is a major port of entry to Zanzibar. In addition, this study includes a larger set of SNPs, providing more robustness for analyses such as PCA and IBD. Therefore, the conclusions of this paper are well supported by data.

Weaknesses:

Some points need to be clarified:<br /> 1) SNPs in linkage disequilibrium (LD) can introduce bias in PCA and IBD analysis. Were SNPs in LD filtered out prior to these analyses?<br /> 2) Many IBD algorithms do not handle polyclonal infections well, despite an increasing number of algorithms that are able to handle polyclonal infections and multiallelic SNPs. How polyclonal samples were handled for IBD analysis?

Reviewer #1 (Public Review):

The overall tone of the rebuttal and lack of responses on several questions was surprising. Clearly, the authors did not appreciate the phrase 'no smoking gun' and provided a lengthy repetition of the fair argument about 'ticking boxes' on the classic list of criteria. They also make repeated historical references that descriptions of neurotransmitters include many papers, typically over decades, e.g. in the case of ACh and its discovery by Sir Henry Dale. While I empathize with the authors' apparent frustration (I quote: '...accept the reality that Rome was not built in a single day and that no transmitter was proven by a one single paper') I am a bit surprised at the complete brushing away of the argument, and in fact the discussion. In the original paper, the notion of a receptor was mentioned only in a single sentence and all three reviewers brought up this rather obvious question. The historical comparisons are difficult: Of course many papers contribute to the identification of a neurotransmitter, but there is a much higher burden of proof in 2023 compared to the work by Otto Loewi and Sir Henry Dale: most, if not all, currently accepted neurotransmitter have a clear biological function at the level of the brain and animal behavior or function - and were in fact first proposed to exist based on a functional biological experiment (e.g. Loewi's heart rate change). This, and the isolation of the chemical that does the job, were clear, unquestionable 'smoking guns' a hundred years ago. Fast forward 2023: Creatine has been carefully studied by the authors to tick many of the boxes for neurotransmitters, but there is no clear role for its function in an animal. The authors show convincing effects upon K+ stimulation and electrophysiological recordings that show altered neuronal activity using the slc6a8 and agat mutants as well as Cr application - but, as has been pointed out by other reviewers, these effects are not a clear-cut demonstration of a chemical transmitter function, however many boxes are ticked. The identification of a role of a neurotransmitter for brain function and animal behavior has reasonably more advanced possibilities in 2023 than a hundred years ago - and e.g. a discussion of approaches for possible receptor candidates should be possible.

Again, I reviewed this positively and agree that a lot of cumulative data are great to be put out there and allow the discovery to be more broadly discussed and tested. But I have to note, that the authors simply respond with the 'Rome was not built in a single day' statement to my suggestions on at least 'have some lead' how to approach the question of a receptor e.g. through agonists or antagonists (while clearly stating 'I do not think the publication of this manuscript should not be made dependent' on this). Similarly, in response to reviewer 2's concerns about a missing receptor, the authors' only (may I say snarky) response is ' We have deleted this sentence, though what could mediate postsynaptic responses other than receptors?' The bullet point by reviewer 3 ' • No candidate receptor for creatine has been identified postsynaptically.' is the one point by that reviewer that is simply ignored by the authors completely. Finally, I note that my reivew question on the K stimulation issues (e.g. 35 neurons that simply did not respond at all) was: ' Response: To avoid the disadvantage of K stimulation, we also performed optogenetic experiments recently and obtained encouraging preliminary results.' No details, not data - no response really.

In sum, I find this all a bit strange and the rebuttal surprising - all three reviewers were supportive and have carefully listed points of discussion that I found all valid and thoughtful. In response, the authors selectively responded scientifically to some experimental questions, but otherwise simply rather non-scientifically dismissed questions with 'Rome was not built in a day'-type answers, or less. I my view, the authors have disregarded the review process and the effort of three supportive reviewers, which should be part of the permanent record of this paper.

Reviewer #1 (Public Review):

Bolumar et al. isolated and characterized EV subpopulations, apoptotic bodies (AB), Microvesicles (MV), and Exosomes (EXO), from endometrial fluid through the female menstrual cycle. By performing DNA sequencing, they found the MVs contain more specific DNA sequences than other EVs, and specifically, more mtDNA were encapsulated in MVs. They also found a reduction of mtDNA content in the human endometrium at the receptive and post-receptive period that is associated with an increase in mitophagy activity in the cells, and a higher mtDNA content in the secreted MVs was found at the same time. Last, they demonstrated that the endometrial Ishikawa cell-derived EVs could be taken by the mouse embryos and resulted in altered embryo metabolism.

This is a very interesting study and is the first one demonstrating the direct transmission of maternal mtDNA to embryos through EVs.

Reviewer #1 (Public Review):

Summary:<br /> The study examines the role of release site clearance in synaptic transmission during repetitive activity under physiological conditions in two types of central synapses, calyx of Held and hippocampal CA1 synapses. After the acute block of endocytosis by pharmacology, deeper synaptic depression or less facilitation was observed in two types of synapses. Acute block of CDC42 and actin polymerization, which possibly inhibits the activity of Intersectin, affected synaptic depression at the calyx synapse, but not at CA1 synapses. The data suggest an unexpected, fast role of the site clearance in counteracting synaptic depression.

Strengths:<br /> The study uses an acute block of the molecular targets with pharmacology together with precise electrophysiology. The experimental results are clear-cut and convincing. The study also examines the physiological roles of the site clearance using action potential-evoked transmission at physiological Ca and physiological temperature at mature animals. This condition has not been examined.

Weaknesses:<br /> Pharmacology may have some off-target effects, though acute manipulation should be appreciated. Although this is a hard question and difficult to address experimentally, reagents may affect synaptic vesicle mobilization to the release sites directly in addition to blocking endocytosis.

Reviewer #1 (Public Review):

Summary:

o A well-executed series of experiments that will likely be of immense interest to (a) vector-borne disease researchers and (b) gram-negative sepsis/bacteremia researchers. The study uses comparative transcriptomics to begin probing what makes Peromyscus leucopus a unique host for numerous pathogens across the tree of life. Authors responded well to concerns raised in peer review and have produced an excellent second version of the manuscript.

Strengths:

o Use of outbred M. musculus is a commendable choice for the studies here.<br /> o Use of both LPS and B. hermsii allows analysis of multiple different signaling pathways that may differ between the species.<br /> o Upload of analyzed data onto Dryad is appreciated.

Weaknesses:

o None noted beyond the authors own limitation discussion section

Reviewer #1 (Public Review):

The manuscript by Lin, Sosnick et al investigates the functional conformational dynamics of two members of the SLC26 family of anion transporters (Prestin and SLC26A9). A key aspect of the work is that the authors use HDX-MS to convincingly identify that the folding of the unstable anion binding site is related to the fast electromechanical changes that are important for the function of Prestin. In good apparent agreement, such folding-related changes upon anion binding are absent in the related non-piezoelectric SLC26A9 that does not exhibit similar electro-motile transport. Overall, I find the work very interesting and generally well carried out - and it should be of considerable interest to researchers studying transmembrane transporters or just membrane proteins in general.

Reviewer #1 (Public Review):

This study provides compelling evidence that RAR, rather than its obligate dimerization partner RXR, is functionally limiting for chromatin binding. This manuscript provides a paradigm for how to dissect the complicated regulatory networks formed by dimerizing transcription factor families.

Dahal and colleagues use advanced SMT techniques to revisit the role of RXR in DNA-binding of the type-2 nuclear receptor (T2NR) RAR. The dominant consensus model for regulated DNA binding of T2NRs posits that they compete for a limited pool of RXR to form an obligate T2NR-RXR dimer. Using advanced SMT and proximity-assisted photoactivation technologies, Dahal et al. now test the effect of manipulating the endogenous pool size of RAR and RXR on heterodimerization and DNA-binding in live U2OS cells. Surprisingly, it turns out that RAR, rather than RXR, is functionally limiting for heterodimerization and chromatin binding. By inference, the relative pool size of various T2NRs expressed in a given cell, rather than RXR, is likely to determine chromatin binding and transcriptional output.

The conclusions of this study are well supported by the experimental results and provide unexpected novel insights into the functioning of the clinically important class of T2NR TFs. Moreover, the presented results show how the use of novel technologies can put long-standing theories on how transcription factors work upside down. This manuscript provides a paradigm for how to further dissect the complicated regulatory networks formed by T2NRs or other dimerizing TFs. I found this to be a complete story that does not require additional experimental work. However, I do have some suggestions for the authors to consider.

Reviewer #1 (Public Review):

Major concerns:

1. Is the direct binding of MCAK to the microtubule cap important for its in vivo function?

a. The authors claim that their "study provides mechanistic insights into understanding the end-binding mechanism of MCAK". I respectfully disagree. My concern is that the paper offers limited insights into the physiological significance of direct end-binding for MCAK activity, even in vitro. The authors estimate that in the absence of other proteins in vitro, ~95% of MCAK molecules arrive at the tip by direct binding in the presence of ~ physiological ATP concentration (1 mM). In cells, however, the major end-binding pathway may be mediated by EB, with the direct binding pathway contributing little to none. This is a reasonable concern because the apparent dissociation constant measured by the authors shows that MCAK binding to microtubules in the presence of ATP is very weak (69 uM). This concern should be addressed by 1) calculating relative contributions of direct and EB-dependent pathways based on the affinities measured in this and other published papers and estimated intracellular concentrations. Although there are many unknowns about these interactions in cells, a modeling-based analysis may be revealing. 2) the recapitulation of these pathways using purifying proteins in vitro is also feasible. Ideally, some direct evidence should be provided, e.g. based on MCAK function-separating mutants (GDP-Pi tubulin binding vs. catalytic activity at the curled protofilaments) that contribution from the direct binding of MCAK to microtubule cap in EB presence is significant.

b. As mentioned in the Discussion, preferential MCAK binding to tubulins near the MT tip may enhance MCAK targeting of terminal tubulins AFTER the MCAK has been "delivered" to the distal cap via the EB-dependent mechanism. This is a different targeting mechanism than the direct MCAK-binding. However, the measured binding affinity between MCAK and GMPCPP tubulins is so weak (69 uM), that this effect is also unlikely to have any impact because the binding events between MCAK and microtubule should be extremely rare. Without hard evidence, the arguments for this enhancement are very speculative.

2. The authors do not provide sufficient justification and explanation for their investigation of the effects of different nucleotides in MCAK binding affinity. A clear summary of the nucleotide-dependent function of MCAK (introduction with references to prior affinity measurements and corresponding MCAK affinities), the justifications for this investigation, and what has been learned from using different nucleotides (discussion) should be provided. My take on these results is that by far the strongest effect on microtubule wall and tip binding is achieved by adding any adenosine, whereas differences between different nucleotides are relatively minor. Was this expected? What can be learned from the apparent similarity between ATP and AMPPNP effects in some assays (Fig 1E, 4C, etc) but not others (Fig 1D,F, etc)?

3. It is not clear why the authors decided to use these specific mutant MCAK proteins to advance their arguments about the importance of direct tip binding. Both mutants are enzymatically inactive. Both show roughly similar tip interactions, with some (minor) differences. Without a clear understanding of what these mutants represent, the provided interpretations of the corresponding results are not convincing.

4. GMPCPP microtubules are used in the current study to represent normal dynamic microtubule ends, based on some published studies. However, there is no consensus in the field regarding the structure of growing vs. GMPCPP-stabilized microtubule ends, which additionally may be sensitive to specific experimental conditions (buffers, temperature, age of microtubules, etc). To strengthen the authors' argument, Taxol-stabilized microtubules should be used as a control to test if the effects are specific. Additionally, the authors should consider the possibility that stronger MCAK binding to the ends of different types of microtubules may reflect MCAK-dependent depolymerization events on a very small scale (several tubulin rows). These nano-scale changes to tubulins and the microtubule end may lead to the accumulation of small tubulin-MCAK aggregates, as is seen with other MAPs and slowly depolymerizing microtubules. These effects for MCAK may also depend on specific nucleotides, further complicating the interpretation. This possibility should be addressed because it provides a different interpretation than presented in the manuscript.

5. It would be helpful if the authors provided microtubule polymerization rates and catastrophe frequencies for assays with dynamic microtubules and MCAK in the presence of different nucleotides. The video recordings of microtubules under these conditions are already available to the authors, so it should not be difficult to provide these quantifications. They may reveal that microtubule ends are different (or not) under the examined conditions. It would also help to increase the overall credibility of this study by providing data that are easy to compare between different labs.

6. Are there other published studies that report MCAK binding affinity to microtubules? I find it quite surprising that the authors have reported the apparent dissociation constant for MCAK as 1mM. Such a high Kd value suggests no interaction under normal conditions, given that the intracellular concentrations of most proteins are orders of magnitude lower. If this information is inaccurate, it raises questions about the accuracy of other quantifications in the study.

7. Experimental and data analysis techniques are described superficially, and in some cases, only references to the prior work by others are provided. More direct evidence for these techniques and the corresponding controls should be provided.

Reviewer #1 (Public Review):

This work provides new mechanistic insights into the competitive inhibition in the mammalian P2X7 receptors using structural and functional approaches. The authors solved the structure of panda (pd) P2X7 in the presence of the classical competitive antagonists PPNDS and PPADS. They find that both drugs bind to the orthosteric site employed by the physiological agonist ATP. However, owing to the presence of a single phosphate group, they prevent movements in the flipper domain required for channel opening. The authors performed structure-based mutational analysis together with electrophysiological characterization to understand the subtype-specific binding of these drugs. It is known from previous studies that P2X1 and P2X3 are more sensitive to these drugs as compared to P2X7, hence, the residues adjacent to the ATP binding site in pdP2X7 were mutated to those present in P2X1. They observed that mutations of Q143, I214, and Q248 into lysine (hP2X1) increased the P2X7 sensitivity to PPNDS, whereas in P2X1, mutations of these lysines to alanine reduced sensitivity to PPNDS, suggesting that these key residues contribute to the subunit-specific sensitivity to these drugs. Similar experiments were done in hP2X3 to demonstrate its higher sensitivity to PPNDS. This preprint provides a useful framework for developing subtype-specific drugs for the family of P2X receptor channels, an area that is currently relatively unexplored.

The conclusions of the paper are mostly well supported, but need some clarification for the following:

1) Why was the crystallization construct of panda P2X7 used for structural studies instead of rat P2X7 with the cytoplasmic ballast which is a more complete receptor that is closely related to the human receptor? Can the authors provide a justification for this choice?

2) Was there a good reason why hP2X1 and hP2X3 currents were recorded in perforated patches, whereas pdP2X7 currents were recorded using the whole-cell configuration? It seems that the extent of rundown is less of a problem with perforated patch recordings. Can the authors comment and perhaps provide a justification? It would also be good to present data for repeated applications of ATP alone using protocols similar to those for testing antagonists so the reader can better appreciate the extent of run down with different recording configurations for the different receptors.

3) The data in Fig. S1, panel A shows multiple examples where the currents activated by ATP after removal of the antagonist are considerably smaller than the initial ATP application. Is this due to rundown or incomplete antagonist unbinding? It is interesting that this wasn't observed with hP2X1 and hP2X3 even though they have a higher affinity for the antagonist. Showing examples of rundown without antagonist application would help to distinguish these distinct phenomena and it would be good for the authors to comment on this in the text. It is also curious why a previous study on pdP2X7 did not seem to have problems with rundown (see Karasawa and Kawate. eLife, 2016).

4) The written presentation could be improved as there are many instances where the writing lacks clarity and the reader has to guess what the authors wish to communicate.

Reviewer #1 (Public Review):

Summary:<br /> This work describes the mechanism of protein disaggregation by the ClpL AAA+ protein of Listeria monocytogenes. Using several model subtrate proteins the authors first show that ClpL possesses a robust disaggregase activity that does not further require the endogenous DnaK chaperone in vitro. In addition, they found that ClpL is more thermostable than the endogenous L. monocytogenes DnaK and has the capacity to unfold tightly folded protein domains. The mechanistic basis for the robust disaggregase activity of ClpL was also dissected in vitro and in some cases, supported by in vivo data performed in chaperone-deficient E. coli strains. The data presented show that the two AAA domains, the pore-2 site and the N-terminal domain (NTD) of ClpL are critical for its disaggregase activity. Remarkably, grafting the NTD of ClpL to ClpB converted ClpB into an autonomous disaggregase, highlighting the importance of such a domain in the DnaK-independent disaggregation of proteins. The role of the ClpL NTD domain was further dissected, identifying key residues and positions necessary for aggregate recognition and disaggregation. Finally, using sets of SEC and negative staining EM experiments combined with conditional covalent linkages and disaggregation assays the authors found that ClpL shows significant structural plasticity, forming dynamic hexameric and heptameric active single rings that can further form higher assembly states via their middle domains.

Strengths:<br /> The manuscript is well-written and the experimental work is well executed. It contains a robust and complete set of in vitro data that push further our knowledge of such important disaggregases. It shows the importance of the atypical ClpL N-terminal domain in the disaggregation process as well as the structural malleability of such AAA+ proteins. More generally, this work expands our knowledge of heat resistance in bacterial pathogens.

Weaknesses:<br /> There is no specific weakness in this work, although it would have helped to have a drawing model showing how ClpL performs protein disaggregation based on their new findings. The function of the higher assembly states of ClpL remains unresolved and will need further extensive research. Similarly, it will be interesting in the future to see whether the sole function of the plasmid-encoded ClpL is to cope with general protein aggregates under heat stress.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, Schmassmann et al. present a study on the immune microenvironment of grade 4 gliomas using single-cell RNA-seq data from the tumor center, periphery, and peripheral blood of patients. This manuscript is overall well written and reads easily. The approach to studying the TME at various spatial locations is innovative and interesting, and the dataset presented has the potential to become a useful resource for the community. However, the size of the dataset, notably in the context of the important inter-patient variability on key clinical information, hinders the generalizability of the results. The analysis presented by the authors seems at times somewhat shallow as compared to other studies in the literature, being almost solely based on the analysis of a single dataset with extremely limited biological validation of the observations, and some claims made by the authors do not seem appropriately backed by the data they present. While I appreciate the vast analysis effort undertaken by the authors, it seems more work is required to make the most of this interesting dataset and substantiate the conclusions.

Strengths:<br /> The authors have provided useful insights into diverse GBMs (IDH mutant and IDH wild-type) that provide a deep assessment of individual tumors with spatial information.

Weaknesses:<br /> A larger set of tumors will need to be explored before general principles of immune biology and GBM immune evasion can be uncovered. This is a descriptive study that provides some interesting new hypotheses - but these will need deeper functional exploration.

