Reviewer #2 (Public Review):

Summary:<br /> In this paper, the authors investigated the admixture history of domestic cattle since they were introduced into Iberia, by studying genomic data from 24 ancient samples dated to ~2000-8000 years ago and comparing them to modern breeds. They aimed to (1) test for introgression from (local) wild aurochs into domestic cattle; (2) characterize the pattern of admixture (frequency, extent, sex bias, directionality) over time; (3) test for correlation between genetic ancestry and stable isotope levels (which are indicative of ecological niche); and (4) test for the hypothesized higher aurochs ancestry in a modern breed of fighting bulls.

Strengths:<br /> Overall, this study collects valuable new data that are useful for testing interesting hypotheses, such as admixture between domestic and wild populations, and correlation between genome-wide aurochs ancestry and aggressiveness.

Weaknesses:<br /> Most conclusions are partially supported by the data presented. The presence of admixed individuals in prehistorical periods supports the hypothesized introgression, although this conclusion needs to be strengthened with an analysis of potential contamination. The frequency, sex-bias, and directionality of admixture remain highly uncertain due to limitations of the data or issues with the analysis. There is considerable overlap in stable isotope values between domestic and wild groups, indicating a shared ecological niche, but variation in classification criteria for domestic vs wild groups and in skeletal elements sampled for measurements significantly weakens this claim. Lastly, the authors presented convincing evidence for relatively constant aurochs ancestry across all modern breeds, including the Lidia breed which has been bred for aggressiveness for centuries. My specific concerns are outlined below.

Contamination is a common concern for all ancient DNA studies. Contamination by modern samples is perhaps unlikely for this specific study of ancient cattle, but there is still the possibility of cross-sample contamination. The authors should estimate and report contamination estimates for each sample (based on coverage of autosomes and sex chromosomes, or heterozygosity of Y or MT DNA). Such contamination estimates are particularly important to support the presence of individuals with admixed ancestry, as a domestic sample contaminated with a wild sample (or vice versa) could appear as an admixed individual.

A major limitation of this study is uncertainty in the "population identity" for most sampled individuals (i.e., whether an individual belonged to the domesticated or wild herd when they were alive). Based on chronology, morphology, and genetic data, it is clear the Mesolithic samples from the Artusia and Mendandia sites are bona fide aurochs, but the identities of individuals from the other two sites are much less certain. Indeed, archeological and morphological evidence from El Portalon supports the presence of both domestic animals and wild aurochs, which is echoed by the inter-individual heterogeneity in genetic ancestry. Based on results shown in Fig 1C and Fig 2 it seems that individuals moo017, moo020, and possibly moo012a are likely wild aurochs that had been hunted and brought back to the site by humans. Although the presence of individuals (e.g., moo050, moo019) that can only be explained by two-source models strongly supports that interbreeding happened (if cross-contamination is ruled out), it is unclear whether these admixed individuals were raised in the domestic population or lived in the wild population and hunted.

Such uncertainty in "population identity" limits the authors' ability to make conclusions regarding the frequency, sex bias, and directionality of gene flow between domestic and wild populations. For instance, the wide range of ancestry estimates in Neolithic and Chalcolithic samples could be interpreted as evidence of (1) frequent recent gene flow or (2) mixed practices of herding and hunting and less frequent gene flow. Similarly, the statement about "bidirection introgression" (on pages 8 and 11) is not directly supported by data. As the genomic, morphological, and isotope data cannot confidently classify an individual as belonging to the domesticated or wild population, it seems impossible to conclude the direction of gene flow (if by "bidirection introgression" the authors mean something other than "bidirectional gene flow", they need to clearly explain this before reaching the conclusion.)

The f4 statistics shown in Fig 3B are insufficient to support the claim regarding sex-biased hybridization, as the f4 statistic values are not directly comparable between the X chromosome and autosomes. Because the effective population size is different for the X chromosome and autosomes (roughly 3:4 for populations with equal numbers of males and females), the expected amount of drift is different, hence the fraction of allele sharing (f4) is expected to be different. In fact, the observation that moo004 whose autosomal genome can be modeled as 100% domestic ancestry still shows a higher f4 value for the X chromosome than autosomes hints at this issue. A more robust metric to test for sex-biased admixture is the admixture proportion itself, which can be estimated by qpAdm or f4-ratio (see Patterson et al 2012). However, even with this method, criticism has been raised (e.g., Lazaridis and Reich 2017; Pfennig and Lachance, 2023). In general, detecting sex-bias admixture is a tough problem.

In general, the stable isotope analysis seems to be very underpowered, due to the issues of variation in classification criteria and skeletal sampling location discussed by the authors in supplementary material. The authors claimed a significant difference in stable nitrogen isotope between (inconsistently defined) domestic cattle and wild aurochs, but no figures or statistics are presented to support this claim. Please describe the statistical method used and the corresponding p-values. The authors can consider including a figure to better show the stable isotope results.

Reviewer #2 (Public Review):

Summary:<br /> Wang and collaborators have evaluated the impact of inflammation on bone loss induced by Doxorubicin, which is commonly used in chemotherapy to treat various cancers. In mice, they show that a single injection of Doxorubicin induces systemic inflammation, leukopenia, and significant bone loss associated with increased bone-resorbing osteoclast numbers. In vitro, the authors show that Doxorubicin activates the AIM2 and NLRP3 inflammasomes in macrophages and neutrophils. Importantly, they show that the full knockouts (germline deletions) of AIM2 (Aim2-/-) and NLRP3 (Nlrp3-/-) and Caspase 1 (Casp1-/-) limit (but do not completely abolish) bone loss induced 4 weeks after a single injection of Doxorubicin in mice. From these results, they conclude that Doxorubicin activates inflammasomes to cause inflammation-associated bone loss.

Strengths:<br /> While numerous studies have reported that Doxorubicin activates the inflammasome in myeloid cells and various other cell types, that Doxorubicin induces systemic inflammation, and that both the systemic inflammation and Doxorubicin treatment leads to bone loss, functional experiments demonstrating that NRLP3 and/or AIM2 loss-of-functions, and thus the systemic impairment of the inflammatory response, may prevent bone-loss induced by Doxorubicin were lacking. The strength of this manuscript is that it provides these missing data.

Weaknesses:<br /> However, one could argue that most of the conclusions drawn from the data presented here have been previously reported and that it was very much expected that reducing systemic inflammation in treated animals (in Aim2-/- and/or Nlrp3-/- mice) would preserve bone homeostasis to some extent, similarly to what has been reported in the context of cardiotoxicity induced by Doxorubicin.

Since the manuscript focuses on therapeutic considerations aiming to preserve bone homeostasis in animals treated with Doxorubicin, additional experiments evaluating and comparing various therapeutic options could improve the impact of the study. Drugs targeting the inflammasomes could be tested in addition to the genetic mouse models. Since increased osteoclast numbers (and likely bone resorption) are associated with Doxorubicin-induced bone loss, antiresorptive drugs such as Bisphosphonates or anti-RANKL antibodies could be tested and compared to anti-inflammatory drugs. Since autophagy and senescence have been shown to contribute to bone loss induced by Doxorubicin, it would be interesting to use the pharmacologic inhibitors (targeting autophagy or senescence) used in these previous studies to evaluate the relative impact of these different cellular mechanisms, on bone loss induced by Doxorubicin.

Moreover, the cellular and molecular mechanisms by which Doxorubicin induces bone loss in vivo could be further evaluated. Doxorubicin has been reported to directly affect bone-making osteoblasts and bone-resorbing osteoclasts. It would be important to determine the relative importance of the activation of the AIM2 and NLRP3 inflammasomes in these cells compared to macrophages and neutrophils. Floxed mouse lines exist for both Aim2 and Nlrp3, as well as relevant cell-specific Cre lines. Thus, cell-specific conditional knockouts could have been used in the current study, instead of using global knockout animals. Genetic tools also exist to induce the specific ablation of macrophages or neutrophils and could be used. Furthermore, it is unclear whether local inflammation is induced in the bone marrow of Doxorubicin-treated mice, and what is the relative impact of local versus systemic inflammation in bone loss in these mice. Markers of the inflammasomes, pyroptosis, and NETosis could be evaluated on bone sections, and on bone and bone marrow samples. The effect of Doxorubicin on osteoblast numbers in vivo and on bone resorption (not just osteoclast numbers) should be evaluated as well. These mechanistic aspects are important and needed to better understand the cytotoxic mechanisms triggered by Doxorubicin, and define the best therapeutic approaches to preserve bone integrity in chemotherapy.

Finally, it would be important to assess the bone mass of Doxorubicin-treated control, Aim2-/-, Nlrp3-/- and Cas1-/- mice at a later time point than 4 weeks post-injection. Nlrp3 knockout has been reported to increase the density of the cortical and trabecular bones. The bone mass of Aim2-/-, Nlrp3-/- and Cas1-/- mice at baseline may be higher than that of control mice, and it may take slightly longer for Doxorubicin to reduce bone mass to the same extent than in controls. It would be also interesting to do similar experiments using animals treated multiple times with Doxorubicin instead of using a single injection, since patients receive their chemotherapy multiple times.

Reviewer #2 (Public Review):

Summary and strengths:

Early career funding success has an immense impact on later funding success and faculty persistence, as evidenced by well-documented "rich-get-richer" or "Matthew effect" phenomena in science (e.g., Bol et al., 2018, PNAS). In this study the authors examined publicly available data on the distribution of the National Institutes of Health's K99/R00 awards - an early career postdoc-to-faculty transition funding mechanism - and showed that although 89% of K99 awardees successfully transitioned into faculty, disparities in subsequent R01 grant obtainment emerged along three characteristics: researcher mobility, gender, and institution. Men who moved to a top-25 NIH funded institution in their postdoc-to-faculty transition experienced the shortest median time to receiving a R01 award, 4.6 years, in contrast to the median 7.4 years for women working at less well-funded schools who remained at their postdoc institutions.

Amongst the three characteristics, the finding that researcher mobility has the largest effect on subsequent funding success is key and novel. Other data supplement this finding: for example, although the total number of R00 awards has increased, most of this increase is for awards to individuals moving to different institutions. In 2010, 60% of R00 awards were activated at different institutions compared to 80% in 2022. These findings enhance previous work on the relationship between mobility and ones' access to resources, collaborators, or research objects (e.g., Sugimoto and Larivière, 2023, Equity for Women in Science (Harvard University Press)).

These results empirically demonstrate that even after receiving a prestigious early career grant, researchers with less mobility belonging to disadvantaged groups at less-resourced institutions continue to experience barriers that delay them from receiving their next major grant. This result has important policy implications aimed at reducing funding disparities - mainly that interventions that focus solely on early career or early stage investigator funding alone will not achieve the desired outcome of improving faculty diversity.

The authors also highlight two incredible facts: No postdoc at a historically Black college or university (HBCU) has been awarded a K99 since the program's launch. And out of all 2,847 R00 awards given thus far, only two have been made to faculty at HBCUs. Given the track record of HBCUs for improving diversity in STEM contexts, this distribution of awards is a massive oversight that demands attention.

At no fault of the authors, the analysis is limited to only examining K99 awardees and not those who applied but did not receive the award. This limitation is solely due to the lack of data made publicly available by the NIH. If this data were available, this study would have been able to compare the trajectory of winners versus losers and therefore could potentially quantify the impact of the award itself on later funding success, much like the landmark paper by Bol et al. (PNAS; 2018) that followed the careers of an early career grant scheme in the Netherlands. Such an analysis would also provide new insights that would inform policy.

Although data on applications versus awards for the K99/R00 mechanism are limited, there exists data for applicant race and ethnicity for the 2007-2017 period, which were made available by a Freedom of Information Act request through the now defunct Rescuing Biomedical Research Initiative (https://web.archive.org/web/20180723171128/http://rescuingbiomedicalresearch.org/blog/examining-distribution-k99r00-awards-race/). These results are highly relevant given the discussion of K99 award impacts on the sociodemographic composition of U.S. biomedical faculty. During the 2007-2017 period, the K99 award rate for white applicants was 31% compared to 26.7% for Asian applicants and 16.2% for Black applicants. In terms of award totals, these funding rates amount to 1,384 awards to white applicants, 610 to Asian applicants, and 25 to Black applicants. However, the work required to include these data may be beyond the scope of the study.

The conclusions are well-supported by the data, and limitations of the data and the name-gender matching algorithm are described satisfactorily.

Reviewer #2 (Public Review):

This paper makes important and novel advances that significantly enhance our understanding of the ClC-2 channel. The EM data are of high quality, and the most important argument, concerning the role of the N-terminus of the protein as an occluding inactivation gate, is very well supported by both structural, computational, and functional data (some of which is previously published). The proposal that the "run up" observed in patch clamp experiments represents relief of inactivation is interesting and compelling. The model predicts that mutations at the hairpin binding site should influence this "run up", which should be tested in the near future. Finally, the confirmation of the AK-42 binding site further solidifies evidence that this is a pore-blocking compound; the authors' argument about determinants of specificity is convincing.

Reviewer #2 (Public Review):

Tian et al. performed a meta-analysis of 113 genome-wide origin profile datasets in humans to assess the reproducibility of experimental techniques and shared genomics features of origins. Techniques to map DNA replication sites have quickly evolved over the last decade, yet little is known about how these methods fare against each other (pros and cons), nor how consistent their maps are. The authors show that high-confidence origins recapitulate several known features of origins (e.g., correspondence with open chromatin, overlap with transcriptional promoters, CTCF binding sites). However, surprisingly, they find little overlap between ORC/MCM binding sites and origin locations.

Overall, this meta-analysis provides the field with a good assessment of the current state of experimental techniques and their reproducibility, but I am worried about: (a) whether we've learned any new biology from this analysis; (b) how binding sites and origin locations can be so mismatched, in light of numerous studies that suggest otherwise; and (c) some methodological details described below.

-- I understand better the inclusion/exclusion logic for the samples. But I'm still not sure about the fragments. As the authors wrote, there is both noise and stochasticity; the former is not important but the latter is essential to include. How can these two be differentiated, and what may be the expected overlap as a function of different stochasticity rates?

-- Many of the major genomic features analyzed have already been found to be associated with origin sites. For example, the correspondence with TSS has been reported before:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320713/<br /> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547456/

-- Line 250: The most surprising finding is that there is little overlap between ORC/MCM binding sites and origin locations. The authors speculate that the overlap between ORC1 and ORC2 could be low because they come from different cell types. Equally concerning is the lack of overlap with MCM. If true, these are potentially major discoveries that butts heads with numerous other studies that have suggested otherwise.

The key missing dataset is ORC1 and ORC2 CHiP-seq from the same cell type. This shouldn't be too expensive to perform, and I hope someone performs this test soon. Without this, I remain on the fence about how much existing datasets are "junk" vs how much the prevailing hypothesis about replication needs to be revisited. Nonetheless, the authors do perform a nice analysis showing that existing techniques should be carefully used and interpreted.

Reviewer #2 (Public Review):

In this paper Sasani, Quinlan and Harris present a new method for identifying genetic factors affecting germline mutation, which is particularly applicable to genome sequence data from mutation accumulation experiments using recombinant inbred lines. These are experiments where laboratory organisms are crossed and repeatedly inbred for many generations, to build up a substantial number of identifiable germline mutations. The authors apply their method to such data from mice, and identify two genetic factors at two separate genetic loci. Clear evidence of such factors has been difficult to obtain, so this is an important finding. They further show evidence of an epistatic interaction between these factors (meaning that they do not act independently in their effects on the germline mutation process). This is exciting because such interactions are difficult to detect and few if any other examples have been studied.

The authors present a careful comparison of their method to another similar approach, quantitative trait locus (QTL) analysis, and demonstrate that in situations such as the one analysed it has greater power to detect genetic factors with a certain magnitude of effect. They also test the statistical properties of their method using simulated data and permutation tests. Overall the analysis is rigorous and well motivated, and the methods explained clearly.

The main limitation of the approach is that it is difficult to see how it might be applied beyond the context of mutation accumulation experiments using recombinant inbred lines. This is because the signal it detects, and hence its power, is based on the number of extra accumulated mutations linked to (i.e. on the same chromosome as) the mutator allele. In germline mutation studies of wild populations the number of generations involved (and hence the total number of mutations) is typically small, or else the mutator allele becomes unlinked from the mutations it has caused (due to recombination), or is lost from the population altogther (due to chance or perhaps selection against its deleterious consequences).

Nevertheless, accumulation lines are a common and well established experimental approach to studying mutation processes in many organisms, so the new method could have wide application and impact on our understanding of this fundamental biological process.

The evidence presented for an epistatic interaction is convincing, and the authors suggest some plausible potential mechanisms for how this interaction might arise, involving the DNA repair machinery and based on previous studies of the proteins implicated. However as with all such findings, given the higher degree of complexity of the proposed model it needs to be treated with greater caution, perhaps until replicated in a separate dataset or demonstrated in follow-up experiments exploring the pathway itself.

Reviewer #2 (Public Review):

Pinos et al present five atherosclerosis studies in mice to investigate the impact of dietary supplementation with b-carotene on plaque remodeling during resolution. The authors use either LDLR-ko mice or WT mice injected with ASO-LDLR to establish diet-induced hyperlipidemia and promote atherogenesis during 16 weeks, and then they promote resolution by switching the mice for 3 weeks to a regular chow, either deficient or supplemented with b-carotene. Supplementation was successful, as measured by hepatic accumulation of retinyl esters. As expected, chow diet led to reduced hyperlipidemia, and plaque remodeling (both reduced CD68+ macs and increased collagen contents) without actual changes in plaque size. But, b-carotene supplementation resulted in further increased collagen contents and, importantly, a large increase in plaque regulatory T-cells (TREG). This accumulation of TREG is specific to the plaque, as it was not observed in blood or spleen. The authors propose that the anti-inflammatory properties of these TREG explain the atheroprotective effect of b-carotene, and found that treatment with anti-CD25 antibodies (to induce systemic depletion of TREG) prevents b-carotene-stimulated increase in plaque collagen and TREG.

An obvious strength is the use of two different mouse models of atherogenesis, as well as genetic and interventional approaches. The analyses of aortic root plaque size and contents are rigorous and included both male and female mice (although the data was not segregated by sex). Unfortunately, the authors did not provide data on lesions in en face preparations of the whole aorta.

Overall, the conclusion that dietary supplementation with b-carotene may be atheroprotective via induction of TREG is reasonably supported by the evidence presented. Other conclusions put forth by the authors (e.g., that vitamin A production favors TREG production or that BCO1 deficiency reduces plasma cholesterol), however, will need further experimental evidence to be substantiated.

The authors claim that b-carotene reduces blood cholesterol, but data shown herein show no differences in plasma lipids between mice fed b-carotene-deficient and -supplemented diets (Figs. 1B, 2A, and S3A). Also, the authors present no experimental data to support the idea that BCO1 activity favors plaque TREG expansion (e.g., no TREG data in Fig 3 using Bco1-ko mice).

As the authors show, the treatment with anti-CD25 resulted in only partial suppression of TREG levels. Because CD25 is also expressed in some subpopulation of effector T-cells, this could potentially cloud the interpretation of the results. Data in Fig 4H showing loss of b-carotene-stimulated increase in numbers of FoxP3+GFP+ cells in the plaque should be taken cautiously, as they come from a small number of mice. Perhaps an orthogonal approach using FoxP3-DTR mice could have produced a more robust loss of TREG and further confirmation that the loss of plaque remodeling is indeed due to loss of TREG.

Reviewer #2 (Public Review):

Summary:

This work elaborates on a combined therapeutic approach comprising ionizing radiation and CCR5i/αPD1 immunotherapy as a promising strategy in pancreatic cancer. Previous research has established that NK cell-derived CCL5 and XCL1 play a crucial role in recruiting cDC1 cells to the tumor microenvironment, contributing to tumor control. In this study, by using a murine pancreatic cancer model, the authors propose that the addition of radiation therapy to CCR5i and αPD1 immunotherapy could upregulate CD8+ T cells and a subgroup of NK cells within the tumor and result in better tumor control. They further analyzed human single-cell sequencing data from pancreatic cancer patients and identified one subgroup of NK cells (NK C1) with tissue-resident features. Subsequent cell-cell contact analysis reveals the NK-cDC1-CD8 cell axis in pancreatic cancer. By analyzing TCGA data, they found that high NK C1 signature levels were associated with better survival in pancreatic cancer patients. Thus, radiotherapy could benefit the outcome of patients bearing low NK C1 signatures. Importantly, the positive correlation between NK C1 score with survival extends beyond pancreatic cancer, showing potential applicability across various solid cancers.

Strengths:

This study could add new insight into the clinical practice by introducing such novel combined therapy and shed light on the underlying immune cell dynamics. These findings hold potential for more effective and targeted treatment in the future. Mouse experiments nicely confirmed that such combined therapy could significantly reduce tumor volume. The elegant use of single-cell sequencing analysis and human database examination enriches the narrative and strengthens the study's foundation. Additionally, the notion that NK C1 signature correlates with patient survival in various solid cancers is of high interest and relevance.

Weaknesses:

1. The role of CCR5i requires further clarification. While the authors demonstrated its capacity to reduce Treg in murine tumors, its impact on other cell populations, including NK cells and CD8+ T cells, was not observed. Nevertheless, the effect of CCR5i on tumor growth in Figure 2B should be shown. If the combination of radiotherapy and αPD1 already can achieve good outcomes as shown in Figure 3A, the necessity to include CCR5i is questioned. Overall, a more comprehensive elucidation of the roles of CCL5 and CCR5i in this context would be good.

2. In line with this, spatial plots in Figure 4 did not include the group with only radiotherapy and αPD1. This inclusion would facilitate a clearer comparison and better highlight the essential role of CCR5i.

3. NK C1 cells should be also analyzed in the mouse model. The authors suggest that NKNKG2D-ve could be the cell population. Staining of inhibitory markers should be considered, for example, TIGIT and TIM3 as presented in Figure 5B.

4. While the cell-cell contact analysis generated from single-cell sequencing data is insightful, extending this analysis to the mouse model under therapy would be highly informative. NK and CD8 cells in the tumor increased upon the combined therapy. However, cDC1 was not characterized. Analysis regarding cDC1 would provide more information on the NK/cDC1/CD8 axis.

5. Human database analysis showed a positive correlation between NK C1 score and CCL5 in pancreatic cancer. Furthermore, radiotherapy could benefit the outcome of patients bearing low NK C1 scores. It would be interesting to test if radiotherapy could also benefit patients with low CCL5 levels in this cohort.

Reviewer #3 (Public Review):

Prior studies in humans and in chickens suggested that TMEM263 could play an important role in longitudinal bone growth, but a definitive assessment of the function and potential mechanism of action of this species-conserved plasma membrane protein has been lacking. Here, the authors create a TMEM263 null mouse model and convincingly show dramatic cessation of post-natal growth, which becomes apparent by day PND21. They report proportional dwarfism, highly significant bone and related phenotypes, as well as notable alterations of hepatic GH signaling to IGF1. A large body of prior work has established an essential role for GH and it's stimulation of IGF1 production in liver and other tissues in post-natal growth. On this basis, the authors conclude that the observed decrease in serum IGF1 seen in TMEM263-KO mice is causal for the growth phenotype, which seems likely. Moreover, they ascribe the low serum IGF1 to the observed decreases in hepatic GH receptor (GHR) expression and GHR/JAK2/STAT5 signaling to IGF1, which is plausible but not proven by the experiments presented.

The finding that TMEM263 is essential for normal hepatic GHR/IGF1 signaling is an important, and unexpected finding, one that is likely to stimulate further research into the underlying mechanisms of TMEM263 action, including the distinct possibility that these effects involve direct protein-protein interactions between GHR and TMEM263 on the plasma membrane of hepatocytes, and perhaps on other mouse cell types and tissues as well, where TMEM263 expression is up to 100-fold higher (Fig. 1C).

An intriguing finding of this study, which is under emphasized and should be noted in the Abstract, is the apparent feminization of liver gene expression in male TMEM263-KO mice, where many male-biased genes are downregulated, and many female-biased genes are upregulated. Further investigation of these liver gene responses by comparison to public datasets could be very useful, as it could help determine: (1) whether the TMEM263 liver phenotype is similar to that of hypophysectomized male mouse liver, where GH and GHR/STAT5/IGF1 signaling are both totally ablated; or alternatively, (2) whether the phenotype is more similar to that of a male mouse given GH as a continuous infusion, which induces widespread feminization of gene expression in the liver, and is perhaps similar to the gene responses seen in the TMEM263-KO mice. Answering this question could provide critical insight into the mechanistic basis for the hepatic effects of TMEM263 gene KO.

Comments on revised version:

The authors have addressed a majority of the concerns raised during the initial review. The evidence supporting the whole-body growth and skeletal phenotypes, as well as the disruption of GH/IGF1 signaling seen in TMEM263-KO mice, is convincing. However, there is insufficient evidence to definitively conclude that the observed alteration of hepatic GH/IGF1 signaling is causative of the body growth and skeletal phenotypes.

Reviewer #2 (Public Review):

Summary:<br /> Anil Verma et al. have performed prime-boost HIV vaccination to enhance HIV-1 Env antibodies in the rhesus macaques model. The authors used two different adjuvants, a cationic liposome-based adjuvant (CAF01) and a monophosphoryl lipid A (MPLA)+QS-21 adjuvant. They demonstrated that these two adjuvants promote different transcriptomes in the GC-TFH subsets. The MPLA+QS-21 adjuvant induces abundant GC TFH1 cells expressing CXCR3 at first priming, while the CAF01 adjuvant predominantly induced GC TFH1/17 cells co-expressing CXCR3 and CCR6. Both adjuvants initiate comparable Env antibody responses. However, MPLA+QS-21 shows more significant IgG1 antibodies binding to gp140 even after 30 weeks.<br /> The enhancement of memory responses by MPLA+QS-21 consistently associates with the emergence of GC TFH1 cells that preferentially produce IFN-γ.

