Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors delineate the crucial role of the SIRT2-ACSS2 axis in ACSS2 degradation. They demonstrate that SIRT2 acts as an ACSS2 deacetylase specifically under nutrient stress conditions, notably during amino acid deficiency. The SIRT2-mediated deacetylation of ACSS2 at K271 consequently triggers its proteasomal degradation. Additionally, they illustrate that acetylation of ACSS2 at K271 enhances ACSS2 protein levels, thereby promoting De Novo lipogenesis.

Strengths:

The findings presented in this manuscript are clearly interesting.

Weaknesses:

Further support is required for the model put forward by the authors.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Bell et al. provide an exhaustive and clear description of the diversity of a new class of predicted type IV restriction systems that the authors denote as CoCoNuTs, for their characteristic presence of coiled-coil segments and nuclease tandems. Along with a comprehensive analysis that includes phylogenetics, protein structure prediction, extensive protein domain annotations, and in-depth investigation of encoding genomic contexts, they also provide detailed hypothesis about the biological activity and molecular functions of the members of this class of predicted systems. This work is highly relevant, it underscores the wide diversity of defence systems that are used by prokaryotes and demonstrates that there are still many systems to be discovered. The work is sound and backed up by a clear and reasonable bioinformatics approach.

Strengths:

The analysis provided by the authors is extensive and covers the three most important aspects that can be covered computationally when analysing a new family/superfamily: phylogenetics, genomic context analysis, and protein-structure-based domain content annotation. With this, one can directly have an idea about the superfamily of the predicted system and infer about their biological role. The bioinformatics approach is sound and makes use of the most current advances in the fields of protein evolution and structural bioinformatics.

Weaknesses:

It is not clear how coiled-coil segments were assigned if only based on AF2-predicted models or also backed by sequence analysis, as no description is provided in the methods. The structure prediction quality assessment is based solely on the average pLDDT of the obtained models (with a threshold of 80 or better). However, this is not enough, particularly when multimeric models were used. The PAE matrix should be used to evaluate relative orientations, particularly in the case where there is a prediction that parts from 2 proteins are interacting. In the case of multimers, interface quality scores, as the ipTM or pDockQ, should also be considered and, at minimum, reported.

These weaknesses were addressed during revision, and the results provided by the authors support their conclusions. The data resulting from this work will be useful for the general life sciences community, particularly the prokaryotic defense and microbiology communities. It also underscores the high range of functionally unknowns in sequenced genomes that are now much easier to find and interpret due to the success of deep-learning based methods and automated robust bioinformatics pipelines.

Reviewer #1 (Public Review):

Summary:

Campbell et al investigated the effects of light on the human brain, in particular the subcortical part of the hypothalamus during auditory cognitive tasks. The mechanisms and neuronal circuits underlying light effects in non-image forming responses are so far mostly studied in rodents but are not easily translated in humans. Therefore, this is a fundamental study aiming to establish the impact light illuminance has on the subcortical structures using the high-resolution 7T fMRI. The authors found that parts of the hypothalamus are differently responding to illuminance. In particular, they found that the activity of the posterior hypothalamus increases while the activity of the anterior and ventral parts of the hypothalamus decreases under high illuminance. The authors also report that the performance of the 2-back executive task was significantly better in higher illuminance conditions. However, it seems that the activity of the posterior hypothalamus subpart is negatively related to the performance of the executive task, implying that it is unlikely that this part of the hypothalamus is directly involved in the positive impact of light on performance observed. Interestingly, the activity of the posterior hypothalamus was, however, associated with an increased behavioural response to emotional stimuli. This suggests that the role of this posterior part of the hypothalamus is not as simple regarding light effects on cognitive and emotional responses. This study is a fundamental step towards our better understanding of the mechanisms underlying light effects on cognition and consequently optimising lighting standards.

Strengths:

While it is still impossible to distinguish individual hypothalamic nuclei, even with the high-resolution fMRI, the authors split the hypothalamus into five areas encompassing five groups of hypothalamic nuclei. This allowed them to reveal that different parts of the hypothalamus respond differently to an increase in illuminance. They found that higher illuminance increased the activity of the posterior part of the hypothalamus encompassing the MB and parts of the LH and TMN, while decreasing the activity of the anterior parts encompassing the SCN and another part of TMN. These findings are somewhat in line with studies in animals. It was shown that parts of the hypothalamus such as SCN, LH, and PVN receive direct retinal input in particular from ipRGCs. Also, acute chemogenetic activation of ipRGCs was shown to induce activation of LH and also increased arousal in mice.

Weaknesses:

While the light characteristics are well documented and EDI calculated for all of the photoreceptors, it is not very clear why these irradiances and spectra were chosen. It would be helpful if the authors explained the logic behind the four chosen light conditions tested. Also, the lights chosen have cone-opic EDI values in a high correlation with the melanopic EDI, therefore we can't distinguish if the effects seen here are driven by melanopsin and/or other photoreceptors. In order to provide a more mechanistic insight into the light-driven effects on cognition ideally one would use a silent substitution approach to distinguish between different photoreceptors. This may be something to consider when designing the follow-up studies.

Reviewer #1 (Public Review):

Summary:

Major findings or outcomes include a genome for the wasp, characterization of the venom constituents and teratocyte and ovipositor expression profiles, as well as information about Trichopria ecology and parasitism strategies. It was found that Trichopria cannot discriminate among hosts by age, but can identify previously parasitized hosts. The authors also investigated whether superparasitism by Trichopria wasps improved parasitism outcomes (it did), presumably by increasing venom and teratocyte concentrations/densities. Elegant use of Drosophila ectopic expression tools allowed for functional characterization of venom components (Timps), and showed that these proteins are responsible for parasitoid-induced delays in host development. After finding that teratocytes produce a large number of proteases, experiments showed that these contribute to digestion of host tissues for parasite consumption.<br /> The discussion ties these elements together by suggesting that genes used for aiding in parasitism via different parts of the parasitism arsenal arise from gene duplication and shifts in tissue of expression (to venom glands or teratocytes).

Strengths:

The strength of this manuscript is that it describes the parasitism strategies used by Trichopria wasps at a molecular and behavioral level with broad strokes. It represents a large amount of work that in previous decades might have been published in several different papers. Including all of these data in a manuscript together makes for a comprehensive and interesting study.

Weaknesses:

The weakness is that the breadth of the study results in fairly shallow mechanistic or functional results for any given facet of Trichopria's biology. Although none of the findings are especially novel given results from other parasitoid species in previous publications, integrating results together provides significant information about Trichopria biology.

Reviewer #1 (Public Review):

Summary:

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that does not respond to immunotherapy. This work represents an extension of the authors' prior observation that PI3Ka deletion in an orthotopic KPC pancreatic tumor model confers susceptibility to immune-mediated elimination. The authors' major claims in the present manuscript are as follows:

(1) PI3Ka (Pik3ca) knockout in KPC pancreatic tumor cells induces clonal T cell expansion.

(2) Genome-wide LOF screen in aKPC cells to identify tumor-intrinsic determinants of PI3Ka-KO-enhanced T cell response identified Pccb.

(3) When Pccb is knocked out in the context of Pi3ka knockout KPC, anti-tumor T cell response is reduced as measured by<br /> a. Increased tumor progression<br /> b. Decreased survival<br /> c. T cells are still clonally expanded but less functional

(4) ICB is able to "reactivate" clonally expanded T cells.

(5) Conclusion: Pccb modulates the activity of T cells in PDAC.

Overall, the experiments were appropriately executed and technically sound, albeit underpowered for single-cell analyses. Upon careful consideration of the data, the biggest weakness of the paper is the authors' interpretations of results, particularly for claims 1 and 4 (see below for details). Much of the data is correlative and does not delve into causation, leaving this reviewer wishing for experiments that would clearly demonstrate that Pccb in tumor cells directly impacts T cell anti-tumor activity.

Strengths:

(1) Tumor intrinsic determinants of intratumoral T cell infiltration in PDAC are less commonly evaluated as combination therapies for ICB. This is a point of conceptual innovation and importance.

(2) A sensitized CRISPR screen to identify mutations that rescue KPC/PI3Ka-KO tumors from immune-mediated killing is an elegant method to better understand the molecular mechanisms contributing to KPC immunosurveillance. Further, one screen candidate (Pccb) was experimentally validated.

(3) Single-cell clonotype analyses hold promise for identifying tumor-reactive T cells (though authors never demonstrated that specific clones were tumor antigen specific).

Weaknesses:

(1) "Clonal expansion of cytotoxic T cells infiltrating the pancreatic αKO tumors"<br /> a. Only two tumor-bearing hosts were evaluated by single-cell TCR sequencing, thus limiting conclusions that may be drawn regarding repertoire diversity and expansion.<br /> b. High abundance clones in the TME do not necessarily have tumor specificity, nor are they necessarily clonally expanded. They may be clones which are tissue-resident or highly chemokine-responsive and accumulate in larger numbers independent of clonal expansion. Please consider softening language to clonal enrichment or refer to clone size as clonal abundance throughout the paper.<br /> c. The whole story would be greatly strengthened by cytotoxicity assays of abundant TCR clones to show tumor antigen specificity.

(2) "A genome-wide CRISPR gene-deletion screen to identify molecules contributing to Pik3ca-mediated pancreatic tumor immune evasion"<br /> a. CRISPR mutagenesis yielded outgrowth of only 2/8 tumors. A more complete screen with an increased total number of tumors would yield much stronger gene candidates with better statistical power. It is unsurprising that candidates were observed in only one of the two tumors. Nevertheless, the authors moved forward successfully with Pccb.

(3) T cells infiltrate p-αKO tumors with increased expression of immune checkpoints<br /> a. In Figure 4D, cell counts are not normalized to totalCD8+ T cell counts making it difficult to directly compare aKO to p-aKO tumors. Based on quantifications from Figure 4D, I suspect normalization will strengthen the conclusion that CD8+ infiltrate is more exhausted in p-aKO tumors.<br /> b. Flow cytometric analysis to further characterize the myeloid compartment is incomplete (single replicate) and does not strengthen the argument that p-aKO TME is more immunosuppressive.<br /> c. It could, however, strengthen the argument that TIL has less anti-tumor potential if effector molecule expression in CD8+ infiltrating cells were quantified.

(4) Inhibition of PD1/PD-L1 checkpoint leads to elimination of most p-αKO tumors<br /> a. It is reasonable to conclude that p-aKO tumors are responsive to immune checkpoint blockade. However, there is no data presented to support the statement that checkpoint blockade reactivates an existing anti-tumor CD8+ T cell response and does not instead induce a de novo response.<br /> b. The discussion of these data implies that anti-PD-1 would not improve aKO tumor control, but these data are not included. As such, it is difficult to compare the therapeutic response in aKO versus p-aKO. Further, these data are at best an indirect comparison of the T cell responsiveness against tumor, as the only direct comparison is infiltrating cell count in Figure 4 and there are no public TCR clones with confirmed anti-tumor specificity to follow in the aKO versus p-aKO response.

Reviewer #1 (Public Review):

Summary:

This study has as its goal to determine how the structure and function of the circuit that stabilizes gaze in the larval zebrafish depends on the presence of the output cells, the motor neurons. A major model of neural circuit development posits that the wiring of neurons is instructed by their postsynaptic cells, transmitting signals retrogradely on which cells to contact and, by extension, where to project their axons. Goldblatt et al. remove the motor neurons from the circuit by generating null mutants for the phox2a gene. The study then shows that, in this mutant that lacks the isl1-labelled extraocular motor neurons, the central projection neurons have 1) largely normal responses to vestibular input; 2) normal gross morphology; 3) minimally changed transcriptional profiles. From this, the authors conclude that the wiring of the circuit is not instructed by the output neurons, refuting the major model.

Strengths:

I found the manuscript to be exceptionally well-written and presented, with clear and concise writing and effective figures that highlight key concepts. The topic of neural circuit wiring is central to neuroscience, and the paper's findings will interest researchers across the field, and especially those focused on motor systems.

The experiments conducted are clever and of a very high standard, and I liked the systematic progression of methods to assess the different potential effects of removing phox2a on circuit structure and function. Analyses (including statistics) are comprehensive and appropriate and show the authors are meticulous and balanced in most of the conclusions that they draw. Overall, the findings are interesting, and with a few tweaks, should leave little doubt about the paper's main conclusions.

Weaknesses:

The main point is the incomplete characterisation of the effects of removing phox2a on the extra-ocular motor neurons. Are these cells no longer there, or are they there but no longer labelled by isl1:GFP? If they are indeed removed, might they have developed early on, and subsequently lost? These questions matter as the central focus of the manuscript is whether the presence of these cells influences the connectivity and function of their presynaptic projection neurons. Therefore, for the main conclusions to be fully supported by the data, the authors would need to test whether 1) the motor neurons that otherwise would have been labelled by the isl1:GFP line are physically no longer there; 2) that this removal (if, indeed, it is that) is developmental. If these experiments are not feasible, then the text should be adjusted to take this into account. A further point to address is the context of the manipulation. If the phox2a removal does indeed take out the extra-ocular motor neurons, what percentage of postsynaptic neurons to the projection neurons are still present? In other words, how does the postsynaptic nMLF output relate to the motor neurons? If, for instance, the nMLF (which, as the authors state, are likely still innervated by the projection neurons) are the main output of the projections neurons, then this would affect the interpretation of the results.

Reviewer #1 (Public Review):

Though the Norrin protein is structurally unrelated to the Wnt ligands, it can activate the Wnt/β-catenin pathway by binding to the canonical Wnt receptors Fzd4 and Lrp5/6, as well as the tetraspanin Tspan12 co-receptor. Understanding the biochemical mechanisms by which Norrin engages Tspan12 to initiate signaling is important, as this pathway plays an important role in regulating retinal angiogenesis and maintaining the blood-retina-barrier. Numerous mutations in this signaling pathway have also been found in human patients with ocular diseases. The overarching goal of the study is to define the biochemical mechanisms by which Tspan12 mediates Norrin signaling. Using purified Tspan12 reconstituted in lipid nanodiscs, the authors conducted detailed binding experiments to document the direct, high-affinity interactions between purified Tspan12 and Norrin. To further model this binding event, they used AlphaFold to dock Norrin and Tspan12 and identified four putative binding sites. They went on to validate these sites through mutagenesis experiments. Using the information obtained from the AlphaFold modeling and through additional binding competition experiments, it was further demonstrated that Tspan12 and Fzd4 can bind Norrin simultaneously, but Tspan12 binding to Norrin is competitive with other known co-receptors, such as HSPGs and Lrp5/6. Collectively, the authors proposed that the main function of Tspan12 is to capture low concentrations of Norrin at the early stage of signaling, and then "hand over" Norrin to Fzd4 and Lrp5/6 for further signal propagation. Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Strengths:

• Biochemical reconstitution of Tspan12 and Fzd4 in lipid nanodiscs is an elegant approach for testing the direct binding interaction between Norrin and its co-receptors. The proteins used for the study seem to be of high purity and quality.

• The various binding experiments presented throughout the study were carried out rigorously. In particular, BLI allows accurate measurement of equilibrium binding constants as well as on and off rates.

• It is nice to see that the authors followed up on their AlphaFold modeling with an extensive series of mutagenesis studies to experimentally validate the potential binding sites. This adds credence to the AlphaFold models.

• Table S1 is a further testament to the rigor of the study.

• Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Suggestions for improvement:

• It would be helpful to show Coomassie-stained gels of the key mutant Norrin and Tspan12 proteins presented in Figures 2E and 2F.

• Many Norrin and Tspan12 mutations have been identified in human patients with FEVR. It would be interesting to comment on whether any of the mutations might affect the Norrin-Tspan12 binding sites described in this study.

• Some of the negative conclusions (e.g. the lack of involvement of Tspan12 in the formation of the Norrin-Lrp5/6-Fzd4-Dvl signaling complex) can be difficult to interpret. There are many possible reasons as to why certain biological effects are not recapitulated in a reconstitution experiment. For instance, the recombinant proteins used in the experiment may not be presented in the correct configurations, and certain biochemical modifications, such as phosphorylation, may also be missing.

Reviewer #1 (Public Review):

Summary:<br /> Bendzunas, Byrne et al. explore two highly topical areas of protein kinase regulation in this manuscript. Firstly, the idea that Cys modification could regulate kinase activity. The senior authors have published some standout papers exploring this idea of late, and the current work adds to the picture of how active site Cys might have been favoured in evolution to serve critical regulatory functions. Second, BRSK1/2 are understudied kinases listed as part of the "dark kinome" so any knowledge of their underlying regulation is of critical importance to advancing the field.

Strengths:<br /> In this study, the author pinpoints highly-conserved, but BRSK-specific, Cys residues as key players in kinase regulation. There is a delicate balance between equating what happens in vitro with recombinant proteins relative to what the functional consequence of Cys mutation might be in cells or organisms, but the authors are very clear with the caveats relating to these connections in their descriptions and discussion. Accordingly, by extension, they present a very sound biochemical case for how Cys modification might influence kinase activity in cellular environs.

Comments on revised version:

The authors have satisfactorily addressed my concerns.

Reviewer #1 (Public Review):

Summary:

The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

Strengths:

The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

Weaknesses:

(1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

(2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

(3) Fig.1B-C: To give a standard deviation from 2 data points has no statistical significance. In this case it would be better to define as range/difference of the 2 data points.

Reviewer #1 (Public Review):

Summary:

Tuberculous meningitis (TBM) is one of the most severe form of extrapulmonary TB. TBM is especially prevalent in people who are immunocompromised (e.g. HIV-positive). Delays in diagnosis and treatment could lead to severe disease or mortality. In this study, the authors performed the largest ever host whole blood transcriptomics analysis on a cohort of 606 Vietnamese participants. The results indicated that TBM mortality is associated with increased neutrophil activation and decreased T and B cell activation pathways. Furthermore, increased angiogenesis was also observed in HIV-positive patients who died from TBM, whereas activated TNF signaling and down-regulated extracellular matrix organisation were seen in the HIV-negative group. Despite similarities in transcriptional profiles between PTB and TBM compared to healthy controls, inflammatory genes were more active in HIV-positive TBM. Finally, 4 hub genes (MCEMP1, NELL2, ZNF354C and CD4) were identified as strong predictors of death from TBM.

Strengths:

This is a really impressive piece of work, both in terms of the size of the cohort which took years of effort to recruit, sample and analyse and also the meticulous bioinformatics performed. The biggest advantage of obtaining a whole blood signature is that it allows an easier translational development into test that can be used in the clinical with a minimally invasive sample. Furthermore, the data from this study has also revealed important insights in the mechanisms associated with mortality and the differences in pathogenesis between HIV-positive and HIV-negative patients, which would have diagnostic and therapeutic implications.

Weaknesses:

The authors have addressed all the weaknesses in the revised version.

Reviewer #2 (Public Review):

Summary:

This study from Bamgbose et al. identifies a new and important interaction between H4K20me and Parp1 that regulates inducible genes during development and heat stress. The authors present convincing experiments that form a mostly complete manuscript that significantly contributes to our understanding of how Parp1 associates with target genes to regulate their expression.

Strengths:

The authors present 3 compelling experiments to support the interaction between Parp1 and H4K20me, including:

(1) PR-Set7 mutants remove all K4K20me and phenocopy Parp mutant developmental arrest and defective heat shock protein induction.

(2) PR-Set7 mutants have dramatically reduced Parp1 association with chromatin and reduced poly-ADP ribosylation.

(3) Parp1 directly binds H4K20me in vitro.

Reviewer #1 (Public Review):

Summary:

The endocannabinoid system (ECS) components are dysregulated within the lesion microenvironment and systemic circulation of endometriosis patients. Using endometriosis mouse models and genetic loss of function approaches, Lingegowda et al. report that canonical ECS receptors, CNR1 and CNR2, are required for disease initiation, progression, and T-cell dysfunction.

Strengths:

The approach uses genetic approaches to establish in vivo causal relationships between dysregulated ECS and endometriosis pathogenesis. The experimental design incorporates bulk RNAseq approaches, as well as imaging mass spectrometry to characterize the mouse lesions. The identification of immune-related and T-cell-specific changes in the lesion microenvironment of CNR1 and CNR2 knockout (KO) mice represents a significant advance

Weaknesses:

Although the mouse phenotypic analyses involve a detailed molecular characterization of the lesion microenvironment using genomic approaches, detailed measurements of lesion size/burden and histopathology would provide a better understanding of how CNR1 or CNR2 loss contributes to endometriosis initiation and progression. The cell or tissue-specific effects of the CNR1 and CNR2 are not incorporated into the experimental design of the studies. Although this aspect of the approach is recognized as a major limitation, global CNR1 and CNR2 KO may affect normal female reproductive tract function, ovarian steroid hormone levels, decidualization response, or lead to preexisting alterations in host or donor tissues, which could affect lesion establishment and development in the surgically induced, syngeneic mouse model of endometriosis.

Reviewer #1 (Public Review):

Strengths

The paper has shown the expression of RGS10 is related to the molecular subtype, distant metastasis, and survival status of breast cancer. The study utilizes bioinformatic analyses, human tissue samples, and in vitro and in vivo experiments which strengthen the data. RGS10 was validated to inhibit EMT through a novel mechanism dependent on LCN2 and miR-539-5p, thereby reducing cancer cell proliferation, colony formation, invasion, and migration. The study elaborated the function of RGS10 in influencing the prognosis and biological behavior which could be considered as a potential drug target in breast cancer.

Weakness<br /> The mechanism by which the miR-539-5p/RGS10/LCN2 axis may be related to the prognosis of cancer patients still needs to be elucidated. In addition, the sample size used is relatively limited. Especially, if further exploration of the related pathways and mechanisms of LCN2 can be carried out by using organoid models, as well as the potential of RGS10 as a biomarker for further clinical translation to verify its therapeutic target effect, which will make the data more convincing.

Reviewer #1 (Public Review):

Summary:

This is an interesting study that utilizes a novel epigenome profiling technology (single molecule imaging) in order to demonstrate its utility as a readout of therapeutic response in multiple DIPG cell lines. Two different drugs were evaluated, singly and in combination. Sulfopin, an inhibitor of a component upstream of the MYC pathway, and Vorinostat, an HDAC inhibitor. Both drugs sensitised DIPG cells, but high (>10 micromolar) concentrations were needed to achieve half-maximal effects. The combination seemed to have some efficacy in vivo, but also produced debilitating side-effects that precluded the measurement of any survival benefit.

Strengths:

Interesting use of a novel epigenome profiling technology (single molecule imaging).

Weaknesses:

The use of this novel imaging technology ultimately makes up only a minor part of the study. The rest of the results, i.e. DIPG sensitivity to HDAC and MYC pathway inhibition, have already been demonstrated by others (Grasso Monje 2015; Pajovic Hawkins 2020, among others). The drugs have some interesting opposing effects at the level of the epigenome, demonstrated through CUT&RUN, but this is not unexpected in any way. The drugs evaluated here also didn't have higher efficacy, or efficacy at especially low concentrations, than inhibitors used in previous reports. The combination therapy attempted here also caused severe side effects in mice (dehydration/deterioration), such that an effect on survival could not be determined. I'm not sure this study advances knowledge of targeted therapy approaches in DIPGs, or if it iterates on previous findings to deliver new, or more efficient, mechanistic or therapeutic/pharmaclogic insights. It is a translational report evaluating two drugs singly and in combination, finding that although they sensitise cells in vitro, efficacy in vivo is limited at best, as this particular combination cannot progress to human translation.

Reviewer #1 (Public Review):

Summary:

The authors want to explore how much two known minibinder protein domains against the Spike protein of SARS-CoV-2 can function as a binding domain of 2 sets of synthetic receptors (SNIPR and CAR); the authors also want to know how some modifications of the linkers of these new receptors affect their activation profile.

Major strengths and weaknesses of the methods and results:

- Strengths include: analysis of synthetic receptor function for 2 classes of synthetic receptors, with robust and appropriate assays for both kinds of receptors. The modifications of the linkers are also interesting and the types of modifications that are often used in the field.

- Weaknesses include: none of the data analysis provides statistical interpretation of the results (that I could find). One dataset is confusing: Figures 5A and C, are said to be the same assay with the same constructs, but the results are 30% in A, and 70% in C.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

Given the open-ended nature of the goal (implicit in it being an exploration), it is hard to say if the authors have reached their aims; they have done an exploration for sure; is it big enough an exploration? This reviewer is not sure.