Reviewer #1 (Public Review):

Summary:<br /> Kwong et al. present evidence that two actin-filament based cytoskeletal structures regulate the clockwise and anticlockwise rotation of the cytoplasm. These claims are based on experiments using cells plated on micropatterned substrates (circles). Previous reports have shown that the actomyosin network that forms on the dorsal surface of a cell plated on a circle drives a rotational or swirling pattern of movement in the cytoplasm. This actin network is composed of a combination of non-contractile radial stress fibers (AKA dorsal stress fibers) which are mechanically coupled to contractile transverse actin arcs (AKA actin arcs). The authors claim that directionality of the rotation of the cytoplasm (i.e., clockwise or anticlockwise) depends on either the actin arcs or radial fibers, respectively. While this would interesting, the authors are not able to remove either actin-based network without effecting the other. This is not surprising, as it is likely that the radial fibers require the arcs to elongate them, and the arcs require the radial fibers to stop them from collapsing. As such, it is difficult to make simple interpretations such as the clockwise bias is driven by the arcs and anticlockwise bias is driven by the radial fibers.

Weaknesses:<br /> There are also multiple problems with how the data is displayed and interpreted. First, it is difficult to compare the experimental data with the controls as the authors do not include control images in several of the figures. For example, Figure 6 has images showing myosin IIA distribution, but Figure 5 has the control image. Each figure needs to show controls. Otherwise, it will be difficult for the reader to understand the differences in localization of the proteins shown. This could be accomplished by either adding different control examples or by combining figures.

It is important that the authors should label the range of gray values of the heat maps shown. It is difficult to know how these maps were created. I could not find a description in the methods, nor have previous papers laid out a standardized way of doing it. As such, the reader needs some indication as to whether the maps showing different cells were created the same and show the same range of gray levels. In general, heat maps showing the same protein should have identical gray levels. The authors already show color bars next to the heat maps indicating the range of colors used. It should be a simple fix to label the minimum (blue on the color bar) and the maximum (red on the color bar) gray levels on these color bars. The profiles of actin shown in Figure 3 and Figure 3- figure supplement 3 were useful for interpretating the distribution of actin filaments. Why did not the authors show the same for the myosin IIa distributions?<br /> Line 189 "This absence of radial fibers is unexpected". The authors should clarify what they mean by this statement. The claim that the cell in Figure 3B has reduced radial stress fiber is not supported by the data shown. Every actin structure in this cell is reduced compared to the cell on the larger micropattern in Figure 3A. It is unclear if the radial stress fibers are reduced more than the arcs. Are the authors referring to radial fiber elongation?<br /> The choice of the small molecule inhibitors used in this study is difficult to understand, and their results are also confusing. For example, sequestering G actin with Latrunculin A is a complicated experiment. The authors use a relatively low concentration (50 nM) and show that actin filament-based structures are reduced and there are more in the center of the cell than in controls (Figure 3E). What was the logic of choosing this concentration? Using a small molecule that binds the barbed end (e.g., cytochalasin) could conceivably be used to selectively remove longer actin filaments, which the radial fibers have compared to the lamellipodia and the transverse arcs. The authors should articulate how the actin cytoskeleton is being changed by latruculin treatment and the impact on chirality. Is it just that the radial stress fibers are not elongating? There seems to be more radial stress fibers than in controls, rather than an absence of radial stress fibers. Similar problems arise from the other small molecules as well. LPA has more effects than simply activating RhoA. Additionally, many of the quantifiable effects of LPA treatment are apparent only after the cells are serum starved, which does not seem to be the case here. Furthermore, inhibiting ROCK with, Y-27632, effects myosin light chain phosphorylation and is not specific to myosin IIA. Are the two other myosin II paralogs expressed in these cells (myosin IIB and myosin IIC)? If so, the authors' statements about this experiment should refer to myosin II not myosin IIa. None of the uses of the small molecules above have supporting data using a different experimental method. For example, backing up the LPA experiment by perturbing RhoA tho.<br /> The use of SMIFH2 as a "formin inhibitor" is also problematic. SMIFH2 also inhibits myosin II contractility, making interpreting its effects on cells difficult to impossible. The authors present data of mDia2 knockdown, which would be a good control for this SMIFH2. However, the authors claim that mDia2 "typically nucleates tropomyosin-decorated actin filaments, which recruit myosin II and anneal endwise with α-actinin- crosslinked actin filaments." There is no reference to this statement and the authors own data shows that both arcs and radial fibers are reduced by mDia2 knockdown. Overall, the formin data does not support the conclusions the authors report.<br /> The data in Figure 7 does not support the conclusion that myosin IIa is exclusively on top of the cell. There are clear ventral stress fibers in A (actin) that have myosin IIa localization. The authors simply chose to not draw a line over them to create a height profile.

Reviewer #1 (Public Review):

Schmit et al. analyze and compare different strategies for the allocation of funding for insecticide-treated nets (ITNs) to reduce the global burden of malaria. They use previously published models of Plasmodium falciparum and Plasmodium vivax malaria transmission to quantify the effect of ITN distribution on clinical malaria numbers and the population at risk. The impact of different resource allocation strategies on the reduction of malaria cases or a combination of malaria cases and achieving pre-elimination is considered to determine the optimal strategy to allocate global resources to achieve malaria eradication.

Strengths:

Schmit et al. use previously published models and optimization for a rigorous analysis and comparison of the global impact of different funding allocation strategies for ITN distribution. This provides evidence of the effect of three different approaches: the prioritization of high-transmission settings to reduce the disease burden, the prioritization of low-transmission settings to "shrink the malaria map", and a resource allocation proportional to the disease burden.

Weaknesses:

The analysis and optimization which provide the evidence for the conclusions and are thus the central part of this manuscript necessitate some simplifying assumptions which may have important practical implications for the allocation of resources to reduce the malaria burden. For example, seasonality, mosquito species-specific properties, stochasticity in low transmission settings, and changing population sizes were not included. Other challenges to the reduction or elimination of malaria such as resistance of parasites and mosquitoes or the spread of different mosquito species as well as other beneficial interventions such as indoor residual spraying, seasonal malaria chemoprevention, vaccinations, combinations of different interventions, or setting-specific interventions were also not included. Schmit et al. clearly state these limitations throughout their manuscript.

This work considers different ITN distribution strategies, other interventions are not considered. It also provides a global perspective but an analysis of the specific local setting (as also noted by Schmit et al.) and different interventions as well as combinations of interventions should also be taken into account for any decisions. Nonetheless, the rigorous analysis supports the authors' conclusions and provides evidence that supports the prioritization of funding of ITNs for settings with high Plasmodium falciparum transmission. Overall, this work may contribute to making evidence-based decisions regarding the optimal prioritization of funding and resources to achieve a reduction in the malaria burden.

Reviewer #1 (Public Review):

Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.

Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.

Weaknesses: Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation. Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

Reviewer #1 (Public Review):

Anderson, Henikoff and Ahmad et al. performed a series of genomics assays to study Drosophila spermatogenesis. Their main approaches include (1) Using two different genetic mutants that arrest male germ cell differentiation at distinct stages, bam and aly mutant, they performed CUT&TAG using H3K4me2, a histone modification for active promoters and enhancers; (2) Using FACS sorted pure spermatocytes, they performed CUT&TAG using antibodies against RNA PolII phosphorylated Ser 2, H4K16ac, H3K9me2, H3K27me3, and ubH2AK118. They also compare these chromatin profiling results with the published single-cell and single-nucleus RNA-seq data. Their analyses are across the genome but the major conclusions are about the chromatin features of the sex chromosomes. For example, the X chromosome is lack of dosage compensation as well as inactivation in spermatocytes, while Y chromosome is activated but enriched with ubH2A in spermatocytes. Overall, this work provides high quality epigenome data in testes and in purified germ cells. The analyses are very informative to understand and appreciate the dramatic chromatin structure change during spermatogenesis in Drosophila.

Reviewer #1 (Public Review):

Suarez-Freire et al. analyzed here the function of the exocyst complex in the secretion of the glue proteins by the salivary glands of the Drosophila larva. This is a widely used, genetically accessible system in which the formation, maturation and precisely timed exocytosis of the glue secretory granules can be beautifully imaged. Using RNAi, the authors show that all units of the exocyst complex are required for exocytosis. They show that not just granule fusion with the plasma membrane is affected (canonical role), but also, with different penetrance, that glue protein is retained in the ER, secretory granules fail to fuse homo-typically or fail to acquire maturation features. The authors document these phenotypes and postulate specific roles for the exocyst in these additional processes to explain them: exocyst as an ER-Golgi tether and exocyst as a granule-granule tether. However, the evidence for these highly novel, potentially interesting roles would need to be more compelling to support direct involvement. For instance, the localization of exocyst to Golgi or to granule-granule contact sites does not seem substantial. Instead, it is possible that defects in Golgi traffic and granule homotypic fusion are not due to direct involvement of the exocyst in these processes, but secondary to a defect in canonical exocyst roles at the plasma membrane. A block in the last step of glue exocytosis could perhaps propagate backward in the secretory pathway to disrupt Golgi complexes or cause poor cellular health due to loss of cell polarity or autophagy. In the absence of stronger evidence for these other exocyst roles, I would suggest focusing the study on the canonical role (interesting, as it was previously reported that Drosophila exocyst had no function in the salivary gland and limited function elsewhere [DOI: 10.1034/j.1600-0854.2002.31206.x]), and leave the alternative roles for discussion and deeper study in the future.

Reviewer #1 (Public Review):

Summary:

There is a long-believed dogma in the malaria field; a mosquito infected with a single oocyst is equally infectious to humans as another mosquito with many oocysts. This belief has been used for goal setting (and modeling) of malaria transmission-blocking interventions. While recent studies using rodent malaria suggest that the dogma may not be true, there was no such study with human P. falciparum parasites. In this study, the numbers of oocysts and sporozoite in the mosquitoes and the number of expelled sporozoites into artificial skin from the infected mosquito was quantified individually. There was a significant correlation between sporozoite burden in the mosquitoes and expelled sporozoites. In addition, this study showed that highly infected mosquitoes expelled sporozoites sooner.

Strengths:

• The study was conducted using two different parasite-mosquito combinations; one was lab-adapted parasites with Anopheles stephensi and the other was parasites, which were circulated in infected patients, with An. coluzzii. Both combinations showed statistically significant correlations between sporozoite burden in mosquitoes and the number of expelled sporozoites.

• Usually, this type of study has been done in group bases (e.g., count oocysts and sporozoites at different time points using different mosquitoes from the same group). However, this study determined the numbers in individual bases after multiple optimization and validation of the approach. This individual approach significantly increases the power of correlation analysis.

Weaknesses:

• In a natural setting, most mosquitoes have less than 5 oocysts. Thus, the conclusion is more convincing if the authors perform additional analysis for the key correlations (Fig 3C and 4D) excluding mosquitoes with very high total sporozoite load (e.g., more than 5-oocyst equivalent load).

• As written as the second limitation of the study, this study did not investigate whether all expelled sporozoites were equally infectious. For example, Day 9 expelled sporozoites may be less infectious than Day 11 sporozoites, or expelled sporozoites from high-burden mosquitoes may be less infectious because they experience low nutrient conditions in a mosquito. Ideally, it is nice to test the infectivity by ex vivo assays, such as hepatocyte invasion assay, and gliding assay at least for salivary sporozoites. But are there any preceding studies where the infectivity of sporozoites from different conditions was evaluated? Citing such studies would strengthen the argument.

• Since correlation analyses are the main points of this paper, it is important to show 95%CI of Spearman rank coefficient (not only p-value). By doing so, readers will understand the strengths/weaknesses of the correlations. The p-value only shows whether the observed correlation is significantly different from no correlation or not. In other words, if there are many data points, the p-value could be very small even if the correlation is weak.

Reviewer #1 (Public Review):

Summary:

The present study by Mikati et al demonstrates an improved method for in-vivo detection of enkephalin release and studies the impact of stress on the activation of enkephalin neurons and enkephalin release in the nucleus accumbens (NAc). The authors refine their pipeline to measure met and leu enkephalin using liquid chromatography and mass spectrometry. The authors subsequently measured met and leu enkephalin in the NAc during stress induced by handling, and fox urine, in addition to calcium activity of enkephalinergic cells using fiber photometry. The authors conclude that this improved tool for measuring enkephalin reveals experimenter handling stress-induced enkephalin release in the NAc that habituates and is dissociable from the calcium activity of these cells, whose activity doesn't habituate. The authors subsequently show that NAc enkephalin neuron calcium activity does habituate to fox urine exposure, is activated by a novel weigh boat, and that fox urine acutely causes increases in met-enk levels, in some animals, as assessed by microdialysis.

Strengths:

A new approach to monitoring two distinct enkephalins and a more robust analytical approach for more sensitive detection of neuropeptides. A pipeline that potentially could help for the detection of other neuropeptides.

Weaknesses:

Some of the interpretations are not fully supported by the existing data or would require further testing to draw those conclusions. This can be addressed by appropriately tampering down interpretations and acknowledging other limitations the authors did not cover brought by procedural differences between experiments.

Reviewer #1 (Public Review):

Summary:<br /> Mitochondria is the power plant of the cells including neurons. Thomas et al. characterized the distribution of mitochondria in dendrites and spines of L2/3 neurons from the ferret visual cortex, for which visually driven calcium responses of individual dendritic spines were examined. The authors analyzed the relationship between the position of mitochondria and the morphology or orientation selectivity of nearby dendrite spines. They found no correlation between mitochondrion location and spine morphological parameters associated with the strength of synapses, but correlation with the spine-somatic difference in orientation preference and local heterogeneity in preferred orientation of nearby spines. Moreover, they reported that the spines that have a mitochondrion in the head or neck are larger in size and have stronger orientation selectivity. Therefore, they proposed that "mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs."

Strengths:<br /> This paper attempted to address a fundamental question: whether the distribution of the mitochondria along the dendrites of visual cortical neurons is associated with the functions of the spines, postsynaptic sites of excitatory synapses. Two state of the art techniques (2 photon Ca imaging of somata and spines and EM reconstructions of cortical pyramidal neurons) had been used on the same neurons, which provides a great opportunity to examine and correlate the functional properties of spine ultrastructure and spatial distribution of dendritic mitochondria. The conclusion that dendritic mitochondria support functional diversity of spines, but not synaptic strength is surprising and will inspire rethinking the role of mitochondria in synaptic functions.

Weaknesses:<br /> Overall, the findings are intriguing. However, the interpretations of these findings need extra cautions due to the limitations of experimental designs and tools in this study. Neurons in L2/3 of visual cortex are highly diverse in functional properties, which is represented by not only orientation selectivity, but also direction selectivity and spatial/temporal frequency selectivity, etc. The orientation tuning with fixed spatial and temporal frequency may not be the optimal way of stimulating individual synaptic inputs to evaluate synaptic strengths. And the correlation between mitochondria distribution and spine activity evoked by other visual stimulation parameters is worth exploration. Moreover, GCaMP6s measures only spine Ca signals mediated by NMDA and voltage-gated Ca channels, but not sodium currents mediated by ligand-gated or voltage-gated channels. Thus, it reports only some aspects of synaptic properties. Future studies with new tools might help resolve those issues.

Reviewer #1 (Public Review):

Ahn and Amrein characterize the expression of members of the Gr28 family of gustatory receptors in taste neurons in the Drosophila melanogaster larva, define the behaviorally-relevant ligands for these receptors, and use chemogenetic experiments to show, strikingly, that different neurons have opposite behavioral responses to the chemogenetic ligand. They go on to show what neurons need to be silenced to lose responses to bitters, and very nicely show what subunits of the Gr28 bitter receptors are necessary and sufficient for responses to bitters. This is a nice piece of work, rigorously carried out, that tackles the neurons and receptors that drive innate responses to tastants in Drosophila larvae.

The authors have revised the paper to address all of my recommendations. The new cartoons are extremely clear and I appreciate the more measured language when discussing the hypothetical structure and stoichiometry of the functional GR complex.

Joint Public Review:

The work is of fundamental importance and is a useful structural resource to the SARS-CoV2 proteome. The work relies on large-scale SARS-CoV2 genomes and extracts frequent mutations in two key proteins NSP16 and NSP10. The impact of these mutations was studied using x-ray crystallogrpahy, biophysical assays and simulations to propose structural changes. The evidence is, therefore, convincing to suggest NSP10 conformational changes are limited. More analysis on functional implications would be useful to understand the underlying reasons of limited structural variability. The questions raised during the review of the original submission have been addressed by the authors.

Reviewer #1 (Public Review):

Summary:<br /> This study addressed an alternative hypothesis to temporal binding phenomena. In temporal binding, two events that are separated in time are "pulled" towards one another, such that they appear more coincidental. Previous research has shown evidence of temporal binding events in the context of actions and multisensory events. In this context, the author revisits the well-known Libet clock paradigm, in which subjects view a moving clock face, press a button at a time of their choosing to stop the clock, a tone is played (after some delay), and then subjects move the clock dial to the point where the one occurred (or when the action occurred). Classically, the reported clock time is a combination of the action and sound times. The author here suggests that attention can explain this by a mechanism in which the clock dial leads to a roving window of spatiotemporal attention (that is, it extends in both space and time around the dial). To test this, the author conducted a number of experiments where subjects performed the Libet clock experiment, but with a variety of different stimulus combinations. Crucially, a visual detection task was introduced by flashing a disc at different positions along the clock face. The results showed that detection performance was also "pulled" towards the action event or sensory event, depending on the condition. A model of roving spatiotemporal attention replicated these effects, providing further evidence of the attentional window.

Strengths:<br /> The study provides a novel explanation for temporal binding phenomena, with clear and cleverly designed experiments. The results provide a nice fit to the proposed model, and the model itself is able to recapitulate the observed effects.

Weaknesses:<br /> Despite the above, the paper could be clearer on why these effects are occurring. In particular, the control experiment introduced in Experiment 3 is not well justified. Why should a tactile stimulus not lead to a similar effect? There are possibilities here, but the author could do well to lay them out. Further, from a perspective related to the attentional explanation, other alternatives are not explored. The author cites and considers work suggesting that temporal binding relies on a Bayesian cue combination mechanism, in which the estimate is pulled towards the stimulus with the lowest variance, but this is not discussed. None of this necessarily detracts from the findings, but otherwise makes the case for attention less clear.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Millard and colleagues investigated if the analgesic effect of nicotine on pain sensitivity, assessed with two pain models, is mediated by Peak Alpha Frequency (PAF) recorded with resting state EEG. The authors found indeed that nicotine (4 mg, gum) reduced pain ratings during phasic heat pain but not cuff pressor algometry compared to placebo conditions. Nicotine also increased PAF (globally). However, mediation analysis revealed that the reduction in pain ratings elicited by the phasic heat pain after taking nicotine was not mediated by the changes in PAF. Also, the authors only partially replicated the correlation between PAF and pain sensitivity at baseline (before nicotine treatment). At the group-level no correlation was found, but an exploratory analysis showed that the negative correlation (lower PAF, higher pain sensitivity) was present in males but not in females. The authors discuss the lack of correlation.<br /> In general, the study is rigorous, methodology is sound and the paper is well-written. Results are compelling and sufficiently discussed.

