Reviewer #1 (Public Review):

Summary:<br /> Using a state-of-the-art image analysis pipeline the authors report that muscle cell hypertrophy in mice and humans occurs primarily through an increase in the number of myofibrils (myofibrillogenesis) and not myofibril hypertrophy.

Strengths:<br /> A strength of the study is the development and validation of an automated image analysis pipeline to quantify myofibril size and abundance in mouse and human muscle cells. In addition to the pipeline, which requires relatively readily available microscopy equipment (an additional strength) is the development of a methodology to optimally prepare muscle samples for high-resolution imaging.

Weaknesses:<br /> A weakness of the study was that only one time-point was assessed during hypertrophy. As mentioned by the authors, this precluded an assessment of the myofibril splitting mechanism. The second weakness was the criteria (aspect ratio of <2.5:1) used to identify a myofibril which excluded a significant number of myofibrils from analysis. How might the inclusion of these odd-shaped myofibrils impact the outcome of the study?

Reviewer #1 (Public Review):

Summary:<br /> This study investigated the phosphoryl transfer mechanism of the enzyme adenylate kinase, using SCC-DFTB quantum mechanical/molecular mechanical (QM/MM) simulations, along with kinetic studies exploring the temperature and pH dependence of the enzyme's activity, as well as the effects of various active site mutants. Based on a broad free energy landscape near the transition state, the authors proposed the existence of wide transition states (TS), characterized by the transferring phosphoryl group adopting a meta-phosphate-like geometry with asymmetric bond distances to the nucleophilic and leaving oxygens. In support of this finding, kinetic experiments were conducted with Ca2+ ions (instead of Mg2+) at different temperatures, which revealed a negative entropy of activation. Overall, in its present form, the manuscript has more weaknesses in terms of interpretation of the simulation results than strengths, which need to be addressed by the authors.

There are several major concerns:

First, the authors' claim that the catalytic mechanism of adenylate kinase (Adk) has not been previously studied by QM/MM free energy simulations is somewhat inaccurate. In fact, two different groups have previously investigated the catalytic mechanism of Adk. The first study, cited by the authors themselves, used the string method to determine the minimum free energy profile, but resulted in an unexpected intermediate; note that they obtained a minimum free energy profile, not a minimum energy profile. The second study (Ojedat-May et al., Biochemistry 2021 and Dulko-Smith et al., J Chem Inf Model 2023) overlaps substantially with the present study, but its main conclusions differ from those of the present study. Therefore, a thorough discussion comparing the results of these studies is needed.

Second, the interpretation of the TS ensemble needs deeper scrutiny. In general, the TS is defined as the hypersurface separating the reactant and product states. Consequently, if a correct reaction coordinate is defined, trajectories initiated at the TS should have equal probabilities of reaching either the reactant or product state; if an approximate reaction coordinate, such as the distance difference used in this study, is used, recrossing may be introduced as a correction into the probabilities. Thus, in order to establish the presence of a wide TS region, it is necessary to characterize the TS ensemble through a commitment analysis across the TS region.

The relatively flat free energy surface observed near TS in Figures 1c and 2a, may be attributed to the cleavage and formation of P-O bonds relative to the marginally stable phosphorane intermediate, as described in Zhou et al.'s work (Chem Rev 1998, 98:991). This scenario is clearly different from a wide TS ensemble concept. In addition, given the inherent similarity in reactivity of the two oxygens towards the phosphoryl atom, it is reasonable to expect a single TS as shown in Figure 1 - supplement 9, rather than two TSs with a marginally stable intermediate as shown in Figure 1c. Consequently, it remains uncertain whether the elongated P-O bonds observed near the TS and their asymmetry are realistic or potentially an artifact of the pulling/non-equilibrium MD simulations. Further validation in this regard is required.

Third, there are several inconsistencies in the free energy results and their discussion. First, the data from Kerns et al. (Kerns, NSMB, 2015, 22:124) indicate that the ATP/AMP -> ADP/ADP reaction proceeds at a faster rate than the ADP/ADP -> ATP/AMP reaction, suggesting that the ADP/ADP state has a lower free energy (approximately -1.0 kcal/mol) compared to the ATP/ATP state. This contrasts with Figure 1c, which shows a higher free energy of 6.0 kcal/mol for the ATP/ADP state. This discrepancy needs to be discussed. Furthermore, the barrier for ATP/AMP -> ADP/ADP, calculated to be 20 kcal/mol for the fully charged state, exceeds the corresponding barrier for the monoprotonated state. This cautions against the conclusion that the fully charged state is the reactive state. In addition, the difference in the barrier for the no-Mg2+ system compared to the barriers with Mg2+ is substantially too large (21 kcal/mol from the calculation versus 7 kcal/mol from the experimental values). These inconsistencies raise questions as to their origins, whether they result from the use of the pulling/non-equilibrium MD simulation approach, which may yield unrealistic TS geometries, or from potential issues related to the convergence of the determined free energy values. To address this issue, a comparison of results obtained by umbrella sampling and similar methodologies is necessary.

Reviewer #1 (Public Review):

Summary:<br /> This work is an extension of the authors' earlier work published in Sci Adv in 2001, wherein the authors showed that DTD2 deacylates N-ethyl-D-aminoacyl-tRNAs arising from acetaldehyde toxicity. The authors in this study, investigate the role of archaeal/plant DTD2 in the deacylation/detoxification of D-Tyr-tRNATyr modified by multiple other aldehydes and methylglyoxal (produced by plants). Importantly, the authors take their biochemical observations to plants, to show that deletion of DTD2 gene from a model plant (Arabidopsis thaliana) makes them sensitive to the aldehyde supplementation in the media especially in the presence of D-Tyr. These conclusions are further supported by the observation that the model plant shows increased tolerance to the aldehyde stress when DTD2 is overproduced from the CaMV 35S promoter. The authors propose a model for the role of DTD2 in the evolution of land plants. Finally, the authors suggest that the transgenic crops carrying DTD2 may offer a strategy for stress-tolerant crop development. Overall, the authors present a convincing story, and the data are supportive of the central theme of the story.

Strengths:<br /> Data are novel and they provide a new perspective on the role of DTD2, and propose possible use of the DTD2 lines in crop improvement.

Weaknesses:<br /> (a) Data obtained from a single aminoacyl-tRNA (D-Tyr-tRNATyr) have been generalized to imply that what is relevant to this model substrate is true for all other D-aa-tRNAs (term modified aa-tRNAs has been used synonymously with the modified Tyr-tRNATyr). This is not a risk-free extrapolation. For example, the authors see that DTD2 removes modified D-Tyr from tRNATyr in a chain-length dependent manner of the modifier. Why do the authors believe that the length of the amino acid side chain will not matter in the activity of DTD2?<br /> (b) While the use of EFTu supports that the ternary complex formation by the elongation factor can resist modifications of L-Tyr-tRNATyr by the aldehydes or other agents, in the context of the present work on the role of DTD2 in plants, one would want to see the data using eEF1alpha. This is particularly relevant because there are likely to be differences in the way EFTu and eEF1alpha may protect aminoacyl-tRNAs (for example see description in the latter half of the article by Wolfson and Knight 2005, FEBS Letters 579, 3467-3472).

Reviewer #1 (Public Review):

The authors aimed to investigate if 2-hydroxybutyrate (2HB), a metabolite induced by exercise, influences physiological changes, particularly metabolic alterations post-exercise training. They treated young mice and cultured myoblasts with 2HB, conducted exercise tests, metabolomic profiling, gene expression analysis, and knockdown experiments to understand 2HB's mechanisms. Their findings indicate that 2HB enhances exercise tolerance, boosts branch chain amino acid (BCAA) enzyme gene expression in skeletal muscles, and increases oxidative capacity. They also highlight the role of SIRT4 in these effects. This study establishes 2HB, once considered a waste product, as a regulator of exercise-induced metabolic processes. The study's strength lies in its consistent results across in vitro, in vivo, and ex vivo analyses. The authors propose a mechanism in which 2HB inhibits BCAA breakdown, raises NAD+/NADH ratio, activates SIRT4, increases ADP ribosylation, and controls gene expression.

However, some questions remain unclear based on these findings:

This study focused on the effects of short-term exercise (1 or 5 bouts of treadmill running) and short-term 2HB treatment (1 or 4 days of treatment). Adaptations to exercise training typically occur progressively over an extended period. It's important to investigate the effects of long-term 2HB treatment and whether extended combined 2HB treatment and exercise training have independent, synergistic, or antagonistic effects.

Exercise training leads to significant mitochondrial changes, including increased mitochondrial biogenesis in skeletal muscle. It would be valuable to compare the impact of 2HB treatment on mitochondrial content and oxidative capacity in treated mice to that in exercised mice.

The authors demonstrate that 2-ketobutyrate (2KB) can serve as an oxidative fuel, suggesting a role for the intact BCAA catabolic pathway. However, it's puzzling that the knockout of BCKDHA, a subunit crucial for the second step of BCAA catabolism, did not result in changes in oxidative capacity in cultured myoblasts.

Nevertheless, this innovative model of metabolic signaling during exercise will serve as a valuable reference for informing future.

Reviewer #1 (Public Review):

Summary:<br /> The OSCA/TMEM63 channels have recently been identified as mechanosensitive channels. In a previous study, the authors found that OSCA subtypes (1, 2, and 3) respond differently to stretch and poke stimuli. For example, OSCA1.2 is activated by both poke and stretch, while OSCA3.1, responds strongly to stretch but poorly to poke stimuli. In this study, the authors use cryo-EM, mutagenesis, and electrophysiology to dissect the mechanistic determinants that underlie the channels' ability to respond to poke and stretch stimuli.

The starting hypothesis of the study is that the mechanical activation of OSCA channels relies on the interactions between the protein and the lipid bilayer and that the differential responses to poke and stretch might stem from variations in the lipid-interacting regions of OSCA proteins. The authors specifically identify the amphipathic helix (AH), the fenestration, and the Beam Like Domain (BLD) as elements that might play a role in mechanosensing.

The strength of this paper lies in the technically sound data - the structural work and electrophysiology are both very well done. For example, the authors produce a high-resolution OSCA3.1 structure which will be a useful tool for many future studies. Also, the study identifies several interesting mutants that seemingly uncouple the OSCA1.2 poke and stretch responses. These might be valuable in future studies of OSCA mechanosensation.

However, the experimental approach employed by the authors to dissect the molecular mechanisms of poke and stretch falls short of enabling meaningful mechanistic conclusions. For example, we are left with several unanswered questions surrounding the role of AH and the fenestration lipids in mechanosensation: Is the AH really important for the poke response if mutating residues conserved between OSCA1.2 and OSCA3.1 disrupts the OSCA1.2 ability to respond to poke but mutating the OSCA1.2 AH to resemble that of OSCA3.1 results in no change to its "pokability"? Similar questions arise in response to the study of the fenestration-lining residues.

Reviewer #1 (Public Review):

This manuscript by Tan et al is using cryo-electron tomography to investigate the structure of yeast nucleosomes both ex vivo (nuclear lysates) and in situ (lamellae and cryosections). The sheer number of experiments and results are astounding and comparable with an entire PhD thesis. However, as is always the case, it is hard to prove that something is not there. In this case, canonical nucleosomes. In their path to find the nucleosomes, the authors also stumble over new insights into nucleosome arrangement that indicates that the positions of the histones is more flexible than previously believed.

Major strengths and weaknesses:

Personally, I am not ready to agree with their conclusion that heterogenous non-canonical nucleosomes predominate in yeast cells, but this reviewer is not an expert in the field of nucleosomes and can't judge how well these results fit into previous results in the field. As a technological expert though, I think the authors have done everything possible to test that hypothesis with today's available methods. One can debate whether it is necessary to have 35 supplementary figures, but after working through them all, I see that the nature of the argument needs all that support, precisely because it is so hard to show what is not there. The massive amount of work that has gone into this manuscript and the state-of-the art nature of the technology should be warmly commended. I also think the authors have done a really great job with including all their results to the benefit of the scientific community. Yet, I am left with some questions and comments:

Could the nucleosomes change into other shapes that were predetermined in situ? Could the authors expand on if there was a structure or two that was more common than the others of the classes they found? Or would this not have been found because of the template matching and later reference particle used?

Could it simply be that the yeast nucleoplasm is differently structured than that of HeLa cells and it was harder to find nucleosomes by template matching in these cells? The authors argue against crowding in the discussion, but maybe it is just a nucleoplasm texture that side-tracks the programs?