Strengths:<br /> The strength of this manuscript is that all experiments have been done in the rhesus macaques model with great care. This manuscript beautifully indicated that MPLA+QS-21 would be a promising adjuvant to induce the memory B cell response in the HIV vaccine.

Weaknesses:<br /> The authors did not provide clear evidence to indicate the functional relevance of GC TFH1 in IgG1 class-switch and B cell memory responses.

Reviewer #2 (Public Review):

Summary:<br /> Planctomycetes encompass a group of bacteria with unique biological traits, the compartmentalized cells make them appear to be organisms in between prokaryotes and eukaryotes. However, only few of the Planctomycetes bacteria are cultured thus far, and hampers insight into the biological traits of this evolutionary important organisms.

This work reports the methodology details of how to isolate the deep-sea bacteria that could be recalcitrant to laboratory cultivation, and further reveals the distinct characteristics of the new species of a deep-sea Planctomycetes bacterium, such as the chronic phage release without breaking the host and promote the host and related bacteria in nitrogen utilization. Therefore, the finding of this work is of importance in extending our knowledge on bacteria.

Strengths:

Through combination of microscopic, physiological, genomics and molecular biological approaches, this reports isolation and comprehensively investigation of the first anaerobic representative of the deep-sea Planctomycetes bacterium, in particular in that of the budding division, and release phage without lysis the cells. Most of results and conclusions are supported by the experimental evidences.

Reviewer #2 (Public Review):

Theta-nested gamma oscillations (TNGO) play an important role in hippocampal memory and cognitive processes and are disrupted in pathology. Deep brain stimulation has been shown to affect memory encoding. To investigate the effect of pulsed CA1 neurostimulation on hippocampal TNGO the authors coupled a physiologically realistic model of the hippocampus comprising EC, DG, CA1, and CA3 subfields with an abstract theta oscillator model of the medial septum (MS). Pathology was modeled as weakened theta input from the MS to EC simulating MS neurodegeneration known to occur in Alzheimer's disease. The authors show that if the input from the MS to EC is strong (the healthy state) the model autonomously generates TNGO in all hippocampal subfields while a single neurostimulation pulse has the effect of resetting the TNGO phase. When the MS input strength is weaker the network is quiescent but the authors find that a single CA1 neurostimulation pulse can switch it into the persistent TNGO state, provided the neurostimulation pulse is applied at the peak of the EC theta. If the MS theta oscillator model is supplemented by an additional phase-reset mechanism a single CA1 neurostimulation pulse applied at the trough of EC theta also produces the same effect. If the MS input to EC is weaker still, only a short burst of TNGO is generated by a single neurostimulation pulse. The authors investigate the physiological origin of this burst and find it results from an interplay of CAN and M currents in the CA1 excitatory cells. In this case, the authors find that TNGO can only be rescued by a theta frequency train of CA1 pulses applied at the peak of the EC theta or again at either the peak or trough if the MS oscillator model is supplemented by the phase-reset mechanism.

The main strength of this model is its use of a fairly physiologically detailed model of the hippocampus. The cells are single-compartment models but do include multiple ion channels and are spatially arranged in accordance with the hippocampal structure. This allows the understanding of how ion channels (possibly modifiable by pharmacological agents) interact with system-level oscillations and neurostimulation. The model also includes all the main hippocampal subfields. The other strength is its attention to an important topic, which may be relevant for dementia treatment or prevention, which few modeling studies have addressed.

The work has several weaknesses. First, while investigations of hippocampal neurostimulation are important there are few experimental studies from which one could judge the validity of the model findings. All its findings are therefore predictions. It would be much more convincing to first show the model is able to reproduce some measured empirical neurostimulation effect before proceeding to make predictions. Second, the model is very specific. Or if its behavior is to be considered general it has not been explained why. For example, the model shows bistability between quiescence and TNGO, however what aspect of the model underlies this, be it some particular network structure or particular ion channel, for example, is not addressed. Similarly for the various phase reset behaviors that are found. We may wonder whether a different hippocampal model of TNGO, of which there are many published (for example [1-6]) would show the same effect under neurostimulation. This seems very unlikely and indeed the quiescent state itself shown by this model seems quite artificial. Some indication that particular ion channels, CAN and M are relevant is briefly provided and the work would be much improved by examining this aspect in more detail. In summary, the work would benefit from an intuitive analysis of the basic model ingredients underlying its neurostimulation response properties. Third, while the model is fairly realistic, considerable important factors are not included and in fact, there are much more detailed hippocampal models out there (for example [5,6]). In particular, it includes only excitatory cells and a single type of inhibitory cell. This is particularly important since there are many models and experimental studies where specific cell types, for example, OLM and VIP cells, are strongly implicated in TNGO. Other missing ingredients one may think might have a strong impact on model response to neurostimulation (in particular stimulation trains) include the well-known short-term plasticity between different hippocampal cell types and active dendritic properties. Fourth the MS model seems somewhat unsupported. It is modeled as a set of coupled oscillators that synchronize. However, there is also a phase reset mechanism included. This mechanism is important because it underlies several of the phase reset behaviors shown by the full model. However, it is not derived from experimental phase response curves of septal neurons of which there is no direct measurement. The work would benefit from the use of a more biologically validated MS model.

[1] Hyafil A, Giraud AL, Fontolan L, Gutkin B. Neural cross-frequency coupling: connecting architectures, mechanisms, and functions. Trends in neurosciences. 2015 Nov 1;38(11):725-40.

[2] Tort AB, Rotstein HG, Dugladze T, Gloveli T, Kopell NJ. On the formation of gamma-coherent cell assemblies by oriens lacunosum-moleculare interneurons in the hippocampus. Proceedings of the National Academy of Sciences. 2007 Aug 14;104(33):13490-5.

[3] Neymotin SA, Lazarewicz MT, Sherif M, Contreras D, Finkel LH, Lytton WW. Ketamine disrupts theta modulation of gamma in a computer model of hippocampus. Journal of Neuroscience. 2011 Aug 10;31(32):11733-43.

[4] Ponzi A, Dura-Bernal S, Migliore M. Theta-gamma phase-amplitude coupling in a hippocampal CA1 microcircuit. PLOS Computational Biology. 2023 Mar 23;19(3):e1010942.

[5] Bezaire MJ, Raikov I, Burk K, Vyas D, Soltesz I. Interneuronal mechanisms of hippocampal theta oscillations in a full-scale model of the rodent CA1 circuit. Elife. 2016 Dec 23;5:e18566.

[6] Chatzikalymniou AP, Gumus M, Skinner FK. Linking minimal and detailed models of CA1 microcircuits reveals how theta rhythms emerge and their frequencies controlled. Hippocampus. 2021 Sep;31(9):982-1002.

Reviewer #2 (Public Review):

In this study, Fleck and colleagues investigate the effects of auxin exposure on Drosophila melanogaster adults, focusing their analysis on feeding behavior, fatty acid metabolism, and oogenesis. The motivation for the study is that auxin-inducible transcription systems are now being used by Drosophila researchers to drive transcription using the Gal4-UAS system as a complement to Gal80ts versions of the system. I found the study to be carefully done. This study will be of interest for researchers using the Drosophila system, especially those focusing on fatty acid metabolism or physiology. The authors have adequately addressed all the minor points I raised in my review of the first submission.

Reviewer #2 (Public Review):

This study visualizes a specific localized form of cell-to-cell communication and conveys very well with what dynamics and sensitivity this biological phenomenon occurs.<br /> Using a FRET-based PKA biosensor, the authors observed that radial localized kinase activity changes spontaneously occur in adjacent cells of certain cell density. This phenomenon of radial propagation of PKA activity changes in groups of cells was further mechanistically elucidated and characterized. Interestingly, the authors found that individual cells in the cell groups form spontaneous Ca2+ transients, which at a certain strength can trigger the biosynthesis and release of prostaglandin E2 (PGE2). PGE2 then acts on the neighboring cells and triggers the increase of cAMP levels and the associated activation of the PKA via G-protein-coupled receptors (EP2 and EP4). In systematic, well-structured experiments, it was then found that the frequency of occurrence of such radial activations depends not only on the cell density but also on the activation state of the ERK MAP kinase pathway.

Strength<br /> In this study, the authors skillfully used various modern genetically encoded biosensors and other tools such as optogenetic tools to visualize and characterize an interesting biological phenomenon of cell-to-cell communication. The insights gained with these investigations produce a better understanding of the dynamics, sensitivity, and spatial extent with which such communications can occur in a cell network. It is also worth noting that the authors have not limited the studies to 2D cell culture in vitro, but were also able to confirm the findings in an animal model.

Weakness<br /> The work is hardly conclusive as to the actual biological significance of the phenomenon. It would be interesting to know more under which physiological and pathological conditions PGE2 triggers such radical PKA activity changes. It is not well explained in which tissues and organs and under what conditions this type of cell-to-cell communication could be particularly important.<br /> The authors also do not explain further why in certain cells of the cell clusters Ca2+ signals occur spontaneously and thus trigger the phenomenon. What triggers these Ca2+ changes? And why could this be linked to certain cell functions and functional changes?<br /> What explains the radius and the time span of the radial signal continuation? To what extent are these factors also related to the degradation of PGE2? The work could be stronger if such questions and their answers would be experimentally integrated and discussed.

Reviewer #2 (Public Review):

In this work, Boor and colleagues explored the role of microbial food cues in the regulation of neuroendocrine controlled foraging behavior. Consistent with previous reports, the authors find that C. elegans foraging behavior is regulated by the neuroendocrine TGFβ ligand encoded by daf-7. In addition to its known role in the neuroendocrine/sensory ASI neurons, Boor and colleagues show that daf-7 expression is dynamically regulated in the ASJ sensory neurons by microbial food cues - and that this regulation is important for exploration/exploitation balance during foraging. They identify at least two independent pathways by which microbial cues regulate daf-7 expression in ASJ: a gustatory pathway that promotes daf-7 expression and an opposing interoceptive pathway, also chemosensory in nature but which requires microbial ingestion to inhibit daf-7 expression via ASIC channels, encoded by del-3/del-7. In contrast, the authors show that the conserved PDF neuropeptide signaling pathway likely functions via the gustatory pathway to promote daf-7 expression. They further identify a novel role for the C. elegans ALK orthologue encoded by scd-2, which acts in interneurons to regulate daf-7 expression and foraging behavior. These results together imply that distinct cues from microbial food are used to regulate the balance between exploration and exploitation via conserved signaling pathways.

Strengths:<br /> The findings that gustatory and interoceptive inputs into foraging behavior are separable and opposing are novel and interesting, which they have shown most clearly in Figure 1 and Figure 3. These data clarify how these parallel chemosensory pathways can be integrated at the level of daf-7 expression.

It is also clear from their results that removal of the interoceptive cue (via transfer to non-digestible food) results in rapid induction of daf-7::gfp in ASJ - suggesting that this pathway is likely chemosensory and not simply nutritive in nature. They have also shown that daf-7 in ASJ plays an important role in the regulation of foraging behavior.

The role of the hen-1/scd-2 pathway in mediating the effects of ingested food is also compelling and well-interpreted, with a few small caveats, described below. This implies that important elements of this food sensing pathway may be conserved in mammals.

Weaknesses:<br /> Although not a weakness of this work per se, the roles of the 5-HT and hen-1/scd-2 pathway remain a bit unclear, likely reflecting their complex genetic contributions to foraging and daf-7 expression. Future work should clarify how these signals are integrated and whether the integration of these pathways improve exploration/exploitation balance to regulate animal fitness.

Reviewer #2 (Public Review):

Language skills are traditionally associated with a network of brain regions in the left hemisphere. In this intriguing study, Esteban Villar-Rodríguez and collaborators examined whether atypical hemispheric lateralization for language determines the functional and structural organisation of the network for inhibitory control as well as its relationship with schizotypy and autistic spectrum traits. The results suggest that individuals who have atypical lateralisation of the language function have also an atypical (mirrored) lateralisation of the inhibitory control network, compared to the typical group (individuals with left-lateralised language function). Furthermore, the atypical organization of language production is associated with a greater white matter volume of the corpus callosum, and atypical lateralization of inhibitory control is related to a higher interhemispheric functional coupling of the IFC, suggesting a link between atypical functional lateralisation (language and inhibitory control) and structural and functional changes in the brain.

This study also provides interesting evidence on how atypical language lateralisation impacts some aspects of language behaviour (reading), i.e., atypical lateralization predicts worse reading accuracy. Furthermore, the results suggest an association between atypical lateralization and increased schizotypy and autistic traits.

Reviewer #2 (Public Review):

The manuscript presents a valuable investigation of genetic associations related to plant resistance against the turnip mosaic virus (TuMV) using Arabidopsis thaliana as a model. The study infects over 1,000 A. thaliana inbred lines with both ancestral and evolved TuMV and assesses four disease-related traits: infectivity, disease progress, symptom severity, and necrosis. The findings reveal that plants infected with the evolved TuMV strain generally exhibited more severe disease symptoms than those infected with the ancestral strain. However, there was considerable variation among plant lines, highlighting the complexity of plant-virus interactions.

A major genetic locus on chromosome 2 was identified, strongly associated with symptom severity and necrosis. This region contained several candidate genes involved in plant defense against viruses. The study also identified additional genetic loci associated with necrosis, some common to both viral isolates and others specific to individual isolates. Structural variations, including transposable element insertions, were observed in the genomic region linked to disease traits.

Surprisingly, the minor allele associated with increased disease symptoms was geographically widespread among the studied plant lines, contrary to typical expectations of natural selection limiting the spread of deleterious alleles. Overall, this research provides valuable insights into the genetic basis of plant responses to TuMV, highlighting the complexity of these interactions and suggesting potential avenues for improving crop resilience against viral infections.

Overall, the manuscript is well-written, and the data are generally high-quality. The study is generally well-executed and contributes to our understanding of plant-virus interactions.

Reviewer #2 (Public Review):

Murata et al have characterized a transcription activator previously identified in an earlier genetic screen by Russell et al (named Fd2; for female-defective 2), here named PFG. The authors show solid evidence that PFG is a partner of the previously described transcription factor AP2-FG and describe three sets of genes: genes activated by PFG or AP2-FG alone and genes activated by the complex. The authors also show differential binding to the target DNA sequences by AP2-FG to either a 10bp, if in a complex with PFG or a 5bp motif if alone. In all, this is a useful study which further elucidates the underlying regulatory network that drives development of sexual stages and ultimately transmission to mosquitoes. The data presented are clear and solid and the conclusions drawn are mostly supported by the results shown.

A few comments:

Given that the transcriptional programme is so dynamic, the timing of the ChIP-seq experiments is crucial. Could the authors clarify the timings of the different ChIP-seq experiments (AP2-FG, PFG, PFG in AP2-FG-, AP2-FG in PFG-, ...)

Fig 4c is an example of great overlap of peaks, but it would be helpful if the authors could quantify the overlaps between experiments (and describe the overlap parameters used).

It remains unclear if AP2-FG and PFG interact directly or if they bind sequentially in the transcriptional activation process. Perhaps they are part of a larger complex? Immunoprecipitation followed by mass spectrometry of the GFP-tagged version of PFG in the presence and absence of AP2-FG would be highly informative.

Sourcing? Look this up.<br /> https://www.a5-size.com/history/

Reviewer #2 (Public Review):

The paper by Huan, Yong, et al. studies epithelial cell extrusion in MDCK monolayers grown on sinusoidally wavy surfaces in varying media osmolarities, finding that both curvature and osmolarity-mediated basal hydraulic stress spatially regulate extrusion events. The authors fabricated wavy substrates of varying periods and amplitude out of PDMS (and PA hydrogels) and monitored monolayer evolution and cell extrusion over time, by combining live-cell imaging with a convolutional network-based algorithm for automatic detection of extrusions.

In general, the study has been elegantly designed, starting with convincing evidence for enhanced extrusion rates in concave valleys with respect to convex hills. Next, the authors showed that hyper-osmotic medium reduced cell extrusion rate, which was demonstrated in a variety of different media compositions (e.g. with sucrose, DMSO, or NaCl), while hypo-osmotic medium increased cell extrusion rate. Additionally, the authors applied reflection interference contrast microscopy to reveal fluid spaces between the substrate and the basal side of the monolayer, which were found to grow when media composition was altered from hyper-osmotic to normal osmotic conditions. Using a 3D traction force microscopy approach, the authors demonstrated that cells on convex regions apply a downward pointing force on the substrate, opposite to cells on the concave regions. This was linked to a larger basal separation on the concave valleys as opposed to the convex hills. Finally, the authors focussed on the FAK-Akt pathway to explore the hypothesis that basal hydraulic stress interferes with focal adhesions, leading to differences in cell extrusion rates in media of different osmolarity and on convex or concave surfaces.

Comments on the revised version:

My previous comments were reasonably answered. In response to the comment that "experiments that are currently underway" for "Recommendation 5 - reviewer #1", I would also suggest the authors to either add the additional data or alter the emphasis on the FAK-AKT pathway in the manuscript accordingly if additional data is not presented.

Reviewer #2 (Public Review):

The authors convincingly show multiple inner and outer leaflet non-protein (lipid) densities in a cryo-EM closed state structure of GLIC, a prokaryotic homologue of canonical pentameric ligand-gated ion channels, and observe lipids in similar sites during extensive simulations at both resting and activating pH. The simulations not only corroborate structural observations but also suggest the existence of a state-dependent lipid intersubunit site only occupied in the open state. These important findings will be of considerable interest to the ion channel community and provide new hypotheses about lipid interactions in conjunction with channel gating.

Comments on the revised version:

The authors have addressed all of my comments.

Reviewer #2 (Public Review):

This work recorded neurons in the parahippocampal regions of the medial entorhinal cortex (MEC) and pre- and para-subiculum (PrS, PaS) during a visually guided navigation task on a 'tree maze'. They found that many of the neurons reflected in their firing the visual cue (or the associated correct behavioral choice of the animal) and also the absence of reward in inbound passes (with increased firing rate). Rate remapping explained best these firing rate changes in both conditions for those cells that exhibited place-related firing. This work used a novel task, and the increased firing rate at error trials in these regions is also novel.

The limitation is that cells in these regions were analyzed together.

Comments on the revised submission:

I accept the authors' response that histological differentiation of these regions was not possible.

Reviewer #2 (Public Review):

The authors investigated a central component of adaptive and flexible human behaviour: our ability to stop ongoing action plans. This ability is under prefrontal control, with an important contribution of the right inferior prefrontal gyrus (rIFG). This is a well-studied system, yet providing causal evidence, especially at an electrophysiological level, has proven challenging. In this study the authors use a novel non-invasive brain stimulation technique, transcranial ultrasonic stimulation (TUS), to selectively stimulate the rIFG and record behavioural and electrophysiological changes in the context of a stop-signal task.

The principal finding of this work is that following TUS over rIFG, participants are faster to respond to a stop signal when successfully inhibiting a planned action program. This faster stop-inhibition was reflected both in behaviour and evoked responses as measured with electroencephalography.

The spatial specificity of the TUS stimulation allows strong inferences on selective targeting. The inclusion of two control groups, one receiving stimulation over an active control site, and the other receiving a non-stimulating sham condition, makes the specificity of the observed effect convincing.

The EEG analyses are advanced, exploiting robust data-cleaning and selection approaches to allow strong inferences for analyses in sensor space. Through careful trial-matching and dynamic time-warping, the effects of primary interest - responses evoked by stopping behaviour - could be isolated from those evoked by the go-cue and go-response.

The manuscript focusses on the latency of the electrophysiological response (ERP). Indeed, an earlier P300 ERP is expected considering that TUS over rIFG led to an earlier stop-signal-reaction time (SSRT). However, as the SSRT is inferred from a model fit on the probability of go-responses as a function of the stop-signal delay (more often failing to inhibit go-responses when the stop-signal arrives late), the empirical observation of a latency shift in the closely related P300 ERP is valuable.

It is less clear how the P300 ERP itself relates to the TUS stimulation over rIFG, considering that this ERP has a well-established mid-frontal topology, while rIFG is in the lateral prefrontal cortex. The authors suggest that in the context of stopping control, rIFG is positioned upstream from the mid-frontal regions. However, previous work has revealed an inverse temporal and causal relationship, where rIFG contributions follow those of preSMA (e.g. Neubert et al., 2010, PNAS).

Behavioural changes, especially those dependent on attention and a speeded response, are commonly driven by non-specific cues, such as auditory, somatosensory, or multi-modal cues. This is a major confounding factor for all brain stimulation paradigms. TUS is no exception. Pulsed TUS protocols, such as the 1000 Hz pulsed protocol employed here, are very likely to be accompanied by an auditory confound. In the condition of interest in this experiment, TUS is delivered together with the visual stop-signal, creating a multimodal cue. In the main analyses (figures 3 and 4) this is only contrasted against conditions where the stop-signal is unimodal (visual) only, creating a multi-modal vs. uni-modal contrast.

Indeed, the critical comparison to allow the strongest inference is not between stop-TUS vs. go-TUS, nor between stop-TUS vs. no-TUS, but between the two TUS sites: rIFG-TUS vs rS1-TUS in the stop condition. The inclusion of the S1-TUS condition in this study is therefore highly valuable, although this contrast was implemented as a between-group design, and no assessment of confound matching between rIFG-TUS and S1-TUS is reported. Perhaps more importantly, the main analyses and figures (e.g. figure 3), do not include this comparison. In fact, the data from the TUS control-site group are not included in any analyses of evoked potentials (EEG) at all (e.g. figure 4), even though this is the main focus of the study.

The title of the study is "Transcranial focused ultrasound to rIFG improves response inhibition through modulation of the P300 onset latency". The discussion reads "P300 latency modulation occurred only in the rIFG group". It is not straightforward to see how this conclusion is supported without including a control site in the analyses. Further, the reported difference in onset latency is based on a visual inspection of the data, not on a quantified statistical analysis ("visually contrasting SS-US difference waveforms across tFUS conditions (Fig. 4B, upper right) revealed P300 onsets shifted earlier during Stop-tFUS"). Visual inspection of the same figure might also highlight a clear difference in ERP amplitude, in addition to latency. Lastly, the suggestion of a directional mediation effect ("improves response inhibition through modulation of the P300 onset latency") is only supported by a correlational analysis relating P300 onset latency with the estimated stop-signal-reaction-time.

In summary, by advancing transcranial ultrasonic stimulation to study prefrontal control, this work signifies a paradigm shift towards using interventional tools in cognitive neuroscience. The specificity and precision that ultrasound stimulation provides, with reduced discomfort as compared to TMS, are urgently needed to support a refined and causal understanding of the neural circuits underlying human cognition. The central claims of this study are partially supported by the data presented and might benefit from quantitatively comparing the effects of TUS over the region of interest and the control site.

Reviewer #2 (Public Review):

In this study, authors identified the complex TOR, HOG and CWI signaling networks-involved genes that relatively modulate the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation.

They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially found that the conserved site K19 of FKBP3 plays a key role in regulating the aflatoxin biosynthesis. In general, there is heavy workload task carried in this study and the findings are interesting and important for understanding or controlling the aflatoxin biosynthesis. However, findings have not been deeply explored and conclusions are mostly are based on parallel phenotypic observations. In addition, there are some concerns for the conclusions.

Reviewer #3 (Public Review):

Summary:<br /> In this paper presented by Liu et al, native MS on the lipid A transporter MsbA was used to obtain thermodynamic insight into protein-lipid interactions. By performing the analyses at different lipid A concentrations and temperatures, dissociation constants for 2-3 lipid A binding sites were determined, as well as enthalpies were calculated using non-linear van't Hoff fitting.

Strengths:<br /> This is an extensive high quality native MS dataset that provides unique opportunities to gain insights into the thermodynamic parameters underlying lipid A binding. In addition, it provides coupling energies between mutations introduced into MsbA, that are implicated in lipid A binding.

Weaknesses:<br /> It remains elusive, which KD values belong to which of the possible lipid A binding sites.

Appraisal:<br /> The authors convincingly addressed the concerns raised by the reviewers.

Reviewer #2 (Public Review):

Summary:

The authors investigated the molecular evolution of members of the gasdermin (GSDM) family. By adding the evolutionary time axis of animals, they created a new molecular phylogenetic tree different from previous ones. The analyzed result verified that non-mammalian GSDMAs and mammalian GSDMAs have diverged into completely different and separate clades. Furthermore, by biochemical analyses, the authors demonstrated non-mammalian GSDMA proteins are cleaved by the host-encoded caspase-1. They also showed mammalian GSDMAs have lost the cleavage site recognized by caspase-1. Instead, the authors proposed that the newly appeared GSDMD is now cleaved by caspase-1.

Through this study, we have been able to understand the changes in the molecular evolution of GSDMs, and by presenting the cleavage of GSDMAs through biochemical experiments, we have become able to grasp the comprehensive picture of this family molecules. However, there are some parts where explanations are insufficient, so supplementary explanations and experiments seem to be necessary.

Strengths:

It has a strong impact in advancing ideas into the study of pyroptotic cell death and even inflammatory responses involving caspase-1.

Weaknesses:

Based on the position of mammalian GSDMA shown in the molecular phylogenetic tree (Figure 1), it may be difficult to completely agree with the authors' explanation of the evolution of GSDMA.

1) Focusing on mammalian GSDMA, this group and mammalian GSDMD diverged into two clades, and before that, GSDMA/D groups and mammalian GSDMC separated into two, more before that, GSDMB, and further before that, non-mammalian GSDMA, when we checked Figure 1. In the molecular phylogenetic tree, it is impossible that GSDMA appears during evolution again. Mammalian GSDMAs are clearly paralogous molecules to non-mammalian GSDMAs in the figure. If they are bona fide orthologous, the mammalian GSDMA group should show a sub-clade in the non-mammalian GSDMA clade. It is better to describe the plausibility of the divergence in the molecular evolution of mammalian GSDMA in the Discussion section.