Strengths:<br /> Strengths of this study are the pre-registration, proper sample size calculation, and data analysis. But also the presence of the analgesic effect of nicotine and the change in PAF.

Weaknesses:<br /> It would even be more convincing if they had manipulated PAF directly.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript introduced a new behavioral apparatus to regulate the animal's behavioral state naturally. It is a thermal maze where different sectors of the maze can be set to different temperatures; once the rest area of the animal is cooled down, it will start searching for a warmer alternative region to settle down again. They recorded with silicon probes from the hippocampus in the maze and found that the incidence of SWRs was higher at the rest areas and place cells representing a rest area were preferentially active during rest-SWRs as well but not during non-REM sleep.

Strengths:<br /> The maze can have many future applications, e.g., see how the duration of waking immobility can influence learning, future memory recall, or sleep reactivation. It represents an out-of-the-box thinking to study and control less-studies aspects of the animals' behavior.

Weaknesses:<br /> The impact is only within behavioral research and hippocampal electrophysiology.

Reviewer #1 (Public Review):

Summary: The study introduces and validates the Cyclic Homogeneous Oscillation (CHO) detection method to precisely determine the duration, location, and fundamental frequency of non-sinusoidal neural oscillations. Traditional spectral analysis methods face challenges in distinguishing the fundamental frequency of non-sinusoidal oscillations from their harmonics, leading to potential inaccuracies. The authors implement an underexplored approach, using the auto-correlation structure to identify the characteristic frequency of an oscillation. By combining this strategy with existing time-frequency tools to identify when oscillations occur, the authors strive to solve outstanding challenges involving spurious harmonic peaks detected in time-frequency representations. Empirical tests using electrocorticographic (ECoG) and electroencephalographic (EEG) signals further support the efficacy of CHO in detecting neural oscillations.

Strengths:

1. The paper puts an important emphasis on the 'identity' question of oscillatory identification. The field primarily identifies oscillations through frequency, space (brain region), and time (length, and relative to task or rest). However, more tools that claim to further characterize oscillations by their defining/identifying traits are needed, in addition to data-driven studies about what the identifiable traits of neural oscillations are beyond frequency, location, and time. Such tools are useful for potentially distinguishing between circuit mechanistic generators underlying signals that may not otherwise be distinguished. This paper states this problem well and puts forth a new type of objective for neural signal processing methods.

2. The paper uses synthetic data and multimodal recordings at multiple scales to validate the tool, suggesting CHO's robustness and applicability in various real-data scenarios. The figures illustratively demonstrate how CHO works on such synthetic and real examples, depicting in both time and frequency domains. The synthetic data are well-designed, and capable of producing transient oscillatory bursts with non-sinusoidal characteristics within 1/f noise. Using both non-invasive and invasive signals exposes CHO to conditions which may differ in extent and quality of the harmonic signal structure. An interesting followup question is whether the utility demonstrated here holds for MEG signals, as well as source-reconstructed signals from non-invasive recordings.

3. This study is accompanied by open-source code and data for use by the community.

Weaknesses:

1. Due to the proliferation of neural signal processing techniques that have been designed to tackle issues such as harmonic activity, transient and event-like oscillations, and non-sinusoidal waveforms, it is naturally difficult for every introduction of a new tool to include exhaustive comparisons of all others. Here, some additional comparisons may be considered for the sake of context, a selection of which follows, biased by the previous exposure of this reviewer. One emerging approach that may be considered is known as state-space models with oscillatory and autoregressive components (Matsuda 2017, Beck 2022). State-space models such as autoregressive models have long been used to estimate the auto-correlation structure of a signal. State-space oscillators have recently been applied to transient oscillations such as sleep spindles (He 2023). Therefore, state-space oscillators extended with auto-regressive components may be able to perform the functions of the present tool through different means by circumventing the need to identify them in time-frequency. Another tool that should be mentioned is called PAPTO (Brady 2022). Although PAPTO does not address harmonics, it detects oscillatory events in the presence of 1/f background activity. Lastly, empirical mode decomposition (EMD) approaches have been studied in the context of neural harmonics and non-sinusoidal activity (Quinn 2021, Fabus 2022). EMD has an intrinsic relationship with extrema finding, in contrast with the present technique. In summary, the existence of methods such as PAPTO shows that researchers are converging on similar approaches to tackle similar problems. The existence of time-domain approaches such as state-space oscillators and EMD indicates that the field of time-series analysis may yield even more approaches that are conceptually distinct and may theoretically circumvent the methodology of this tool.

2. The criteria that the authors use for neural oscillations embody some operating assumptions underlying their characteristics, perhaps informed by immediate use cases intended by the authors (e.g., hippocampal bursts). The extent to which these assumptions hold in all circumstances should be investigated. For instance, the notion of consistent auto-correlation breaks down in scenarios where instantaneous frequency fluctuates significantly at the scale of a few cycles. Imagine an alpha-beta complex without harmonics (Jones 2009). If oscillations change phase position within a timeframe of a few cycles, it would be difficult for a single peak in the auto-correlation structure to elucidate the complex time-varying peak frequency in a dynamic fashion. Likewise, it is unclear whether bounding boxes with a pre-specified overlap can capture complexes that maneuver across peak frequencies.

3. Related to the last item, this method appears to lack implementation of statistical inferential techniques for estimating and interpreting auto-correlation and spectral structure. In standard practice, auto-correlation functions and spectral measures can be subjected to statistical inference to establish confidence intervals, often helping to determine the significance of the estimates. Doing so would be useful for expressing the likelihood that an oscillation and its harmonic has the same auto-correlation structure and fundamental frequency, or more robustly identifying harmonic peaks in the presence of spectral noise. Here, the authors appear to use auto-correlation and time-frequency decomposition more as a deterministic tool rather than an inferential one. Overall, an inferential approach would help differentiate between true effects and those that might spuriously occur due to the nature of the data. Ultimately, a more statistically principled approach might estimate harmonic structure in the presence of noise in a unified manner transmitted throughout the methodological steps.

4. As with any signal processing method, hyperparameters and their ability to be tuned by the user need to be clearly acknowledged, as they impact the robustness and reproducibility of the method. Here, some of the hyperparameters appear to be: a) number of cycles around which to construct bounding boxes and b) overlap percentage of bounding boxes for grouping. Any others should be highlighted by the authors and clearly explained during the course of tool dissemination to the community, ideally in tutorial format through the Github repository.

5. Most of the validation demonstrations in this paper depict the detection capabilities of CHO. For example, the authors demonstrate how to use this tool to reduce false detection of oscillations made up of harmonic activity and show in simulated examples how CHO performs compared to other methods in detection specificity, sensitivity, and accuracy. However, the detection problem is not the same as the 'identity' problem that the paper originally introduced CHO to solve. That is, detecting a non-sinusoidal oscillation well does not help define or characterize its non-sinusoidal 'fingerprint'. An example problem to set up this question is: if there are multiple oscillations at the same base frequency in a dataset, how can their differing harmonic structure be used to distinguish them from each other? To address this at a minimum, Figure 4 (or a followup to it) should simulate signals at similar levels of detectability with different 'identities' (i.e. different levels and/or manifestations of harmonic structure), and evaluate CHO's potential ability to distinguish or cluster them from each other. Then, does a real-world dataset or neuroscientific problem exist in which a similar sort of exercise can be conducted and validated in some way? If the "what" question is to be sufficiently addressed by this tool, then this type of task should be within the scope of its capabilities, and validation within this scenario should be demonstrated in the paper. This is the most fundamental limitation at the paper's current state.

References:

Beck AM, He M, Gutierrez R, Purdon PL. An iterative search algorithm to identify oscillatory dynamics in neurophysiological time series. bioRxiv. 2022. p. 2022.10.30.514422. doi:10.1101/2022.10.30.514422

Brady B, Bardouille T. Periodic/Aperiodic parameterization of transient oscillations (PAPTO)-Implications for healthy ageing. Neuroimage. 2022;251: 118974.

Fabus MS, Woolrich MW, Warnaby CW, Quinn AJ. Understanding Harmonic Structures Through Instantaneous Frequency. IEEE Open J Signal Process. 2022;3: 320-334.

Jones SR, Pritchett DL, Sikora MA, Stufflebeam SM, Hämäläinen M, Moore CI. Quantitative analysis and biophysically realistic neural modeling of the MEG mu rhythm: rhythmogenesis and modulation of sensory-evoked responses. J Neurophysiol. 2009;102: 3554-3572.

He M, Das P, Hotan G, Purdon PL. Switching state-space modeling of neural signal dynamics. PLoS Comput Biol. 2023;19: e1011395.

Matsuda T, Komaki F. Time Series Decomposition into Oscillation Components and Phase Estimation. Neural Comput. 2017;29: 332-367.

Quinn AJ, Lopes-Dos-Santos V, Huang N, Liang W-K, Juan C-H, Yeh J-R, et al. Within-cycle instantaneous frequency profiles report oscillatory waveform dynamics. J Neurophysiol. 2021;126: 1190-1208.

Reviewer #1 (Public Review):

Summary:<br /> This important study from Godneeva et al. establishes a Drosophila model system for understanding how the activity of Tif1 proteins is modified by SUMO. The authors convincingly show that Bonus, like homologous mammalian Tif1 proteins, is a repressor, and that it interacts with other co-repressors Mi-2/NuRD and SetDB1 in Drosophia ovaries and S2 cells. They also show that Bonus is SUMOylated by Su(var)2-10 on one lysine at its N-terminus to promote its interaction with SetDB1. By combining biochemistry with an elegant reporter gene approach, they show that SUMOylation is important for Bonus interaction with SetDB1, and that this SUMO-dependent interaction triggers high levels of H3K9me3 deposition and gene silencing. While there are still major questions of how SUMO molecularly promotes this process, the authors conducted several experiments that will guide future work. For example, they showed that SUMOylation likely indirectly promotes Bon interaction with SetDB1 because mostly unSUMOylated Bon copurifies with SetDB1. They also show that SUMOylated and unSUMOylated Bon differentially localize within the cell, and preventing Bon SUMOylation alters its subcellular localization. These important experiments disfavor a simple model where SUMO bridges the Bon/SetDB1 interaction and hint at a more complex multi-step assembly process that regulates Bon-dependent transcriptional silencing.

Reviewer #1 (Public Review):

In their study, Zhou et al. unveil the pivotal role of ULK4 in conjunction with STK36, shedding light on their collective impact on GLI2 phosphorylation and the subsequent activation of the SHH pathway. The research delves deep into the intricate interactions between ULK4 and various components of the SHH pathway within the primary cilium.

The main strength of the study lies in the careful and systematic sequence of logical methods. The authors apply the expression of a range of different deletion and mutation constructs and carry out a comprehensive biochemical study of the consequences of depletion and reintroduction of various components in the context of STK36 and ULK4.

Their findings reveal that ULK4 forms dynamic interactions with a complex composed of STK36 and GLI2. It is proposed that ULK4 acts as a scaffold, facilitating the essential interaction between STK36 and GLI2, thereby driving GLI2 phosphorylation by STK36. Notably, the research reveals that the N-terminal pseudokinase domain of ULK4 binds to Stk36, while the C-terminal regulatory domain of ULK4 interacts with Gli2. Moreover, the study presents compelling evidence for co-localization of ULK4 and STK36 with GLI2 at the ciliary tip within NIH 3T3 cells. Importantly, ULK4 and STK36 mutually rely on each other for their accumulation at this ciliary tip.

This intricate mechanism, orchestrated by ULK4, brings to light the nuanced modulation of the SHH pathway. The research is substantiated by rigorous Co-IP experiments, kinase assays, and confocal imaging localization studies. To unravel the fine details of GLI2 phosphorylation at the primary cilium tip, the authors meticulously employ a diverse array of mutated and wild-type constructs of STK36 and ULK4.

In summary, the studiy provide compelling insights into the intricate regulation of signaling pathways. Zhou et al.'s work on ULK4 and STK36 in the SHH pathway deepen our understanding of these complex processes, offering potential avenues for drug development, particularly in the context of cancer therapeutics.

Reviewer #1 (Public Review):

This work provides a new dataset of 71,688 images of different ape species across a variety of environmental and behavioral conditions, along with pose annotations per image. The authors demonstrate the value of their dataset by training pose estimation networks (HRNet-W48) on both their own dataset and other primate datasets (OpenMonkeyPose for monkeys, COCO for humans), ultimately showing that the model trained on their dataset had the best performance (performance measured by PCK and AUC). In addition to their ablation studies where they train pose estimation models with either specific species removed or a certain percentage of the images removed, they provide solid evidence that their large, specialized dataset is uniquely positioned to aid in the task of pose estimation for ape species.

The diversity and size of the dataset make it particularly useful, as it covers a wide range of ape species and poses, making it particularly suitable for training off the shelf pose estimation networks or for contributing to the training of a large foundational pose estimation model. In conjunction with new tools focused on extracting behavioral dynamics from pose, this dataset can be especially useful in understanding the basis of ape behaviors using pose.

Overall this work is a terrific contribution to the field, and is likely to have a significant impact on both computer vision and animal behavior.

Strengths:<br /> - Open source dataset with excellent annotations on the format, as well as example code provided for working with it<br /> - Properties of the dataset are mostly well described<br /> - Comparison to pose estimation models trained on humans vs monkeys, finding that models trained on human data generalized better to apes than the ones trained on monkeys, in accordance with phylogenetic similarity. This provides evidence for an important consideration in the field: how well can we expect pose estimation models to generalize to new species when using data from closely or distantly related ones.<br /> - Sample efficiency experiments reflect an important property of pose estimation systems, which indicates how much data would be necessary to generate similar datasets in other species, as well as how much data may be required for fine tuning these types of models (also characterized via ablation experiments where some species are left out)<br /> - The sample efficiency experiments also reveal important insights about scaling properties of different model architectures, finding that HRNet saturates in performance improvements as a function of dataset size sooner than other architectures like CPMs (even though HRNets still perform better overall).

Reviewer #1 (Public Review):

Summary: The study provides valuable insights into the role of PfMORC in Plasmodium's epigenetic regulation, backed by a comprehensive methodological approach. The overarching goal was to understand the role of PfMORC in epigenetic regulation during asexual blood stage development, particularly its interactions with ApiAP2 TFs and its potential involvement in the regulation of genes vital for Plasmodium virulence. To achieve this, they conducted various analyses. These include a proteomic analysis to identify nuclear proteins interacting with PfMORC, a study to determine the genome-wide localization of PfMORC at multiple developmental stages, and a transcriptomic analysis in PfMORCHA-glmS knockdown parasites. Taken together, this study suggests that PfMORC is involved in chromatin assemblies that contribute to the epigenetic modulation of transcription during the asexual blood stage development.

Strengths: The study employed a multi-faceted approach, combining proteomic, genomic, and transcriptomic analyses, providing a holistic view of PfMORC's role. The proteomic analysis successfully identified several nuclear proteins that may interact with PfMORC. The genome-wide localization offered valuable insights into PfMORC's function, especially its predominant recruitment to subtelomeric regions. The results align with previous findings on PfMORC's interaction with ApiAP2 TFs. Notably, the authors meticulously contextualized their findings with prior research, including pre-prints, adding credibility to their work.

Weaknesses: While the study identifies potential interacting partners and loci of binding, direct functional outcomes of these interactions remain an inference. The authors heavily rely on past research for some of their claims. While it strengthens some assertions, it might indicate a lack of direct evidence in the current study for particular aspects. The declaration that PfMORC may serve as an attractive drug target is substantial. While the data suggests its involvement in essential processes, further studies are required to validate its feasibility as a drug target.

Reviewer #1 (Public Review):

Summary: Hansen et al. dissect the molecular mechanisms of bacterial ice nucleating proteins mutating the protein systematically. They assay the ice nucleating ability for variants changing the R-coils as well as the coil capping motifs. The ice nucleation mechanism depends on the integrity of the R-coils, without which the multimerization and formation of fibrils are disrupted.

Strengths: The effects of mutations are really dramatic, so there is no doubt about the effect. The variants tested are logical and progressively advance the story. The authors identify an underlying mechanism involving multimerization, which is plausible and compatible with EM data. The model is further shown to work in cells by tomography.

Weaknesses: The theoretical model presented for how the proteins assemble into fibrils is simple, but not supported by much data.

Reviewer #1 (Public Review):

Summary:

Very systematic generation of phosphosite-specific antisera to monitor FFA2 phosphorylation in native cells and tissues. Provides evidence that FFA2 phosphorylation is tissue-specific.

Strengths:

Technical tour de force, rigorous experimental approaches taking advantage of wt and DREADD versions of FFA2 to make sure that ligand-and receptor-dependent phosphorylations are indeed specific to FFA2.

Weaknesses:

In this reviewer's opinion, the only shortcoming is that the implications of tissue-selective phosphorylation barcoding remain unexplored. However, I understand that tool development is required before tools are used to provide insight into the functional outcomes of receptor regulation by phosphorylation. The study is a technical tour de force to generate highly valuable tools. I have no major criticisms but suggest adding an additional aspect to the discussion as specified below.

Arrestins are highly flexible and dynamic phosphate sensors. If two arrestins have to recognize 800 different phosphorylated GPCRs, is it possible that any barcode serves the same purpose: arrestin recognition followed by signal arrest and internalization? Because phosphorylation barcoding is linked to G protein-independent signaling, which is claimed by some but is experimentally unsupported, and because arrestins don't transduce receptor signals on their own (they only scaffold signaling components and shuttle receptors within cellular compartments), I would also include this option in the discussion, i.e. that the different barcodes are a way nature may have chosen to regulate the location of 800 GPCRs by only 2 arrestins.

Reviewer #1 (Public Review):

This is an interesting, informative, and well-designed study that combines theoretical and experimental methodologies to tackle the phenomenon of higher-resolution structures/substructures in model biomolecular condensates. The results should be published. However, there is significant room for improvement in the presentation and interpretation of the results. As it stands, the precise definition of "frustration," which is a main theme of this manuscript (as emphasized in the title), is not sufficiently well articulated. This situation should be rectified to avoid "frustration" becoming a "catch-all" term without a clear perimeter of applicability rather than a precise, informative description of the physical state of affairs. There are also a few other concerns, e.g., regarding interpretation of correlation of phase-separation critical temperature and transfer free energy of amino acid residues as well as the difference between critical temperature and onset temperature, and the way the simulated configurations are similar to that of gyroids. Accordingly, the manuscript should be revised to address the following:

1. It is accurately pointed out on p.4 that elastin-like polypeptides (ELPs) undergo heat-induced phase separation and therefore exhibit lower critical solution temperatures (LCSTs). But it is not entirely clear how this feature is reproduced by the authors' simulation. A relationship between simulated surface tension and "transition temperature" is provided in Fig.1C; but is the "transition temperature" (authors cited ref.41 by Urry) the same as critical temperature? Apparently, Urry's Tt is "critical onset temperature", the temperature when phase separation happens at a given polymer concentration. This is different from the (global) critical temperature LCST - though the two may be correlated-or not-depending on the shape of the phase boundary. Moreover, is the MOFF coarse-grained forcefield (first step in the multi-scale simulation), by itself, capable of reproducing heat-induced phase separation in a way similar to the forcefield of Dignon et al., ACS Cent Sci 5, 821-230 (2019)? Or is this temperature-dependent effect appearing only subsequently, after the implementation of the MARTINI and/or all-atom steps? Clarification is needed. To afford a more informative context for the authors' introductory discussion, the aforementioned Dignon et al. work and the review by Cinar et al. [Chem Eur J 25, 13049-13069 (2019)], both touching upon the physical underpinning of the LCST feature of elastin, should also be cited along with refs.41-43.