The title of the paper is not well reflected in the main figures. The title of Figure 2 says "Canonical nucleosomes are rare in wild-type cells", but that is not shown/quantified in that figure. Rare is comparison to what? I suggest adding a comparative view from the HeLa cells, like the text does in lines 195-199. A measure of nucleosomes detected per volume nucleoplasm would also facilitate a comparison.

If the cell contains mostly non-canonical nucleosomes, are they really non-canonical? Maybe a change of language is required once this is somewhat sure (say, after line 303).

The authors could explain more why they sometimes use conventional the 2D followed by 3D classification approach and sometimes "direct 3-D classification". Why, for example, do they do 2D followed by 3D in Figure S5A? This Figure could be considered a regular figure since it shows the main message of the paper.

Figure 1: Why is there a gap in the middle of the nucleosome in panel B? The authors write that this is a higher resolution structure (18Å), but in the even higher resolution crystallography structure (3Å resolution), there is no gap in the middle.

Reviewer #1 (Public Review):

This manuscript by Tan et al is using cryo-electron tomography to investigate the structure of yeast nucleosomes both ex vivo (nuclear lysates) and in situ (lamellae and cryosections). The sheer number of experiments and results are astounding and comparable with an entire PhD thesis. However, as is always the case, it is hard to prove that something is not there. In this case, canonical nucleosomes. In their path to find the nucleosomes, the authors also stumble over new insights into nucleosome arrangement that indicates that the positions of the histones is more flexible than previously believed.

Major strengths and weaknesses:

Personally, I am not ready to agree with their conclusion that heterogenous non-canonical nucleosomes predominate in yeast cells, but this reviewer is not an expert in the field of nucleosomes and can't judge how well these results fit into previous results in the field. As a technological expert though, I think the authors have done everything possible to test that hypothesis with today's available methods. One can debate whether it is necessary to have 35 supplementary figures, but after working through them all, I see that the nature of the argument needs all that support, precisely because it is so hard to show what is not there. The massive amount of work that has gone into this manuscript and the state-of-the art nature of the technology should be warmly commended. I also think the authors have done a really great job with including all their results to the benefit of the scientific community. Yet, I am left with some questions and comments:

Could the nucleosomes change into other shapes that were predetermined in situ? Could the authors expand on if there was a structure or two that was more common than the others of the classes they found? Or would this not have been found because of the template matching and later reference particle used?

Could it simply be that the yeast nucleoplasm is differently structured than that of HeLa cells and it was harder to find nucleosomes by template matching in these cells? The authors argue against crowding in the discussion, but maybe it is just a nucleoplasm texture that side-tracks the programs?

The title of the paper is not well reflected in the main figures. The title of Figure 2 says "Canonical nucleosomes are rare in wild-type cells", but that is not shown/quantified in that figure. Rare is comparison to what? I suggest adding a comparative view from the HeLa cells, like the text does in lines 195-199. A measure of nucleosomes detected per volume nucleoplasm would also facilitate a comparison.

If the cell contains mostly non-canonical nucleosomes, are they really non-canonical? Maybe a change of language is required once this is somewhat sure (say, after line 303).

The authors could explain more why they sometimes use conventional the 2D followed by 3D classification approach and sometimes "direct 3-D classification". Why, for example, do they do 2D followed by 3D in Figure S5A? This Figure could be considered a regular figure since it shows the main message of the paper.

Figure 1: Why is there a gap in the middle of the nucleosome in panel B? The authors write that this is a higher resolution structure (18Å), but in the even higher resolution crystallography structure (3Å resolution), there is no gap in the middle.

Reviewer #1 (Public Review):

The goal of this study was to determine whether short (1 month) internships for biomedical science trainees (mostly graduate students but some post-docs) were beneficial for the trainees, their mentors, and internship hosts. Over a 5 year period, the outcomes of trainees who completed internships were compared with peers who did not. Both quantitative results in terms of survey responses and qualitative results obtained from discussion groups were provided. Overall, the data suggest that internships aid graduate students in multiple ways and do not harm progress on dissertation projects. 'Buy-in' from mentors and prospective mentors appeared to increase over time, and hosts also gained from the contributions of the interns even in a short time period. While the program also appeared valuable for post-doctoral trainees, it was less favorably considered by post-doc mentors.

Strengths:

The internship program that was examined here appears to have been very well designed in terms of availability to students, range of internship offerings, length of time away from PhD lab, and assessments.<br /> Having a built-in peer control group of graduate students who did not do internships was valuable for much of the quantitative analyses. However, as the authors acknowledge, those who did opt for internships are a self-selected group who may have character traits that would help them overcome the potential negative impacts of the internship.<br /> The quantitative data is convincing and addresses important considerations for all stakeholders.<br /> The manuscript is well-constructed to individually address the impact of the program on each set of stakeholders, while also showcasing areas of mutual benefit.<br /> The discussion of challenges and limitations, from the perspectives of participating stakeholders, program leaders, and also institutions, is comprehensive and very thoughtful.

Weaknesses:

The qualitative data that resulted from the 'focus groups' of faculty mentors was somewhat difficult to evaluate given the very limited number of participants (n=7).

Overall, the data support the authors' conclusions with respect to the utility of internship programs for all stakeholders. As the authors note, the data relate to a specific program where internship length was defined, costs were covered by a grant or institutional funding, and there were multiple off-site internship hosts available. Thus, the results here may not replicate for other programs with different criteria.

This work provides a valuable assessment of how relatively short internships can impact graduate students, both in terms of their graduate tenure and in their decision-making for careers post-graduation. As more graduate programs are heeding calls from funding agencies and professional societies to increase knowledge about, and familiarity with, multiple career paths beyond academia for PhD students, there is a need to evaluate the best ways to accomplish that goal. Hands-on internships are valuable across many spheres so it makes sense that they would be for life science graduates too. However, the fear that time-to-degree and/or productivity would be negatively impacted is important to acknowledge. By providing clear data that this is not the case, these investigators have increased the likelihood that internships could be considered by more institutions. The one big drawback, and one that the authors discuss at some length, is the funding model that could enable internship programs to be used more widely.

Reviewer #1 (Public Review):

Summary:<br /> Huang and colleagues present a method for approximation of linkage disequilibrium (LD) matrices. The problem of computing LD matrices is the problem of computing a correlation matrix. In the cases considered by the authors, the number of rows (n), corresponding to individuals, is small compared to the number of columns (m), corresponding to the number of variants. Computing the correlation matrix has cubic time complexity [O(nm^2)], which is prohibitive for large samples. The authors approach this using three main strategies: 1. they compute a coarsened approximation of the LD matrix by dividing the genome into variant-wise blocks which statistics are effectively averaged over; 2. they use a trick to get the coarsened LD matrix from a coarsened genomic relatedness matrix (GRM), which, with O(n^2 m) time complexity, is faster when n << m; 3. they use the Mailman algorithm to improve the speed of basic linear algebra operations by a factor of log(max(m,n)). The authors apply this approach to several datasets.

Strengths:<br /> - the authors demonstrate that their proposed method performs in line with theoretical explanations<br /> - the coarsened LD matrix is useful for describing global patterns of LD, which do not necessarily require variant-level resolution<br /> - they provide an open-source implementation of their software

Weaknesses:<br /> - the coarsened LD matrix is of limited utility outside of analyzing macroscale LD characteristics<br /> - the method still essentially has cubic complexity--albeit the factors are smaller and Mailman reduces this appreciably. It would be interesting if the authors were able to apply randomized or iterative approaches to achieve more fundamental gains. The algorithm remains slow when n is large and/or the grid resolution is increased.

Joint Public Review:

Summary:

In this paper, the authors point out that the standard approach of estimating LD is inefficient for datasets with large numbers of SNPs, with a computational cost of O(nm^2), where n is the number of individuals and m is the number of SNPs. Using the known relationship between the LD matrix and the genomic-relatedness matrix, they can calculate the mean level of LD within the genome or across genomic segments with a computational cost of O(n^2m). Since in most datasets, n<<br /> Strengths:

Generally, for computational papers like this, the proof is in the pudding, and the authors have been successful at their aim of producing an efficient computational tool. The most compelling evidence of this in the paper are Figure 2 and Supplementary Figure S2. In Figure 2, they report how well their X-LD estimates of LD compare to estimates based on the standard approach using PLINK. They appear to have very good agreement. In Figure S2, they report the computational runtime of X-LD vs PLINK, and as expected X-LD is faster than PLINK as long as it is evaluating LD for more than 8000 SNPs.

Weakness:

This method seems to be limited to calculating average levels of LD in broad regions of the genome. While it would be possible to make the regions more fine-grained, doing so appears to make this approach much less efficient. As such, applications of this method may be limited to those proposed in the paper, for questions where average LD of large chromosomal segments is informative.

Impact:

This approach seems to produce real gains for settings where broad average levels of LD are useful to know, but it will likely have less of an impact in settings where fine-grained levels are LD are necessary (e.g., accounting for LD in GWAS summary statistics).

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 it 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):

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 is an interesting and somewhat unusual paper supporting the idea that creatine is a neurotransmitter in the central nervous system of vertebrates. The idea is not entirely new, and the authors carefully weigh the evidence, both past and newly acquired, to make their case. The strength of the paper lies in the importance of the potential discovery - as the authors point out, creatine ticks more boxes on criteria of neurotransmitters than some of the ones listed in textbooks - and the list of known transmitters (currently 16) certainly is textbook material. A further strength of the manuscript is the careful consideration of a list of criteria for transmitters and newly acquired evidence for four of these criteria: 1. evidence that creatine is stored in synaptic vesicles, 2. mutants for creatine synthesis and a vesicular transporter show reduced storage and release of creatine, 3. functional measurement that creatine release has an excitatory or inhibitory (here inhibitory) effect in vivo, and 4. ATP-dependence. The key weakness of the paper is that there is no single clear 'smoking gun', like a postsynaptic creatine receptor, that would really demonstrate the function as a transmitter. Instead, the evidence is of a cumulative nature, and not all bits of evidence are equally strong. On balance, I found the path to discovery and the evidence assembled in this manuscript to establish a clear possibility, positive evidence, and to provide a foundation for further work in this direction.

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):

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. The authors rightfully point out the complexities of the adaptation process and that the truth must lie somewhere between two extreme models of evolutionary genetics, i.e. a population genetics view of large effect loci and a quantitative genetics model. The authors take great care in acknowledging and investigating the multiple biases inherent to the used methods (GEA and GWAS) and use a conservative approach to draw their conclusions. The multiplicity of analyses and their interdependence make them slightly hard to understand and the manuscript would benefit from more careful explanations of concepts and logical links throughout. This work will be of interest to evolutionary biologists and population geneticists in particular, and constitutes an additional applied example to the comparative local adaptation literature.

Some thoughts on the last paragraph of the discussion (L481-497): I think it would be fine to have some more thoughts here on the processes that could contribute to the presence/absence of inversions, maybe in an "Ideas and Speculation" subsection. To me, your results point to the fact that though inversions are often presented as important for local adaptation, they seem to be highly contingent on the context of adaptation in each species. First, repeatability results are only at the window/gene level in your results, the specific mutations are not under scrutiny. Is it possible that inversions are only necessary when sets of small effect mutations are used, opposite to a large effect mutation in other species? Additionally, in a model with epistasis, fitness effects of mutations are dependent on the genomic background and it is possible that inversions were necessary in only certain contexts, even for the same mutations, i.e. some adaptive path contingency. Finally, do you have specific demographic history knowledge in this system that maps to the observations of the presence of inversions or not? For example, have the species "using" inversions been subject to more gene flow compared to others?

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:

The work by Combrisson and colleagues investigates the degree to which reward and punishment learning signals overlap in the human brain using intracranial EEG recordings. The authors used information theory approaches to show that local field potential signals in the anterior insula and the three sub regions of the prefrontal cortex encode both reward and punishment prediction errors, albeit to different degrees. Specifically, the authors found that all four regions have electrodes that can selectively encode either the reward or the punishment prediction errors. Additionally, the authors analyzed the neural dynamics across pairs of brain regions and found that the anterior insula to dorsolateral prefrontal cortex neural interactions were specific for punishment prediction errors whereas the ventromedial prefrontal cortex to lateral orbitofrontal cortex interactions were specific to reward prediction errors. This work contributes to the ongoing efforts in both systems neuroscience and learning theory by demonstrating how two differing behavioral signals can be differentiated to a greater extent by analyzing neural interactions between regions as opposed to studying neural signals within one region.

Strengths:

The experimental paradigm incorporates both a reward and punishment component that enables investigating both types of learning in the same group of subjects allowing direct comparisons.

