Joint Public Review:

The manuscript highlights a mechanistic insight into meiotic initiation in budding yeast. In this study, the authors analyzed the genetic link between the mitotic cell cycle regulator SBF (the Swi4-Swi6 complex) and a meiosis inducing regulator Ime1 in the context of meiotic initiation. The authors' comprehensive analyses with cytology, imaging, RNA-seq using mutant strains lead to the conclusion that Swi4 levels regulates Ime1-Ume6 interaction to activate expression of early meiosis genes for meiotic initiation.

The authors first show a down regulation of Swi4 at the protein level upon meiosis entry and then investigate downstream consequences. This study reveals several regulations: 1) Mutations in CLN1 and 2, which are targets of Swi4, allow rescuing the delay in meiotic entry observed when Swi4 is overexpressed; 2) Ime1 activity is antigonized by Swi4, and more specifically its interaction with Ume6. 3) Expression of SWI4 is regulated by LUTI-based transcription at the SWI4 locus that impedes expression of canonical SWI4 transcripts 4) The expression of SWI4 LUTI is likely negatively regulated by the Ime1-Ume6 complex 5) Whi5 restrict SBF activity during meiotic entry, thereby ensuring Cyclin repression.

The important implication in this paper is that meiotic initiation is regulated by the balance of mitotic cell cycle regulator and meiosis-specific transcription factor.

Joint Public Review:

Summary<br /> Sender et al describe a model to estimate what fraction of DNA becomes cell-free DNA in plasma. This is of great interest to the community, as the amount of DNA from a certain tissue (for example, a tumor) that becomes available for detection in the blood has important implications for disease detection.

Strengths<br /> The question asked by the authors has potentially important implications for disease diagnosis. Understanding how genomic DNA degrades in the human circulation can guide towards ways to enrich for DNA of interest or may lead to unexpected methods of conserving cell-free DNA. Thus, the question "how much genomic DNA becomes cfDNA" is of great interest to the scientific and medical community. I believe this manuscript has the potential to be a widely used resource. As more data is collected on cell-free DNA yields and cellular turnover in the body, this work will only increase in importance.

Appraisal<br /> At this stage of the manuscript (second submission), I think the authors provide important evidence and analysis that aim to answer their research question. Previous concerns about methodology have been addressed.

Impact<br /> This manuscript will be highly impactful on the community. The field of liquid biopsies (non-invasive diagnostics) has the potential to revolutionize the medical field (and has already in certain areas, such as prenatal diagnostics). Yet, there is a lack of basic science questions in the field. This manuscript is an important step forward in asking more "basic science" questions that seek to answer a fundamental biological question.

No feedback! This is great! I love how you touched on so many abiotic factors in one post! If you wanted to expand on any of them, you certainly could, but you have plenty of information already!

This introduction is so much fun! I love the unique way you chose to format this post.

The images are great too, but it might be helpful to add captions with a brief explanation.

Awesome explanation, very clear and informational!

Even though I'm already excited to read this, it may be good to add a little hook/intro paragraph to illuminate why this is so interesting!

Reviewer #1 (Public Review):

Summary:<br /> In the first half of this study, Pham et al. investigate the regulation of TEAD via ubiquitination and PARylation, identifying an E3 ubiquitin ligase, RNF146, as a negative regulator of TEAD activity through an siRNA screen of ubiquitin-related genes in MCF7 cells. The study also finds that depletion of PARP1 reduced TEAD4 ubiquitination levels, suggesting a certain relationship between TEAD4 PARylation and ubiquitination which was also explored through an interesting D70A mutation. Pham et al. subsequently tested this regulation in D. melanogaster by introducing Hpo loss-of-function mutations and rescuing the overgrowth phenotype through RNF146 overexpression.

In the second half of this study, Pham et al. designed and assayed several potential TEAD degraders with a heterobifunctional design, which they term TEAD-CIDE. Compounds D and E were found to effectively degrade pan-TEAD, an effect which could be disrupted by treatment with TEAD lipid pocket binders, proteasome inhibitors, or E1 inhibitors, demonstrating that the TEAD-CIDEs operate in a proteasome-dependent manner. These TEAD-CIDEs could reduce cell proliferation in OVCAR-8, a YAP-deficient cell line, but not SK-N-FI, a Hippo pathway independent cell line. Finally, this study also utilizes ATAC-seq on Compound D to identify reductions in chromatin accessibility at the regions enriched for TEAD DNA binding motifs.

Strengths:<br /> The study provides compelling evidence that the E3 ubiquitin ligase RNF146 is a novel negative regulator of TEAD activity. The authors convincingly delineate the mechanism through multiple techniques and approaches. The authors also describe the development of heterobifunctional pan-degraders of TEAD, which could serve as valuable reagents to more deeply study TEAD biology.

Weaknesses:<br /> The scope of this study is extremely broad. The first half of the paper highlights the mechanisms underlying TEAD degradation; however, the connection to the second half of the paper on small molecule degraders of TEAD is jarring, and it seems as though two separate stories were combined into this single massive study. In my opinion, the study would be stronger if it chose to focus on only one of these topics and instead went deeper.

Additionally, the figure clarity needs to be substantially improved, as readability and interpretation were difficult in many panels. Lastly, there are numerous typos and poor grammar throughout the text that need to be addressed.

Reviewer #1 (Public Review):

Summary:<br /> The current manuscript provides an extensive in vivo analysis of two guidance pathways identifying multiple mechanisms that shape the bifurcation of DRG axons when forming the dorsal funiculus in the DREZ.

Strengths:<br /> Multiple mouse mutant lines were used, together with complementary techniques; the results are very clear and compelling.<br /> The findings are very significant and clearly move forward our understanding of the regulation of axonal development at the DREZ.

Weaknesses:<br /> No major weaknesses were found. As it is I have no recommendations that would increase the clarity or quality of the manuscript.

Reviewer #1 (Public Review):

Yu et al. investigated Fusarium oxysporum f. sp. lycopersici SIX effectors structure using experimental and computational approaches, and while doing so, the authors identified several SIX effectors as member of the FOLD family, and expanded the known diversity of the SIX effectors. A very interesting and novel finding is the presence of FOLD putative effectors in other Ascomycetes secretome, sharing structural similarities with SIX effectors Avr1, Avr3 and SIX6.

By performing technically sound predictions and experimental confirmation, the authors also confirmed co-operative interactions between Fol effectors, something that was previously known for different pairs of proteins, expanding this observation for new SIX effectors. In addition, the authors contributed to the understanding of the interaction Fol effectors, specifically FOLD and LARS effectors, - I receptors to suppress immunity by structurally similar effectors.

The conclusions of this paper are supported by data and I think it is a pioneer study analyzing the correspondence between AlphaFold predictions and experimentally derived structures, highlighting that models can answer the scientific questions in some cases but could not be enough in others.

Reviewer #1 (Public Review):

The author found the nifuroxazide has the potential to augment the efficacy of radiotherapy in HCC by reducing PD-L1 expression. This effect may be attributed to increased degradation of PD-L1 through the ubiquitination-proteasome pathway. These evidences support the future application of nifuroxazide in the treatment of HCC.

Reviewer #1 (Public Review):

Summary:

The authors developed a deep learning method called H3-OPT, which combines the strength of AF2 and PLM to reach better prediction accuracy of antibody CDR-H3 loops than AF2 and IgFold. These improvements will have an impact on antibody structure prediction and design.

Strengths:

The training data are carefully selected and clustered, the network design is simple and effective.

The improvements include smaller average Ca RMSD, backbone RMSD, side chain RMSD, more accurate surface residues and/or SASA, and more accurate H3 loop-antigen contacts.

The performance is validated from multiple angles.

The revised manuscript has cleared my previous concerns.

Reviewer #1 (Public Review):

Summary:<br /> This study presents a valuable finding on the increased activity of two well-studied signal transduction pathways - STAT-3 and TGF-Beta in a specific subtype of pancreatic cancer. Specifically, SMAD4 deficient tumors (commonly observed in pancreatic cancer) are well differentiated in the presence of STAT3. Yet surprisingly, in the presence of SMAD4 in a STAT-3 deficient pancreatic cancer, the phenotype is poorly differentiated in the background of KRASGD12D. The evidence in the animal models supporting the authors' claims is solid, although including TCGA data and/or a larger number of patients would have strengthened the study. The work will be of interest to medical biologists working on pancreatic cancer and potentially the broader field.

Strengths:<br /> Strengths are the animal models and the lead author's expertise in STAT3 signaling.

Weaknesses:<br /> Weaknesses are the absence of correlation between the results from the animal studies and human pancreatic cancers.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript by Chen et al. presents a detailed metabolic characterization of male and female WT and CTRP10 knockout mice. The main finding is that female KO mice become obese on both low-fat and high-fat diets but without evidence of marked insulin resistance, hepatic steatosis, dyslipidemia, or increased inflammatory markers. The authors performed a detailed transcriptomic analysis and identified differentially expressed genes that distinguish high-fat diet-fed CTRP10 KO from WT control mice. They further show that this set of genes exhibits cross-correlation in human tissues, and that this is greater in females than in males. The data indicate that the CTRP10 KO model may be useful to understand how obesity and metabolic dysfunction are coupled to each other, and how this occurs by a sex-biased mechanism.