2. "Frustration" and "frustrated" are used prominently in the manuscript to characterize certain observed molecular configurations (11 times total, in both the title and in the abstract). Apparently, it is the most significant conceptual pronouncement of this work, hence its precise meaning is of central importance to the authors' thesis. Whereas one should recognize that the theoretical and experimental observations are striking without invocation of the "frustration" terminology, usage of the term can be useful if it offers a unifying conceptual framework. However, as it stands, a clear definition of the term "frustration" is lacking, leaving readers to wonder what molecular configurations are considered "frustrated" and what are not (i.e., is the claim of observation of frustration falsifiable?). For instance, "frustrated microphase separation" appears in both the title and abstract. A logical question one may ask is: "Are all microphase separations frustrated"? If the answer is in the affirmative, does invocation of the term "frustration" add anything to our physical insight? If the answer is not in the affirmative, then how does one distinguish between microphase separations that are frustrated from those that are not frustrated? Presumably all simulated and experimental molecular configurations in the present study are those of lowest free energy for the given temperature. In other words, they are what they are. In the discussion about frustrated phase separation on p.13, for example, the authors appear to refer to the fact that chain connectivity is preventing hydrophobic residues to come together in a way to achieve the most favorable interactions as if there were no chain connectivity (one may imagine in that case all the hydrophobic residues will form a large cluster without microphase separation). Is this what the authors mean by "frustration"? If that's true, isn't that merely stating the obvious, at least for the observed microphase separation? In general, does "frustration" always mean deviation of actual, physical molecular configurations from certain imagined/hypothetical/reference molecular configurations, and therefore dependent upon the choice of the imagined reference configuration? If this is how the authors apply the term "frustration" in the present work, what is the zero-frustration reference state/configuration for microphase separation? And, similarly, what is the zero-frustration reference state/configuration when frustrated EPS-water interactions are discussed (~p.14-p.15, Fig.5)? How do non-frustrated water-protein interactions look like? Is the classic clathrate-like organization of water hydrogen bonds around small nonpolar solute "frustrated"?

3. In the discussion about the correlation of various transfer free energy scales for amino acids and Urry's critical onset temperature (ref.41) on p.11 and Fig.4, is there any theoretical relationship to be expected between the interactions among amino acids of ELPs and their critical onset temperatures? While a certain correlation may be intuitively expected if the free energy scale "is working", is there any theoretical insight into the mathematical form of this relationship? A clarifying discussion is needed because it bears logically on whether the observed correlation or lack thereof for different transfer energy scales is a good indication of the adequacy of the energy scales in describing the actual physical interactions at play. This question requires some prior knowledge of the expected mathematical relationship between interaction parameters and onset temperature.

4. To provide a more comprehensive context for the present study, it is useful to compare the microphase separation seen in the authors' simulation with the micelle-like structures observed in recent simulated condensed/aggregated states of hydrophobic-polar (HP) model sequences in Statt et al., J Chem Phys 152, 075101 (2020) [see esp. Fig.6] and Wessén et al., J Phys Chem B 126, 9222-9245 (2022) [see, e.g., Fig.10].

5. "Gyroid-like morphology" is mentioned several times in the manuscript (p.4, p.8, p.17, Fig.S3). This is apparently an interesting observation, but a clear explanation is lacking. A more detailed and specific discussion, perhaps with additional graphical presentations, should be provided to demonstrate why the simulated condensed-phase ELP configurations are similar to the classical description of gyroid as in, e.g., Terrones & Mackay, Chem Phys Lett 207, 45-50 (1993) and Lambert et al., Phil Trans R Soc A 354, 2009-2023 (1996).

Reviewer #1 (Public Review):

This remarkable and creative study from the Asbury lab examines the extent to which mechanical coupling can coordinate the growth of two microtubules attached to isolated kinetochores. The concept of mechanical coupling in kinetochores was proposed in the mid-1990s and makes sense intuitively (as shown in Fig. 1B). But intuitive concepts still need experimental validation, which this study at long last provides. The experiments described in this paper will serve as a foundation for the transition of an intuitive concept into a robust, quantitative, and validated model.

The introduction cites at least 5 papers that proposed mechanical coupling in kinetochores, as well as 5 theoretical studies on mechanical coupling within microtubule bundles, so it's clear that this manuscript will be of considerable interest to the field. The intro is very well written (as is the manuscript in general), but I recommend that the authors include a brief review of the variable size of k-fibers across species, to help the reader contextualize the problem. For example, budding yeast kinetochores are built around a single microtubule (Winey 1995), so mechanical coupling is not relevant for this species.

Indeed, the use of yeast kinetochores to study mechanical coupling is an odd fit, because these structures did not evolve to support such coupling. There is no doubt that yeast kinetochores are useful for demonstrating mechanical coupling and for measuring the stiffnesses necessary to achieve coupling, but I recommend that the authors include a caveat somewhere in the manuscript, perhaps in the place where they discuss their use of simple elastic coupling as compared to viscoelastic coupling or strain-stiffening. It's easy to imagine that kinetochores with large k-fibers might require complex coupling mechanisms, for example. And is mechanical coupling relevant for holocentric kinetochores like those found in C. elegans?

The paper shows considerable rigour in terms of experimental design, statistical analysis, and presentation of results. My only comment on this topic relates to the bandwidth of the dual-trap assay, which I recommend describing in the main text in addition to the methods. For example, the authors note that the stage position is updated at 50 Hz. The authors should clearly explain that this bandwidth is sufficiently fast relative to microtubule growth speeds.

After describing their measurements, the authors use Monte Carlo simulations to show that pauses are essential to a quantitative explanation of their coupling data. Apparently, there is a history of theoretical approaches to coupling, as the introduction cites 5 theoretical studies.

Overall, this paper is rigorous, creative, and thought-provoking. The unique experimental approach developed by the Asbury lab shows great promise, and I very much look forward to future iterations.

Reviewer #1 (Public Review):

The paper offers interesting insight into the allosteric communication pathways of the CTFR protein. A mutation to this protein can cause cystic fibrosis and both synthetic and endogenous ligands exert allosteric control of the function of this pivotal enzyme. The current study utilizes Gaussian Network Models (GNMs) of various substrate and mutational states of CFTR to quantify and characterize the role of individual residues in contributing to two main quantities that the authors deem important for allostery: transfer entropy (TE) and cross correlation. I found the TE of the Apo system and the corresponding statistical analysis particularly compelling. The authors updated the manuscript nicely to include the limitations of the chosen model (GNM) and thus allow the reader to assess the limitations of the results. I appreciated the comprehensive discussion of a proposed mechanism by which allostery is achieved in the protein (though I would have put that in the introduction and had it motivate the choice of methods). This discussion allows the reader to place the allosteric mechanism of this protein in the broader context of protein allostery.

Reviewer #1 (Public Review):

The authors have previously employed micrococcal nuclease tethered to various Mcm subunits to the cut DNA to which the Mcm2-7 double hexamers (DH) bind. Using this assay, they found that Mcm2-7 DH are located on many more sites in the S. cerevisiae genome than previously shown. They then demonstrated that these sites have characteristics consistent with origins of DNA replication, including the presence of ARS consensus sequences, the location of very inefficient sites of initiation of DNA replication in vivo, and for the most part are free of nucleosomes. They contain a G-C skew and they locate to intergenic regions of the genome. The authors suggest, consistent with published single molecule results, that there are many more potential origins in the S. cerevisiae genome than previously annotated, but also conclude that many of the newly discovered Mcm2-7 DH are very infrequently used as active origins of DNA replication.

The results are convincing and are consistent with prior observations. The analysis of the origin associated features is informative.

Specific Comments:

1. Page 8. The addition of an estimate of the most active origins using Southern blotting is fine for highly active origins, but how was Southern blotting used to calculate that 1-2% of cells in the eight cohort have an Mcm complex loaded.

Reviewer #1 (Public Review):

This paper describes the discovery, functional analysis and structure of TcaP, a protein encoded by the Vibrio phage satellite PLE, that forms a size-determining scaffold around PLE procapsids made from helper phage ICP1 structural proteins.

The system displays a fascinating similarity to the P2/P4 system, which had previously been unique in its use of a dominant, size-determining external scaffolding protein (Sid). An interesting observation is that PLE appears to be dependent on small capsids for efficient transduction, a phenomenon not previously seen in headful packaging phage/satellite pairs. It is not clear why this is the case.

The work is interesting, comprehensive and of high quality. The reconstruction and modeling statistics are good; unfortunately, although the map has clear alpha-helical density around the threefold axes, the TcaP model does not include this critical region. The comparison to Sid provides an illustration of probable convergent evolution.

The paper constitutes an important contribution to the field of phage and virus structure and assembly, with implications for understanding the evolution of phage satellites and for macromolecular assembly processes in general.

Joint Public Review:

In this study, Wang et al extend on their previous finding of a novel quality control pathway, the MAGIC pathway. This pathway allows misfolded cytosolic proteins to become imported into mitochondria and there they are degraded by the LON protease. Using a screen, they identify Snf1 as a player that regulates MAGIC. Snf1 inhibits mitochondrial protein import via the transcription factor Hap4 via an unknown pathway. This allows cells to adapt to metabolic changes, upon high glucose levels, misfolded proteins become imported and degraded, while during low glucose growth conditions, import of these proteins is prevented, and instead import of mitochondrial proteins is preferred.

This is a nice and well-structured manuscript reporting on important findings about a regulatory mechanism of a quality control pathway. The findings are obtained by a combination of mostly fluorescent protein-based assays. Findings from these assays support the claims well.

While this study convincingly describes the mechanisms of a mitochondria-associated import pathway using mainly model substrates, my major concern is that the physiological relevance of this pathway remains unclear: what are endogenous substrates of the pathway, to which extent are they imported and degraded, i.e. how much does MAGIC contribute to overall misfolded protein removal (none of the experiments reports quantitative "flux" information). Lastly, it remains unclear by which mechanism Snf1 impacts on MAGIC or whether it is "only" about being outcompeted by mitochondrial precursors.

Reviewer #1 (Public Review):

Summary:<br /> Sumarac et al investigate differences in globus pallidus internus (GPi) spike activity and short- and long-term plasticity of direct pathway projections in patients with Parkinson's disease (PD) and dystonia. Their main claims are that GPi neurons exhibit distinct characteristics in these two disorders, with PD associated with specific power-frequency oscillations and dystonia showing lower firing rates, increased burstiness, and less regular activity. Additionally, long-term plasticity and synaptic depression appear to differ between the two conditions. The authors suggest that these findings support the concept of hyperfunctional GPi output in PD and hypofunctional output in dystonia, possibly driven by variations in the plasticity of striato-pallidal synapses. Overall enthusiasm is relatively high, but I think the discussion omits discussing findings that don't align well with standard models.

Strengths:<br /> These types of studies are valuable as the data arise from patients who have dystonia or PD. This could provide unique insights into disease pathophysiology that might not be recapitulated in animal systems work.

Weaknesses:<br /> - The rate model and indirect/direct pathway ideas lack explanatory power; too much of the hypothesis generation and discussion in this manuscript is set in the context of these old ideas. Their data in my view emphasize this somewhat emphatically. Most patients with the 'hypokinetic' movement disorder PD have dystonia as a part of their motor features. Dystonia is a form of excessive muscle activation that on the one hand is 'hyperkinetic' but on the other usually decreases the speed of motor tasks, even in patients with primary dystonia. Similarly, PD patients display a bewildering variety of hyperkinetic manifestations as well (rest tremor, dystonia, dyskinesia). If these are truly independent classifications, i.e. hyper- versus hypo-kinetic, the authors must acknowledge that there is considerable overlap in the spike activity across groups - numerous dystonia patients display higher discharge rates than the majority of the PD sample. Based on the firing rate alone, it would not be possible to distinguish these groups.

- If beta power is pathognomonic of parkinsonism, the authors found no differences in beta-related spike discharges across the groups. One would have predicted greater beta power in PD than in primary dystonia. This should be discussed explicitly and an interpretation should be provided.

- The study lacks a healthy control group, making it challenging to differentiate disease-specific findings from normal variations in GPi activity and plasticity. Although this is acknowledged in the discussion, this complicates the interpretation of the results. The sample sizes for PD and dystonia patients are relatively small, and the study combines various forms of dystonia, potentially masking subtype-specific differences. A larger and more homogenous sample could enhance the study's reliability.

- While they mention that data are available on request, sharing data openly would increase transparency and allow for independent validation of the results. It is unclear how sharing deidentified data would compromise patient privacy or present ethical issues of any kind, as claimed by the authors.

- They appropriately acknowledge several limitations, such as the inability to use pharmacological interventions and the need for further research in the chronic setting.

- The manuscript highlights differences in GPi activity and plasticity between PD and dystonia but could provide more context on the clinical implications of these findings, particularly regarding what the implications would be novel paradigms for deep brain stimulation.

- While statistical tests are mentioned, the manuscript could benefit from a more detailed presentation of statistical methods, including correction for multiple comparisons and effect sizes. Did the authors consider different recording sites within each patient as independent observations? I think this is not appropriate if that was the case.

- The manuscript could elaborate on the potential mechanisms underlying the observed differences in GPi activity and plasticity and their relevance to the pathophysiology of PD and dystonia.

Reviewer #1 (Public Review):

Summary:<br /> Rigor in the design and application of scientific experiments is an ongoing concern in preclinical (animal) research. Because findings from these studies are often used in the design of clinical (human) studies, it is critical that the results of the preclinical studies are valid and replicable. However, several recent peer-reviewed published papers have shown that some of the research results in cardiovascular research literature may not be valid because their use of key design elements is unacceptably low. The current study is designed to expand on and replicate previous preclinical studies in nine leading scientific research journals. Cardiovascular research articles that were used for examination were obtained from a PubMed Search. These articles were carefully examined for four elements that are important in the design of animal experiments: use of both biological sexes, randomization of subjects for experimental groups, blinding of the experimenters, and estimating the proper size of samples for the experimental groups. The findings of the current study indicate that the use of these four design elements in the reported research in preclinical research is unacceptably low. Therefore, the results replicate previous studies and demonstrate once again that there is an ongoing problem in the experimental design of preclinical cardiovascular research.

Strengths:<br /> This study selected four important design elements for study. The descriptions in the text and figures of this paper clearly demonstrate that the rate of use of all four design elements in the examined research articles was unacceptably low. The current study is important because it replicates previous studies and continues to call attention once again to serious problems in the design of preclinical studies, and the problem does not seem to lessen over time.

Weaknesses:<br /> The current study uses both descriptive and inferential statistics extensively in describing the results. The descriptive statistics are clear and strong, demonstrating the main point of the study, that the use of these design elements is quite low, which may invalidate many of the reported studies. In addition, inferential statistical tests were used to compare the use of the four design elements against each other and to compare some of the journals. The use of inferential statistical tests appears weak because the wrong tests may have been used in some cases. However, the overall descriptive findings are very strong and make the major points of the study.

Reviewer #1 (Public Review):

Summary:<br /> This work successfully identified and validated TRLs in hepatic metastatic uveal melanoma, providing new horizons for enhanced immunotherapy. Uveal melanoma is a highly metastatic cancer that, unlike cutaneous melanoma, has a limited effect on immune checkpoint responses, and thus there is a lack of formal clinical treatment for metastatic UM. In this manuscript, the authors described the immune microenvironmental profile of hepatic metastatic uveal melanoma by sc-RNAseq, TCR-seq, and PDX models. Firstly, they identified and defined the phenotypes of tumor-reactive T lymphocytes (TRLs). Moreover, they validated the activity of TILs by in vivo PDX modeling as well as in vitro co-culture of 3D tumorsphere cultures and autologous TILs. Additionally, the authors found that TRLs are mainly derived from depleted and late-activated T cells, which recognize melanoma antigens and tumor-specific antigens. Most importantly, they identified TRLs-associated phenotypes, which provide new avenues for targeting expanded T cells to improve cellular and immune checkpoint immunotherapy.

Strengths:<br /> Jonas A. Nilsson, et al. has been working on new therapies for melanoma. The team has also previously performed the most comprehensive genome-wide analysis of uveal melanoma available, presenting the latest insights into metastatic disease. In this work, the authors performed paired sc-RNAseq and TCR-seq on 14 patients with metastatic UM, which is the largest single-cell map of metastatic UM available. This provides huge data support for other studies of metastatic UM.

Weaknesses:<br /> Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated. That is, insufficient analyses are performed to fully support the key claims in the manuscript by the data presented. In particular：

The author's description of the overall results of the article should be logical, not just a description of the observed phenomena. For example, the presentation related to the results of TRLs lacked logic. In addition, the title of the article emphasizes the three subtypes of hepatic metastatic UM TRLs, but these three subtypes are not specifically discussed in the results as well as the discussion section. The title of the article is not a very comprehensive generalization and should be carefully considered by the authors.

The authors' claim that they are the first to use autologous TILs and sc-RNAseq to study immunotherapy needs to be supported by the corresponding literature to be more convincing. This can help the reader to understand the innovation and importance of the methodology. In addition, the authors argue that TILs from metastatic UM can kill tumor cells. This is the key and bridging point to the main conclusion of the article. Therefore, the credibility of this conclusion should be considered. Metastatic UM1 and UM9 remain responsive to autologous tumors under in vitro conditions with their autologous TILs. In contrast, UM22, also as a metastatic UM, did not respond to TIL treatment. In particular, the presence of MART1-responsive TILs. The reliability of the results obtained by the authors in the model of only one case of UM22 liver metastasis should be considered. The authors should likewise consider whether such a specific cellular taxon might also exist in other patients with metastatic UM, producing an immune response to tumor cells. The results would be more comprehensive if supported by relevant data.

In addition, the authors in that study used previously frozen biopsy samples for TCR-seq, which may be associated with low-quality sequencing data, high risk of outcome indicators, and unfriendly access to immune cell information. The existence of these problems and the reliability of the results should be considered. If special processing of TCR-seq data from frozen samples was performed, this should also be accounted for.