2) Regarding (1), it is recommended that the authors reconsider the validity of estimates of divergence dates by focusing on mammalian species divergence. Because the validity of this estimation requires recheck of the molecular phylogenetic tree, including alignment.

3) If GSDMB and/or GSDMC between non-mammalian GSDMA and mammalian GSDMD as shown in the molecular phylogenetic tree would be cleaved by caspase-1, the story of this study becomes clearer. The authors should try that possibility.

Reviewer #2 (Public Review):

Summary:<br /> The authors described cell type mapping was conducted for both WT and fracture types. Through this, unique cell populations specific to fracture conditions were identified. To determine these, the most undifferentiated cells were initially targeted using stemness-related markers and CytoTrace scoring. This led to the identification of SSPC differentiating into fibroblasts. It was observed that the fibroblast cell type significantly increased under fracture conditions, followed by subsequent increases in chondrocytes and osteoblasts.

Strengths:<br /> This study presented the injury-induced fibrogenic cell (IIFC) as a characteristic cell type appearing in the bone regeneration process and proposed that the IIFC is a progenitor undergoing osteochondrogenic differentiation.

Weaknesses:<br /> This study endeavored to elucidate the role of IIFC through snRNAseq analysis and in vivo observation. However, such validation alone is insufficient to confirm that IIFC is an osteochondrogenic progenitor, and additional data presentation is required.

Reviewer #2 (Public Review):

In this study, Aso and Rubin generated new split-GAL4 lines to label Drosophila mushroom body output neurons (MBONs) that previously lacked specific GAL4 drivers. The MBONs represent the output channels for the mushroom body (MB), a computational center in the fly brain. Prior research identified 21 types of typical MBONs whose dendrites exclusively innervate the MB and 14 types of atypical MBONs whose dendrites also innervate brain regions outside the MB. These MBONs transmit information from the MB to other brain areas and form recurrent connections to dopaminergic neurons whose axonal terminals innervate the MB. Investigating the functions of the MBONs is crucial to understanding how the MB processes information and regulates behavior. The authors previously established a collection of split-GAL4 lines for most of the typical MBONs and one atypical MBON. That split-GAL4 collection has been an invaluable tool for researchers studying the MB. This work extends their previous effort by generating additional driver lines labeling the MBON types not covered by the previous split-GAL4 collection. Using these new driver lines, the authors also activated the labeled MBONs using optogenetics and assessed their role in learning, locomotion, and valence coding. The expression patterns of the new split-GAL4 lines and the behavioral analysis presented in this manuscript are convincing. I believe that these new lines will be a valuable resource for the fly community.

Reviewer #2 (Public Review):

It has been widely proposed that the neural circuit uses a copy of motor command, an efference copy, to cancel out self-generated sensory stimuli so that intended movement is not disturbed by the reafferent sensory inputs. However, how quantitatively such an efference copy suppresses sensory inputs is unknown. Here, Canelo et al. tried to demonstrate that an efference copy operates in an all-or-none manner and that its amplitude is independent of the amplitude of the sensory signal to be suppressed. Understanding the nature of such an efference copy is important because animals generally move during sensory processing, and the movement would devastatingly distort that without a proper correction. The manuscript is concise and written very clearly. However, experiments do not directly demonstrate if the animal indeed uses an efference copy in the presented visual paradigms and if such a signal is indeed non-scaled. As it is, it is not clear if the suppression of behavioral response to the visual background is due to the act of an efference copy (a copy of motor command) or due to an alternative, more global inhibitory mechanism, such as feedforward inhibition at the sensory level or attentional modulation. To directly uncover the nature of an efference copy, physiological experiments are necessary. If that is technically challenging, it requires finding a behavioral signature that unambiguously reports a (copy of) motor command and quantifying the nature of that behavior.

Reviewer #2 (Public Review):

This paper is an attempt to extend or augment muscle synergy and motor primitive analyses and ideas with addition of task-driven measures. The authors' idea is to use information metrics (mutual information, co-information) in 'synergy' constraint creation that includes task information directly. By using task related information and muscle information sources and then sparsification, the methods construct task relevant network communities among muscles, together with task redundant communities, and task irrelevant communities. This process of creating network communities may then constrain and help to guide subsequent synergy identification using the authors published sNM3F algorithm to detect spatial and temporal synergies. The revised paper is now much clearer and examples are helpful in various ways.

The impact of the information theoretic constraints developed as network communities on subsequent synergy separation are posited to be benign and to improve separation and identification of synergies over other methods (e.g., NNMF). However, not fully addressed are the possible impacts of the methods on the resulting compositionality and its links with physiological bases: the possibility remains that the methods here sometimes will instead lead to modules that represent more descriptive ML frameworks for task description, and resulting 'synergies' that may not support physiological work easily. Accordingly, there is a caveat for users of this framework. This is recognized and acknowledged by the authors in their rebuttal letters responding to prior reviews. It will remain for other work to explore this issue, likely through testing on detailed high degree of freedom artificial neuromechanical models and tasks. This possible issue and caveat with the strategy proposed by the authors likely should be more fully acknowledged in the paper.

The approach of the methods seeks to identify task relevant coordinative couplings. This identification is a meta problem for more classical synergy analyses. Classical/prior analyses seek compositional elements stable across tasks. These elements may then be explored in causal experiments and in generative simulations of coupling and control strategies. However, task-based understanding of synergy roles and functional uses as captured in the proposed methods are significant, and the field is clearly likely to be aided by methods in this study.<br /> Information based separation has been used in muscle synergy analyses previously, by using infomax ICA, to discover physiological primitives. Though linear mixing of sources is assumed in ICA, minimized mutual information among source (synergy) drives is the basis of the separation and can detect low variance synergy contributions (e.g., see Yang, Logan, Giszter, 2019). In the work in the current paper, instead, mutual information approaches are used to cluster muscles and task features into network communities preceding the SNM3F algorithm use for separation, rather than using minimized information in the separation process directly. This contrast of an accretive or agglomerative mutual information strategy in the paper here, which is used to cluster into networks, versus a minimizing mutual information source separation used in infomax ICA epitomizes a key difference in approach. Indeed, physiological causal testing of synergy ideas is neglected in the literature reviews presented in the paper. Although these are only in animal work (e.g., Hart and Giszter, 2010; Takei and Seki, 2017), the clear connection of muscle synergy analysis choices to physiology is important, and eventually these issues need to be better managed and understood in relation to the new methods proposed here, even if not in this paper. Analyses of synergies using the methods the paper has proposed will likely be very much dependent on the number and quality of task variables included and how these are chosen, and the impacts of these on the ensuing sparsification and network communities used prior to SNM3F has already been noted. The authors acknowledge this in their responses. It would be useful in the future to explore the approach described with a range of simulated data to better understand the caveats, and optimizations for best practices in applications of this approach.

A key component of the authors' arguments here is their 'emergentist' view presented in the work, but perhaps not made fully explicit. Through the reductionist lens, which was used in the other physiological work noted above, muscle groupings are the units (primitives or 'building blocks' with informational separations) of coordinated movement and thus the space of these intermuscular unit interactions and controls is of particular interest for understanding movement construction and underlying physiology. This may allow representation of a hierarchy or heterarchy of neural control elements with clear physiological bases at spinal, brainstem and cortical levels. On the other hand, the emergentist view utilized by the authors here suggests that muscle groupings emerge from interactions between many constituent parts in a more freeform fashion with potentially larger task synergy assemblies (also quantified here using information tools). Information methods are applied differently using the two different lenses. The emergentist lens may potentially obscure fundamental neural controls and make them harder to explore in the descriptions resulting. Nonetheless, the different approaches to muscle synergy research, seeking different sorts of explanation and description of 'synergy', can be complementary and beneficial for the field overall going forward, so long as the caveats and concerns noted here are employed by readers in the interpretation of this new method.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Brotherton et al. describes a structural study of connexin-26 (Cx26) gap junction channel mutant K125E, which is designed to mimic the CO2-inhibited form of the channel. In the wild-type Cx26, exposure to CO2 is presumed to close the channel through carbamylation of the residue K125. The authors mutated K125 to a negatively charged residue to mimic this effect, and they observed by cryo-EM analysis of the mutated channel that the pore of the channel is constricted. The authors were able to observe conformations of the channel with resolved density for the cytoplasmic loop (in which K125 is located). Based on the observed conformations and on the position of the N-terminal helix, which is involved in channel gating and in controlling the size of the pore, the authors propose the mechanisms of Cx26 regulation.

Strengths:<br /> This is a very interesting and timely study, and the observations provide a lot of new information on connexin channel regulation. The authors use the state of the art cryo-EM analysis and 3D classification approaches to tease out the conformations of the channel that can be interpreted as "inhibited", with important implications for our understanding of how the conformations of the connexin channels controlled.

Weaknesses:<br /> My fundamental question to the premise of this study is: to what extent can K125 carbamylation by recapitulated by a simple K125E mutation? Lysine has a large side chain, and its carbamylation would make it even slightly larger. While the authors make a compelling case for E125-induced conformational changes focusing primarily on the negative charge, I wonder whether they considered the extent to which their observation with this mutant may translate to the carbamoylated lysine in the wild-type Cx26, considering not only the charge but also the size of the modified side-chain.

Reviewer #2 (Public Review):

In this manuscript, the authors set out to identify transporters that must exist in Stramenophiles due to the fact that the second half of glycolysis appears to be conducted in the mitochondria. They hypothesize that a Stramenophile-specific clade of transporters related to the dicarboxylate carriers is likely the relevant family and then go on to test two proteins from Blastocystis due to the infectious disease relevance of this organism. They show rather convincingly that these two proteins are expressed and are localized to the mitochondria in the native organism. The purified proteins bind to glycolytic intermediates and one of them, GIC-2, transports several glycolytic intermediates in vitro. This is a very solid and well-executed study that clearly demonstrates that bCIC-2 can transport glycolytic intermediates.

1. The major weakness is that the authors aren't able to show that this protein actually has this function in the native organism. This could be impossible due to the lack of genetic tools in Blastocystis, but it leaves us without absolute confidence that bGIC-2 is the important glycolytic intermediate mitochondrial transporter (or even that it has this function in vivo).

2. It's atypical that the figures and figure panels don't really follow the order of their citation in the text. It's not a big deal, but mildly annoying to have to skip around in the figures (e.g. Figure 3D-E are described in the same paragraph as Figure 5). In addition, to facilitate the flow and a proper understanding I would encourage a reordering between figures 5D and 6 since Figure 6 is needed to understand the results shown in panel 5D, which may lead to confusion.

3. My impression is that the authors under-emphasize the fact that the hDIC also binds (and is stabilized by) glycolytic intermediates (G3P and 3PG). In the opinion of this reviewer, this might change the interpretation about the uniqueness of the bGIC proteins. They act on additional glycolytic intermediates, but it's not unique.

Reviewer #2 (Public Review):

This manuscript reports the discovery and analysis of a large protein complex that controls mating type and sexual reproduction of the model ciliate Tetrahymena thermophila. In contrast to many organisms that have two mating types or two sexes, Tetrahymena is multi-sexual with 7 distinct mating types. Previous studies identified the mating type locus, which encodes two transmembrane proteins called MTA and MTB that determine the specificity of mating type interactions. In this study, mutants are generated in the MTA and MTB genes and mutant isolates are studied for mating properties. Cells missing either MTA or MTB failed to co-stimulate wild-type cells of different mating types. Moreover, a mixture of mutants lacking MTA or MTB also failed to stimulate. These observations support the conclusion that MTA and MTB may form a complex that directs mating-type identity. To address this, the proteins were epitope-tagged and subjected to IP-MS analysis. This revealed that MTA and MTB are in a physical complex, and also revealed a series of 6 other proteins (MRC1-6) that together with MTA/B form the mating type recognition complex (MTRC). All 8 proteins feature predicted transmembrane domains, three feature GFR domains, and two are predicted to function as calcium transporters. The authors went on to demonstrate that components of the MTRC are localized on the cell surface but not in the cilia. They also presented findings that support the conclusion that the mating type-specific region of the MTA and MTB genes can influence both self- and non-self-recognition in mating.

Taken together, the findings presented are interesting and extend our understanding of how organisms with more than two mating types/sexes may be specified. The identification of the six-protein MRC complex is quite intriguing. It would seem important that the function of at least one of these subunits be analyzed by gene deletion and phenotyping, similar to the findings presented here for the MTA and MTB mutants. A straightforward prediction might be that a deletion of any subunit of the MRC complex would result in a sterile phenotype. The manuscript was very well written and a pleasure to read.

Reviewer #2 (Public Review):

The current manuscript undoubtedly demonstrates that JAG1 can induce osteogenesis via non-canonical signaling. Using the mouse-calvarial critical defect model, the authors have clearly shown the anabolic regenerative effect of JAG1 via non-canonical pathways. Exploring the molecular mechanisms, the authors have shown that non-canonically JAG1 regulates multiple pathways including STAT5, AKT, P38, JNK, NF-ĸB, and p70 S6K, which together possibly culminate in the activation of p70 S6K. More analysis is required to strongly conclude the role of the JAG1-p70 S6K pathway in the process. In summary, these findings have significant implications for designing new approaches for bone regenerative research.

Reviewer #2 (Public Review):

This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress, and causes the accumulation of mitochondrial precursor proteins in the cytosol.<br /> The data shown are of high quality and well controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. The last part of the study is less convincing. The authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hex-resistant TOM mutant was not identified. Moreover, it is not clear whether the concentrations of t-2-hex in this study are physiological. This is, however, a critical aspect. The literature is full of studies claiming the toxic effects of compounds such as H2O2; even if such studies are technically sound, they are misleading if non-physiological concentrations of such compounds were used.<br /> Nevertheless, this is an interesting study of high quality. A few specific aspects should be addressed.

Reviewer #2 (Public Review):

In the manuscript by Weber and colleagues, the authors investigated the role of a DEAD-box helicase DDX6 in regulating mRNA stability upon ribosome slowdown in human cells. The authors knocked out DDX6 KO in HEK293T cells and showed that the half-life of a reporter containing a rare codon repeat is elongated in the absence of DDX6. By analogy to the proposed function of fission yeast Dhh1p (DDX6 homolog) as a sensor for slow ribosomes, the authors demonstrated that recombinant DDX6 interacted with human ribosomes. The interaction with the ribosome was mediated by the FDF motif of DDX6 located in its RecA2 domain, and rescue experiments showed that DDX6 requires the FDF motif as well as its interaction with the CCR4-NOT deadenylase complex and ATPase activity for degrading a reporter mRNA with rare codons. To identify endogenous mRNAs regulated by DDX6, they performed RNA-Seq and ribosome footprint profiling. The authors focused on mRNAs whose stability is increased in DDX6 KO cells with high local ribosome density and validated that such mRNA sequences induced mRNA degradation in a DDX6-dependent manner.

The experiments were well-performed, and the results clearly demonstrated the requirement of DDX6 in mRNA degradation induced by slowed ribosomes. However, in some cases, the authors interpreted their data in a biased way, possibly influenced by the yeast study, and drew too strong conclusions. In addition, the authors should have cited important studies about codon optimality in mammalian cells. This lack of information hinders placing their important discoveries in a correct context.

1) Although the authors concluded that DDX6 acts as a sensor of the slowed ribosome, it is not clear if DDX6 indeed senses the ribosome speed. What the authors showed is a requirement of DDX6 for mRNA decay induced by rare codons, and DDX6 binds to the ribosome to exert this role. For example, DDX6 may bridge the sensor and decay machinery on the ribosome. Without structural or biochemical data on the recognition of the slowed ribosome by DDX6, the role of DDX6 as a sensor remains one of the possible models. It should be described in the discussion section.

2) It is not clear if DDX6 directly binds the ribosome. The authors used ribosomes purified by sucrose cushion, but ribosome-associating and FDF motif-interacting factors might remain on ribosomes, even after RNaseI treatment. Without structural or biochemical data of the direct interaction between the ribosome and DDX6, the authors should avoid description as if DDX6 directly binds to the ribosome.

3) Although the authors performed rigorous reporter assays recapitulating the effect of ribosome-retardation sequences on mRNA stability, this is not the first report showing that codon optimality determines mRNA stability in human cells. The authors did not cite important previous studies, such as Wu et al., 2019 (PMID: 31012849), Hia et al., 2019 (PMID: 31482640), Narula et al., 2019 (PMID: 31527111), and Forrest et al., 2020 (PMID: 32053646). These milestone papers should be cited in the Introduction, Results, and Discussion.

4) While both DDX6 and deadenylation by the CCR4-NOT were required for mRNA decay by the slowed ribosome, whether DDX6 is required for deadenylation was not investigated. Given that the CCR4-NOT deadenylate complex directly interacts with the empty ribosome E-site in yeast and humans (Buschauer et al., 2020 PMID: 32299921 and Absmeier et al., 2023 PMID: 37653243), whether the loss of DDX6 also affected the action of the CCR4-NOT complex is an important point to investigate, or at least should be discussed in this paper.

Reviewer #3 (Public Review):

Goetz, Akl and Dixit investigated the heterogeneity in the fidelity of sensing the environment by individual cells in a population using computational modeling and analysis of experimental data for two important and well-studied mammalian signaling pathways: (insulin-like growth factor) IGF/FoxO and (epidermal growth factor) EFG/EFGR mammalian pathways. They quantified this heterogeneity using the conditional mutual information between the input (eg. level of IGF) and output (eg. level of FoxO in the nucleus), conditioned on the "state" variables which characterize the signaling pathway (such as abundances of key proteins, reaction rates, etc.) First, using a toy stochastic model of a receptor-ligand system - which constitutes the first step of both signaling pathways - they constructed the population average of the mutual information conditioned on the number of receptors and maximized over the input distribution and showed that it is always greater than or equal to the usual or "cell state agnostic" channel capacity. They constructed the probability distribution of cell state dependent mutual information for the two pathways, demonstrating agreement with experimental data in the case of the IGF/FoxO pathway using previously published data. Finally, for the IGF/FoxO pathway, they found the joint distribution of the cell state dependent mutual information and two experimentally accessible state variables: the response range of FoxO and total nuclear FoxO level prior to IGF stimulation. In both cases, the data approximately follow the contour lines of the joint distribution. Interestingly, high nuclear FoxO levels, and therefore lower associated noise in the number of output readout molecules, is not correlated with higher cell state dependent mutual information, as one might expect. This paper contributes to the vibrant body of work on information theoretic characterization of biochemical signaling pathways, using the distribution of cell state dependent mutual information as a metric to highlight the importance of heterogeneity in cell populations. The authors suggest that this metric can be used to infer "bottlenecks" in information transfer in signaling networks, where certain cell state variables have a lower joint distribution with the cell state dependent mutual information.

The utility of a metric based on the conditional mutual information to quantify fidelity of sensing and its heterogeneity (distribution) in a cell population is supported in the comparison with data. Some aspects of the analysis and claims in the main body of the paper and SI need to be clarified and extended.

Remaining Comments:

- I think Review Figure 2 which is currently in the SI would improve the main body of the paper if moved there. In that case, the discussion of this figure in the main text would have to address more than it currently does, namely "the same cell's FoxO responses to the same input were found to have significantly less variation compared to the variation within the population".

Reviewer #2 (Public Review):

Summary: In this study, the authors performed a screening for PDXP inhibitors to identify compounds that could increase levels of pyridoxal 5'- phosphate (PLP), the co-enzymatically active form of vitamin B6. For the screening of inhibitors, they first evaluated a library of about 42,000 compounds for activators and inhibitors of PDXP and secondly, they validated the inhibitor compounds with a counter-screening against PGP, a close PDXP relative. The final narrowing down to 7,8-DHF was done using PLP as a substrate and confirmed the efficacy of this flavonoid as an inhibitor of PDXP function. Physiologically, the authors show that, by acutely treating isolated wild-type hippocampal neurons with 7,8-DHF they could detect an increase in the ratio of PLP/PL compared to control cultures. This effect was not seen in PDXP KO neurons.

Strengths: The screening and validation of the PDXP inhibitors have been done very well because the authors have performed crystallographic analysis, a counter screening, and mutation analysis. This is very important because such rigor has not been applied to the original report of 7,8 DHF as an agonist for TrkB. Which is why there is so much controversy on this finding.

Weaknesses: As mentioned in the summary report the study may benefit from some in vivo analysis of PLP levels following 7,8-DHF treatment, although I acknowledge that it may be challenging because of the working out of the dosage and timing of the procedure.

Reviewer #2 (Public Review):

Summary:

In this study, Kashio et al examined the role of TRPV4 in regulating perspiration in mice. They find coexpression of TRPV4 with the chloride channel ANO1 and aquaporin 5, which implies possible coupling of heat sensing through TRPV4 to ion and water excretion through the latter channels. Calcium imaging of eccrine gland cells revealed that the TRPV4 agonist GSK101 activates these cells in WT mice, but not in TRPV4 KO. This effect is reduced with cold-stimulating menthol treatment. Temperature-dependent perspiration in mouse skin, either with passive heating or with ACh stimulation, was reduced in TRPV4 KO mice. Functional studies in mice - correlating the ability to climb a slippery slope to properly regulate skin moisture levels - reveal potential dysregulation of foot pad perspiration in TRPV4 KO mice, which had fewer successful climbing attempts. Lastly, a correlation of TRPV4 to hypohydrosis in humans was shown, as anhidrotic skin showed reduced levels of TRPV4 expression compared to normohidrotic or control skin.

Strengths:

The functional studies of mice climbing slippery slopes is a novel method to determine mechanisms of functional perspiration in mice. Since mice do not perspire for thermoregulation, other functional readouts are needed to study perspiration in mice.

Weaknesses:

1. The coexpression data needs additional controls. In the TRPV4 KO mice, there appears to be staining with the TRPV4 Ab in TRPV4 KO mice below the epidermis. This pattern appears similar to that of the location of the secretory coils of the sweat glands (Fig 1A). Is the co-staining the authors note later in Figure 1 also seen in TRPV4 KOs? This control should be shown, since the KO staining is not convincing that the Ab doesn't have off-target binding.

2. Are there any other markers besides CGRP for dark cells in mice to support the conclusion that mouse secretory cells have clear cell and dark cell properties?

3. The authors utilize menthol (as a cooling stimulus) in several experiments. In the discussion, they interpret the effect of menthol as potentially disrupting TRPV4-ANO1 interactions independent of TRPM8. Yet, the role of TRPM8, such as in TRPM8 KO mice, is not evaluated in this study.

4. Along those lines, the authors suggest that menthol inhibits eccrine function, which might lead to a cooling sensation. But isn't the cooling sensation of sweating from evaporative cooling? In which case, inhibiting eccrine function may actually impair cooling sensations.

5. The climbing assay is interesting and compelling. The authors note performing this under certain temperature and humidity conditions. Presumably, there is an optimal level of skin moisture, where skin that is too dry has less traction, but skin that is too wet may also have less traction. It would bolster this section of the study to perform this assay under hot conditions (perhaps TRPV4 KO mice, with impaired perspiration, would outperform WT mice with too much sweating?), or with pharmacologic intervention using TRPV4 agonists or antagonists to more rigorously evaluate whether this model correlates to TRPV4 function in the setting of different levels of perspiration.

6. There are other studies (PMID 33085914, PMID 31216445) that have examined the role of TRPV4 in regulating perspiration. The presence of TRPV4 in eccrine glands is not a novel finding. Moreover, these studies noted that TRPV4 was not critical in regulating sweating in human subjects. These prior studies are in contradiction to the mouse data and the correlation to human anhidrotic skin in the present study. Neither of these studies is cited or discussed by the authors, but they should be.

Reviewer #2 (Public Review):

Summary:

In the paper, the authors use a cellular Potts model to investigate muscle regeneration. The model is an attempt to combine many contributors to muscle regeneration into one coherent framework. I believe the resulting model has the potential to be very useful in investigating the complex interplay of multiple actors contributing to muscle regeneration.

Strengths:

The manuscript identified relevant model parameters from a long list of biological studies. This collation of a large amount of literature into one framework has the potential to be very useful to other authors. The mathematical methods used for parameterization and validation are transparent.

Weaknesses:

I have a few concerns which I believe need to be addressed fully.

My main concerns are the following:

1) The model is compared to experimental data in multiple results figures. However, the actual experiments used in these figures are not described. To me as a reviewer, that makes it impossible to judge whether appropriate data was chosen, or whether the model is a suitable descriptor of the chosen experiments. Enough detail needs to be provided so that these judgements can be made.

2) Do I understand it correctly that all simulations are done using the same initial simulation geometry? Would it be possible to test the sensitivity of the paper results to this geometry? Perhaps another histological image could be chosen as the initial condition, or alternative initial conditions could be generated in silico? If changing initial conditions is an unreasonably large request, could the authors discuss this issue in the manuscript?

3) Cytokine knockdowns are simulated by 'adjusting the diffusion and decay parameters' (line 372). Is that the correct simulation of a knockdown? How are these knockdowns achieved experimentally? Wouldn't the correct implementation of a knockdown be that the production or secretion of the cytokine is reduced? I am not sure whether it's possible to design an experimental perturbation which affects both parameters.

4) The premise of the model is to identify optimal treatment strategies for muscle injury (as per the first sentence of the abstract). I am a bit surprised that the implemented experimental perturbations don't seem to address this aim. In Figure 7 of the manuscript, cytokine alterations are explored which affect muscle recovery after injury. This is great, but I don't believe the chosen alterations can be done in experimental or clinical settings. Are there drugs that affect cytokine diffusion? If not, wouldn't it be better to select perturbations that are clinically or experimentally feasible for this analysis? A strength of the model is its versatility, so it seems counterintuitive to me to not use that versatility in a way that has practical relevance. - I may well misunderstand this though, maybe the investigated parameters are indeed possible drug targets.