Strengths:<br /> The work presents a large amount of data, which has been carefully acquired and is convincing. The transcriptomic analysis will further help to define what pathways are associated with obesity, but not necessarily with metabolic dysfunction. The manuscript will be of interest to investigators studying metabolic diseases, and to those studying sex-specific differences in metabolic physiology. The limitations of the study are acknowledged, including that a whole-body knockout was used. The cause of the increased body weight is not entirely clear, despite the careful and detailed analysis that was performed. Notwithstanding these limitations, the phenotype is interesting, and this work will establish a basis for further work to understand the mechanisms that are involved.

Weaknesses:<br /> Genes identified as DEGs in the mouse RNAseq data set were used to identify a set of human orthologous transcripts and the abundances of these transcripts were correlated with each other in Figure 10. This identified a greater correlation ("connectivity") in subQ adipose compared to other tissues, and in females compared to males. The description of how this analysis was done could be clearer. In some cases, the text refers to the software that was used without describing the goal of the analysis. In other instances, specialized terminology was used (e.g. "biweight midcorrelation") without defining what this means.

Reviewer #1 (Public Review):

Summary:

Bartolome et al. report adaptation of proximity labeling using BirA and TurboID fusions to proteasome subunits to identify the proteasome-proximal proteome both in cultured cells and also in a newly developed mouse model. Using this approach, the authors demonstrate identification of many known proteasome-interacting proteins, as well as several new proteins, some of which are validated directly. The authors further evaluate the proteasome-proximal proteome in most mouse organs, and find substantial agreement with the proteome identified from cultured cells, as well as between tissues. This represents one of the first studies of the "proteasome-ome" in vivo, and sets the stage for addressing numerous important future questions regarding how the proteasome's environment changes over time, in response to different stimuli, and in distinct disease conditions.

Strengths:

Generally speaking, the approach provided is rigorous and supported by several complementary lines of evidence, such as demonstration that the interactome is enriched for known proteasome-binding proteins and co-purification or co-elution experiments. Similarly, the high agreement between the outcomes in cultured cells and in the mouse model developed by the authors provides further confidence in the results.

Weaknesses:

The major weakness of the work is arguably the choice of proteasome subunits for tagging with biotinylating enzymes. In most cases, the subunits and termini chosen for tagging are known to either protrude toward functionally important regions (such as the substrate-processing pore of the ATPase component), to have important functional roles likely to be disrupted via tagging, or are subunits known to be substituted by others in some conditions. Thus, the interactome reported may conflate those of normal proteasomes with those harboring tag-induced functional or structural defects. Although the authors made a commendable attempt to demonstrate minimal impacts of tagging, the conclusions would be greatly further strengthened by contrasting the impacts of tagging subunits less likely to cause perturbations and by more rigorously demonstrating normal proteolysis of a broader array of known proteasome substrates.

Reviewer #1 (Public Review):

Summary:<br /> Zhang et al. describe novel roles for the centriolar protein CEP44, namely that it is required for centriole engagement (and thus inhibition of centriole reduplication) and that it promotes microtubule stability. While a function of CEP44 in centriole engagement is somehow convincingly shown, the data do not support a role for CEP44 in microtubule stabilization.

Strengths:<br /> The finding that centriole engagement relies on CEP44 is novel and of great interest to the centriole field. Interestingly, the authors correlate reduced CEP44 expression levels with the occurrence of breast carcinoma, which makes this study also very interesting for a broad audience.

Weaknesses:<br /> The paper has important findings, but unfortunately, the main claims are only partially supported.

1) The role of CEP44 in microtubule stability is not clear from the presented data:<br /> - Fig. 7A and S6 A, there is no visible difference in microtubule density/intensity between the different groups of cells. In Fig. 7C, the CEP44 S324A spindle looks even brighter than the WT spindle. The authors need to indicate how many cells were analyzed. This information is actually lacking in all the experiments.

2) Several figure parts are not properly labelled.

3) Several of the experiments (WBs) likely miss proper controls: How did the authors detect proteins that run at very similar sizes: 55 kDa (alpha-tubulin), 44 kDa (Cep44), and 57 kDa (Cep57 and Cep57L)? The loading control needs to be detected in the same lane as the protein of interest. Did the authors strip and reprobe membranes? If so, this needs to be indicated and included in the methods section.

4) It is not clear how such a low CEP44-FLAG expression (Fig. 5A) can rescue a CEP44 KO.

Reviewer #1 (Public Review):

Summary:<br /> In their manuscript, Zhou et al. analyze the factors controlling the activation and maintenance of a sustained cell cycle block in response to persistent DNA DSBs. By conditionally depleting components of the DDC using auxin-inducible degrons, the authors verified that some DDC proteins are only required for the activation (e.g., Dun1) or the maintenance (e.g., Chk1) of the DSB-dependent cell cycle arrest, while others such as Ddc2, Rad24, Rad9 or Rad53 are required for both processes. Notably, they further demonstrate that after a prolonged arrest (>24 h) in a strain carrying two DSBs, the DDC becomes dispensable and the mitotic block is then maintained by SAC proteins such as Mad1, Mad2, or the mitotic exit network (MEN) component Bub2.

Strengths:<br /> The manuscript dissects the specific role that different components of the DDC and the SAC have during the induction of a cell cycle arrest induced by DNA damage, as well as their contribution to the short-term and long-term maintenance of a DNA DSB-induced mitotic block. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented. The conclusions drawn are also generally well supported by the experimental data. The observations contribute to drawing a clearer picture of the relative contribution of these factors to the maintenance of genome stability in cells exposed to permanent DNA damage.

Weaknesses:<br /> The main weakness of the study is that it is fundamentally based only on the use of the auxin-inducible degron (AID) strategy to deplete proteins. This is a widely used method that allows a very efficient depletion of proteins. However, the drawback is that a tag is added to the protein, which can affect the functionality of the targeted protein or modify its capacity to interact with others. In fact, three of the proteins that are depleted using the AID systems are shown to be clearly hypomorphic. Verification of at least some of the results using an alternative manner to eliminate the proteins would help to strengthen the conclusions of the manuscript.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes the identification and characterization of rice SCC3, including the generation and characterization of plants containing apparently lethal null mutations in SCC3 as well as mutant plants containing a c-terminal frame-shift mutation. The weak scc3 mutants showed both vegetative and reproductive defects. Specifically, mitotic chromosomes appeared to partially separate during prometaphase, while meiotic chromosomes were diffuse during early meiosis and showed alterations in sister chromatid cohesion, homologous chromosome pairing, and recombination. The authors suggest that SCC3 acts as a cohesin subunit in mitosis and meiosis, but also plays more functions other than just cohesion.

Strengths:<br /> The manuscript contains a large amount of generally high-quality data.

Weaknesses:<br /> Several of the conclusions drawn in the manuscript are not supported by the data. There are many examples where the authors either draw conclusions or make statements that are just not justified based on the data presented or present a conclusion as a new finding, which has already been demonstrated in the past by others. For example, they claim that SCC3 functions in the maintenance of replication. From my reading of the manuscript, nowhere did the authors examine DNA replication. Likewise, several of the conclusions drawn are in direct contrast with what is known about SCC3 in other organisms. Therefore, the conclusions are either groundbreaking or incorrect.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript describes a deficiency in nuclear pore complexes (NPCs) to maintain proper compartmentalization between the nucleus and cytoplasm in a mouse model of AD-related Aβ pathology. Experiments demonstrate NPC dysfunction in cultured neurons and mouse tissue as a result of intracellular Aβ, which may cause reduced levels of certain nucleoporins, leading to a reduced number of NPCs, and their dysfunction in nuclear protein import and maintaining nucleocytoplasmic compartmentalization. In addition, the authors also report a potential mechanism for how NPC dysfunction may result in increased vulnerability to inflammation-induced necroptosis, where core components are reportedly activated via phosphorylation through nucleocytoplasmic shutting. Overall, the study is interesting and well conducted and reveals striking NCT defects in a Aβ pathology disease model that may have important implications for our understanding of AD pathology.

Strengths:<br /> Previous studies have found nucleocytoplasmic transport (NCT) defects in other models of age-related neurodegenerative diseases, including Huntington's disease, tauopathy, C9orf72-linked frontotemporal dementia / amyotrophic lateral sclerosis (FTD/ALS), and TDP-43 proteinopathy in FTD/ALS. Typically, NCT defects have been linked mechanistically to aberrant co-aggregation of nucleoporins with e.g. TDP-43 and tau found in disease models and sometimes also human autopsy tissue. This study is novel, in that it describes NCT defects that are caused by Alzheimer's disease (AD) related Aβ pathology, using a human APP knock-in mouse model (AppNL-G-F/NL-G-F) that exhibits robust Aβ pathology in the CNS. The main focus of this study is on the barrier dysfunction of the NPCs leading to compartmentalization defects, while previous publications in the field have focused more on active protein import and RNA export defects. This is of considerable interest since an age-dependent decline in NPC barrier function has been observed in transdifferentiated neurons derived from normal-aged fibroblasts (Mertens et al., 2015). The potential link of NPC dysfunction to an increased vulnerability to inflammation-induced necroptosis may also be relevant to other neurodegenerative disorders with NCT dysfunction. Experiments are largely focused on either dissociated neuronal cultures, or studies using mouse tissue at different stages of disease progression. Experiments are mostly based on immunocytochemistry (ICC) and histochemistry (IHC) of nucleoporins to show morphological NPC defects and fluorescent reporter constructs and dyes of defined MW to show NPC dysfunction. The experiments using an anti-nuclear pore O-linked glycoprotein antibody [RL1], which recognizes multiple metazoan nucleoporins that are modified via post-translational O-GlcNAcylation, show a very striking reduction in staining intensity that is also replicated with antibodies specific for the FG-motif rich Nup98 and the very stable and essential NPC component Nup107. Taken together, the fluorescence microscopy studies convincingly support the claim of NPC dysfunction leading to defective compartmentalization between the nucleus and cytoplasm.