Joint Public Review:

Murphy, Fancy and Skene performed a reanalysis of snRNA-seq data from Alzheimer Disease (AD) patients and healthy controls published previously by Mathys et al. (2019), arriving at the conclusion that many of the transcriptional differences described in the original publication were false positives. This was achieved by revising the strategy for both quality control and differential expression analysis. With this re-analysis, the authors aim to raise awareness of the impact of data analysis choices for scRNA-seq data and to caution focus on putatively wrongly identified genes in the AD research community. The revised manuscript has been improved by separating QC and DE analysis, which makes interpretation of both steps more straightforward.

STRENGTHS:

The authors demonstrate that the choice of data analysis strategy can have a vast impact on the results of a study, which in itself may not be obvious to many researchers.

The authors apply a pseudobulk-based differential expression analysis strategy (essentially, adding up counts from all cells per individual and comparing those counts with standard RNA-seq differential expression tests), which is (a) in line with latest community recommendations, (b) different from the "default options" in most popular scRNA-seq analysis suites, and (c) explains the vastly different number of DEGs identified by the authors and the original publication. The recommendation of this approach together with a detailed assessment of the DEGs found by both methodologies could potentially be a useful finding for the research community. Unfortunately, it is currently not sufficiently substantiated.

All code and data used in this study are publicly available to the readers.

WEAKNESSES:

The authors interpret the fact that they found fewer DEGs with their method than the original paper as a good thing by making the assumption that all genes that were not found were false positives. However, they do not prove this, and it is likely that at least some genes were not found due to a lack of statistical power and not because they were actually "incorrect". The original paper also had performed independent validations of some genes that were not found here. I had raised this weakness in my first review, but it was not explicitly addressed and still pertains to the revised manuscript. The authors have added an analysis that shows that "pseudoreplication" is prone to false positive (FP) discoveries for high cell numbers (Fig. 1f), but this does not prove that all of Mathys' DEGs were wrong.

I am concerned that almost all DEGs found by the authors are in the rare cell types, foremost the rare microglia (see Fig. 1e). Indeed, there is a weak negative correlation between cell counts and numbers of DEGs (Fig. 1e), if the correlation analysis is to be believed (see next point). It is unclear to me how many cells the pseudo-bulk counts were based on for these cell types, but it seems that (a) there were few and (b) there were quite few reads per cells. If both are the case, the pseudobulk counts for these cell populations might be rather noisy and the DEG results liable to outliers with extreme fold changes. Supp. Fig. 3b now shows three examples of DEGs, of which one (EGR1) looks like the DE call is indeed largely driven by four outliers, while Supp. Fig 3a shows at least one gene (BEX1) that could be FP of the pseudobulk approach due to insufficient statistical power. The authors go on to cite two papers (one is their own, published in a journal with suspected lack of appropriate quality assurance measures https://predatoryreports.org/the-predatory-journals-1), to support that the finding of DEGs in microglia "makes more sense" (l. 127). In summary, neither the presented examples nor the supporting literature are convincing. Lastly, the authors even show themselves that their approach is liable to FPs if applied with very low cell numbers in the range of those for microglia and OPCs (Fig. 1g).

The correlation analysis between cell counts and number of DEGs found is weak. In all three cases (Fig. 1c, d, e) the correlation is largely driven by a single outlier data point.

The authors claim they improved the quality control of the dataset but offer no objective metric to assess this putative improvement. The authors' QC procedure removes some 20k cells that had not been filtered out by Mathys' et al. As the authors state themselves, this difference is mostly due to the removal of cells with a high mitochondrial read content. Murphy et al use a fixed threshold for the mitochondrial percentage of reads, while the original paper had removed cell clusters with an "abnormally high" mitochondrial read fraction. That also seems reasonable, given that some cells might have a higher mitochondrial read content for reasons other than being "low quality". Simply stating that Mathys' approach was ineffective at removing cells with high mitochondrial read content is a self-fulfilling prophecy given the difference in approach, and itself not proof that the original QC procedure was inferior.

Batch correction: "Dataset integration has become a common step in single-cell RNA-Seq protocols and is recommended to remove confounding sources of variation" (l. 38). While it is true that many authors now choose to perform an integration step as part of their analysis workflow, this is by no means uncontroversial as there is a risk of "over-integration" and loss of true biological differences. I had raised this point previously, but the authors chose not to address it (quoted text and line numbers updated). Given that there is controversy in the literature and "community opinion" on the topic of data integration, this is another example of the authors claiming superiority in analysis without showing proof.

Due to a lack of comparison with other methods and due to the fact that the author's methodology was only applied to a single dataset, the paper presents merely a case study, which could be useful but falls short of providing a general recommendation for a best practice workflow.

APPRAISAL:

The manuscript could help to increase awareness of data analysis choices in the community, but only if the superiority of the methodology was clearly demonstrated. However, the authors only show that there are differences but have no convincing (orthogonal) evidence that their methodology was indeed better. This applies to both QC and DE analysis.

Reviewer #1 (Public Review):

This paper presents a highly compelling and novel hypothesis for how the brain could generate signals to guide navigation toward remembered goals. Under this hypothesis, which the authors call "Endotaxis", the brain co-opts its ancient ability to navigate up odor gradients (chemotaxis) by generating a "virtual odor" that grows stronger the closer the animal is to a goal location. This idea is compelling from an evolutionary perspective and a mechanistic perspective. The paper is well-written and delightful to read.

The authors develop a detailed model of how the brain may perform "Endotaxis", using a variety of interconnected cell types (point, map, and goal cells) to inform the chemotaxis system. They tested the ability of this model to navigate in several state spaces, representing both physical mazes and abstract cognitive tasks. The Endotaxis model performed reasonably well across different environments and different types of goals.

The authors further tested the model using parameter sweeps and discovered a critical level of network gain, beyond which task performance drops. This critical level approximately matched analytical derivations.

Overall, this paper provides a very compelling model for how neural circuits may have evolved the ability to navigate towards remembered goals, using ancient chemotaxis circuits.

This framework will likely be very important for understanding how the hippocampus (and other memory/navigation-related circuits) interfaces with other processes in the brain, giving rise to memory-guided behavior.

Reviewer #1 (Public Review):

Cell death plays a critical role on regulating organogenesis. During tooth morphogenesis, apoptosis of embryonic dental tissue plays critical roles on regulating tooth germ development. The current study focused on ferroptosis, another way of cell death which has rarely been investigated in tooth development, and showed it may also play an important role on regulating the tooth dimension. The topic is novel and interesting, but the experimental design has some flaws which compromised the study.

The entire study was based on ex vivo tooth germ explant culture. I hope the authors can continue working on this direction with more convincing transgenic models.

Reviewer #1 (Public Review):

Summary:<br /> In the present manuscript, the authors present the results of a well-designed, thoughtful, and well-motivated study, targeting the role of angular gyrus in insight-based memory gains. The study is well conducted, timely, and presents clear-cut behavioral results. However, the analysis of the EEG-data lacks clarity and leaves many open questions - especially with regard to the representational similarity analyses. (Nevertheless, analogous concerns with regard to the focus on the three-way interaction and the comparison of linked vs. non-linked events pertain similarly to the connectivity analyses.)

Strengths:<br /> - Well-conducted study with a proper sham-controlled TMS design.<br /> - Clever insight-based memory task.<br /> - Interesting behavioral findings.

Weaknesses:<br /> - "We then calculated Pearson's correlations to compare the power patterns across theta frequency between the time points of linked events (A with B), as well as between the time points of non-linked events (A with X) for the pre- and the post-phase separately, separately for stories linked via imagination and via observation." (p.34)

The RSA basically asks on the lowest level, whether neural activation patterns (as measured by EEG) are more similar between linked events compared to non-linked events. At least this is the first question that should be asked. However, on page 11 the authors state: "We examined insight-induced effects on neural representations for linked events [...]". Hence, the critical analysis reported in the manuscript fully ignores the non-linked events and their neural activation patterns. However, the non-linked events are a critical control. If the reported effects do not differ between linked and non-linked events, there is no way to claim that the effects are due to experimental manipulation - neither imagination nor observation. Hence, instead of immediately reporting on group differences (sham vs. control) in a two-way interaction (pre vs. post X imagination vs. observation), the authors should check (and report) first, whether the critical experimental manipulation had any effect on the similarity of neural activation patterns in the first place.

Overall, the focus on the targeted three-way interaction is poorly motivated. Also, a functional interpretation is largely missing.

- "Interestingly, we observed a different pattern of insight-related representational pattern changes for non-linked events."

It is not sufficient to demonstrate that a given effect is present in one condition (linked events) but not the other (non-linked events). To claim that there are actually different patterns, the authors would need to compare the critical conditions directly (Nieuwenhuis et al., 2011).

- "This analysis yielded a negative cluster (p = 0.032, ci-range = 0.00, SD = 0.00) in the parieto-temporal region (electrodes: T7, Tp7, P7; Fig. 3B)." (p. 11)

The authors report results with specificity for certain topographical locations. However, this is in stark contrast to the fact that the authors derived time X time RSA maps.

"These theta power values were then combined to create representational feature vectors, which consisted of the power values for four frequencies (4-7 Hz) × 41 time points (0-2 seconds) × 64 electrodes. We then calculated Pearson's correlations to compare the power patterns across theta frequency between the time points of linked events (A with B), as well as between the time points of non-linked events (A with X) for the pre- and the post-phase separately, separately for stories linked via imagination and via observation. To ensure unbiased results, we took precautions not to correlate the same combination of stories twice, which prevented potential inflation of the data. To facilitate statistical comparisons, we applied a Fisher z-transform to the Pearson's rho values at each time point. This yielded a global measure of similarity on each electrode site. We, thus, obtained time × time similarity maps for the linked events (A and B) and the non-linked events (A and X) in the pre- and post-phases, separately for the insight gained through imagination and observation." (p. 34+35)

If RSA values were calculated at each time point and electrode, the Pearson correlations would have been computed effectively between four samples only, which is by far not enough to derive reliable estimates (Schönbrodt & Perugini, 2013). The problem is aggravated by the fact that due to the time and frequency smoothing inherent in the time-frequency decomposition of the EEG data, nearby power values across neighboring theta frequencies are highly similar to start with. (e.g., Schönauer et al., 2017; Sommer et al., 2022)

Alternative approaches would be to run the correlations across time for each electrode (resulting in the elimination of the time dimension) or to run the correlations at each time point across electrodes (resulting in the elimination of topographic specificity).

At least, the authors should show raw RSA maps for linked and non-linked events in the pre- and post-phases separately for the insight gained through imagination and observation in each group, to allow for assessing the suitability of the input data (in the supplements?) before progressing to reporting the results of three-way interactions.

References:<br /> Nieuwenhuis, S., Forstmann, B. U., & Wagenmakers, E.-J. (2011). Erroneous analyses of interactions in neuroscience: A problem of significance. Nature Neuroscience, 14(9), 1105-1107. https://doi.org/10.1038/nn.2886<br /> Schönauer, M., Alizadeh, S., Jamalabadi, H., Abraham, A., Pawlizki, A., & Gais, S. (2017). Decoding material-specific memory reprocessing during sleep in humans. Nature Communications, 8(1), 15404. https://doi.org/10.1038/ncomms15404<br /> Schönbrodt, F. D., & Perugini, M. (2013). At what sample size do correlations stabilize? Journal of Research in Personality, 47(5), 609-612. https://doi.org/10.1016/j.jrp.2013.05.009<br /> Sommer, V. R., Mount, L., Weigelt, S., Werkle-Bergner, M., & Sander, M. C. (2022). Spectral pattern similarity analysis: Tutorial and application in developmental cognitive neuroscience. Developmental Cognitive Neuroscience, 54, 101071. https://doi.org/10.1016/j.dcn.2022.101071

Reviewer #1 (Public Review):

Microglia are increasingly recognized as playing an important role in shaping the synaptic circuit and regulating neural dynamics in response to changes in their surrounding environment and in brain states. While numerous studies have suggested that microglia contribute to sleep regulation and are modulated by sleep, there has been little direct evidence that the morphological dynamics of microglia are modulated by the sleep/wake cycle. In this work, Gu et al. applied a recently developed miniature two-photon microscope in conjunction with EEG and EMG recording to monitor microglia surveillance in freely-moving mice over extended period of time. They found that microglia surveillance depends on the brain state in the sleep/wake cycle (wake, non-REM, or REM sleep). Furthermore, they subjected the mouse to acute sleep deprivation, and found that microglia gradually assume an active state in response. Finally, they showed that the state-dependent morphological changes depend on norepinephrine (NE), as chemically ablating noradrenergic inputs from locus coeruleus abolished such changes; this is in agreement with previous publications. The authors also showed that the effect of NE is partially mediated by β2-adrenergic receptors, as shown with β2-adrenergic receptor knock-out mice. Overall, this study is a technical tour de force, and its data add valuable direct evidence to the ongoing investigations of microglial morphological dynamics and its relationship with sleep. Nevertheless, microglial morphodynamics likely reflect the integrated influence of neighboring neuronal activities and neuromodulatory factors; the pan-tissue β2AR knockout mouse model may also broadly affect the animal's physiology and sleep behavior. Therefore, future studies are needed to address the specific role of microglial β2AR on its morphodynamics in sleep.

Reviewer #1 (Public Review):

The authors performed an RNAi screen to identify epigenetic regulators involved in oxygen-glucose deprivation (OGD)-induced neuronal injury using immortalized mouse hippocampal neuronal cell line HT-22. They identified PRMT5 as a novel negative regulator of neuronal cell survival after OGD. Both in vitro and in vivo experiments were then performed to evaluate the roles of PRMT5 in OGD and ischemic stroke-induced injury. The authors found that genetic and pharmacological inhibition of PRMT5 protected against neuronal cell death in both in vitro and in vivo models. Furthermore, they found that in response to OGD and ischemia, PRMT5 was translocated from the cytosol to the nucleus, where PRMT5 bound to the chromatin and promoter regions of targeted genes to repress the expression of downstream genes. Further, they showed that silencing PRMT5 significantly altered the OGD-induced changes for a large-scale of genes. In a mouse model of middle cerebral artery occlusion (MCAO), PRMT5 inhibitor EPZ015666 protected against neuronal death in vivo. This study reveals a potential therapeutic target for the treatment of ischemic stroke. Overall, the authors have done elegant work showing the role of PRMT5 in neuronal cell survival. However, the essential mechanisms underlying PRMT5 nuclear translocation have not been investigated, and the in vivo animal studies should be further strengthened.

Reviewer #1 (Public Review):

The present work establishes 14-3-3 proteins as binding partners of spastin and suggests that this binding is positively regulated by phosphorylation of spastin. The authors show evidence that 14-3-3 - spastin binding prevents spastin ubiquitination and final proteasomal degradation, thus increasing the availability of spastin. The authors measured microtubule severing activity in cell lines and axon regeneration and outgrowth as a prompt to spastin activity. By using drugs and peptides that separately inhibit 14-3-3 binding or spastin activity, they show that both proteins are necessary for axon regeneration in cell culture and in vivo models in rats.<br /> The following is an account of the major strengths and weaknesses of the methods and results.

Major strengths<br /> -The authors performed pulldown assays on spinal cord lysates using GST-spastin, then analyzed pulldowns via mass spectrometry and found 3 peptides common to various forms of 14-3-3 proteins. In co-expression experiments in cell lines, recombinant spastin co-precipitated with all 6 forms of 14-3-3 tested. The authors could also co-immunoprecipitate spastin-14-3-3 complexes from spinal cord samples and from primary neuronal cultures.<br /> -By protein truncation experiments they found that the Microtubule Binding Domain of spastin contained the binding capability to 14-3-3. This domain contained a putative phosphorylation site, and substitutions that cannot be phosphorylated cannot bind to 14-3-3.<br /> -Overexpression of GFP-spastin shows a turn-over of about 12 hours when protein synthesis is inhibited by cycloheximide. When 14-3-3 is co-overexpressed, GFP-spastin does not show a decrease by 12 hours. When S233A is expressed, a turn-over of 9 hours is observed, suggesting that phosphorylation increases the stability of the protein. In support of that notion, the phospho-mimetic S233D makes it more stable, lasting as much as the over-expression of 14-3-3.<br /> -By combining FCA with Spastazoline, authors claim that FCA increased regeneration is due to increased spastin activity in various models of neurite outgrowth and regeneration in cell culture and in vivo, the authors show impressive results on the positive effect of FCA in regeneration, and that this is abolished when spastin is inhibited.

Major weaknesses<br /> 1- The present manuscript suggests that 14-3-3 and spastin work in the same pathway to promote regeneration. Although the manuscript contains valuable evidence in support for a role of 14-3-3 and spasting in regeneration, the conclusive evidence is difficult to generate, and is missing in the present manuscript. For example, there are simpler explanations for the combined effect of FC-A and spastazoline. The FC-A mechanism of action can be very broad, since it will increase the binding of all 14-3-3 proteins with presumably all their substrates, hence the pathways affected can rise to the hundreds. The fact that spastazoline abolishes FC-A effect, may not be because of their direct interaction, but because spastin is a necessary component of the execution of the regeneration machinery further downstream, in line with the fact that spastazoline alone prevented outgrowth and regeneration, and in agreement with previous work showing that normal spastin activity is necessary for regeneration.<br /> With this in mind, I consider the title and most major conclusions of the manuscript related to these two proteins acting together for the observed effects are overstated.

2- Authors show that S233D increases MT severing activity, and explain that it is related to increased binding to 14-3-3. An alternative explanation is that phosphorylation at S233 by itself could increase MT severing activity. The authors could test if purified spastin S233D alone could have more potent enzymatic activity.

3- The interpretation of the authors cannot explain how Spastin can engage in MT severing while bound to 14-3-3 using its Microtubule Binding Domain.

4- Also, the term "microtubule dynamics", which is present in the title and in other major conclusions, is overstated. Although authors show, in cell lines, changes in microtubule content, it is far from evidence for changes in "MT dynamics" in the settings of interest (i.e. injured axons).

5- In the same lines, the manuscript lacks evidence for the changes of MT content and/dynamics as a function of the proposed 14-3-3 - Spastin pathway.

Reviewer #1 (Public Review):

This study extends the previous interesting work of this group to address the potentially differential control of movement and posture. Their earlier work explored a broad range of data to make the case for a downstream neural integrator hypothesized to convert descending velocity movement commands into postural holding commands. Included in that data were observations from people with hemiparesis due to stroke. The current study uses similar data but pushes into a different, but closely related direction, suggesting that these data may address the independence of these two fundamental components of motor control. I find the logic laid out in the second sentence of the abstract ("The paretic arm after stroke is notable for abnormalities both at rest and during movement, thus it provides an opportunity to address the relationships between control of reaching, stopping, and stabilizing") less than compelling, but the study does make some interesting observations. Foremost among them, is the relation between the resting force postural bias and the effect of force perturbations during the target hold periods, but not during movement. While this interesting observation is consistent with the central mechanism the authors suggest, it seems hard to me to rule out other mechanisms, including peripheral ones.