5) A similar comment applies to Figure 5 and 6: Should I think of these results as experimentally testable predictions? Are any of the results surprising or new, for example in the sense that one would not have expected other cytokines to be affected as described in Figure 6?

6) In figure 4, there were differences between the experiments and the model in two of the rows. Are these differences discussed anywhere in the manuscript?

7) The variation between experimental results is much higher than the variation of results in the model. For example, in Figure 3 the error bars around experimental results are an order of magnitude larger than the simulated confidence interval. Do the authors have any insights into why the model is less variable than the experimental data? Does this have to do with the chosen initial condition, i.e. do you think that the experimental variability is due to variation in the geometries of the measured samples?

8) Is figure 2B described anywhere in the text? I could not find its description.

Reviewer #2 (Public Review):

Summary:

Fertilization is a crucial event in sexual reproduction, but the molecular mechanisms underlying egg-sperm fusion remain elusive. Elofsson et al. used AlphaFold to explore possible synapse-like assemblies between sperm and egg membrane proteins during fertilization. Using a systematic search of protein-protein interactions, the authors proposed a pentameric complex of three sperm (IZUMO1, SPACA6, and TMEM81) and two egg (JUNO and CD9) proteins, providing a new structural model to be used in future structure-function studies.

Strengths:

1. The study uses the AlphaFold algorithm to predict higher-order assemblies. This approach could offer insights into a highly transient protein complex, which is challenging to detect experimentally.<br /> 2. The article predicts a pentameric complex between proteins involved in fertilization, shedding light on the architectural aspects of the egg-sperm fusion synapse.

Weaknesses:

1. The procedures and discriminator scores used to evaluate specific from non-specific complexes were developed previously by the same authors. Therefore, in this manuscript, they are not contributing a new method.<br /> 2. The proposed model, which is a prediction from a modeling algorithm, lacks experimental validation of the identity of the components and the predicted contacts.

It is noteworthy that in an independent study, Deneke et al. provide experimental evidence of the interaction between IZUMO1/SPACA6/TMEM81 in zebrafish. This is an important element that supports the findings presented in this manuscript.

Reviewer #2 (Public Review):

The setting of planar cell polarity in epithelial tissues involves a complex interplay of chemical interactions. While local interactions can spontaneously give rise to cell polarity, planar cell polarity also involves tissue scale gradients whose effects are not clear. To understand their role, the authors built a minimal mechanistic model in considering two atypical cadherins, Fat (Ft) and Dachsous (Ds) which can associate at cell-cell interfaces to form hetero-dimers in which monomers belong to adjacent cells. This association can be seen as a local interaction between cells and is also sensitive to overall concentration gradients. From their model which appears to capture diverse experimental observations, the authors conclude that tissue-scale gradients provide to planar cell polarity a directional cue and some robustness to cellular stochasticity. While this model comes after similar works reaching similar predictions, the quality of this model is in its simplicity, its convenience for experimental testing, and the diversity of experimental observations it recapitulates.

A strength of this work is to recapitulate many experimental observations made on planar cell polarity. It, for example, seems to capture the response of tissues to perturbations such as local downregulation of some important proteins, and the polarity patterns observed in the presence of noise in synthesis or cell-to-cell heterogeneity. It also gives a mechanistic description of planar cell polarity, making its experimental interpretation simple. Finally, the simplicity of the model facilitates its exploration and makes it easily testable because of the reduced amount of free model parameters.

A weakness of this work is that it comes after several models with similar hypotheses and similar predictions. Another weakness is that some conclusions of this work rely on visual appreciation rather than quantification. This is particularly true for what concerns 2D patterns. An argument of the authors is for example that their model reproduces a variety of known spatial patterns, but the comparison with experiments is only visual and would be more convincing in being more quantitative.

Reviewer #2 (Public Review):

In this study, Leiba et al. aim at establishing the developing zebrafish embryo as a suitable infection model to study Salmonella persistence in vivo. Under environmental stress (ex: macrophage phagosomes) a proportion of bacteria switch to a slow/arrested growth state confering increased resistance to antibiotic treatments. Persisters are getting increasingly linked to infection relapses. Understanding how persistent infections emerge and bacteria survive in an organism for long time without replicating before switching back to a replicative state is essential. Zebrafish represents an alternative model to mice offering the possibility to image the whole organism and capture persistency with an amazing spatio-temporal resolution.

In this paper, the authors demonstrate that persistent infections of Salmonella can be reproduced in the developing zebrafish. The kinetics of infection have been well characterized and shows a very nice heterogeneity between animals demonstrating the complex host-pathogen interactions (Fig 1). From the perspective of persistence, the presence of Salmonella survivors to host clearing is reported until 14dpi demonstrating the possibility to induce persistent infection in this model. Through the manuscript, the authors have used a variety of state-of-the-art technics illustrating the flexibility of this model including microscopy and imaging of specific immune populations, various transgenic animals and selective depletion of macrophages or neutrophils to assess their relative contributions. Overall, the conclusions of the authors are well supported by the presented data. This said, the authors should strengthen the conclusions of the paper by providing a better characterization of the infection.

Major comments:<br /> 1- Figure 1: What is the general life-spam of the fish?

2- Figure 2: It would be nice to clearly state what infection scenario we are looking at. Have the authors studied "high proliferation", "infected" or "cleared" zebrafish?

3- Figure 3 and 4: It would be very informative if the authors can tell us what proportion of Salmonella is associated with macrophages and neutrophils. From panel C and D (Figure 3) and Figure 4 C and D and Suppl Fig 1, it seems that a lot of bacteria are extracellular. Maybe an EM image of the tissue would help to understand if the bacteria is "all" intracellular or intracellular.

4- Figure 3 and 4: It would be very useful if the authors can tell us if the intracellular bacteria are mainly found individually (like in Figure 3C) or does host cells harbor many intracellular bacteria. Looking at figure 4G: it is not clear to me how many intracellular bacteria can be counted on this image.

5- Figure 3 and 4: The authors should also perform an experiment with a Salmonella strain harboring a growth reporter to quantify the amount of replicating and non-replicating bacteria. This experiment is not absolutely necessary for the story, but if possible, it would provide a very nice add-up to the story and impact to the paper.

6- Figure 6: The authors should provide in suppl. the flow cytometry scatter plots used to delineate the different subpopulations.

7- Figure 6: A specific characterization of macrophages harboring Salmonella persisters at 4dpi is missing. As shown by the authors in Figure 6, the tnfa- populations of macrophages at 4dpi are very similar for both infected and non-infected larvae. Persisters should indeed reside within tnfa- macrophages but they should also induce a specific signature through the actions of Salmonella effectors. Measuring this signature will allow a direct comparison with published data in mice and assess how accurately the zebrafish model recapitulates the manipulation of macrophages by Salmonella

Reviewer #2 (Public Review):

This paper uses two-photon imaging of mouse ganglion cells responding to chromatic natural scenes along with convolutional neural network (CNN) models fit to the responses of a large set of ganglion cells. The authors analyze CNN models to find the most effective input (MEI) for each ganglion cell as a novel approach to identifying ethological function. From these MEIs they identify chromatic opponent ganglion cells, and then further perform experiments with natural stimuli to interpret the ethological function of those cells. They conclude that a type of chromatic opponent ganglion cell is useful for the detection of the transition from the ground to the sky across the horizon. The experimental techniques, data, and fitting of CNN models are all high quality. However, there are conceptual difficulties with both the use of MEIs to draw conclusions about neural function and the ethological interpretations of experiments and data analyses, as well as a lack of comparison with standard approaches. These bear directly both on the primary conclusions of the paper and on the utility of the new approaches.

1. Claim of feature detection. The color opponent cells are cast as a "feature detector" and the term 'detector' is in the title. However insufficient evidence is given for this, and it seems likely a mischaracterization. An example of a ganglion cell that might qualify as a feature detector is the W3 ganglion cell (Zhang et al., 2012). These cells are mostly silent and only fire if there is differential motion on a mostly featureless background. Although this previous work does not conduct a ROC analysis, the combination of strong nonlinearity and strong selectivity are important here, giving good qualitative support for these cells as participating in the function of detecting differential motion against the sky. In the present case, the color opponent cells respond to many stimuli, not just transitions across the horizon. In addition, for the receiver operator characteristic (ROC) analysis as to whether these cells can discriminate transitions across the horizon, the area under the curve (AUC) is on average 0.68. Although there is not a particular AUC threshold for a detector or diagnostic test to have good discrimination, a value of 0.5 is chance, and values between 0.5 and 0.7 are considered poor discrimination, 'not much better than a coin toss' (Applied Logistic Regression, Hosmer et al., 2013, p. 177). The data in Fig. 6F is also more consistent with a general chromatic opponent cell that is not highly selective. These cells may contribute information to the problem of discriminating sky from ground, but also to many other ethologically relevant visual determinations. Characterizing them as feature detectors seems inappropriate and may distract from other functional roles, although they may participate in feature detection performed at a higher level in the brain.

2. Appropriateness of MEI analysis for interpretations of the neural code. There is a fundamental incompatibility between the need to characterize a system with a complex nonlinear CNN and then characterizing cells with a single MEI. MEIs represent the peak in a complex landscape of a nonlinear function, and that peak may or may not occur under natural conditions. For example, MEIs do not account for On-Off cells, On-Off direction selectivity, nonlinear subunits, object motion sensitivity, and many other nonlinear cell properties where multiple visual features are combined. MEIs may be a useful tool for clustering and distinguishing cells, but there is not a compelling reason to think that they are representative of cell function. This is an open question, and thus it should not be assumed as a foundation for the study. This paper potentially speaks to this issue, but there is more work to support the usefulness of the approach. Neural networks enable a large set of analyses to understand complex nonlinear effects in a neural code, and it is well understood that the single-feature approach is inadequate for a full understanding of sensory coding. A great concern is that the message that the MEI is the most important representative statistic directs the field away from the primary promise of the analysis of neural networks and takes us back to the days when only a single sensory feature is appreciated, now the MEI instead of the linear receptive field. It is appropriate to use MEI analyses to create hypotheses for further experimental testing, and the paper does this (and states as much) but it further takes the point of view that the MEI is generally informative as the single best summary of the neural code. The representation similarity analysis (Fig. 5) acts on the unfounded assumption that MEIs are generally representative and conveys this point of view, but it is not clear whether anything useful can be drawn from this analysis, and therefore this analysis does not support the conclusions about changes in the representational space. Overall this figure detracts from the paper and can safely be removed. In addition, in going from MEI analysis to testing ethological function, it should be made much more clear that MEIs may not generally be representative of the neural code, especially when nonlinearities are present that require the use of more complex models such as CNNs, and thus testing with other stimuli are required.

3. Usefulness of MEI approach over alternatives. It is claimed that analyzing the MEI is a useful approach to discovering novel neural coding properties, but to show the usefulness of a new tool, it is important to compare results to the traditional technique. The more standard approach would be to analyze the linear receptive field, which would usually come from the STA of white noise measurement, but here this could come from the linear (or linear-nonlinear) model fit to the natural scene response, or by computing an average linear filter from the natural scene model. It is important to assess whether the same conclusion about color opponency can come from this standard approach using the linear feature (average effective input), and whether the MEIs are qualitatively different from the linear feature. The linear feature should thus be compared to MEIs for Fig. 3 and 4, and the linear feature should be compared with the effects of natural stimuli in terms of chromatic contrast (Fig. 6b). With respect to the representation analysis (Fig. 5), although I don't believe this is meaningful for MEIs, if this analysis remains it should also be compared to a representation analysis using the linear feature. In fact, a representation analysis would be more meaningful when performed using the average linear feature as it summarizes a wider range of stimuli, although the most meaningful analysis would be directly on a broader range of responses, which is what is usually done.

4. Definition of ethological problem. The ethological problem posed here is the detection of the horizon. The stimuli used do not appear to relate to this problem as they do not include the horizon and only include transitions across the horizon. It is not clear whether these stimuli would ever occur with reasonable frequency, as they would only occur with large vertical saccades, which are less common in mice. More common would be smooth transitions across the horizon, or smaller movements with the horizon present in the image. In this case, cells which have a spatial chromatic opponency (which the authors claim are distinct from the ones studied here) would likely be more important for use in chromatic edge detection or discrimination. Therefore the ethological relevance of any of these analyses remains in question.

It is further not clear if detection is even the correct problem to consider. The horizon is always present, but the problem is to determine its location, a conclusion that will likely come from a population of cells. This is a distinct problem from detecting a small object, such as a small object against the background of the sky, which may be a more relevant problem to consider.

5. Difference in cell type from those previously described. It is claimed that the chromatic opponent cells are different from those previously described based on the MEI analysis, but we cannot conclude this because previous work did not perform an MEI analysis. An analysis should be used that is comparable to previous work, the linear spatiotemporal receptive field should be sufficient. However, there is a concern that because linear features can change with stimulus statistics (Hosoya et al., 2005), a linear feature fit to natural scenes may be different than those from previous studies even for the same cell type. The best approach would likely be presenting a white noise stimulus to the natural scenes model to compute a linear feature, which still carries the assumption that this linear feature from the model fit to a natural stimulus would be comparable to previous studies. If the previous cells have spatial chromatic opponency and the current cells only have chromatic opponency in the center, there should be both types of cells in the current data set. One technical aspect relating to this is that MEIs were space-time separable. Because the center and surround have a different time course, enforcing this separability may suppress sensitivity in the surround. Therefore, it would likely be better if this separability were not enforced in determining whether the current cells are different than previously described cells. As to whether these cells are actually different than those previously described, the authors should consider the following uncited work; (Ekesten Gouras, 2005), which identified chromatic opponent cells in mice in approximate numbers to those here (~ 2%). In addition, (Yin et al., 2009) in guinea pigs and (Michael, 1968) in ground squirrels found color-opponent ganglion cells without effects of a spatial surround as described in the current study.

Reviewer #3 (Public Review):

This study succeeds to highlight and address important gaps in our understanding of plant-insect interactions mediating pest control in a widely known agro-ecological system for sustainable intensification, push-pull agriculture. In particular, the authors present a large amount of data on plant volatile emission, thought to be critical for the functioning of these systems, in reasonable and relevant contexts, as well as on other traits of the plants in the system relevant for pest control. These data come from plants grown both in controlled and field environments, which is unusual. The arguments on mechanism are further supported by insect behavioral assays, which seem to be thoughtfully designed, but also use some non-standard approaches that could be better explained. While most or all of the authors' results pre-date some relevant recent publications in this field, they do incorporate comparisons to current literature in order to better place their findings in the current state of the art.

Reviewer #2 (Public Review):

This study by Du et. al addressed the role and regulation of proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) in Aristolochic acid Nephropthathy (AAN) and immune defense. PSTPIP2 expression is downregulated in AAN. Conditional knock-in of PSTPIP2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Reducing the expression of PSTPIP2 to normal levels in knock-in mouse using shRNA promoted kidney injury. Using in vivo model, the role of PSTPIP2 in AAN injury and renal function, apoptosis, neutrophil infiltration and NET formation is established. Using in vitro models, a PSTPIP2/NFkB-mediated NET formation via IL-19-IL20-beta Receptor pathway is shown to induce inflammation and apoptosis in AAN. The studies are well presented.

Reviewer #2 (Public Review):

For about four decades it has been known that RNA molecules can increase the rate of chemical reactions, just like the much more prevalent protein enzymes. Some have suggested that RNA enzymes, also called "ribozymes" were very important at the beginning of life, but that the importance was mostly erased when ribosomal protein synthesis emerged through evolution. The ribosome and spliceosome are two important examples of modern biological functions known to be catalyzed by RNA. In addition to these large RNA machines, the genomes of humans, and all domains of life, also contain a class of small ribozymes that catalyze self-cleavage of the RNA backbone. However, unlike RNA cleaving proteins that are well studied, there exists little evidence that the self-cleaving of RNA by ribozymes has important downstream consequences. This new paper provides evidence that a ribozyme found in all mammals has an important role in memory formation. The authors found a way to block the ribozyme activity and then observe the effect on memory formation in mice, and in the expression of genes in neurons that are known to underly this memory formation process. The authors found that blocking the ribozyme activity in mouse brains actually improved their performance in a memory task. In addition, they found that blocking the ribozyme changed the expression of the gene in which the ribozyme is found (a gene called CPEB3), suggesting that the way the ribozyme effects memory is through controlling the expression of the gene where it is found. The paper confirms the biological importance of this ribozyme, and encourages further investigation into self-cleaving ribozymes in general. Interestingly, the ribozyme found in humans is in fact slower cleaving than most mammals, similar to the blocked ribozyme in these experiments, which brings up the intriguing possibility that the CPEB3 ribozyme is a part of what makes us human!

Reviewer #2 (Public Review):

The authors examine the use of metformin in the treatment of hepatic ischemia/reperfusion injury (HIRI) and suggest the mechanism of action is mediated in part by the gut microbiota and changes in hepatic ferroptosis. While the concept is intriguing, the experimental approaches are inadequate to support these conclusions.

The histological and imaging studies were considered a strength and reveal a significant impact of metformin post-HIRI.

Weaknesses largely stem from the experimental design. The impact of metformin on the microbiota is profound resulting in changes in bile acid, lipid, and glucose homeostasis. Throughout the manuscript no comparisons are made with metformin alone which would better capture the metformin-specific effects. With the pathology and metabolic disturbances resulting from HIRI, it is important to understand if metformin is providing beneficial effects from reported mechanisms such as changes in bile acid, glucose, and/or lipid metabolism, or are these changes the result of a new unappreciated mechanism. A comparison of the reported and the new pathways is not included.

Overall, while the concept is interesting and has potential to better understand the pleiotropic functions of metformin, the limitations with the experimental design and lack of key controls make it challenging to support the conclusions.

Reviewer #2 (Public Review):

Summary:

This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

Strengths:

The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

Weaknesses:

The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

Reviewer #2 (Public Review):

In this study, Kajtor et al investigated the use of a single-animal trial-based behavioral assay for the assessment of subtle changes in the locomotor behavior of different genetic models of Parkinson's disease of Drosophila. Different genotypes used in this study were Ddc-GAL4>UAS-Parkin-275W and UAS- α-Syn-A53T. The authors measured Drosophila's response to predator-mimicking passing shadow as a threatening stimulus. Along with these, various dopamine (DA) receptor mutants, Dop1R1, Dop1R2 and DopEcR were also tested.<br /> The behavior was measured in a custom-designed apparatus that allows simultaneous testing of 13 individual flies in a plexiglass arena. The inter-trial intervals were randomized for 40 trials within 40 minutes duration and fly responses were defined into freezing, slowing down, and running by hierarchical clustering. Most of the mutant flies showed decreased reactivity to threatening stimuli, but the speed-response behavior was genotype invariant.<br /> These data nicely show that measuring responses to the predator-mimicking passing shadows could be used to assess the subtle differences in the locomotion parameters in various genetic models of Drosophila.

The understanding of the manifestation of various neuronal disorders is a topic of active research. Many of the neuronal disorders start by presenting subtle changes in neuronal circuits and quantification and measurement of these subtle behavior responses could help one delineate the mechanisms involved. The data from the present study nicely uses the behavioral response to predator-mimicking passing shadows to measure subtle changes in behavior. However, there are a few important points that would help establish the robustness of this study.<br /> 1) The visual threat stimulus for measuring response behavior in Drosophila is previously established for both single and multiple flies in an arena. A comparative analysis of data and the pros and cons of the previously established techniques (for example, Gibson et al., 2015) with the technique presented in this study would be important to establish the current assay as an important advancement.<br /> 2) Parkinson's disease mutants should be validated with other GAL-4 drivers along with Ddc-GAL4, such as NP6510-Gal4 (Riemensperger et al., 2013). This would be important to delineate the behavioral differences due to dopaminergic neurons and serotonergic neurons and establish the Parkinson's disease phenotype robustly.<br /> 3) The DopEcR mutant genotype used for behavior analysis is w1118; PBac{PB}DopEcRc02142/TM6B, Tb1. Balancer chromosomes, such as TM6B,Tb can have undesirable and uncharacterised behavioral effects. This could be addressed by removing the balancer and testing the DopEcR mutant in homozygous (if viable) or heterozygous conditions.<br /> 4) The height of the arena is restricted to 1mm. However, for the wild-type flies (Canton-S) and many other mutants, the height is usually more than 1mm. Also, a 1 mm height could restrict the fly movement. For example, it might not allow the flies to flip upside down in the arena easily. This could introduce some unwanted behavioral changes. A simple experiment with an arena of height at least 2.5mm could be used to verify the effect of 1mm height.<br /> 5) The detailed model for Monte Carlo simulation for speed-response simulation is not described. The simulation model and its hyperparameters need to be described in more depth and with proper justification.<br /> 6) The statistical analysis in different experiments needs revisiting. It wasn't clear to me if the authors checked if the data is normally distributed. A simple remedy to this would be to check the normality of data using the Shapiro-Wilk test or Kolmogorov-Smirnov test. Based on the normality check, data should be further analyzed using either parametric or non-parametric statistical tests. Further, the statistical test for the age-dependent behavior response needs revisiting as well. Using two-way ANOVA is not justified given the complexity of the experimental design. Again, after checking for the normality of data, a more rigorous statistical test, such as split-plot ANOVA or a generalized linear model could be used.<br /> 7) The dopamine receptor mutants used in this study are well characterized for learning and memory deficits. In the Parkinson's disease model of Drosophila, there is a loss of DA neurons in specific pockets in the central brain. Hence, it would be apt to use whole animal DA receptor mutants as general DA mutants rather than the Parkinson's disease model. The authors may want to rework the title to reflect the same.

Reviewer #2 (Public Review):

Summary:

The generation of functional extranumerary inner hair cells (IHCs) in postnatal mice, particularly with virus-mediated knockdown of Cldn9 mRNA expression in the neonatal cochlear duct, is an important observation. It is significant because not many studies exist that report molecular manipulations of the neonatal organ of Corti that result in the generation of new hair cells that remain functional and appear to be intact for an extended time, here more than one year. Overall, this is a carefully conducted study; the observations are clear, and the methods are solid. Two independent methods for reducing the expression of Cldn9 mRNA were used: a conditional transgenic model and AAV-mediated knockdown with shRNA. The lack of a functional explanation of how the reduced expression of Cldn9 specifically leads to the formation of extranumerary IHCs leaves open questions. For example, it is not clear whether there is indeed a fate change happening and whether Cldn9 reduction affects developmental processes. The discussion of how Cldn9 reduction potentially affects Notch signaling, without hard evidence, is handwaving.

Strengths:

It is a very interesting observation and somewhat unexpected in its specificity for inner hair cells. Using two different approaches to manipulate Cldn9 expression provides a strong experimental foundation. The study is conducted quantitatively and with care.

Weaknesses:

The lack of mechanistic insight results in an open-ended story where at least the potential interaction of Cldn9 reduction with known and well-characterized signaling pathway components should have been investigated. This missed opportunity limits the scope of the study and should be addressed: How does Cldn9 downregulation affect the expression levels of other known genes linked to hair cell production and cell fate decisions? Quantitative RT-PCR is working well for the authors, and a comparison of the expression of Notch or other known pathway components could provide mechanistic insight.

It is unclear how P21 inner hair cells were identified for the patch clamp experiments shown in Fig 4E-H. This is a challenging endeavor without the possibility of using specific markers.

Please also address the numerous minor points outlined below; it will improve the paper's readability.

Please include page numbers and line numbers in a revised manuscript.

Reviewer #2 (Public Review):

Summary,<br /> The paper aimed to examine the effect of co-ablating Substance P and CGRPα peptides on pain using Tac1 and Calca double knockout (DKO) mice. The authors observed no significant changes in acute, inflammatory, and neuropathic pain. These results suggest that Substance P and CGRPα peptides do not play a major role in mediating pain in mice. Moreover, they reveal that the lack of behavioral phenotype cannot be explained by the redundancy between the two peptides, which are often co-expressed in the same neuron

Strengths,<br /> The paper uses a straightforward approach to address a significant question in the field. The authors confirm the absence of Substance P and CGRPα peptides at the levels of DRG, spinal cord, and midbrain. Subsequently, they employ a comprehensive battery of behavioral tests to examine pain phenotypes, including acute, inflammatory, and neuropathic pain. Additionally, they evaluate neurogenic inflammation by measuring edema and extravasation, revealing no changes in DKO mice. The data are compelling, and the study's conclusions are well-supported by the results. The manuscript is succinct and well-presented.

Reviewer #2 (Public Review):

Summary<br /> This study examines the construct of "cognitive spaces" as they relate to neural coding schemes present in response conflict tasks. The authors use a novel experimental design in which different types of response conflict (spatial Stroop, Simon) are parametrically manipulated. These conflict types are hypothesized to be encoded jointly, within an abstract "cognitive space", in which distances between task conditions depend only on the similarity of conflict types (i.e., where conditions with similar relative proportions of spatial-Stroop versus Simon conflicts are represented with similar activity patterns). Authors contrast such a representational scheme for conflict with several other conceptually distinct schemes, including a domain-general, domain-specific, and two task-specific schemes. The authors conduct a behavioral and fMRI study to test whether prefrontal cortex activity is correlated to one of these coding schemes. Replicating the authors' prior work, this study demonstrates that sequential behavioral adjustments (the congruency sequence effect) are modulated as a function of the similarity between conflict types. In fMRI data, univariate analyses identified activation in left prefrontal and dorsomedial frontal cortex that was modulated by the amount of Stroop or Simon conflict present, and representational similarity analyses that identified coding of conflict similarity, as predicted under the cognitive space model, in right lateral prefrontal cortex.