The results are extremely clearly presented, both in the figures and in the text, both for the methods and the results. The claims put forward (with limited exceptions see below) are very solidly supported by the presented data.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

The work may stimulate others to consider minibinders as potential binding domains for synthetic receptors. The modifications that are presented although not novel, do provide a starting point for larger-scale analysis.

It is not clear how much this is generalizable to other binders (the authors don't make such claims though). The claims are very focused on the tested modifications, and the 2 receptors and minibinder used, a scope that I would define as narrow; the take-home message if one wants to try it with other minibinders or other receptors seems to be: test a few things, and your results may surprise you.

Any additional context you think would help readers interpret or understand the significance of the work:

We are at the infancy stage of synthetic receptors optimization and next-generation derivation; there is a dearth of systematic studies, as most focus is on developing a few ones that work. This work is an interesting attempt to catalyze more research with these new minibinders. Will it be picked up based on this? Not sure.

Reviewer #1 (Public Review):

Summary:

This paper explores the contribution of transgenerational effects to phenotypic variation in twenty-five phenotypes and transcript variation in the heart, liver, pituitary, whole embryo, and placenta. The authors use a powerful design, exploiting the use of consomics, and argue that there are no observable changes attributable to the differences in the parental origin of the four chromosomes they examine.

Strengths:<br /> It's good to see a use for consomics. This is a powerful and useful design to address the problem they are tackling.

Weaknesses:<br /> The difficulty faced by the authors is that they have interrogated only a small portion of the genome, using bulk RNA sequencing and a set of correlated phenotypes, thus restricting the conclusions they can draw from the absence of significant findings.

Reviewer #1 (Public Review):

In this study, the authors explored how the reduced growth fitness, resulting from genome reduction, can be compensated through evolution. They conducted an evolution experiment with a strain of Escherichia coli that carried a reduced genome, over approximately 1,000 generations. The authors carried out sequencing, and found no clear genetic signatures of evolution across replicate populations. They carry out transcriptomics and a series of analyses that lead them to conclude that there are divergent mechanisms at play in individual evolutionary lineages. The authors used gene network reconstruction to identify three gene modules functionally differentiated, correlating with changes in growth fitness, genome mutation, and gene expression, respectively, due to evolutionary changes in the reduced genome.

I think that this study addresses an interesting question. Many microbial evolution experiments evolve by loss of function mutations, but presumably a cell that has already lost so much of its genome needs to find other mechanisms to adapt. Experiments like this have the potential to study "constructive" rather than "destructive" evolution.

Comments on revised version:

I think the authors have carefully gone through the manuscript and addressed all of my concerns.

Reviewer #1 (Public Review):

⍺-synuclein (syn) is a critical protein involved in many aspects of human health and disease. Previous studies have demonstrated that post-translational modifications (PTMs) play an important role in regulating the structural dynamics of syn. However, how post-translational modifications regulate syn function remains unclear. In this manuscript, Wang et al. reported an exciting discovery that N-acetylation of syn enhances the clustering of synaptic vesicles (SVs) through its interaction with lysophosphatidylcholine (LPC). Using an array of biochemical reconstitution, single vesicle imaging, and structural approaches, the authors uncovered that N-acetylation caused distinct oligomerization of syn in the presence of LPC, which is directly related to the level of SV clustering. This work provides novel insights into the regulation of synaptic transmission by syn and might also shed light on new ways to control neurological disorders caused by syn mutations.

Reviewer #1 (Public Review):

Summary:

The mammalian Shieldin complex consisting of REV7 (aka MAD2L2, MAD2B) and SHLD1-3 affects pathway usage in DSB repair favoring non-homologous endjoining (NHEJ) at the expense of homologous recombination (HR) by blocking resection and/or priming fill-in DNA synthesis to maintain or generate near blunt ends suitable for NHEJ. While the budding yeast Saccharomyces cerevisiae does not have homologs to SHLD1-3, it does have Rev7, which was identified to function in conjunction with Rev3 in the translesion DNA polymerase zeta. Testing the hypothesis that Rev7 also affects DSB resection in budding yeast, the work identified a direct interaction between Rev7 and the Rad50-Mre11-Xrs2 complex by two-hybrid and direct protein interaction experiments. Deletion analysis identified that the 42 amino acid C-terminal region was necessary and sufficient for the 2-hybrid interaction. Direct biochemical analysis of the 42 aa peptide was not possible. Rev7 deficient cells were found to be sensitive to HU only in synergy with G2 tetraplex forming DNA. Importantly, the 42 aa peptide alone suppressed this phenotype. Biochemical analysis with full-length Rev7 and a C-terminal truncation lacking the 42 aa region shows G4-specific DNA binding that is abolished in the C-terminal truncation and with a substrate containing mutations to prevent G4 formation. Rev7 lacks nuclease activity but inhibits the dsDNA exonuclease activity of Mre11. The C-terminal truncation protein lacking the 42 aa region also showed some inhibition suggesting the involvement of additional binding sites besides the 42 aa region. Also, the Mre11 ssDNA endonuclease activity is inhibited by Rev7 but not the degradation of linear ssDNA. Rev7 does not affect ATP binding by Rad50 but inhibits in a concentration-dependent manner the Rad50 ATPase activity. The C-terminal truncation protein lacking the 42 aa region also showed some inhibition but significantly less than the full-length protein.

Using an established plasmid-based NHEJ assay, the authors provide strong evidence that Rev7 affects NEHJ, showing a four-fold reduction in this assay. The mutations in the other Pol zeta subunits, Rev3 and Rev1, show a significantly smaller effect (~25% reduction). A strain expressing only the Rev7 C-terminal 42 aa peptide showed no NHEJ defect, while the truncation protein lacking this region exhibited a smaller defect than the deletion of REV7. The conclusion that Rev7 supports NHEJ mainly through the 42 aa region was validated using a chromosomal NHEJ assay. The effect on HR was assessed using a plasmid:chromosome system containing G4 forming DNA. The rev7 deletion strain showed an increase in HR in this system in the presence and absence of HU. Cells expressing the 42 aa peptide were indistinguishable from the wild type as were cells expressing the Rev7 truncation lacking the 42 aa region. The authors conclude that Rev7 suppresses HR, but the context appears to be system-specific and the conclusion that Rev7 abolished HR repair of DSBs is unwarranted and overly broad.

Strength:

This is a well-written manuscript with many well-executed experiments that suggest that Rev7 inhibits MRX-mediated resection to favor NEHJ during DSB repair. This finding is novel and provides insight into the potential mechanism of how the human Shieldin complex might antagonize resection.

Weaknesses:

The nuclease experiments were conducted using manganese as a divalent cation, and it is unclear whether there is an effect with the more physiological magnesium cation. Additional controls for the ATPase and nuclease experiments to eliminate non-specific effects would be helpful. Evidence for an effect on resection in cells is lacking. The major conclusion about the role of Rev7 in regulating the choice between HR and NHEJ is not justified, as only a highly specialized assay is used that does not warrant the broad conclusion drawn. Specifically, the results that the Rev7 C-terminal truncation lacking the 42 aa region still suppresses HR is unexpected and unexplained. The effect of Rev7 on G4 metabolism is underdeveloped and distracts from the main results that Rev7 modulated MRX activity. The authors should consider removing this part and develop a more complete story on this later.

Reviewer #1 (Public Review):

Summary:

Extracellular ATP represents a danger-associated molecular pattern associated to tissue damage and can act also in an autocrine fashion in macrophages to promote proinflammatory responses, as observed in a previous paper by the authors in abdominal sepsis. The present study addresses an important aspect possibly conditioning the outcome of sepsis that is the release of ATP by bacteria. The authors show that sepsis-associated bacteria do in fact release ATP in a growth dependent and strain-specific manner. However, whether this bacterial derived ATP play a role in the pathogenesis of abdominal sepsis has not been determined. To address this question, a number of mutant strains of E. coli has been used first to correlate bacterial ATP release with growth and then, with outer membrane integrity and bacterial death. By using E. coli transformants expressing the ATP-degrading enzyme apyrase in the periplasmic space, the paper nicely shows that abdominal sepsis by these transformants results in significantly improved survival. This effect was associated with a reduction of peritoneal macrophages and CX3CR1+ monocytes, and an increase in neutrophils. To extrapolate the function of bacterial ATP from the systemic response to microorganisms, the authors exploited bacterial OMVs either loaded or not with ATP to investigate the systemic effects devoid of living microorganisms. This approach showed that ATP-loaded OMVs induced degranulation of neutrophils after lysosomal uptake, suggesting that this mechanism could contribute to sepsis severity.

Strengths:

A strong part of the study is the analysis of E. coli mutants to address different aspects of bacterial release of ATP that could be relevant during systemic dissemination of bacteria in the host.

Weaknesses:

As pointed out in the limitations of the study whether ATP-loaded OMVs provide a mechanistic proof of the pathogenetic role of bacteria-derived ATP independently of live microorganisms in sepsis is interesting but not definitively convincing. It could be useful to see whether degranulation of neutrophils is differentially induced by apyrase-expressing vs control E. coli transformants. Also, the increase of neutrophils in bacterial ATP-depleted abdominal sepsis, which has better outcomes than "ATP-proficient" sepsis, seems difficult to correlate to the hypothesized tissue damage induced by ATP delivered via non-infectious OMVs. Are the neutrophils counts affected by ATP delivered via OMVs? A comparison of cytokine profiles in the abdominal fluids of E. coli and OMV treated animals could be helpful in defining the different responses induced by OMV-delivered vs bacterial-released ATP. The analyses performed on OMV treated versus E. coli infected mice are not closely related and difficult to combine when trying to draw a hypothesis for bacterial ATP in sepsis. Also it was not clear why lung neutrophils were used for the RNAseq data generation and analysis.

Joint Public Review

This work investigates the evolutionary conservation and functional significance of FoxO transcription factors in the response of airway epithelia to diverse stressors, ranging from hypoxia to temperature fluctuations and oxidative stress. Utilizing a comprehensive approach encompassing Drosophila, murine models, and human samples, the study investigates FoxO's role across species. The authors demonstrate that hypoxia triggers a dFOXO-dependent immune response in Drosophila airways, with subsequent nuclear localization of dFOXO in response to various stressors. Transcriptomic analysis reveals differential regulation of crucial gene categories in respiratory tissues, highlighting FoxO's involvement in metabolic pathways, DNA replication, and stress resistance mechanisms.

The study underscores FoxO's importance in maintaining homeostasis by revealing reduced stress resistance in dFOXO Drosophila mutants, shedding light on its protective role against stressors. In mammalian airway cells, FoxO exhibits nuclear translocation in response to hypoxia, accompanied by upregulation of cytokines with antimicrobial activities. Intriguingly, mouse models of asthma show FoxO downregulation, which is also observed in sputum samples from human asthma patients, implicating FoxO dysregulation in respiratory pathologies.

Overall, the manuscript suggests that FoxO signaling plays a critical role in preserving airway epithelial cell homeostasis under stress conditions, with implications for understanding and potentially treating respiratory diseases like asthma. By providing compelling evidence of FoxO's involvement across species and its correlation with disease states, the study underscores the importance of further exploration into FoxO-mediated mechanisms in respiratory health.

Strengths

(1) This study shows that FoxO transcription factors are critical for regulating immune and inflammatory responses across species, and for orchestrating responses to various stressors encountered by airway epithelial cells, including hypoxia, temperature changes, and oxidative stress. Understanding the intricate regulation of FoxO transcription factors provides insights into modulating immune and inflammatory pathways, offering potential avenues for therapeutic interventions against respiratory diseases and other illnesses.

(2) The work employs diverse model systems, including Drosophila, murine models, and human samples, thereby establishing a conserved role for FoxOs in airway epithelium and aiding translational relevance to human health.

(3) The manuscript establishes a strong correlation between FoxO expression levels and respiratory diseases such as asthma. Through analyses of both murine models of asthma and asthmatic human samples, the study demonstrates a consistent reduction in FoxO expression, indicating its potential involvement in the pathogenesis of respiratory disorders. This correlation underscores the clinical relevance of FoxO dysregulation and opens avenues for developing treatments for respiratory conditions like asthma, COPD, and pulmonary fibrosis, addressing significant unmet clinical needs.

(4) The study unveils intriguing mechanistic details regarding FoxO regulation and function. Particularly noteworthy is the observation of distinct regulatory mechanisms governing dFOXO translocation in response to different stressors. The independence of hypoxia-induced dFOXO translocation from JNK signaling adds complexity to our understanding of FoxO-mediated stress responses. Such mechanistic insights deepen our understanding of FoxO biology and pave the way for future investigations into the intricacies of FoxO signaling pathways in airway epithelial cells.

Weaknesses

(1) The manuscript does not distinguish between FoxO expression levels and FoxO activation status. While FoxO nuclear localization is observed in Drosophila and murine models, it remains unclear whether this reflects active FoxO signaling or merely FoxO expression, limiting the mechanistic understanding of FoxO regulation.

(2) The manuscript utilizes various stressors across different experiments without providing a clear rationale for their selection. This lack of coherence in stressor choice complicates the interpretation of results and diminishes the ability to draw meaningful comparisons across experiments.

(3) The manuscript frequently refers to "FoxO signaling" without providing specific signaling readouts. This ambiguity undermines the clarity of the conclusions drawn from the data and hinders the establishment of clear cause-and-effect relationships between FoxO activation and cellular responses to stress.

(4) Many conclusions drawn in the manuscript rely heavily on the quantification of immunostaining images for FoxO nuclear localization. While this is an important observation, it does not provide a sufficient mechanistic understanding of FoxO expression or activation regulation.

(5) The primary weakness in the Drosophila experiments is the analysis of dFoxO in homozygous dFoxO mutant animals, which precludes determining the specific role of dFoxO in airway cells. Despite available tools for tissue-specific gene manipulation, such as tissue-specific RNAi and CRISPR techniques, these approaches were not employed, limiting the precision of the findings.

(6) In mammalian experiments, the results are primarily correlative, lacking causal evidence. While changes in FoxO expression are observed under pathological conditions, the absence of experiments on FoxO-deficient cells or tissues precludes establishing a causal relationship between FoxO dysregulation and respiratory pathologies.

(7) Although the evidence suggests a critical role for FoxO in airway tissues, the precise nature of this role remains unclear. With gene expression changes analyzed only in Drosophila, the extent of conservation in downstream FoxO-mediated pathways between mammals and Drosophila remains uncertain. Additionally, the functional consequences of FoxO deficiency in airway cells were not determined, hindering comparisons between species and limiting insights into FoxO's functional roles in different contexts.

Reviewer #1 (Public Review):

In this manuscript, entitled " Merging Multi-OMICs with Proteome Integral Solubility Alteration Unveils Antibiotic Mode of Action", Dr. Maity and colleagues aim to elucidate the mechanisms of action of antibiotics through combined approaches of omics and the PISA tool to discover new targets of five drugs developed against Helicobacter pylori.

Strengths:

Using transcriptomics, proteomic analysis, protein stability (PISA), and integrative analysis, Dr. Maity and colleagues have identified pathways targeted by five compounds initially discovered as inhibitors against H. pylori flavodoxin. This study underscores the necessity of a global approach to comprehensively understanding the mechanisms of drug action. The experiments conducted in this paper are well-designed and the obtained results support the authors' conclusions.

Weaknesses:

This manuscript describes several interesting findings. A few points listed below require further clarification:

(1) Compounds IVk exhibits markedly different behavior compared to the other compounds. The authors are encouraged to discuss these findings in the context of existing literature or chemical principles.

(2) The incubation time for treating H. pylori with the drugs was set at 4 hours for transcriptomic and proteomic analyses, compared to 20 min for PISA analysis. The authors need to explain the reason for these differences in treatment duration.

(3) The PISA method facilitates the identification of proteins stabilized by drug treatment. DnaJ and Trigger factor (tig), well-known molecular chaperones, prevent protein aggregation under stress. Their enrichment in the soluble fraction is expected and does not necessarily indicate direct stabilization by the drugs. The possibility that their stabilization results from binding to other proteins destabilized by the drugs should be considered. To prevent any misunderstanding, the authors should clarify that their methodology does not solely identify direct targets. Instead, the combination of their findings sheds light on various pathways affected by the treatment.

(4) At the end of the manuscript, the authors conclude that four compounds "strongly interact with CagA". However, detailed molecule/protein interaction studies are necessary to definitively support this claim. The authors should exercise caution in their statement. As the authors mentioned, additional research (not mandated in the scope of this current paper) is necessary to determine the drug's binding affinity to the proposed targets.

(5) The authors should clarify the PISA-Express approach over standard PISA. A detailed explanation of the differences between both methods in the main text is important.

Reviewer #1 (Public Review):

Summary:

This manuscript provides an initial characterization of three new missense variants of the PLCG1 gene associated with diverse disease phenotypes, utilizing a Drosophila model to investigate their molecular effects in vivo. Through the meticulous creation of genetic tools, the study assesses the small wing (sl) phenotype - the fly's ortholog of PLCG1 - across an array of phenotypes from longevity to behavior in both sl null mutants and variants. The findings indicate that the Drosophila PLCG1 ortholog displays aberrant functions. Notably, it is demonstrated that overexpression of both human and Drosophila PLCG1 variants in fly tissue leads to toxicity, underscoring their pathogenic potential in vivo.

Strengths:

The research effectively highlights the physiological significance of sl in Drosophila. In addition, the study establishes the in vivo toxicity of disease-associated variants of both human PLCG1 and Drosophila sl.

Weaknesses:

The study's limitations include the human PLCG1 transgene's inability to compensate for the Drosophila sl null mutant phenotype, suggesting potential functional divergence between the species. This discrepancy signals the need for additional exploration into the mechanistic nuances of PLCG1 variant pathogenesis, especially regarding their gain-of-function effects in vivo.

Overall:

The study offers compelling evidence for the pathogenicity of newly discovered disease-related PLCG1 variants, manifesting as toxicity in a Drosophila in vivo model, which substantiates the main claim by the authors. Nevertheless, a deeper inquiry into the specific in vivo mechanisms driving the toxicity caused by these variants in Drosophila could significantly enhance the study's impact.

Reviewer #1 (Public Review):

Summary:

Casas-Tinto et al. present convincing data that injury of the adult Drosophila CNS triggers transdifferentiation of glial cells and even the generation of neurons from glial cells. This observation opens up the possibility of getting a handle on the molecular basis of neuronal and glial generation in the vertebrate CNS after traumatic injury caused by Stroke or Crush injury. The authors use an array of sophisticated tools to follow the development of glial cells at the injury site in very young and mature adults. The results in mature adults revealing a remarkable plasticity in the fly CNS and dispels the notion that repair after injury may be only possible in nerve cords which are still developing. The observation of so-called VC cells which do not express the glial marker repo could point to the generation of neurons by former glial cells.

Conclusion:

The authors present an interesting story that is technically sound and could form the basis for an in-depth analysis of the molecular mechanism driving repair after brain injury in Drosophila and vertebrates.

Strengths:

The evidence for transdifferentiation of glial cells is convincing. In addition, the injury to the adult CNS shows an inherent plasticity of the mature ventral nerve cord which is unexpected.

Weaknesses:

Traumatic brain injury in Drosophila has been previously reported to trigger mitosis of glial cells and generation of neural stem cells in the larval CNS and the adult brain hemispheres. Therefore this report adds to but does not significantly change our current understanding. The origin and identity of VC cells is unclear.

Good example with capitalization.

Reviewer #1 (Public Review):

In this manuscript, Huang and colleagues explored the role of iron in bacterial therapy for cancer. Using proteomics, they revealed the upregulation of bacterial genes that uptake iron, and reasoned that such regulation is an adaptation to the iron-deficient tumor microenvironment. Logically, they engineered E. Coli strains with enhanced iron-uptake efficiency, and showed that these strains, together with iron scavengers, suppress tumor growth in a mouse model. Lastly, they reported the tumor suppression by IroA-E. Coli provides immunological memory via CD8+ T cells. In general, I find the findings in the manuscript novel and the evidence convincing.

(1) Although the genetic and proteomic data are convincing, would it be possible to directly quantify the iron concentration in (1) E. Coli in different growth environments, and (2) tumor microenvironment? This will provide functional consequence of upregulating genes that import iron into the bacteria.

(2) Related to 1, the experiment to study the synergistic effect of CDG and VLX600 (lines 139-175) is very nice and promising, but one flaw here is a lack of the measurement of iron concentration. Therefore, a possible explanation could be that CDG acts in another manner, unrelated to iron uptake, that synergizes with VLX600's function to deplete iron from cancer cells. Here, a direct measurement of iron concentration will show the effect of CDG on iron uptake, thus complementing the missing link.

(3) Lines 250-268: Although statistically significant, I would recommend the authors characterize the CD8+ T cells a little more, as the mechanism now seems quite elusive. What signals or memories do CD8+ T cells acquire after IroA-E. Coli treatment to confer their long-term immunogenicity?

(4) Perhaps this goes beyond the scope of the current manuscript, but how broadly applicable is the observed iron-transport phenomenon in other tumor models? I would recommend the authors to either experimentally test it in another model, or at least discuss this question.

Reviewer #1 (Public Review):

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.

REVISED VERSION: while the authors have partially addressed my concerns, I do not feel they have addressed them all. I do not feel they have addressed the weight instability and concerns about the stacked regression models satisfactorily. I also must say that I agree with Reviewer 3 about the limitations of the brain age and brain cognition methods conceptually. In particular that the regression model used to predict fluid cognition will by construction explain more variance in cognition than a brain age model that is trained to predict age. This suffers from the same problem the authors raise with brain age and would indeed disappear if the authors had a separate measure of cognition against which to validate and were then to regress this out as they do for age correction. I am aware that these conceptual problems are more widespread than this paper alone (in fact throughout the brain age literature), so I do not believe the authors should be penalised for that. However, I do think they can make these concerns more explicit and further tone down the comments they make about the utility of brain cognition. I have indicated the main considerations about these points in the recommendations section below.

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative that explains more unique variance in the downstream regression.

This is a reasonably good paper and the use of a commonality analysis is a nice contribution to understanding variance partitioning across different covariates. I have some comments that I believe the authors ought to address, which mostly relate to clarity and interpretation

First, from a conceptual point of view, the authors focus exclusively on cognition as a downstream outcome. I would suggest the authors nuance their discussion to provide broader considerations of the utility of their method and on the limits of interpretation of brain age models more generally.

Second, from a methods perspective , there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand how the stacked regression models were constructed. I would request that the authors provide more information to enable the reader to better understand the stacked regression models used to ensure that these models are not overfit.

Please also provide an indication of the different regression strengths that were estimated across the different models and cross-validation splits. Also, how stable were the weights across splits?

Please provide more details about the task designs, MRI processing procedures that were employed on this sample in addition to the regression methods and bias correction methods used. For example, there are several different parameterisations of the elastic net, please provide equations to describe the method used here so that readers can easily determine how the regularisation parameters should be interpreted.

Reviewer #1 (Public Review):

Summary

A new method, tCFS, is introduced to offer richer and more efficient measurement of interocular suppression. It generates a new index, the suppression depth, based on the contrast difference between the up-ramped contrast for the target to breakthrough suppression and the down-ramped contrast for the target to disappear into suppression. A uniform suppression depth regardless of image types (e.g., faces, gratings and scrambles) was discovered in the paper, favoring an early-stage mechanism involving CFS. Discussions about claims of unconscious processing and the related mechanisms.

Strength

The tCFS method adds to the existing bCFS paradigms by providing the (re-)suppression threshold and thereafter the depression depth. Benefiting from adaptive procedures with continuous trials, the tCFS is able to give fast and efficient measurements. It also provides a new opportunity to test theories and models about how information is processed outside visual awareness.

Weakness:

This paper reports the surprising finding of uniform suppression depth over a variety of stimuli. This is novel and interesting. But given the limited samples being tested, the claim of uniformity suppression depth needs to be further examined, with respect to different complexities and semantic meanings.

From an intuitive aspect, the results challenged previous views about "preferential processing" for certain categories, though it invites further research to explore what exactly could suppression depth tell us about unconscious visual processing.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Abe and colleagues employ in vivo 2-photon calcium imaging of dopaminergic axons in the mPFC. The study reveals that these axons primarily respond to unconditioned aversive stimuli (US) and enhance their responses to initially-neutral stimuli after classical association learning. The manuscript is well-structured and presents results clearly. The utilization of a refined prism-based imaging technique, though not entirely novel, is well-implemented. The study's significance lies in its contribution to the existing literature by offering single-axon resolution functional insights, supplementing prior bulk measurements of calcium or dopamine release. Given the current focus on neuromodulator neuron heterogeneity, the work aligns well with current research trends and will greatly interest researchers in the field.