Weaknesses:<br /> However, the molecular mechanisms leading to NPC dysfunction and the cellular consequences of resulting compartmentalization defects are not as thoroughly explored. Results from complementary key experiments using western blot analysis are less impressive than microscopy data and do not show the same level of reduction. The antibodies recognizing multiple nucleoporins (RL1 and Mab414) could have been used to identify specific nucleoporins that are most affected, while the selection of Nup98 and Nup107 is not well explained. There is also no clear hypothesis on how Aβ pathology may affect nucleoporin levels and NPC function. All functional NCT experiments are based on reporters or dyes, although one would expect widespread mislocalization of endogenous proteins, likely affecting many cellular pathways. The second part of this manuscript reports that in App KI neurons, disruption in the permeability barrier and nucleocytoplasmic transport may enhance activation of key components of the necrosome complex that include receptor-interacting kinase 3 (RIPK3) and mixed lineage kinase domain1 like (MLKL) protein, resulting in an increase in TNFα-induced necroptosis. While this is of potential interest, it is not well integrated in the study. This potential disease pathway is not shown in the very simple schematic (Fig. 8) and is barely mentioned in the Discussion section, although it would deserve a more thorough examination.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript aimed at elucidating the substrate specificity of two M23 endopeptidase Lysostaphin (LSS) and LytM in S. aureus. Endopeptidases are known to cleave the glycine-bridges of staphylococcal cell wall peptidoglycan (PG). To address this question, various glycine-bridge peptides were synthesized as substrates, the catalytic domain of LSS and LytM were recombinantly expressed and purified, and the reactions were analyzed using solution-state NMR. The major finding is that LytM is not only a Gly-Gly endopeptidase, but also cleaves D-Ala-Gly. Technically, the advantage of using real-time NMR was emphasized in the manuscript. The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM. However, the biological significance and relevance of the conclusions remain clear, as the results are mostly from synthetic substrates.

Strengths:<br /> The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM.

Weaknesses:<br /> 1. Significance: while the current study provided a detailed analysis of various substrates, the conclusions are mainly based on synthesized peptides. One experiment used purified muropeptides (Fig. 3H); however, the results were unclear from this figure. The results from synthesized peptides may not necessarily correlate with their biological functions in vivo. Secondly, the study used only the catalytic domain of both proteins. It is known that the substrate specificity of these enzymes is regulated by their substrate-binding domains. There is no mention of other domains in the manuscript and no justification of why only the catalytic domain was studied. In short, the relevance of the results from the current study to the enzymes' actual physiological functions remains to be addressed, which attenuated the significance of the study.

2. Impact and novelty: (1) the current study provided evidence suggesting the novel function of LytM in cleaving D-Ala-Gly. The impact of this finding is unclear. The manuscript discussed Enterococcus faecalis EnpA. But how about other M23 endopeptidases? What is biological relevance? (2) A very similar study published recently showed that the activity of LSS and LytM is regulated by PG cross-linking: LSS cleaves more cross-linked PG and LytM cleaves less cross-linked PG (Razew, A., Laguri, C., Vallet, A., et al. Staphylococcus aureus sacculus mediates activities of M23 hydrolases. Nat Commun 14, 6706 (2023). The results of this paper are different from the current study whereby both LSS and LytM prefer cross-linked substrates (Fig, 2JKL). Moreover, no D-Ala-Gly cleavage was observed by LytM using purified PG substrate from Razew A et al. An explanation of inconsistent results is needed here. In my opinion, the knowledge generated from the current study has not been fully settled. If the results can be validated, the contribution to the field is incremental, but not substantial. (3) The authors emphasized a few times in the text that it is superior to use NMR technology. In my opinion, NMR has certain advantages, such as measuring the efficacy of cleavage, but it is not that superior. It should be complementary to other methods such as mass spectrometry. In addition, more relevant solid-state NMR using intact PG or bacterial cells was not discussed in the study. I am of the opinion that the corresponding text should be revised.

3. The conclusions are not fully supported by the data<br /> As mentioned above, the conclusions from synthesized peptide substrates may not necessarily reveal physiological functions. The conclusions need to be validated by more physiological substrates.

4. There are some issues with the presentation of the figures, text, and formatting.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript by Dubey et al. examines the function of the acetyltransferase Tip60. The authors show that (auto)acetylation of a lysine residue in Tip60 is important for its nuclear localization and liquid-liquid-phase-separation (LLPS).

The main observations are: (i) Tip60 is localized to the nucleus, where it typically forms punctate foci. (ii) An intrinsically disordered region (IDR) within Tip60 is critical for the normal distribution of Tip60. (iii) Within the IDR the authors show that a lysine residue (K187), that is auto-acetylated, is critical. Mutation of that lysine residue to a non-acetylable arginine abolishes the behavior. (iv) biochemical experiments show that the formation of the punctate foci may be consistent with LLPS.

Strengths:<br /> The experiments are largely convincing and appear to be well executed.

Weaknesses:<br /> The main concern I have is that all in vivo (i.e. in cells) experiments are done with overexpression in Cos-1 cells, in the presence of the endogenous protein. No attempt is made to use e.g. cells that would be KO for Tip60 in order to have a cleaner system or to look at the endogenous protein. It would be reassuring to know that what the authors observe with highly overexpressed proteins also takes place with endogenous proteins.

Also, it is not clear how often the experiments have been repeated and additional quantifications (e.g. of western blots) would be useful.

In addition, regarding the LLPS description (Figure 1), it would be important to show the wetting behavior and the temperature-dependent reversibility of the droplet formation.

On balance, this is an interesting study that describes the role of acetylation of Tip60 in controlling its biochemical behavior as well as its localization and function in cells. The authors mention in their Discussion section other examples showing that acetylation can change the behavior of proteins with respect to LLPS; depending on the specific context, acetylation can promote (as here for Tip60) or impair LLPS.

Reviewer #1 (Public Review):

This study shows that SET7 and LSD1 regulate the dynamic methylation of EZH2 at K20, which is recognized by L3MBTL3 promoting protein degradation via the DCAF5-CRL4 E3 ubiquitin ligase. K20 methylation negatively regulates S21 phosphorylation and vice versa, modulating EZH2 functions. Mice harboring the K20 methylation-deficient mutant (K20R) exhibit hematopoietic defects. Overall, this is an interesting study elucidating a novel mechanism of EZH2 regulation. The methodologies are sound and the conclusions are largely supported by the data provided. However, there are some questions regarding the overall model and some contradictory results.

Reviewer #1 (Public Review):

The study by Schmehl and colleagues asks an important question, i.e. how are multiple objects/stimuli represented in the visual system despite broad tuning properties of neurons along multiple different dimensions (e.g. space, features). This is a continuation of an impactful and highly significant line of work from the Groh lab and their collaborators. In previous work, they showed that fluctuations in firing patterns may be critical in representing multiple objects and parse them in time. In this particular study, the authors ask three specific questions to extend these observations: (i) Are such fluctuations widespread in the visual system?; (ii) Are they related to the perceptual distinction of objects?; (iii) And how are they related to the functional specialization of neuronal populations along feature dimensions (e.g. faces, motion).

It seems to me that there is ample evidence for the first two questions from previous work by these authors. For (i), fluctuations in firing patterns related to multiple stimuli have been shown in the auditory (e.g. inferior colliculus, Caruso et al., 2018) and multiple areas of the visual system (i.e. V1, V4, and the face patch system; Caruso et al., 2018; Jun et al., 2022). The present study adds data from MT to this increasing evidence. For (ii), Jun et al., 2022 already showed that fluctuations are not related to stimuli perceived as merged, or not distinct. Thus, the main contribution appears to be related to functional specialization. I suggest clarifying the major novelty of the present report and to focus the introduction on it.

The present work analyzed three different data sets acquired in different areas (V1, V4, MT, IT face network), using different feature stimuli (motion, faces), obtained under various attention conditions/states (passive fixation, actively ignored). Many of the results are nice confirmations and minor extensions of previous work. The conceptual advance and novelty of the findings are therefore limited.

There is a growing literature on fluctuating neural firing patterns that is not considered in this report. The scholarship appears a bit impoverished with only 19 references, many of which point to work from this group of collaborators. I suggest that the authors consider the present work in the context of the wider literature more scholarly, even if not all the relations of these different lines of work can be conclusively connected at this point. For a few examples, there is work by Kienitz and colleagues on fluctuating neural patterns in V4 evoked by competing grating stimuli. Also, the work by Engel, Moore, and colleagues on 'on' and 'off' states in the context of selective attention seems relevant, or the work by Fiebelkorn and Kastner on rhythmic perception and attention.

I love this introduction! You do a marvelous job of drawing in the reader, especially with these photographs!

Reviewer #1 (Public Review):

Summary:<br /> In this study, the authors attempt to reinvestigate an old question in population genetics regarding the age of alleles that have experienced different strengths (and directions) of natural selection. Under simple population genetic models, alleles that are positively selected are expected to change frequency in populations faster than neutral alleles. So the naïve expectation is that if you look at alleles that are the same population frequency, those that have been evolving neutrally should have been segregating in the population longer than those that have been experiencing natural selection. While this is exactly what the authors find for alleles inferred to be experiencing negative selection (i.e. they tend to be younger than alleles inferred to be neutral that are at the same frequency), the authors find the opposite for alleles inferred to be under positive selection: they tend to be older than alleles inferred to be neutral. The authors argue that this pattern can be explained by a model where positively selected mutations experience a phase of balancing selection that can dramatically extend the period of time that these alleles segregate in the population.