Strengths

This study addresses an important question regarding how conflict or difficulty might be encoded in the brain within a computationally efficient representational format. Relative to the other models reported in the paper, the evidence in support of the cognitive space model is solid. The ideas postulated by the authors are interesting and valuable ones, worthy of follow-up work that provides additional necessary scrutiny of the cognitive-space account.

Weaknesses

Future, within-subject experiments will be necessary to disentangle the cognitive space model from confounded task variables. A between-subjects manipulation of stimulus orientation/location renders the results difficult to interpret, as the source and spatial scale of the conflict encoding on cortex may differ from more rigorous (and more typical) within-subject manipulations.

Results are also difficult to interpret because Stroop and Simon conflict are confounded with each other. For interpretability, these two sources of conflict need to be manipulated orthogonally, so that each source of conflict (as well as their interaction) could be separately estimated and compared in terms of neural encoding. For example, it is therefore not clear whether the RSA results are due to encoding of only one type of conflict (Stroop or Simon), to a combination of both, and/or to interactive effects.

Finally, the motivation for the use of the term "cognitive space" to describe results is unclear. Evidence for the mere presence of a graded/parametric neural encoding (i.e., the reported conflict RSA effects) would not seem to be sufficient. Indeed, it is discussed in the manuscript that cognitive spaces/maps allow for flexibility through inference and generalization. Future work should therefore focus on linking neural conflict encoding to inference and generalization more directly.

Reviewer #2 (Public Review):

Summary:<br /> Sullivan and Bashaw delve into the mechanisms that drive neural circuit assembly, and specifically, into the regulation of cell surface proteins that mediate axon pathfinding. During nervous system development, axons must traverse a molecularly and physically complex extracellular milieu to reach their synaptic targets. A fundamental, conserved repulsive signaling pathway is initiated by the Slit-Robo ligand-receptor pair. Robo, expressed on axon growth cones, binds Slit, secreted by midline cells, to prevent "pre-crossing" and "re-crossing" of axons at the midline. To control this repulsion, Robo surface levels are tightly regulated. In Drosophila, Commissureless (Comm) downregulates Robo surface levels and is required for axon crossing at the midline. Several studies suggest that PY motifs in Comm are required to localize Robo to endosomes. PY motifs have been shown to bind WW-domain containing proteins including the ubiquitin ligase Nedd4 family, so the authors propose that Comm may regulate Robo through Nedd4 interactions. Previous studies have hinted at a role for Nedd4-mediated ubiquitination of Comm in the regulation of Robo localization, but there have also been conflicting data. For example, Comm mutants that are unable to be ubiquitinated mimic wild-type Comm, suggesting that ubiquitination of Comm is not required for regulation of Robo. The current study utilizes a suite of genetic analyses in Drosophila to resolve discrepancies pertaining to the mode of Comm-dependent regulation of Robo1 and proposes that Comm acts as an adapter for the Nedd4 ubiquitin ligase to recognize Robo1 as a substrate. The authors also demonstrate that Nedd4 is indeed required for midline crossing.

Strengths:<br /> While this work is more incremental rather than field-shifting, it is nonetheless an excellent example of a rigorous, thorough analysis that culminates in enriching our mechanistic understanding of how neurons regulate cell-surface receptors in a spatiotemporal manner to control fundamental steps of circuit wiring. The experimental approach is thorough, and the manuscript is extremely well-written.

Weaknesses:<br /> Some key experiments (eg. complex formation) were performed in cell culture in an overexpression background. Also, there was a missed opportunity to bolster the model proposed by using Comm PY mutants in several experiments. Finally, Comm PY domains are required for proper Comm localization in neurons, but corresponding Robo localization was not analyzed.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, L&S investigates the important general question of how humans achieve invariant behavior over stimuli belonging to one category given the widely varying input representation of those stimuli and more specifically, how they do that in arbitrary abstract domains. The authors start with the hypothesis that this is achieved by invariance transformations that observers use for interpreting different entries and furthermore, that these transformations in an arbitrary domain emerge with the help of the transformations (e.g. translation, rotation) within the spatial domain by using those as "scaffolding" during transformation learning. To provide the missing evidence for this hypothesis, L&S used behavioral category learning studies within and across the spatial, auditory, and visual domains, where rotated and translated 4-element token sequences had to be learned to categorize and then the learned transformation had to be applied in new feature dimensions within the given domain. Through single- and multiple-day supervised training and unsupervised tests, L&S demonstrated by standard computational analyses that in such setups, space and spatial transformations can, indeed, help with developing and using appropriate rotational mapping whereas the visual domain cannot fulfill such a scaffolding role.

Strengths:<br /> The overall problem definition and the context of spatial mapping-driven solution to the problem is timely. The general design of testing the scaffolding effect across different domains is more advanced than any previous attempts clarifying the relevance of spatial coding to any other type of representational codes. Once the formulation of the general problem in a specific scientific framework is done, the following steps are clearly and logically defined and executed. The obtained results are well interpretable, and they could serve as a good stepping stone for deeper investigations. The analytical tools used for the interpretations are adequate. The paper is relatively clearly written.

Weaknesses:<br /> Some additional effort to clarify the exact contribution of the paper, the link between analyses and the claims of the paper, and its link to previous proposals would be necessary to better assess the significance of the results and the true nature of the proposed mechanism of abstract generalization.

1) Insufficient conceptual setup: The original theoretical proposal (the Tolman-Eichenbaum-Machine, Whittington et al., Cell 2020) that L&S relate their work to proposes that just as in the case of memory for spatial navigation, humans and animals create their flexible relational memory system of any abstract representation by a conjunction code that combines on the one hand, sensory representation and on the other hand, a general structural representation or relational transformation. The TEM also suggests that the structural representation could contain any graph-interpretable spatial relations, albeit in their demonstration 2D neighbor relations were used. The goal of L&S's paper is to provide behavioral evidence for this suggestion by showing that humans use representational codes that are invariant to relational transformations of non-spatial abstract stimuli and moreover, that humans obtain these invariances by developing invariance transformers with the help of available spatial transformers. To obtain such evidence, L&S use the rotational transformation. However, the actual procedure they use actually solved an alternative task: instead of interrogating how humans develop generalizations in abstract spaces, they demonstrated that if one defines rotation in an abstract feature space embedded in a visual or auditory modality that is similar to the 2D space (i.e. has two independent dimensions that are clearly segregable and continuous), humans cannot learn to apply rotation of 4-piece temporal sequences in those spaces while they can do it in 2D space, and with co-associating a one-to-one mapping between locations in those feature spaces with locations in the 2D space an appropriate shaping mapping training will lead to the successful application of rotation in the given task (and in some other feature spaces in the given domain). While this is an interesting and challenging demonstration, it does not shed light on how humans learn and generalize, only that humans CAN do learning and generalization in this, highly constrained scenario. This result is a demonstration of how a stepwise learning regiment can make use of one structure for mapping a complex input into a desired output. The results neither clarify how generalizations would develop in abstract spaces nor the question of whether this generalization uses transformations developed in the abstract space. The specific training procedure ensures success in the presented experiments but the availability and feasibility of an equivalent procedure in a natural setting is a crucial part of validating the original claim and that has not been done in the paper.

2) Missing controls: The asymptotic performance in experiment 1 after training in the three tasks was quite different in the three tasks (intercepts 2.9, 1.9, 1.6 for spatial, visual, and auditory, respectively; p. 5. para. 1, Fig 2BFJ). It seems that the statement "However, our main question was how participants would generalise learning to novel, rotated exemplars of the same concept." assumes that learning and generalization are independent. Wouldn't it be possible, though, that the level of generalization depends on the level of acquiring a good representation of the "concept" and after obtaining an adequate level of this knowledge, generalization would kick in without scaffolding? If so, a missing control is to equate the levels of asymptotic learning and see whether there is a significant difference in generalization. A related issue is that we have no information on what kind of learning in the three different domains was performed, albeit we probably suspect that in space the 2D representation was dominant while in the auditory and visual domains not so much. Thus, a second missing piece of evidence is the model-fitting results of the ⦰ condition that would show which way the original sequences were encoded (similar to Fig 2 CGK and DHL). If the reason for lower performance is not individual stimulus difficulty but the natural tendency to encode the given stimulus type by a combo of random + 1D strategy that would clarify that the result of the cross-training is, indeed, transferring the 2D-mapping strategy.

Reviewer #2 (Public Review):

Summary:<br /> This is an unusual, but interesting approach to link the "taste" of plants and plant extracts to their therapeutic use in ancient Graeco-Roman culture. The authors used a panel of 11 trained tasters to test ~700 different medicinal plants and describe them in terms of 22 "taste" descriptors. They correlated these descriptors with the plant's medical use as reported in the De Materia Medica (DMM 1st Century, CE). Correcting for some of the plants' evolutionary phylogenetic relationships, the authors found that taste descriptors along with intensity measures were correlated with the "versatility" and/or a specific therapeutic use of the medicine. For example, simple but intense tastes were correlated with versatility of a medicine. Specific intense tastes were linked to versatility while others were not; intense bitter, starchy, musky, sweet, cooling and soapy were associated with versatility, but sour and woody were negatively associated. Also some specific tastes could be associated with specific uses - both positive and negative associations. Some of these findings make sense immediately, but others are somewhat surprising, and the authors propose some links between taste and medicinal use (both historical and modern use) in the discussion. The authors state that this study allows for a re-evaluation of pre-scientific knowledge, pointing toward a central role for taste in medicine.

Strengths:<br /> The real strength of this study is the novelty of this approach - using modern day tasters to evaluate ancient medicinal plants to understand the potential relationships between taste and therapeutic use, lending some support to the idea that the "taste" of a medicine is linked to its effectiveness as a treatment.

Weaknesses:<br /> Because of the limitations of time and the type of botanicals being tested, there is an inherent difficulty in assessing taste intensity. However, because these botanicals are tested by multiple panelists and sometimes tested repeatedly by individual panelists, this helps support the author's analyses.

Reviewer #2 (Public Review):

Erk2 is an essential element of the MAP kinase signaling cascade and directly controls cell proliferation, migration, and survival. Therefore, it is one of the most important drug targets for cancer therapy. The catalytic subunit of Erk2 has a bilobal architecture, with the small lobe harboring the nucleotide-binding pocket and the large lobe harboring the substrate-binding cleft. Several studies by the Ahn group revealed that the catalytic domain hops between (at least) two conformational states: active (R) and inactive (L), which exchange in the millisecond time scale based on the chemical shift mapping. The R state is a signature of the double phosphorylated Erk2 (2P-Erk2), while the L state has been associated with the unphosphorylated kinase (0P-Erk2). Interestingly, the X-ray structures reveal only minimal differences between these two states, a feature that led to the conclusion that active and inactive states are structurally similar but dynamically very different. The Ahn group also found that ATP-competitive inhibitors can steer the populations of Erk2 either toward the R or the L state, depending on their chemical nature. The latter opens up the possibility of modulating the activity of this kinase by changing the chemistry of the ATP-competitive inhibitor. To prove this point, the authors present a set of nineteen compounds with diverse chemical substituents. From their combined NMR and HDX-Mass Spec analyses, fourteen inhibitors drive the kinase toward the R state, while four compounds keep the kinase hopping between the R and L states. Based on these data, the authors rationalize the effects of these inhibitors and the importance of the nature of the substituents on the central scaffold to steer the kinase activity. While all these inhibitors target the ATP binding pocket, they display diverse structural and dynamic effects on the kinase, selecting a specific structural state. Although the inhibited kinase is no longer able to phosphorylate substrates, it can initiate signaling events functioning as scaffolds for other proteins. Therefore, by changing the chemistry of the inhibitors it may be possible to affect the MAP cascade in a predictable manner. This concept, recently introduced as proof of principle, finds here its significance and practical implications. The design of the next-generation inhibitors must be taken into account for these design principles.<br /> The research is well executed, and the data support the author's conclusions.

Reviewer #2 (Public Review):

In this manuscript the authors present and characterize LOVdeg, a modified version of the blue-light sensitive AsLOV2 protein, which functions as a light-inducible degron in Escherichia coli. Light has been shown to be a powerful inducer in biological systems as it is often orthogonal and can be controlled in both space and time. Many optogenetic systems target regulation of transcription, however in this manuscript the authors target protein degradation to control protein levels in bacteria. This is an important advance in bacteria, as inducible protein degradation systems in bacteria have lagged behind eukaryotic systems due to protein targeting in bacteria being primarily dependent on primary amino acid sequence and thus more difficult to engineer. In this manuscript, the authors exploit the fact that the J-alpha helix of AsLOV2, which unwinds into a disordered domain in response to blue light, contains an E-A-A amino acid sequence which is very similar to the C-terminal L-A-A sequence in the SsrA tag which is targeted by the unfoldases ClpA and ClpX. They truncate AsLOV2 to create AsLOV2(543) and combine this truncation with a mutation that stabilizes the dark state to generate AsLOV2*(543) which, when fused to the C-terminus of mCherry, confers light-induced degradation. The authors do not verify the mechanism of degradation due to LOVdeg, but evidence from deletion mutants contained in the supplemental material hints that there is a ClpA dominated mechanism. The LOVdeg is able to target mCherry for protein degradation across different phases of bacterial growth, which is important for regulating processes at stationary phase and a potential additional advantage over transcriptional repression systems. They demonstrate modularity of this LOVdeg by using it to degrade the LacI repressor, CRISPRa activation through degradation of MCP-SoxS, and the AcrB protein which is part of the AcrAB-TolC multidrug efflux pump. In all cases, measurement of the effect of the LOVdeg is indirect as the authors measure reduction in LacI repression, reduction in CRISPRa activation, and drug resistance rather than directly measuring protein levels. Nevertheless the evidence is convincing, although seemingly less effective than in the case of mCherry degradation, although it is hard to compare due to the different endpoints being measured. The authors further modify LOVdeg to contain a known photocycle mutation that slows its reversion time in the dark, so that LOVdeg is more sensitive to short pulses of light which could be useful in low light conditions or for very light sensitive organisms. They also demonstrate that combining LOVdeg with a blue-light transcriptional repression system (EL222) can decrease protein levels an additional 23-fold (relative to 7-fold with LOVdeg alone). Finally, the authors apply LOVdeg to a metabolic engineering task, namely reducing expression of octanoic acid by regulating the enzyme CpFatB1, an acyl-ACP thioesterase. The authors show that tagging CpFatB1 with LOVdeg allows light induced reduction in octanoic acid titer over a 24 hour fermentation. In particular, by comparing control of CpFatB1 with EL222 transcriptional repression alone, LOVdeg, or both the authors show that light-induced protein degradation is more effective than light-induced transcriptional repression. The authors suggest that this is because transcriptional repression is not effective when cells are at stationary phase (and thus there is no protein dilution due to cell division). Overall, the authors have generated a modular, light-activated degron tag for use in Escherichia coli that is likely to be a useful tool in the synthetic biology and metabolic engineering toolkit.

Reviewer #2 (Public Review):

In this study, the authors hypothesized that the balance of mTOR complexes in Sertoli cells may also play a significant role in age-dependent changes in the sperm epigenome. To test this hypothesis, the authors use transgenic mice with manipulated activity of mTOR complexes in Sertoli cells. These results suggest that the mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.

The authors attempt to demonstrate that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. The authors have effectively met their research objectives, and their conclusions are supported by the data presented.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors work to extend their previous observation that galectin-9 interacts with arabinogalactans of Mtb in their EMBO reports 2021 manuscript. Here they provide evidence that the CARD2 domain of galectin-9 can inhibit the growth of Mtb in culture. In addition, antibodies that also bind to AG appear to inhibit Mtb growth in culture. These data indicate that independent of the common cell-associated responses to galectin-9 and antibodies, the interaction of these proteins with AG of mycobacteria may have consequences for bacterial growth.

Strengths:

The authors provided several lines of evidence in culture media that the introduction of galectin-9 proteins and antibodies inhibits the growth rate of Mtb.

Weaknesses:

In light of other observations that cleaved galectin-9 levels in the plasma is a biomarker for severe infection (Padilla A et al Biomolecules 2021 and Iwasaki-Hozumi H et al. Biomoleucles 2021) it is difficult to reconcile the author's interpretation that the elevated gal-9 in Active TB patients (Figure 1E) contributes to the maintenance of latent infection in humans. The authors should consider incorporating these observations in the interpretation of their own results.

The anti-AG titers were measured only in individuals with active TB (Figure 3C), generally thought to be a less protective immunological state. The speculation that individuals with anti-AG titers have some protection is not founded. Further only 2 mAbs were tested to demonstrate restriction of Mtb in culture. It is possible that clones of different affinities for AG present within a patient's polyclonal AG-antibody responses may or may not display a direct growth restriction pressure on Mtb in culture. The authors should soften the claims about the presence of AG-titers in TB patients being indicative of protection.

Reviewer #2 (Public Review):

Summary:

The authors generated a DNA methylation score in cord blood for detecting exposure to cigarette smoke during pregnancy. They then asked if it could be used to predict height, weight, BMI, adiposity, and WHR throughout early childhood.

Strengths:

The study included two cohorts of European ancestry and one of South Asian ancestry.

Weaknesses:

1. The number of mothers who self-reported any smoking was very low, much lower than in the general population and practically non-existent in the South Asian population. As a result, all analyses appeared to have been underpowered. It is possibly for this reason that the authors chose to generate their DNA methylation model using previously published summary statistics. The resulting score is not of great value in itself due to the low-powered dataset used to estimate covariance between CpG sites. In fact, a score was generated for a much larger, better-powered dataset several years ago (Reese, EHP, 2017, PMID 27323799).

2. The conclusion that "even minimal smoking exposure in South Asian mothers who were not active smokers showed a DNAm signature of small body size and low birthweight in newborns" is not warranted because no analyses were performed to show that the association between DNA methylation and birth size/weight was driven by maternal smoking.

3. Although it was likely that some mothers were exposed to second-hand smoke and/or pollution, data on this was either non-existent or not included in this study. Including this would have allowed a more novel investigation of the effects of smoke exposure on the pregnancies of non-smoking mothers.

4. One of the European cohorts and half of the South Asian cohort had DNA methylation measured on only 2500 CpG sites. This set of sites included only 125 sites previously linked to prenatal smoking. The resulting model of prenatal smoking was small (only 11 CpG sites). It is possible that a large model may have been more powerful.

5. The health outcomes investigated are potentially interesting but there are other possibly more important outcomes of interest such as birth complications, asthma, and intellectual impairment which are known to be associated with prenatal smoking.

Reviewer #2 (Public Review):

Summary:<br /> Peled et al identified HER2 mutations in connection with resistance to the anti-HER2 antibody Pertuzumab-mediated therapy. After constructing a yeast display library of Pertuzumab variants with 3.86×1011 sequences for targeted screening of variant combinations in chosen 6 out of 14 residues, the authors performed experimental screening to obtain the clones that bind to HER2 WT and/or mutants (S310Y and S310F), and then combined new variations to obtain antibodies with a broad spectrum binding to both WT and two HER2 mutants. These are interesting studies of clinical impact and translational potential.

Strengths:<br /> 1. Deep computational analyses of large datasets of clinical data provide useful information about HER2 mutations and their potential relevance to antibody therapy resistance.

2. There is valuable information analyzing the residues within or near the interface between the antigen HER2 and the Pertuzumab antibody (heavy chain). The experimental antibody library screening obtained 90+ clones from 3.86×1011 sequences for further functional validation.

Weaknesses:<br /> 1. There is a lack of assessment for antibody variant functions in cancer cell phenotypes in vitro (proliferation, cell death, motility) or in vivo (tumor growth and animal survival). The only assay was the western blotting of phosphopho-HER3 in Figure 4. However, HER2 levels and phosphor-HER2 were not analyzed.

2. There is a misleading impression from the title of computational engineering of a therapeutic antibody and the statement in the abstract "we designed a multi-specific version of Pertuzumab that retains original function while also bindings these HER2 variants" for a few reasons:<br /> a. The primary method used for variant antibody identification for HER2 mutant binding is rather traditional experimental screening based on yeast display instead of the computational design of a multi-specific version of Pertuzumab.<br /> b. There is insufficient or lack of computational power in the antibody design or prioritization in choosing variant residues for the library construction of 3.86×1011 sequences. It seems random combinations from 6 residues out of 4 groups with 20 amino acid options.<br /> c. The final version of the tri-binding variant is a combination of screened antibody clones instead of computation design from scratch.<br /> d. There is incomplete experimental evidence about the therapeutic values of newly obtained antibody clones.

3. Figures can be improved with better labeling and organization. Some essential pieces of data such as Supplementary Figure 1B on HER2 mutations in S310 that abrogated its binding to Pertuzumab should be placed in the main figures.

4. It is recommended to provide a clear rationale or flowchart overview into the main Figure 1. Figure 2A can be combined with Figure 1 to the list of targeted residues.

5. The quality of Figures such as Figure 2B-C flow data needs to be improved.

Reviewer #2 (Public Review):

Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells by acting on the Wnt/b-catenin signaling pathway. They used various models, such as the piglet model, mouse model, and intestinal organoids to address how A. muciniphila and B. fragilis offer protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling. While a large body of work has been performed in this study, there are quite a few questions to be addressed.

Major comments:

- The similar protective effect of FMT with fecal samples, A. muciniphila or B. fragilis is perhaps not that surprising, considering that FMT likely restores microbiota-mediated colonization resistance against ETEC infection. While FMT with fecal samples increases SCFAs, it is unclear whether/how FMT with A. muciniphila or B. fragilis alter the microbiota composition/abundance as well as metabolites in the current models in a way that offers protection.

- Does ETEC infection in piglets/mice cause histological damage in the intestines? These data should be shown.

- Line 447, "ETEC adheres to intestinal epithelial cells". However, there is no data showing the adherence (or invasion) of ETEC to intestinal epithelial cells, irrespective of piglets/mouse/organoids.

- In both basal-out and apical-out intestinal organoid models, A. muciniphila protects organoids against ETEC infection. Did ETEC enter into intestinal epithelial cells at all after only one hour of infection? Is the protection through certain A. muciniphila metabolites?

Reviewer #2 (Public Review):

In this manuscript, Tiedje and colleagues longitudinally track changes in parasite numbers across four time points as a way of assessing the effect of malaria control interventions in Ghana. Some of the study results have been reported previously, and in this publication, the authors focus on age-stratification of the results. Malaria prevalence was lower in all age groups after IRS. Follow-up with SMC, however, maintained lower parasite prevalence in the targeted age group but not the population as a whole. Additionally, they observe that diversity measures rebounds more slowly than prevalence measures. Overall, I found these results clear, convincing, and well-presented. They add to a growing literature that demonstrates the relevance of asymptomatic reservoirs.

There is growing interest in developing an expanded toolkit for genomic epidemiology in malaria, and detecting changes in transmission intensity is one major application. As the authors summarize, there is no one-size-fits-all approach, and the Bayesian MOIvar estimate developed here has the potential to complement currently used methods. I find its extension to a calculation of absolute parasite numbers appealing as this could serve as both a conceptually straightforward and biologically meaningful metric. However, I am not fully convinced the current implementation will be applied meaningfully across additional studies.

1. I find the term "census population size" problematic as the groups being analyzed (hosts grouped by age at a single time point) do not delineate distinct parasite populations. Separate parasite lineages are not moving through time within these host bins. Rather, there is a single parasite population that is stochastically divided across hosts at each time point. I find this distinction important for interpreting the results and remaining mindful that the 2,000 samples at each time point comprise a subsample of the true population. Instead of "census population size", I suggest simplifying it to "census count" or "parasite lineage count".

It would be fascinating to use the obtained results to model absolute parasite numbers at the whole population level (taking into account, for instance, the age structure of the population), and I do hope this group takes that on at some point even if it remains outside the scope of this paper. Such work could enable calculations of absolute---rather than relative---fitness and help us further understand parasite distributions across hosts.

2. I'm uncertain how to contextualize the diversity results without taking into account the total number of samples analyzed in each group. Because of this, I would like a further explanation as to why the authors consider absolute parasite count more relevant than the combined MOI distribution itself (which would have sample count as a denominator). It seems to me that the "per host" component is needed to compare across age groups and time points---let alone different studies.

3. Thinking about the applicability of this approach to other studies, I would be interested in a larger treatment of how overlapping DBLa repertoires would impact MOIvar estimates. Is there a definable upper bound above which the method is unreliable? Alternatively, can repertoire overlap be incorporated into the MOI estimator?

Smaller comments:<br /> - Figure 1 provides confidence intervals for the prevalence estimates, but these aren't carried through on the other plots (and Figure 5 has lost CIs for both metrics). The relationship between prevalence and diversity is one of the interesting points in this paper, and it would be helpful to have CIs for both metrics when they are directly compared.

Reviewer #2 (Public Review):

Summary:<br /> In the manuscript by Luo et al, the authors investigated the nature and function of TRAIL-HS binding for the regulation of TRAIL-mediated apoptosis in cancer cells. The authors discovered that TRAIL binds to 12mer HS and identified the amino acid residues critical for the binding. The authors further nicely showed that 12mer HS binds to TRAIL homotrimer and larger HS can further promote the formation of larger TRAIL oligomers. Structural analyses were conducted to characterize the binding of TRAIL/HS complexes. At functional level, the authors demonstrated that HS promotes the cell surface binding of TRAIL to enhance TRAIL-mediated apoptosis in a variety of cancer cells. Moreover, the ability of TRAIL to induce apoptosis is correlated with cell surface HS level. Lastly, the authors showed that HS forms complex with TRAIL and its receptor DR5 and promotes DR5 internalization.