On the other hand, the relation between force bias and the well-recognized flexor synergy seems rather self-evident, and I don't see that these results add much to that story. I am also struck by what seems to be a contradiction between the conclusions of the current and former studies: "These findings in stroke suggest that moving and holding still are functionally separable modes of control" and "the commands that hold the arm and finger at a target location depend on the mathematical integration of the commands that moved the limb to that location." The former study is mentioned here only in passing, in a single phrase in the discussion, with no consideration of the relation between the two studies. This is odd and should be addressed.

A minor wording concern I had is that the term "holding still" is frequently hard to parse. A couple of examples: "These findings in stroke suggest that moving and holding still are functionally separable modes of control." This example is easily read, "moving and holding [continue to be] functionally separable". Another: "...active reaching and holding still in the same workspace, " could be "...active reaching and holding [are] still in the same workspace." Simply "holding", "posture" or "posture maintenance" would all be better options.

Reviewer #1 (Public Review):

Summary:

Walsh and colleagues investigated how cued probabilistic expectations about future stimuli may influence different stages of decision-making as implemented in the human brain. In their study, participants were provided with cues that could correctly (or incorrectly) cue which visual stimulus would be presented. These cues also predicted the motor action that would likely produce a correct judgment for that trial. In addition, a 'neutral' cue was included that did not predict any particular stimulus. They report that measures of steady-state visual evoked potentials (SSVEPs, proposed to index the magnitude of visual neural activity in favour of the correct response) were smaller when the cue incorrectly predicted the upcoming image, compared to when an accurate cue or a neutral cue was presented. Their primary finding adds to an ongoing debate in the field of decision-making research about how cued expectations may influence how we make decisions.

Strengths:

This study uses a carefully constructed experiment design and decision-making task that allows separation of multiple electroencephalographic (EEG) signals thought to track different stages of decision-making. For example, the steady-state visual evoked potential measures can be cleanly dissociated from more anterior beta-band activity over the motor cortex. They also allow evaluation of how cued expectancy effects may unfold over a number of testing sessions. This is important because the most consistent evidence of expectation-related modulations of electrophysiological measures (using EEG, local field potentials, or single neuron firing rates) is from studies of non-human primates that involved many days of cue-stimulus contingency learning, and there is a lack of similar work using several testing sessions in humans. Although there were several experimental conditions included in the study, careful trial-balancing was conducted to minimise biases due to incidental differences in the number of trials included for analyses across each condition. Performance for each individual was also carefully calibrated to maximise the possibility of identifying subtle changes in task performance by expectation and avoid floor or ceiling effects.

Weaknesses:

Although the experiment and analysis methods are cohesive and well-designed, there are some shortcomings that limit the inferences that can be drawn from the presented findings.

The first relates to the measures of SSVEPs and their relevance for decision-making in the task. In order to eliminate the influence of sporadic pulses of contrast changes that occurred during stimulus presentation, a time window of 680-975 ms post-stimulus onset was used to measure the SSVEPs. The mean response times for the valid and neutral cues were around 850-900 ms for correct responses, and within the same time window for errors in the invalid cue condition. In addition, a large portion of response times in perceptual decision-making tasks are substantially faster than the mean due to right-skewed response time distributions that are typically observed. As it has also been estimated to require 70-100 ms to execute a motor action (e.g., a keypress response) following the commitment to a decision. This raises some concerns about the proportion of trials in which the contrast-dependent visual responses (indexed by the SSVEPs) indexed visual input that was actually used to make the decision in a given trial. Additional analyses of SSVEPs that take the trial-varying pulses into account could be run to determine whether expectations influenced visual responses earlier in the trial. Presenting response time quantile plots may also help to determine the proportions of motor responses (used to report a decision) that occurred during or after the SSVEP measurement window.

In addition, an argument is made for changes in the evidence accumulation rate (called the drift rate) by stimulus expectancy, corresponding to the observed changes in SSVEP measures and differences in the sensory encoding of the stimulus. This inference is limited by the fact that evidence accumulation models (such as the Diffusion Decision Model) were not used to test for drift rate changes as could be determined from the behavioural data (by modelling response time distributions). There appear to be ample numbers of trials per participant to test for drift rate changes in addition to the starting point bias captured in earlier models. Due to the very high number of trials, models could potentially be evaluated for each single participant. This would provide more direct evidence for drift rate changes than the findings based on the SSVEPs, particularly due to the issues with the measurement window relating to the response times as mentioned above.

Reviewer #1 (Public Review):

Summary:

The results in this manuscript show that after the same injury, axon regeneration of three types of sensory neurons and motor neurons differs. In addition, they analyzed their transcriptomic profiles with or without injury. Finally, they also pinpoint a molecular candidate that might regulate axon regeneration in PNS.

Strengths:

With four different transgenic lines to label different populations of PNS axons, the authors show that nociceptors have the greatest regeneration, followed by motoneurons, and then cutaneous mechanoreceptors and proprioceptors.

These transgenic tools were further used in RNA profiling analysis. They identified signatures of these different populations in intact and injured states, implicating that differentially activated regenerative programs might be a contributing factor to different regenerative outcomes.

They showed that Med12 is induced in proprioceptors and down-regulated in mechanoreceptors and nociceptors. Further, knockout down Med12 with shRNA increased neurite growth.

Weaknesses:

While in vivo injury was used to assess regeneration from subsets of PNS neurons, different in vitro neurite growth or explant assays were used for further assessments. However, the authors did not assess whether the differential "regenerative" responses in vivo could be recapitulated in vitro. Such results will be important in interpreting the results.

Intriguingly, even in individual groups of PNS neurons, not all neurons regenerate to the same extent. It is known that the distance between the cell body and the lesion site affects neuronal injury responses. It would be interesting to test this in the observed regeneration.

Fig 1: The authors quantified the number of regenerating axons at two different time points. However, the total numbers of neurons/axons in each subset are different. The authors should use these numbers to normalize their regenerative axons.

Fig 2-5: In explaining differential regeneration of individual groups of neurons, there are at least two possibilities: (1). Each group of neurons has different injury/regenerative responses; (2). The same set of injury/regenerative responses are differentially activated. Some data in this manuscript suggested the latter possibility. But some other data point in the opposite direction. It would be informative for the authors to analyze/discuss this further.

Fig 6: Is it possible to assess the regenerative effects of knockdown Med12 after in vivo injury?

Reviewer #1 (Public Review):

Summary:<br /> In this paper, Weber et al. investigate the role of 4 dopaminergic neurons of the Drosophila larva in mediating the association between an aversive high-salt stimulus and a neutral odor. The 4 DANs belong to the DL1 cluster and innervate non-overlapping compartments of the mushroom body, distinct from those involved in appetitive associative learning. Using specific driver lines, they show that activation of the DAN-g1 is sufficient to mimic an aversive memory and it is also necessary to form a high-salt memory of full strength, although optogenetic silencing of this neuron only partially affects the performance index. The authors use calcium imaging to show that the DAN-g1 is not the only one that responds to salt. DAN-c1 and d1 also respond to salt, but they seem to play no role in the assays tested. DAN-f1, which does not respond to salt, is able to lead to the formation of memory (if optogenetically activated), but it is not necessary for the salt-odor memory formation in normal conditions. However, silencing of DAN-f1 together with DAN-g1, enhances the memory deficit of DAN-g1.

Strengths:<br /> The paper therefore reveals that also in the Drosophila larva as in the adult, rewards and punishments are processed by exclusive sets of DANs and that a complex interaction between a subset of DANs mediates salt-odor association.<br /> Overall, the manuscript contributes valuable results that are useful for understanding the organization and function of the dopaminergic system. The behavioral role of the specific DANs is accessed using specific driver lines which allow for testing of their function individually and in pairs. Moreover, the authors perform calcium imaging to test whether DANs are activated by salt, a prerequisite for inducing a negative association with it. Proper genetic controls are carried across the manuscript.

Weaknesses:<br /> The authors use two different approaches to silence dopaminergic neurons: optogenetics and induction of apoptosis. The results are not always consistent, and the authors could improve the presentation and interpretation of the data. Specifically, optogenetics seems a better approach than apoptosis, which can affect the overall development of the system, but apoptosis experiments are used to set the grounds of the paper.

The physiological data would suggest the role of a certain subset of DANs in salt-odor association, but a different partially overlapping set seems to be necessary. This should be better discussed and integrated into the author's conclusion. The EM data analysis reveals a non-trivial organization of sensory inputs into DANs and it is hard to extrapolate a link to the functional data presented in the paper.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript by Ishii et al utilizes a classical, but extremely understudied, female self-paced assay to directly address aspects of female sexual motivation independent from the male's behavior. This allowed for a clear separation of appetitive and consummatory events, of which whole brain unbiased activity was mapped. Mating completion in females was then focused on the medial preoptic nucleus where the authors performed a rigorous set of single-cell GCaMP recordings in populations marked by Vglut2 and Vgat, finding the latter display stronger and prolonged activity after the onset of mating completion. Finally, they demonstrate function to these Fos-TRAPPED completion cells demonstrating their capacity to suppress female sexual behavior.

Strengths:<br /> This manuscript sought to explicitly explore the female mating drive as dictated by the female, a very rare angle for those studying mating behavior which almost always is controlled by the male's behavior. To achieve this, the authors went back to old literature and modified a classical paradigm in which a measurable approach and avoidance of male conspecifics can be measured in female mice using a self-paced mating assay. Strengths include a detailed quantification of female behaviors demonstrating a robust attenuated sexual motivation in females after mating completion. To determine the neural basis behind this, a brain-wide analysis of cells responding to mating completion in the female brain was conducted which revealed numerous anatomical regions displaying increased Fos activity, including the MPOA, of which the authors concentrated the remaining of their study. Employing microendoscopic imaging, the authors discovered that this mating completion signal was strongly represented in the MPOA. The single cell data analyses are of very high quality as is the number of individual cells resolved. While they identified both excitatory and inhibitory cell types that were activated by mating completion, they found the latter exhibited stronger and more persistent activity. Segmentation into individual mating behaviors reinforced the importance of GABAergic completion cells, which display prolonged activity late after the onset of mating completion. This information provides a potential mechanism for how female mice suppress further mating activity following completion. The authors then definitively demonstrate this function by TRAP'ping completion cells with chemogenetic actuators and show that CNO-induced activation of these cells specifically and strongly suppresses female sexual behavior. All experiments were extremely well-designed and performed carefully and expertly with the necessary controls solidifying the conclusions.

Weaknesses:<br /> While there are no glaring weaknesses in this study, it should be noted that a great deal of literature has pinpointed the MPOA (and specifically inhibitory cells in this area) as being critical to sexual behavior, including female mating. However, no study to my knowledge has explored self-paced female mating with such fine control over manipulating and monitoring cellular activity in this region. In addition, this study may act to inspire others to further explore the additional brain regions found to show upregulation of neural activity (Fos) during mating completion in the female using the data sets generated here.

Reviewer #1 (Public Review):

Summary:<br /> In this study, the authors evaluated a novel eIF2B activator, DNL343, in two mouse models representing different forms of the integrated stress response (ISR). They first assessed the pharmacokinetics of DNL343, demonstrating its ability to cross the blood-brain barrier and exhibit good bioavailability. In an acute ISR model induced by optic nerve crush (ONC) injury, DNL343 treatment reduced ISR-induced transcriptional changes and neuronal loss, demonstrating neuroprotective effects. Next, the authors generated an eIF2B loss-of-function mice model by knocking in disease-causing Eif2b5 variants. The model presents a chronic ISR and mimics vanishing white matter disease (VWMD). DNL343 treatment from the pre-symptomatic stage improved body weight and motor functions corrected transcriptional changes, and reversed proteomic and metabolomic alterations in the brain and cerebrospinal fluid. DNL343 treatment initiated at an advanced disease stage also showed positive effects, restoring body weight gain, suppressing ISR, reducing neurodegeneration biomarkers, and extending lifespan. These findings highlight DNL343 as an effective ISR inhibitor with potential applications in treating VWMD and other neurodegenerative disorders involving ISR.

Strengths:<br /> The study's findings regarding the novel compound DNL343 offer significant promise in addressing VWMD, a condition currently lacking disease-modifying treatment. DNL343 directly targets eIF2B, the disease-causing complex in VWMD, and demonstrates notable efficacy in reversing the integrated stress response (ISR) and mitigating neurodegeneration in a VWMD mouse model. These results raise hope for the potential application of DNL343 in VWMD treatment, a development eagerly anticipated by patients and the VWMD research community. Moreover, the study hints at the broader potential of DNL343 in treating other ISR-related neurodegenerative disorders, such as amyotrophic lateral sclerosis, a prospect that holds broader interest. Additionally, the study's identification of potential biomarkers for VWMD represents a notable strength, potentially leading to improved disease progression assessment pending further confirmation in future research.

Weaknesses:<br /> There are a couple of notable concerns in this study. Firstly, while the in vivo evidence strongly supports the efficacy of DNL343 in mitigating ISR and neurodegeneration, there is a lack of direct biochemical evidence to confirm its activity in eIF2B activation. Secondly, the potential for cardiovascular toxicity, which has been reported for a related eIF2B activator in a canine model (as mentioned in the manuscript), has not been evaluated for DNL343 in this study. This data gap regarding toxicity could be crucial for informing the future development of DNL343 for potential human use. Further investigation into these areas would be valuable for a comprehensive understanding of the compound's mechanisms and safety profile.

Reviewer #1 (Public Review):

Summary:<br /> In this work, the authors use an OT setup to measure the DNA gripping and DNA slipping dynamics of phage lambda terminase motor interaction with DNA. They discover major differences in the dynamics of these two events, in comparison to the phage T4 motor, which they previously investigated. They attribute these differences to the presence of the TerS (small terminase) subunit of the motor complex of phage lambda in addition to the TerL (large terminase) subunit in phage, while in T4 only the TerL subunit is present. By exposing the stalled phage lambda procapsid-DNA complex (stalled with ATP-gammaS) to solutions containing 1) no nucleotide, 2) poorly hydrolyzed ATP*, and 3) ADP, they found that the gripping persistence is strongest with ATP*, weaker with ADP, and weakest with no nucleotide. This demonstrates nucleotide-dependent DNA gripping and friction of the motor. However, both persistence of gripping and friction are dramatically stronger than in the T4 TerL motor, due to the presence of the TerS subunit. While TerS was believed to be essential for the initiation of packaging in vivo, its role during DNA translocation was unclear. This study reveals the key role played by TerS in DNA gripping and DNA-motor friction, highlighting its role in DNA translocation where TerS acts as a "sliding clamp".

The study also provides a method to investigate factors affecting the stability of the initiation complex in viral packaging motors.

Strengths:<br /> The experiments are well carried out and the conclusions are justified. These findings are of great significance and advance our understanding of viral motor function in the DNA packaging process and packaging dynamics.

Weaknesses:<br /> While the collected OT data is quantitative, therefore is no further quantitative analysis of the motor packaging dynamics with regard to different motor subunit functions and the presence of nucleotides.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Pan DY et al. discovered that the clearance of senescent osteoclasts can lead to a reduction in sensory nerve innervation. This reduction is achieved through the attenuation of Netrin-1 and NGF levels, as well as the regulation of H-type vessels, resulting in a decrease in pain-related behavior. The experiments are well-designed. The results are clearly presented, and the legends are also clear and informative. Their findings represent a potential treatment for spine pain utilizing senolytic drugs.

Strengths:<br /> Rigorous data, well-designed experiments as well as significant innovation make this manuscript stand out.

Weaknesses:<br /> Quantification of histology and detailed statistical analysis will further strengthen this manuscript.

Reviewer #1 (Public Review):

In this article, the authors found a distinct fibroblast subpopulation named AG fibroblasts, which are capable of regulating myeloid cells, T cells and ILCs, and proposed that AG fibroblasts function as a previously unrecognized surveillant to orchestrate chronic gingival inflammation in periodontitis. Generally speaking, this article is innovative and interesting.

Reviewer #1 (Public Review):

The manuscript by Lolicato and colleagues characterizes the role of FGF2 dimerization in unconventional secretion of this signaling molecule using a combination of cell-based and in vitro assays. FGF2 is a signaling molecule secreted from the cell via an unconventional mechanism because it lacks a signal sequence. Previous studies by the same group have established a compelling model in which FGF2 forms an oligomer in a PIP2 dependent manner at the plasma member, which drives its translocation to the cell exterior. The same group also reports two cysteine residues that are critical for FGF2 oligomerization and secretion.

In this study, the authors analyzed the impact of single Cysteine to Alanine substitution on oligomerization and secretion of FGF2. They found that C95 but not C77 is required for PIP2 dependent membrane binding, FGF2 oligomerization and secretion. On the other hand, C77 is required for the interaction of FGF2 with the plasma membrane Na, K-ATPase, which is thought to enhance the FGF2-PIP2 interaction. Using a set of bi-functional crosslinkers, the authors were able to capture a fraction of the FGF2 homo-dimer, which is dependent on C95. They propose that FGF2 forms a disulfide-bridged dimer via C95, which serves as the building block for FGF2 oligomerization in the plasma membrane.

The revised manuscript has carefully addressed my concerns. I should clarify that when I inquired about evidence for a disulfide-linked FGF2 dimer, I referred to in vivo evidence. I was aware of the authors' previous in vitro study, which demonstrated that FGF2 indeed can form a disulfide dimer under an in vitro condition. Although the new manuscript still contains no in vivo data on this issue, the authors have added numerous controls. In particular, the fact that the FGF2 C95S mutant is severely defective in secretion does provide strong support for the involvement of the thiol group of C95 in FGF2 secretion. The additional discussions on other examples of cytosolically-localized disulfide proteins and those in proximity to membranes further alleviates my concern.

Reviewer #1 (Public Review):

Terzioglu and co-workers tested the provocative hypothesis that mitochondria maintain an internal temperature considerably higher than cytosolic/external environmental temperature due to the inherent thermodynamic inefficiency of mitochondrial oxidative phosphorylation. As a follow-up to a prior paper from some of the same authors, the goal of this study was to conduct additional experiments to assess mitochondrial temperature in cultured cells. Consistent with the prior work, the authors provide consistent evidence that the temperature of mitochondria in four different types of cultured mammalian cells, as well as cells from Drosophila (poikilotherms), is 15oC or more above the external temperature at which cells are maintained (e.g., 37oC). Additional evidence shows that mitochondria maintain higher temperatures under several different types of cellular metabolic stresses predicted to decrease the dependence on OxPhos, adding to the notion that natural thermodynamic inefficiency and heat generation may be an important, and potentially regulated, characteristic of mitochondrial metabolism.