Strengths:<br /> The question that the authors address is very interesting and thought provoking. When confronted with a counter-intuitive finding, the authors describe an interesting hypothesis to explain it. The authors investigate a number of interesting sub analyses to corroborate their findings.

Weaknesses:<br /> While there are some intriguing hypotheses in this manuscript, I struggle to be convinced. The main point that the authors argue is that positively selected alleles are older than their neutral counterparts at the same frequency. They argue that this may be because the positively selected alleles are stuck in some form of balancing selection for a long time before they switch to a more classical form of directional selection. The form of balancing selection they argue is one caused by linkage to deleterious alleles, which takes time for the beneficial alleles to recombine onto a more neutral background. I would really like to see some simulations that demonstrate this can actually occur on average. Reading this paper brought back memories of the classic Birky and Walsh (1988; PMCID: PMC281982) paper that argued that linkage amongst selected alleles does not impact the substitution rate of linked neutral alleles, but does reduce the substitution rate among beneficial alleles. Their simple simulations in 1988 illuminated how this works, and they developed a simple mathematical model that helped us understand how it works. In the current paper, it seems the authors are arguing for a similar effect, but rather than focus on beneficial alleles that fix, they are focusing on beneficial alleles that are still segregating. These seem like similar stories, but without simulations or a mathematical model, I struggle to gain any insight into why the observation is the way it is (and not simply due to a number of possible confounding effects noted below).<br /> There are a number of elements to the methods and interpretation that could use clarification.<br /> • Genetic data. One of the biggest weaknesses of this analysis is the choice of genetic data. The authors use the UK10k dataset, and reference the 2015 paper. Looking at that paper, it seems that the data may be composed of low coverage whole genome sequencing data (7x) and high coverage exome sequence data (80x). It appears that these data were integrated into a single VCF file, similar to the 1000 Genomes Project Phase 3 data. If these are the data that was used, then there are substantial differences between the coding and non-coding variants that are compared. However, it is possible that the authors chose to restrict the analysis to the low coverage WGS data and neglected to indicate it in the methods section. I will assume that this is the case for the rest of the review, but the authors should clarify.<br /> • Recombination rates. I believe the authors use an LD-based recombination map. While these maps are correlated at the longer physical distances with pedigree maps, there are substantial differences at shorter physical scales. These differences have been argued to be due to the action of natural selection skewing patterns of LD. If that is the case, then some of the observations in this paper are circular. Please confirm similar findings with a pedigree-based recombination map.<br /> • Recombination rates, pt 2. The authors compare patterns of non-synonymous coding variants to a set of non-coding, non-regulatory SNPs. They argue "these will necessarily have experienced similar mutational and recombinational processes". I don't know that this is true. There are both distinct recombination patterns and mutational patterns in genes vs non-coding regions of the genome. It would be important to more carefully match coding and non-coding variants based on both recombination as well as the type of nucleotide change. There are substantial differences in CpG composition in coding vs non-coding regions for example. While the authors say "Analyses thought to be sensitive to CpG high mutability were limited to SNPs that did not occur as part of a CpG", it is quite unclear what where CpGs were included vs excluded.<br /> • Identifying ancestral vs derived alleles. It is unclear how the authors identified ancestral vs derived alleles (they say "inferred ancestral sequence from Ensembl (1) and a maximum likelihood estimator". Several studies have shown that ancestral misidentification can cause skews in the site frequency spectrum. If the ancestral state of some fraction of alleles were misidentified, then the estimated allele age would be incorrect. Figure 1B shows that the mean frequency of the alleles with the largest delta-EP tend to be very low. This makes me think that ancestral misidentification may have impacted the results.<br /> • Figure 2B and C. I do not understand how the median can be so far outside the mean and error bars. The legend does not specify what the error bars are, but I feel the distribution must be shown if it is so skewed that the mean and any definition of error does not include the median.<br /> • Inferring allele ages. The authors use two methods for estimating allele ages, but focus on GEVA. They use the default parameter of effective population size 10,000. How sensitive is the model to this assumption? It has been shown that different regions of the genome (particularly coding vs neutral non-coding) experience different rates of deleterious mutations, and therefore different rates of background selection. Simple models of background selection would suggest that these regions will therefore have different effective population sizes.<br /> • Fst analysis. The authors look at Fst among 3 populations as a function of delta-EP compared to frequency-matched control SNPs. They find there is no statistical support for different levels of Fst in any pairwise comparison for any delta-EP bin. It seems strange that alleles with large delta-EP would not show increased Fst compared to control SNPs... If they are indeed positively selected, the assumption must be that they are then positively selected in all populations, which seems unlikely. Alternatively, by considering only narrow allele frequency bins, it is possible that Fst is also being controlled, and therefore this analysis is non-informative. A simulation would help understand what the expected pattern is here.<br /> • It would be great to show more figures like 2A. You can place the x-axis on a log-scale so that it is easier to view the lower allele frequencies. This plot clearly shows differences among the 3 categories. I am very surprised at the much shorter error bars for negative delta-EP at high frequency compared to positive delta-EP variants... Shouldn't there be very few negative delta-EP alleles at such high frequency?

Reviewer #1 (Public Review):

Summary:<br /> Herein, Blaeser et al. explored the impact of migraine-related cortical spreading depression (CSD) on the calcium dynamics of meningeal afferents that are considered the putative source of migraine-related pain. Critically previous studies have identified widespread activation of these meningeal afferents following CSD; however, most studies of this kind have been performed in anesthetized rodents. By conducting a series of technically challenging and compelling calcium imaging experiments in conscious head fixed mice they find in contrast that a much smaller proportion of meningeal afferents are persistently activated following CSD. Instead, they identify that post-CSD responses are differentially altered across a wide array of afferents, including increased and decreased responses to mechanical meningeal deformations and activation of previously non-responsive afferents following CSD. Given that migraine is characterized by worsening head pain in response to movement, the findings offer a potential mechanism that may explain this clinical phenomenon.

Strengths:<br /> Using head fixed conscious mice overcomes the limitations of anesthetized preps and the potential impact of anaesthesia on meningeal afferent function which facilitated novel results when compared to previous anesthetized studies. Further, the authors used a closed cranial window preparation to maximize normal physiological states during recording, although the introduction of a needle prick to induce CSD will have generated a small opening in the cranial preparation, rendering it not fully closed as suggested. However, technical issues with available AAV's and alternate less invasive triggering methodologies necessitate the current approach.

Weaknesses:<br /> Although this is a well conducted technically challenging study that has added valuable knowledge on the response of meningeal afferents the study would have benefited from the inclusion of more female mice. Migraine is a female dominant condition and an attempt to compare potential sex-differences in afferent responses would undoubtedly have improved the outcome. The authors report potential sex-specific effects on AAV transfection rates between males and females which have contributed to this imbalance.

The authors imply that the current method shows clear differences when compared to older anaesthetized studies; however, many of these were conducted in rats and relied on recording from the trigeminal ganglion. Attempts to address this point have proven difficult due to limited GCaMP signalling in anaesthetised mice, meaning that technical differences cannot be ruled out.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sex-biased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of non-synonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sex-biased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of non-synonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

Reviewer #1 (Public Review):

The apicoplast, a non-photosynthetic vestigial chloroplast, is a key metabolic organelle for the synthesis of certain lipids in apicomplexan parasites. Although it is clear metabolite exchange between the parasite cytosol and the apicoplast must occur, very few transporters associated with the apicoplast have been identified. The current study combines data from previous studies with new data from biotin proximity labeling to identify new apicoplast resident proteins including two putative monocarboxylate transporters termed MCT1 and MCT2. The authors conduct a thorough molecular phylogenetic analysis of the newly identified apicoplast proteins and they provide compelling evidence that MCT1 and MCT2 are necessary for normal growth and plaque formation in vitro along with maintenance of the apicoplast itself. They also provide indirect evidence for a possible need for these transporters in isoprenoid biosynthesis and fatty acid biosynthesis within the apicoplast. Finally, mouse infection experiments suggest that MCT1 and MCT2 are required for normal virulence, with MCT2 completely lacking at the administered dose. Overall, this study is generally of high quality, includes extensive quantitative data, and significantly advances the field by identifying several novel apicoplast proteins together with establishing a critical role for two putative transporters in the parasite. The study, however, could be further strengthened by addressing the following aspects:

Main comments:

1. The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of IPP is only supported by indirect measurements (effects on host GFP uptake or trafficking, possibly due to effects on IPP dependent proteins such as rabs, and mitochondrial membrane potential, possibly due to effects on IPP dependent ubiquinone). This conclusion would be more strongly supported by directly measuring levels of IPP. If their or technical limitations that prevent direct measurement of IPP then the author should note such limitations and acknowledge in the discussion that the conclusion is based on indirect evidence.

2. The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of fatty acids is also poorly supported by the data. The authors do not distinguish between the lower fatty acid levels being due to reduced synthesis of fatty acids, reduced salvage of host fatty acids, or both. Indeed, the authors provide evidence that parasite endocytosis of GFP is dependent on AMT1 and AMT2. Host GFP likely enters the parasite within a membrane bound vesicle derived from the PVM. The PVM is known to harbor host-derived lipids. Hence, it is possible that some of the decrease in fatty acid levels could be due to reduced lipid salvage from the host. Experiments should be conducted to measure the synthesis and salvage of fatty acids (e.g., by metabolic flux analysis), or the authors should acknowledge that both could be affected.