The use of intracranial EEG signals provides much needed insight into the timing of when reward and punishment prediction errors signals emerge in the studied brain regions.

Information theory methods provide important insight into the interregional dynamics associated with reward and punishment learning and allows the authors to assess that reward versus punishment learning can be better dissociated based on interregional dynamics over local activity alone.

Weaknesses:

The analysis presented in the manuscript focuses solely on gamma band activity. The presence and potential relevance of other frequency bands is not discussed. It is possible that slow oscillations, which are thought to be important for coordinating neural activity across brain regions could provide additional insight.

The data is averaged across all electrodes which could introduce biases if some subjects had many more electrodes than others. Controlling for this variation in electrode number across subjects would ensure that the results are not driven by a small subset of subjects with more electrodes.

The potential variation in reward versus punishment learning across subjects is not included in the manuscript. While the time course of reward versus punishment prediction errors is symmetrical at the group level, it is possible that some subjects show faster learning for one versus the other type which can bias the group average. Subject level behavioral data along with subject level electrode numbers would provide more convincing evidence that the observed effects are not arising from these potential confounds.

It is unclear if the findings in Figures 3 and 4 truly reflect the differential interregional dynamics in reward versus punishment learning or if these results arise as a statistical byproduct of the reward vs punishment bias observed within each region. For instance, the authors show that information transfer from anterior insula to dorsolateral prefrontal cortex is specific to punishment prediction error. However, both anterior insula and dorsolateral prefrontal cortex have higher prevalence of punishment prediction error selective electrodes to begin with. Therefore the findings in Fig 3 may simply be reflecting the prevalence of punishment specificity in these two regions above and beyond a punishment specific neural interaction between the two regions. Either mathematical or analytical evidence that assesses if the interaction effect is simply reflecting the local dynamics would be important to make this result convincing.

Reviewer #1 (Public Review):

Summary:

The current work by Kulich et al. examines the dynamic relocalization of NGR1 (LAZY2) a member of the LAZY protein family which is key for auxin redistribution during gravitropic responses. After gravistimulation of the triple mutant ngr123 (lazy234), the PIN3 activating kinase D6PK is not polarized in the columella cells.

Strengths:

The authors show a thorough characterization of NGR1 relocalization dynamics after gravistimulation.

Weaknesses:

Genetically the relocalization of D6PK depends on the LAZY protein family, but some essential details are missing in this study. On the one hand, NGR1-GFP does not associate with the BFA compartments and maintains its association with the PM and amyloplasts. On the other hand, D6PK relies on GNOM, via vesicle trafficking sensitive to BFA, suggesting that D6PK follows a different relocalization route than NGR1 which is BFA-insensitive. Based on these observations, D6PK relocalization requires the LAZY proteins, but D6PK and NGR1 relocalize through independent routes. How can this be interpreted or reconciled?

Two other works (now published) provide valuable and fundamental findings related to the mechanism examined in the current manuscript and display complementary and similar results to the ones shown in the current manuscript. Given the similarities in the examined mechanisms, these preprints should be referenced, recognized, and discussed in the manuscript under review. It is assumed that the three projects were independently developed, but the results of these previous works should be addressed and taken into account at least during the discussion and when drawing any conclusions. This does not mean that this work is less relevant. On the contrary, some of the observations that seem to be redundant are more solid, and firm conclusions can now be drawn from them.

Reviewer #1 (Public Review):

Summary:<br /> "Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock" is a convincing manuscript that delves into the structural and biochemical aspects of FRQ and the FFC under both LLPS and non-LLPS conditions. Circadian clocks serve as adaptations to the daily rhythms of sunlight, providing a reliable internal representation of local time.

All circadian clocks are composed of positive and negative components. The FFC contributes negative feedback to the Neurospora circadian oscillator. It consists of FRQ, CK1, and FRH. The FFC facilitates close interaction between CK1 and the WCC, with CK1-mediated phosphorylation disrupting WCC:c-box interactions necessary for restarting the circadian cycle.

Despite the significance of FRQ and the FFC, challenges associated with purifying and stabilizing FRQ have hindered in vitro studies. Here, researchers successfully developed a protocol for purifying recombinant FRQ expressed in E. coli.

Armed with full-length FRQ, they utilized spin-labeled FRQ, CK1, and FRH to gain structural insights into FRQ and the FFC using ESR. These studies revealed a somewhat ordered core and a disordered periphery in FRQ, consistent with prior investigations using limited proteolysis assays. Additionally, p-FRQ exhibited greater conformational flexibility than np-FRQ, and CK1 and FRH were found in close proximity within the FFC. The study further demonstrated that under LLPS conditions in vitro, FRQ undergoes phase separation, encapsulating FRH and CK1 within LLPS droplets, ultimately diminishing CK1 activity within the FFC. Intriguingly, higher temperatures enhanced LLPS formation, suggesting a potential role of LLPS in the fungal clock's temperature compensation mechanism.

Biological significance was supported by live imaging of Neurospora, revealing FRQ foci at the periphery of nuclei consistent with LLPS. The amino acid sequence of FRQ conferred LLPS properties, and a comparison of clock repressor protein sequences in other eukaryotes indicated that LLPS formation might be a conserved process within the negative arms of these circadian clocks.

In summary, this manuscript represents a valuable advancement with solid evidence in the understanding of a circadian clock system that has proven challenging to characterize structurally due to obstacles linked to FRQ purification and stability. The implications of LLPS formation in the negative arm of other eukaryotic clocks and its role in temperature compensation are highly intriguing.

Strengths:<br /> The strengths of the manuscript include the scientific rigor of the experiments, the importance of the topic to the field of chronobiology, and new mechanistic insights obtained.

Weaknesses:<br /> This reviewer had questions regarding some of the conclusions reached.

Reviewer #1 (Public Review):

Single-molecule visualization of chromatin remodelers on long chromatin templates-a long sought-after goal-is still in its infancy. This work describes the behaviors of two remodelers RSC and ISW2, from SWI/SNF and ISWI families respectively, with well-conducted experiments and rigorous quantitative analysis, thus representing a significant advance in the field of chromatin biology and biophysics. Overall, the conclusions are supported by the data and the manuscript is clearly written. However, there are a few occasions where the strength of the conclusion suffers from low statistics. Some of the statements are too strong given the evidence presented.

Specific comments:

1. It is confusing what is the difference between the "non-diffusive" behavior of the remodeler upon nucleosome encounter and the nucleosome-translocating behavior in the presence of ATP. For example, in Figure 3F, readers can see a bit of nucleosome translocation in the first segment. Is the lower half-life of "non-diffusive" ISW2 with ATP on a nucleosome array because it is spending more time translocating nucleosomes? The solid and dashed green lines in Figure 3F and 3G are not explained. It is also not explained why Figure 3H and 3I are fit by double exponentials.<br /> 2. What is the fraction of 1D vs. 3D nucleosome encountered by the remodelers? This is an important parameter to compare between RSC and ISW2.<br /> 3. A major conclusion stated repeatedly in the manuscript is that nucleosome translocation by a remodeler is terminated by a downstream nucleosome. But this is based on a total of 4 events. The problem of dye photobleaching was mentioned, which is a bit surprising considering that the green excitation was already pulsed. The authors should try to get more events by lowering the laser power or toning down the conclusion that translocation termination is prominently due to blockage by a downstream nucleosome. Quantifying the translocation distances before termination, in addition to the durations (Figure 4G and 4H), would also be helpful.<br /> 4. The claim on nucleosome translocation directionality is also based on a small number of events, particularly for RSC. 6/9 is hardly over 50% if one considers the Poisson counting error (RSC was also found to switch directions.) If the authors would like to make a firm statement to support the "push-pull" model, they should obtain more events.<br /> 5. At 5 pN of tether tension, the outer wrap of nucleosomes is destabilized, which could impact nucleosome translocation dynamics. Additionally, a low buffer flow was kept on during data acquisition, which could bias remodeler diffusion behavior. The authors should rule out or at a minimum discuss these possibilities.

Reviewer #1 (Public Review):

Trebino et al. investigated the BRAF activation process by analysing the interactions of BRAF N-terminal regulatory regions (CRD, RBD and BSR) with the C-terminal kinase domain and with the upstream regulators HRAS and KRAS. To this end, they generated four constructs comprising different combinations of N-terminal domains of BRAF and analysed their interaction with HRAS as well as conformational changes that occur. By HDX-MS they confirmed that the RBD is indeed the main mediator of interaction with HRAS. Moreover, they observed that HRAS binding leads to conformational changes exposing the BSR to the environment. Next, the authors used OpenSPR to determine the binding affinities of HRAS to the different BRAF constructs. While BSR+RBD, RBD+CRD and RBD bound HRAS with nanomolar affinity, no binding was observed with the construct comprising all three domains. Based on these experiments, the authors concluded that BSR and CRD negatively regulate binding to HRAS and hypothesised that BSR may confer some RAS isoform specificity. They corroborated this notion by showing that KRAS bound to BRAF-NT1 (BSR+RBD+CRD) while HRAS did not. Next, the authors analysed the autoinhibitory interaction occurring between the N-terminal regions and the kinase domain. Through pulldown and OpenSPR experiments, they confirm that it is mainly the CRD that makes the necessary contacts with the kinase domain. In addition, they show that the BSR stabilizes these interactions and that the addition of HRAS abolishes them. Finally, the D594G mutation within the KD of BRAF is shown to destabilise these autoinhibitory interactions, which could explain its oncogenic potential.

Overall, the in vitro study provides new insights into the regulation of BRAF and its interactions with HRAS and KRAS through a comprehensive in vitro analysis of the BRAF N-terminal region. Also, the authors report the first KD values for the N- and C-terminal interactions of BRAF and show that the BSR might provide isoform specificity towards KRAS. While these findings could be useful for the development of a new generation of inhibitors, the overall impact of the manuscript could probably be enhanced if the authors were to investigate in more detail how the BSR-mediated specificity of BRAF towards certain RAS isoforms is achieved. Moreover, though the very "clean" in vitro approach is appreciated, it also seems useful to examine whether the observed interactions and conformational changes occur in the full-length BRAF molecule and in more physiological contexts. Some of the results could be compared with studies including full length constructs.

Reviewer #1 (Public Review):

Summary:<br /> Mutational analysis of diffuse midline glioma (DMG) found that ACVR1 mutations, which up-regulate the BMP signaling pathway are found in most H3.1K27M, but not H3.3K27M DMG cases. In this manuscript, Huchede et al attempted to determine whether the BMP signaling pathway has any role in H3.3K27M DMG tumors. They found that the BMP signaling is activated to a similar level in H3.3K27M DMG cells with wild-type ACVR1 compared to ACVR1 DMG cells, likely due to the expression of BMP7 or BMP2. They went on to test whether cells treated with BMP7 or BMP2 treatments affected the gene expression and cell fitness of tumor cells with H3.3K27M mutation. They concluded that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. The major issue for this conclusion is that the authors did not use the right models/controls to obtain results to support this conclusion as detailed below. Therefore, in order to strengthen the conclusion, the authors need to address the major concerns below.

Strength:<br /> This paper addresses an important question in the DMG field.

Major concerns/weakness:<br /> 1) All the results in Fig. 2 utilized two glioma lines SF188 and Res259. The authors should repeat all these experiments in a couple of H3.3K27M DMG lines by deleting the H3.3K27M mutation first.<br /> 2) Fig. 3. The experiments of BMP2 treatment should be repeated in other H3.3K27M DMG lines using H3.1K27M ACVR1 mutant tumor lines as controls.

Minor concerns<br /> Fig.2A. BMP2 expression increased in H3.3K27M SF188 cells. Therefore, the statement "whereas BMP2 and BMP4 expressions are not significantly modified (Figure 2A and Figure 2-figure supplement A-B)" is not accurate.

Reviewer #1 (Public Review):

Summary:<br /> The paper titled "GABRD promotes the progression of breast cancer through CDK1-dependent cell cycle regulation" investigates the role of GABRD, a subunit of the GABAA receptor, in breast cancer progression and its potential association with CDK1-dependent cell cycle regulation. The study is commendable for shedding light on the role of GABRD in breast cancer, but a few areas can be further improved to enhance the significance and completeness of the research.

Strengths:<br /> The study presents valuable insights into the role of GABRD and its potential interaction with CDK1 in breast cancer progression.

Recent literature suggests that the neurotransmitter GABA and its receptors play a vital role in regulating various tumors. The paper's innovation lies in revealing GABRD as the most relevant subunit within the GABA receptor family concerning breast cancer and exploring its potential mechanisms in regulating breast cancer progression, including the proposed GABRD-CDK1 axis.