Comment on the revised version:

In my opinion, the authors did a great job with the revision of the manuscript.

Reviewer #1 (Public Review):

Summary:

Previous work in humans and non-human animals suggests that during offline periods following learning, the brain replays newly acquired information in a sequential manner. The present study uses an MEG-based decoding approach to investigate the nature of replay/reactivation during a cued recall task directly following a learning session, where human participants are trained on a new sequence of 10 visual images embedded in a graph structure. During retrieval, participants are then cued with two items from the learned sequence, and neural evidence is obtained for the simultaneous or sequential reactivation of future sequence items. The authors find evidence for both sequential and clustered (i.e., simultaneous) reactivation. Replicating previous work, low-performing participants tend to show sequential, temporally segregated reactivation of future items, whereas high-performing participants show more clustered reactivation. Adding to previous work, the authors show that an image's reactivation strength varies depending on its proximity to the retrieval cue within the graph structure.

Strengths:

As the authors point out, work on memory reactivation has largely been limited to the retrieval of single associations. Given the sequential nature of our real-life experiences, there is clearly value in extending this work to structured, sequential information. State-of-the-art decoding approaches for MEG are used to characterize the strength and timing of item reactivation. The manuscript is very well written with helpful and informative figures in the main sections. The task includes an extensive localizer with 50 repetitions per image, allowing for stable training of the decoders and the inclusion of several sanity checks demonstrating that on-screen items can be decoded with high accuracy.

Weaknesses:

Of major concern, the experiment is not optimally designed for analysis of the retrieval task phase, where only 4 min of recording time and a single presentation of each cue item are available for the analyses of sequential and non-sequential reactivation. In their revision, the authors include data from the learning blocks in their analysis. These blocks follow the same trial structure as the retrieval task, and apart from adding more data points could also reveal a possible shift from sequential to clustered reactivation as learning of the graph structure progresses. The new analyses are not entirely conclusive, maybe given the variability in the number of learning blocks that participants require to reach criterion. In principal, they suggest that reactivation strength increases from learning (pre-rest) to final retrieval (post-rest).

On a more conceptual note, the main narrative of the manuscript implies that sequential and clustered reactivation are mutually exclusive, such that a single participant would show either one or the other type. With the analytic methods used here, however, it seems possible to observe both types of reactivation. For example, the observation that mean reactivation strength (across the entire trial, or in a given time window of interest) varies with graph distance does not exclude the possibility that this reactivation is also sequential. In fact, the approach of defining one peak time window of reactivation may bias towards simultaneous, graded reactivation. It would be helpful if the authors could clarify this conceptual point. A strong claim that the two types of reactivation are mutually exclusive would need to be substantiated by further evidence, for instance a suitable metric contrasting "sequenceness" vs "clusteredness".

On the same point, the non-sequential reactivation analyses use a time window of peak decodability that is determined based on the average reactivation of all future items, irrespective of graph distance. In a sequential forward cascade of reactivations, it could be assumed that the reactivation of near items would peak earlier than the reactivation of far items. In the revised manuscript, the authors now show the "raw" timecourses of item decodability at different graph distances, clearly demonstrating their peak reactivation times, which show convincingly that reactivation for near and far items occurs at very similar time points. The question that remains, therefore, is whether the method of pre-selecting a time window of interest described above could exert a bias towards finding clustered reactivation.

Reviewer #1 (Public Review):

Summary:

In this study, Jellinger et al. performed engram-specific sequencing and identified genes that were selectively regulated in positive/negative engram populations. In addition, they performed chronic activation of the negative engram population over 3 months and observed several effects on fear/anxiety behavior and cellular events such as upregulation of glial cells and decreased GABA levels.

Strengths:

They provide useful engram-specific GSEA data and the main concept of the study, linking negative valence/memory encoding to cellular level outcomes including upregulation of glial cells, is interesting and valuable.

Weaknesses:

A number of experimental shortcomings make the conclusion of the study largely unsupported. In addition, the observed differences in behavioral experiments are rather small, inconsistent, and the interpretation of the differences is not compelling.

Major points for improvement:

(1) Lack of essential control experiments

With the current set of experiments, it is not certain that the DREADD system they used was potent and stable throughout the 3 months of manipulations. Basic confirmatory experiments (e.g., slice physiology at 1m vs. 3m) to show that the DREADD effects on these vHP are stable would be an essential bottom line to make these manipulation experiments convincing.

Furthermore, although the authors use the mCherry vector as a control, they did not have a vehicle/saline control for the hM3Dq AAV. Thus, the long-term effects such as the increase in glial cells could simply be due to the toxicity of DREADD expression, rather than an induced activity of these cells.

(2) Figure 1 and the rest of the study are disconnected

The authors used the cFos-tTA system to label positive/negative engram populations, while the TRAP2 system was used for the chronic activation experiments. Although both genetic tools are based on the same IEG Fos, the sensitivity of the tools needs to be validated. In particular, the sensitivity of the TRAP2 system can be arbitrarily altered by the amount of tamoxifen (or 4OHT) and the administration protocols. The authors should at least compare and show the percentage of labeled cells in both methods and discuss that the two experiments target (at least slightly) different populations. In addition, the use of TRAP2 for vHP is relatively new; the authors should confirm that this method actually captures negative engram populations by checking for reactivation of these cells during recall by overlap analysis of Fos staining or by artificial activation.

(3) Interpretation of the behavior data

In Figures 3a and b, the authors show that the experimental group showed higher anxiety based on time spent in the center/open area. However, there were no differences in distance traveled and center entries, which are often reduced in highly anxious mice. Thus, it is not clear what the exact effect of the manipulation is. The authors may want to visualize the trajectories of the mice's locomotion instead of just showing bar graphs.

In addition, the data shown in Figure 4b is somewhat surprising - the 14MO control showed more freezing than the 6MO control, which can be interpreted as "better memory in old". As this is highly counterintuitive, the authors may want to discuss this point. The authors stated that "Mice typically display increased freezing behavior as they age, so these effects during remote recall are expected" without any reference. This is nonsense, as just above in Figure 4a, older mice actually show less freezing than young mice.

Overall, the behavioral effects are rather small and random. I would suggest that these data be interpreted more carefully.

(4) Lack of citation and discussion of relevant study

Khalaf et al. 2018 from Gräff lab showed that experimental activation of recall-induced populations leads to fear attenuation. Despite the differences in experimental details, the conceptual discrepancy should be discussed.

Reviewer #1 (Public Review):

Summary:

This important study investigated the role of oxytocin (OT) neurons in the paraventricular nucleus (PVN) and their projections to the medial prefrontal cortex (mPFC) in regulating pup care and infanticide behaviors in mandarin voles. The researchers used techniques like immunofluorescence, optogenetics, OT sensors, and peripheral OT administration. Activating OT neurons in the PVN reduced the time it took pup-caring male voles to approach and retrieve pups, facilitating pup-care behavior. However, this activation had no effect on females. Interestingly, this same PVN OT neuron activation also reduced the time for both male and female infanticidal voles to approach and attack pups, suggesting PVN OT neuron activity can promote pup care while inhibiting infanticide behavior. Inhibition of these neurons promoted infanticide. Stimulating PVN->mPFC OT projections facilitated pup care in males and in infanticide-prone voles, activation of these terminals prolonged latency to approach and attack. Inhibition of PVN->mPFC OT projections promoted infanticide. Peripheral OT administration increased pup care in males and reduced infanticide in both sexes. However, some results differed in females, suggesting other mechanisms may regulate female pup care.

Strengths:

This multi-faceted approach provides converging evidence, strengthens the conclusions drawn from the study, and makes them very convincing. Additionally, the study examines both pup care and infanticide behaviors, offering insights into the mechanisms underlying these contrasting behaviors. The inclusion of both male and female voles allows for the exploration of potential sex differences in the regulation of pup-directed behaviors. The peripheral OT administration experiments also provide valuable information for potential clinical applications and wildlife management strategies.

Weaknesses:

While the study presents exciting findings, there are several weaknesses that should be addressed. The sample sizes used in some experiments, such as the Fos study and optogenetic manipulations, appear to be small, which may limit the statistical power and generalizability of the results. Effect sizes are not reported, making it difficult to evaluate the practical significance of the findings. The imaging parameters and analysis details for the Fos study are not clearly described, hindering the interpretation of these results (i.e., was the entire PVN counted?). Also, does the Fos colocalization align with previous studies that look at PVN Fos and maternal/ paternal care? Additionally, the study lacks electrophysiological data to support the optogenetic findings, which could provide insights into the neural mechanisms underlying the observed behaviors.

The study has several limitations that warrant further discussion. Firstly, the potential effects of manipulating OT neurons on the release of other neurotransmitters (or the influence of other neurochemicals or brain regions) on pup-directed behaviors, especially in females, are not fully explored. Additionally, it is unclear whether back-propagation of action potentials during optogenetic manipulations causes the same behavioral effect as direct stimulation of PVN OT cells. Moreover, the authors do not address whether the observed changes in behavior could be explained by overall increases or decreases in locomotor activity.

The authors do not specify the percentage of PVN->mPFC neurons labeled that were OT-positive, nor do they directly compare the sexes in their behavioral analysis (or if they did, it is not clear statistically). While the authors propose that the sex difference in pup-directed behaviors is due to females having greater OT expression, they do not provide evidence to support this claim from their labeling data. It is also uncertain whether more OT neurons were manipulated in females compared to males. The study could benefit from a more comprehensive discussion of other factors that could influence the neural circuit under investigation, especially in females.

Reviewer #1 (Public Review):

Summary:

The present paper introduces Oscillation Component Analysis (OCA), in analogy to ICA, where source separation is underpinned by a biophysically inspired generative model. It puts the emphasis on oscillations, which is a prominent characteristic of neurophysiological data.

Strengths:

Overall, I find the idea of disambiguating data-driven decompositions by adding biophysical constrains useful, interesting and worth-pursuing. The model incorporates both a component modelling of oscillatory responses that is agnostic about the frequency content (e.g.. doesn't need bandpass filtering or predefinition of bands) and a component to map between sensor and latent-space. I feel these elements can be useful in practice.

Weaknesses:

Lack of empirical support: I am missing empirical justification of the advantages that are theoretically claimed in the paper. I feel the method needs to be compared to existing alternatives.

How to bring forward the voices, stories of people that come from places that have been marginalised

Reviewer #1 (Public Review):

Summary:

The paper examined livestock abortion, as it is an important disease syndrome that affects productivity and livestock economies. If livestock abortion remains unexamined it poses risks to public health.

Several pathogens are associated with livestock abortions across Africa however the livestock disease surveillance data rarely include information from abortion events, little is known about the aetiology and impacts of livestock abortions, and data are not available to inform prioritisation of disease interventions. Therefore the current study seeks to examine the issue in detail and proposes some solutions.

The study took place in 15 wards in northern Tanzania spanning pastoral, agropastoral, and smallholder agro-ecological systems. The key objective is to investigate the causes and impacts of livestock abortion.

The data collection system was set up such that farmers reported abortion cases to the field officers of the Ministry of Livestock and Fisheries livestock.

The reports were made to the investigation teams. The team only included abortion of those that the livestock field officers could attend to within 72 hours of the event occurring.

Also, a field investigation was carried out to collect diagnostic samples from aborted materials. In addition, aborting dams and questionnaires were administered to collect data on herd/flock management. Laboratory diagnostic tests were carried out for a range of abortigenic pathogens

Over the period of the study, 215 abortion events in cattle (n=71), sheep 48 (n=44), and goats (n=100) were investigated. All 49 investigated cases varied widely across wards. The aetiological attribution, achieved for 19.5% of cases through PCR-based diagnostics, was significantly affected by delays in the field investigation.

The result also revealed that vaginal swabs from aborting dams provided a practical and sensitive source of diagnostic material for pathogen detection.

Livestock abortion surveillance can generate valuable information on causes of zoonotic disease outbreaks, and livestock reproductive losses and can identify important pathogens that are not easily captured through other forms of livestock disease surveillance. The study demonstrated the feasibility of establishing an effective reporting and investigation system that could be implemented across a range of settings, including remote rural areas,

Strengths:

The paper combines both science and socio-economic methodology to achieve the aim of the study. The methodology was well presented and the sequence was great. The authors explain where and how the data was collected. Figure 2 was used to describe the study area which was excellently done. The section on the investigation of cases was well written. The sample analysis was also well-written. The authors devoted a section to summarizing the investigated cases and description of the livestock 221-study population. The logit model was well-presented.

Reviewer #1 (Public Review):

In this manuscript, Naseri et al. present a new strategy for identifying human genetic variants with recessive effects on disease risk by the genome-wide association of phenotype with long runs-of-homozygosity (ROH). The key step of this approach is the identification of long ROH segments shared by many individuals (termed "shared ROH diplotype clusters" by the authors), which is computationally intensive for large-scale genomic data. The authors circumvented this challenge by converting the original diploid genotype data to (pseudo-)haplotype data and modifying the existing positional Burrow-Wheeler transformation (PBWT) algorithms to enable an efficient search for haplotype blocks shared by many individuals. With this method, the authors identified over 1.8 million ROH diplotype clusters (each shared by at least 100 individuals) and 61 significant associations with various non-cancer diseases in the UK Biobank dataset.

Overall, the study is well-motivated, highly innovative, and potentially impactful. Previous biobank-based studies of recessive genetic effects primarily focused on genome-wide aggregated ROH content, but this metric is a poor proxy for homozygosity of the recessive alleles at causal loci. Therefore, searching for the association between phenotype and specific variants in the homozygous state is a key next step towards discovering and understanding disease genes/alleles with recessive effects. That said, I have some concerns regarding the power and error rate of the methods, for both identification of ROH diplotype clusters and subsequent association mapping. In addition, some of the newly identified associations need further validation and careful consideration of potential artifacts (such as cryptic relatedness and environment sharing).

(1) Identification of ROH diplotype clusters.<br /> The practice of randomly assigning heterozygous sites to a homozygous state is expected to introduce errors, leading to both false positives and false negatives. An advantage that the authors claim for this practice is to reduce false negatives due to occasional mismatch (possibly due to genotyping error, or mutation), but it's unclear how much the false positive rate is reduced compared to traditional ROH detection algorithm. The authors also justified the "random allele drawing" practice by arguing that "the rate of false positives should be low" for long ROH segments, which is likely true but is not backed up with quantitative analysis. As a result, it is unclear whether the trade-off between reducing FNs and introducing FPs makes the practice worthwhile (compared to calling ROHs in each individual with a standard approach first followed by scanning for shared diplotypes across individuals using BWT). I would like to see a combination of back-of-envelope calculation, simulation (with genotyping errors), and analysis of empirical data that characterize the performance of the proposed method.

In particular, I find the high number of ROH clusters in MHC alarming, and I am not convinced that this can be fully explained by a high density of SNPs and low recombination rate in this region. The authors may provide further support for their hypothesis by examining the genome-wide relationship between ROH cluster abundance and local recombination rate (or mutation rate).

(2) Power of ROH association. Given that the authors focused on long segments only (which is a limitation of the current method), I am concerned about the power of the association mapping strategy, because only a small fraction of causal alleles are expected to be present in long, homozygous haplotypes shared by many individuals. It would be useful to perform a power analysis to estimate what fraction of true causal variants with a given effect size can be detected with the current method. To demonstrate the general utility of this method, the authors also need to characterize the condition(s) under which this method could pick up association signals missed by standard GWAS with recessive effects considered. I suspect some variants with truly additive effects can also be picked up by the ROH association, which should be discussed in the manuscript to guide the interpretation of results.

(3) False positives of ROH association. GWAS is notoriously prone to confounding by population and environmental stratification. Including leading principal components in association testing alleviates this issue but is not sufficient to remove the effects of recent demographic structure and local environment (Zaidi and Mathieson 2020 eLife). Similar confounding likely applies to homozygosity mapping and should be carefully considered. For example, it is possible that individuals who share a lot of ROH diplotypes tend to be remotely related and live near each other, thus sharing similar environments. Such scenarios need to be excluded to further support the association signals.

(4) Validation of significant associations. It is reassuring that some of the top associations are indirectly corroborated by significant GWAS associations between the same disease and individual SNPs present in the ROH region (Tables 1 and 2). However, more sanity checks should be done to confirm consistency in direction of effect size (e.g., risk alleles at individual SNPs should be commonly present in risk-increasing ROH segment, and vice versa) and the presence of dominance effect.

Reviewer #1 (Public Review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings. Moreover, it enables the visualization of actual cell locations, allowing for the examination of spatial properties (e.g., Figure 4G).

Weaknesses:

There is a notable deviation from several observations obtained through conventional electrophysiological recordings. Particularly, as mentioned below in detail, the considerable differences in baseline firing rates and no observations of ripple-triggered firing patterns raise some concerns about potential artifacts from imaging and analsyis, such as cell toxicity, abnormal excitability, and false detection of spikes. While these findings are intriguing if the validity of these methods is properly proven, accepting the current results as new insights is challenging.

Reviewer #1 (Public Review):

In this study, the authors offer a fresh perspective on how visual working memory operates. They delve into the link between anticipating future events and retaining previous visual information in memory. To achieve this, the authors build upon their recent series of experiments that investigated the interplay between gaze biases and visual working memory. In this study, they introduce an innovative twist to their fundamental task. Specifically, they disentangle the location where information is initially stored from the location where it will be tested in the future. Participants are tasked with learning a novel rule that dictates how the initial storage location relates to the eventual test location. The authors leverage participants' gaze patterns as an indicator of memory selection. Intriguingly, they observe that microsaccades are directed towards both the past encoding location and the anticipated future test location. This observation is noteworthy for several reasons. Firstly, participants' gaze is biased towards the past encoding location, even though that location lacks relevance to the memory test. Secondly, there's a simultaneous occurrence of an increased gaze bias towards both the past and future locations. To explore this temporal aspect further, the authors conduct a compelling analysis that reveals the joint consideration of past and future locations during memory maintenance. Notably, microsaccades biased towards the future test location also exhibit a bias towards the past encoding location. In summary, the authors present an innovative perspective on the adaptable nature of visual working memory. They illustrate how information relevant to the future is integrated with past information to guide behavior.

Reviewer #1 (Public Review):

Using A. carterae as a model system, this work investigates the properties of the trans-spliced SL leader sequences and the dinoflagellate eIF4E protein family members.

Analysis was performed to identify the 5' cap type of the SL leader. Variation in the SL leader sequence and an abundance of modified bases was documented.

Various aspects of the sequence and expression of the eIF4E family members were examined. This included phylogeny, mRNA, and protein expression levels in A. carterae, and the ability of eIF4E proteins to bind cap structures. Differences in expression levels and cap-binding capacity were characterized, leading to the proposition that eIF4E-1a serves as the major cap-binding protein in A. carterae.

A major discussion point is the potential for differential eIF4E binding to specific SL leader sequences as a regulatory mechanism, which is an exciting prospect. However, despite indications of sequence variability and the presence of various nucleotide modifications in the SL, and the several eIF4E variants, direct evidence to support this hypothesis is lacking.

It is an extensive and highly descriptive study. The work is presented clearly, although it is rather lengthy and contains repetition across the introduction, results, and discussion sections. Its style leans more towards a review format. As a non-expert in the field, I appreciated the extensive background however I do believe the paper would benefit from a more concise format.

Reviewer #1 (Public Review):

Summary:

The authors want to determine the role of the sperm hook of the house mouse sperm in movement through the uterus. The authors are trying to distinguish between two hypotheses put forward by others on the role of the sperm hook: (1) the sperm cooperation hypothesis (the sperm hook helps to form sperm trains) vs (2) the migration hypothesis (that the sperm hook is needed for sperm movement through the uterus). They use transgenic lines with fluorescent labels to sperm proteins, and they cross these males to C57BL/6 females in pathogen-free conditions. They use 2-photon microscopy on ex vivo uteri within 3 hours of mating and the appearance of a copulation plug. There are a total of 10 post-mating uteri that were imaged with 3 different males. They provide 10 supplementary movies that form the basis for some of the quantitative analysis in the main body figures. Their data suggest that the role of the sperm hook is to facilitate movement along the uterine wall.

Strengths:

Ex vivo live imaging of fluorescently labeled sperm with 2-photon microscopy is a powerful tool for studying the behavior of sperm.

Weaknesses:

The paper is descriptive and the data are correlations.

The data are not properly described in the figure legends.

When statistical analyses are performed, the authors do not comment on the trend that sperm from the three males behave differently from each other. This weakens confidence in the results. For example, in Figure 1 the sperm from male 3613 (blue squares) look different from male 838 (red circles), but all of these data are considered together. The authors should comment on why sperm across males are considered together when the individual data points appear to be different across males.

Movies S8-S10 are single data points and no statistical analyses are performed. Therefore, it is unclear how penetrant the sperm movements are.

Movies S1B - did the authors also track the movement of sperm located in the middle of the uterus (not close to the wall)? Without this measurement, they can't be certain that sperm close to the uterus wall travels faster.

Movie S5A - is of lower magnitude (200 um scale bar) while the others have 50 and 20 uM scale bars. Individual sperm movement can be observed in the 20 uM (Movie 5SC). If the authors went to prove that there is no upsucking movement of sperm by the uterine contractions, they need to provide a high magnification image.

Movie S8 - if the authors want to make the case that clustered sperm do not move faster than unclustered sperm, then they need to show Movie S8 at higher magnification. They also need to quantify these data.

Movie S9C - what is the evidence that these sperm are dead or damaged?

MovIe S10 - both slow- and fast-moving sperm are seen throughout the course of the movie, which does not support the authors' conclusion that sperm tails beat faster over time.

Reviewer #1 (Public Review):

The authors have performed all-atom MD simulations to study the working mechanism of hsPepT2. It is widely accepted that conformational transitions of proton-coupled oligopeptide transporters (POTs) are linked with gating hydrogen bonds and salt bridges involving protonatable residues, whose protonation triggers gate openings. Through unbiased MD simulations, the authors identified extra-cellular (H87 and D342) and intra-cellular (E53 and E622) triggers. The authors then validated these triggers using free energy calculations (FECs) and assessed the engagement of the substrate (Ala-Phe dipeptide). The linkage of substrate release with the protonation of the ExxER motif (E53 and E56) was confirmed using constant-pH molecular dynamics (CpHMD) simulations and cell-based transport assays. An alternating-access mechanism was proposed. The study was largely conducted properly, and the paper was well-organized. However, I have a couple of concerns for the authors to consider addressing.

(1) As a proton-coupled membrane protein, the conformational dynamics of hsPepT2 are closely coupled to protonation events of gating residues. Instead of using semi-reactive methods like CpHMD or reactive methods such as reactive MD, where the coupling is accounted for, the authors opted for extensive non-reactive regular MD simulations to explore this coupling. Note that I am not criticizing the choice of methods, and I think those regular MD simulations were well-designed and conducted. But I do have two concerns.

a) Ideally, proton-coupled conformational transitions should be modelled using a free energy landscape with two or more reaction coordinates (or CVs), with one describing the protonation event and the other describing the conformational transitions. The minimum free energy path then illustrates the reaction progress, such as OCC/H87D342-  OCC/H87HD342H  OF/H87HD342H as displayed in Figure 3. Without including the protonation as a CV, the authors tried to model the free energy changes from multiple FECs using different charge states of H87 and D342. This is a practical workaround, and the conclusion drawn (the OCCOF transition is downhill with protonated H87 and D342) seems valid. However, I don't think the OF states with different charge states (OF/H87D342-, OF/H87HD342-, OF/H87D342H, and OF/H87HD342H) are equally stable, as plotted in Figure 3b. The concern extends to other cases like Figures 4b, S7, S10, S12, S15, and S16. While it may be appropriate to match all four OF states in the free energy plot for comparison purposes, the authors should clarify this to ensure readers are not misled.

b) Regarding the substrate impact, it appears that the authors assumed fixed protonation states. I am afraid this is not necessarily the case. Variations in PepT2 stoichiometry suggest that substrates likely participate in proton transport, like the Phe-Ala (2:1) and Phe-Gln (1:1) dipeptides mentioned in the introduction. And it is not rigorous to assume that the N- and C-termini of a peptide do not protonate/deprotonate when transported. I think the authors should explicitly state that the current work and the proposed mechanism (Figure 8) are based on the assumption that the substrates do not uptake/release proton(s).