Strengths:<br /> Overall, this is a nicely executed study providing both mechanistic and functional insight for TRAIL-mediated apoptosis. It conducted detailed characterization on the direct binding between HS and TRAIL and provided solid evidence supporting the role of such interaction for the regulation of TRAIL-induced apoptosis. The experiments were well-designed with proper controls included. The data interpretation is accurate. The manuscript was clearly written and easy to follow by general readers.

Weaknesses:<br /> There is no major weakness identified from this study. As the authors pointed out, the current relationship between cell surface HS level and sensitivity to TRAIL-mediated apoptosis is still correlative and will need further investigation in the future.

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1.

The authors have sufficiently addressed prior comments and added new experiments to provide details on possible mechanisms.

Reviewer #2 (Public Review):

Summary:

In their manuscript, the authors address the question of whether the inversion polymorphism in D. melanogaster can be explained by sexually antagonistic selection. They designed a new simulation tool to perform computer simulations, which confirmed their hypothesis. They also show a tradeoff between male reproduction and survival. Furthermore, some inversions display sex-specific survival.

Strengths:<br /> It is an interesting idea on how chromosomal inversions may be maintained

Weaknesses:<br /> General points:<br /> The manuscript lacks clarity of writing. It is impossible to fully grasp what the authors did in this study and how they reached their conclusions. Therefore, I will highlight some cases that I found problematic.<br /> Although this is an interesting idea, it clearly cannot explain the apparent influence of seasonal and clinal variation on inversion frequencies.

Specific points:<br /> The simulations are highly specific and make very strong assumptions, which are not well-justified.

Reviewer #2 (Public Review):

Ehring et al. analyze contributions of Dispatched, Scube2, serum lipoproteins and Sonic Hedgehog lipid modifications to the generation of different Shh release forms. Hedgehog proteins are anchored in cellular membranes by N-terminal palmitate and C-terminal cholesterol modifications, yet spread through tissues and are released into the circulation. How Hedgehog proteins can be released, and in which form, remains controversial. The authors systematically dissect contributions of several previously identified factors, and present evidence that Disp, Scube2 and lipoproteins concertedly act to release a novel Shh variant that is cholesterol-modified but not palmitoylated. The results provide new insights into the function of Disp and Scube2 in Hedgehog release. The findings concerning the function of lipoproteins and cholesterol in Hedgehog release are largely confirmatory (PMID 23554573, 20685986). However, in light of the multitude of competing models for Hedgehog release, the present study is a valuable contribution that provides further insights into the relevance of lipoproteins in this process.

A novel and surprising finding of the present study is the differential removal of Shh N- or C-terminal lipid anchors depending on the presence of HDL and/or Disp. In particular, the identification of a non-palmitoylated but cholesterol-modified Shh variant that associates with lipoproteins is potentially important. The authors use RP-HPLC and defined controls to assess the properties of processed Shh forms, but their precise molecular identify remains to be defined. A caveat is the strong reliance on over-expression of Shh in a single cell line. The authors detect Shh variants that are released independently of Disp and Scube2 in secretion assays, which however are excluded from interpretation as experimental artifacts. Thus, it would be important to demonstrate key findings in cells that secrete Shh endogenously.

Reviewer #2 (Public Review):

By mapping the sites of the Mcm2-7 replicative helicase loading across the budding yeast genome using high-resolution chromatin endogenous cleavage or ChEC, Bedalov and colleagues find that these markers for origins of DNA replication are much more broadly distributed than previously appreciated. Interestingly, this is consistent with early reconstituted biochemical studies that showed that the ACS was not essential for helicase loading in vitro (e.g. Remus et al., 2009, PMID: 19896182). To accomplish this, they combined the results of 12 independent assays to gain exceptionally deep coverage of Mcm2-7 binding sites. By comparing these sites to previous studies mapping ssDNA generated during replication initiation, they provide evidence that at least a fraction of the 1600 most robustly Mcm2-7-bound sequences act as origins. A weakness of the paper is that the group-based (as opposed to analyzing individual Mcm2-7 binding sites) nature of the analysis prevents the authors from concluding that all of the 1,600 sites mentioned in the title act as origins. The authors also show that the location of Mcm2-7 location after loading are highly similar in the top 500 binding sites, although the mobile nature of loaded Mcm2-7 double hexamers prevents any conclusions about the location of initial loading. Interestingly, by comparing subsets of the Mcm2-7 binding sites, they find that there is a propensity of at least a subset of these sites to be nucleosome depleted, to overlap with at least a partial match to the ACS sequence (found at all of the most well-characterized budding yeast origins), and a GC-skew centered around the site of Mcm loading. Each of these characteristics is related to previously characterized S. cerevisiae origins of replication.

Overall, this manuscript greatly broadens the number of sites that are capable of loading Mcm2-7 in budding yeast cells and shows that a subset of these additional sites act as replication origins. Although these studies show that the sequence specificity of S. cerevisiae replication origins still sets it apart from metazoan origins, the ability to license and initiate replication from sites with increasing sequence divergence suggests a previously unappreciated versatility.

Reviewer #2 (Public Review):

This is a thorough and convincing body of work that represents an incremental but significant improvement on iterations of this method of CRISPR-based Sterile Insect Technique ('pgSIT'). In this version, compared to previous, the authors target more genes than previously, in order to induce both female inviability (targeting the genes intersex and doublesex, compared to fem-myo previously) and male sterility (targeting a beta-tubulin, as previously in the release generation.

The characterization of the lines is extensive and this data will be useful to the field. However, what is lacking is some context as to how this formulation compares to the previous iteration. Mention is made of the possible advantage of removing most females, compared to just making them flightless (as previously) but there is no direct comparison, either experimental, or theoretical i.e. imputing the life history traits into a model. For me this is a weakness, yet easily addressed. In a similar vein, much is made in alluding to the 'safety concerns of gene drive' and how this is a more palatable half-way house, just because it has CRISPR component within it; it is not. It would be much more sensible, and more informative, to compare this pgSIT technology to RIDL (release of insects carrying a dominant lethal), which is essentially a transgene-based version of the Sterile Insect Technique, as is the work presented here.

The authors achieve impressive results and show that these strains, under a scenario of high levels of release ratios compared to WT, could achieve significant local suppression of mosquito populations. The sensitivity analysis that examines the effect of changing different biological or release parameters is well performed and very informative.

The authors are honest in acknowledging that there are still challenges in bringing this to field release, namely in developing sexing strains and optimizing release strategies.

Reviewer #2 (Public Review):

In this work, Vollenweider et al. examine the effectiveness of using natural products, specifically molecules that chelate iron, to treat infectious agents. Through the purification of 320 environmental isolates, 25 potential candidates were identified from natural products based on inhibition assays and were further screened. The structural information and chemical composition were determined.

The paper is well-structured and thorough; targeting virulence factors in this manner is a great idea. My enthusiasm is dampened by the mediocre effects of the compounds. The lack of a dose-response curve in the survivability assays suggests a limited scope for these molecules. While it is encouraging that the best survivability occurred at the lowest toxicity level, it opens questions as to how effective such molecules can be. Either the reduction in mortality was offset by using higher concentrations, which was not observed in the compound-alone test, or there is no dose-response curve. The latter would suggest to me that the variation in survivability is not due to the addition of siderophores.

I would also like to see how these molecules compare to other iron-chelating molecules. Desferoxamine is a bacteria-derived siderophore that is FDA-approved. However, it is not used to treat infections. Would the author consider comparing their candidate molecules to well-studied molecules? This also raises questions about the novelty of this work; I think the authors could rephrase the discussion to better reflect that bioprospecting for iron-chelating molecules has previously occurred and been successful.

Finally, I am concerned about the few mutations reported in the resistance study. Looking at the SI, it appears that very few mutations were seen. It is unclear what filtering the authors used to arrive at such a low number of mutations. Even filtering against mutations that were selected by adaptation to the media, it seems low that only a handful of clones had distinct mutations.

This paper has a lot of strengths. The workflow is logical and well-executed; the only significant weakness is the effect of the molecules and the lack of an explanation for a dose-response curve in the survivability assay, especially when compared to the data reported in Figure 3. As the authors describe in lines 214-217.

Reviewer #2 (Public Review):

Summary:<br /> Dr. Khulman and colleagues present a very interesting experimental and mathematical modeling work on IS608 transposition. The system has a number of unique advantages that create experimental possibilities utilized here to investigate transposition dynamics. This kind of approach is badly missing from the field and I believe the experiments and modeling work shown here and the type of results that can be derived are groundbreaking and certainly deserve high visibility.

Strengths:<br /> The attempt to measure and model transposition dynamics in cells.

Weaknesses:<br /> - Lack of controls using an active site mutant of TnpB.<br /> - Lack of control in RecA- cells as transposon restoration is proposed to be dependent on homologous recombination.<br /> - Lack of consideration of the levels of ωRNA present.

Reviewer #2 (Public Review):

Hagihara et al. conducted a study investigating the correlation between decreased brain pH, increased brain lactate, and poor working memory. They found altered brain pH and lactate levels in animal models of neuropsychiatric and neurodegenerative disorders. Their study suggests that poor working memory performance may predict higher brain lactate levels.

However, the study has some significant limitations. One major concern is that the authors examined whole-brain pH and lactate levels, which might not fully represent the complexity of disease states. Different brain regions and cell types may have distinct protein and metabolite profiles, leading to diverse disease outcomes. For instance, certain brain regions like the hippocampus and nucleus accumbens exhibit opposite protein/signaling pathways in neuropsychiatric disease models.

Moreover, the memory tests used in the study are specific to certain brain regions, but the authors did not measure lactate levels in those regions. Without making lactate measurements in brain-regions and cell types involved in these diseases, any conclusions regarding the role of lactate in CNS diseases is premature.

Additionally, evidence suggests that exogenous treatment with lactate has positive effects, such as antidepressant effects in multiple disease models (Carrard et al., 2018, Carrard et al., 2021, Karnib et al., 2019, Shaif et al., 2018). It also promotes learning, memory formation, neurogenesis, and synaptic plasticity (Suzuki et al., 2011, Yang et al., 2014, Weitian et al., 2015, Dong et al., 2017, El Hayek et al. 2019, Wang et al., 2019, Lu et al., 2019, Lev-Vachnish et a.l, 2019, Descalzi G et al., 2019, Herrera-López et al., 2020, Ikeda et al., 2021, Zhou et al., 2021,Roumes et al., 2021, Frame et al., 2023, Akter et al., 2023).

In conclusion, the relevance of total brain pH and lactate levels as indicators of the observed correlations is controversial, and evidence points towards lactate having more positive rather than negative effects. It is important that the authors perform studies looking at brain-region-specific concentrations of lactate and that they modulate lactate levels (decrease) in animal models of disease to validate their conclusions. It is also important to consider the above-mentioned studies before concluding that "altered brain pH and lactate levels are rather involved in the underlying pathophysiology of some patients with neuropsychiatric disorders" and that "lactate can serve as a potential therapeutic target for neuropsychiatric disorders".

Reviewer #2 (Public Review):

The study is novel and valuable to the field and provides new and important insights into the role of lipid mediators in VML injuries. By expanding our understanding of the mechanisms that regulate muscle regeneration following VML injuries, the study has the potential to guide the development of novel therapeutic interventions that promote tissue repair and recovery. The data presented in the manuscript is of good quality. The findings and conclusions are supported by a variety of different analyses (e.g., gene expression, histology, flow cytometry).

Despite the strengths of the study, some limitations are identified. Specifically, the impact of maresin 1 on macrophage phenotypes (M1/M2) could have been explored in more detail using histological or protein expression analysis. Moreover, additional data are needed to substantiate the claims about increased muscle regeneration. Lastly, the study does not address myofiber innervation, myofiber-type transitions, or motor unit remodeling.

Reviewer #2 (Public Review):

In this study, the goal is to leverage the power of Bayesian inference to estimate online the probability that any given arbitrarily chosen strategy is being used by the decision-maker. By computing the trial-by-trial MAP and variance of the posterior distribution for each candidate strategy, the authors can not only see which strategy is primarily being used at every given time during the task and when strategy changes occur but also detect when the target rule of a learning task becomes the front-running strategy, i.e., when successful learning occurs.

Strengths:<br /> 1. The proposed approach adds to recent methods for capturing the dynamics of decision-making at finer temporal resolution (trials) (Roy et al., 2021; Ashwood et al., 2022) but it is novel and differs from these in that it is suited especially well for analyzing when learning occurs, or when a rule switches and learning must recommence, and it does not necessitate large numbers of trials.

2. The manuscript starts with a validation of the approach using synthetic data and then is applied to datasets of trial-based two-alternative forced choice tasks ranging from rodent to non-human primate to human, providing solid evidence of its utility.

3. Compared to classic procedures for identifying when an animal has learned a contingency which typically needs to be conservative in favor of better accuracy, this method retrieves signs of learning happening earlier (~30 trials earlier on average). This is achieved by identifying the moment (trial) when the posterior probability of the correct "target" rule surpasses the probability of all other strategies. Having greater temporal precision in detecting when learning happens may have a very significant impact on studies of the neural mechanisms of learning.

4. This approach seems amenable to testing many different strategies depending on the purpose of the analysis. In the manuscript, the authors test target versus non-target strategies (correct versus incorrect) and also in another version of the analysis, they test what they call "exploratory" strategies.

5. One of the main appeals of this method is its apparent computational simplicity. It necessitates only updating on every trial the parameters of a beta distribution (prior distribution for a given strategy) with the evidence that the behavior on trial was either consistent or inconsistent with the strategy. Two scalars, the mode of the posterior (MAP) and the inverse of the variance, are all that are required for identifying the decision criterion (highest MAP and if tied lowest variance) and the learning criterion (first trial where MAP for target strategy is higher than chance).

Weaknesses:<br /> 1. It seems like a limitation of this approach is that the candidate strategies to arbitrate between must be known ex-ante. It is not clear how this approach could be applied to uncover latent strategies that are not mixtures of the strategies selected.

2. Different strategies may be indistinguishable from each other and thus it may not be possible to distinguish between them. Similarly, the fact that two strategies seem to be competing for the highest MAP doesn't necessarily mean that those are correct strategies and perhaps interchangeable as the manuscript seems to suggest.

3. The decay parameter is a necessary component to make the strategy selection non-stationary and accommodate data sets where the rules are changing throughout the task. However, the choice of the decay parameter value bounds does not seem very principled. Having this parameter as a free-parameter adds a flexibility that seems to have significant effects on when the strategy switch is detected and how stable the detected switch is.

4. This method is a useful approach for arbitrating between strategies and describing the behavior with a temporal precision that may prove important for studies attempting to tie these precise events to changes in neural activity. However, it seems limited in its explanatory power. In its current form, this method does not provide a prediction of the probability to transition from one strategy to another. And, because the MAP of different strategies may be close at any given moment, it is hard to imagine using this approach to tease out the different "mental states" that represent each strategy being at play.

Reviewer #2 (Public Review):

Working memory is not error free. Behavioral reports of items held in working memory display several types of bias, including contraction bias and serial dependence. Recent work from Akrami and colleagues demonstrates that inactivating rodent PPC reduces both forms of bias, raising the possibility of a common cause.

In the present study, Boboeva, Pezotta, Clopath, and Akrami introduce circuit and descriptive variants of a model in which the contents of working memory can be replaced samples from recent sensory history. This volatility manifests as contraction bias and serial dependence in simulated behavior, parsimoniously explaining both sources of bias. The authors validate their model by showing that it can recapitulate previously published and novel behavioral results in rodents and neurotypical and atypical humans.

Both the modeling and the experimental work is rigorous, providing convincing evidence that a model of working memory in which reports sometimes sample past experience can produce both contraction bias and serial dependence, and that this model is consistent with behavioral observations across rodents and humans in the parametric working memory (PWM) task.

These efforts constitute an admirable initial validation of the proposed model, and the authors present several novel predictions that will allow for further tests in future experiments. First, the authors note that their circuit model predicts a bimodal error distribution in delayed estimation paradigms (due to noisy sampling of sensory history on a subset of trials) that merges into a unimodal distribution when recent sensory history and the current to-be-reported stimulus have very similar values (Fig. 5c). Analysis of extent delayed estimation datasets (e.g., https://osf.io/jmkc9/) or new experiments will provide the opportunity for a straightforward test of this hypothesis.

Second, the bulk of the modeling efforts presented here are devoted to a circuit-level description of how putative posterior parietal cortex (PPC) and working-memory (WM) related networks may interact to produce such volatility and biases in memory. This effort is extremely useful because it allows the model to be constrained by neural observations and manipulations in addition to behavior, and the authors begin this line of inquiry here (by showing that the circuit model can account for effects of optogenetic inactivation of rodent PPC). As the authors note, population electrophysiology in PPC and WM-related areas and single-trial analyses will play an important role in the ultimate validation of this model.

Finally, it is noteworthy that, in the spirit of moving away from an overreliance on p-values (e.g., Amrhein et al., PeerJ 2017), the authors eschew conventional hypothesis testing when reporting their experimental results. The p-values aren't missed, in large part thanks to excellent visualizations and apparently large effect sizes. It's unclear how well this approach would generalize to other questions and datasets; nevertheless, this study provides an interesting data point in the ongoing conversation around reproducibility and rigor.

Reviewer #2 (Public Review):

Schmid et al present a lovely study looking at the effect of passive auditory exposure on learning a categorization task.<br /> The authors utilize a two-alternative choice task where mice have to discriminate between upward and downward moving frequency sweeps. Once mice learn to discriminate easy stimuli, the task is made psychometric and additional intermediate stimuli are introduced (as is standard in the literature). The authors introduce an additional two groups of animals, one that was passively exposed to the task stimuli before any behavioral shaping, and one that had passive exposure interleaved with learning. The major behavioral finding is that passive exposure to sounds improves learning speed. The authors show this in a number of ways through linear fits to the learning curves. Additionally, by breaking down performance based on the "extreme" vs "psychometric" stimuli, the authors show that passive exposure can influence responses to sounds that were not present during the initial training period. One limitation here is that the presented analysis is somewhat simplistic, does not include any detailed psychometric analysis (bias, lapse rates etc), and primarily focuses on learning speed. Ultimately though, the behavioral results are interesting and seem supported by the data.

To investigate the neural mechanisms that may underlie their behavioral findings, the authors turn to a family of artificial neural network models and evaluate the consequences of different learning algorithms and schedules, network architectures, and stimulus distributions, on the learning outcomes. The authors work through five different architectures that fail to recapitulate the primary behavior findings before settling on a final model, utilizing a combination of supervised and unsupervised learning, that was capable of reproducing the key aspects of the experiments. Ultimately, the behavioral results presented are consistent with network models that build latent representations of task-relevant features that are determined by statistical properties of the input distribution.

Reviewer #2 (Public Review):

This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however the biological insight gained is rather limited.

Reviewer #2 (Public Review):

The authors attempt to address a long-standing controversy in the study of the neural correlates of visual awareness, namely whether neurons in prefrontal cortex are necessarily involved in conscious perception. Several leading theories of consciousness propose a necessary role for (at least some sub-regions of) PFC in basic perceptual awareness (e.g., global neuronal workspace theory, higher order theories), while several other leading theories posit that much of the previously reported PFC contributions to perceptual awareness may have been confounded by task-based cognition that co-varied between the aware and unaware reports (e.g., recurrent processing theory, integrated information theory). By employing intracranial EEG in human patients and a threshold detection task on low-contrast visual stimuli, the authors assessed the timing and location of neural populations in PFC that are differentially activated by stimuli that are consciously perceived vs. not perceived. Overall, the reported results support the view that certain regions of PFC do contribute to visual awareness, but at time-points earlier than traditionally predicted by GNWT and HOTs.

Major strengths of this paper include the straightforward visual threshold detection task including the careful calibration of the stimuli and the separate set of healthy control subjects used for validation of the behavioral and eye tracking results, the high quality of the neural data in six epilepsy patients, the clear patterns of differential high gamma activity and temporal generalization of decoding for seen versus unseen stimuli, and the authors' interpretation of these results within the larger research literature on this topic. This study appears to have been carefully conducted, the data were analyzed appropriately, and the overall conclusions seem warranted given the main patterns of results.

Weaknesses include the saccadic reaction time results and the potential flaws in the design of the reporting task. As the authors acknowledge, this is not a "no report" paradigm, rather, it's a paradigm aimed at balancing the post-perceptual cognitive and motor requirements between the seen and unseen trials. On each trial, subjects/patients either perceived the stimulus or not, and had to briefly maintain this "yes/no" judgment until a fixation cross changed color, and the color change indicated how to respond (saccade to the left or right). Differences in saccadic RTs (measured from the time of the fixation color change to moving the eyes to the left or right response square) were evident between the seen and unseen trials (faster for seen). In the discussion, the authors summarize several alternative explanations of the saccade results and limitations of their report paradigm that will help guide future research.

The current results help advance our understanding of the contribution of PFC to visual awareness. These results, when situated within the larger context of the rapidly developing literature on this topic provide converging evidence that some sub-regions of PFC contribute to visual awareness, but at latencies earlier than originally predicted by proponents of, especially, global neuronal workspace theory. Three recent studies that used "no report paradigms", but with clearly visible stimuli, reported very similar results in PFC (Vishne et al., 2023; Broday-Dvir et al., 2023; Cogitate et al., 2023). The current study uses a report paradigm, but with consciously seen vs. unseen conditions, to fill the gap left by these previous studies, i.e., it remained unclear whether the PFC results from the previous studies were related to conscious or unconscious processing. Taken as a whole, evidence appears to be converging for a limited and early-in-time (200-300ms) contribution of PFC to visual awareness, after task and motor confounds are minimized.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediators of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from post-mortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the pre-symptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied with the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function. This is an expected result, given the fairly focal approach of ephrinB2 knockdown in C3-C5 spinal segments.

The major limitation of the study is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many other that comprise the ventral gray matter, have not been investigated in this study. Nonetheless, there is convincing evidence to suggest astrocytes play a significant role, as compared to oligodendrocytes in promoting ALS pathogenesis.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript by Martin-Flores et al. examines the role of DKK3 in Alzheimer's disease, focusing on the regulation of synaptic number. Using human AD brain databases and tissue samples, the authors demonstrate increased levels of DKK3 protein and mRNA in the brains of AD patients. DKK3 is expressed in excitatory neurons in WT mouse brains and accumulates at atrophic neurites around amyloid plaques in AD mouse brains. Interestingly, the secretion of DKK3 appears to be regulated by NMDAR antagonists, as well as chemical LTD. Through gain and loss of function studies, the authors reveal that DKK3 regulates the number of both excitatory and inhibitory synapses with distinct downstream pathways. Finally, the authors investigate the contribution of DKK3 to synaptic changes in AD and find that DKK3 loss of function rescues both excitatory and inhibitory synaptic defects, resulting in improved memory function in J20 mice.

Strengths:<br /> Overall, the data is clearly presented and deals with the novel roles of DKK3 in controlling excitatory and inhibitory synapses. The finding that shRNA expression of DKK3 in AD model mice rescues synaptic phenotypes and memory impairment is potentially interesting and may provide a new strategy for AD treatment.

Weaknesses:<br /> There are no major weaknesses.

Reviewer #3 (Public Review):

Summary:<br /> Onck and co-workers present in this work the identification of binding partners and sites of polyPR on various nuclear transport components and elucidate how polyPR might potentially influence the transport process. It's interesting to note that some interaction sites on transport components also serve as their inherent/functional binding sites (Figure 3). The difference in the effects between short polyPR (PR7) and long polyPR (PR50) is also evident, although the authors might need to clarify the mechanisms better. Overall, I find this manuscript well organized and concisely written, and it would greatly enhance our understanding of the toxicity induced by polyPR.

Strengths:<br /> The 1-bead per atom force field model used in the study is well-tuned for studying the interactions between polyPR and proteins, as the essential cation-pi interactions (between Arg and Phe/Tyr/Trp) was included using a 8-6 LJ model.

Weaknesses:<br /> To cite the author's response: "At the moment, accurately capturing the binding of NCT components to their native binding targets and the competition with polyPR are best resolved by all-atom molecular dynamics simulations, which come with significant computational demands. This level of detail and computation-intensive analyses is beyond the scope of the current study."

Reviewer #2 (Public Review):

Summary:<br /> Chang et al. investigate neuronal activity firing patterns across various cortical regions in an interesting context-dependent tactile vs visual detection task, developed previously by the authors (Chevee et al., 2021; doi: 10.1016/j.neuron.2021.11.013). The authors report the important involvement of a medial frontal cortical region (MM, probably a similar location to wM2 as described in Esmaeili et al., 2021 & 2022; doi: 10.1016/j.neuron.2021.05.005; doi: 10.1371/journal.pbio.3001667) in mice for determining task rules.

Strengths:<br /> The experiments appear to have been well carried out and the data well analysed. The manuscript clearly describes the motivation for the analyses and reaches clear and well-justified conclusions. I find the manuscript interesting and exciting!

Weaknesses:<br /> I did not find any major weaknesses.

Reviewer #2 (Public Review):

Summary:<br /> The authors' long-term goals are to understand the utility of precisely phased cortex stimulation regimes on recovery of function after spinal cord injury (SCI). In prior work, the authors explored the effects of contralesion cortex stimulation. Here, they explore ipsilesion cortex stimulation in which the corticospinal fibers that cross at the pyramidal decussation are spared. The authors explore the effects of such stimulation in intact rats and rats with a hemisection lesion at the thoracic level ipsilateral to the stimulated cortex. The appropriately phased microstimulation enhances contralateral flexion and ipsilateral extension, presumably through lumbar spinal cord crossed-extension interneuron systems. This microstimulation improves weight bearing in the ipsilesion hindlimb soon after injury, before any normal recovery of function would be seen. The contralateral homologous cortex can be lesioned in intact rats without impacting the microstimulation effect on flexion and extension during gait. In two rats ipsilateral flexion responses are noted, but these are not clearly demonstrated to be independent of the contralateral homologous cortex remaining intact.