The methods in the study are sufficiently documented to allow replication studies and the quality of the figures and tables is very satisfactory.

In general, this manuscript is well-crafted and addresses a compelling and pertinent topic.

Weaknesses:<br /> The following minor issues should be addressed:

1. While the study demonstrates the impact of GABRD expression on patient overall survival, it would be beneficial to supplement this with additional survival indicators. Analyzing other survival metrics, such as disease-free survival or progression-free survival, could provide a more comprehensive understanding of GABRD's clinical relevance in breast cancer.

2. The manuscript alludes to GABRD's regulation of the cell cycle through its interaction with CDK1. Elaborating on the specific binding mechanisms and molecular interactions involved in this regulation would provide a more detailed insight into the proposed GABRD-CDK1 axis.

3. The criteria for high and low expression of GABRD In Table 1 and Fig. 1D should be clearly defined.

4. It would be helpful to explain the reason for classifying the tumor size with 3cm (not 2 or 5cm) in Table 2. It would also be helpful to explain whether the differences in GABRD expression in breast cancer subtypes with different HR and HER-2 expression statuses were analyzed.

Reviewer #1 (Public Review):

Summary: Crohn's disease is a prevalent inflammatory bowel disease that often results in patient relapse post anti-TNF blockades. This study employs a multifaceted approach utilizing single-cell RNA sequencing, flow cytometry, and histological analyses to elucidate the cellular alterations in pediatric Crohn's disease patients pre and post-anti-TNF treatment and comparing them with non-inflamed pediatric controls. Utilizing an innovative clustering approach, the research distinguishes distinct cellular states that signify the disease's progression and response to treatment. Notably, the study suggests that the anti-TNF treatment pushes pediatric patients towards a cellular state resembling adult patients with persistent relapses. This study's depth offers a nuanced understanding of cell states in CD progression that might forecast the disease trajectory and therapy response.

Robust Data Integration: The authors adeptly integrate diverse data types: scRNA-seq, histological images, flow cytometry, and clinical metadata, providing a holistic view of the disease mechanism and response to treatment.

Novel Clustering Approach: The introduction and utilization of ARBOL, a tiered clustering approach, enhances the granularity and reliability of cell type identification from scRNA-seq data.

Clinical Relevance: By associating scRNA-seq findings with clinical metadata, the study offers potentially significant insights into the trajectory of disease severity and anti-TNF response; which might help with the personalized treatment regimens.

Treatment Dynamics: The transition of the pediatric cellular ecosystem towards an adult, more treatment-refractory state upon anti-TNF treatment is a significant finding. It would be beneficial to probe deeper into the temporal dynamics and the mechanisms underlying this transition.

Comparative Analysis with Adult CD: The positioning of on-treatment biopsies between treatment-naïve pediCD and on-treatment adult CD is intriguing. A more in-depth exploration comparing pediatric and adult cellular ecosystems could provide valuable insights into disease evolution.

Areas of improvement:<br /> 1. The legends accompanying the figures are quite concise. It would be beneficial to provide a more detailed description within the legends, incorporating specifics about the experiments conducted and a clearer representation of the data points.

2. Statistical significance is missing from Fig. 1c WBC count plot, Fig. 2 b-e panels. Please provide it even if it's not significant. Also, the legend should have the details of stat test used.

3. In the study, the NOA group is characterized by patients who, after thorough clinical evaluations, were deemed to exhibit milder symptoms, negating the need for anti-TNF prescriptions. This mild nature could potentially align the NOA group closer to FIGD-a condition intrinsically defined by its low to non-inflammatory characteristics. Such an alignment sparks curiosity: is there a marked correlation between these two groups? A preliminary observation suggesting such a relationship can be spotted in Figure 6, particularly panels A and B. Given the prevalence of FIGD among the pediatric population, it might be prudent for the authors to delve deeper into this potential overlap, as insights gained from mild-CD cases could provide valuable information for managing FIGD.

4. Furthermore, Figure 7 employs multi-dimensional immunofluorescence to compare CD, encompassing all its subtypes, with FIGD. If the data permits, subdividing CD into PR, FR, and NOA for this comparison could offer a more nuanced understanding of the disease spectrum. Such a granular perspective is invaluable for clinical assessments. The key question then remains: do the sample categorizations for the immunofluorescence study accommodate this proposed stratification?

5. The study's most captivating revelation is the proximity of anti-TNF-treated pediatric CD (pediCD) biopsies to adult treatment-refractory CD. Such an observation naturally raises the question: How does this alignment compare to a standard adult colon, and what proportion of this similarity is genuinely disease-specific versus reflective of an adult state? To what degree does the similarity highlight disease-specific traits?<br /> Delving deeper, it will be of interest to see whether anti-TNF treatment is nudging the transcriptional state of the cells towards a more mature adult stage or veering them into a treatment-resistant trajectory. If anti-TNF therapy is indeed steering cells toward a more adult-like state, it might signify a natural maturation process; however, if it's directing them toward a treatment-refractory state, the long-term therapeutic strategies for pediatric patients might need reconsideration.

Reviewer #1 (Public Review):

Ye et al. used Mendelian randomization method to evaluate the causative association between circulating immune cells and periodontitis and finally screened out three risk immune cells related to periodontitis. Overall, this is an important and novel piece of work that has the potential to contribute to our understanding of the causal relationship between circulating immune cells related to periodontitis. However, there are still some concerns that need to be addressed.

1. The authors used 1e-9 as the threshold to select effective instrumental variables (IVs), which should give the corresponding references. Meanwhile, the authors should test and discuss the potential impact of inconsistent thresholds for exposure (1e-9, 5e-6 were selected by the author respectively) and outcome IVs (5e-8) on the robustness of the results.<br /> 2. What is the reference for selecting Smoking, Fasting plasma glucose, and BMI as covariates? They do not seem to be directly related to immune cells as confounding factors.<br /> 3. It is not entirely clear about the correction of P-value for the total number of independent statistical tests.<br /> 4. The author used whole blood data to apply FUSION algorithm. Although whole blood is a representative site, the authors should add FUSION testing of periodontally relevant tissues, such as oral mucosa.<br /> 5. The authors chose gingival hyperplasia as a secondary validation phenotype of periodontitis in this study. However, gingival recession, as another important phenotype associated with periodontitis, should also be tested and discussed.<br /> 6. This study used GLIDE data as a replicated validation, but the results were inconsistent with FinnGen's dataset.

Reviewer #1 (Public Review):

Summary<br /> The authors use an elegant but somewhat artificial heterodimerisation approach to activate the isolated cytoplasmic domains of different receptor kinases (RKs) including the receptor kinase BRI1 and EFR. The developmental RK BRI1 is known to be activated by the co-receptor BAK1. Active BRI1 is then able to phosphorylate downstream substrates. The immune receptor EFR is also an active protein kinase also activated by the co-receptor BAK1. EFR however appears to have little or no kinase activity but seems to use an allosteric mechanism to in turn enable BAK1 to phosphorylate the substrate kinase BIK1. EFR tyrosine phosphorylation by BAK1 appears to trigger a conformational change in EFR, activating the receptor. Likewise, kinase activating mutations can cause similar conformational transitions in EFR and also in BAK1 in vitro and in planta.

Strengths: I particularly liked The HDX experiments coupled with mutational analysis (Fig. 2) and the design and testing of the kinase activating mutations (Fig. 3), as they provide novel mechanistic insights into the activation mechanisms of EFR and of BAK1. These findings are nicely extended by the large-scale identification of EFR-related RKs from different species with potentially similar activation mechanisms (Fig. 5).

Weaknesses: In my opinion, there are currently two major issues with the present manuscript. (1) Due o the small effect sizes it is absolutely critical that the EFRD849N mutant is indeed 100% inactive and based on previous reports from the same group I am not certain it is (https://pubmed.ncbi.nlm.nih.gov/34531323/) (Fig. 1). Along these lines quantitative enzyme kinetic assays and additional controls in the immune assays could help to improve and substantiate the different trans-phosphorylation events depicted in Fig.1 (2) How the active-like conformation of EFR is in turn activating BAK1 is poorly characterized, but appears to be the main step in the activation of the receptor complex. Extending the HDX analyses to resting and Rap-activated receptor complexes could be a first step to address this question.

Overall this is an interesting study that aims to advance our understanding of the activation mechanisms of different plant receptor kinases with important functions in plant immunity.

Reviewer #1 (Public Review):

Summary:<br /> Gao et al. have demonstrated that the pesticide emamectin benzoate (EB) treatment of brown planthopper (BPH) leads to increased egg-laying in the insect, which is a common agricultural pest. The authors hypothesize that EB upregulates JH titer resulting in increased fecundity.

Strengths:<br /> The finding that a class of pesticide increases the fecundity of brown planthopper is interesting.

Weaknesses:<br /> 1. EB is an allosteric modulator of GluCl. That means EB physically interacts with GluCl initiating a structural change in the cannel protein. Yet the authors' central hypothesis here is about how EB can upregulate the mRNA of GluCl. I do not know whether there is any evidence that an allosteric modulator can function as a transcriptional activator for the same receptor protein. The basic premise of the paper sounds counterintuitive. This is a structural problem and should be addressed by the authors by giving sufficient evidence about such demonstrated mechanisms before.

2. I am surprised to see a 4th instar larval application or treatment with EB results in the upregulation of JH in the adult stages. Complicating the results further is the observation that a 4th instar EB application results in an immediate decrease in JH titer. There is a high possibility that this late JH titer increase is an indirect effect.

3. The writing quality of the paper needs improvement. Particularly with respect to describing processes and abbreviations. In several instances the authors have not adequately described the processes they have introduced, thus confusing readers.

4. In the section 'EB promotes ovarian development' the authors have shown that EB treatment results in increased detention of eggs which contradicts their own results which show that EB promotes egg laying. Again, this is a serious contradiction that nullifies their hypothesis.

5. Furthermore, the results suggest that oogenesis is not affected by EB application. The authors should devote a section to discussing how they are observing increased egg numbers in EB-treated insects while not impacting Oogenesis.

6. Met is the receptor of JH and to my understanding, remains mostly constant in terms of its mRNA or protein levels throughout various developmental periods in many different insects. Therefore, the presence of JH becomes the major driving factor for physiological events and not the presence of the receptor Met. Here the authors have demonstrated an increase in Met mRNA as a result of EB treatment. Their central hypothesis is that EB increases JH titer to result in enhanced fecundity. JH action will not result in the activation of Met. Although not contradictory to the hypothesis, the increase in mRNA content of Met is contrary to the findings of the JH field thus far.

7. As pointed out before, it is hard to rationalize how a 4th instar exposure to EB can result in the upregulation of key genes involved in JH synthesis at the adult stage. The authors must consider providing a plausible explanation and discussion in this regard.

8. I have strong reservations against such an irrational hypothesis that Met (the receptor for JH) and JH-Met target gene Kr-h1 regulate JH titer (Line 311, Fig 3 supplemental 2D). This would be the first report of such an event on the JH field and therefore must be analysed in depth. I strongly suggest the authors remove such claims from the manuscript without substantiating it.

9. Kr-h1 is JH/Met target gene. The authors demonstrate that silencing of Kr-h1 results in inhibition of FAMeT, which is a gene involved in JH synthesis. A feedback loop in JH synthesis is unreported. It is the view of this reviewer that the authors must go ahead with a mechanistic detail of Kr-h1 mediated JH upregulation before this can be concluded. Mere qPCR experiments are not sufficient to substantiate a claim that is completely contrary to the current understanding of the JH signalling pathway.

10. The authors have performed knockdowns of JHAMT, Met, and Kr-h1 to demonstrate the effect of these factors on fecundity in BPH. Additionally, they have performed rescue experiments with EB application on these knockdown insects (Figure 3K-M). This, I believe, is a very flawed experiment. The authors demonstrate EB works through JHAMT in upregulating JH titer. In the absence of JHAMT, EB application is not expected to rescue the phenotype. But the authors have reported a complete rescue here. In the absence of Met, the receptor of JH, either EB or JH is not expected to rescue the phenotype. But a complete rescue has been reported. These two experimental results contradict their own hypothesis.

11. A significant section of the paper deals with how EB upregulates JH titer. JH is a hormone synthesized in the Corpora Allata. Yet the authors have chosen to use the whole body for all of their experiment. Changes in the whole body for mRNA of those enzymes involved in JH synthesis may not reflect the situation in Corpora Allata. Although working with Corpora Allata is challenging, discarding the abdomen and thorax region and working with the head and neck region of the insect is easily doable. Results from such sampling are always more convincing when it comes to JH synthesis studies.