(2) I have more serious concerns about the CpHMD employed in the study.

a) The CpHMD in AMBER is not rigorous for membrane simulations. The underlying generalized Born model fails to consider the membrane environment when updating charge states. In other words, the CpHMD places a membrane protein in a water environment to judge if changes in charge states are energetically favorable. While this might not be a big issue for peripheral residues of membrane proteins, it is likely unphysical for internal residues like the ExxER motif. As I recall, the developers have never used the method to study membrane proteins themselves. The only CpHMD variant suitable for membrane proteins is the membrane-enabled hybrid-solvent CpHMD in CHARMM. While I do not expect the authors to redo their CpHMD simulations, I do hope the authors recognize the limitations of their method.

b) It appears that the authors did not make the substrate (Ala-Phe dipeptide) protonatable in holo-simulations. This oversight prevents a complete representation of ligand-induced protonation events, particularly given that the substrate ion pairs with hsPepT2 through its N- & C-termini. I believe it would be valuable for the authors to acknowledge this potential limitation.

Reviewer #1 (Public Review):

Summary:

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies.

Strengths:

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of the USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8.

Weaknesses:

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the ups-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs.

Reviewer #1 (Public Review):

Summary:

The authors found that the loss of cell-ECM adhesion leads to the formation of giant monocular vacuoles in mammary epithelial cells. This process takes place in a macropinocytosis-like process and involves PI3 kinase. They further identified dynamin and septin as essential machinery for this process. Interestingly, this process is reversible and appears to protect cells from cell death.

Strengths:

The data are clean and convincing to support the conclusions. The analysis is comprehensive, using multiple approaches such as SIM and TEM. The discussion on lactation is plausible and interesting.

Weaknesses:

As the first paper describing this phenomenon, it is adequate. However, the elucidation of the molecular mechanisms is not as exciting as it does not describe anything new. It is hoped that novel mechanisms will be elucidated in the future. In particular, the molecules involved in the reversing process could be quite interesting. Additionally, the relationship to conventional endocytic compartments, such as early and late endosomes, is not analyzed.

Reviewer #1 (Public Review):

Summary:

The authors demonstrated the phenomenon of p130Cas, a protein primarily localized at focal adhesions, and its formation of condensates. They identified the constituents within the condensates, which include other focal adhesion proteins, paxillin, and RNAs. Furthermore, they proposed a link between p130Cas condensates and translation.

Strengths:

Adhesion components undergo rapid exchange with the cytoplasm for some unclear biological functions. Given that p130Cas is recognized as a prominent mechanical focal adhesion component, investigating its role in condensate formation, particularly its impact on the translation process, is intriguing and significant.

Weaknesses:

The authors identified the disordered region of p130Cas and investigated the formation of p130Cas condensate. They attempted to demonstrate that p130Cas condensates inhibit translation, but the results did not fully support this assertion. There are several comments below:

(1) Despite isolating p130Cas-GFP protein using GFP-trap beads, the authors cannot conclusively eliminate the possibility of isolating p130Cas from focal adhesions. While the characterization of the GFP-tagged pulls can reveal the proteins and RNAs associated with p130Cas, they need to clarify their intramolecular mechanism of localization within p130Cas droplets. Whether the protein condensates retain their liquid phase or these GFP-p130Cas pulls represent protein aggregate remains uncertain.

(2) The authors utilized hexanediol and ammonium acetate to highlight the phenomenon of p130Cas condensates. Although hexanediol is an inhibitor for hydrophobic interactions and ammonium acetate is a salt, a more thorough explanation of the intramolecular mechanisms underlying p130Cas protein-protein interaction is required. Additionally, given that the size of p130Cas condensates can exceed >100um2, classification is needed to differentiate between p130Cas condensates and protein aggregation.

(3) The connection between p130Cas condensates and translation inhibition appears tenuous. The data only suggests a correlation between p130Cas expression and translation inhibition. Further evidence is required to bolster this hypothesis.

Reviewer #1 (Public Review):

The paper 'Structural Analysis of the Dynamic Ribosome-Translocon Complex,' authored by Lewis et al., meticulously explores various conformations and states of the ribosome-translocon complex. Employing advanced techniques such as cryoEM structural determination and AlphaFold modeling, the study delves into the dynamic nature of the ribosome-translocon complex. The findings from these analyses unveil crucial insights, significantly advancing our understanding of the co-translational translocation process in cellular mechanisms.

To begin with, the authors employed a construct comprising the first two transmembrane domains of rhodopsin as a model for studying protein translocation. They conducted in vitro translation, followed by the purification of the ribosome-translocon complex, and determined its cryoEM structures. An in-depth analysis of their ribosome-translocon complex structure revealed that the nascent chain can pass through the lateral gate of translocon Sec61, akin to the behavior of a Signaling Peptide. Additionally, Sec61 was found to interact with 28S rRNA helix 24 and the ribosomal protein uL24. In summary, their structural model aligns with the through-pore model of insertion, contradicting the sliding model.

Secondly, the authors successfully identified RAMP4 in their ribosome-translocon complex structure. Notably, the transmembrane domain of RAMP4 mimics the binding of a Signaling Peptide at the lateral gate of Sec61, albeit without unplugging. Intriguingly, RAMP4 is exclusively present in the non-multipass translocon ribosome-translocon complex, not in those containing multipass translocon. This observation suggests that co-translational translocation specifically occurs in the Sec61 channel that includes bound RAMP4. Additionally, the authors discovered an interaction between the C-tail of ribosomal proteins uL22 and the translocon Sec61, providing valuable insights into the nascent chain's behavior.

Moving on to the third point, the focused classification unveiled TRAP complex interactions with various components. The authors propose that the extra density observed in their novel ribosome-translocon complex can be attributed to calnexin, a major binder of TRAP according to previous studies. Furthermore, the new structure reveals a TRAP-OSTA interaction. This newly identified TRAP-OSTA interaction offers a potential explanation for why patients with TRAP delta defects exhibit congenital disorders of glycosylation.

In conclusion, this paper presents a robust contribution to the field with its thorough structural and modeling analyses. The significance of the findings is evident, providing valuable insights into the intricate mechanisms of protein co-translational translocation. The well-crafted writing, meticulous analyses, and clear figures collectively contribute to the overall strength of the paper.

Major points:

(1) The identification of RAMP4 is a pivotal discovery in this paper. The sophisticated AlphaFold prediction, de novo model building of RAMP4's RBD domain, and sequence analyses provide strong evidence supporting the inclusion of RAMP4 in the ribosome-translocon complex structure.

However, it is crucial to ensure the presence of RAMP4 in the purified sample. Particularly, a validation step such as western blotting for RAMP4 in the purified samples would strengthen the assertion that the ribosome-translocon complex indeed contains RAMP4. This is especially important given the purification steps involving stringent membrane solubilization and affinity column pull-down.

(2) Despite the comprehensive analyses conducted by the authors, it is challenging to accept the assertion that the extra density observed in TRAP class 1 corresponds to calnexin. The additional density in TRAP class 1 appears to be less well-resolved, and the evidence for assigning it as calnexin is insufficient. The extra density there can be any proteins that bind to TRAP. It is recommended that the authors examine the density on the ER lumen side. An investigation into whether calnexin's N-globular domain and P-domain are present in the ER lumen in TRAP class 1 would provide a clearer understanding.

(3) In the section titled 'TRAP competes and cooperates with different translocon subunits,' the authors present a compelling explanation for why TRAP delta defects can lead to congenital disorders of glycosylation. To enhance this explanation, it would be valuable if the authors could provide additional analyses based on mutations mentioned in the references. Specifically, examining whether these mutations align with the TRAP delta-OSTA structure models would strengthen the link between TRAP delta defects and the observed congenital disorders of glycosylation.

Joint Public Review:

Summary:

This study presents an immunotherapeutic strategy for treating mouse cutaneous squamous cell carcinoma (mCSCC) using serum from mice inoculated with mCSCC. The author hypothesizes that antibodies in the generated serum could aid the immune system in tumor volume reduction. The study results showed a reduction in tumor volume and altered expression of several cancer markers (p53, Bcl-xL, NF-κB, Bax) suggesting the potential effectiveness of this approach.

Strengths:

The approach shows potential effect on preventing tumor progression, from both the tumor size and the cancer biomarker expression levels bringing attention to the potential role of antibodies and B cell responses in cancer therapy.

Weaknesses:

These are some of the specific things that the author could consider to strengthen the evidence supporting the claims in their study.

(1) The study fails to provide evidence of the specific effect of mCSCC-antibodies on mCSCC. The study utilized serum which also contains many immune response factors like cytokines that could contribute to tumor reduction. There is no information on serum centrifugation conditions, which makes it unclear whether immune components like antigen-specific T cells, activated NK cells, or other immune cells were removed from the serum. The study does not provide evidence of neutralizing antibodies through isolation, analysis of B cell responses, or efficacy testing against specific cancer epitopes. To affirm the specific antibodies' role in the observed immune response, isolating antibodies rather than employing whole serum could provide more conclusive evidence. Purifying the serum to isolate mCSCC-binding antibodies, such as through protein A purification, and ELISA would have been more useful to quantify the immune response. It would be interesting to investigate the types of epitopes targeted following direct tumor cell injection. A more thorough characterization of the antibodies, including B cell isolation and/or hybridoma techniques, would strengthen the claim.

(2) In the study design, the control group does not account for the potential immunostimulatory effects of serum injection itself. A better control would be tumor-bearing mice receiving serum from healthy non-mCSCC-exposed mice. Additionally, employing a completely random process for allocating the treatment groups would be preferable. Also, the study does not explain why intravenous injection of tumor cells would produce superior antibodies compared to those naturally generated in mCSCC-bearing mice.

(3) In Figure 2B, it would be more helpful if the author could provide raw data/figures of the tumor than just the bar graph. Similarly in Figure 3, the author should show individual data points in addition to the error bar to visualize the actual distribution.

(4) The author mentioned that different stages of tumor cells have different surface biomarkers. Therefore, experimenting with injecting tumor cells at various stages could reveal the most immunogenic stage. Such an approach would allow for a comparative analysis of immune responses elicited by tumor cells at different stages of development.

(5) In the abstract the author mentioned that using mCSCC is a proof-of-concept for this potential cancer treatment strategy. The discussion session should extend to how this strategy might apply to other cancer types beyond carcinoma.

Reviewer #1 (Public Review):

Original review:<br /> The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain. Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Comments on revision:<br /> I acknowledge the significant changes made by the authors in the revised manuscript. However, I remain puzzled by the data concerning the interaction between BIN1 and PRC1. While I agree with the authors that even weak interactions among proteins can be significant, I am hesitant to accept a priori that the lack of clear evidence of colocalization between proteins can be justified solely by their low affinity.

Moreover, the possibility that other mitotic proteins may be potential partners of BIN1 does not inherently support an interaction between BIN1 and PRC1. I suggest that the authors present the interaction with PRC1 as a potential event and emphasize that further studies are needed to definitively establish it.

Joint Public Review:

Zhang et. al. presents compelling results that support the identification of epigenetically mediated control for the recognition of dihydropyrimidine dehydrogenase (DPYD) gene expression that is linked with cancer treatment resistance 5-fluorouracil. The experimental approach was developed and pursued with in vitro and in vivo strategies. Combining molecular, cellular, and biochemical approaches, the authors identify a germline variant with compromised enhancer control. Several lines of evidence were presented that are consistent with increased CEBP recruitment to the DPYD regulatory domain with consequential modifications in promoter-enhancer interactions that are associated with compromised 5-fluorouracil resistance. Functional identification of promoter and enhancer elements was validated by CRISPRi and CRISPRa assays. ChIP and qPCR documented histone marks that can account for the control of DPYD gene expression were established. Consistency with data from patient-derived specimens and direct assessment of 5-fluorouracil sensitivity provides confidence in the proposed mechanisms. The model is additionally supported by genome data from a population with high "compromised allele frequency". It can be informative to directly demonstrate DPYD promoter-enhancer interactions. However, the genetic variants support the integration of regulatory activities.

Reviewer #1 (Public Review):

Gap junction channels establish gated intercellular conduits that allow the diffusion of solutes between two cells. Hexameric connexin26 (Cx26) hemichannels are closed under basal conditions and open in response to CO2. In contrast, when forming a dodecameric gap-junction, channels are open under basal conditions and close with increased CO2 levels. Previous experiments have implicated Cx26 residue K125 in the gating mechanism by CO2, which is thought to become carbamylated by CO2. Carbamylation is a labile post-translational modification that confers negative charge to the K125 side chain. How the introduction of a negative charge at K125 causes a change in gating is unclear, but it has been proposed that carbamylated K125 forms a salt bridge with the side chain at R104, causing a conformational change in the channel. It is also unclear how overall gating is controlled by changes in CO2, since there is significant variability between structures of gap-junction channels and the cytoplasmic domain is generally poorly resolved. Structures of WT Cx26 gap-junction channels determined in the presence of various concentrations of CO2 have suggested that the cytoplasmatic N-terminus changes conformation depending on the concentration of the gas, occluding the pore when CO2 levels are high.

In the present manuscript, Deborah H. Brotherton and collaborators use an intercellular dye-transfer assay to show that Cx26 gap-junction channels containing the K125E mutation, which mimics carbamylation caused by CO2, is constitutively closed even at CO2 concentrations where WT channels are open. Several cryo-EM structures of WT and mutant Cx26 gap junction channels were determined at various conditions and using classification procedures that extracted more than one structural class from some of the datasets. Together, the features on each of the different structures are generally consistent with previously obtained structures at different CO2 concentrations and support the mechanism that is proposed in the manuscript. The most populated class for K125E channels determined at high CO2 shows a pore that is constricted by the N-terminus, and a cytoplasmic region that was better resolved than in WT channels, suggesting increased stability. The K125E structure closely resembles one of the two major classes obtained for WT channels at high CO2. These findings support the hypothesis that the K125E mutation biases channels towards the closed state, while WT channels are in an equilibrium between open and closed states even in the presence of high CO2. Consistently, a structure of K125E obtained in the absence of CO2 appeared to also represent a closed state but at a lower resolution, suggesting that CO2 has other effects on the channel beyond carbamylation of K125 that also contribute to stabilizing the closed state. Structures determined for K125R channels, which are constitutively open because arginine cannot be carbamylated, and would be predicted to represent open states, yielded apparently inconclusive results.

A non-protein density was found to be trapped inside the pore in all structures obtained using both DDM and LMNG detergents, suggesting that the density represents a lipid rather than a detergent molecule. It is thought that the lipid could contribute to the process of gating, but this remains speculative. The cytoplasmic region in the tentatively closed structural class of the WT channel obtained using LMNG was better resolved. An additional portion of the cytoplasmic face could be resolved by focusing classification on a single subunit, which had a conformation that resembled the AlphaFold prediction. However, this single-subunit conformation was incompatible with a C6-symmetric arrangement. Together, the results suggest that the identified states of the channel represent open states and closed states resulting from interaction with CO2. Therefore, the observed conformational changes illuminate a possible structural mechanism for channel gating in response to CO2.

Reviewer #1 (Public Review):

Muscle models are important tools in the fields of biomechanics and physiology. Muscle models serve a wide variety of functions, including validating existing theories, testing new hypotheses, and predicting forces produced by humans and animals in health and disease. This paper attempts to provide an alternative to Hill-type muscle models that includes contributions of titin to force enhancement over multiple time scales. Due to the significant limitations of Hill-type models, alternative models are needed and therefore the work is important and timely.

The effort to include a role for titin in muscle models is a major strength of the methods and results. The results clearly demonstrate the weaknesses of Hill models and the advantages of incorporating titin into theoretical treatments of muscle mechanics. Another strength is to address muscle mechanics over a large range of time scales.

The authors succeed in demonstrating the need to incorporate titin in muscle models, and further show that the model accurately predicts in situ force of cat soleus (Kirsch et al. 1994; Herzog & Leonard, 2002) and rabbit posts myofibrils (Leonard et al. 2010). However, it remains unclear whether the model will be practical for use with data from different muscles or preparations. Several ad hoc modifications were described in the paper, and the degree to which the model requires parameter optimization for different muscles, preparations and experiment types remains unclear.

I think the authors should state how many parameters require fitting to the data vs the total number of model parameters. It would also be interesting for the authors to discuss challenges associated with modeling ex vivo and in vivo data sets, due to differences in means of stimulation vs. model inputs.

Reviewer #1 (Public Review):

Summary:

The authors' earlier deep mutational scanning work observed that allosteric mutations in TetR (the tetracycline repressor) and its homologous transcriptional factors are distributed across the structure instead of along the presumed allosteric pathways as commonly expected. Especially, in addition, the loss of the allosteric communications promoted by those mutations, was rescued by additional distributed mutations. Now the authors develop a two-domain thermodynamic model for TetR that explains these compelling data. The model is consistent with the in vivo phenotypes of the mutants with changes in parameters, which permits quantification. Taken together their work connects intra- and inter-domain allosteric regulation that correlate with structural features. This leads the authors to suggest broader applicability to other multidomain allosteric proteins.

Here the authors follow their first innovative observations with a computational model that captures the structural behavior, aiming to make it broadly applicable to multidomain proteins. Altogether, an innovative and potentially useful contribution.

Weaknesses:

None that I see, except that I hope that in the future, if possible, the authors would follow with additional proteins to further substantiate the model and show its broad applicability. I realize however the extensive work that this would entail.

Reviewer #2 (Public Review):

In this revised manuscript Aguillon and collaborators convincingly demonstrating that CLK is required for free-running behavioral rhythms under constant conditions in the Cnidarian Nematostella. The results also convincingly show that CLK impacts rhythmic gene expression in this organism. This original work thus demonstrate that CLK was recruited very early during animal evolution in the circadian clock mechanism to optimize behavior and gene expression with the time-of-day. The manuscript could still benefit from some improvements so that it is more accessible for a wide readership.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Ruichen Yang et al. investigated the importance of BMP signaling in preventing microtia. Authors showed that Cre recombinase mediated deletion of Bmpr1a using skeletal stem specific Cre Prx1Cre leads to microtia in adult and young mice. In these mice, distal auricle is more affected than middle and proximal. In these Bmpr1a floxed Prx1Cre mice, auricle chondrocyte start to differentiate into osteoblasts through increase in PKA signaling. The authors showed human single-cell RNA-Seq data sets where they observed increased PKA signaling in microtia patient which resembles their animal model experiments.

Strengths:

Although the importance of BMP signaling in skeletal tissues has been previously reported, the importance of its role in microtia prevention is novel and very promising to study in detail. The authors satisfied the experimental questions by performing correct methods and explaining the results in detail.

Reviewer #1 (Public Review):

In this study, the authors address a fundamental unresolved question in cerebellar physiology: do synapses between granule cells (GCs) and Purkinje cells (PCs) made by the ascending part of the axon (AA) have different synaptic properties from those made by parallel fibers? This is an important question, as GCs integrate sensorimotor information from numerous brain areas with a precise and complex topography.

Summary:<br /> The authors argue that CGs located close to PCs essentially contact PC dendrites via the ascending part of their axons. They demonstrate that joint high-frequency (100 Hz) stimulation of distant parallel fibers and local CGs potentiates AA-PC synapses, while parallel fiber-PC synapses are depressed. On the basis of paired-pulse ratio analysis, they concluded that evoked plasticity was postsynaptic. When individual pathways were stimulated alone, no LRP was observed. This associative plasticity appears to be sensitive to timing, as stimulation of parallel fibers first results in depression, while stimulation of the AA pathway has no effect. NMDA, mGluR1 and GABAA receptors are involved in this plasticity.

Strengths:<br /> Overall, the associative modulation of synaptic transmission is convincing, and the experiments carried out support this conclusion. However, weaknesses limit the scope of the results.

Weaknesses:<br /> One of the main weaknesses of this study is the suggestion that high-frequency parallel-fiber stimulation cannot induce long term potentiation unless combined with AA stimulation. Although we acknowledge that the stimulation and recording conditions were different from those of other studies, according to the literature (e.g. Bouvier et al 2016, Piochon et al 2016, Binda et al, 2016, Schonewille et al 2021 and others), high-frequency stimulation of parallel fibers leads to long-term postsynaptic potentiation under many different experimental conditions (blocked or unblocked inhibition, stimulation protocols, internal solution composition). Furthermore, in vivo experiments have confirmed that high-frequency parallel fibers are likely to induce long-term potentiation (Jorntell and Ekerot, 2002; Wang et al, 2009). This article provides further evidence that long-term plasticity (LTP and LTD) at this connection is a complex and subtle mechanism underpinned by many different transduction pathways. It would therefore have been interesting to test different protocols or conditions to explain the discrepancies observed in this dataset.<br /> Another important weakness is the lack of evidence that the AAs were stimulated. Indeed, without filling the PC with fluorescent dye or biocytin during the experiment, and without reconstructing the anatomical organization, it is difficult to assess whether the stimulating pipette is positioned in the GC cluster that is potentially in contact with the PC with the AAs. According to EM microscopy, AAs account for 3% of the total number of synapses in a PC, which could represent a significant number of synapses. Although the idea that AAs repeatedly contact the same Purkinje cell has been propagated, to the best of the review author's knowledge, no direct demonstration of this hypothesis has yet been published. In fact, what has been demonstrated (Walter et al 2009; Spaeth et al 2022) is that GCs have a higher probability of being connected to nearby PCs, but are not necessarily associated with AAs.

My application won't fix this but it's a tool - it's for the teacher and learner but again it won't assist if there aren't deeper incentives to inspire and create a better system - WICKED PROBLEM

Reviewer #1 (Public Review):

Summary:

The manuscript proposes a series of steps using the FIJI environment, the authors have created a plugin for the initial steps of the process, merging images into an RGB stack, conversion to HSV, and then using brightness for reference and hue to distinguish the phases of the cycle. Then, the well-known Trackmate plugin was used to identify single cells and extract intensities. The data was further post-processed in R, where a series of steps, smoothing, scaling, and addressing missing frames were used to train a random forest. Hard-coded values of hue were used to distinguish G1, S, and G2/M. The process was validated with a score comparing the quality of the tracks and the authors reported the successful measure of the cell cycles.

Strengths:

The implementation of the pipeline seems easy, although it requires two separate platforms: Fiji and R. A similar approach could be implemented in a single programming environment like Python or Matlab and there would not be any need to export from one to the other. However, many labs have similar setups and that is not necessarily a problem.

Weaknesses:

I found two important weaknesses in the proposal:

(1) The pipeline relies on a large number of hard-coded conditions: size of Gaussian blur (Gaussian should be written in uppercase), values of contrast, size of filters, levels of intensity, etc. Presumably, the authors followed a heuristic approach and tried values of these and concluded that the ones proposed were optimal. A proper sensitivity analysis should be performed. That is, select a range of values of the variables and measure the effect on the output.

(2) Linked to the previous comments. Other researchers that want to follow the pipeline would have either to have exactly the same acquisition conditions as the manuscript or start playing with values and try to compensate for any difference in their data (cell diameter, fluorescent intensity, etc.) to see if they can match the results of the manuscript.

Reviewer #1 (Public Review):

• A summary of what the authors were trying to achieve.

The authors cultured pre- and Post-vaccine PBMCs with overlapping peptides encoding S protein in the presence of IL-2, IL-7, and IL-15 for 10 days, and extensively analyzed the T cells expanded during the culture; by including scRNAseq, scTCRseq, and examination of reporter cell lines expressing the dominant TCRs. They were able to identify 78 S epitopes with HLA restrictions (by itself represents a major achievement) together with their subset, based on their transcriptional profiling. By comparing T cell clonotypes between pre- and post-vaccination samples, they showed that a majority of pre-existing S-reactive CD4+ T cell clones did not expand by vaccinations. Thus, the authors concluded that highly-responding S-reactive T cells were established by vaccination from rare clonotypes.

• An account of the major strengths and weaknesses of the methods and results.

Strengths

• Selection of 4 "Ab sustainers" and 4 "Ab decliners" from 43 subjects who received two shots of mRNA vaccinations.<br /> • Identification of S epitopes of T cells together with their transcriptional profiling. This allowed the authors to compare the dominant subsets between sustainers and decliners.