Strengths

Demonstration that both a fluorescent (Mito Thermo Yellow) and a genetic-based (mito-gTEMP) mitochondrial targeted temperature probe elicit similar quantitative changes in mitochondrial temperature under different experimental conditions is a strength. The addition of the genetic probe to the current study supports prior findings using the fluorescent probe and thus achieves a primary objective of the study.

The experiments are well designed and executed. Specific attention given to potential artifacts affecting probe signal and/or non-specific effects from the different experimental interventions is a strength.

The use of different cultured cell lines from different organisms provides additional evidence of elevated temperature as a general property of functioning mitochondria, representing additional validation.

Weakness:

While the findings and potential interpretations put forward by the authors are intriguing, the severity of the interventions (e.g., mitochondrial complex-specific inhibitors, inhibition of protein synthesis) and the absence of simultaneous or parallel measurements of other key bioenergetic parameters (i.e., membrane potential, oxygen consumption rate, etc.) limits the ability to interpret potential cause and effect - whether the thermogenesis aspect of OxPhos is being sensed and regulated, or whether temperature changes are more of a biproduct of adjustments in OxPhos flux under the experimental circumstances. In other words, the physiological relevance of the findings remains unclear.

Related, several of the interventions are employed to either increase or decrease dependence on OxPhos flux, but no outcome measures are reported to document whether the intended objective was achieved (e.g., increased OxPhos flux in low glucose plus galactose, decreased ATP demand-OxPhos flux with anisomycin, etc.).

Reviewer #1 (Public Review):

This manuscript by Xu and colleagues addresses the important question of how multi-modal associations are encoded in the rodent brain. They use behavioral protocols to link stimuli to whisker movement and discover that the barrel cortex can be a hub for associations. Based on anatomical correlations, they suggest that structural plasticity between different areas can be linked to training. Moreover, they provide electrophysiological correlates that link to behavior and structure. Knock-down of nlg3 abolishes plasticity and learning.

This study provides an important contribution as to how multi-modal associations can be formed across cortical regions.

Reviewer #1 (Public Review):

Soudi, Jahani et al. provide a valuable comparative study of local adaptation in four species of sunflowers, and investigate the repeatability of observed genomic signals of adaptation and their link to haploblocks, known to be numerous and important in this system. The study builds on previous work in sunflowers that have investigated haploblocks in those species and on methodologies developed to look at repeated signals of local adaptations. The authors provide solid evidence of both genotype-environment associations (GEA) and genome-wide association study (GWAS), as well as phenotypic correlations with the environment, to show that part of the local adaptation signal is repeatable and significantly co-occur in regions harboring haploblocks. Results also show that part of the signal is species specific and points to high genetic redundancy. This work will be of interest to evolutionary biologists in general and population geneticists in particular, and constitutes a good example of comparative local adaptation. Importantly, this study helps in advancing our understanding of the genetic architecture implicated in the adaptation process.

Strenghts: The authors take great care in acknowledging and investigating the multiple biases inherent to the used methods (GEA and GWAS) and use conservative and well thought statistical approaches to draw their conclusions. Additionally, I appreciated the nuanced discussion and can only agree with the authors that the adaptation process is complex and does not fully fit the classic simplified genetics models of either few large effect genes or only infinitesimal quantitative traits. I find the added Summary figure of this revised version (S1) extremely helpful in better understanding the different analysis steps and how they relate to the different questions.

Weaknesses: After those revisions, I did not find any major weakness and am satisfied with the authors responses.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript of Davidsen and Sullivan describes an improved tRNA-seq protocol to determine aminoacyl-tRNA levels. The improvements include: (i) optimizing the Whitfeld or oxidation reaction to select aminoacyl-tRNAs from oxidation-sensitive non-acylated tRNAs; (ii) using a splint-assisted ligation to modify the tRNAs' ends for the following RT-PCR reaction; (iii) using an error-tolerating mapping algorithm to map the tRNA sequencing reads that contain mismatches at modified nucleotides.

Strengths:<br /> The two steps, the oxidation, and the splint-assisted ligation are yield-diminishing steps, thus the protocol of Davidsen and Sullivan is an important improvement of the current protocols to enhance the quantification of aminocyl-tRNAs.

Weaknesses:<br /> The oxidation and the selection of aminoacyl-tRNA is the first step in all protocols. Thereafter they differ on whether blunt ligation, hairpin (DM-tRNA-seq, YAMAT-seq, QuantM-seq, mim tRNA-seq, LOTTE tRNA-seq), or splint ligation is used and finally what detection method is applied (i-tRAP, tRNA microarrays). What is the correlation to those alternative approaches (e.g. i-tRAP (PMID 36283829), tRNA microarrays (PMID: 31263264) etc.)? What is the correlation with other approaches with which this improved protocol shares some steps (DM-tRNA-seq, mim-tRNA-seq)?

Reviewer #1 (Public Review):

Summary:<br /> The authors present a neural network (NN)-based approach to computationally cheaper emulation of simulations of biophysically relatively detailed cardiac cell models based on systems of ordinary differential equations. Relevant case studies are used to demonstrate the performance in the prediction of standard action potentials, as well as action potentials manifesting early depolarizations. Application to the "reverse problem" (inferring the effect of pharmacological compounds on ion channels based on action potential data before and after drug treatment) is also explored, which is a task of generally high interest.

Strengths:<br /> This is a well-designed study, which explores an area that many in the cardiac simulation community will be interested in. The article is well written and I particularly commend the authors on transparency of methods description, code sharing, etc. - it feels rather exemplary in this regard and I only wish more authors of cardiac simulation studies took such an approach. The training speed of the network is encouraging and the technique is accessible to anyone with a reasonably strong GPU, not needing specialized equipment.

Weaknesses:<br /> Below are several points that I consider to be weaknesses and/or uncertainties of the work:

1. I am not convinced by the authors' premise that there is a great need for further acceleration of cellular cardiac simulations - it is easy to simulate tens of thousands of cells per day on a workstation computer, using simulation conditions similar to those of the authors. I do not really see an unsolved task in the field that would require further speedup of single-cell simulations.

At the same time, simulations offer multiple advantages, such as the possibility to dissect mechanisms of the model behaviour, and the capability to test its behaviour in a wide array of protocols - whereas a NN is trained for a single purpose/protocol, and does not enable a deep investigation of mechanisms. Therefore, I am not sure the cost/benefit ratio is that strong for single-cell emulation currently.

An area that is definitely in need of acceleration is simulations of whole ventricles or hearts, but it is not clear how much potential for speedup the presented technology would bring there. I can imagine interesting applications of rapid emulation in such a setting, some of which could be hybrid in nature (e.g. using simulation for the region around the wavefront of propagating electrical waves, while emulating the rest of the tissue, which is behaving more regularly/predictable, and is likely to be emulated well), but this is definitely beyond of the scope of this article.

2. The authors run a cell simulation for 1000 beats, training the NN emulator to mimic the last beat. It is reported that the simulation of a single cell takes 293 seconds, while emulation takes only milliseconds, implying a massive speedup. However, I consider the claimed speedup achieved by emulation to be highly context-dependent, and somewhat too flattering to the presented method of emulation. Two specific points below:

First, it appears that a not overly efficient (fixed-step) numerical solver scheme is used for the simulation. On my (comparable, also a Threadripper) CPU, using the same model ("ToR-ORd-dyncl"), but a variable step solver ode15s in Matlab, a simulation of a cell for 1000 beats takes ca. 50 seconds, rather than 293 of the authors. This can be further sped up by parallelization when more cells than available cores are simulated: on 32 cores, this translates into ca. 2 seconds amortized time per cell simulation (I suspect that the NN-based approach cannot be parallelized in a similar way?). By amortization, I mean that if 32 models can be simulated at once, a simulation of X cells will not take X*50 seconds, but (X/32)*50. (with only minor overhead, as this task scales well across cores).

Second, and this is perhaps more important - the reported speed-up critically depends on the number of beats in the simulation - if I am reading the article correctly, the runtime compares a simulation of 1000 beats versus the emulation of a single beat. If I run a simulation of a single beat across multiple simulated cells (on a 32-core machine), the amortized runtime is around 20 ms per cell, which is only marginally slower than the NN emulation. On the other hand, if the model was simulated for aeons, comparing this to a fixed runtime of the NN, one can get an arbitrarily high speedup.

Therefore, I'd probably emphasize the concrete speedup less in an abstract and I'd provide some background on the speedup calculation such as above, so that the readers understand the context-dependence. That said, I do think that a simulation for anywhere between 250 and 1000 beats is among the most reasonable points of comparison (long enough for reasonable stability, but not too long to beat an already stable horse; pun with stables was actually completely unintended, but here it is...). I.e., the speedup observed is still valuable and valid, albeit in (I believe) a somewhat limited sense.

3. It appears that the accuracy of emulation drops off relatively sharply with increasing real-world applicability/relevance of the tasks it is applied to. That said, the authors are to be commended on declaring this transparently, rather than withholding such analyses. I particularly enjoyed the discussion of the not-always-amazing results of the inverse problem on the experimental data. The point on low parameter identifiability is an important one and serves as a warning against overconfidence in our ability to infer cellular parameters from action potentials alone. On the other hand, I'm not that sure the difference between small tissue preps and single cells which authors propose as another source of the discrepancy will be that vast beyond the AP peak potential (probably much of the tissue prep is affected by the pacing electrode?), but that is a subjective view only. The influence of coupling could be checked if the simulated data were generated from 2D tissue samples/fibres, e.g. using the Myokit software.

Given the points above (particularly the uncertain need for further speedup compared to running single-cell simulations), I am not sure that the technology generated will be that broadly adopted in the near future. However, this does not make the study uninteresting in the slightest - on the contrary, it explores something that many of us are thinking about, and it is likely to stimulate further development in the direction of computationally efficient emulation of relatively complex simulations.

Reviewer #1 (Public Review):

In this study, the authors demonstrated a new model that B cell contraction after antigen encountering was dependent on N-WASP-branched actin polymerization. This statement is achieved by a systemic comparison of genetic modified mice vs wild type mice or inhibitor treated cells vs control cells. By imaging how B cells interact with antigen-coated planar lipid bilayer, the authors further suggested that the contraction event may provide B cells a channel to dismiss downstream kinase for a purpose to attenuate B cell activation signaling.

In this revised version, the authors have fully addressed my concerns raised against the initial submission of their studies.

Reviewer #1 (Public Review):

Summary:

The investigators sought to determine whether Marco regulates the levels of aldosterone by limiting uptake of its parent molecule cholesterol in the adrenal gland. Instead, they identify an unexpected role for Marco on alveolar macrophages in lowering the levels of angiotensin-converting enzyme in the lung. This suggests an unexpected role of alveolar macrophages and lung ACE in the production of aldosterone.

Strengths:

The investigators suggest an unexpected role for ACE in the lung in the regulation of systemic aldosterone levels.<br /> The investigators suggest important sex-related differences in the regulation of aldosterone by alveolar macrophages and ACE in the lung.<br /> Studies to exclude a role for Marco in the adrenal gland are strong, suggesting an extra-adrenal source for the excess Marco observed in male Marco knockout mice.

Weaknesses:

While the investigators have identified important sex differences in the regulation of extrapulmonary ACE in the regulation of aldosterone levels, the mechanisms underlying these differences are not explored.<br /> The physiologic impact of the increased aldosterone levels observed in Marco -/- male mice on blood pressure or response to injury is not clear.<br /> The intracellular signaling mechanism linking lung macrophage levels with the expression of ACE in the lung is not supported by direct evidence.

Joint Public Review:

This paper aimed to assess the link between genetic and environmental factors on psychotic-like experiences and the potential mediation through cognitive ability. This study was based on data from the ABCD cohort, including 6,602 children aged 9-10 years. The authors report a mediating effect, suggesting that cognitive ability is a key mediating pathway in linking several genetic and environmental (risk and protective) factors to psychotic-like experiences.

Strengths of the methods: The authors use a wide range of validated (genetic, self- and parent-reported, and cognitive) measures in a large dataset with a 2-year follow-up period. The statistical methods have the potential to address key limitations of previous research.

Weaknesses of the methods: Not the largest or most recent GWASes were used to generate PGSes.

Strengths of the results: The authors included a comprehensive array of analyses.

Weaknesses of the results: Results are only sometimes clearly described and presented.

Appraisal: The authors suggest that their findings provide evidence for policy reforms (e.g., targeting residential environments, family SES, parenting, and schooling).

Impact: Immediate impact is limited given the short follow-up period (2 years), possibly concerns for selection bias and attrition in the data, and some methodological concerns. The authors are transparent about most of these limitations.

Reviewer #1 (Public Review):

Summary:

The study conducted on mice establishes a noteworthy connection between dietary protein intake and resistance exercise impact on metabolic health and muscle development. In sedentary mice, a diet rich in protein resulted in excessive fat accumulation and compromised blood sugar regulation in comparison to a diet low in protein. Intriguingly, when mice followed the high protein diet alongside progressive resistance training, they exhibited protection against surplus fat gain, though blood glucose regulation remained impaired. The research also revealed that resistance training notably enhanced muscle hypertrophy induced by exercise, particularly in mice on the high protein diet. Although the maximum strength achieved was similar across diets, this highlights the potential synergy between high protein consumption and resistance exercise in promoting skeletal muscle growth.

Strengths:

The study possesses several significant strengths. Firstly, it combines controlled dietary manipulations with resistance exercise, providing a comprehensive understanding of their combined effects on metabolic health and muscle growth. The use of mouse models, while not directly translatable to humans, offers a controlled experimental environment, enabling precise measurements and observations. Moreover, the study reveals nuanced outcomes such as the differential impact of high protein intake on adiposity and muscle hypertrophy. The emphasis on both positive and negative findings lends balance to the conclusions, enhancing the overall credibility of the study. Additionally, the clear delineation of diet-exercise interactions contributes to the broader understanding of dietary and exercise recommendations for metabolic health and muscle development.

Weaknesses:

Certain limitations warrant consideration. Firstly, the study's exclusive reliance on mice might limit the generalizability of the findings to humans due to inherent physiological differences. Additionally, the absence of direct investigation into the underlying molecular mechanisms responsible for the observed outcomes leaves room for speculation. Moreover, the research's concentration on male and young mice raises questions about the applicability of these findings to female and older subjects. Lastly, the study's duration and the specific resistance exercise protocol utilized might not fully reflect long-term human scenarios, underscoring the need for further research in more diverse populations and over extended timeframes.

Reviewer #1 (Public Review):

The paper from Hsu and co-workers describes a new automated method for analyzing the cell wall peptidoglycan composition of bacteria using liquid chromatography and mass spectrometry (LC/MS) combined with newly developed analysis software. The work has great potential for determining the composition of bacterial cell walls from diverse bacteria in high-throughput, allowing new connections between cell wall structure and other important biological functions like cell morphology or host-microbe interactions to be discovered. A downside to the method is that it does require some prior knowledge of an organisms peptidoglycan composition to generate the database for automated analysis. Nevertheless, the automation will allow rapid analysis of peptidoglycan composition under a variety of conditions and/or between closely related organisms once the general peptidoglycan structure is known. The methodology described will therefore be useful for the field.

The potential connection between the structure of different cell walls from bifidobacteria and cell stiffness proposed in the report is weak. The cells analyzed are from different strains such that there are many possible reasons for the change in physical measurements made by AFM. Conclusions relating cell wall composition to stiffness would be best drawn from a single strain of bacteria genetically modified to have an altered content of 3-3 crosslinks.

Reviewer #1 (Public Review):

The manuscript describes that cultured mammalian cells adapt to chronic stress by increasing their size and protein translation through Hsp90. The authors extensively use Hsp90 knockout cells and mass spectrometry to provide solid evidence that chronic heat shock response is accompanied by cell size changes and stress resistance in large cells. The major strength of the work is the authors ability to document the heat shock response in detail. The increased stress resistance of large cells is conceptually important and provides one potential explanation why cells need to control their size. This work adds to our understanding of how cellular stress is managed, and while stress responses have been observed previously in relation to cell size, this work provides evidence for increased stress resistance in larger cells.

Reviewer #1 (Public Review):

The manuscript by Sejour et al. is testing "translational ramp" model described previously by Tuller et al. in S. cerevisiae. Authors are using bioinformatics and reporter based experimental approaches to test whether "rare codons" in the first 40 codons of the gene coding sequences increase translation efficiency and regulate abundance of translation products in yeast cells. Authors conclude that "translation ramp" model does not have support using a new set of reporters and bioinformatics analyses. The strength of bioinformatic evidence and experimental analyses (even very limited) of the rare codons insertion in the reporter make a compelling case for the authors claims. However the major weakness of the manuscript is that authors do not take into account other models that previously disputed "rare or slow codon" model of Tuller et al. and overstate their own results that are rather limited. This maintains to be the weak part of the manuscript even in the revised form.

The studies that authors do not mention argue with "translation ramp" model and show more thorough analyses of translation initiation to elongation transition as well as early elongation "slow down" in ribosome profiling data. Moreover several studies have used bioinformatical analyses to point out the evolution of N-terminal sequences in multiple model organisms including yeast, focusing on either upstream ORFs (uORFs) or already annotated ORFs. The authors did not mention multiple of these studies in their revised manuscript and did not comment on their own results in the context of these previous studies. As such the authors approach to data presentation, writing and data discussion makes the manuscript rather biased, focused on criticizing Tuller et al. study and short on discussing multiple other possible reasons for slow translation elongation at the beginning of the protein synthesis. This all together makes the manuscript at the end very limited.

Reviewer #1 (Public Review):

Zhang et al. investigate the hypothesis that tRNA methyl transferase 1 (TRMT1) is cleaved by NSP5 (nonstructural protein 5 or MPro), the SARS-CoV-2 main protease, during SARS-CoV-2 infection. They provide solid evidence that TRMT1 is a substrate of Nsp5, revealing an Nsp5 target consensus sequence and evidence of TRMT1 cleavage in cells. Their conclusions are exceptionally strong given the co-submission by D'Oliveira et al showing cleavage of TRMT1 in vitro by Nsp5. Separately, the authors convincingly demonstrate widespread downregulation of RNA modifications during CoV-2 infection, including a requirement for TRMT1 in efficient viral replication. This finding is congruent with the authors' previous work defining the impact of TRMT1 and m2,2g on global translation, which is most likely necessary to support infection and virion production. What still remains unclear is the functional relevance of TRMT1 cleavage by Nsp5 during infection. Based on the data provided here, TRMT1 cleavage may be an act by CoV-2 to self-limit replication, as the expression of a non-cleavable TRMT1 (versus wild-type TRMT1) supports enhanced viral RNA expression at certain MOIs. Theoretically, TRMT1 cleavage should inactivate the modification activity of TRMT1, which the authors thoroughly and elegantly investigate with rigorous biochemical assays. However, only a minority of TRMT1 undergoes cleavage during infection in this study and thus whether TRMT1 cleavage serves an important functional role during CoV-2 replication will be an important topic for future work. The authors fairly assess their work in this regard. This study pushes forward the idea that control of tRNA expression and functionality is an important and understudied area of host-pathogen interaction.