12. The phenomenon reported was specific to BPH and not found in other insects. This limits the implications of the study.

13. Overall, the molecular experiments are very poorly designed and can at best be termed superficial. There are several contradictions within the paper and no discussion or explanation has been provided for that.

Reviewer #1 (Public Review):

In this study the authors attempt to describe alterations in gene expression, protein expression, and protein phosphorylation as a consequence of chronic adenylyl cyclase 8 overexpression in a mouse model. This model is claimed to have resilience to cardiac stress.

Major strengths of the study include 1) the large dataset generated which will have utility further scientific inquiry for the authors and others in the field, 2) the innovative approach of using cross-analyses linking transcriptomic data to proteomic and phosphoproteomic data. One weakness is the lack of a focused question and clear relevance to human disease. These are all critical biological pathways that the authors are studying and essentially, they have compiled a database that could be surveyed to generate and test future hypotheses.

Reviewer #1 (Public Review):

Summary:<br /> The authors characterize S. enterica WbaP biochemically and structurally. The enzyme catalyzes the initial step in O antigen biosynthesis by transferring a phospho-galactosyl unit from UDP-galactose to undecaprenyl-phosphate. This initial primer is then extended by other glycosyltransferases to form the O antigen repeat unit.

To preserve the biologically functional unit of WbaP, the authors chose a 'detergent-free' purification method based on membrane extraction using SMALP polymers. The obtained material was characterized biochemically and by single-particle cryo-electron microscopy.

Strengths:<br /> The authors were able to isolate WbaP in a catalytically active and oligomeric form and determined a low-resolution cryo-EM structure of the dimeric complex. Using a disulfide cross-linking approach and other biophysical methods, the authors validated an AlphaFold predicted WbaP model used to interpret the experimental cryo-EM map.

Weaknesses:<br /> The rationale for using SMALP to extract WbaP from the membrane was to 'preserve' the native lipid bilayer surrounding the protein. However, the physical properties of the lipids co-purifying with the protein are unclear. The volume of the EM map assigned to the SMALP polymers suggests a more micellar character.

Overall, the obtained cryo-EM map appears to be at fairly low resolution. Based on Figure 6, individual helices are not resolved, suggesting an overall resolution significantly below the stated 4.1 Å. Thus, the presented structure is the one of an AlphaFold WbaP model.

I believe the UMP titration analysis could be improved. The authors assume that a 'domain of unknown function (DUF)' binds UMP and regulates the enzyme's activity. UMP, a reaction product of WbaP, may also inhibit the enzyme competitively. Therefore, deleting the DUF for the UMP inhibition studies could help with data interpretation.

• for: interesting fact - many Albertans are from America

• interesting fact

  • 3 million people settled the Canadian Praries between 1880 and 1913
  • 30% of them were fromNebrask and Montana

Reviewer #1 (Public Review):

Martinez-Gutierrez and colleagues presented a timeline of important bacteria and archaea groups in the ocean and based on this they correlated the emergence of these microbes with GOE and NOE, the two most important geological events leading to the oxygen accumulation of the Earth. The whole study builds on molecular clock analysis, but unfortunately, the clock analysis contains important errors in the calibration information the study used, and is also oversimplified, leaving many alternative parameters that are known to affect the posterior age estimates untested. Therefore, the main conclusion that the oxygen availability and redox state of the ocean is the main driver of marine microbial diversification is not convincing.

Basically, what the molecular clock does is to propagate the temporal information of the nodes with time calibrations to the remaining nodes of the phylogenetic tree. So, the first and the most important step is to set the time constraints appropriately. But four of the six calibrations used in this study are debatable and even wrong.

(1) The record for biogenic methane at 3460 Ma is not reliable. The authors cited Ueno et al. 2006, but that study was based on carbon isotope, which is insufficient to demonstrate biogenicity, as mentioned by Alleon and Summons 2019.

(2) Three calibrations at Aerobic Nitrososphaerales, Aerobic Marinimicrobia, and Nitrite oxidizing bacteria have the same problem - they are all assumed to have evolved after the GOE where the Earth started to accumulate oxygen in the atmosphere, so they were all capped at 2320 Ma. This is an important mistake and will significantly affect the age estimates because maximum constraint was used (maximum constraint has a much greater effect on age estimates and minimum constraint), and this was used in three nodes involving both Bacteria and Archaea. The main problem is that the authors ignored the numerous evidence showing that oxygen can be produced far before GOE by degradation of abiotically-produced abundant H2O2 by catalases equipped in many anaerobes, also produced by oxygenic cyanobacteria evolved at least 500 Ma earlier than the onset of GOE (2500 Ma), and even accumulated locally (oxygen oasis). It is well possible that aerobic microbes could have evolved in the Archaean.

Once the phylogenetic tree is appropriately calibrated with fossils and other time constraints, the next important step is to test different clock models and other factors that are known to significantly affect the posterior age estimates. For example, different genes vary in evolutionary history and evolutionary rate, which often give very different age estimates. So it is very important to demonstrate that these concerns are taken into account. These are done in many careful molecular dating studies but missing in this study.

Reviewer #1 (Public Review):

Martinez-Gutierrez and colleagues presented a timeline of important bacteria and archaea groups in the ocean and based on this they correlated the emergence of these microbes with GOE and NOE, the two most important geological events leading to the oxygen accumulation of the Earth.

The following suggestion is very important and requires additional clock analysis.

"Three calibrations at Aerobic Nitrososphaerales, Aerobic Marinimicrobia, and Nitrite oxidizing bacteria have the same problem - they are all assumed to have evolved after the GOE where the Earth started to accumulate oxygen in the atmosphere, so they were all capped at 2320 Ma. This is an important mistake and will significantly affect the age estimates because maximum constraint was used (maximum constraint has a much greater effect on age estimates and minimum constraint), and this was used in three nodes involving both Bacteria and Archaea. The main problem is that the authors ignored the numerous evidence showing that oxygen can be produced far before GOE by degradation of abiotically-produced abundant H2O2 by catalases equipped in many anaerobes, also produced by oxygenic cyanobacteria evolved at least 500 Ma earlier than the onset of GOE (2500 Ma), and even accumulated locally (oxygen oasis). It is well possible that aerobic microbes could have evolved in the Archaean."

Joint Public Review:

In the manuscript by Rajan et al., the authors have highlighted the direct interaction between Dbp5 and tRNA, wherein Dbp5 serves as a mediator for tRNA export. This export process is subject to spatial regulation, as Dbp5 ATPase activation occurs specifically at nuclear pore complexes. Notably, this regulation is independent of the Los1-mediated pre-tRNA export route and instead relies on Gle1.

The manuscript is well constructed and nicely written. The authors have addressed the concerns as raised by the previous reviewers and added additional experiments.

I have a few comments for polishing the manuscript.

Major comments:<br /> 1. In their previous paper (Lari et al, 2019; Azra Lari Arvind Arul Nambi Rajan Rima Sandhu Taylor Reiter Rachel Montpetit Barry P Young Chris JR Loewen Ben Montpetit (2019) A nuclear role for the DEAD-box protein Dbp5 in tRNA export eLife 8:e48410.) as well as in the current manuscript the authors states that Dbp5 is involved in the export of tRNA that is independent of and parallel to Los1. They state that Dbp5 binds to the tRNA independent of known tRNA export proteins. The obtained conclusion is both intriguing and innovative, since it suggests that there are other variables, beyond the ones previously identified as tRNA factors, that might interact with Dbp5 to facilitate the export process. In order to find out additional factors aiding this process the authors may employ total RNA‐associated protein purification (TRAPP) experiments ( Shchepachevto et al., 2019; Shchepachev V, Bresson S, Spanos C, Petfalski E, Fischer L, Rappsilber J, Tollervey D. Defining the RNA interactome by total RNA-associated protein purification. Mol Syst Biol. 2019 Apr 8;15(4):e8689. doi: 10.15252/msb.20188689. PMID: 30962360; PMCID: PMC6452921) to identify extra factors involved in conjunction with Dbp5. The process elucidates hitherto uninvestigated tRNA export components that function in conjunction with Dbp5.

2. Various reports suggest that eukaryotic translation elongation factor 1 eEF1A is involved tRNA export Bohnsack et al., 2002 (Bohnsack MT, Regener K, Schwappach B, Saffrich R, Paraskeva E, Hartmann E, Görlich D. Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm. EMBO J. 2002 Nov 15;21(22):6205-15. doi: 10.1093/emboj/cdf613. PMID: 12426392; PMCID: PMC137205), Grosshans etal., 2002; Grosshans H, Hurt E, Simos G. An aminoacylation-dependent nuclear tRNA export pathway in yeast. Genes Dev. 2000 Apr 1;14(7):830-40. PMID: 10766739; PMCID: PMC316491). The presence of mutations in eEF1A has been seen to hinder the nuclear export process of all transfer RNAs (tRNAs). eEF1A has been shown to interact with Los1 aiding in tRNA export. The authors can also explore the crosstalk between Dbp5 and eEF1A in this study. Additionally, suppressor screening analysis in dbp5R423A , los1∆dbp5R423A los1∆msn∆dbp5R423A could shed more light on this.

3. Unfortunately, this article is not significantly different from that published in eLife in 2018. In fact, it raises more questions than it brings answers by not identifying a transporter for export and not identifying a role for the helicase activity of Dbp5. The addition of Gle1 is potentially novel but it's unclear why the authors didn't address the potential involvement of IP6.

Reviewer #1 (Public Review):

The authors have shown the following:<br /> 1. SY1 aggregation enhances (in terms of number of aggregates) when Sphingolipid biosynthesis is blocked.<br /> 2. In a normal cell (where sphingolipid biosynthesis is not hampered), the aggregate of SY1 (primarily the Class I aggregate) is localized only on the mitochondrial endomembrane system.<br /> 3. The localization is due to the association of SY1 (aggregates) with mitochondrial proteins like Tom70, Tim44, etc. (Is the localization completely lost? What happens to the toxicity when the aggregates are not localized on mitochondria?)<br /> 4. This fuels the loss of mitochondrial function.<br /> 5. Mitochondrial function is further abrogated when there is a block in sphingolipid biosynthesis.<br /> 6. A similar phenomenon is conserved in mammalian cell lines.

However, my major concern is that the role of sphingolipid in the mitochondrial association of the aggregates is not proven beyond doubt. I am also missing the importance of mitochondrial association in the context of IB maturation and cellular toxicity.

Reviewer #1 (Public Review):

Summary:<br /> This study investigated the role of CD47 and TSP1 in extramedullary erythropoiesis by utilization of both global CD47-/- mice and TSP1-/- mice.

Strengths:<br /> Flow cytometry combined with spleen bulk and single-cell transcriptomics were employed. The authors found that stress-induced erythropoiesis markers were increased in CD47-/- spleen cells, particularly genes that are required for terminal erythroid differentiation. Moreover, CD47 dependent erythroid precursors population was identified by spleen scRNA sequencing. In contrast, the same cells were not detected in TSP1-/- spleen. These findings provide strong evidence to support the conclusion that the differential role of CD47 and TSP1 in extramedullary erythropoiesis in mouse spleen.

Weaknesses:<br /> Methods and data analysis are appropriate. However, some clarifications are required. The discussion section needs to be expanded.<br /> 1). The sex of mice that were used in the study is unknown.<br /> 2). In the method of Single-cell RNA sequencing (page 10), it mentioned that single cell suspensions from mouse spleens were depleted of all mature hematopoietic cell lineages by passing through CD8a microbeads and CD8a+ T cell isolation Kit. As described, it is confusing what cell types are obtained for performing scRNAseq. More information is required for clarity.<br /> 3). The constitutive CD47 knockout mouse model is utilized in this study. The observed accumulation of erythroid precursors in the spleens of CD47-/- mice suggests a chronic effect of CD47 on spleen function. Can the current findings be extrapolated to acute scenarios involving CD47 knockdown or loss, as this may have more direct relevance to the potential side effects associated with anti-CD47-mediated cancer therapy? Please expand on this topic in the discussion section.