Weaknesses were adequately addressed in the revised manuscript, and I do not have any additional concerns.

Reviewer #1 (Public Review):

Summary:

Wang et al. generate XAP5 and XAP5L knockout mice and find that they are male infertile due to meiotic arrest and reduced sperm motility, respectively. RNA-Seq was subsequently performed and the authors concluded that XAP5 and XAP5L are antagonistic transcription factors of cilliogenesis (in XAP5-KO P16 testis: 554 genes were unregulated and 1587 genes were downregulated; in XAP5L-KO sperm: 2093 genes were unregulated and 267 genes were downregulated).

Strengths:

Knockout mouse models provided strong evidence to indicate that XAP5 and XAP5L are critical for spermatogenesis and male fertility.

Weaknesses:

The key conclusions are not supported by evidence. First, the authors claim that XAP5 and XAP5L transcriptionally regulate sperm flagella development; however, detailed molecular experiments related to transcription regulation are lacking. How do XAP5 and XAP5L regulate their targets? Only RNA-Seq is not enough. Second, the authors declare that XAP5 and XAP5L are antagonistic transcription factors; however, how do XAP5 and XAP5L regulate sperm flagella development antagonistically? Only RNA-Seq is not enough. Third, I am concerned about whether XAP5 really regulates sperm flagella development. XAP5 is specifically expressed in spermatogonia and XAP5-cKO mice are in meiotic arrest, indicating that XAP5 regulates meiosis rather than sperm flagella development.

Reviewer #1 (Public Review):

Summary:

Babosha et al. deeply investigate the N-terminal region of the Drosophila dosage compensation protein MSL1. Much of the prior research into the dosage compensation complex has focused on the male-specific MSL2 protein. However, the authors point out prior evidence that the N-terminus of MSL1 is important for protein function, including interaction with MSL2. Through a series of transgenic deletions and substitutions, the authors pinpoint two regions: N-terminal amino acids 3-7 and 41-65, which are critical for the binding of MSL1 to the X-chromosome and recruitment of MSL2. To deepen these observations, the authors perform well-controlled immunoprecipitation experiments to test the interaction of mutant MSL1 proteins with the lncRNA roX2, which is critical for the stability and localization of the dosage compensation complex. Through immunoprecipitation, the authors discover that the interaction of their mutant MSL1 proteins with roX2 is compromised. They suggest that the roX-MSL1 interaction is mediated by the N-terminal amino acids and is also critical for interaction with MSL2 and X-specific localization. This agrees with previous models that MSL1 and MSL2 directly interact through other regions.

This work lays the foundation for future investigations into the overall structure of the dosage compensation machinery, which allows this unique complex to specifically target the X-chromosome through still unclear mechanisms.

Strengths:

The data provided by the authors is of high quality and supports the authors' conclusions, which are nicely contextualized in the text with previous models. The novelty of this study is specifically pinpointing the amino acid regions of MSL1 that interact with roX. The authors point out that, surprisingly, the N-terminal region of MSL1 is not particularly well conserved, indicating that the interactions outlined in this study might be Drosophila/Diptera-specific.

The major strength of this study is that the authors find agreement between multiple dimensions of experimentation: the regions of MSL1 that are required for roX2 interaction (immunoprecipitation experiments) are also the regions that are critical for MSL1 localization to polytene chromosomes in an artificial female in vivo system, which are also critical for male-specific survival. The authors later suggest that it is the roX2 interaction that is responsible for the latter observations, although there is no direct evidence for this suggestion.

Weaknesses:

A minor weakness of the study is that it largely supports, and incrementally expands, the existing model in the field: that roX RNAs mediate the assembly of the complex on chromatin. I hesitate to call this a weakness, as supporting an existing model is still strong scientifically. However, the current study does not dramatically push the model forward.

Reviewer #1 (Public Review):

Summary:

This paper focuses on the effects of a L114P mutation in the TALK-1 channel on islet function and diabetes. This mutation is clinically relevant and a cause of MODY diabetes. This work employs a mouse model with heterozygous and homozygous mutants. The homozygous mice are homozygous lethal from severe hyperglycemia. The work shows that the mutation increases K+ currents and inhibits insulin secretion. This is a very nice paper with mechanistic insight and clear clinical importance. It is generally well written and the data is well presented.

Comments on revision:

I have no further comments to add at this time. The authors have adequately addressed my concerns.

Reviewer #1 (Public Review):

Summary:

In this study the authors demonstrated that ablation of astrocytes in lumbar spinal cord not only reduced neuropathic pain but also caused microglia activation. Furthermore, RNA sequencing and bioinformatics revealed an activation of STING/type I IFNs signal pathway in spinal cord microglia after astrocyte ablation.

Strengths:

The findings are novel and interesting and provide new insights into astrocyte-microglia interaction in neuropathic pain. This study may also offer a new therapeutic strategy for the treatment of debilitating neuropathic pain in patients with SCI.

Weaknesses:

More details are needed to justify the sample size, statistics, and sex of animals.

Reviewer #1 (Public Review):

Summary:

This work revealed an important finding that the blood-brain barrier (BBB) functionality changes with age and is more pronounced in males. The authors applied a non-invasive, contrast-agent-free approach of MRI called diffusion-prepared arterial spin labeling (DP-pCASL) to a large cohort of healthy human volunteers. DP-pCASL works by tracking the movement of magnetically labeled water (spins) in blood as it perfuses brain tissue. It probes the molecular diffusion of water, which is sensitive to microstructural barriers, and characterizes the signal coming from fast-moving spins as blood and slow-moving spins as tissue, using different diffusion gradients (b-values). This differentiation is then used to assess the water exchange rates (kw) across the BBB, which acts as a marker for BBB functionality. The main finding of the authors is that kw decreases with age, and in some brain regions, kw decreases faster in males. The neuroprotective role of the female sex hormone, estrogen, on BBB function is discussed as one of the explanations for this finding, supported by literature. The study also shows that BBB function remains stable until the early 60s and remarkably decreases thereafter.

Strengths:

The two main strengths of the study are the MRI method used and the amount of data. The authors employed a contrast-agent-free MRI method called ASL, which offers the opportunity to repeat such experiments multiple times without any health risk - a significant advantage of ASL. Since ASL is an emerging field that requires further exploration and testing, a study evaluating blood-brain barrier functionality is of great importance. The authors utilized a large dataset of healthy humans, where volunteer data from various studies were combined to create a substantial pool. This strategy is effective for statistically evaluating differences in age and gender.

Weaknesses:

Gender-related differences are only present in some brain regions, not in the whole brain or gray matter - which is usually the assumption unless stated otherwise. From the title, this was not clear. Including simulations could increase readers' understanding related to model fitting and the interdependence of parameters, if present. The discussion follows a clear line of argument supported by literature; however, focusing solely on AQP4 channels and missing a critical consideration of other known/proven changes in transport mechanisms through the BBB and their effects substantially weakens the discussion.

Reviewer #1 (Public Review):

Updated summary:

Glenn et al. present solid evidence that both lab and clinical Salmonella enterica serovars rapidly migrate towards human serum using an exciting approach that combines microfluidics, structural biology and genotypic analysis. The authors succeed in bringing to light a novel context for the role of serine as a bacterial chemoattractant as well as documenting what is likely to be a key step in bloodstream entry for some of the main sepsis-associated pathogens during gastrointestinal bleeding. They illustrate the generality of their findings through phylogenetic analysis, testing additional species within the Enterobacteriaceae family and showing attraction towards swine and equine serum. Their interdisciplinary approach here greatly increases the scope of their findings.<br /> I would also like to note that, whilst I enjoyed the interdisciplinary scope of this study, I am personally not well placed to review the protein structural aspects of this work.

Additional strengths of the revised manuscript:

All weaknesses raised in my review of the original manuscript have been satisfactorily addressed in the revised manuscript. It is interesting to note that the accumulation pattern of the bacteria 50-75 um from the source of serum could, as the author's now note, be due to the avoidance of bactericidal serum elements. Alternative explanations, however, could include chemoreceptor saturation (i.e. close to the serum source, high ligand concentrations could saturate chemoreceptors preventing further chemotaxis) or Weber's Law considerations (cell's ability to detect a given change in chemical concentrations diminishes with increasing background concentrations - thus, as cells get closer to the serum source, their ability to chemotax decreases).

The authors have also added new experimental data and analyses and these constitute major new strengths of the revised manuscript:<br /> - The authors show that the competitive advantage of WT cells relative to a tsr mutant is removed when serum is treated with serine-racemase and this provides strong evidence that chemotaxis towards serine is responsible for the reduced attraction of the tsr mutant towards serum (i.e. rather than any possible pleiotropic effects).<br /> - New experimental data showing Salmonella enterica is also attracted to swine and equine serum (including an ex vivo swine model) is a useful addition that hints at the potential generality of the response reported here.<br /> - The authors now include additional data to back up the intriguing lack of a movement response towards norepinephrine and DHMA reported here.

Additional weaknesses of the revised manuscript:

- The addition of an ex vivo swine model is an exciting new inclusion in the updated manuscript. However, information regarding biological and technical replication here is currently unclear or missing.

Reviewer #1 (Public Review):

Summary:

The presented study by Centore and colleagues investigates the inhibition of BAF chromatin remodeling complexes. The study is well-written, and includes comprehensive datasets, including compound screens, gene expression analysis, epigenetics, as well as animal studies. This is an important piece of work for the uveal melanoma research field, and sheds light on a new inhibitor class, as well as a mechanism that might be exploited to target this deadly cancer for which no good treatment options exist.

Strengths:

This is a comprehensive and well-written study.

Weaknesses:

There are minimal weaknesses.

Reviewer #1 (Public Review):

Summary:

In this study, the authors investigate the potential therapeutic effects of the PEGylated PDZ peptide, derived from the ZO-1 protein, in suppressing LPS-induced systemic inflammation. The authors found that the pretreatment of PEGylated PDZ peptide led to a restoration of tissue injuries in the kidney, liver, and lung, and diminished alterations in biochemical plasma markers induced by LPS. This was accompanied by decreased production of pro-inflammatory cytokines in the plasma and lung BALF of the PDZ-administered mice.

Strengths:

- The data presented here is solid and the results provide the groundwork for developing novel anti-inflammatory therapeutic strategies.<br /> - The authors employ various cells and in vivo models to test the efficacy of the peptide.

Weaknesses:<br /> The mechanism of action remains largely unknown.

Reviewer #1 (Public Review):

In this study, Alejandro Rosell et al. uncovers the immunoregulation functions of RAS-p110α pathway in macrophages, including the extravasation of monocytes from the bloodstream and subsequent lysosomal digestion. Disrupting RAS-p110α pathway by mouse genetic tools or by pharmacological intervention, hampers the inflammatory response, leading to delayed resolution and more severe acute inflammatory reactions. The authors proposed that activating p110α using small molecules could be a promising approach for treating chronic inflammation. This study provides insights into the roles and mechanisms of p110α on macrophage function and the inflammatory response, while some conclusions are still questionable because of several issues described below.

(1) Fig. 1B showed that disruption of RAS-p110α causes the decrease in the activation of NF-κB, which is a crucial transcription factor that regulates the expression of proinflammatory genes. However, the authors observed that disruption of RAS-p110α interaction results in an exacerbated inflammatory state in vivo, in both localized paw inflammation and systemic inflammatory mediator levels. Also, the authors introduced that "this disruption leads to a change in macrophage polarization, favouring a more proinflammatory M1 state" in introduction according to reference 12. The conclusions drew from the signaling and the models seemed contradictory and puzzling. Besides, it is not clear why the protein level of p65 was decreased at 10' and 30'. Was it attributed to the degradation of p65 or experimental variation?

(2) In Fig 3, the authors used bone-marrow derived macrophages (BMDMs) instead of isolated monocytes to evaluate the ability of monocyte transendothelial migration, which is not sufficiently convincing. In Fig. 3B, the authors evaluated the migration in Pik3caWT/- BMDMs, and Pik3caWT/WT BMDMs treated with BYL-719'. Given that the dose effect of gene expression, the best control is Pik3caWT/- BMDMs treated with BYL-719.

(3) In Fig. 4E-4G, the authors observed that elevated levels of serine 3 phosphorylated Cofilin in Pik3caRBD/- BMDMs both in unstimulated and in proinflammatory conditions, and phosphorylation of Cofilin at Ser3 increase actin stabilization, it is not clear why disruption of RAS-p110α binding caused a decrease in the F-actin pool in unstimulated BMDMs?

Reviewer #1 (Public Review):

Summary:

This paper reports a number of somewhat disparate findings on a set of colorectal tumour and infiltrating T-cells. The main finding is a combined machine-learning tool which combines two previous state-of-the-art tools, MHC prediction, and T-cell binding prediction to predict immunogenicity. This is then applied to a small set of neoantigens and there is a small-scale validation of the prediciton at the end.

Strengths:

The prediction of immunogenic neoepitopes is an important and unresolved question.

Weaknesses:

The paper contains a lot of extraneous material not relevant to the main claim. Conversely, it lacks important detail on the major claim.

(1) The analysis of T cell repertoire in Figure 2 seems irrelevant to the rest of the paper. As far as I could ascertain, this data is not used further.

(2) The key claim of the paper rests on the performance of the ML algorithm combining NETMHC and pmtNET. In turn, this depends on the selection of peptides for training. I am unclear about how the negative peptides were selected. Are they peptides from the same databases as immunogenic petpides but randomised for MHC ? It seems as though there will be a lot of overlap between the peptides used for testing the combined algorithm, and the peptides used for training MHCNet and pmtMHC. If this is so, and depending on the choice of negative peptides, it is surely expected that the tools perform better on immunogenic than on non-immunogenic peptides in Figure 3. I don't fully understand panel G, but there seems very little difference between the TCR ranking and the combined. Why does including the TCR ranking have such a deleterious effect on sensitivity?

(3) The key validation of the model is Figure 5. In 4 patients, the authors report that 6 out 21 neo-antigen peptides give interferon responses > 2 fold above background. Using NETMHC alone (I presume the tool was used to rank peptides according to bding to the respecitve HLAs in each individual, but this is not clear), identified 2; using the combined tool identified 4. I don't think this is significant by any measure. I don't understand the score shown in panel E but I don't think it alters the underlying statistic.

In conclusion, the paper demonstrates that combining MHCNET and pmtMHC results in a modest increase in the ability to discriminate 'immunogenic' from 'non-immunogenic' peptide; however, the strength of this claim is difficult to evaluate without more knowledge about the negative peptides. The experimental validation of this approach in the context of CRC is not convincing.

Joint Public Review:

Summary:

In their paper Li et al. investigate the transcriptome of satellite cells obtained from different muscle types including hindlimb, diaphragm and extraocular muscles (EOM) from wild type and G93A transgenic mice (end stage ALS) in order to identify potential factors involved in the maintenance of the neuromuscular junction. The underlying hypothesis being that since EOMs are largely spared from this debilitating disease, they may secrete NMJ-protective factors. The results of their transcriptome analysis identified several axon guidance molecules including the chemokine Cxcl12, which are particularly enriched in EOM-derived satellite cells. Transduction of hindlimb-derived satellite cells with AAV encoding Cxcl12 reverted hindlimb-derived myotubes from the G93A mice into myotubes sharing phenotypic characteristics similar to those of EOM-derived satellite cells. Additionally, the authors were able to demonstrate that EOM-derived satellite cell myotube cultures are capable of enhancing axon extensions and innervation in co-culture experiments.

Strengths:

The strength of the paper is that the authors successfully isolated and purified different populations of satellite cells, compared their transcriptomes, identified specific factors release by EOM-derived satellite cells, overexpressed one of these factors (the chemokine Cxcl12) by AAV-mediated transduction of hindlimb-derived satellite cells. The transduced cells were then able to support axon guidance and NMJ integrity. They also show that administration of Na butyrate to mice decreased NMJ denervation and satellite cell-depletion of hind limbs. Furthermore, addition of Na Butyrate to hindlimb derived satellite cell myotube cultures increased Cxcl12 expression. These are impressive results providing important insights for the development of therapeutic targets to slow the loss on neuromuscular function characterizing ALS.

Comments on latest version:

The authors have sufficiently acknowledged and discussed the limitations of experiments involving NaBu treatment. The authors have also addressed the use of AAV-mediated delivery of Cxcl12.

Reviewer #1 (Public Review):

In this manuscript, Ngo et al. report a peculiar effect where a single base mismatch (CC) can enhance the mechanical stability of a nucleosome. In previous studies, the same group used a similar state-of-the-art fluorescence-force assay to study the unwrapping dynamics of 601-DNA from the nucleosome and observed that force-induced unwrapping happens more slowly for DNA that is more bendable because of changes in sequence or chemical modification. This manuscript appears to be a sequel to this line of projects, where the effect of CC is tested. The authors confirmed that CC is the most flexible mismatch using the FRET-based cyclization assay and found that unwrapping becomes slower when CC is introduced at three different positions in the 601 sequence. The CC mismatch only affects the local unwrapping dynamics of the outer turn of nucleosomal DNA.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Ketaren, Mast, Fridy et al. assessed the ability of a previously generated llama nanobody library (Mast, Fridy et al. 2021) to bind and neutralize SARS-CoV-2 delta and omicron variants. The authors identified multiple nanobodies that retain neutralizing and/or binding capacity against delta, BA.1 and BA.4/5. Nanobody epitope mapping on spike proteins using structural modeling revealed possible mechanisms of immune evasion by viral variants as well as mechanisms of cross-variant neutralization by nanobodies. The authors additionally identified two nanobody pairs involving non-neutralizing nanobodies that exhibited synergy in neutralization against the delta variant. These results enabled the refinement of target epitopes of the nanobody repertoire and the discovery of several pan-variant nanobodies for further preclinical development.

Strengths:

Overall, this study is well executed and provides a valuable framework for assessing the impact of emerging SARS-CoV-2 variants on nanobodies using a combination of in vitro biochemical and cellular assays as well as computational approaches. There are interesting insights generated from the epitope mapping analyses, which offer possible explanations for how delta and omicron variants escape nanobody responses, as well as how some nanobodies exhibit cross-variant neutralization capacity. These analyses laid out a clear path forward for optimizing these promising next-gen therapeutics, particularly in the face of rapidly emerging SARS-CoV-2 variants. This work will be of interest to researchers in the fields of antibody/nanobody engineering, SARS-CoV-2 therapeutics, and host-virus interaction.

Weaknesses:

A main weakness of the study is that the efficacy statement is not thoroughly supported. While the authors comprehensively characterized the neutralizing ability of nanobodies in vitro, there is no animal data involving mice or hamsters to demonstrate the real protective efficacy in vivo. Yet, in the title and throughout the manuscript, the authors repeatedly used phrases like "retains efficacy" or "remains efficacious" to describe the nanobodies' neutralization or binding capacities. This claim is not well supported by the data and underestimates the impact of variants on the nanobodies, especially the omicron sublineages. For example, the authors showed that S1-RBD-15 had a ~100-fold reduction in neutralization titer against Omicron, with an IC50 at around 1 uM. This is much higher than the IC50 value of a typical anti-ancestral RBD nanobody reported in the previous study (Mast, Fridy et al. 2021). In fact, the authors themselves ascribe nanobodies with an IC50 above 1 uM as weak neutralizers. And there were many in the range of 0.1-1 uM. Furthermore, many nanobodies selected for affinity measurement against BA.4/5 had no detectable binding. Without providing in vivo protection data or including monoclonal antibodies that are known to be efficacious against variants in the in vitro assays as a benchmark, it is difficult to evaluate the efficacy just with the IC50 values.

Comments post revision:

The authors are to be commended for their comprehensive response to the referees' comments. In the revised manuscript, the authors made extensive changes throughout the texts and added new figures that greatly improved their clarity. While the manuscript is still limited in solely relying on in vitro data for efficacy assessment, it nicely demonstrates how the combination of experimental and computational techniques could lead to the discovery of broadly neutralizing nanobody candidates for further lead optimization.

Reviewer #1 (Public Review):

Summary:

The Roco proteins are a family of GTPases characterized by the conserved presence of an ROC-COR tandem domain. How GTP binding alters the structure and activity of Roco proteins remains unclear. In this study, Galicia C et al. took advantage of conformation-specific nanobodies to trap CtRoco, a bacterial Roco, in an active monomeric state and determined its high-resolution structure by cryo-EM. This study, in combination with the previous inactive dimeric CtRoco, revealed the molecular basis of CtRoco activation through GTP-binding and dimer-to-monomer transition.

Strengths:

The reviewer is impressed by the authors' deep understanding of the CtRoco protein. Capturing Roco proteins in a GTP-bound state is a major breakthrough in the mechanistic understanding of the activation mechanism of Roco proteins and shows similarity with the activation mechanism of LRRK2, a key molecule in Parkinson's disease. Furthermore, the methodology the authors used in this manuscript - using conformation-specific nanobodies to trap the active conformation, which is otherwise flexible and resistant to single-particle average - is highly valuable and inspiring.

Reviewer #1 (Public Review):

In the presented manuscript, the authors investigate how neural networks can learn to replay presented sequences of activity. Their focus lies on the stochastic replay according to learned transition probabilities. They show that based on error-based excitatory and balance-based inhibitory plasticity networks can self-organize towards this goal. Finally, they demonstrate that these learning rules can recover experimental observations from song-bird song learning experiments.

Overall, the study appears well-executed and coherent, and the presentation is very clear and helpful. However, it remains somewhat vague regarding the novelty. The authors could elaborate on the experimental and theoretical impact of the study, and also discuss how their results relate to those of Kappel et al, and others (e.g., Kappel et al (doi.org/10.1371/journal.pcbi.1003511)). Overall, the work could benefit if there was either (A) a formal analysis or derivation of the plasticity rules involved and a formal justification of the usefulness of the resulting (learned) neural dynamics; and/or (B) a clear connection of the employed plasticity rules to biological plasticity and clear testable experimental predictions. Thus, overall, this is a good work with some room for improvement.

Reviewer #1 (Public Review):

Summary:

The aim of the present work is to evaluate the role of BMP9 and BMP10 in liver by depleting Bmp9 and Bmp10 from the main liver cell types (endothelial cells (EC), hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes (H)) using cell-specific cre recombinases. They show that HSCs are the main source of BMP9 and BMP10 in the liver. Using transgenic ALK1 reporter mice, they show that ALK1, the high affinity type 1 receptor for BMP9 and BMP10, is expressed on KC and EC. They have also performed bulk RNAseq analyses on whole liver, and cell-sorted EC and KC, and showed that loss of Bmp9 and Bmp10 decreased KC signature and that KC are replaced by monocyte-derived macrophages. EC derived from these Bmp9fl/flBmp10fl/flLratCre mice also lost their identity and transdifferentiated into continuous ECs. Liver iron metabolism and metabolic zonation were also affected in these mice. In conclusion, this work supports that BMP9 and BMP10 produced by HSC play a central role in mediating liver cell-cell crosstalk and liver homeostasis.

Strengths:

This work further supports the role of BMP9 and BMP10 in liver homeostasis. Using a specific HSC-Cre recombinase, the authors show for the first time that it is the BMP9 and BMP10 produced by HSC that play a central role in mediating liver cell-cell crosstalk to maintain a healthy liver. Although the overall message of the key role of BMP9 in liver homeostasis has been described by several groups, the role of hepatic BMP10 has not been studied before. Thus, one of the novelties of this work is to have used liver cell specific Cre recombinase to delete hepatic Bmp9 and Bmp10. The second novelty is the demonstration of the role of BMP9 and BMP10 in KC Differentiation/homeostasis which has already been slightly addressed by this group by knocking out ALK1, the high affinity receptor of BMP9 and BMP10 (Zhao et al. JCI, 2022).

Weaknesses:

This work remains rather descriptive and the molecular mechanisms are barely touched upon and could have been more explored.<br /> Some references should be added; In particular, a work that has already demonstrated, using a different approach (in situ hybridization RNAscope), that in the liver BMP9 and BMP10 are expressed by HSC (Tillet et al., J Biol Chem 2018). Another publication (Bouvard et al., Cardiovasc Res, 2021) has previously showed that deletion of Bmp9 and Bmp10 leads to liver fibrosis and could have thus been cited. There is also a reference that is not correctly cited. Ref 26 (Herrera et al., 2014) does not say that "BMP10 is mostly expressed in the heart, followed by the liver" or that "BMP9 and BMP10 also bind to ALK2" as cited in the manuscript.<br /> The gating strategies for cell sorting which is used for bulk RNAseq and FACS analyses should be better described in order to better follow the manuscript. This point is particularly important for KC gating as the authors show that Tim4 is very strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2c), yet, it seems that this marker is used for gating macrophages (Suppl fig4). Same question with F4/80 which is strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2d) and also used for gating. It is important to show the gating strategy for both Control and Bmp9fl/flBmp10fl/flLratCre mice.<br /> The authors should explain how they selected the genes shown on each heatmaps and add references that can justify the choice of the genes.<br /> Quantifications of Immunostaining and FACS data should be added as well as statistical analyses.