Reviewer #1 (Public Review):

The manuscript addresses a fundamental question about how different types of communication signals differentially affect brain state and neurochemistry. In addition, their manuscript highlights the various processes that modulate brain responses to communication signals, including prior experience, sex, and hormonal status. Overall, the manuscript is well-written and the research is appropriately contextualized.

That being said, it remains important for the authors to think more about their analytical approaches. In particular, the effect of normalization and the explicit outlining and interpretations of statistical models. As mentioned in the original review, the normalization of neurochemical data seems unnecessary given the repeated-measures design of their analysis and by normalizing all data to the baseline data and including this baseline data in the repeated measures analysis, one artificially creates a baseline period with minimal variation that dramatically differs in variance from other periods (akin to heteroscedasticity). If the authors want to analyze how a stimulus changes neurochemical concentrations, they could analyze the raw data but depict normalized data in their figures (similar to other papers). Or they could analyze group differences in the normalized data of the two stimulus periods (i.e., excluding the baseline period used for normalization).

It would also be useful for the authors to provide further discussion of the potential contributions of different types of experiences (mating vs. restraint) to the change in behavior and neurochemical responses to the vocalization playbacks and to try to disentangle sensory and motor contributions to neurochemical changes.

Reviewer #1 (Public Review):

Summary:<br /> This paper describes a comparison of different statistical methods for model comparison and covariate selection in neural encoding models. It shows in particular that issues arising from temporal autocorrelation and missing variables can lead to statistical tests with substantially higher false positive rates than expected from theory. The paper proposes methods for overcoming these problems, in particular cross-validation with cyclical shift permutation tests. The results are timely, important, and likely to have a broad impact. In particular, the paper shows that cell tuning classification can vary dramatically with the testing procedure, which is an important lesson for the field as a whole.

Strengths:<br /> - Novel and important comparison of different methods for variable selection in nested models.

Weaknesses:<br /> - Does not (yet) examine effect sizes<br /> - Does not motivate/explain key methods clearly enough in the main text.

General Comments:<br /> 1. My first general comment is that the paper in its current form focuses on the "null hypothesis significance testing" (NHST) paradigm. That is, it is focused on binary tests about what variables to include (or not include) in a regression model, and the false-positive rates of such tests. However, the broader statistics community has recently seen a shift away from NHST and towards a statistical reporting paradigm focused on effect sizes. See for example:<br /> - "Scientists rise up against statistical significance". Nature, March 2019.<br /> - Moving to a World Beyond "p < 0.05". RL Wasserstein, AL Schirm, NA Lazar. The American Statistician, 2019.

In light of this shift, I think the paper would be substantially strengthened if the authors could add a description of effect sizes for the statistical procedures they consider. Thus, for example, in cases where a procedure selects the wrong model (e.g., by selecting a variable that should not be included), how large is the inferred regression weight, and/or how large is the improvement in prediction performance (e.g. test log-likelihood) from including the erroneous regressor? How strong is the position tuning ascribed to a MEC cell that is inappropriately classified as having position tuning under one of the sub-optimal procedures? (Figure 7 shows some example place maps, but it would be nice to see a more thorough and rigorous analysis).

My suspicion would be that even when the hypothesis test gives a false positive, the effect sizes tend to remain small... but it is certainly possible that I'm mistaken, or that inferred effect sizes are more accurate for some procedures than others.

2. My only other major criticism relates to clarity and readability: in particular, the various procedures discussed in the paper ("forward selection", "maxT correction", "permutation test with cyclic shifts") are not clearly explained in the main paper, but are relegated to the Methods. Although I think it is useful to keep many of the mathematical details in the methods section, it would benefit the reader to have a general and intuitive explanation of the key methods within the flow of the main paper. The first paragraph of the Results section is particularly underdeveloped and hard to read and could benefit from a substantial revision to introduce and motivate the terms and procedures more clearly. I would recommend moving much of the text from the Methods into the Results section, or at the very least adding a paragraph describing the general idea/motivation for each method in Results.

Reviewer #1 (Public Review):

Summary:<br /> Zhu et al. set out to better understand the neural mechanisms underlying Drosophila larval escape behavior. The escape behavior is comprised of several sequenced movements, including a lateral roll motion followed by fast crawling. The authors specifically were looking to identify neurons important for the roll-to-crawl transition.

Strengths:<br /> This paper is clearly written. The experiments are logical and complementary. They support the author's main claim that SeIN128 is a type of descending neuron that is both necessary and sufficient to modulate the termination of rolling.

Weaknesses:<br /> -This manuscript is narrowly focused on Drosophila larval escape behavior. It would be more accessible to a broader audience if this work was put into a larger context of descending control.<br /> -In general, the rigor is high. However, a few control experiments are missing.

Reviewer #1 (Public Review):

This valuable study demonstrates a novel mechanism by which implicit motor adaptation saturates for large visual errors in a principled normative Bayesian manner. Additionally, the study revealed two notable empirical findings: visual uncertainty increases for larger visual errors in the periphery, and proprioceptive shifts/implicit motor adaptation are non-monotonic, rather than ramp-like. This study is highly relevant for researchers in sensory cue integration and motor learning. However, I find some areas where statistical quantification is incomplete, and the contextualization of previous studies to be puzzling.

Issue #1: Contextualization of past studies.

While I agree that previous studies have focused on how sensory errors drive motor adaptation (e.g., Burge et al., 2008; Wei and Kording, 2009), I don't think the PReMo model was contextualized properly. Indeed, while PReMo should have adopted clearer language - given that proprioception (sensory) and kinaesthesia (perception) have been used interchangeably, something we now make clear in our new study (Tsay, Chandy, et al. 2023) - PReMo's central contribution is that a perceptual error drives implicit adaptation (see Abstract): the mismatch between the felt (perceived) and desired hand position. The current paper overlooks this contribution. I encourage the authors to contextualize PReMo's contribution more clearly throughout. Not mentioned in the current study, for example, PReMo accounts for the continuous changes in perceived hand position in Figure 4 (Figure 7 in the PReMo study).

There is no doubt that the current study provides important additional constraints on what determines perceived hand position: Firstly, it offers a normative Bayesian perspective in determining perceived hand position. PReMo suggests that perceived hand position is determined by integrating motor predictions with proprioception, then adding a proprioceptive shift; PEA formulates this as the optimal integration of these three inputs. Secondly, PReMo assumed visual uncertainty to remain constant for different visual errors; PEA suggests that visual uncertainty ought to increase (but see Issue #2).

Issue #2: Failed replication of previous results on the effect of visual uncertainty.

2a. A key finding of this paper is that visual uncertainty linearly increases in the periphery; a constraint crucial for explaining the non-monotonicity in implicit adaptation. One notable methodological deviation from previous studies is the requirement to fixate on the target: Notably, in the current experiments, participants were asked to fixate on the target, a constraint not imposed in previous studies. In a free-viewing environment, visual uncertainty may not attenuate as fast, and hence, implicit adaptation does not attenuate as quickly as that revealed in the current design with larger visual errors. Seems like this current fixation design, while important, needs to be properly contextualized considering how it may not represent most implicit adaptation experiments.

2b. Moreover, the current results - visual uncertainty attenuates implicit adaptation in response to large, but not small, visual errors - deviates from several past studies that have shown that visual uncertainty attenuates implicit adaptation to small, but not large, visual errors (Tsay, Avraham, et al. 2021; Makino, Hayashi, and Nozaki, n.d.; Shyr and Joshi 2023). What do the authors attribute this empirical difference to? Would this free-viewing environment also result in the opposite pattern in the effect of visual uncertainty on implicit adaptation for small and large visual errors?

2c. In the current study, the measure of visual uncertainty might be inflated by brief presentation times of comparison and referent visual stimuli (only 150 ms; our previous study allowed for a 500 ms viewing time to make sure participants see the comparison stimuli). Relatedly, there are some individuals whose visual uncertainty is greater than 20 degrees standard deviation. This seems very large, and less likely in a free-viewing environment.

2d. One important confound between clear and uncertain (blurred) visual conditions is the number of cursors on the screen. The number of cursors may have an attenuating effect on implicit adaptation simply due to task-irrelevant attentional demands (Parvin et al. 2022), rather than that of visual uncertainty. Could the authors provide a figure showing these blurred stimuli (gaussian clouds) in the context of the experimental paradigm? Note that we addressed this confound in the past by comparing participants with and without low vision, where only one visual cursor is provided for both groups (Tsay, Tan, et al. 2023).

Issue #3: More methodological details are needed.

3a. It's unclear why, in Figure 4, PEA predicts an overshoot in terms of perceived hand position from the target. In PReMo, we specified a visual shift in the perceived target position, shifted towards the adapted hand position, which may result in overshooting of the perceived hand position with this target position. This visual shift phenomenon has been discovered in previous studies (e.g., (Simani, McGuire, and Sabes 2007)).

3b. The extent of implicit adaptation in Experiment 2, especially with smaller errors, is unclear. The implicit adaptation function seems to be still increasing, at least by visual inspection. Can the authors comment on this trend, and relatedly, show individual data points that help the reader appreciate the variability inherent to these data?

3c. The same participants were asked to return for multiple days/experiments. Given that the authors acknowledge potential session effects, with attenuation upon re-exposure to the same rotation (Avraham et al. 2021), how does re-exposure affect the current results? Could the authors provide clarity, perhaps a table, to show shared participants between experiments and provide evidence showing how session order may not be impacting results?