Reviewer #1 (Public Review):

The work analyzes how centrosomes mature before cell division. A critical aspect is the accumulation of pericentriolar material (PCM) around the centrioles to build competent centrosomes that can organize the mitotic spindle. The present work builds on the idea that the accumulation of PCM is catalyzed either by the centrioles themselves (leading to a constant accumulation rate) or by enzymes activated by the PCM itself (leading to autocatalytic accumulation). These ideas are captured by a previous model derived for PCM accumulation in C. elegans (ref. 8) and are succinctly summarized by Eq. 1. The main addition of the present work is to allow the activated enzymes to diffuse in the cell, so they can also catalyze the accumulation of PCM in other centrosomes (captured by Eqs. 2-4). The authors claim that this helps centrosomes to reach the same size, independent of potential initial mismatches.

A strength of the paper is the simplicity of the equations, which are reduced to the bare minimum and thus allow a detailed inspection of the physical mechanism. One shortcoming of this approach is that all equations assume that the diffusion of molecules is much faster than any of the reactive time scales, although there is no experimental evidence for this.

Another shortcoming of the paper is that it is not clear what species the authors are investigating and how general the model is. There are huge differences in centrosome maturation and the involved proteins between species. However, this is not mentioned in the abstract or introduction. Moreover, in the main body of the paper, the authors mention C. elegans on pages 2 and 3, but refer to Drosophila on page 4, switching back to C. elegans on page 5, and discuss Drosophila on page 6. This is confusing and looks as if they are cherry-picking elements from various species. The original model in ref. 8 was constructed for C. elegans and it is not clear whether the autocatalytic model is more general than that. In any case, a more thorough discussion of experimental evidence would be helpful.

The authors show convincingly that their model compensates for initial size differences in centrosomes and leads to more similar final sizes. These conclusions rely on numerical simulations, but it is not clear how the parameters listed in Table 1 were chosen and whether they are representative of the real situation. Since all presented models have many parameters, a detailed discussion on how the values were picked is indispensable. Without such a discussion, it is not clear how realistic the drawn conclusions are. Some of this could have been alleviated using a linear stability analysis of the ordinary differential equations from which one could have gotten insight into how the physical parameters affect the tendency to produce equal-sized centrosomes.

The authors use the fact that their model stabilizes centrosome size to argue that their model is superior to the previously published one, but I think that this conclusion is not necessarily justified by the presented data. The authors claim that "[...] none of the existing quantitative models can account for robustness in centrosome size equality in the presence of positive feedback." (page 1; similar sentence on page 2). This is not shown convincingly. In fact, ref 8. already addresses this problem (see Fig. 5 in ref. 8) to some extent. More importantly, the conclusion seems to largely be based on the analysis shown in Fig. 2A, but the parameters going into this figure are not clear (see the previous paragraph). In particular, the initial size discrepancy of 0.1 µm^3 seems quite large, since it translates to a sphere of a radius of 300 nm. A similarly large initial discrepancy is used on page 3 without any justification. Since the original model itself already showed size stability, a careful quantitative comparison would be necessary.

The analysis of the size discrepancy relies on stochastic simulations (e.g., mentioned on pages 2 and 4), but all presented equations are deterministic. It's unclear what assumptions go into these stochastic equations, and how they are analyzed or simulated. Most importantly, the noise strength (presumably linked to the number of components) needs to be mentioned. How is this noise strength determined? What are the arguments for this choice? This is particularly crucial since the authors quote quantitative results (e.g., "a negligible difference in steady-state size (∼ 2% of mean size)" on page 4).

Moreover, the two sets of testable predictions that are offered at the end of the paper are not very illuminative: The first set of predictions, namely that the model would anticipate an "increase in centrosome size with increasing enzyme concentration, the ability to modify the shape of the sigmoidal growth curve, and the manipulation of centrosome size scaling patterns by perturbing growth rate constants or enzyme concentrations.", are so general that they apply to all models describing centrosome growth. Consequently, these observations do not set the shared enzyme pool apart and are thus not useful to discriminate between models. The second part of the first set of predictions about shifting "size scaling" is potentially more interesting, although I could not discern whether "size scaling" referred to scaling with cell size, total amount of material, or enzymatic activity at the centrioles. The second prediction is potentially also interesting and could be checked directly by analyzing published data of the original model (see Fig. 5 of ref. 8). It is unclear to me why the authors did not attempt this.

Taken together, I think the shared enzyme pool is an interesting idea, but the experimental evidence for it is currently lacking. Moreover, the model seems to make little testable predictions that differ from previous models.

Reviewer #1 (Public Review):

This important study from Jahncke et al. demonstrates inhibitory synaptic defects and elevated seizure susceptibility in multiple models of dystroglycanopathy. A strength of the paper is the use of a wide range of genetic models to disrupt different aspects of dystroglycan protein or glycosylation in forebrain neurons. The authors use a combination of immunohistochemistry and electrophysiology to identify cellular migration, lamination, axonal targeting, synapse formation/function, and seizure phenotypes in forebrain neurons. This is an elegant study with extensive data supporting the conclusions. The role of dystroglycan and the dystrophin glycoprotein complex (DGC) in cellular migration and synapse formation are of broad interest.<br /> A strength of this paper is the use of several transgenic mouse lines with mutations in genes involved in glycosylation of dystroglycan. Knockout of POMT2 abolishes the majority of dystroglycan glycosylation, while point mutations in B4GAT and FKRP presumably produce more minor changes in glycosylation. This is a powerful approach to investigate the role of glycosylation in dystroglycan function.

Reviewer #1 (Public Review):

Mature mammalian olfactory sensory neurons (OSN) express only one of the hundreds of possible odor receptors (ORs) encoded in the genome. The process of selecting this OR in each OSN is the consequence of both deterministic developmental processes involving transcription factors, and more stochastic processes. How this balance is implemented is a major problem in molecular neuroscience, one whose solution has significant systems-level implications for odor coding. In Bashkirova et al the authors substantially revise the canonical view of how this process works. By querying single cell transcriptomes and genetic architecture across OSN development, the authors demonstrate that OSN progenitors express ORs for their zone and for more dortsal zones, and that the degree of heterochromatinization of non-expressed ORs varies as a function of which zone a given OSN resides in. Through additional genetic experiments (including knockouts of transcription factors that seem to be associated with zonal identity, and the clever use of OR transgenes) they synthesize these findings into a model in which progenitors co-express many ORs - both ORs that are appropriate for their zone and ORs that are dorsal to their zone - and that this expression both facilitates heterochromatinzation of non-selected and extra-zonal ORs, and enables singular OR selection. The experiments are careful and the data are novel, and definitely revise our simplistic current view of how this process works; as such this work will have significant impact on the field. As presented the model requires additional experiments to fully flesh it out, and to definitively demonstrate that i.e., precocious expression leads to gene silencing, but with some additional clarifications in the discussion this paper both breaks new ground and sets the stage for future work exploring mechanisms of OSN development and OR selection.

Reviewer #1 (Public Review):

In this study, Jiamin Lin et al. investigated the potential positive feedback loop between ZEB2 and ACSL4, which regulates lipid metabolism and breast cancer metastasis. They reported a correlation between high expression of ZEB2 and ACSL4 and poor survival of breast cancer patients, and showed that depletion of ZEB2 or ACSL4 significantly reduced lipid droplets abundance and cell migration in vitro. The authors also claimed that ZEB2 activated ACSL4 expression by directly binding to its promoter, while ACSL4 in turn stabilized ZEB2 by blocking its ubiquitination.

Reviewer #1 (Public Review):

In order to find small molecules capable of enhancing regenerative repair, this study employed a high throughput YAP-activity screen method to query the ReFRAME library, identifying CLK2 inhibitor as one of the hits. Further studies showed that CLK2 inhibition leads to AMOTL2 exon skipping, rendering it unable to suppress YAP.

The novelty of the study is that it showed that inhibition of a kinase not previously associated with the HIPPO pathway can influence YAP activity through modification of mRNA splicing. The major arguments appear solid.

In the revised manuscript, additional discussion was provided regarding drug concentration and molecular mechanisms, which helps clear some of the confusing points in the original manuscript.

Reviewer #1 (Public Review):

The work in this paper is in general done carefully. Reconstructions are done appropriately and the effects of statistical uncertainty are quantified properly. I was glad to see that the tree and alignment are now deposited.

The paper identifies which mutations are crucial for the functional differences between the ancestors tested. This is done quite carefully - the authors even show that the same substitutions also work in extant proteins.<br /> These substitutions very slightly lower the affinity and increase the cooperativity of the C-terminal peptide binding to the alpha crystallin domain - a key oligomeric interaction. These relatively minor changes nevertheless apparently affect the subunit exchange behaviour and oligomerization of the sHSP.

Lastly, the authors use likelihood methods to test for signatures of selection. This reviewer is not a fan of these methods, as they are easily misled by common biological processes (see PMID 37395787 for a recent critique). The paper is relatively careful in the interpretation of this test though, and I think the importance of the other findings does not depend on the action of selection along this branch.

Reviewer #1 (Public Review):

The goal of this paper is to characterize the molecular mechanisms that lead to lung cyst formation in a murine model wherein the Bmpr1a receptor gene has been inactivated in the fetal lung mesenchyme. In this context, it is important to note that very little is known regarding how lung cysts form, and generally the presumption has been that these pathological structures result from dysregulated events in the epithelium. Thus, the emphasis in this paper on derangements in a fundamental developmental signaling pathway in the lung mesenchyme that results in cyst formation is both novel and significant.

In this manuscript, the authors seek to understand how abnormal lung development leads to the formation of cysts in the lung. Cysts are enlarged pathological balloon-like structures that interfere with normal gas exchange and characterize a variety of pediatric and adult lung diseases. To date, the molecular signals underlying cyst formation are poorly understood. Using genetically modified mice, the focus herein is on how inactivation of a specific gene known to transduce key developmental signals (Bmpr1a) leads to the development of cysts. One novelty of this work is that the gene inactivation has been targeted to a set of primitive fetal lung cells that give rise to structural and contractile cells supporting bronchial airways. Alterations in the function of this particular cell type has not previously been examined in the context of lung cyst pathogenesis.

Notably, the experiments and models are state-of-art and the authors are careful in their interpretations. It should be noted, however, that there are also several concerns that limit enthusiasm at this time. These include a lack of data evaluating relative histological similarities and differences between cysts generated in their murine model and human lung cysts, and whether there is information implicating a role for the gene studied in this paper in human cysts. Secondly, despite an abundance of data, at the end of the day, the key molecular signals are not clearly identified.

Additional Feedback

Overall, this is a well-executed paper that addresses how derangements in signals emanating from the fetal lung mesenchyme in embryonic life lead to cyst formation. This work, therefore, seeks to fill in basic deficiencies in knowledge since the pathogenesis of lung cyst formation is poorly understood and the role of altered mesenchymal cell activity in this process has not been carefully addressed. For the most part, the experiments are clearly presented, and the models are relevant and state-of-the art. Although enthusiasm is high, there are several overriding concerns, which the authors should consider.

While the paper seeks to understand the key molecular events leading to cyst formation and a plethora of data is provided, this goal is largely not clearly met. As a result, the paper ends up being descriptive. Further, without these data, a so-called definitive rescue experiment is not possible at this time. In addition, the experiments, particularly toward the end of the manuscript are not well integrated with the overall body of the Results. This is particularly true for figure 7. While interesting, the results in some of these latter figures are insufficiently linked to the primary observations. This issue further contributes to concerns that the manuscript is largely descriptive.

Importantly, it would be useful to have provided more detailed information on the structure and histological properties of the murine cysts and how such findings relate to human lung cysts. Also, the authors should examine whether there is any information on Bmpr1a in human cyst formation (i.e GWAS data).

Throughout the paper, there is a lack of quantification for the histological findings. Littermate controls should also be clearly defined genetically,

Specific Concerns by figure

Figure 1 suppl: "Doxycycline" is misspelled.

Figure1c Suppl: Hard to discern clear-cut expression of Bmpr1a protein in mesenchyme in WT. Comparable images with similar sizes of airways should be used.

Figure 2a: Expression of several genes studied and altered should be identified on scatter plot.

Figure 2c: Authors should also consider staining for other smooth muscle markers.

Figure 3: ELN expression should be defined in a clear quantitative manner.

Figure 4: Additional information on p38 dependent signaling (? Including in vivo studies) would potentially help to understand key molecular events and perhaps could help to address key mechanistic events, including their location and identity.

Figure 6: Would be helpful to know whether Bmpr1a receptor is expressed in Myocd KO.

Figure 7: Not clear how these findings, though interesting, relate to the body of studies and the pathogenesis of cyst formation. Other points: 1) The authors should re-examine/repeat co-staining in the KO mouse lung (right 2 images in the top group of 4) for Foxj1, Sox2, and CDH (right 2 images, Figure 7A). For one thing, the cadherin stain in the 2 KO images seems localized to the lumen. Secondly, the pattern of cadherin staining looks exactly the same in both KO images, suggesting an error and/or duplication 2) authors should place arrows on the heat map showing the location of SPC, Sox2, Sox9, and FoxJ1 bands 3) figure 7D graph needs numbers on y axis.