Reviewer #1 (Public Review):

This study holds significant importance as it assessed antibody levels arising from both COVID-19 vaccination and natural infection in a representative population-based sample. The analysis was conducted with thoughtfulness and rigor. The sampling methodology ensured the representation of the broader Canadian population, including minorities and indigenous communities. Findings suggest, that despite a substantial number of individuals having been previously infected, especially following the first omicron wave, repeat booster vaccination is essential to ensure that individuals develop an optimal antibody response against new exposures to infection, given the waning of antibodies over time. The study findings carry global significance as it informs decisions about the relevance of booster vaccination for reducing infection incidence amid the ongoing challenge of vaccine hesitancy and the continual emergence of new variants.

Among the weaknesses of the study, from my perspective, is the lack of explicit clarification that one objective of achieving repeat booster vaccination is to impart a robust level of protection against acquiring infections. Previous studies have demonstrated that the effectiveness of even only primary-series vaccination against COVID-19 severe disease was high, with slow waning over time. However, even when effectiveness against severity is high, infections may still present a risk for progression to severe COVID-19 among older individuals and those with comorbidities. Another limitation is that the study did not investigate whether there were variations in spike levels based on the last vaccine type administered. Furthermore, it is important to comment on the generalizability of the findings considering that individuals who participated in the research may have been different from those who did not participate and therefore residual confounding cannot be eliminated.

Reviewer #1 (Public Review):

Authors investigated the role of OBOX4 in the zygotic genome activation (ZGA) in mice. Obox4 genes form an array of duplicated genes they were identified as a candidate ZGA factor based on expression patterns during early development. The role of OBOX4 was subsequently studied in embryonic stem cells and early embryos. It was found that transcriptional activation mediated by OBOX4 has similar features as that of DUX, which was previously identified as a zygotic transcription factor involved in ZGA and a major activator of the zygotic expression program. It was, however, unexpected that Dux knock-out did not impair embryonic development. The work by Guo et al. provides several lines of evidence that OBOX4-mediated activation of gene expression considerably overlaps with that of DUX and this redundancy might explain the loss of early developmental phenotype in Dux mutants. Consistent with this model, double mutants of Obox4 and Dux show impaired development. Given the difficulties with investigating details of the genetic model in double mutants at the preimplantation embryo stage, authors not only crossed genetic mutants, but also used (1) nuclear transfer of mutated nuclei of ESCs, which could be characterized on their own in separate experiments, and (2) antisense oligonucleotides (ASO) microinjection, which included a rescue control demonstrating that reintroducing OBOX4 is sufficient to rescue the phenotype caused by blocking both, Dux and Obox4.

This work is important for the field because it reveals functional redundancy and plasticity of the zygotic genome activation in mammals, where the mouse model stands as a remarkable example of genome activation, which massively integrated long terminal repeat (LTR)-derived enhancers from retrotransposons and now two of the key activating zygotic factors appear to be encoded by tandemly duplicated clusters of different phylogenetic age. Identification of OBOX4 as a second factor partially redundant with DUX now allows us to decipher what constitutes the essential part of the ZGA program.

Reviewer #1 (Public Review):

Summary:

This study examines how blood vessels exposed to the cytokine VEGF respond to vascular leakage when the VEGF receptor NRP1 is targeted. This study compares results in in two different body sites of the dermis and in a different organ, the trachea. The authors refer to the two different sites of the dermis as two different organs, but the dermis is one organ. The authors report that vascular leakage is differentially affected by NRP1 targeting in the ear skin compared to the trachea and back skin. They attribute these differences to NRP1 presence in cells other than the vascular endothelium, especially in the ear skin, where they observe higher perivascular NRP1 staining.

The manuscript states that the aim was to uncover the role of NRP1 in VEGF-mediated vascular permeability. This was misleading, because a lot is already known on NRP1 in this pathway, as is evidenced by a large number of publications the authors themselves quote (and sometimes misquote). The main information they wish to add is the possibility that NRP1 may also play a role in other cells to regulate permeability, as they previously suggested for blood vessel growth. Several technical issues and experimental limitations call into question whether the above conclusion can be reached with the data provided.

Strengths:

It is an interesting concept that NRP1 regulates vascular permeability by acting in perivascular cells.

Weaknesses:

(A) Technical limitations due to assay type:

A direct comparison of the skin in two body sites is not warranted given that the authors used different methods to study the two sites. Below is a list of differences reported in their methods section:

(A1) Different tracers were used to visualize VEGF165-induced leakage in different sites.<br /> Ear skin assay: 2 kDa FITC and two different dextrans, 10 kDa TRITC dextran, and another dextran whose molecular weight is not specified. It is not explained why 3 different tracers were used. Figures 1 and 2 report data with 2 kDa TRITC dextran.<br /> Back skin assay: They describe the Miles assay using Evans Blue, which binds to albumin, making it a 67 kDa tracer. However, Figure 1 suggests that 2 kDa dextran was used, and perhaps Evans Blue was only used for the supplemental data. This is relevant because current knowledge suggests that small dyes use the junctional pathway, whereas larger proteins such as albumin can use vesicular transport. The former is thought to be a fast pathway (hence, the authors measured dye extravasation 3 min after VEGF165 injection). The latter pathway is a slower one (hence, measured 30 min after VEGF165 injection in the Miles assay).

Quantification: For ear skin, the number of leakage sites and lag period is quantified, as well as leakage over time. For back skin, the amount of extravasated dye is quantified at a fixed time point. Such different measurements do not allow for direct comparison.

(A2) Mice were prepared in different ways for the different body sites studied:<br /> Ear skin assay: general anesthesia with ketamine-xylazine.<br /> Back skin assay: No anesthesia is described for the back skin Miles assay. This would be a concern because intradermal injections are considered to be painful. For back skin histology, they do report to have used isoflurane anesthesia before perfusion fixation. However, it is not advisable to use used isoflurane anesthesia for perfusion fixation if this has been done via the conventional cardiac route, because opening the chest cavity to access the heart for perfusion causes lung collapse, meaning that the mice cannot breathe the anaesthetic, and there is a risk of them regaining consciousness. The authors should clarify what exactly they have done, for ethical reasons and also because the type of anesthesia can affect vascular studies, for example, see PMID 36418078.

(A3) Differential histamine use:<br /> Back skin assay: uses anti-histamine, as is advised with intradermal injections to minimize vascular leakage due to histamine release after local trauma.<br /> Ear skin assay: no anti-histamine was used, so histamine-induced background leakage might have been present, independently of VEGF165. The authors suggest that the ear skin injection does not cause trauma, but it is unclear how this is possible, given that skin needs to be disrupted for the needle to enter the tissue.

(A4) Different VEGF165 concentration used:<br /> The ear skin assay uses 10 ng VEGF per injection, and the back skin assay 80 ng.

Given all these differences in experimental protocols, as well as different knockdown efficiency (see below), the results for the different sites are not directly comparable. Hence it cannot presently be concluded that the role of NRP1 in both sites is different, and further work is required to make a firm conclusion. In addition, the conflicts between the reported methods and figures need to be resolved.

(B) It is unclear whether appropriate controls were used:

(B1) What genotype and treatment are the control mice for NRP1 targeting? The ideal control would be wild-type mice with the same CreER, injected with tamoxifen according to the same timeline, to account for vehicle, tamoxifen, and tamoxifen-induced CreER toxicity (https://doi.org/10.1038/s44161-022-00125-6). This could be a littermate mouse or, alternatively, a separate experiment should be shown comparing wild-type mice carrying the same CreER as used for the ablation studies and injected with tamoxifen, versus wild-type mice injected with tamoxifen, to demonstrate that the induction regime does not in itself cause phenotypes.

(B2) Has a PBS injection been performed to compare baseline leakage between genotypes, independently of VEGF165 injections? This is an essential control.

(B3) The experimental protocol assays 4 days after 5 consecutive tamoxifen injections, which does not allow much time for drug washout. Moreover, this is a lot of tamoxifen (80 mg x 5 = 400 mg tamoxifen per kg). Due to the possibility that tamoxifen-induced effects might still be present and cause sex-differential effects, the corresponding sex for each individual data point should be indicated in all graphs.

(B4) i.p. peanut oil is used in undefined volumes; this vehicle was shown to cause inflammation if administered i.p. (PMID 33139505). Therefore, inflammation might be present, which might affect different body sites differently.

(C) Validation of NRP1 targeting:<br /> The authors have not performed an NRP1 knockout in the endothelium, as they repeatedly claim. In the lung, there is a good knockdown of around 75%; this may or may not be due to complete EC knockdown with preservation of NRP1 in other cell types. In the trachea, ear skin, and back skin, knockdown was not quantified, although qualitative comparisons by NRP1 immunostaining in Supplementary Figure 1 suggest that the back skin targeting worked better than the ear skin targeting, which would confound results, but in any case, it was neither a knockdown nor knockout. The staining for global targeting looks fainter than for the other genotypes, and the single-channel images seem to have different intensities than the overlays in Supplementary Figure 1 A.

(D) Systemic permeability studies:<br /> Organs have very different baseline permeability, due to the properties of the vascular barrier, i.e. tight barriers in the brain and retina and permeable endothelium in the liver and kidney. In this assay, VEGF is not delivered from the tissue side, as would be typical during inflammation but is delivered through the circulation, which has been shown to differentially affect the VEGF response, at least in some tissues (PMID 25175707). Nevertheless, this is a helpful readout, especially given that PBS controls appear not to have been performed above to establish baseline leakage between genotypes and tissues.

Figure Supplement 3 shows that VEGF induces vascular leakage in all body sites examined, independently of the size of the tracer used, and agreeing with current literature. An additional set of panels should be included with data shown without calculating the fold change relative to the control, set to 1, to account for the endothelium in different organs having different baseline vascular permeability. How do the authors explain that VEGF has the same effect in the ear and back skin in this assay, when NRP1 is present, given that they claim a role for perivascular NRP1 in the ear, but not back skin, for reducing VEGF/VEGFR2 signalling?

(E) Comparing results obtained with different tools:

- The endothelial NRP1 knockdown yielded different results for ear and back skin.<br /> - Anti-NRP1 yielded similar results for ear and back skin.<br /> - The global NRP1 ko yielded similar results for ear and back skin.<br /> Because anti-NRP1 and the global NRP1 knockdown gives similar results for all tissues, the authors deduce that the NRP1 acts in cell types other than endothelial cells to regulate permeability. This is an interesting idea, based on the lab's prior work in angiogenesis. In their trans-interaction scenario, NRP1 would have the same role in ECs in all sites, but non-endothelial NRP1 can override the function of the endothelial NRP1 function depending on its expression levels.

Confidence in this conclusion would require additional experiments:<br /> - Show that the endothelial knockdown works equally well in different body sites, via NRP1 staining and/or by checking recombination efficiency with a reporter.<br /> - Using an analogous assay to measure permeability in different body sites.<br /> - Perform a non-endothelial knockdown, i.e. in pericytes, which is hypothesized to be the source of NRP1 that affects vascular leakage signalling in endothelial cells in trans.

(F) Abstract, introduction, and references:<br /> The authors suggest controversy with regard to NRP1's roles in permeability. However, NRP1's function in VEGF signalling has been defined as being an accessory to VEGFR2, with a role in promoting SFK activation. This function relies on the NRP1 cytoplasmic domain, which mediates VEGFR2 trafficking and signalling; the relevant literature for the NRP1 cytoplasmic domain is mentioned for arteriogenesis (PMID 23639442), but not permeability (PMID 28289053). Another paper is mentioned which describes a VEGFR2-independent pathway for a CendR ligand, but this prior study did NOT make the claim that VEGF signalling is NRP1-independent or promotes it (PMID 27117252). In the eye, NRP1 has been implicated in both SEMA3A and VEGF165-induced permeability, which was also corroborated by the Miles assay in two prior studies (PMID 18180379, PMID 28289053). The last sentence in the abstract is incorrect, because differences in ear versus back skin do not constitute organotypic difference (as the organ is the dermis), and the potential role of perivascular cells is only inferred from the global endothelial NRP1 knockdown, which gives the same result as reported for the endothelial NRP1 knockdown in the literature.

(1) Lines 5/.53: The references for VEGF-NRP1 signalling in age-related macular degeneration are not helpful: Raimondi investigated VEGF-independent NRP1 pathways in angiogenesis, Fernandez-Robredo investigated NRP1 pathways in angiogenesis and showed that fewer vessels correlated with less leakage but did not test VEGF signaling specifically. A more suitable reference would have been PMID 28289053.

(2) Lines 63/64 and repeated in 84-89: The references quoted all showed that NRP1 inhibition reduces vascular permeability, and therefore do not provide evidence for the idea that NRP1 inhibition promotes permeability, as the authors report here for the ear skin; the only study supporting them is one using arterial endothelial cells, which are not permeability-relevant.

(3) Lines 106/107: The references used to underpin organ-specific barrier properties are correct, but as stated above, the dermis is the dermis, and therefore, these references would not be useful to provide support for the idea that the ear and back skin behave differently after NRP1 knockdown.

(G) Additional comments on the figures:<br /> Figure 4: The authors show that VEGFR2 is essential for permeability, and VEGF164 effects are VEGFR2 dependent - this is well established for VEGF164 in the Miles assay, including the accessory role of NRP1 (e.g. PMID 28289053). As the proposed trans function of NRP1 cannot make a difference in VEGFR2 signaling when VEGFR2 is not there, this experiment is only confirmatory of prior VEGFR2 knowledge.

Reviewer #1 (Public Review):

Summary:

In this article, Kremser et al set off to explore how local interactions between cells can drive pattern formation by focusing on the French flag problem whereby an initially homogeneous system breaks axial symmetry to form three distinct regions of different cell fates. The authors use a cellular automata model together with evolution searches on possible rules that determine cell state and tissue level patterning. It is assumed that three cell states are possible and that at each time iteration each cell updates its fate according to the current state of itself and its neighbours. The authors use a computational procedure based on evolution algorithms to identify "fit" update rules that can successfully drive patterning into three distinct domains and go on to provide insights with regards to the function of these rules as well as their properties such as robustness and patterning dynamics. The article is generally well-written, the results seem solid, and the analysis and methods are thorough and generally well-explained. A main concern is the lack of connection between the biology that motivated the analysis and the results, this could be improved in the discussion by making the methods somewhat more concise to allow space to make links back to potential biological mechanisms when the results are presented. We raise some general points and some more specific questions and suggestions for clarification below that we hope will help improve the MS and make it more accessible to a wider audience.

General points:

• Although the authors motivate their work on the premise that biological patterns at the tissue level often are driven by local cell-cell interactions, by the end of the analysis any possible connection to the underlying biology is lost. For example, it would have been useful to discuss how the rules that evolved to dominate the patterning process in the results section could be implemented by cells. Is there a connection that could be made back to Notch signalling and its multiple ligands or to morphogens that diffuse only locally? Would the large number of rules possible in the cellular automata context reflect transcriptional feedback? This is an important point to bring the work "home". At the moment, it feels like a nice computational analysis of cellular automata but the links to the systems that motivate the work are lost in the process.

• When growth is considered (p.14-15) a discussion of timescales seems pertinent. Often patterning takes place at a timescale faster than cell division so the system could be allowed to reach a steady state before a new division event takes place. What are the time scales of updating the phenotype compared with the time scales of division in the model and in relevant biological systems? How would different limiting cases impact conclusions, e.g. new cells added and pattern allowed to reach steady state before more growth versus cells added while patterning dynamics are still updating?

• An interesting question is whether certain elements of rules (out of the 27 possible elements for the system with 3 states) are more or less likely to appear together in an evolved final rule. This may give a mechanistic understanding of what combinations of elements are likely to drive the optimal pattern and which combinations are avoided altogether.

Reviewer #2 (Public Review):

In this study, Torcq and colleagues make carefull observations of the cellular morphology of haemogenic endothelium undergoing endothelial to haematopoietic transition (EHT) to become stem cells, using the zebrafish model. To achieve this, the used an extensive array of transgenic lines driving fluorescent markers, markers of apico-basal polarity (podocalixin-FP fusions) or tight junction markers (jamb-FP fusions). The use of the runx truncation to block native Runx1 only in endothelial cells is an elegant tool to achieve something akin to tissue-specific deletion of Runx1. Overall, the imaging data is of excellent quality. They demonstrate that differences in apico-basal polarity are strongly associated with different cellular morphologies of cells undergoing EHT from HE (EHT pol- and EHT pol+) which raises the exciting possibility that these morphological differences reflect heterogeneity of HE (and potentially HSCs, but this is not addressed in this manuscript) at a very early stage. They then overexpress a truncated form of Runx1 (just the runt domain) to block Runx1 function and show that more HE cells abort EHT and remain associated with the embryonic dorsal aorta. The revised version identifies pard3ab as differentially distributed in dtRunx mutants and correlates that distribution with a potential regulatory role on cell polarity. No direct evidence for their role in EHT is presented.

The manuscript has now been streamlined and reference to figures made much clearer. It provides for a clearer reading, and clearly a well thought out discussion of HE, polarity and the regulation of the EHT process. The evidence for the different cellular morphologies of cells undergoing EHT is strong, and the main claim that tuning apico-basal polarity and junctional recycling underlie morphological complexity of EHT (rather than of HSCs) is well supported by the data.

Reviewer #1 (Public Review):

Summary

The authors investigated the antigenic diversity of recent (2009-2017) A/H3N2 influenza neuraminidases (NAs), the second major antigenic protein after haemagglutinin. They used 27 viruses and 43 ferret sera and performed NA inhibition. This work was supported by a subset of mouse sera. Clustering analysis determined 4 antigenic clusters, mostly in concordance with the genetic groupings. Association analysis was used to estimate important amino acid positions, which were shown to be more likely close to the catalytic site. Antigenic distances were calculated and a random forest model used to determine potential important sites.

This revision has addressed many of my concerns of inconsistencies in the methods, results and presentation. There are still some remaining weaknesses in the computational work.

Strengths

(1) The data cover recent NA evolution and a substantial number (43) of ferret (and mouse) sera were generated and titrated against 27 viruses. This is laborious experimental work and is the largest publicly available neuraminidase inhibition dataset that I am aware of. As such, it will prove a useful resource for the influenza community.

(2) A variety of computational methods were used to analyse the data, which give a rounded picture of the antigenic and genetic relationships and link between sequence, structure and phenotype.

(3) Issues raised in the previous review have been thoroughly addressed.

Weaknesses:

Some concerns regarding the robustness of the machine learning model and potential overfitting remain.

Reviewer #2 (Public Review):

Summary:

The manuscript by Kandoi et al. describes a new 3D retinal organoid model of a mono-allelic copy number variant of the rhodopsin gene that in a patient led to autosomal dominant retinitis pigmentosa. The evidence provided here is relatively strong that the rod photoreceptor phenotype observed in an adult patient with RP in vivo is similar to that phenotype observed in human stem cell-derived retinal organoids. Increases in RHO expression were detected by qPCR, RNA-seq, and IHC support this phenotype. Importantly, the amelioration of photoreceptor rhodopsin mislocalization and related defects using the small molecule drug photoregulin demonstrates an important potential clinical application.

Strengths:<br /> - Retinal organoids derived from patient with adRP.<br /> - RHO mislocalization could explain the phenotype in patients.

Weaknesses:

- Organoids at 300 days do not show PR loss.

Additional minor weaknesses

- Bulk RNAseq methods require greater detail, particularly with respect to how total or mRNA was purified, how was it quantified for concentration and integrity (i.e. Nanodrop, Tape station, Bioanalyzer), what reagents were used for library preparation and how many reads were analyzed per sample.

- Fig. 4. The levels of RHO visualized in tissue sections (panels A-C) does not seem to match the general levels shown for the western blots (panel D) which appear to be far higher in RM western blot samples than in the IHC images. Please clarify why there is such a difference.

- Line 186: by what criteria are the authors able to state that " there were no clear visible anatomical changes in apical-basal retinal cell type distribution (data not shown)". Was this based on histological staining with antibodies, nuclear counter-staining or some other evaluation?

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels (stress) over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most robustly related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, change in intrinsic functional connectivity related to changes in diurnal cortisol release and long-term cortisol exposure. These changes are proposed to result from a combination of factors, which is supported by multivariate analyses showing changes across subfields and training content relate to cortisol changes.

The authors demonstrate that mindfulness training programs are a potential avenue for stress interventions that impact hippocampal structure and cortisol, providing a promising approach to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. The authors note the limitations of their approaches and provide detailed information on the data used and analyses conducted. The results provide a strong basis from which future studies can expand using computational approaches or more fine-grained investigations of the impact of mindfulness training on cortisol levels and the hippocampus.

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

The authors start from the premise that neural circuits exhibit "representational drift" -- i.e., slow and spontaneous changes in neural tuning despite constant network performance. While the extent to which biological systems exhibit drift is an active area of study and debate (as the authors acknowledge), there is enough interest in this topic to justify the development of theoretical models of drift.

The contribution of this paper is to claim that drift can reflect a mixture of "directed random motion" as well as "steady state null drift." Thus far, most work within the computational neuroscience literature has focused on the latter. That is, drift is often viewed to be a harmless byproduct of continual learning under noise. In this view, drift does not affect the performance of the circuit nor does it change the nature of the network's solution or representation of the environment. The authors aim to challenge the latter viewpoint by showing that the statistics of neural representations can change (e.g. increase in sparsity) during early stages of drift. Further, they interpret this directed form of drift as "implicit regularization" on the network.

The evidence presented in favor of these claims is concise, but on balance I find their evidence persuasive, at least in artificial network models. This paper includes a brief analysis of four independent experiments in Figure 3, which corroborates the main claims of the paper. Future work should dig deeper into the experimental data to provide a finer grained characterization. For example, in addition to quantifying the overall number of active units, it would be interesting to track changes in the signal-to-noise ratio of each place field, the widths of the place fields, et cetera.

To establish the possibility of implicit regularization in artificial networks, the authors cite convincing work from the machine learning community (Blanc et al. 2020, Li et al., 2021). Here the authors make an important contribution by translating these findings into more biologically plausible models and showing that their core assumptions remain plausible. The authors also develop helpful intuition in Figure 5 by showing a minimal model that captures the essence of their result.

Reviewer #1 (Public Review):

In this manuscript, Nagel et al. sought to characterize the composition of urinary compounds, some of which are putative chemosignals. They used urines from adult males and females in three different strains, including one wild-derived strain. By performing mass spectrometry of two classes of compounds: volatile organic compounds and proteins, they found that urines from inbred strains are qualitatively similar to those of a wild strain. This finding is significant because there is a high degree of diversity in different inbred strains and wild mice, with respect to the polymorphisms of chemosensory receptor genes and expression of vomeronasal ligands previously identified. Notably, their study did not characterize steroids, which represent a major class of urinary chemosignals activating vomeronasal neurons. Therefore, important future studies should address the strain dependence of steroid composition in urines.

In the second part of this work, the authors used calcium imaging to monitor the pattern of vomeronasal neuron responses to these urines. By performing pairwise comparisons, the authors found a large degree of strain-specific response and a relatively minor response to sex-specific urinary stimuli. This is a finding generally in agreement with previous calcium imaging work by Ron Yu and colleagues in 2008. The authors extend the previous work by using urines from wild mice. They further report that the concentration diversity of urinary compounds in different urine batches is largely uncorrelated with the activity profiles of these urines. In addition, the authors found that the patterns of vomeronasal neuron response to urinary cues are not identical when measured using different recipient strains.

The pitfalls of this study are the omission of steroids for the mass spectrometry experiments and the indirect (correlational) nature of their mass spectrometry data and activity data. Whether the urinary compounds identified in this study activate vomeronasal neurons were not tested.