3d. The number of trials per experiment should be detailed more clearly in the Methods section (e.g., Exp 4). Moreover, could the authors please provide relevant code on how they implemented their computational models? This would aid in future implementation of these models in future work. I, for one, am enthusiastic to build on PEA.

3f. In addition to predicting a correlation between proprioceptive shift and implicit adaptation on a group level, both PReMo and PEA (but not causal inference) predict a correlation between individual differences in proprioceptive shift and proprioceptive uncertainty with the extent of implicit adaptation (Tsay, Kim, et al. 2021). Interestingly, shift and uncertainty are independent (see Figures 4F and 6C in Tsay et al, 2021). Does PEA also predict independence between shift and uncertainty? It seems like PEA does predict a correlation.

References:

Avraham, Guy, Ryan Morehead, Hyosub E. Kim, and Richard B. Ivry. 2021. "Reexposure to a Sensorimotor Perturbation Produces Opposite Effects on Explicit and Implicit Learning Processes." PLoS Biology 19 (3): e3001147.<br /> Makino, Yuto, Takuji Hayashi, and Daichi Nozaki. n.d. "Divisively Normalized Neuronal Processing of Uncertain Visual Feedback for Visuomotor Learning."<br /> Parvin, Darius E., Kristy V. Dang, Alissa R. Stover, Richard B. Ivry, and J. Ryan Morehead. 2022. "Implicit Adaptation Is Modulated by the Relevance of Feedback." BioRxiv. https://doi.org/10.1101/2022.01.19.476924.<br /> Shyr, Megan C., and Sanjay S. Joshi. 2023. "A Case Study of the Validity of Web-Based Visuomotor Rotation Experiments." Journal of Cognitive Neuroscience, October, 1-24.<br /> Simani, M. C., L. M. M. McGuire, and P. N. Sabes. 2007. "Visual-Shift Adaptation Is Composed of Separable Sensory and Task-Dependent Effects." Journal of Neurophysiology 98 (5): 2827-41.<br /> Tsay, Jonathan S., Guy Avraham, Hyosub E. Kim, Darius E. Parvin, Zixuan Wang, and Richard B. Ivry. 2021. "The Effect of Visual Uncertainty on Implicit Motor Adaptation." Journal of Neurophysiology 125 (1): 12-22.<br /> Tsay, Jonathan S., Anisha M. Chandy, Romeo Chua, R. Chris Miall, Jonathan Cole, Alessandro Farnè, Richard B. Ivry, and Fabrice R. Sarlegna. 2023. "Implicit Motor Adaptation and Perceived Hand Position without Proprioception: A Kinesthetic Error May Be Derived from Efferent Signals." BioRxiv. https://doi.org/10.1101/2023.01.19.524726.<br /> Tsay, Jonathan S., Hyosub E. Kim, Darius E. Parvin, Alissa R. Stover, and Richard B. Ivry. 2021. "Individual Differences in Proprioception Predict the Extent of Implicit Sensorimotor Adaptation." Journal of Neurophysiology, March. https://doi.org/10.1152/jn.00585.2020.<br /> Tsay, Jonathan S., Steven Tan, Marlena Chu, Richard B. Ivry, and Emily A. Cooper. 2023. "Low Vision Impairs Implicit Sensorimotor Adaptation in Response to Small Errors, but Not Large Errors." Journal of Cognitive Neuroscience, January, 1-13.

Reviewer #2 (Public Review):

Summary:<br /> This study seeks to understand the connection between protein sequence and function in disordered regions enriched in polar amino acids (specifically Q, N, S and T). While the authors suggest that specific motifs facilitate protein-enhancing activities, their findings are correlative, and the evidence is incomplete. Similarly, the authors propose that the re-assignment of stop codons to glutamine-encoding codons underlies the greater user of glutamine in a subset of ciliates, but again, the conclusions here are, at best, correlative. The authors perform extensive bioinformatic analysis, with detailed (albeit somewhat ad hoc) discussion on a number of proteins. Overall, the results presented here are interesting but are unable to exclude competing hypotheses.

Strengths:<br /> Following up on previous work, the authors wish to uncover a mechanism associated with poly-Q and SCD motifs explaining proposed protein expression-enhancing activities. They note that these motifs often occur IDRs and hypothesize that structural plasticity could be capitalized upon as a mechanism of diversification in evolution. To investigate this further, they employ bioinformatics to investigate the sequence features of proteomes of 27 eukaryotes. They deepen their sequence space exploration uncovering sub-phylum-specific features associated with species in which a stop-codon substitution has occurred. The authors propose this stop-codon substitution underlies an expansion of ploy-Q repeats and increased glutamine distribution.

Weaknesses:<br /> The authors were provided with a series of suggested changes to improve clarity, and a series of concerns raised. Some of these have been addressed but many have not. At this point, I do not see my role as telling the authors how to re-write their manuscript, but many of the concerns raised in my original review remain, and the authors have done little to allay those concerns in their revisions.

Reviewer #2 (Public Review):

The authors have greatly expanded their helpful hippocampome.org resource for the community regarding hippocampal cell types and their interactions from many perspectives. The many updates from v1.0 to v1.12 are nicely summarized in Table 1.

With v2.0, they now achieve the original vision of their project - to enable data-driven spiking neural network simulations of rodent hippocampal circuits. This work thus moves hippocampome.org from not only being a useful resource but also being able to launch simulations in which the models have direct links to the experimental literature. This will not only be of interest to the vast hippocampal community, but also to the diverse computational neuroscience community as theoretical models can potentially be "experimentally tested" with v2.0 to allow theoretical insights to be more biologically applicable.

Reviewer #1 (Public Review):

The authors investigate the function of the PTB domain containing adaptor protein Numb in skeletal muscle structure and function. In particular, the effects of reduced Numb expression in aging muscle is proposed as a mechanism for reduced contractile function associated with sarcopenia. Using ex-vivo analysis of conditional Numb and Numblike knockout muscle the authors demonstrate that loss of Numb but not the related Numblike gene expression perturbs muscle force generation. In order to explore the molecular mechanisms involved, Numb interacting proteins were identified in C2C12 cell cultured myotubes by immunoprecipitation and LC-MS/MS. The authors identify Septin 7 as well as Septin 2, 9 and 10 as a Numb binding proteins and demonstrate that loss of Numb/Numblike in myofibers causes changes in Septin 7 subcellular localization. Of note, whether additional septins form a complex or are also disrupted by Numb/Numblike loss remains an interesting area for further investigation. Additional investigation of the specificity and mapping of the Numb-Septin 7 (or another Septin) interaction would be of interest and provide an approach for future studies to demonstrate the biological relevance and specificity of the Numb-Septin 7 interaction in skeletal muscle

Reviewer #1 (Public Review):

In this manuscript, Davidsen and coworkers describe the development of a novel aspartate biosensor jAspSNFR3. This collaborative work supports and complements what was reported in a recent preprint by Hellweg et al., (bioRxiv.; doi: 10.1101/2023.05.04.537313). In both studies, the newly engineered aspartate sensor was developed from the same glutamate biosensor previously developed by the authors of this manuscript. This coincidence is not casual but is the result of the need to find tools capable of measuring aspartate levels in vivo. Therefore, it is undoubtedly a relevant and timely work carried out by groups experienced in aspartate metabolism and in the generation of metabolite biosensors.

Reviewer #1 (Public Review):

Summary:

In this work the authors provide evidence to show that an increase in Kv7 channels in hilar mossy cells of Fmr1 knock out mice results in a marked decrease in their excitability. The reduction in excitatory drive onto local hilar interneurons produces an increased excitation/inhibition ratio in granule cells. Inhibiting Kv7 channels can help normalize the excitatory drive in this circuit, suggesting that they may represent a viable target for targeted therapeutics for fragile-x syndrome.

Strengths:

The work is supported by a compelling and thorough set of electrophysiological studies. The authors do an excellent job of analysing their data and present a very complete data set.

Weaknesses:

There are no significant weaknesses in the experimental work, however the complexity of the data presentation and the lack of a schematic showing the organizational framework of this circuit make the data less accessible to non-experts in the field. I highly encourage a graphical abstract and network diagram to help individuals understand the implications of this work.

The work is important as it identifies a unique regional and cell specific abnormality in Fmr1 KO mice, showing how the loss of one gene can result in region specific changes in brain circuits.

Reviewer #1 (Public Review):

Summary:<br /> The authors measured the oxygen stable isotope ratios in six orangutan teeth using a state of the art micro-sampling technique (SHRIMP SI) to gather substantial multi-year isotopic data for six modern and five fossil orangutan individuals from Borneo and Sumatra. This fine-scale sampling technique allowed to address the fundamental question if breastfeeding affects the oxygen isotope ratios in teeth forming in the first one to two years of life, during which orangutans can be assumed to largely depend on breastmilk. The authors provide compelling evidence that the consumption of milk does not appear to affect the overall isotopic profile in early forming teeth. They conclude that this allows us to use these teeth as terrestrial/arboreal isotopic proxies in paleoenvironmental research, which would provide an invaluable addition to otherwise largely marine climate records in this regions.

Strengths:<br /> The overall large sample size of orangutan dental isotope records as well as the rigorous dating of the fossil specimens provide a strong dataset for addressing the outlined questions. The direct comparison of modern and fossil orangutan specimens provides a valuable evaluation of the use of these modern and past environmental proxies, with some discussion of the implications for the environmental conditions during the expansion of early modern humans into this region of the world.