Strengths:<br /> This paper adds to prior data on cortical microstimulation by the laboratory in interesting ways. First, the strong effects of the spared crossed fibers from the ipsi-lesional cortex in parts of the ipsi-lesion leg's step cycle and weight support function are solidly demonstrated. This raises the interesting possibility that stimulating the contra-lesion cortex as reported previously may execute some of its effects through callosal coordination with the ipsi-lesion cortex tested here. This is not fully discussed by the authors but may represent a significant aspect of these data. The authors demonstrate solidly that ablation of the contra-lesional cortex does not impede the effects reported here. I believe this has not been shown for the contra-lesional cortex microstimulation effects reported earlier, but I may be wrong.

Effects and neuroprosthetic control of these effects are explored well in the ipsi-lesion cortex tests here.

Weaknesses:<br /> Some data is based on very few rats. For example (N=2) for ipsilateral flexion effects of microstimulation. N=3 for homologous cortex ablation, and only ipsi extension is tested it seems. There is no explicit demonstration that the ipsilateral flexion effects in only 2 rats reported can survive the contra-lateral cortex ablation.

Some improvements in clarity and precision of descriptions are needed, as well as fuller definitions of terms and algorithms.

Likely Impacts:<br /> This data adds in significant ways to prior work by the authors, and an understanding of how phased stimulation in cortical neuroprosthetics may aid in recovery of function after SCI, especially if a few ambiguities in writing and interpretation are fully resolved.

Reviewer #2 (Public Review):

Summary:<br /> This is a fundamental and elegant study showing the role of BMP signaling in cerebellar development. This is an important question because there are multiple diseases, including aggressive childhood cancers, which involve granule cell precursors. Thus understanding of the factors that govern the formation of the granule cell layer is important both from a basic science and a disease perspective.

Overall, the manuscript is clear and well-written. The figures are extremely clear, wonderfully informative, and overall quite beautiful.

Figures 1-3 show the experimental design and report how BMP activity is altered over development in both the chick and the human developing cerebellum. Both data are very impressive and convincing.

They then go on to modulate BMP activity in the developing chick, using a complex electroporation paradigm that allows them to label cells with GFP as well as with cell-specific reporters of BMP activity levels. They bidirectionally modulate BMP levels and then can look at both cell-specific and non-specific alterations in the formation of the external and internal granule cell layer, across different developmental timepoints. These are really elegant and rigorous experiments, as they look at both sagittal and transverse sections to collect this data. This makes the data extremely compelling. With these rigorous techniques, they show that BMP signaling serves more than one function across development: it is involved in the initial tangential migration from the rhombic lip, but at a later time, both up- and down-regulation of BMP activity reduces the density of amplifying cells in the external granule cell layer.

Strengths:<br /> Overall, I think the paper is interesting and important and the data is strong. The use of both chick and human tissue strengthens the findings. They are extremely rigorous, analyzing data from multiple planes at multiple ages, which also really strengthens their findings. The dual electroporation approach is extremely elegant, providing beautiful visual representations of their findings.

Weaknesses:<br /> I find no significant weaknesses.

Reviewer #2 (Public Review):

Summary:

H2S is a gas that is toxic to many animals and causes avoidance in animals such as C. elegans. The authors show that H2S increases the frequency of turning and the speed of locomotion. The response was shown to be modulated by a number of neurons and signaling pathways as well as by ambient oxygen concentrations. The long-term adaptation involved gene expression changes that may be related to iron homeostasis as well as the homeostasis of mitochondria.

Strengths:

Overall, the authors provide many pieces that will be important for solving how H2S signals through neuronal circuits to change gene expression and physiological programs. The experiments rely mostly on a behavioral assay that measures the increase of locomotion speed upon exposure to H2S. This assay is then combined with manipulations of environmental factors, different wild-type strains, and mutants. The mutants analyzed were obtained as candidates from the literature and from transcriptional profiling that the authors carried out in worms that were exposed to H2S. These studies imply several genetic signaling pathways, some neurons, and metabolism-related factors in the response to H2S. Hence the data provided should be useful for the field.

Weaknesses:

On the other hand, many important aspects of the underlying mechanisms remain unsolved and the reader is left with many loose ends. For example, it is not clear how H2S is actually sensed, how sensory neurons are activated and signal to downstream circuits, and what the role of ciliated and RMG neurons is in this circuit. It remains unclear how signals lead to gene expression and physiological changes such as metabolic rewiring. Solving all this would clearly be beyond the scope of a single manuscript. Yet, the manuscript also does not focus on understanding one of these central aspects and rather is all over the place, which makes it harder to understand for readouts that are not in this core field. Multiple additional methods and approaches exist to dig deeper into these mechanisms in the future, such as neuronal calcium imaging, optogenetics, and metabolic analysis. To generate a story that will be interesting to a broad readership substantial additional experimentation would be required. Further, in the current manuscript, it is often difficult to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved in terms of documentation, narration/explanation, and visualization.

Reviewer #2 (Public Review):

Summary:<br /> Authors hypothesized that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. They test this hypothesis in a mouse model of TBI induced by controlled cortical impact in wild-type and kcnt1 knock-out (KO) mice. The authors performed a series of experiments including behavioral assessment, electrographic recordings in vivo and in vitro, western blotting, and immunofluorescence imaging with the goal of investigating the contributory role of KNa1.1 channel to post-traumatic epileptogenesis.

Strengths:<br /> The hypothesis is innovative, focusing on the specific role of the KNa1.1 channel in the development of PTE post-TBI. The use of a comprehensive set of techniques, including EEG, whole cell patch clamp, western blot, immunofluorescence imaging, and behavioral assessments, provides a diverse data set. The study makes initial steps in correlating specific molecular changes with functional outcomes in TBI models, offering potential pathways for therapeutic intervention.

Weaknesses:<br /> 1) The study presents interesting findings on early changes in protein expression and electrophysiological properties following TBI. However, I would like to draw attention to the timeline of EEG and cellular assessments that require further clarification or consideration. The patch clamp recordings and other assays were conducted within 14 days post-TBI, while EEG recordings with or without PTZ testing were performed at 3 months post-injury. This temporal gap leaves a period where changes in electrophysiological properties and PTE status are not accounted for. Since epileptogenesis post-TBI involves a dynamic process spanning from hyperacute and acute phases to chronic development, capturing these changes continuously or at least at more frequent intervals (on and off bi-weekly) could provide a more comprehensive understanding of this progression. In the current study design, the one-week duration of EEG recordings at the 3-month timepoint raises the possibility that some seizures might have occurred undetected between the early post-injury phase and the EEG recording period. This gap could potentially affect the interpretation of results, especially when correlating early post-injury cellular changes with later seizure activity and thresholds and hence is a significant limitation to data interpretation. Experiments using western blots, immunofluorescence, and patch clamp were done at an early timepoint hence the relevance of these datasets to PTE status outcome is not established.

2) While referencing Nichols et al., 2015, to justify the 14-day timeline for characterizing seizures is understandable, it is important to consider differences in animal models (juvenile rats in Nichols vs. adult mice in the present study) which might influence the generalizability of the findings.

3) Behavior: Authors performed behavioral assays using the rotarod technique evaluating the hanging time of mice with different severity of TBI (mild, moderate, severe). The purpose of the testing is explained as 'to sort out an appropriate TBI model'. The authors also measured mortality rates 'to attain a stable model'. It is not clear what is assumed by the terms 'appropriate' or 'stable' model. Furthermore, the relevance of this to post-traumatic epileptogenesis is unclear. Additionally, the mortality in the Sham group within 2 weeks of craniectomy is not explained.

4) Seizure assessment: authors report seizure severity in PTZ-induced seizures but no mention about the severity of spontaneous seizures between different TBI severity modalities. When characterizing the PTZ-induced seizures, the mild TBI group does not have generalized seizures. Does this mean that al all 6 tested animals in the mild TBI group had exclusively focal seizures? What about the spontaneous seizure occurrence: were all seizures generalized or were any focal too? Did that differ between mild, moderate, and severe TBI?

5) Experiments with KCNT1 KO mice: in all experiments with a mutant mouse line, authors only used them in TBI group. Without the Sham group, it is difficult to discern whether any observed changes in seizure susceptibility in the KCNT1 KO TBI group are due to gene deletion, the TBI, or a combination of both. This group would provide a crucial comparison point to isolate the effects of the KCNT1 knockout from those of TBI. This limits the ability to make comprehensive conclusions about the role of the KCNT1 gene in seizure susceptibility following TBI.

6) While the current study showed interesting data about the KNa1.1 changes early after TBI, the study design and disconnect between early and late electrophysiology experiments timeline, does not establish a correlative or causative link between KNa1.1 and post-traumatic epileptogenesis since it remained unresolved whether KCNT1 KO mice developed no PTE (or less severe/ less frequent seizures at 3 months) compared with WT mice and what are the seizure properties of KCNT1 KO Sham mice compared to WT TBI and Sham groups. The hypothesis was that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. The part about 'promotes the occurrence of PTE' was not established.

7) NeuN is not the best marker of neurons in the context of TBI since TBI affects its expression patterns which will influence the interpretation of co-localization results. Unlike NeuN, Nissl staining is less likely to be affected by factors that alter protein expression, such as TBI. Therefore, it can be a more stable marker for identifying neurons in injured brain tissue.

8) Statistics: Authors report only SEM, which shows the precision of the mean and it will decrease as the sample size increases and does not reflect the data variability.

Reviewer #2 (Public Review):

In this study, Leighton et al performed remarkable experiments by combining in-vivo patch-clamp recording with two-photon dendritic Ca2+ imaging. The voltage-clamp mode is a major improvement over the pioneer versions of this combinatorial experiment that has led to major breakthroughs in the neuroscience field for visualizing and understanding synaptic input activities in single cells in-vivo (sharp electrodes: Svoboda et al, Nature 1997, Helmchen et al, Nature Neurosci 1999; whole-cell current-clamp: Jia et al, Nature 2010, Chen et al, Nature 2011. I suggest that these papers would be cited). This is because in voltage-clamp mode, despite the full control of membrane voltage in-vivo not being realistic, is nevertheless most effective in preventing back-propagation action potentials, which would severely confound the measurement of individual synaptically-induced Ca2+ influx events. Furthermore, clamping the cell body at a strongly depolarized potential (here the authors did -30mV) also facilitates the detection of synaptically-induced Ca2+ influx. As a result, the authors successfully recorded high-quality Ca2+ imaging data that can be used for precise analysis. To date, even in view of the rapid progress of voltage-sensitive indicators and relevant imaging technologies in recent years, this very old 'art' of combining single-cell electrophysiology and two-photon imaging (ordinary, raster-scanned, video-rate imaging) of Ca2+ signals still enables measurements of the best-level precision.

On the other hand, the interpretation of data in this study is a bit narrow-minded and lacks a comprehensive picture. Some suggestions to improve the manuscript are as follows:

1. The authors made a segregation of 'spine synapse' and 'shaft synapse' based solely on the two-photon images in-vivo. However, caution shall be taken here, because the optical resolution under in-vivo imaging conditions like this cannot reliably tell apart whether a bright spot within or partially overlapping a segment of the dendrite is a spine on top of (or below) it. Therefore, what the authors consider as a 'shaft synapse' (by detecting Ca2+ hotspots) has an unknown probability of being just a spine on top or below the dendrite. If there is other imaging data of higher axial resolution to validate or calibrate, the authors shall take some further considerations or analysis to check the consistency of their data, as the authors do need such a segregation between spine and shaft synapses to show how they evolve over the brain development stages.

2. The use of terminology 'bursts of spontaneous inputs' for describing voltage-clamp data seems improper. Conventionally, 'burst' refers to suprathreshold spike firing events, but here, the authors use 'burst' to refer to inward synaptic currents collected at the cell body. Not every excitatory synaptic input (or ensemble of inputs) activation will lead to spike firing under naturalistic conditions, therefore, these two concepts are not equivalent. It is recommended to use 'barrage of inputs' instead of 'burst of inputs'. Imagine a full picture of the entire dendritic tree, the fact that the authors could always capture spontaneous Ca2+ events here and there within a few pieces of dendrites within an arbitrary field-of-view suggests that, the whole dendritic tree must have many more such events going on as a barrage while the author's patch electrode picks up the summed current flow from the whole dendritic tree.

3. Following the above issue, an analysis of the temporal correlation between synaptic (not segregating 'spine' or 'shaft') Ca2+ events and EPSCs is absent. Again, the authors drew arbitrary time windows to clump the events for statistical analysis. However, the demonstrated example data already shows that the onset times of individual synaptic Ca2+ events do not necessarily align with the beginning of a 'barrage' inward current event.

4. The authors claim that "these observations indicate that the activity patterns investigated here are not or only slightly affected by low-level anesthesia". It would be nice to show some of the recordings in this work without any anesthesia to support this claim.

Reviewer #2 (Public Review):

Summary: The study by Jaime-Tobon & Moser is a truly major effort to bridge the gap between classical observations on how auditory neurons respond to sounds and the synaptic basis of these phenomena. The so-called spiral ganglion neurons (SGNs) are the primary auditory neurons connecting the brain with hair cells in the cochlea. They all respond to sounds increasing their firing rates, but also present multiple heterogeneities. For instance, some present a low threshold to sound intensity, whereas others have high threshold. This property inversely correlates with the spontaneous rate, i.e., the rate at which each neuron fires in the absence of any acoustic input. These characteristics, along with others, have been studied by many reports over the years. However, the mechanisms that allow the hair cells-SGN synapses to drive these behaviors are not fully understood.

Strengths:<br /> The level of experimental complexity described in this manuscript is unparalleled, producing data that is hardly found elsewhere. The authors provide strong proof for heterogeneity in transmitter release thresholds at individual synapses and they do so in extremely complex experimental settings. In addition, the authors found other specific differences such as in synaptic latency and max EPSCs. A reasonable effort is put into bridging these observations with those extensively reported in in vivo SGNs recordings. Similarities are many and differences are not particularly worrying as experimental conditions cannot be perfectly matched, despite the authors' efforts in minimizing them.

Weaknesses:<br /> Some concern surges in relation to mismatches with previous reports of IHC-SGN synapses function. EPSCs at these synapses present a peculiar distribution of amplitudes, shapes, and rates. These characteristics are well-established and some do not seem to be paralleled in this study. Here, amplitude distributions are drastically shifted to smaller values, and rates of events are very low, all compared with previous evidence. The reasons for these discrepancies are unclear. The rate at which spontaneous EPSCs appear is an especially sensitive matter. A great part of the conclusions relies on the definition of which of the SGNs (or should say synapses) belong to the low end and which to the high end in the spectrum of spontaneous rates. The data presented by the authors seem a bit off and the criteria used to classify recordings are not well justified. The authors should clarify the origin of these differences since they do not seem to come from obvious reasons such as animal ages, recording techniques, mouse strain, or even species.

Reviewer #2 (Public Review):

Summary:

In this paper, Sang et al. set out to identify gustatory receptors involved in salt taste sensation in Drosophila melanogaster. In a two-choice assay screen of 30 Ir mutants, they identified that Ir60b is required for avoidance of high salt. In addition, they demonstrate that activation of Ir60b neurons is sufficient for gustatory avoidance using either optogenetics or TRPV1 to specifically activate Ir60b neurons. Then, using tip recordings of labellar gustatory sensory neurons and proboscis extension response behavioral assays in Ir60b mutants, the authors demonstrate that Ir60b is dispensable for labellar taste neuron responses to high salt and the suppression of proboscis extension by high salt. Since external gustatory receptor neurons (GRNs) are not implicated, they look at Poxn mutants, which lack external chemosensory sensilla but have intact pharyngeal GRNs. High salt avoidance was reduced in Poxn mutants but was still greater than Ir60b mutants, suggesting that pharyngeal gustatory sensory neurons alone are sufficient for high salt avoidance. The authors use a new behavioral assay to demonstrate that Ir60b mutants ingest a higher volume of sucrose mixed with high salt than control flies do, suggesting that the action of Ir60b is to limit high salt ingestion. Finally, they identify that Ir60b functions within a single pair of gustatory sensory neurons in the pharynx, and that these neurons respond to high salt but not bitter tastants.

Strengths:

A great strength of this paper is that it rigorously corroborates previously published studies that have implicated specific Irs in salt taste sensation. It further introduces a new role for Ir60b in limiting high salt ingestion, demonstrating that Ir60b is necessary and sufficient for high salt avoidance and convincingly tracing the action of Ir60b to a particular subset of gustatory receptor neurons. Overall the authors have achieved their aim by identifying a new gustatory receptor involved in limiting high salt ingestion. They use rigorous genetic, imaging, and behavioral studies to achieve this aim, often confirming a given conclusion with multiple experimental approaches. They have further done a great service to the field by replicating published studies and corroborating the roles of a number of other Irs in salt taste sensation. An aspect of this study that merits further investigation is how the same gustatory receptor neurons and Ir in the pharynx can be responsible for regulating the ingestion of both appetitive (sugar) and aversive tastants (high salt).

Weaknesses:

There are several weaknesses that, if addressed, could greatly improve this work.<br /> 1) The authors combine the results and discussion but provide a very limited interpretation of their results. More discussion of the results would help to highlight what this paper contributes, how the authors interpret their results, and areas for future study.<br /> 2) The authors rename previously studied populations of labellar GRNs to arbitrary letters, which makes it difficult to understand the experiments and results in some places. These GRN populations would be better referred to according to the gustatory receptors they are known to express.<br /> 3) The conclusion that GRNs responsible for high salt aversion may be inhibited by those that function in low salt attraction is not well substantiated. This conclusion seems to come from the fact that overexpression of Ir60b in salt attraction and salt aversion sensory neurons still leads to salt aversion, but there need not be any interaction between these two types of sensory neurons if they act oppositely on downstream circuits.<br /> 4) The authors rely heavily on a new Droso-X behavioral apparatus that is not sufficiently described here or in the previous paper the authors cite. This greatly limits the reader's ability to interpret the results.

Reviewer #2 (Public Review):

Summary:<br /> McSwiggen et al present a high throughput platform for SPT that allows them to identify pharmaceutics interactions with the diffusional behavior of receptors and in turn to identify potent new ligands and cellular mechanisms. The manuscript is well written, it provides a solid new mentor and a proper experimental foundation

Strengths:<br /> The method capitalizes and extends to existing high throughput toolboxes and is directly applied to multiple receptors and ligands. The outcomes are important and relevant for society. 10^6 cells and >400 ligands per is a significant achievement.

The method can detect functionally relevant changes in transcription factor dynamics and accurately differentiate the ligand/target specificity directly within the cellular environment. This will be instrumental in screening libraries of compounds to identify starting points for the development of new therapeutics. Identifying hitherto unknown networks of biochemical signaling pathways will propel the field of single-particle live cell and quantitative microscopy in the area of diagnostics. The manuscript is well-written and clearly conveys its message.

Weaknesses:<br /> There are a few elements, that if rectified would improve the claims of the manuscript.

The authors claim that they measure receptor dynamics. In essence, their readout is a variation in diffusional behavior that correlates to ligand binding. While ligand binding can result in altered dynamics or /and shift in conformational equilibrium, SPT is not recording directly protein structural dynamics, but their effect on diffusion. They should correct and elaborate on this.

L 148 What do the authors mean 'No correlation between diffusion and monomeric protein size was observed, highlighting the differences between cellular protein dynamics versus purified systems'. This is not justified by data here or literature reference. How do the authors know these are individual molecules? Intensity distributions or single bleaching steps should be presented.

Along the same lines, the data in Figs 2 and 4 show that not only the immobile fraction is increased but also that the diffusion coefficient of the fast-moving (attributed to free) is reduced. The authors mention this and show an extended Fig 5 but do not provide an explanation. How do potential transient ligand binding and the time-dependent heterogeneity in motion (see comment above) contribute to this? Also, in line 216 the authors write "with no evidence" of transient diffusive states. How do they define transient diffusive states? While there are toolboxes to directly extract the existence and abundance of these either by HMM analysis or temporal segmentation, the authors do not discuss or use them.

The authors discuss the methods for extracting kinetic information of ligand binding by diffusion. They should consider the temporal segmentation of heterogenous diffusion. There are numerous methods published in journals or BioRxiv based on analytical or deep learning tools to perform temporal segmentation. This could elevate their analysis of Kon and Koff.

Reviewer #2 (Public Review):

Summary:

In the present manuscript, Zhang et al utilize single-nuclei RNA-Seq to investigate the heterogeneity of perirenal adipose tissue. The perirenal depot is interesting because it contains both brown and white adipocytes, a subset of which undergo functional "whitening" during early development. While adipocyte thermogenic transdifferentiation has been previously reported, there remain many unanswered questions regarding this phenomenon and the mechanisms by which it is regulated.

Strengths:

The combination of UCP1-lineage tracing with the single nuclei analysis allowed the authors to identify four populations of adipocytes with differing thermogenic potential, including a "whitened" adipocyte (mPRAT-ad2) that retains the capacity to rapidly revert to a brown phenotype upon cold exposure. They also identify two populations of white adipocytes that do not undergo browning with acute cold exposure.

Anatomically distinct adipose depots display interesting functional differences, and this work contributes to our understanding of one of the few brown depots present in humans.

Weaknesses:

The most interesting aspect of this work is the identification of a highly plastic mature adipocyte population with the capacity to switch between a white and brown phenotype. The authors attempt to identify the transcriptional signature of this ad2 subpopulation, however, the limited sequencing depth of single nuclei somewhat lessens the impact of these findings. Furthermore, the lack of any form of mechanistic investigation into the regulation of mPRAT whitening limits the utility of this manuscript. However, the combination of well-executed lineage tracing with comprehensive cross-depot single-nuclei presented in this manuscript could still serve as a useful reference for the field.

Reviewer #2 (Public Review):

This study by Matsuo-Takasaki et al. reported the development of a novel suspension culture system for hiPSC maintenance using Wnt/PKC inhibitors. The authors showed elegantly that inhibition of the Wnt and PKC signaling pathways would repress spontaneous differentiation into neuroectoderm and mesendoderm in hiPSCs, thereby maintaining cell pluripotency in suspension culture. This is a solid study with substantial data to demonstrate the quality of the hiPSC maintained in the suspension culture system, including long-term maintenance in >10 passages, robust effect in multiple hiPSC lines, and a panel of conventional hiPSC QC assays. Notably, large-scale expansion of a clinical grade hiPSC using a bioreactor was also demonstrated, which highlighted the translational value of the findings here. In addition, the author demonstrated a wide range of applications for the IWR1+LY suspension culture system, including support for freezing/thawing and PBMC-iPSC generation in suspension culture format. The novel suspension culture system reported here is exciting, with significant implications in simplifying the current culture method of iPSC and upscaling iPSC manufacturing.

Another potential advantage that perhaps wasn't well discussed in the manuscript is the reported suspension culture system does not require additional ECM to provide biophysical support for iPSC, which differentiates from previous studies using hydrogel and this should further simplify the hiPSC culture protocol.

Interestingly, although several hiPSC suspension media are currently available commercially, the content of these suspension media remained proprietary, as such the signaling that represses differentiation/maintains pluripotency in hiPSC suspension culture remained unclear. This study provided clear evidence that inhibition of the Wnt/PKC pathways is critical to repress spontaneous differentiation in hiPSC suspension culture.

I have several concerns that the authors should address, in particular, it is important to benchmark the reported suspension system with the current conventional culture system (eg adherent feeder-free culture), which will be important to evaluate the usefulness of the reported suspension system. Also, the manuscript lacks a clear description of a consistent robust effect in hiPSC maintenance across multiple cell lines. There are also several minor comments that should be addressed to improve readability, including some modifications to the wording to better reflect the results and conclusions.

Reviewer #2 (Public Review):

Summary:

The authors have developed novel Ovalbumin model (OTII) peptide that can be labeled with a site-specific FlAsH dye to track agonist peptides both in vitro and in vivo. The utility of this tool could allow better tracking of activated polyclonal T cells particularly in novel systems. The authors have provided solid evidence that peptides are functional, capable of activating OTII T cells, and these peptides can undergo trogocytosis by cognate T cells only.

Strengths:<br /> -An extensive array of in vitro and in vivo studies are used to assess peptide functionality.<br /> -Nice use of cutting edge intravital imaging,<br /> -internal controls such as multiple non-cogate T cells were used to improve robustness of the results<br /> -One of the strengths is the direct labeling of the peptide, and the potential utility in other systems.

Weaknesses:<br /> -Peptide labeling specificity and efficiency is not clear. High levels of background labeling. While it was sufficient for demonstrating the system works, it may pose problems depending on the peptide sequence, and/or use at lower dose.<br /> -Only one peptide system was tested, namely OVA323-339 region.<br /> -Limited novel biological findings. This study mostly describes a new tool that may have exciting potential.

Reviewer #2 (Public Review):

Summary:

The goal of this study was to examine the role of FNDC5 in the response of the murine skeleton to either lactation or a calcium-deficient diet. The authors find that female FNDC5 KO mice are somewhat protected from bone loss and osteocyte lacunar enlargement caused by either lactation or a calcium-deficient diet. In contrast, male FNDC5 KO mice lose more bone and have a greater enlargement of osteocyte lacunae than their wild-type controls. Based on these results, the authors conclude that in males irisin protects bone from calcium deficiency but that in females it promotes calcium removal from bone for lactation.

While some of the conclusions of this study are supported by the results, it is not clear that the modest effects of FNDC5 deletion have an impact on calcium homeostasis or milk production.

Specific comments:

1. The authors sometimes refer to FNDC5 and other times to irisin when describing causes for a particular outcome. Because irisin was not measured in any of the experiments, the authors should not conclude that lack of irisin is responsible. Along these lines, is there any evidence that either lactation or a calcium-deficient diet increases the production of irisin in mice?