Nevertheless, the major contribution of this work is the identification of specific molecules in mouse urines. This work is likely to be of significant interest to researchers in chemosensory signaling in mammals and could provide a systematic avenue to exhaustively identify additional pheromones in mice.

Reviewer #1 (Public Review):

Summary:

This important study investigated the role of the PHOX2B transcription factor in neurons in the key brainstem chemosensory structure, the retrotrapezoid nucleus (RTN), for maintaining proper CO2 chemoreflex responses of breathing in the adult rat in vivo. PHOX2B has an important transcriptional role in neuronal survival and/or function, and mutations of PHOX2B severely impair the development and function of the autonomic nervous system and RTN, resulting in the developmental genetic disease congenital central hypoventilation syndrome (CCHS) in neonates, where the RTN may not form and is functionally impaired. The function of the wild-type PHOX2B protein in adult RTN neurons that continue to express PHOX2B is unknown. By utilizing a viral PHOX2B-shRNA approach for the knockdown of PHOX2B specifically in RTN neurons, the authors' solid results show impaired ventilatory responses to elevated inspired CO2, measured by whole-body plethysmography in freely behaving adult rats, that develop progressively over a four-week period in vivo, indicating effects on RTN neuron transcriptional activity and associated blunting of the CO2 ventilatory response. The RTN neuronal mRNA expression data presented suggests the impaired hypercapnic ventilatory response is possibly due to the decreased expression of key proton sensors in the RTN. This study will be of interest to neuroscientists studying respiratory neurobiology as well as the neurodevelopmental control of motor behavior.

Strengths:

(1) The authors used a shRNA viral approach to progressively knock down the PHOX2B protein, specifically in RTN neurons, to determine whether PHOX2B is necessary for the survival and/or chemosensory function of adult RTN neurons in vivo.

(2) To determine the extent of PHOX2B knockdown in RTN neurons, the authors combined RNAScope® and immunohistochemistry assays to quantify the subpopulation of RTN neurons expressing PHOX2B and Neuromedin B (Nmb), which has been proposed to be key chemosensory neurons in the RTN.

(3) The authors demonstrate that knockdown efficiency is time-dependent, with a progressive decrease in the number of Nmb-expressing RTN neurons that co-express PHOX2B over a four-week period.

(4) Their results convincingly show hypoventilation, particularly in 7.2% CO2 only, for PHOX2B-shRNA RTN-injected rats after four weeks compared to naïve and non-PHOX2B-shRNA targeted (NT-shRNA) RTN-injected rats, suggesting a specific impairment of chemosensitive properties in RTN neurons with PHOX2B knockdown.

(5) Analysis of the association between PHOX2B knockdown in RTN neurons and the<br /> attenuation of the hypercapnic ventilatory response (HCVR), by evaluating the correlation between the number of Nmb+/PHOX2B+ or Nmb+/PHOX2B- cells in the RTN and the resulting HCVR, showed a significant correlation between HCVR and number of Nmb+/PHOX2B+ and Nmb+/PHOX2B- cells, suggesting that the number of PHOX2B-expressing cells in the RTN is a predictor of the chemoreflex response and the reduction of PHOX2B protein impairs the CO2-chemoreflex.

(6) The data presented indicate that PHOX2B knockdown reduces the HCVR and the expression of Gpr4 and Task2 mRNAs. This suggests that PHOX2B knockdown affects RTN neurons' transcriptional activity and decreases the CO2 response, possibly by reducing the expression of key proton sensors in the RTN.

(7) This study's results show that independent of its role during development, PHOX2B is still required to maintain proper CO2 chemoreflex responses in the adult brain, and its reduction in CCHS may contribute to the respiratory impairment in this disorder.

Weaknesses:

(1) The authors found a significant decrease in the total number of Nmb+ RTN neurons (i.e., Nmb+/PHOX2B+ plus Nmb+/ PHOX2B-) in NT-shRNA rats at two weeks post viral injection, and also at the four-week period where the impairment of the chemosensory function of the RTN became significant, suggesting some inherent cell death possibly due to off-target toxic effects associated with shRNA procedures.

(2) The tissue sampling procedures for quantifying numbers of cells expressing proteins/mRNAs throughout the extended RTN region bilaterally have not been completely validated to accurately represent the full expression patterns in the RTN under the experimental conditions.

(3) The inferences about RTN neuronal expression of NMB, GPR4, or TASK2 are based on changes in mRNA levels, so it remains speculation that the observed reduction in Gpr4 and Task2 mRNA translates to a reduction in the protein levels and associated reduction of RTN neuronal chemosensitive properties.

Reviewer #1 (Public Review):

Summary:

The aim of the study described in this paper was to test whether visual stimuli that pulse synchronously with the systole phase of the cardiac cycle are suppressed compared with stimuli that pulse in the diastole phase. To this end, the authors employed a binocular rivalry task and used the duration of the perceived image as the metric of interest. The authors predicted that if there was global suppression of the visual stimulus during systole then the durations of the stimulus that were pulsing synchronously with systole should be of shorter duration than those pulsing in diastole. However, the results observed were the opposite of those predicted. The authors speculate on what this facilitation effect might mean for the baroreceptor suppression hypothesis.

Strengths:

This is an interesting and timely study that uses a clever paradigm to test the baroreceptor suppression hypothesis in vision. This is a refreshingly focussed paper with interesting and seemingly counterintuitive results.

Weaknesses:

The paper could benefit from a clearer explanation of the predicted results. For those not experts in binocular rivalry, it would be useful to explain the predicted results. Does pulsing stimuli in this way change durations in such a task? If there is global suppression of visual stimuli why would this lead to shorter/longer durations in the systole compared to the diastole conditions? In addition, the duration lengths in both conditions seem to be longer than one cardiac cycle. If the cardiac cycle modulates duration it would be interesting to discuss why this occurs on some cycles but not on others. If there is a facilitation effect why does it only occur on some cycles?

Reviewer #1 (Public Review):

Summary:

Pham and colleagues provide an illuminating investigation of aquaporin-4 water flux in the brain utilizing ex vivo and in vivo techniques. The authors first show in acute brain slices, and in vivo with fiber photometry, SRB-loaded astrocytes swell after inhibition of AQP4 with TGN-020, indicative of tonic water efflux from astrocytes in physiological conditions. Excitingly, they find that TGN-020 increases the ADC in DW-MRI in a region-specific manner, potentially due to AQP4 density. The resolution of the DW-MRI cannot distinguish between intracellular or extracellular compartments, but the data point to an overall accumulation of water in the brain with AQP4 inhibition. These results provide further clarity on water movement through AQP4 in health and disease.

Overall, the data support the main conclusions of the article, with some room for more detailed treatment of the data to extend the findings.

Strengths:

The authors have a thorough investigation of AQP4 inhibition in acute brain slices. The demonstration of tonic water efflux through AQP4 at baseline is novel and important in and of itself. Their further testing of TGN-020 in hyper- and hypo-osmotic solutions shows the expected reduction of swelling/shrinking with AQP4 blockade.

Their experiment with cortical spreading depression further highlights the importance of water efflux from astrocytes via AQP4 and transient water fluxes as a result of osmotic gradients. Inhibition of AQP4 increases the speed of tissue swelling, pointing to a role in the efflux of water from the brain.

The use of DW-MRI provides a non-invasive measure of water flux after TGN-020 treatment.

Weaknesses:

The authors specifically use GCaMP6 and light sheet microscopy to image their brain sections in order to identify astrocytic microdomains. However, their presentation of the data neglects a more detailed treatment of the calcium signaling. It would be quite interesting to see whether these calcium events are differentially affected by AQP4 inhibition based on their cellular localization (ie. processes vs. soma vs. vascular end feet which all have different AQP4 expressions).

The authors show the inhibition of AQP4 with TGN-020 shortens the onset time of the swelling associated with cortical spreading depression in brain slices. However, they do not show quantification for many of the other features of CSD swelling, (ie. the duration of swelling, speed of swelling, recovery from swelling).

Significance:

AQP4 is a bidirectional water channel that is constitutively open, thus water flux through it is always regulated by local osmotic gradients. Still, characterizing this water flux has been challenging, as the AQP4 channel is incredibly water-selective. The authors here present important data showing that the application of TGN-020 alone causes astrocytic swelling, indicating that there is constant efflux of water from astrocytes via AQP4 in basal conditions. This has been suggested before, as the authors rightfully highlight in their discussion, but the evidence had previously come from electron microscopy data from genetic knockout mice.

AQP4 expression has been linked with the glymphatic circulation of cerebrospinal fluid through perivascular spaces since its rediscovery in 2012 [1]. Further studies of aging[2], genetic models[3], and physiological circadian variation[4] have revealed it is not simply AQP4 expression but AQP4 polarization to astrocytic vascular endfeet that is imperative for facilitating glymphatic flow. Still, a lingering question in the field is how AQP4 facilitates fluid circulation. This study represents an important step in our understanding of AQP4's function, as the basal efflux of water via AQP4 might promote clearance of interstitial fluid to allow an influx of cerebrospinal fluid into the brain. Beyond glymphatic fluid circulation, clearly, AQP4-dependent volume changes will differentially alter astrocytic calcium signaling and, in turn, neuronal activity.

(1) Iliff, J.J., et al., A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid β. Sci Transl Med, 2012. 4(147): p. 147ra111.<br /> (2) Kress, B.T., et al., Impairment of paravascular clearance pathways in the aging brain. Ann Neurol, 2014. 76(6): p. 845-61.<br /> (3) Mestre, H., et al., Aquaporin-4-dependent Glymphatic Solute Transport in the Rodent Brain. eLife, 2018. 7.<br /> (4) Hablitz, L., et al., Circadian control of brain glymphatic and lymphatic fluid flow. Nature Communications, 2020. 11(1).

Reviewer #1 (Public Review):

Yun et al. examined the molecular and neuronal underpinnings of changes in Drosophila female reproductive behaviors in response to social cues. Specifically, the authors measure the ejaculate-holding period, which is the amount of time females retain male ejaculate after mating (typically 90 min in flies). They find that female fruit flies, Drosophila melanogaster, display shorter holding periods in the presence of a native male or male-associated cues, including 2-Methyltetracosane (2MC) and 7-Tricosene (7-T). They further show that 2MC functions through Or47b olfactory receptor neurons (ORNs) and the Or47b channel, while 7-T functions through ppk23 expressing neurons. Interestingly, their data also indicates that two other olfactory ligands for Or47b (methyl laurate and palmitoleic acid) do not have the same effects on the ejaculate-holding period. By performing a series of behavioral and imaging experiments, the authors reveal that an increase in cAMP activity in pC1 neurons is required for this shortening of the ejaculate-holding period and may be involved in the likelihood of remating. This work lays the foundation for future studies on sexual plasticity in female Drosophila.

The conclusions of this paper are mostly supported by the data, but aspects of the lines used for individual pC1 subtypes and visual contributions as well as the statistical analysis need to be clarified.

(1) The pC1 subtypes (a - e) are delineated based on their morphology and connectivity. While the morphology of these neurons is distinct, they do share a resemblance that can be difficult to discern depending on the imaging performed. Additionally, genetic lines attempting to label individual neurons can easily be contaminated by low-level expression in off-target neurons in the brain or ventral nerve cord (VNC), which could contribute to behavioral changes following optogenetic manipulations. In Figures 5C - D, the authors generated and used new lines for labeling pC1a and pC1b+c. The line for pC1b+c was imaged as part of another recent study (https://doi.org/10.1073/pnas.2310841121). However, similar additional images of the pC1a line (i.e. 40x magnification and VNC expression) would be helpful in order to validate its specificity.

(2) The author's experiments examining olfactory and gustatory contributions to the holding period were well controlled and described. However, the experiments in Figure 1D examining visual contributions were not sufficiently convincing as the line used (w1118) has previously been shown to be visually impaired (Wehner et al., 1969; Kalmus 1948). Using another wild-type line would have improved the authors' claims.

(3) When comparisons between more than 2 groups are shown as in Figures 1E, 3D, and 5E, the comparisons being made were not clear. Adding in the results of a nonparametric multiple comparisons test would help for the interpretation of these results.

Reviewer #1 (Public Review):

Summary:

Olfactory sensory neurons (OSNs) in the olfactory epithelium detect myriads of environmental odors that signal essential cues for survival. OSNs are born throughout life and thus represent one of the few neurons that undergo life-long neurogenesis. Until recently, it was assumed that OSN neurogenesis is strictly stochastic with respect to subtype (i.e. the receptor the OSN chooses to express).

However, a recent study showed that olfactory deprivation via naris occlusion selectively reduced birthrates of only a fraction of OSN subtypes and indicated that these subtypes appear to have a special capacity to undergo changes in birthrates in accordance with the level of olfactory stimulation. These previous findings raised the interesting question of what type of stimulation influences neurogenesis, since naris occlusion does not only reduce the exposure to potentially thousands of odors but also to more generalized mechanical stimuli via preventing airflow.

In this study, the authors set out to identify the stimuli that are required to promote the neurogenesis of specific OSN subtypes. Specifically, they aim to test the hypothesis that discrete odorants selectively stimulate the same OSN subtypes whose birthrates are affected. This would imply a highly specific mechanism in which exposure to certain odors can "amplify" OSN subtypes responsive to those odors suggesting that OE neurogenesis serves, in part, an adaptive function.

To address this question, the authors focused on a family of OSN subtypes that had previously been identified to respond to musk-related odors and that exhibit higher transcript levels in the olfactory epithelium of mice exposed to males compared to mice isolated from males. First, the authors confirm via a previously established cell birth dating assay in unilateral naris occluded mice that this increase in transcript levels actually reflects a stimulus-dependent birthrate acceleration of this OSN subtype family. In a series of experiments using the same assay, they show that one specific subtype of this OSN family exhibits increased birthrates in response to juvenile male exposure while a different subtype shows increased birthrates to adult mouse exposure. In the core experiment of the study, they finally exposed naris occluded mice to a discrete odor (muscone) to test if this odor specifically accelerates the birth rates of OSN types that are responsive to this odor. This experiment reveals a complex relationship between birth rate acceleration and odor concentrations showing that some muscone concentrations affect birth rates of some members of this family and do not affect two unrelated OSN subtypes.

Strengths:

The scientific question is valid and opens an interesting direction. The previously established cell birth dating assay in naris occluded mice is well performed and accompanied by several control experiments addressing potential other interpretations of the data.

Weaknesses:

(1) The main research question of this study was to test if discrete odors specifically accelerate the birth rate of OSN subtypes they stimulate, i.e. does muscone only accelerate the birth rate of OSNs that express muscone-responsive ORs, or vice versa is the birthrate of muscone-responsive OSNs only accelerated by odors they respond to?

This question is only addressed in Figure 5 of the manuscript and the results only partially support the above claim. The authors test one specific odor (muscone) and find that this odor (only at certain concentrations) accelerates the birth rate of some musk-responsive OSN subtypes, but not two other unrelated control OSN subtypes. This does not at all show that musk-responsive OSN subtypes are only affected by odors that stimulate them and that muscone only affects the birthrate of musk-responsive OSNs, since first, only the odor muscone was tested and second, only two other OSN subtypes were tested as controls, that, importantly, are shown to be generally stimulus-independent OSN subtypes (see Figure 2 and S2).

As a minimum the authors should have a) tested if additional odors that do not activate the three musk-responsive subtypes affect their birthrate b) choose 2-3 additional control subtypes that are known to be stimulus-dependent (from their own 2020 study) and test if muscone affects their birthrates.

(2) The finding that Olfr1440 expressing OSNs do not show any increase in UNO effect size under any muscone concentration (Figure 5D, no significance in line graph for UNO effect sizes, middle) seems to contradict the main claim of this study that certain odors specifically increase birthrates of OSN subtypes they stimulate. It was shown in several studies that olfr1440 is seemingly the most sensitive OR for muscone, yet, in this study, muscone does not further increase birthrates of OSNs expressing olfr1440. The effect size on birthrate under muscone exposure is the same as without muscone exposure (0%).

In contrast, the supposedly second most sensitive muscone-responsive OR olfr235 shows a significant increase in UNO effect size between no muscone exposure (0%) and 0.1% as well as 1% muscone.

(3) The authors introduce their choice to study this particular family of OSN subtypes with first, the previous finding that transcripts for one of these musk-responsive subtypes (olfr235) are downregulated in mice that are deprived of male odors. Second, musk-related odors are found in the urine of different species. This gives the misleading impression that it is known that musk-related odors are indeed excreted into male mouse urine at certain concentrations. This should be stated more clearly in the introduction (or cited, if indeed data exist that show musk-related odors in male mouse urine) because this would be a very important point from an ethological and mechanistic point of view.

In addition, this would also be important information to assess if the chosen muscone concentrations fall at all into the natural range.

Related: If these are male-specific cues, it is interesting that changes in OR transcripts (Figure 1) can already be seen at the age of P28 where other male-specific cues are just starting to get expressed. This should be discussed.

(4) Figure 5: Under muscone exposure the number of newborn neurons on the closed sides fluctuates considerably. This doesn't seem to be the case in other experiments and raises some concerns about how reliable the naris occlusion works for strong exposure to monomolecular odors or what other potential mechanisms are at play.

(5) In contrast to all other musk-responsive OSN types, the number of newborn OSNs expressing olfr1437 increases on the closed side of the OE relative to the open in UNO-treated male mice (Figure 1). This seems to contradict the presented theory and also does not align with the bulk RNAseq data (Figure S1).

(6) The authors hypothesize in relation to the accelerated birthrate of musk-responsive OSN subtypes that "the acceleration of the birthrates of specific OSN subtypes could selectively enhance sensitivity to odors detected by those subtypes by increasing their representation within the OE". However, for two other OSN subtypes that detect male-specific odors, they hypothesize the opposite "By contrast, Olfr912 (Or8b48) and Olfr1295 (Or4k45), which detect the male-specific non-musk odors 2-sec-butyl-4,5-dihydrothiazole (SBT) and (methylthio)methanethiol (MTMT), respectively, exhibited lower representation and/or transcript levels in mice exposed to male odors, possibly reflecting reduced survival due to overstimulation."

Without any further explanation, it is hard to comprehend why exposure to male-derived odors should, on one hand, accelerate birthrates in some OSN subtypes to potentially increase sensitivity to male odors, but on the other hand, lower transcript levels and does not accelerate birth rates of other OSN subtypes due to overstimulation.

Reviewer #1 (Public Review):

Summary:

In this study, the author aimed to develop a method for estimating neuronal-type connectivity from transcriptomic gene expression data. They sought to develop an interpretable model that could be used to characterize the underlying genetic mechanisms of circuit assembly and connectivity in various neuronal systems.

Strengths:

Many of the proposed suggestions were addressed by the author from the initial review. In general the claims made by the author are more strongly supported by the data and better situated in the literature. A major improvement includes the application of the model to the C. elegans gap junction neuronal system. Despite several key differences in the dataset as compared to the mouse retina data, the proposed model performs comparably to the SCM model currently considered state of the art in the literature (the author should remain cautious about claiming better performance given extremely marginal differences). In section 7.2, the author clearly outlines additional advantages of the proposed model including superior time and space complexity. The overall model performance remains modest, but it learns the same rules as the SCM model as well as other candidate patterns.

As in the initial submission, the bilinear model recapitulates key connectivity motifs for the mouse dataset. The algorithm is shown to converge across several runs affirming its stability/replicability. The model is also extended to predict connectivity on unknown RGC-BC cell type pairs. Without ground truth, the author posits how it should perform based on known functional properties of the RGC type. The hypotheses are confirmed for 8/10 neuronal types with unknown connectivity. The author more clearly describes how this model can be used experimentally for hypothesis testing and presents a more comprehensive future roadmap regarding validation, avenues for improving the model, and incorporation of growing datasets.

Weaknesses:

While the C Elegans dataset is useful because it enables benchmarking to existing models, the dataset is quite different. The gene expression dimensionality is 18 genes as opposed to over 3000 genes in the mouse dataset. It is a strength that the model still works as intended, but a weakness that the bilinear model could not be tested on a similar mouse dataset. This distinction matters because it remains an open question if the PCA methodology would hold up in a dataset with varied distributions of gene expression. Variations of the PCA methodology could be evaluated further with the present dataset to make the generalizability of the model more convincing.

The Gene Ontology analysis requires more methodological explanation. The author claims, "(the linear nature of the model) enables the direct interpretation of gene expressions by examining their associated weights in the model. These weights signify the importance of each gene in determining the connectivity motifs between the BC and RGC types." If I am correctly understanding the methods, the model weights in each dimension are indexing the importance of a gene expression feature as opposed to the importance of a single gene alone, "the gene expression of the BCs in X and the RGCs in Y were featurized by their respective PCs, resulting in matrices of dimensions 22453 × 11323 and 3779 × 3142, respectively." It would be helpful to explain how gene weights are extracted from a gene expression feature once highlighted.

There could be a more rigorous analysis of the predictive capacity of the model even with the current data. The model recapitulates connectivity patterns from the full dataset and a prediction is demonstrated for unknown data. The model is thus championed as a useful tool for predicting how genetic modifications will influence connectivity, but this is not empirically evaluated.

Appraisal of whether the author achieved their aims, and whether results support their conclusions:

In line with the aims of the paper, the author proposed an interpretable bilinear model to learn a shared latent feature space derived from gene expression profiles to predict synaptic connectivity between various neuron types. The model was shown to generalize to two distinct neuronal systems with varying levels of genomic and cellular resolution. While the performance remains modest, the model performs comparably to the existing state of the art despite improved computational complexity.

Discussion of likely impact of the work on the field, and utility of methods and data to the community:

The author has elaborated substantially on the impact of this work, particularly how it could be leveraged in experimental settings. The clear methodology could be implemented by other researchers to test the model on new datasets and for benchmarking novel methods.

Reviewer #1 (Public Review):

The work by Debashish U. Menon, Noel Murcia, and Terry Magnuson brings important knowledge about histone H3.3 dynamics involved in meiotic sex chromosome inactivation (MSCI). MSCI is unique to gametes and failure during this process can lead to infertility. Classically, MSCI has been studied in the context of DNA Damage repair pathways and little is known about the epigenetic mechanisms behind maintenance of the sex body as a silencing platform during meiosis. One of the major strengths of this work is the evidence provided on the role of ARID1A, a BAF subunit, in MSCI through the regulation of H3.3 occupancy in specific genic regions.

Using RNA seq and CUT&RUN and ATAC-seq, the authors show that ARID1A regulates chromatin accessibility of the sex chromosomes and XY gene expression. Loss of ARID1A increases promoter accessibility of XY linked genes with concomitant influx of RNA pol II to the sex body and up regulation of XY-linked genes. This work suggests that ARID1A regulates chromatin composition of the sex body since in the absence of ARID1A, spermatocytes show less enrichment of H3.3 in the sex chromosomes and stable levels of the canonical histones H3.1/3.2. By overlapping CUT&RUN and ATAC-seq data, authors show that changes in chromatin accessibility in the absence of ARID1A are given by redistribution of occupancy of H3.3. Gained open chromatin in mutants corresponds to up regulation of H3.3 occupancy at transcription start sites of genes mediated by ARID1A.

Interestingly, ARID1A loss caused increased promoter occupancy by H3.3 in regions usually occupied by PRDM9. PRDM9 catalyzes histone H3 lysine 4 trimethylation during meiotic prophase I, and positions double strand break (DSB) hotspots. Lack of ARID1A causes reduction in occupancy of DMC1, a recombinase involved in DSB repair, in non-homologous sex regions. These data suggest that ARID1A might indirectly influence DNA DSB repair on the sex chromosomes by regulating the localization of H3.3. This is very interesting given the recently suggested role for ARID1A in genome instability in cancer cells. It raises the question of whether this role is also involved in meiotic DSB repair in autosomes and/or how this mechanism differs in sex chromosomes compared to autosomes.

The fact that there are Arid1a transcripts that escape the Cre system in the Arid1a KO mouse model might difficult the interpretation of the data. The phenotype of the Arid1a knockout is probably masked by the fact that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed. Nonetheless, the authors provide evidence showing that the spermatids observed in cKO testes that progress in spermatogenesis are the ones expressing Arid1a. This work presents enough evidence to include the BAF complex as part of the MSCI process, which increases our knowledge on specific regulation of the sex chromatin during meiosis.