Weaknesses noted:<br /> The detection of the N-terminal TRMT1 fragment by western blot is not robust. The polyclonal antibody used to detect TRMT1 in this work cross-reacts with a non-specific protein product. Unfortunately, this obstructs the visualization of the predicted N-terminal TRMT1 fragment. It is unclear how the authors were able to perform densitometry, given the interference of the non-specific band. Additionally, the replicates in the source data make it clear that the appearance of the N-terminal fragment "wisp" under the non-specific band is not seen in every replicate. Though the disappearance of this wisp with mutant Nsp5 and uncleavable TRMT1 is reassuring, the detection of the N-terminal fragment with the TRMT1 antibody should be assessed critically. Considering this group has strong research interests in TRMT1, I assume that attempts to make other antibodies have proved unfruitful. Additionally, N-terminal tagging of TRMT1 is predicted to disrupt the mitochondrial targeting signal, eliminating the potential for using alternative antibodies to see the N-terminal fragment. These technical issues reiterate the fact that the functional significance of TRMT1 cleavage during CoV-2 infection remains unclear. However, this study demonstrates an important finding that the tRNA modification landscape is altered during CoV-2 infection and that TRMT1 is an important host factor supporting CoV-2 replication.

Reviewer #1 (Public Review):

In this study, the structural characteristics of plant AlaDC and SerDC were analyzed to understand the mechanism of functional differentiation, deepen the understanding of substrate specificity and catalytic activity evolution, and explore effective ways to improve the initial efficiency of theanine synthesis.

On the basis of previous solid work, the authors successfully obtained the X-ray crystal structures of the precursors of theanine synthesis-CsAlaDC and AtSerDC, which are key proteins related to ethylamine synthesis, and found a unique zinc finger structure on these two crystal structures that are not found in other Group II PLP- dependent amino acid decarboxylases. Through a series of experiments, it is pointed out that this characteristic zinc finger motif may be the key to the folding of CsAlaDC and AtSerDC proteins, and this discovery is novel and prospective in the study of theine synthesis.

In addition, the authors identified Phe106 of CsAlaDC and Tyr111 of AtSerDC as key sites of substrate specificity by comparing substrate binding regions and identified amino acids that inhibit catalytic activity through mutation screening based on protein structure. It was found that the catalytic activity of CsAlaDCL110F/P114A was 2.3 times higher than that of CsAlaDC. At the same time, CsAlaDC and AtSerDC substrate recognition key motifs were used to carry out evolutionary analysis of the protein sequences that are highly homologous to CsAlaDC in embryos, and 13 potential alanine decarboxylases were found, which laid a solid foundation for subsequent studies related to theanine synthesis.

In general, this study has a solid foundation, the whole research idea is clear, the experimental design is reasonable, and the experimental results provide strong evidence for the author's point of view. Through a large number of experiments, the key links in the theanine synthesis pathway are deeply studied, and an effective way to improve the initial efficiency of theanine synthesis is found, and the molecular mechanism of this way is expounded. The whole study has good novelty and prospectivity, and sheds light on a new direction for the efficient industrial synthesis of theanine.

Reviewer #1 (Public Review):

D'Oliviera et al. have demonstrated cleavage of human TRMT1 by the SARS-CoV-2 main protease in vitro. Following this, they solved the structure of Mpro-C145A bound to TRMT1 substrate peptide, revealing binding conformation distinct from most viral substrates. Overall, this work enhances our understanding of substrate specificity for a key drug target of CoV2. The paper is well-written and the data is clearly presented. It complements the companion article by demonstrating the interaction between Mpro and TRMT1 and TRMT1 cleavage under isolated conditions in vitro. Importantly, the revelation of flexible substrate binding of Nsp5 is fundamental for understanding Nsp5 as a drug target. Trmt1 cleavage assays revealed similar kinetics for TRMT1 cleavage as compared to the nsp8/9 viral polyprotein cleavage site, however, it would have been more rigorous for the authors to independently reproduce the kinetics reported for nsp8/9 using their specific experimental conditions. The finding that murine TRMT1 lacks a conserved consensus sequence is interesting, but is not experimentally tested here and is reported elsewhere. I am unable to comment critically on the structural analyses as it is outside of my expertise. Overall, I think that these findings are important for confirming TRMT1 as a substrate of Mpro and defining substrate binding and cleavage parameters for an important drug target of SARS-CoV-2.

Joint Public Review:

This study investigates the role of Ikaros, a zinc finger family transcription factor related to Helios and Eos, in T-regulatory (Treg) cell functionality in mice. Through genome-wide association studies and chromatin accessibility studies, the authors find that Ikaros shares similar binding sites to Foxp3. Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibits Th1-like gene expression with abnormal expression of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signalling. Further, two models of inflammatory/ autoimmune diseases - Inflammatory Bowel Disease (IBD) and organ transplantation - are employed to examine the functional role of Ikaros in Treg-mediated immune suppression. The authors provide a detailed analysis of the epigenome and transcriptome of Ikaros-deficient Treg cells.

These studies establish Ikaros as a factor required in Treg for tolerance and the control of inflammatory immune responses. The data are of high quality. Overall, the study is well organized, and reports new data consolidating mechanistic aspects of Foxp3 mediated gene expression program in Treg cells.

Strengths:<br /> The authors have performed biochemical studies focusing on mechanistic aspects of molecular functions of the Foxp3-mediated gene expression program and complemented these with functional experiments using two models of autoimmune diseases, thereby strengthening the study. The studies are comprehensive at both the cellular and molecular levels. The manuscript is well organized and presents a plethora of data regarding the transcriptomic landscape of these cells.

Weakness:<br /> The authors claim that the mice have no pathologic signs of autoimmune disease even at a relatively old age, yet mice have an increased number of activated CD4+ T cells and T-follicular helper cells (even at the age of 6 weeks) as well as reduced naïve T-cells. Thus, immune homeostasis is perturbed in these mice even at a young age and the effect of inflammatory microenvironments on cellular functions cannot be ruled out. Further, clear conclusions from the genome-wide studies are lacking.

Reviewer #1 (Public Review):

Summary:<br /> The Notch signaling pathway plays an important role in many developmental and disease processes. Although well-studied there remain many puzzling aspects. One is the fact that as well as activating the receptor through trans-activation, the transmembrane ligands can interact with receptors present in the same cell. These cis-interactions are usually inhibitory, but in some cases, as in the assays used here, they may also be activating. With a total of 6 ligands and 4 receptors, there is potentially a wide array of possible outcomes when different combinations are co-expressed in vivo. Here the authors set out to make a systematic analysis of the qualitative and quantitative differences in the signaling output from different receptor-ligand combinations, generating sets of "signaling" (ligand expressing) and "receiving" (receptor +/- ligand expressing cells).

The readout of pathway activity is transcriptional, relying on the fusion of GAL4 in the intracellular part of the receptor. Positive ligand interactions result in the proteolytic release of Gal4 that turns on the expression of H2B-citrine. As an indicator of ligand and receptor expression levels, they are linked via TA to H2B mCherry and H2B mTurq expression respectively. The authors also manipulate the expression of the glycosyltransferase Lunatic-Fringe (LFng) that modifies the EGF repeats in the extracellular domains impacting their interactions. The testing of multiple ligand-receptor combinations at varying expression levels is a tour de force, with over 50 stable cell lines generated, and yields valuable insights although as a whole, the results are quite complex.

Strengths:<br /> Taking a reductionist approach to testing systematically differences in the signaling strength, binding strength, and cis-interactions from the different ligands in the context of the Notch1 and Notch 2 receptors (they justify well the choice of players to test via this approach) produces a baseline understanding of the different properties and leads to some unexpected and interesting findings. Notably:

- Jag1 ligand expressing cells failed to activate Notch1 receptor although were capable of activating Notch2. Conversely, Jag2 cells elicited the strongest activation of both receptors. The results with Jag1 are surprising also because it exhibits some of the strongest binding to plate-bound ligands. The failure to activate Notch1 has major functional significance and it will be important in the future to understand the mechanistic basis.

- Jagged ligands have the strongest ciis-inhibitory effects and the receptors differ in their sensitivity to cis-inhibition by Dll ligands. These observations are in keeping with earlier in vivo and cell culture studies. More referencing of those would better place the work in context but it nicely supports and extends previous studies that were conducted in different ways.

- Responses to most trans-activating ligands showed a degree of ultrasensitivity but this was not the case for cis-interactions where effects were more linear. This has implications for the way the two mechanisms operate and for how the signaling levels will be impacted by ligand expression levels.

- Qualitatively similar results are obtained in a second cell line, suggesting they reflect fundamental properties of the ligands/receptors.

Weaknesses:<br /> One weakness is that the methods used to quantify the expression of ligands and receptors rely on the co-translation of tagged nuclear H2B proteins. These may not accurately capture surface levels/correctly modified transmembrane proteins. In general, the multiple conditions tested partly compensate for the concerns - for example, as Jag1 cells do activate Notch2 even if they do not activate Notch1 some Jag1 must be getting to the surface. But even with Notch2, Jag1 activities are on the lower side, making it important to clarify, especially given the different outcomes with the plated ligands. Similarly, is the fact that all ligands "signalled strongest to Notch2" an inherent property or due to differences in surface levels of Notch 2 compared to Notch1? The results would be considerably strengthened by calibration of the ligand/receptor levels (and ideally their sub-cellular localizations). Assessing the membrane protein levels would be relatively straightforward to perform on some of the basic conditions because their ligand constructs contain Flag tags, making it plausible to relate surface protein to H2B, and there are antibodies available for Notch1 and Notch2.

Cis-activation as a mode of signaling has only emerged from these synthetic cell culture assays raising questions about its physiological relevance. Cis-activation is only seen at the higher ligand (Dll1, Dll4) levels, how physiological are the expression levels of the ligands/receptors in these assays? Is it likely that this would make a major contribution in vivo? Is it possible that the cells convert themselves into "signaling" and "receiving" sub-populations within the culture by post-translational mechanism? Again some analysis of the ligand/receptors in the cultures would be a valuable addition to show whether or not there are major heterogeneities.

It is hard to appreciate how much cell-to-cell variability in the "output" there is. For example, low "outputs" could arise from fewer cells becoming activated or from all cells being activated less. As presented, only the latter is considered. That may be already evident in their data, but not easy for the reader to distinguish from the way they are presented. For example, in many of the graphs, data have been processed through multiple steps of normalization. Some discussion/consideration of this point is needed.

Impact:<br /> Overall, cataloguing the outcomes from the different ligand-receptor combinations, both in cis and trans, yields a valuable baseline for those investigating their functional roles in different contexts. There is still a long way to go before it will be possible to make a predictive model for outcomes based on expression levels, but this work gives an idea about the landscape and the complexities. This is especially important now that signaling relationships are frequently hypothesised based on single-cell transcriptomic data. The results presented here demonstrate that the relationships are not straightforward when multiple players are involved.

Reviewer #1 (Public Review):

In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3, by showing that it interacts with an RNA binding protein (IGF2BP2) and, by sequestering it, it regulates the expression of hundreds of genes containing a sequence (11-mer motif) in their untranslated regions (3'-UTR). This sequence is also present in circHIPK3, precisely where IGF2BP2 binds. The study further focuses on one specific case, the STAT3 gene, whose mRNA product is downregulated upon circHIPK3 depletion apparently through sequestering IGF2BP2, which otherwise binds to and stabilizes STAT3 mRNA. The study presents mechanistic insight into the interactions, sequence motifs, and stoichiometries of the molecules involved in this new mode of regulation. Altogether, this new mechanism seems to underlie the effects of circHIPK3 in cancer progression.

Strengths:<br /> The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather by a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of study. They provide both genome-wide analysis and a specific case (STAT3) that is relevant for cancer progression.

Weaknesses:<br /> One of the central conclusions of the manuscript, namely that circHIPK3 sequesters IGF2BP2 and thereby regulates target mRNAs, lacks more direct experimental evidence such as rescue experiments where both species are simultaneously knocked down. CircRNA overexpression lacks a demonstration of circularization efficiencies. There seem to be contradictory effects of circHIPK3 and STAT3 depletion in cancer progression, namely that while circHIPK3 is frequently downregulated in cancer, circHIPK3 downregulation in this study leads to downregulation of STAT3. This does not seem to fit the fact that STAT3 is normally activated in a wide diversity of cancers and is positively associated with cell proliferation. The result is neither consistent with the fact that circHIPK3 expression positively correlates with good clinical outcomes. Overall, the authors have achieved some of their aims but additional controls would be advisable to fully support their conclusions.

Reviewer #1 (Public Review):

Summary:<br /> The authors describe the dynamic distribution of laminin in the olfactory system and forebrain. Using immunohistochemistry and transgenic lines, they found that the olfactory system and adjacent brain tissues are enveloped by BMs from the earliest stages of olfactory system assembly. They also found that laminin deposits follow the axonal trajectory of axons. They performed a functional analysis of the sly mutant to analyse the function of laminin γ1 in the development of the zebrafish olfactory system. Their study revealed that laminin enables the shape and position of placodes to be maintained late in the face of major morphogenetic movements in the brain, and its absence promotes the local entry of sensory axons into the brain and their navigation towards the olfactory bulb.

Strengths:<br /> -They showed that in the sly mutants, no BM staining of laminin and Nidogen could be detected around the OP and the brain. The authors then elegantly used electron microscopy to analyse the ultrastructure of the border between the OP and the brain in control and sly mutant conditions.<br /> -To analyse the role of laminin γ1-dependent BMs in OP coalescence, the authors used the cluster size of Tg(neurog1:GFP)+ OP cells at 22 hpf as a marker. They found that the mediolateral dimension increased specifically in the mutants. However, proliferation did not seem to be affected, although apoptosis appeared to increase slightly at a later stage. This increase could therefore be due to a dispersal of cells in the OP. To test this hypothesis, the authors then analysed the cell trajectories and extracted 3D mean square displacements (MSD), a measure of the volume explored by a cell in a given period of time. Their conclusion indicates that although brain cell movements are increased in the absence of BM during coalescence phases, overall OP cell movements occur within normal parameters and allow OPs to condense into compact neuronal clusters in sly mutants. The authors also analysed the dimensions of the clusters composed of OMP+ neurons. Their results show an increase in cluster size along the dorso-ventral axis. These results were to be expected since, compared with BM, early neurog1+ neurons should compact along the medio-lateral axis, and those that are OMP+ essentially along the dorso-ventral axis. In addition to the DV elongation of OP tissue, the authors show the existence of isolated and ectopic (misplaced) YFP+ cells in sly mutants.<br /> -To understand the origin of these phenotypes, the authors analysed the dynamic behaviour of brain cells and OPs during forebrain flexion. The authors then quantitatively measured brain versus OPs in the sly mutant and found that the OP-brain boundary was poorly defined in the sly mutant compared with the control. Once again, the methods (cell tracks, brain size, and proliferation/apoptosis, and the shape of the brain/OP boundary) are elegant but the results were expected.<br /> -They then analysed the dynamic behaviour of the axon using live imaging. Thus, olfactory axon migration is drastically impaired in sly mutants, demonstrating that Laminin γ1-dependent BMs are essential for the growth and navigation of axons from the OP to the olfactory bulb.<br /> -The authors therefore performed a quantitative analysis of the loss of function of Laminin γ1. They propose that the BM of the OP prevents its deformation in response to mechanical forces generated by morphogenetic movements of the neighbouring brain.

Weaknesses:<br /> - The authors did not analyse neurog1 + axonal migration at the level of the single cell and instead made a global analysis. An analysis at the cell level would strengthen their hypotheses.<br /> - Rescue experiments by locally inducing Laminin expression would have strengthened the paper.<br /> -The paper lacks clarity between the two neuronal populations described (early EONs and late OSNs).<br /> -The authors quantitatively measured brain versus OPs in the sly mutant and found that the OP-brain boundary was poorly defined in the sly mutant compared with the control. Once again, the methods (cell tracks, brain size, proliferation/apoptosis, and the shape of the brain/OP boundary) are elegant but the results were expected.<br /> - A missing point in the paper is the effect of Laminin γ1 on the migration of cranial NCCs that interact with OP cells. The authors could have analysed the dynamic distribution of neural crest cells in the sly mutant.

Reviewer #1 (Public Review):

Most amino acids are stereoisomers in the L-enantiomer, but natural D-serine has also been detected in mammals and its levels shown to be connected to a number of different pathologies. Here, the authors convincingly show that D-serine is transported in the kidney by the neutral amino acid transporter ASCT2 and as a non-canonical substrate for the sodium-coupled monocarboxylate transporter SMCTs. Although both transport D-serine, this important study further shows in a mouse model for acute kidney injury that ASCT2 has the dominant role.

Strengths:<br /> The paper combines proteomics, animal models, ex vivo transport analyses, and in vitro transport assays using purified components. The exhaustive methods employed provide compelling evidence that both transporters can translocate D-serine in the kidney.

Weakness:<br /> In the model for acute kidney injury, the SMCTs proteins were not showing a significant change in expression levels and were rather analysed based on other, circumstantial evidence. Although its clear SMCTs can transport D-serine its physiological role is less obvious compared to ASCT2.

Reviewer #1 (Public Review):

Summary. The authors goal was to map the neural circuitry underlying cold sensitive contraction in Drosophila. The circuitry underlying most sensory modalities has been characterized but noxious cold sensory circuitry has not been well studied. The authors achieve their goal and map out sensory and post-sensory neurons involved in this behavior.

Strengths. The manuscript provides convincing evidence for sensory and post sensory neurons involved in noxious cold sensitive behavior. They use both connectivity data and functional data to identify these neurons. This work is a clear advance in our understanding of noxious cold behavior. The experiments are done with a high degree of experimental rigor.

Positive comments

-Campari is nicely done to map cold responsive neurons, although it doesn't give data on individual neurons.

-Chrimson and TNT experiments are nicely done.

-Cold temperature activates basin neurons, it's a solid and convincing result.

Weaknesses. Among the few weaknesses in this manuscript is the failure to trace the circuit from sensory neuron to motor neuron; and to ignore analysis of the muscles driving, cold induced contraction. Authors also need to elaborate more on the novel aspects of their work in the introduction or abstract.

Major comments.

-Class three sensory neuron connectivity is known, and role in cold response is known (turner 16, 18). Need to make it clearer what the novelty of the experiments are.

-Why focus on premotor neurons in mechano nociceptive pathways? Why not focus on PMNs innervating longitudinal muscles, likely involved in longitudinal larval contraction? Especially since chosen premotor neurons have only weak effects on cold induced contraction?