2. The results of the irisin-rescue experiment shown in figure 2G cannot be appropriately interpreted without normal diet controls. In addition, some evidence that the AAV8-irisin virus actually increased irisin levels in the mice would strengthen the conclusion.

3. There is insufficient evidence to support the idea that the effect of FNDC5 on bone resorption and osteocytic osteolysis is important for the transfer of calcium from bone to milk. Previous studies by others have shown that bone resorption is not required to maintain milk or serum calcium when dietary calcium is sufficient but is critical if dietary calcium is low (Endo. 156:2762-73, 2015). To support the conclusions of the current study, it would be necessary to determine whether FNDC5 is required to maintain calcium levels when lactating mice lack sufficient dietary calcium.

4. The amount of cortical bone loss due to lactation is very similar in both WT and FNDC5 KO mice. The results of the statistical analysis of the data presented in figure 1B are surprising given the very similar effect size of lactation. The key result from the 2-way ANOVA is whether there is an effect of genotype on the effect size of lactation (genotype-lactation interaction). The interaction terms were not provided. Similar concerns are noted for the results shown in figure 1G and H.

5. It is not clear what justifies the term 'primed' or 'activated' for resorption. Is there evidence that a certain level of TRAP expression lowers the threshold for osteocytic osteolysis in response to a stimulus?

Review #2 (Public Review)

In the manuscript Watanuki et al. define the metabolic profile of HSCs in stress/proliferative (myelosuppression with 5-FU), and mitochondrial inhibition and homeostatic conditions. Their conclusions are that during proliferation HSCs rely more on glycolysis (as other cell types) while HSCs in homeostatic conditions are mostly dependent on mitochondrial metabolism. Mitochondrial inhibition is used to demonstrate that blocking mitochondrial metabolism results in similar features of proliferative conditions.

The authors used state-of-the-art technologies that allow metabolic readout in a limited number of cells like rare HSCs. These applications could be of help in the field since one of the major issues in studying HSCs metabolism is the limited sensitivity of the "standard" assays, which make them not suitable for HSC studies.

Reviewer #2 (Public Review):

Summary:<br /> The work by Varadharajan et. al. explored a previously known genetic variant and its pathophysiology in the development of alcohol-associated liver injury. It provides a plausible mechanism for how varying levels of MBOAT7 could impact the lipid metabolomics of the cell, leading to a deleterious phenotype in MBOAT7 knockout. The authors further characterized the impact of the lipidomic changes and raised lysosomal biogenesis and autophagic flux as mechanisms of how MBOAT7 deletion causes the progression of ALD.

Strengths:<br /> Connecting the GWAS data on MBOAT7 variants with plausible pathophysiology greatly enhances the translational relevance of these findings. The global lipidomic profiling of ALD mice is also very informative and may lead to other discoveries related to lipid handling pathways.

Weaknesses:<br /> The rationale of why MBOAT7 metabolites are lower in heavy drinkers than in normal individuals is not well explained. MBOAT7 loss of function drives ALD, but unclear if MBOAT7 deletion also drives preference for alcohol or if alcohol inhibits MBOAT7 function. Presuming most individuals studied here were WT and expressed an appropriate level of MBOAT7?<br /> Also, the discussion of mechanisms of MBOAT7-induced dysregulation of lysosomal biogenesis/autophagy, while very interesting, seems incomplete. It is not clear how MBOAT7 an enzyme involved in membrane phospholipid remodeling increases mTOR which leads to decreased TFEB target gene transcription. Furthermore, given the significant disturbances of global lipidomic profiling in MBOAT7 knockout, many pathways are potentially affected by this deletion. Further in vivo modeling that specifically addresses these pathways (TFEB targeting, mTOR inhibitor) would help strengthen the conclusions of this paper.

Reviewer #2 (Public Review):

Summary:

While a significant portion of immunotherapy research has focused on the pivotal role of T cells in tumor immunity, their effectiveness may be limited by the suppressive nature of the tumor environment. On the other hand, myeloid cells are commonly found within tumors and can withstand these adverse conditions. However, these cells often adopt an immunosuppressive phenotype when infiltrating tumors. Therefore, manipulating myeloid cells could potentially enhance the anti-tumor potential of immunotherapy.

In this manuscript, Farhat-Younes and colleagues have demonstrated that activating the IgM receptor signaling in myeloid cells induces an oxygen burst, the secretion of Granzyme B, and the lysis of adjacent tumor cells. Furthermore, they have outlined a strategy to utilize these features to generate CAR macrophages. However, they have identified a limitation: the expression of scFv in myeloid cells induces ER stress and the degradation of misfolded proteins. To address this issue, chimeric receptors were designed based on the high-affinity FcγRI for IgG. When macrophages transfected with these receptors were exposed to tumor-binding IgG, extensive tumor cell killing, and the release of reactive oxygen species and Granzyme B were observed.

Strengths:<br /> In general, I consider this work to be significant, and the results are compelling. It emphasizes the specific considerations and requirements for successful manipulation in myeloid cells, which could further advance the field of cellular engineering for the benefit of immunotherapy

Weaknesses:

Nevertheless, there are several minor issues that should be addressed:

1- TCR fragments are commonly used to induce ER stress in non-immune cells. Therefore, it would be interesting to investigate whether TCR fragments can be expressed in myeloid cells and if they induce ER stress. Addressing this issue would support the notion that these cells lack the ER chaperones required for folding immunoglobulin variable chains.<br /> 2- It would be valuable to determine whether, after the degradation of scFv fragments by myeloid cells, they are presented on MHC-I and MHC-II.<br /> 3- Some methodological details, such as the vaccination protocol and high-resolution microscopy procedures, are missing from the text.

Reviewer #2 (Public Review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to readers.

The data, for the most part, support the conclusions but I do have concerns about some of the data and the interpretations that should be addressed.

Reviewer #2 (Public Review):

Summary:

This combined experimental-theoretical paper introduces a novel two-domain statistical thermodynamic model (primarily Equation 1) to study allostery in generic systems but focusing here on the tetracycline repressor (TetR) family of transcription factors. This model, building on a function-centric approach, accurately captures induction data, maps mutants with precision, and reveals insights into epistasis between mutations.

Strengths:

The study contributes innovative modeling, successful data fitting, and valuable insights into the interconnectivity of allosteric networks, establishing a flexible and detailed framework for investigating TetR allostery. The manuscript is generally well-structured and communicates key findings effectively.

Weaknesses:

The only minor weakness I found was that I still don't have a better sense into (a) intuition and (b) mathematical derivation of Equation 1, which is so central to the work. I would recommend that the authors provide this early on in the main text.

Reviewer #3 (Public Review):

Summary<br /> In this research advance, the authors purport to show that the unified neutral theory of biodiversity (UNTB) is not a suitable null model for exploring the relationship between macroecological quantities, and additionally that the stochastic logistic growth model (SLM) is a viable replacement. They do this by citing other studies where UNTB was unable to capture individual macroecological quantities, and then demonstrating SLM's strength at predicting the same diversity metrics. They extend this analysis to show SLM's modeling capability at multiple scales of coarse graining, in addition to its failures at predicting these metrics' variances. Finally, authors conduct a similar analysis to Madi et al. (2020) by investigating the relationship between diversity measures within a group and across coarse-grained groups (e.g. genera diversity in one family compared to diversity of families). The authors show that choosing SLM as a null model reveals some previously reported relationships to be no longer "novel", in the sense that the patterns can be adequately captured by the null model. Authors also show that relationships not captured by the null model can be recovered by adding correlations, suggesting interactions are the driving force behind them.

Strengths<br /> 1. Authors make a strong argument that UNTB is not a good null model of macroecological observables and especially relationships between them. Authors convincingly argue that a SLM is a better null since the gamma distribution it predicts is a better description of the empirical Abundance Fluctuation Distributions (AFD).<br /> 2. Authors show that the gamma distribution predicted by SLM is a good fit for the AFD's at many different scales of coarse graining, not just the OTU level as was previously demonstrated. Authors show the same distribution predicted the mean diversity and richness at all scales of coarse graining.<br /> 3. Authors convincingly demonstrate how SLM can be used to test the relevance of interactions to macroecological relationships.

Weaknesses<br /> This reviewer's concerns were convincingly addressed by the revisions.

Overall Impact<br /> The authors present a convincing argument for the use of SLM as a better non-interacting null model for macroecological quantities and relationships.

Reviewer #2 (Public Review):

Summary:

In this study, the authors investigate the effect of ACh on neuronal responses in the auditory cortex of anesthetized rats during an auditory oddball task. The paradigm consisted of two pure tones (selected from the frequency responses at each recording site) presented in a pseudo-random sequence. One tone was presented frequently (the "standard" tone) and the other infrequently (the "deviant" tone). The authors found that ACh enhances the detection of unexpected stimuli in the auditory environment by increasing or decreasing the neuronal responses to deviant and standard tones.

Strengths:

The study includes the use of appropriate and validated methodology in line with the current state-of-the-art, rigorous statistical analysis and the demonstration of the effects of acetylcholine on auditory processing.

Weaknesses:

The study was conducted in anesthetized rats, and further research is needed to determine the behavioral relevance of these findings.

Reviewer #2 (Public Review):

Summary:

This interesting study addresses the ability of Ym1 protein crystals to promote pulmonary type 2 inflammation in vivo, in mice.

Strengths:

The data are extremely high quality, clearly presented, significantly extending previous work from this group on the type 2 immunogenicity of protein crystals.

Weaknesses:

There are no major weaknesses in this study. It would be interesting to see if Ym2 crystals behave similarly to Ym1 crystals in vivo. Some additional text in the Introduction and Discussion would enrich those sections.

Reviewer #2 (Public Review):

Summary:<br /> The work fruitfully adds to the tools to study cannabinoid action and pharmacology specifically, but also this method is applicable to other drugs, in particular, if lipophilic in nature.

Strengths:<br /> The addition of chocolate flavor overcomes aversive reactions which are often experienced in pharmacological treatments, leading to possible caveats in the interpretation of the behavioral outcomes.

Weaknesses:<br /> Certainly, more THC mediated behavioral outcomes could have been tested, but the work presents a proof-of-concept study to investigate acute THC treatment.<br /> It would have been interesting if this application form is also possible for chronic treatment regimen.

Reviewer #3 (Public Review):

Summary: This paper presents evidence of recursive self-embedding in the vocalization structure of orangutans, using fine-grained acoustical analysis. It proves the existence of isochrony nested in isochrony in the motifs produced by a nonhuman vocal system.

Strengths: Very clear written, clear analysis, excellent responses to the Reviewers.

Weaknesses: Jargonous language may be reduced. A video showing the sound as it unfolds and the spectrogram (as in Fig 1A) of the long call could be useful to best exemplify the results.

Reviewer #2 (Public Review):

Summary:<br /> Morucci et al. tested the influence of linguistic prosody long-term priors in forming predictions about simple acoustic rhythmic tone sequences composed of alternating tone duration, by violating context-dependent short-term priors formed during sequence listening. Spanish and Basque participants were selected due to the different rhythmic prosody of the two languages (functor-initial vs. Functor final, respectively), despite a common cultural background. The authors found that neuromagnetic responses to casual tone omissions reflected the linguistic prosody pattern of the participant's dominant language: in Spanish speakers, omission responses were larger to short tones, whereas in Basque speakers, omission responses were larger to long tones. Source localization of these responses revealed this interaction pattern in the left auditory cortex, which the authors interpret as reflecting a perceptual bias due to acoustic cues (inherent linguistic rhythms, rather than linguistic content). Importantly, this pattern was not found when the rhythmic sequence entailed pitch, rather than duration, cues. To my knowledge, this is the first study providing neural signatures of a known behavioral effect linking ambiguous rhythmic tone sequence perceptual organization to linguistic experience.

The conclusions of the study are well supported by the data, albeit weakly by the source analysis, but I have the impression that the rationale of the study and the analyses performed may be missing an important aspect of rhythmic sequence perception, namely the involvement of entrained oscillatory activity to the perceived rhythm, particularly phase alignment to pattern onsets. This view would not change the impact of the results but add depth to their interpretation.

Strengths:<br /> 1) The choice of participants. The bilingual population of the Basque country is perfect for performing studies that need to control for cultural and socio-economic background while having profound linguistic differences. In this sense, having dominant Basque speakers as a sample equates that in Molnar et al. (2016), and thus overcomes the lack of direct behavioral evidence for a difference in rhythmic grouping across linguistic groups. Molnar et al. (2016)'s evidence on the behavioral effect is compelling, and the evidence on neural signatures provided by the present study aligns with it.

2) The experimental paradigm. It is a well-designed acoustic sequence, that considers aspects such as gap length insertion, to be able to analyze omission responses free from subsequent stimulus-driven responses, and which includes a control sequence that uses pitch instead of duration as a cue to rhythmic grouping, which provides a stronger case for the differences found between groups to be due to prosodic duration cues.

3) Data analyses. Sound, state-of-the-art methodology in the event-related field analyses at the sensor level.

Weaknesses:<br /> 1) Despite the evidence provided on neural responses, the main conclusion of the study reflects a known behavioral effect on rhythmic sequence perceptual organization driven by linguistic background (Molnar et al. 2016, particularly). Also, the authors themselves provide a good review of the literature that evidences the influence of long-term priors in neural responses related to predictive activity. Thus, in my opinion, the strength of the statements the authors make on the novelty of the findings may be a bit far-fetched in some instances.

2) Albeit the paradigm is well designed, I fail to see the grounding of the hypotheses laid by the authors as framed under the predictive coding perspective. The study assumes that responses to an omission at the beginning of a perceptual rhythmic pattern will be stronger than at the end. I feel this is unjustified. If anything, omission responses should be larger when the gap occurs at the end of the pattern, as that would be where stronger expectations are placed: if in my language a short sound occurs after a long one, and I perceptually group tone sequences of alternating tone duration accordingly, when I hear a short sound I will expect a long one following; but after a long one, I don't necessarily need to expect a short one, as something else might occur.

3) In this regard, it is my opinion that what is reflected in the data may be better accounted for (or at least, additionally) by a different neural response to an omission depending on the phase of an underlying attentional rhythm (in terms of Large and Jones rhythmic attention theory, for instance) and putative underlying entrained oscillatory neural activity (in terms of Lakatos' studies, for instance). Certainly, the fact that the aligned phase may differ depending on linguistic background is very interesting and would reflect the known behavioral effect.

4) Source localization is performed on sensor-level significant data. The lack of source-level statistics weakens the conclusions that can be extracted. Furthermore, only the source reflecting the interaction pattern is taken into account in detail as supporting their hypotheses, overlooking other sources. Also, the right IFG source activity is not depicted, but looking at whole brain maps seems even stronger than the left. To sum up, source localization data, as informative as it could be, does not strongly support the author's claims in its current state.

Reviewer #2 (Public Review):

Summary:<br /> In this study, Hauser et al investigate the role of amphibian (Xenopus laevis) mast cells in cutaneous immune responses to the ecologically important pathogen Batrachochytrium dendrobatidis (Bd) using novel methods of in vitro differentiation of bone marrow-derived mast cells and in vivo expansion of skin mast cell populations. They find that bone marrow-derived myeloid precursors cultured in the presence of recombinant X. laevis Stem Cell Factor (rSCF) differentiate into cells that display hallmark characteristics of mast cells. They inject their novel (r)SCF reagent into the skin of X. laevis and find that this stimulates the expansion of cutaneous mast cell populations in vivo. They then apply this model of cutaneous mast cell expansion in the setting of Bd infection and find that mast cell expansion attenuates the skin burden of Bd zoospores and pathologic features including epithelial thickness and improves protective mucus production and transcriptional markers of barrier function. Utilizing their prior expertise with expanding neutrophil populations in X. laevis, the authors compare mast cell expansion using (r)SCF to neutrophil expansion using recombinant colony-stimulating factor 3 (rCSF3) and find that neutrophil expansion in Bd infection leads to greater burden of zoospores and worse skin pathology.

Strengths:<br /> The authors report a novel method of expanding amphibian mast cells utilizing their custom-made rSCF reagent. They rigorously characterize expanded mast cells in vitro and in vivo using histologic, morphologic, transcriptional, and functional assays. This establishes solid footing with which to then study the role of rSCF-stimulated mast cell expansion in the Bd infection model. This appears to be the first demonstration of the exogenous use of rSCF in amphibians to expand mast cell populations and may set a foundation for future mechanistic studies of mast cells in the X. laevis model organism.

Weaknesses:<br /> The conclusions regarding the role of mast cell expansion in controlling Bd infection would be stronger with a more rigorous evaluation of the model, as there are some key gaps and remaining questions regarding the data. For example:

1. Granulocyte expansion is carefully quantified in the initial time courses of rSCF and rCSF3 injections, but similar quantification is not provided in the disease models (Figures 3E, 4G, 5D-G). A key implication of the opposing effects of mast cell vs neutrophil expansion is that mast cells may suppress neutrophil recruitment or function. Alternatively, mast cells also express notable levels of csfr3 (Figure 2) and previous work from this group (Hauser et al, Facets 2020) showed rG-CSF-stimulated peritoneal granulocytes express mast cell markers including kit and tpsab1, raising the question of what effect rCSF3 might have on mast cell populations in the skin. Considering these points, it would be helpful if both mast cells and neutrophils were quantified histologically (based on Figure 1, they can be readily distinguished by SE or Giemsa stain) in the Bd infection models.

2. Epithelial thickness and inflammation in Bd infection are reported to be reduced by rSCF treatment (Figure 3E, 5A-B) or increased by rCSF3 treatment (Figure 4G) but quantification of these critical readouts is not shown.

3. Critical time points in the Bd model are incompletely characterized. Mast cell expansion decreases zoospore burden at 21 dpi, while there is no difference at 7 dpi (Figure 3E). Conversely, neutrophil expansion increases zoospore burden at 7 dpi, but no corresponding 21 dpi data is shown for comparison (Figure 4G). Microbiota analysis is performed at a third time point,10 dpi (Figure 5D-G), making it difficult to compare with the data from the 7 dpi and 21 dpi time points. Reporting consistent readouts at these three time points is important to draw solid conclusions about the relationship of mast cell expansion to Bd infection and shifts in microbiota.

4. Although the effect of rSCF treatment on Bd zoospores is significant at 21 dpi (Figure 3E), bacterial microbiota changes at 21 dpi are not (Figure S3B-C). This discrepancy, how it relates to the bacterial microbiota changes at 10 dpi, and why 7, 10, and 21 dpi time points were chosen for these different readouts (Figure 5F-G), is not discussed.

5. The time course of rSCF or rCSF3 treatments relative to Bd infection in the experiments is not clear. Were the treatments given 12 hours prior to the final analysis point to maximize the effect? For example, in Figure 3E, were rSCF injections given at 6.5 dpi and 20.5 dpi? Or were treatments administered on day 0 of the infection model? If the latter, how do the authors explain the effects at 7 dpi or 21 dpi given mast cell and neutrophil numbers return to baseline within 24 hours after rSCF or rCSF3 treatment, respectively?

The title of the manuscript may be mildly overstated. Although Bd infection can indeed be deadly, mortality was not a readout in this study, and it is not clear from the data reported that expanding skin mast cells would ultimately prevent progression to death in Bd infections.

Reviewer #2 (Public Review):

Summary:<br /> This study focuses on the 5-HT3 serotonin receptor, a pentameric ligand-gated ion channel important in chemical neurotransmission. There are many cryo-EM structures of this receptor with diverse ligands bound, however assignment of functional states to the structures remains incomplete. The team applies voltage-clamp fluorometry to measure, at once, both changes in ion channel activity, and changes in fluorescence. Four cysteine mutants were selected for fluorophore labeling, two near the neurotransmitter site, one in the ECD vestibule, and one at the ECD-TMD junction. Agonists, partial agonists, and antagonists were all found to yield similar changes in fluorescence, a proxy for conformational change, near the neurotransmitter site. The strength of the agonist correlated to a degree with propagation of this fluorescence change beyond the local site of neurotransmitter binding. Antagonists failed to elicit a change in fluorescence in the vestibular the ECD-TMD junction sites. The VCF results further turned up evidence supporting intermediate (likely pre-active) states.

Strengths:<br /> The experiments appear rigorous, the problem the team tackles is timely and important, the writing and the figures are for the most part very clear. We sorely need approaches orthogonal to structural biology to annotate conformational states and observe conformational transitions in real membranes- this approach, and this study, get right to the heart of what is missing.

Weaknesses:<br /> The weaknesses in the study itself are overall minor, I only suggest improvements geared toward clarity. What we are still missing is application of an approach like this to annotate the conformation of the part of the receptor buried in the membrane; there is important debate about which structure represents which state, and that is not addressed in the current study.

Reviewer #2 (Public Review):

Summary:<br /> The authors have developed a comprehensive set of tools to describe dynamics within a single time-series or across multiple time-series. The motivation is to better understand interacting networks within the human brain. The time-series used here are from direct estimates of the brain's electrical activity; however the tools have been used with other metrics of brain function and would be applicable to many other fields.

Strengths:<br /> The methods described are principled, based on generative probabilistic models.<br /> This makes them compact descriptors of the complex time-frequency data.<br /> Few initial assumptions are necessary in order to reveal this compact description.<br /> The methods are well described and demonstrated within multiple peer reviewed articles.<br /> This toolbox will be a great asset to the brain imaging community.

Weaknesses:<br /> The only question I had (originally) was how to objectively/quantitatively compare different network models. This has now been addressed by the authors in the latest revision.

Reviewer #2 (Public Review):

Summary:<br /> The authors build a gene expression model based on histone post-translational modifications and find that H3K27ac is correlated with gene expression. They proceed to perturb H3K27ac at 8 gene promoters, and measure gene expression changes to test their model.

Strengths:<br /> The combination of multiple methods to model expression, along with utilizing 6 histone datasets in 13 cell types allowed the authors to build a model that correlates between 0.7-0.79 with gene expression. This group also utilized a tool they are experts in, dCas9-p300 fusions to perturb H3K27ac and monitor gene expression to test their model. Ranked correlations showed some support for the predictions after the perturbation of H3K27ac.

Weaknesses:<br /> The perturbation of only 8 genes, and the only readout being qPCR-based gene expression, as opposed to including H3K27ac, weakened their validation of the computational model. Likewise, the use of six genes that were not expressed being most activated by dCas9-p300 might weaken the correlations vs. looking at a broad range of different gene expressions as the original model was trained on.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript characterizes a chemoattractant response to human serum by pathogenic bacteria, focusing on pathogenic strains of Salmonella enterica (Se). The researchers conducted the chemotaxis assays using a micropipette injection method that allows real-time tracking of bacterial population densities. They found that clinical isolates of several Se strains present a chemoattractant response to human serum. The specific chemoattractant within the serum is identified as L-serine, a highly characterized and ubiquitous chemoattractant, that is sensed by the Tsr receptor. They further show that chemoattraction to serum is impaired with a mutant strain devoid of Tsr. X-ray crystallography is then used to determine the structure of L-serine in the Se Tsr ligand binding domain, which differs slightly from a previously determined structure of a homologous domain. They went on to identify other pathogens that have a Tsr domain through a bioinformatics approach and show that these identified species also present a chemoattractant response to serum.

Strengths and Weaknesses:<br /> This study is well executed and the experiments are clearly presented. These novel chemotaxis assays provide advantages in terms of temporal resolution and the ability to detect responses from small concentrations. That said, it is perhaps not surprising these bacteria respond to serum as it is known to contain high levels of known chemoattractants, serine certainly, but also aspartate. In fact, the bacteria are shown to respond to aspartate and the tsr mutant is still chemotactic. The authors do not adequately support their decision to focus exclusively on the Tsr receptor. Tsr is one of the chemoreceptors responsible for observed attraction to serum, but perhaps, not the receptor. Furthermore, the verification of chemotaxis to serum is a useful finding, but the work does not establish the physiological relevance of the behavior or associate it with any type of disease progression. I would expect that a majority of chemotactic bacteria would be attracted to it under some conditions. Hence the impact of this finding on the chemotaxis or medical fields is uncertain.

The authors also state that "Our inability to substantiate a structure-function relationship for NE/DHMA signaling indicates these neurotransmitters are not ligands of Tsr." Both norepinephrine (NE) and DHMA have been shown previously by other groups to be strong chemoattractants for E. coli (Ec), and this behavior was mediated by Tsr (e.g. single residue changes in the Tsr binding pocket block the response). Given the 82% sequence identity between the Se and Ec Tsr, this finding is unexpected (and potentially quite interesting). To validate this contradictory result the authors should test E. coli chemotaxis to DHMA in their assay. It may be possible that Ec responds to NE and DHMA and Se doesn't. However, currently, the data is not strong enough to rule out Tsr as a receptor to these ligands in all cases. At the very least the supporting data for Tsr being a receptor for NE/DHMA needs to be discussed.

The authors also determine a crystal structure of the Se Tsr periplasmic ligand binding domain bound to L-Ser and note that the orientation of the ligand is different than that modeled in a previously determined structure of lower resolution. I agree that the SeTsr ligand binding mode in the new structure is well-defined and unambiguous, but I think it is too strong to imply that the pose of the ligand in the previous structure is wrong. The two conformations are in fact quite similar to one another and the resolution of the older structure, is, in my view, insufficient to distinguish them. It is possible that there are real differences between the two structures. The domains do have different sequences and, moreover, the crystal forms and cryo-cooling conditions are different in each case. It's become increasingly apparent that temperature, as manifested in differential cooling conditions here, can affect ligand binding modes. It's also notable that full-length MCPs show negative cooperativity in binding ligands, which is typically lost in the isolated periplasmic domains. Hence ligand binding is sensitive to the environment of a given domain. In short, the current data is not convincing enough to say that a previous "misconception" is being corrected.