Weakness:<br /> The authors illustrate that all orangutan individuals sampled, modern and fossil, show a considerable amount of isotopic variation between and within their teeth. Some of this variation is clearly associated with isotopic shifts in precipitation, but some will also be linked to the variation in oxygen isotopes within the forest itself and the many plant foods it produces for the orangutan. In the future, the systematic measurement of oxygen isotopes across orangutan food items, forest canopies and precipitation could help differentiate how much of the observed isotopic variation in teeth is indeed related to climatic shifts alone.

Reviewer #1 (Public Review):

The article offers a comparative study between various methodologies to evaluate the abundance, richness, and diversity of insects from data obtained in a large-scale field experiment. The experiment is impressive in view of the number of locations, its spatial coverage, the number of instruments or methods used, and the data collected appears rich and worthy of multiple publications. The paper focuses on the validation of a novel approach based on optical sensors. These sensors collect the backscattered light from flying insects in their field of view and can retrieve the wingbeat frequency and the body-to-wing backscattering ratios.<br /> Unfortunately, the paper is poorly written and hard to read, with a lack of clear sections, and an overall confusing structure. The methods, metrics, and data analysis are not properly and thoroughly described, making it sometimes difficult to evaluate the validity of the approach.<br /> Most importantly, the methodology to retrieve the richness and diversity from optical sensors seems flawed. While the scope and scale of the experiment is valuable, I do not believe that this article supports the authors' claim. The main criticisms are described in more detail below.

1) The Material and Method section is poorly structured. The article focuses on a series of metrics to evaluate biodiversity from three independent methods: optical sensors, malaise traps, and net sweeping. The authors need to provide a clear and thorough description of what the metrics to be studied are, and how those metrics are evaluated for each method. While it is the main focus of the paper, the term "biodiversity metrics" is never properly defined, it is used in the singular form in both the title and abstract, then in its plural form in the rest of the paper, making the reader further doubt what exactly it means. It is then discussed using the correlation value retrieved when studying richness, so is the biodiversity metric the same as richness? Studying biodiversity remains a complex and sometimes contentious subject and this term, especially when measured by three different methods, is far from obvious. The term "community metrics" is defined as abundance, richness, and diversity; is that the same as biodiversity metrics? In any case, the method section should thoroughly describe how each of those metrics is calculated from the raw data collected by each method. This information is somewhat there, but in a very unorganized way, making it difficult to read. I would recommend organizing this section with multiple and clear sections: 1) describing the metrics that are meant to be studied, 2) the location, dates and time, type of crops, and other general information about the experiment, 3) description and methods around optical sensors, 4) description and methods around malaise traps, 5) description and methods around the sweeping. The last 3 sections should describe how it retrieves the previously defined metrics, potentially using equations.

2) Regarding the calculation of the body-to-wing ratio, sigma is described as a "signal" line 195, then is described as intensity counts in Figure 2; isn't it really the backscattering optical cross-section? It changes significantly over time during the signal, so how is one value of sigma calculated? Is it the average of the whole insect event? The maximum?

3) The "ecosystem services" paragraph is really confusing and needs to be rewritten.

4) Like for the method section, the result section should be structured around the comparison of each metric, abundance, richness, and diversity, or any other properly defined metrics described in the method, so that the result section is consistent with the method section.

5) The abundance is not correlated; interestingly, malaise traps and sweeping are even less correlated which further supports the claims by the authors that new and improved methods are needed. This part of the results could be further developed. A linear fit could be added to Figure 4.

6) Richness and diversity are the most problematic. Again, the method is poorly described, with pieces of explanation spread out throughout the paper, but my understanding is the following: the optical sensor retrieves two features from each insect signal, wbf, and BWR. Clustering is made using DBSCAN which has 2 parameters: minimum number of signals, and merge distance. It is important to note that these two parameters will greatly influence the number of clusters found by DBSCAN. The richness obtained by optical sensors is defined as the number of clusters and the diversity is evaluated from it as well. Hence, both diversity and richness are greatly dependent on the chosen parameters. The DBSCAN parameters are chosen by maximizing the Spearman correlation between richness obtained by the optical sensors and richness by the capture methods. I see a major problem here: if you optimize the parameters, that directly impact the retrieved diversity and richness by optical sensors, to have the best correlation with either the richness or diversity of the other methods, you will automatically create a correlation between the richness and diversity retrieved by the optical sensors and alternative methods. The p-value in Figure 6 does not represent the probability of the correlation hypothesis being false anymore, since the whole process is based on artificially forcing the correlation from the start.

7) In addition, the clustering method provides values higher than 80, which is quite unrealistic with just 2 features, wbf and BWR. It is clear from many studies using optical sensors that the features from optical sensors are subject to variability. Wbf has naturally some variances within the same species, not to mention temperature dependency. Backscattering cross sections will also heavily function on the insect's orientation (facing or sideways) while crossing the cone of light, and, even though it is a ratio, the collection efficiency of the instrument telescope and scattering efficiency of the target will be impacted by the position of the insects within the cone of light, which will also impact the variability on the BWR. While those features can still be used, obtaining 80 clusters from two variables with such statistical fluctuations is simply not credible. Additional features could help, such as the two wavelengths mentioned in the description of the optical sensor but are never mentioned again.

The conclusion then states that the study serves as the first field validation. I disagree; the abundance doesn't correlate, and the richness and diversity evaluations are flawed. While I do think there is great value in the work done by the authors through this impressive field experiment, and in general in their work toward the development of entomological optical sensors, I believe the data analysis and communication of the results do not support the conclusions drawn.

Reviewer #1 (Public Review):

Summary:<br /> The evolution of transporter specificity is currently unclear. Did solute carrier systems evolve independently in response to a cellular need to transport a specific metabolite in combination with a specific ion or counter metabolite, or did they evolve specificity from an ancestral protein that could transport and counter-transport most metabolites? The present study addresses this question by applying selective pressure to Saccharomyces cerevisiae and studying the mutational landscape of two well-characterised amino acid transporters. The data suggest that AA transporters likely evolved from an ancestral transporter and then specific sub-families evolved specificity depending on specific evolutionary pressure.

Strengths:<br /> The work is based on sound logic and the experimental methodology is well thought through. The data appear accurate, and where ambiguity is observed (as in the case of citruline uptake by AGP1), in vitro transport assays are carried out to verify transport function.

Weaknesses:<br /> Although the data and findings are well described, the study lacked additional contextual information that would support a clear take-home message.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript presents a method to infer causality between two genes (and potentially proteins or other molecules) based on the non-genetic fluctuations among cells using a version of the dual-reporter assay as a causal control, where one half of the dual-reporter pair is causally decoupled, as it is inactive. The authors propose a statistical invariant identity to formalize this idea.

Strengths:<br /> The paper outlines a theoretical formalism, which, if experimentally used, can be useful in causal network inference, which is a great need in the study of biological systems.

Weaknesses:<br /> The practical utility of this method may not be straightforward and potentially be quite difficult to execute. Additionally, further investigations are needed to provide evidence of the broad applicability of the method to naturally occurring systems and its scalability beyond the simple circuit in which it is experimentally demonstrated.

Reviewer #1 (Public Review):

Summary:

This manuscript by Bomba-Warczak describes a comprehensive evaluation of long-lived proteins in the ovary using transgenerational radioactive labelled 15N pulse-chase in mice. The transgenerational labeling of proteins (and nucleic acids) with 15N allowed the authors to identify regions enriched in long-lived macromolecules at the 6 and 10-month chase time points. The authors also identify the retained proteins in the ovary and oocyte using MS. Key findings include the relative enrichment in long-lived macromolecules in oocytes, pregranulosa cells, CL, stroma, and surprisingly OSE. Gene ontology analysis of these proteins revealed enrichment for nucleosome, myosin complex, mitochondria, and other matrix-type protein functions. Interestingly, compared to other post-mitotic tissues where such analyses have been previously performed such as the brain and heart, they find a higher fractional abundance of labeled proteins related to the mitochondria and myosin respectively.

Strengths:

A major strength of the study is the combined spatial analyses of LLPs using histological sections with MS analysis to identify retained proteins.

Another major strength is the use of two chase time points allowing assessment of temporal changes in LLPs associated with aging.

The major claims such as an enrichment of LLPs in pregranulosa cells, GCs of primary follicles, CL, stroma, and OSE are soundly supported by the analyses, and the caveat that nucleic acids might differentially contribute to this signal is well presented.

The claims that nucleosomes, myosin complex, and mitochondrial proteins are enriched for LLPs are well supported by GO enrichment analysis and well described within the known body of evidence that these proteins are generally long-lived in other tissues.

Weaknesses:

One small potential weakness is the lack of a mechanistic explanation of if/why turnover may be accelerating at the 6-10 month interval compared to 1-6.

A mild weakness is the open-ended explanation of OSE label retention. This is a very interesting finding, and the claims in the paper are nuanced and perfectly reflect the current understanding of OSE repair. However, if the sections are available and one could look at the spatial distribution of OSE signal across the ovarian surface it would interesting to note if label retention varied by regions such as the CLs or hilum where more/less OSE division may be expected.

Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

Summary:

The current study reports a cryo-EM structure of MFS transporter MelB trapped in an inward-facing state by a conformationally selective nanobody. The authors compare this structure to previously-resolved crystal structures of outward-facing MelB. Additionally, the authors report H/D exchange/ mass spec experiments that identify accessible residues in the protein.

Strengths:

The authors overcame very significant technical challenges to solve the first inward-facing structure of the small, model MFS transporter MelB by cryo-EM. The use of conformation-trapping nanobodies (which had been reported previously by this group) is particularly nice.