Reviewer #1 (Public Review):

Summary:

Asymptomatic malaria infections are frequent during the dry season and have been associated with lower cytoadherence of P. falciparum parasites and lower expression of variant surface antigens. The mechanisms underlying parasite adaptation during the low transmission season remain poorly understood. The authors previously established that members of the non-coding RNA RUF6 gene family, transcribed by RNA pol III, are required for expression of the main variant surface antigens in P. falciparum, PfEMP1, which drive parasite cytoadherence and pathogenicity. In this study, the authors investigated the contribution of RNA pol III transcription in the regulation of PfEMP1 expression in different clinical states, either symptomatic malaria cases during the wet season or asymptomatic infections during the dry season.

By reanalyzing RNAseq data from a previous study in Mali, complemented with RT-qPCR on new samples collected in The Gambia, the authors first report the down-regulation of RNA pol III genes (tRNAs, RUF6) in P. falciparum isolates collected from asymptomatic individuals during the dry season, as compared to isolates from symptomatic (wet season) individuals. They also confirm the down-regulation of var (DBLalpha) gene expression in asymptomatic infection as compared to symptomatic malaria. Plasma analysis in the two groups in the Gambian study reveals higher Magnesium levels in dry season as compared to wet season samples, pointing at a possible role of external factors. The authors tested the effect of MgCl2 supplementation on cultured parasites, as well as three other stimuli (temperature, low glucose, Ile deprivation), and show that Ile deprivation and MgCl2 both induce down-regulation of RNA pol III transcription but not pol I or pol II (except the active var gene). Using RNAseq, they show that MgCl2 supplementation predominantly inhibits RNA pol III-transcribed genes, including the entire RUF6 family. Conditional depletion of Maf1 leads to the up-regulation of RNA pol III gene transcription, confirming that Maf1 is a RNA pol III inhibitor in P. falciparum, as described in other organisms. Quantitative mass spectrometry shows that Maf1 interacts with RNA pol III complex in the nucleus, and with distinct proteins including two phosphatases in the cytoplasm. Using the Maf1 cKD parasites, the authors document that down-regulation of RNA pol III by MgCl2 is dependent on Maf1. Finally, they show that MgCl2 results in decreased cytoadherence of infected erythrocytes, associated with reduced PfEMP1 expression.

Strengths:

-The work is very well performed and presented.<br /> -The study uncovers a novel regulatory mechanism relying on RNA pol III-dependent regulation of variant surface antigens in response to external signals, which could contribute to parasite adaptation during the low transmission season.<br /> -Potential regulators of Maf1 were identified by mass spectrometry, including phosphatases, paving the way for future mechanistic studies.

Weaknesses:

-The signaling pathway upstream of Maf1 remains unknown. In eukaryotes, Maf1 is a negative regulator of RNA pol III and is regulated by external signals via the TORC pathway. Since TORC components are absent in the apicomplexan lineage, one central question that remains open is how Maf1 is regulated in P. falciparum. Magnesium is probably not the sole stimulus involved, as suggested by the observation that Ile deprivation also down-regulates RNA pol III activity.<br /> -The study does not address why MgCl2 levels vary depending on the clinical state. It is unclear whether plasma magnesium is increased during asymptomatic malaria or decreased during symptomatic infection, as the study does not include control groups with non-infected individuals. Along the same line, MgCl2 supplementation in parasite cultures was done at 3mM, which is higher than the highest concentrations observed in clinical samples.<br /> -Although the study provides biochemical evidence of Maf1 accumulation in the parasite nuclear fraction upon magnesium addition, this is not fully supported by the immunofluorescence experiments.

Reviewer #1 (Public Review):

Bian et al showed that biomarker-informed PhenoAgeAccel was consistently related to an increased risk of site-specific cancer and overall cancer within and across genetic risk groups. The results showed that PhenoAgeAccel and genetic liability of a bunch of cancers serve as productive tools to facilitate the identification of cancer-susceptible individuals under an additive model. People with a high genetic risk for cancer may benefit from PhenoAgeAccel-imformed interventions.

As the authors pointed out, the large sample size, the prospective design UK Biobank study, and the effective application of PhenoAgeAccel in predicting the risk of overall cancer are the major strengths of the study. Meanwhile, the CPRS seems to be a solid and comprehensive score based on incidence-weighted site-specific polygenic risk scores across 20 well-powered GWAS for cancers.

Reviewer #1 (Public Review):

Summary:

This manuscript is an extension of previous studies by this group looking at the new drug spectinamide 1599. The authors directly compare therapy with BPaL (bedaquiline, pretomanid, linezolid) to a therapy that substitutes spectinamide for linezolid (BPaS). The Spectinamide is given by aerosol exposure and the BPaS therapy is shown to be as effective as BPaL without adverse effects. The work is rigorously performed and analyses of the immune responses are consistent with curative therapy.

Strengths:

(1) This group uses 2 different mouse models to show the effectiveness of the BPaS treatment.

(2) Impressively the group demonstrates immunological correlates associated with Mtb cure with the BPaS therapy.

(3) Linezolid is known to inhibit ribsomes and mitochondria whereas spectinaminde does not. The authors clearly demonstrate the lack of adverse effects of BPaS compared to BPaL.

Weaknesses:

(1) Although this is not a weakness of this paper, a sentence describing how the spectinamide would be administered by aerosolization in humans would be welcomed.

Reviewer #1 (Public Review):

Summary:

In this work, Huang et al used SMRT sequencing to identify methylated nucleotides (6mA, 4mC, and 5mC) in Pseudomonas syringae genome. They show that the most abundant modification is 6mA and they identify the enzymes required for this modification as when they mutate HsdMSR they observe a decrease of 6mA. Interestingly, the mutant also displays phenotypes of change in pathogenicity, biofilm formation, and translation activity due to a change in gene expression likely linked to the loss of 6mA.

Overall, the paper represents an interesting set of new data that can bring forward the field of DNA modification in bacteria.

Major Concerns:

• Most of the authors' data concern Psph pathovar. I am not sure that the authors' conclusions are supported by the two other pathovars they used in the initial 2 figures. If the authors want to broaden their conclusions to Pseudomonas synringae and not restrict it to Psph, the authors should have stronger methylation data using replicates. Additionally, they should discuss why Pss is so different than Pst and Psph. Could they do a blot to confirm it is really the case and not a sequencing artefact? Is the change of methylation during bacterial growth conserved between the pathovar? The authors should obtain mutants in the other pathovar to see if they have the same phenotype. The authors have a nice set of data concerning Psph but the broadening of the results to other pathovar requires further investigation.

• The authors should include proper statistical analysis of their data. A lot of terms are descriptive but not supported by a deeper analysis to sustain the conclusions. For example, in Figure 4E, we do not know if the overlap is significant or not. Are DEGs more overlapping to 6mA sites than non-DEGs? Here is a non-exhaustive list of terms that need to be supported by statistics: different level (L145), greater conservation (L162), significant conservation (L165), considerable similarity (L175), credible motifs (L189), Less strong (L277) and several "lower" and "higher" throughout the text.

• The authors performed SMRT sequencing of the delta hsdMSR showing a reduction of 6mA. Could they include a description of their results similar to Figures 1-2. How reduced is the 6mA level? Is it everywhere in the genome? Does it affect other methylation marks? This analysis would strengthen their conclusions.

• In Figure 6E to conclude that methylation is required on both strands, the authors are missing the control CAGCN6CGC construct otherwise the effect could be linked to the A on the complementary strand.

Reviewer #1 (Public Review):

Summary:

The manuscript presents a compelling model to explain the impact of mosaicism in preimplantation genetic testing for aneuploidies.

Strengths:

A new view of mosaicism is presented with a computational model, that brings new insights into an "old" debate in our field. It is a very well-written manuscript.

Weaknesses:

Although the manuscript is very well written, this is in a way that assumes that the reader has existing knowledge about specific terms and topics. This was apparent through a lack of definitions and minimal background/context to the aims and conclusions for some of the author's findings.

There is a need for some examples to connect real evidence and scenarios from clinical reports with the model.

Reviewer #1 (Public Review):

Summary:

This study by Park and colleagues uses longitudinal saliva viral load data from two cohorts (one in the US and one in Japan from a clinical trial) in the pre-vaccine era to subset viral shedding kinetics and then use machine learning to attempt to identify clinical correlates of different shedding patterns. The stratification method identifies three separate shedding patterns discriminated by peak viral load, shedding duration, and clearance slope. The authors also assess micro-RNAs as potential biomarkers of severity but do not identify any clear relationships with viral kinetics.

Strengths:

The cohorts are well developed, the mathematical model appears to capture shedding kinetics fairly well, the clustering seems generally appropriate, and the machine learning analysis is a sensible, albeit exploratory approach. The micro-RNA analysis is interesting and novel.

Weaknesses:

The conclusions of the paper are somewhat supported by the data but there are certain limitations that are notable and make the study's findings of only limited relevance to current COVID-19 epidemiology and clinical conditions.

(1) The study only included previously uninfected, unvaccinated individuals without the omicron variant. It has been well documented that vaccination and prior infection both predict shorter duration shedding. Therefore, the study results are no longer relevant to current COVID-19 conditions. This is not at all the authors' fault but rather a difficult reality of much retrospective COVID research.

(2) The target cell model, which appears to fit the data fairly well, has clear mechanistic limitations. Specifically, if such a high proportion of cells were to get infected, then the disease would be extremely severe in all cases. The authors could specify that this model was selected for ease of use and to allow clustering, rather than to provide mechanistic insight. It would be useful to list the AIC scores of this model when compared to the model by Ke.

(3) Line 104: I don't follow why including both datasets would allow one model to work better than the other. This requires more explanation. I am also not convinced that non-linear mixed effects approaches can really be used to infer early model kinetics in individuals from one cohort by using late viral load kinetics in another (and vice versa). The approach seems better for making population-level estimates when there is such a high amount of missing data.

(4) Along these lines, the three clusters appear to show uniform expansion slopes whereas the NBA cohort, a much larger cohort that captured early and late viral loads in most individuals, shows substantial variability in viral expansion slopes. In Figure 2D: the upslope seems extraordinarily rapid relative to other cohorts. I calculate a viral doubling time of roughly 1.5 hours. It would be helpful to understand how reliable of an estimate this is and also how much variability was observed among individuals.

(5) A key issue is that a lack of heterogeneity in the cohort may be driving a lack of differences between the groups. Table 1 shows that Sp02 values and lab values that all look normal. All infections were mild. This may make identifying biomarkers quite challenging.

(6) Figure 3A: many of the clinical variables such as basophil count, Cl, and protein have very low pre-test probability of correlating with virologic outcome.

(7) A key omission appears to be micoRNA from pre and early-infection time points. It would be helpful to understand whether microRNA levels at least differed between the two collection timepoints and whether certain microRNAs are dynamic during infection.

(8) The discussion could use a more thorough description of how viral kinetics differ in saliva versus nasal swabs and how this work complements other modeling studies in the field.

(9) The most predictive potential variables of shedding heterogeneity which pertain to the innate and adaptive immune responses (virus-specific antibody and T cell levels) are not measured or modeled.

(10) I am curious whether the models infer different peak viral loads, duration, expansion, and clearance slopes between the 2 cohorts based on fitting to different infection stage data.

Reviewer #1 (Public Review):

Summary:

The author presents the discovery and characterization of CAPSL as a potential gene linked to Familial Exudative Vitreoretinopathy (FEVR), identifying one nonsense and one missense mutation within CAPSL in two distinct patient families afflicted by FEVR. Cell transfection assays suggest that the missense mutation adversely affects protein levels when overexpressed in cell cultures. Furthermore, conditionally knocking out CAPSL in vascular endothelial cells leads to compromised vascular development. The suppression of CAPSL in human retinal microvascular endothelial cells results in hindered tube formation, a decrease in cell proliferation, and disrupted cell polarity. Additionally, transcriptomic and proteomic profiling of these cells indicates alterations in the MYC pathway.

Strengths:

The study is nicely designed with a combination of in vivo and in vitro approaches, and the experimental results are good quality.

Weaknesses:

My reservations lie with the main assertion that CAPSL is associated with FEVR, as the genetic evidence from human studies appears relatively weak. Further careful examination of human genetics evidence in both patient cohorts and the general population will help to clarify. In light of human genetics, more caution needs to be exercised when interpreting results from mice and cell models and how is it related to the human patient phenotype.

Reviewer #2 (Public Review):

Summary:

An article with lots of interesting ideas and questions regarding the evolution of timing of dormancy, emphasizing mammalian hibernation but also including ectotherms. The authors compare selective forces of constraints due to energy availability versus predator avoidance and requirements and consequences of reproduction in a review of between and within species (sex) differences in the seasonal timing of entry and exit from dormancy.

Strengths:

The multispecies approach including endotherms and ectotherms is ambitious. This review is rich with ideas if not in convincing conclusions. Limitations are discussed yet are impactful, namely that differences among and within species are contrast only for ecological hibernation (the duration of remaining sequestered) and not for "heterothermic hibernation" the period between first and last torpor. Differences between the two can have significant energetic consequences, especially for mammals returning to euthermic levels of body temperature whilst remaining in their cold burrows before emerging, eg. reproductively developing males in spring.

Weaknesses:

The differences between physiological requirements for gameatogenesis between sexes that affect the timing of heterothermy and need for euthermy during mammalian hibernator are significant issues that underlie, but are under discussed, in this contrast of selective pressures that determine seasonal timing of dormancy. Some additional discussion of the effects of rapid rapid climate change on between and within species phenologies of dormancy would have been interesting.

Reviewer #2 (Public Review):

Summary:

This work by Cloarec-Ung et al. sets out to uncover strategies that would allow for the efficient and precision editing of primitive human hematopoietic stem and progenitor cells (HSPCs). Such effective editing of HSPCs via homology directed repair has implications for the development of tractable gene therapy approaches for monogenic hematopoietic disorders as well as precise engineering of these cells for clinical regenerative and/or cell therapy strategies. In the setting of experimental hematology, precision introduction of disease relevant mutations would also open the door to more robust disease modeling approaches. It has been recognized that to encourage HDR, NHEJ as the dominant mode of repair in quiescent HSPCs must be inhibited. Testing editing of human cord blood HSPCs the authors first incorporate a prestimulation phase then identify optimal RNP amounts and donor types/amounts using standard editing culture conditions identifying optimal concentrations of AAV and short single-stranded oligonucleotide donors (ssODNs) that yield minimal impacts to cell viability while still enabling heightened integration efficiency. They then demonstrate the superiority of AZD7648, an inhibitor of NHEJ-promoting DNA-PK, in allowing for much increased HDR with toxicities imparted by this compound reduced substantially by siRNAs against p53 (mean targeting efficiencies at 57 and 80% for two different loci). Although AAV offered the highest HDR frequencies, differing from ssODN by a factor by ~2-fold, the authors show that spacer breaking sequence mutations introduced into the ssODN to better mimic the disruption of the spacer sequence provided by the synthetic intron in the AAV backbone yielded ssODN HDR frequencies equal to that attained by AAV. By examining editing efficiency across specific immunophenotypically identified subpopulations they further suggest that editing efficiency with their improved strategy is consistent across stem and early progenitors and use colony assays to quantify an approximate 4-fold drop in total colony numbers but no skewing in the potentiality of progenitors in the edited HSPC pool. Finally, the authors provide a strategy using mutation-introducing AAV mixed with different ratios of silent ssODN repair templates to enable tuning of zygosity in edited CD34+ cells.

Strengths:

The methods are clearly described and the experiments for the most part also appropriately powered. In addition to using state-of-the-art approaches, the authors also provided useful insights into optimizing the practicalities of the experimental procedures that will aid bench scientists in effectively carrying out these editing approaches, for example avoiding longer handling times inherent when scaling up to editing over multiple conditions.

The sum of the adjustments to the editing procedure have yielded important advances towards minimizing editing toxicity while maximizing editing efficiency in HSPCs. In particular, the significant increase in HDR facilitated by the authors' described application of AZD7648 and the preservation of a pool of targeted progenitors is encouraging that functionally valuable cell types can be effectively edited.

The discovery of the effectiveness of spacer breaking changes in ssODNs allowing for substantially increased targeting efficiency is a promising advance towards democratizing these editing strategies given the ease of designing and synthesizing ssODNs relative to the production of viral donors.

The ability to zygosity tune was convincingly presented and provides a valuable strategy to modify this HDR procedure towards more accurate disease modelling.

Weaknesses:

Despite providing convincing evidence that functional progenitors can be successfully edited by their procedure, as the authors acknowledge it remains to be verified to what degree the survival/self-renewal capacity and in vivo regenerative potential of the more primitive fractions is maintained with their strategy. That said the inclusion of LTC-IC assays that verify the lack of effect on these quite primitive cells is encouraging that functionality of stem cells will be similarly spared.

Reviewer #1 (Public Review):

Summary: The type I ABC importer OpuA from Lactococcus lactis is the best studied transporter involved in osmoprotection. In contrast to most ABC import systems, the substrate binding protein is fused via a short linker to the transmembrane domain of the transporter. Consequently, this moiety is called the substrate binding domain (SBD). OpuA has been studied in the past in great detail and we have a very detailed knowledge about function, mechanisms of activation and deactivation as well as structure.

Strengths: Application of smFRET to unravel transient interactions of the SBDs. The method is applied at a superb quality and the data evaluation is excellent.

Weaknesses: The proposed model is not directly supported by experimental data. Rather alternative models are excluded as they do not fit to the obtained data. However, this is now clearly stated in the manuscript

Reviewer #1 (Public Review):

Building on previous work from the Tansey lab, here Howard et al. characterize transcriptional and translational changes upon WIN site inhibition of WDR5 in MLL-rearranged cancer cells. They first analyze whether C16, a newer generation compound, has the same cellular effects as C6, an early generation compound. Both compounds reduce the expression of WDR5-bound RPGs in addition to the unbound RPG RPL22L1. They then investigate differential translation by ribo-seq and observe that WIN site inhibition reduces the translational RPGs and other proteins related to biomass accumulation (spliceosome, proteasome, mitochondrial ribosome). Interestingly, this reduction adds to the transcriptional changes and is not limited to RPGs whose promoters are bound by WDR5. Quantitative proteomics at two time points confirmed the downregulation of RPGs. Interestingly, the overall effects are modest, but RPL22LA is strongly affected. Unexpectedly, most differentially abundant proteins seem to be upregulated 24 h after C6 (see below). A genetic screen showed that loss of p53 rescues the effect of C6 and C16 and helped the authors to identify pathways that can be targeted by compounds together with WIN site inhibitors in a synergistic way. Finally, the authors elucidated the underlying mechanisms and analyzed the functional relevance of the RPL22, RPL22L1, p53 and MDM4 axis.

Comments on revised version:

The authors have answered my points satisfactorily and the manuscript has become clearer and more meaningful as a result. In particular, the measurement of global translation rate is important and validates the upregulation of a number of proteins following WDR5 inhibitor treatment.

Reviewer #1 (Public Review):

Summary:

The authors use truncations, fragments, and HCN2/4 chimeras to narrow down the interaction and regulatory domains for LRMP inhibition of cAMP-dependent shifts in the voltage dependence of activation of HCN4 channels. They identify the N-terminal domain of HCN4 as a binding domain for LRMP, and highlight two residues in the C-linker as critical for the regulatory effect. Notably, whereas HCN2 is normally insensitive to LRMP, putting the N-terminus and 5 additional C-linker and S5 residues from HCN4 into HCN2 confers LRMP regulation in HCN2.

Strengths:

The work is excellent, the paper well written, and the data convincingly support the conclusions which shed new light on the interaction and mechanism for LRMP regulation of HCN4, as well as identifying critical differences that explain why LRMP does not regulate other isoforms such as HCN2.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

Rigorous data, well-designed experiments as well as significant innovation make this manuscript stand out.

Weaknesses:

All my concerns have been well addressed, no further comments.

Reviewer #1 (Public Review):

Here, using an organoid system, Wong et al generated a new model of hereditary diffuse leukoencephalopathy with axonal spheroids, with which they investigated how CSF1R-mutaions affect the phenotypes of microglia/macrophages, and revealed metabolic changes in microglia/macrophages associated with a proinflammatory phenotype.

In general, this paper is interesting and well-written, and tackles important issues to be addressed.

This study suffers from several major concerns and limitations that dampen the value of the study. As the authors also mentioned, models that perfectly recapitulate the complexity of the HDLS brain the models would be required to better understand the molecular mechanisms of the disease. In this regard, it is unclear how nicely the organoid system in this study can recapitulate the condition in patients with HDLS (e.g. reduced microglia density, downregulated expression of P2YR12, pathological alterations). In addition, the authors used two different models with distinct mutations that could produce different readouts in CSF1R-mediated cellular responses.

Although the reviewer does understand the importance of providing several options/tools to study rare diseases like HDLS and the difficulty of generating stable organoids with less variation, it is unclear if the different outcomes between HD1 and HD2 are generated through different mutations or simply due to different differentiation efficiency from iMacs (e.g. Figure 2B), which needs to be confirmed. Lastly, there is an over-interpretation regarding the results in Figure 6A. There is no difference between isoHD1 iMac control and HD1 Mut iMac.

Reviewer #2 (Public Review):

Summary:

Fertilization is a crucial event in sexual reproduction, but the molecular mechanisms underlying egg-sperm fusion remain elusive. Elofsson A 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 are 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:

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. provides experimental evidence of the interaction between IZUMO1/SPACA6/TMEM81 in zebrafish. This is an important element that supports the findings presented in this manuscript

Regarding the authors response on the question of a global search:<br /> I understand that a global search might be difficult to interpret because a large number of putative false positives. But it is this type of information that is needed to assess the validity of the model and the scoring power in the absence of any experimental validation. At minimum, the search should include a negative control set of proteins known to be unrelated to sperm fertilization or homologous egg-sperm fusion complexes from incompatible species to account for species-specific interactions.

I acknowledge that experimentally validating highly transient complexes presents technical hurdles. However, a high-confidence structural model could enable the design of point mutations specifically disrupting the predicted interactions. Subsequent rescue experiments could then validate the directionality of these interactions. Ultimately, such experiments are crucial for robust model validation.

Reviewer #1 (Public Review):

Summary:

In this study, the authors generated a novel transgenic mouse line OpalinP2A-Flpo-T2A-tTA2 to specifically label mature oligodendrocytes, and at the same time their embryonic origins by crossing with a progenitor cre mouse line. With this clever approach, they found that LGE/CGE-derived OLs make minimum contributions to the neocortex, whereas MGE/POA-derived OLs make a small but lasting contribution to the cortex. These findings are contradictory to the current belief that LGE/CGE-derived OPCs make a sustained contribution to cortical OLs, whereas MGE/POA-derived OPCs are completely eliminated. Thus, this study provides a revised and more comprehensive view on the embryonic origins of cortical oligodendrocytes. To specifically label mature oligodendrocytes, and at the same time their embryonic origins by crossing with a progenitor cre mouse line. With this clever approach, they found that LGE/CGE-derived OLs make minimum contributions to the neocortex, whereas MGE/POA-derived OLs make a small-but-lasting contribution to to cortex. These findings are contradictory to the current belief that LGE/CGE-derived OPCs make a sustained contribution to cortical OLs, whereas MGE/POA-derived OPCs are completely eliminated. Thus, this study has provided a revised and updated view on the embryonic origins of cortical oligodendrocytes.

Strengths:

The authors have generated a novel transgenic mouse line to specifically label mature differentiated oligodendrocytes, which is very useful for tracing the final destiny of mature myelinating oligodendrocytes. Also, the authors carefully compared the distribution of three progenitor cre mouse lines and suggested that Gsh-cre also labeled dorsal OLs, contrary to the previous suggestion that it only marks LGE-derived OPCs. In addition, the author also analyzed the relative contributions of OLs derived from three distinct progenitor domains in other forebrain regions (e.g. Pir, ac). Finally, the new transgenic mouse lines and established multiple combinatorial genetic models will facilitate future investigations of the developmental origins of distinct OL populations and their functional and molecular heterogeneity.

Weaknesses:

Since OpalinP2A-Flpo-T2A-tTA2 only labels mature oligodendrocytes but not OPCs, the authors can not suggest that the lack of LGE/CGE-derived-OLs in the neocortex is less likely caused by competitive postnatal elimination, but more likely due to limited production and/or allocation (line 118-9). It remains possible that LGE/CGE-derived OPCs migrate into the cortex but are later eliminated.

Reviewer #1 (Public Review):

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

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

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

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

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

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

Comments on the revised manuscript:

The authors have adequately addressed my previous concerns.