Reviewer #1 (Public Review):

Summary<br /> This fascinating paper by M. Alfatah et al. describes work to uncover novel genes affecting lifespan in the budding yeast S. cerevisiae, eventually identifying and further characterizing a gene, YBR238C, now named AAG1 by the authors.<br /> The authors began by considering published gene sets pulled from the Saccharomyces genome database that described increases or decreases in either chronological lifespan or replicative lifespan in yeast. They also began with gene sets known to be downregulated upon treatment with the lifespan-extending TOR inhibitor rapamycin.

YBR283C was unique in being largely uncharacterized, downregulated upon rapamycin treatment, and linked to both increased replicative lifespan and increased chronological lifespan upon deletion.

The authors show that YBR283C may act to negatively regulate mitochondrial function, in ways that are both dependent on and independent of the stress-responsive transcription factor Hap4, largely by looking at relative expression levels of relevant mitochondrial genes.

In a hard-to-fully interpret but well-documented series of experiments the authors note that the two paralogues YBR283C and RMD9 (which have ~66% similarity) (a) have opposite effects when acting alone, and (b) appear to interact in that some phenotypes of ybr283c are dependent on RMD9.

A particularly interesting finding in light of the current literature and of the authors' strategy in identifying YBR283C is that changes in electron transport chain genes upon rapamycin treatment appear to be affected via YBR283C.

Based on a series of experiments the authors move to conclude the existence of "a feedback loop between TORC1 and mitochondria (the TORC1-Mitochondria-TORC1 (TOMITO) signaling process) that regulates cellular aging processes."

Strengths<br /> Overall, this study describes a great deal of new data from a large number of experiments, that shed light on the potential specific roles of YBR238C and its paralog RMD9 in aging in yeast, and also underscore the potential of an approach looking for "dark matter" such as uncharacterized genes when seining the increasing deluge of published datasets for new hypotheses to test. This work when revised will become a valuable addition to the field.

Weaknesses<br /> A paralog of YBR283C, RMD9, also exists in the yeast genome. While the authors indicate that part of their interest in YBR283C lies in its uncharacterized nature, its paralogue, RMD9, is not uncharacterized but is named due to its phenotype of Required for Meiotic nuclear Division, which is not mentioned or discussed anywhere in the manuscript currently.

In the context of the current work, in addition to the cited Hillen, H.S et al. and Nouet C. et al, the authors might be very interested in the 2007 Genetics paper "Translation initiation in Saccharomyces cerevisiae mitochondria: functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase and novel protein Rmd9p" (PMID: 17194786), which does not appear to be cited or discussed in the current version of the manuscript.

Reviewer #1 (Public Review):

Summary:<br /> The study compares the transcriptional and epigenetic response of baker's yeast cells to heat shock and ethanol shock. The authors made several interesting observations. In response to heat shock, the transcription factor HSF1 rapidly forms foci, binds upstream elements of heat-shock-response genes, facilitates long-distance genomic contacts between heat-shock-response genes, and the genes are rapidly transcribed. In response to ethanol shock, the transcription factor HSF1 rapidly forms foci, binds upstream elements of heat-shock-response genes, facilitates long-distance genomic contacts between heat-shock-response genes, and yet transcription of the genes is substantially delayed. These insights are potentially important, as current models of eukaryotic gene control predict that physical contact between genes and regulatory elements is necessary, and in some cases sufficient to transcribe a gene. The current study indicates that the two effects are virtually decoupled in response to ethanol shock in yeast cells.

Overall, the conclusions appear appropriately supported by the data, and the data appear of high quality.

Strengths:<br /> The particular strengths of the paper include an impressive combination of genomic and imaging-based approaches and insightful genetically engineered cell systems. The manuscript reports interesting and potentially important findings. The text is generally very well written, the ideas are clearly explained, and the reasoning is easy to follow.

Weaknesses:<br /> The main weakness seems to be that the heat and ethanol shock approaches likely elicit pleiotropic effects, and therefore it is a challenge to test the causal relationship between various observations. Nevertheless, even as indirect effects might contribute to some of the authors' observations, the results are definitively worth reporting. Also, the presentation of some of the data could be improved.

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to compare, from testis tissues at different ages from mice in vivo and after culture, multiple aspects of Leydig cells. These aspects included mRNA levels, proliferation, apoptosis, steroid levels, protein levels, etc. A lot of work was put into this manuscript in terms of experiments, systems, and approaches. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation.

Reviewer #1 (Public Review):

Summary:<br /> This paper provides strong evidence for the roles of JH in an ametabolous insect species. In particular, it demonstrates that:<br /> • JH shifts embryogenesis from a growth mode to a differentiation mode and is responsible for terminal differentiation during embryogenesis. This, and other JH roles, are first suggested as correlations, based on the timing of JH peaks, but then experimentally demonstrated using JH antagonists and rescue thereof with JH mimic. This is a robust approach and the experimental results are very convincing.<br /> • JH redirects ecdysone-induced molting to direct formation of a more mature cuticle<br /> • Kr-h1 is downstream of JH in Thermobia, as it is in other insects, and is a likely mediator of many JH effects<br /> • The results support the proposed model that an ancestral role of JH in promoting and maintaining differentiation was coopted during insect radiations to drive the evolution of metamorphosis. However, alternate evolutionary scenarios should also be considered.

Strengths:<br /> Overall, this is a beautiful, in-depth student. The paper is well-written and clear. The background places the work in a broad context and shows its importance in understanding fundamental questions about insect biology. The researchers are leaders in the field, and a strength of this manuscript is their use of a variety of different approaches (enzymatic assays, gene expression, agonists & antagonists, analysis of morphology using different types of microscopy and detection, and more) to attack their research questions. The experimental data is clearly presented and carefully executed with appropriate controls and attention to detail. The 'multi-pronged' approach provides support for the conclusions from different angles, strengthening conclusions. In sum, the data presented are convincing and the conclusions about experimental outcomes are well-justified based on the results obtained.

Weaknesses:<br /> This paper provides more detail than is likely needed for readers outside the field but also provides sufficient depth for those in the field. This is both a strength and a weakness. I would suggest the authors shorten some aspects of their text to make it more accessible to a broader audience. In particular, the discussion is very long and accompanied by two model figures. The discussion could be tightened up and much of the text used for a separate review article (perhaps along with Figure 11) that would bring more attention to the proposed evolution of JH roles.

Reviewer #1 (Public Review):

Zhang et al. tackle the important topic of primate-specific structural features of the brain and the link with functional specialization. The authors explore and compare gyral peaks of the human and macaque cortex through non-invasive neuroimagery, using convincing techniques that have been previously validated elsewhere. They show that nearly 60% of the macaque peaks are shared with humans, and use a multi-modal parcellation scheme to describe the spatial distribution of shared and unique gyral peaks in both species.

The claim is made that shared peaks are mainly located in lower-order cortical areas whereas unique peaks are located in higher-order regions, however, no systematic comparison is made. The authors then show that shared peaks are more consistently found across individuals than unique peaks, and show a positive but small and non-significant correlation between cross-individual counts of the shared peaks of the human and the macaque i.e. the authors show a non-significant trend for shared peaks that are more consistently found across humans to be those that are also more found across macaques.

In order to identify if unique and shared peaks could be identified based on the structural features of the cortical regions containing them, the authors compared them with t-tests. A correction for multiple comparisons should be applied and t-values reported. Graph-theoretical measures were applied to functional connectivity datasets (resting-state fMRI) and compared between unique and shared peak regions for each species separately. Again the absence of multiple comparison correction and t-values make the results hard to interpret. The same comment applies to the analysis reporting that shared peaks are surrounded by a larger number of brain regions than unique peaks. Finally, the potentially extremely interesting results about differential human gene expression of shared and unique peaks regions are not systematically reported e.g. the 28 genes identified are not listed and the selection procedure of 7 genes is not fully reported.

The paper is well written and the methods used for data processing are very compelling i.e. the peak cluster extraction pipeline and cross-species registration. However, the analysis and especially the reporting of statistics, as they stand now, constitutes the main weakness of the paper. Some aspects of the statistical analysis need to be clarified.

Reviewer #1 (Public Review):

Summary:<br /> This study assumes and weakly tests that auditory rhythm processing is produced by internal oscillating systems, and it evaluates the properties of such putative oscillators across individuals. The authors designed an experiment and performed analyses that address individuals' preferred rate and flexibility, with a special focus on how much past rhythms influence subsequent trials. They find evidence for such historical dependence and show that we adapt less well to new rhythms as we age. While I have important doubts about the entrainment-based interpretation of the results, this work offers a useful contribution to our understanding of individual differences in rhythm processing regardless.

Strengths:<br /> The inclusion of two tasks -- a tapping and a listening task -- complement each other methodologically. By analysing both the production and tracking of rhythms, the authors emphasize the importance of the characteristics of the receiver, the external world, and their interplay. The relationship between the two tasks and components within tasks are explored using a range of analyses. The visual presentation of the results is very clear. The age-related changes in flexibility are useful and compelling.

Weaknesses:<br /> At times, I found it challenging to evaluate the scientific merit of this study from what was provided in the introduction and methods. It is not clear what the experiment assumes, what it evaluates, and which competing accounts or predictions are at play. While some of these questions are answered, clear ordering and argumentative flow is lacking. With that said, I found the Abstract and General Discussion much clearer, and I would recommend reformulating the early part of the manuscript based on the structure of those segments.

Second, in my reading, it is not clear to what extent the study assumes versus demonstrates the entrainment of internal oscillators. I find the writing somewhat ambiguous on this count: on the one hand, an entrainment approach is assumed a priori to design the experiment ("an entrainment approach is adopted") yet a primary result of the study is that entrainment is how we perceive and produce rhythms ("Overall, the findings support the hypothesis that an oscillatory system with a stable preferred rate underlies perception and production of rhythm..."). While one could design an experiment assuming X and find evidence for X, this requires testing competing accounts with competing hypotheses -- and this was not done.

In my view, more evidence is required to bolster the findings as entrainment-based regardless of whether that is an assumption or a result. Indeed, while the effect of previous trials into the behaviour of the current trial is compatible with entrainment hypotheses, it may well be compatible with competing accounts as well. And that would call into question the interpretation of results as uncovering the properties of oscillating systems and age-related differences in such systems. Thus, I believe more evidence is needed to bolster the entrainment hypothesis.

For example, a key prediction of the entrainment model -- which assumes internal oscillators as the mechanism of action -- is that behaviour in the SMT and PTT tasks follows the principles of Arnold's Tongue. Specifically, tapping and listening performance should worsen systematically as a function of the distance between the presented and preferred rate. On a participant-by-participant, does performance scale monotonically with the distance between the presented and preferred rate? Some of the analyses hint at this question, such as the effect of 𝚫IOI on accuracy, but a recontextualization, further analyses, or additional visualizations would be helpful to demonstrate evidence of a tongue-like pattern in the behavioural data. Presumably, non-oscillating models do not follow a tongue-like pattern, but again, it would be very instructive to explicitly discuss that.

Fourth, harmonic structure in behaviour across tasks is a creative and useful metric for bolstering the entrainment hypothesis specifically because internal oscillators should display a preference across their own harmonics. However, I have some doubts that the analyses as currently implemented indicate such a relationship. Specifically, the main analysis to this end involves summing the residuals of the data closest to y=x, y=2*x and y=x/2 lines and evaluating whether this sum is significantly lower than for shuffled data. Out of these three dimensions, y=x does not comprise a harmonic, and this is an issue because it could by itself drive the difference of summed residuals with the shuffled data. I am uncertain whether rerunning the same analysis with the x=y dimension excluded constitutes a simple resolution because presumably there are baseline differences in the empirical and shuffled data that do not have to do with harmonics that would leak into the analysis. To address this, a simulation with ground truths could be helpful to justify analyses, or a different analysis that evaluates harmonic structure could be thought of.

Reviewer #1 (Public Review):

Summary:<br /> The authors conducted two tasks at 300 days of separation. First, a social perception task, where Ps responded whether a pictured person either deserved or needed help. Second, an altruism task, where Ps are offered monetary allocations for themselves and a partner. Ps decide whether to accept, or a default allocation of 20 dollars each. The partners differed in perceived merit, such that they were highly deserving, undeserving, or unknown. This categorisation was decided on the basis of a prisoner's dilemma game the partner played beforehand. "Need" was also manipulated, by altering the probability that the partner must have their hand in cold water at the end of the experiment and this partner can use the money to buy themselves out. These two tasks were conducted to assess the perception of need/merit in the first instance, and how this relates to social behaviour in the second. fMRI data were collected alongside behavioural.

The authors present many analyses of behaviour (including DDM results) and fMRI. E.g., they demonstrate that they could decode across the mentalising network whether someone was making a need or deserving judgement vs control judgement but couldn't decode need vs deserving. And that brain responses during merit inferences (merit - control) systematically covaried with participants' merit sensitivity scores in the rTPJ. They also found relationships between behaviour and rTPJ in the altruism task. And that merit sensitivity in the perception task predicted the influence of merit on social behaviour in the altruism task.

Strengths:<br /> This manuscript represents a sensible model to predict social perceptions and behaviours, and a tidy study design with interesting findings. The introduction introduced the field especially brilliantly for a general audience.

Weaknesses:<br /> 1. The authors do acknowledge right at the end that these are small samples. This is especially the case for the correlational questions. While the limitation is acknowledged at the end, it is not truly acknowledged in the way that the data are interpreted. I.e. much is concluded from absent relationships, where the likelihood of Type II error is high in this scenario. I suggest that throughout the manuscript, authors play down their conclusions about absence of effects.

2. I found the results section quite a marathon, and due to its length I started to lose the thread concerning the overarching aims - which had been established so neatly in the introduction. I am unsure whether all of these analyses were necessary for addressing the key questions or whether some were more exploratory. E.g. it's unclear to me what one would have predicted upfront about the decoding analyses.

3. More specifically, the decoding analyses were intriguing to me. If I understand the authors, they are decoding need vs merit, and need+merit vs control, not the content of these inferences. Do they consider that there is a distributed representation of merit that does not relate to its content but is an abstracted version that applies to all merit judgements? I certainly would not have predicted this and think the analyses raise many questions.

Reviewer #1 (Public Review):

Summary:

This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibit increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:

This report describes a mouse model where the LRRK1 and LRRK2 gene is conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons and dopaminergic (DAergic) neurons in the substantia nigra (SN), and noradrenergic neurons in the locus coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in the LRRK double knockout (DKO) was cell autonomous. To rigorously test this question, the authors have generated a double conditional (cDKO) allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. In my view, this was beyond what is usually required, as most investigators might might combine one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in most DAergic neurons in the SN and that LRRK levers are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DAergic neurons is normal at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death, as well as an increase in activated microglia. The degeneration is limited to DAergic neurons as LC neurons are not lost as this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed rigorously, and the results were statistically sound and compelling.

Weaknesses:

I only have a few minor comments. First is that in PD and other degenerative conditions, loss of axons and terminals occurs prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also the case with the Allen Brain Atlas profile. Thus, authors should discuss the discrepancy as authors seem to imply significant LRRK1 expression in DA neurons.

Reviewer #1 (Public Review):

In this manuscript, the authors identified compound heterozygous mutations in CFAP52 recessively cosegregating with male infertility status in a non-consanguineous family. The Cfap52-mutant patient exhibits a mixed acephalic spermatozoa syndrome (ASS) and multiple morphological abnormalities of the sperm flagella (MMAF) phenotype. The influence of mutations on CFAP52 protein function is well validated by in vitro cell experiments and immunofluorescence staining. Cfap52-KO mice are further constructed and perfectly resemble the Cfap52-mutant patient's infertile phenotype, also showing a mixed ASS and MMAF phenotype. The phenotype and underlying mechanisms of the disruption of sperm head-tail connection and flagella development are carefully analyzed by TEM, Western blotting, and immunofluorescence staining. The data presented revealed a prominent role for CFAP52 in sperm development, suggesting that CFAP52 is a novel diagnostic target for male infertility with defects of sperm head-tail connection and flagella development.

Reviewer #1 (Public Review):

Summary:<br /> TRIP13/Pch2 is a conserved essential regulator of meiotic recombination from yeast to humans. In this manuscript, the authors generated TRIP13 null mice and Flag-tagged TRIP13 knock-in mice to study its role in meiosis. They demonstrate that TRIP13 regulates MORMA domain proteins and is essential for meiotic completion and fertility. The main impact of this manuscript is its clarification of the in vivo function of TRIP13 during mouse meiosis and its previously unrecognized role as a dose-sensitive regulator of meiosis.

Strengths:<br /> Two previously reported Trip13 mutations in mice are both hypomorphic alleles with distinct phenotypes, precluding a conclusion on its function. This study for the first time generated the TRIP13 null mice, definitively revealing the function of TRIP13 in meiosis. The authors also show the novel localization of TRIP13 at SC and its independence from the axial element components. The finding of dose-sensitive regulation of meiosis by TRIP13 has implications in understanding human meiosis and disease phenotypes.

Weaknesses:<br /> This manuscript would be more impactful if more mechanistic advancements could be made. For example, the authors could follow up with one of the new interactors identified by MS to offer new insight into the molecular function of TRIP13.

Reviewer #1 (Public Review):

Summary:<br /> The authors appear to be attempting to describe dynamic changes in the chromatin landscape in spermatogonial cells during postnatal development ranging from prepubertal stages at postnatal days 8 or 15 to adult stages. The authors attempt to relate differences they observe in chromatin accessibility at these different stages to changes in gene expression to better understand the molecular mechanisms regulating this differential gene expression.

Strengths:<br /> The primary strength of the manuscript is that it provides additional datasets describing gene expression and chromatin accessibility patterns in spermatogonial cells at different postnatal ages.

Weaknesses:<br /> There appears to be a lack of basic knowledge of the process of spermatogenesis. For instance, the statement that "During the first week of postnatal life, a population of SCs continues to proliferate to give rise to undifferentiated Asingle (As), Apaired (Apr) and Aaligned (Aal) cells. The remaining SCs differentiate to form chains of daughter cells that become primary and secondary permatocytes around postnatal day (PND) 10 to 12." is inaccurate. The Aal cells are the spermatogonial chains, the two are not distinct from one another. In addition, the authors fail to mention spermatogonial stem cells which form the basis for steady-state spermatogenesis. The authors also do not acknowledge the well-known fact that, in the mouse, the first wave of spermatogenesis is distinct from subsequent waves. Finally, the authors do not mention the presence of both undifferentiated spermatogonia (aka - type A) and differentiating spermatogonia (aka - type B). The premise for the study they present appears to be the implication that little is known about the dynamics of chromatin during the development of spermatogonia. However, there are published studies on this topic that have already provided much of the information that is presented in the current manuscript.

It is not clear which spermatogonial subtype the authors intended to profile with their analyses. On the one hand, they used PLZF to FACS sort cells. This typically enriches for undifferentiated spermatogonia. On the other hand, they report detection in the sorted population of markers such as c-KIT which is a well-known marker of differentiating spermatogonia, and that is in the same population in which ID4, a well-known marker of spermatogonial stem cells, was detected. The authors cite multiple previously published studies of gene expression during spermatogenesis, including studies of gene expression in spermatogonia. It is not at all clear what the authors' data adds to the previously available data on this subject.

The authors analyzed cells recovered at PND 8 and 15 and compared those to cells recovered from the adult testis. The PND 8 and 15 cells would be from the initial wave of spermatogenesis whereas those from the adult testis would represent steady-state spermatogenesis. However, as noted above, there appears to be a lack of awareness of the well-established differences between spermatogenesis occurring at each of these stages.

In general, the authors present observational data of the sort that is generated by RNA-seq and ATAC-seq analyses, and they speculate on the potential significance of several of these observations. However, they provide no definitive data to support any of their speculations. This further illustrates the fact that this study contributes little if any new information beyond that already available from the numerous previously published RNA-seq and ATAC-seq studies of spermatogenesis. In short, the study described in this manuscript does not advance the field.

The phenomenon of epigenetic priming is discussed, but then it seems that there is some expression of surprise that the data demonstrate what this reviewer would argue are examples of that phenomenon. The authors discuss the "modest correspondence between transcription and chromatin accessibility in SCs." Chromatin accessibility is an example of an epigenetic parameter associated with the primed state. The primed state is not fully equivalent to the actively expressing state. It appears that certain histone modifications along with transcription factors are critical to the transition between the primed and actively expressing states (in either direction). The cell types that were investigated in this study are closely related spermatogenic, and predominantly spermatogonial cell types. It is very likely that the differentially expressed loci will be primed in both the early (PND 8 or 15) and adult stages, even though those genes are differentially expressed at those stages. Thus, it is not surprising that there is not a strict concordance between +/- chromatin accessibility and +/- active or elevated expression.

Reviewer #1 (Public Review):

Summary:<br /> Liao et al leveraged two powerful genomics techniques-CUT&RUN and RNA sequencing-to identify genomic regions bound by and activated or inactivated by SMAD1, SMAD5, and the progesterone receptor during endometrial stromal cell decidualization.

Strengths:<br /> The authors utilized powerful next generation sequencing and identified important transcriptional mechanisms of SMAD1/5 and PGR during decidualization in vivo.

Weaknesses:<br /> Overall, the manuscript and study are well structured and provide critical mechanistic updates on the roles of SMAD1/5 in decidualization and preparation of the maternal endometrium for pregnancy. Please consider the following to improve the manuscript:

• Figure 4: A and C show bar graphs, not histograms. Please alter this phrasing.<br /> • What post hoc test was performed on qPCR analyses? (Figure 6). It is evident that any assumptions of equal variance need to be negated due to the wide dispersion in experimental response invalidating the assumptions of a one-way ANOVA.<br /> • Figure 6: what data points are plotted? Are these technical replicates from individual wells or qPCR technical replicates?<br /> • Figure 6: Consider changing graph colors to increase visibility of error bars and data points.<br /> • Figure 6 legend: no histograms are shown in this figure. Refer to all gene names utilizing proper nomenclature and conventions (gene names should be italicized).<br /> • qPCR analyses: qPCR normalization should be done to at least two internal control genes, preferably three according to the MIQE guidelines (PMID: 19246619).<br /> • Supplement figure 2: graphs are bar graphs, not histograms.

Reviewer #1 (Public Review):

Summary:<br /> The authors start out by doing a time-calibrated gene/species tree analysis of the animal gasdermin family, resulting in a dendrogram showing the relationship of the individual gasdermin subfamilies and suggesting a series of gene duplication events (and gene losses) that lead to the gasdermin distribution in extant species. They observe that the GSDMA proteins from birds, reptiles, and amphibians do not form a clade with the mammalian GSDMAs and notice that the non-mammalian GSDMA proteins share a conserved caspase-1 cleavage motif at the predicted activation site. The authors provide several series of experiments showing that the non-mammalian GSDMA proteins can indeed be activated by caspase-1 and that this activation leads to cell death (in human cells). They also investigate the role of the caspase-1 recognition tetrapeptide for cleavage by caspase-1 and for the pathogen-derived protease SpeB.

Strengths:<br /> The evolutionary analysis performed in this manuscript appears to use a broader data basis than what has been used in other published work. An interesting result of this analysis is the suggestion that GSDMA is evolutionarily older than the main mammalian pyroptotic GSDMD, and that birds, reptiles, and amphibians lack GSDMD but use GSDMA for the same purpose. The consequence that bird GSDMA should be activated by an inflammatory caspase (=caspase1) is convincingly supported by the experiments provided in the manuscript.

Weaknesses:<br /> 1) As a non-expert in phylogenetic tree reconstruction, I find the tree resulting from the authors' analysis surprising (in particular the polyphyly of GSDMA) and at odds with several other published trees of this family. The differences might be due to differences in the data being used or due to the tree construction method, but no explanation for this discrepancy is provided.

2) While the cleavability of bird/reptile GSDMA by caspase-1 is well-supported by several experiments, the role of this cleavage for pyroptotic cell killing is addressed more superficially. One cell viability assay upon overexpression of GSDMA-NTD in human HEK293 cells is shown and one micrograph shows pyroptotic morphology upon expression in HeLa cells. It is not clear why these experiments were limited to human cells and why two different cell types were used for the two complementary results.

3) The introduction mentions as a motivation for this work our lack of knowledge of how human GSDMA is activated. This is indeed an interesting and pressing question, but it is not really addressed in the manuscript. This is particularly true when believing the authors' dendrogram results that the bird and mammalian GSDMA families do not form a clade.

As a consequence, the significance of this finding is mostly limited to birds and reptiles.

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):

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):

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).