Weaknesses:

The authors highlight the use of HDX experiments as a measurement of protein conformational dynamics. However, the experiment instead measures the accessibility of different residues. An ideal experiment would trap the transporter in inward- and outward states, but only the inward conformation is trapped here. The outward-facing conformation is instead an ensemble of outward and occluded conformations. It seems obvious that this will be more dynamic than the nanobody-trapped inward state.

Reviewer #1 (Public Review):

Summary: The current study reports a cryo-EM structure of MFS transporter MelB trapped in an inward-facing state by a conformationally selective nanobody. The authors compare this structure to previously-resolved crystal structures of outward-facing MelB. Additionally, the authors report H/D exchange/ mass spec experiments that identify accessible residues in the protein.

Strengths:

The authors overcame very significant technical challenges to solve the first inward-facing structure of the small, model MFS transporter MelB by cryo-EM. The use of conformation-trapping nanobodies (which had been reported previously by this group) is particularly nice.

Weaknesses:

Maps and coordinates were not provided by the authors, which presents a gap in this assessment.

The authors highlight the use of HDX experiments as a measurement of protein conformational dynamics. However, this experiment does not measure the conformational dynamics of the transporter, since in these experiments exchange is not initiated by ligand addition or another trigger. The experiment instead measures the accessibility of different residues, and of course, a freely-exchanging sodium bound transporter would have more exchangeable positions than when a conformation-trapping nanobody is bound. It is not clear what new mechanistic information this provides, since this property of the nanobody has already been established.

Based on the evidence presented, it is somewhat speculative that the structure represents the EIIa-bound regulatory state.

Reviewer #2 (Public Review):

Yanagihara and colleagues investigated the immune cell composition of bronchoalveolar lavage fluid (BALF) samples in a cohort of patients with malignancy undergoing chemotherapy and with with lung adverse reactions including Pneumocystis jirovecii pneumonia (PCP) and immune-checkpoint inhibitors (ICIs) or cytotoxic drug induced interstitial lung diseases (ILDs). Using mass cytometry, their aim was to characterize the cellular and molecular changes in BAL to improve our understanding of their pathogenesis and identify potential biomarkers and therapeutic targets. In this regard, the authors identify a correlation between CD16 expression in T cells and the severity of PCP and an increased infiltration of CD57+ CD8+ T cells expressing immune checkpoints and FCLR5+ B cells in ICI-ILD patients.

The conclusions of this paper are mostly well supported by data, but some aspects of the data analysis need to be clarified and extended.

1) The authors should elaborate on why different set of markers were selected for each analysis step. E.g., Different set of markers were used for UMAP, CITRUS and viSNE in the T cell and myeloid analysis.

2) The authors should state if a normality test for the distribution of the data was performed. If not, non-parametric tests should be used.

3) The authors should explore the correlation between CD16 intensity and the CTCAE grade in T cell subsets such as EMRA CD8 T cells, effector memory CD4, etc as identified in Figure 1B.

4) The authors could use CITRUS to better assess the B cell compartment.

Reviewer #1 (Public Review):

The authors Wang et al. present a study of a mouse model K74R that they claim can extend the life span of mice, and also has some anti-cancer properties in some standrad models of melanoma and hepatocellular carcinoma. Importantly, this mechanism seems to be mediated by the hematopoietic system, and protective effects can be transferred with bone marrow transplantation.

The authors have now adapted their manuscript reflecting the novelties of these studies. Overall, the study is a continuation and also corroboration of previous work, without clinical data yet. The authors have now expanded their work to a second mouse model, which strengthens their data.

Reviewer #1 (Public Review):

Summary:

The authors attempt to fully characterise the immunoglobulin (Ig) heavy (H) chain repertoire of tumor-infiltrating B cells from three different cancer types by identifying the IgH repertoire overlap between these, their corresponding draining lymph nodes (DLNs), and peripheral B cells. The authors claim that B cells from tumors and DLNs have a closer IgH profile than those in peripheral blood and that DLNs are differentially involved with tumor B cells. The claim that tumor-resident B cells are more immature and less specific is made based on the characteristics of the CDR-H3 they express.

Strengths:

The authors show great expertise in developing in-house bioinformatics pipelines, as well as using tools developed by others, to explore the IgH repertoire expressed by B cells as a means of better characterising tumour-associated B cells for the future generation of tumour-reactive antibodies as a therapy.

Weaknesses:

This paper needs major editing, both of the text and the figures, because as it stands it is convoluted and extremely difficult to follow. The conclusions reached are often not obvious from the figures themselves. Sufficient a priori details describing the framework for their analyses are not provided, making the outcome of their results questionable and leaving the reader wondering whether the findings are on solid ground. The authors are encouraged to explain in more detail the premises used in their algorithms, as well as the criteria they follow to define clonotypes, clonal groups, and clonal lineages, which are currently poorly defined and are crucial elements that may influence their results and conclusions. Having excluded the IGHD gene segment from some of their analyses (at least those related to clonal lineage inference and phylogenetic trees), it is not well explained which region of CDR-H3 is responsible for the charge, interaction strength, and Kidera factors, since in some cases the authors mention that the central part of CDR-H3 consists of five amino acids and in others of seven amino acids. How can the authors justify that the threshold for CDR-H3 identity varies according to individual patient data?

Throughout the analyses, the reasons for choosing one type of cancer over another sometimes seem subjective and are not well justified in the text.

Overall, the narrative is fragmented. There is a lack of well-defined conclusions at the end of the results subheadings. The exact same paragraph is repeated twice in the results section. The authors have also failed to synchronise the actual number of main figures with the text, and some panels are included in the main figures that are neither described nor mentioned in the text (Venn diagram Fig. 2A and phylogenetic tree Fig. 5D). Overall, the manuscript appears to have been rushed and not thoroughly read before submission.

Reviewers are forced to wade through, unravel, and validate poorly explained algorithms in order to understand the authors' often bold conclusions.

Reviewer #1 (Public Review):

Summary:<br /> This work explored intra and interspecific niche partitioning along spatial, temporal, and dietary niche partitioning between apex carnivores and mesocarnivores in the Qilian Mountain National Park of China, using camera trapping data and DNA metabarcoding sequencing data. They conclude that spatial niche partitioning plays a key role in facilitating the coexistence of apex carnivore species, spatial and temporal niche partitioning facilitate the coexistence of mesocarnivore species, and spatial and dietary niche partitioning facilitate the coexistence between apex and mesocarnivore species. The information presented in this study is important for wildlife conservation and will contribute substantially to the current understanding of carnivore guilds and effective conservation management in fragile alpine ecosystems.

Strengths:<br /> Extensive fieldwork is evident in the study. Aiming to cover a large percentage of the Qilian Mountain National Park, the study area was subdivided into squares, as a geographical reference to distribute the sampling points where the camera traps were placed and the excreta samples were collected.

They were able to obtain many records in their camera traps and collected many samples of excreta. This diversity of data allowed them to conduct robust analyses. The data analyses carried out were adequate to obtain clear and meaningful results that enabled them to answer the research questions posed. The conclusions of this paper are mostly well supported by data.

The study has demonstrated the coexistence of carnivore species in the landscapes of the Qilian Mountains National Park, complementing the findings of previous studies. The information presented in this study is important for wildlife conservation and will contribute substantially to the current understanding of carnivore guilds and effective conservation management in fragile alpine ecosystems.

Weaknesses:<br /> It is necessary to better explain the methodology because it is not clear what is the total sampling effort. In methodology, they only claim to have used 280 camera traps, and in the results, they mention that there are 319 sampling sites. However, the total sampling effort (e.g. total time of active camera traps) carried out in the study and at each site is not specified.

Reviewer #1 (Public Review):

Summary: This papers performs fine-mapping of the silkworm mutants bd and its fertile allelic version, bdf, narrowing down the causal intervals to a small interval of a handful of genes. In this region, the gene orthologous to mamo is impaired by a large indel, and its function is later confirmed using expression profiling, RNAi, and CRISPR KO. All these experiments are convincingly showing that mamo is necessary for the suppression of melanic pigmentation in the silkworm larval integument.

The authors also use in silico and in vitro assays to probe the potential effector genes that mamo may regulate.

Strengths: The genotype-to-phenotype workflow, combining forward (mapping) and reverse genetics (RNAi and CRISPR loss-of-function assays) linking mamo to pigmentation are extremely convincing.

This revision is a much improved manuscript and I command the authors for many of their edits.

I find the last part of the discussion, starting at "It is generally believed that changes in gene expression patterns are the result of the evolution of CREs", to be confusing.<br /> In this section, I believe the authors sequentially:<br /> - emphasize the role of CRE in morphological evolution (I agree)<br /> - emphasize that TF, and in particular their own CRE, are themselves important mutational targets of evolution (I agree, but the phrasing need to insist the authors are here talking about the CRE found at the TF locus, not the CRE bound by the TF).<br /> - use the stickleback Pel enhancer as an example, which I think is a good case study, but the authors also then make an argument about DNA fragility sites, which is hard to connect with the present study.<br /> - then continue on "DNA fragility" using the peppered moth and butterfly cortex locus. There is no evidence of DNA fragility at these loci, so the connection does not work. "The cortex gene locus is frequently mutated in Lepidoptera", the authors say. But a more accurate picture would be that the cortex locus is repeatedly involved in the generation of color pattern variants. Unlike for Pel fragile enhancer, we don't know if the causal mutations at this locus are repeatedly the same, and the haplotypes that have been described could be collateral rather than causal. Overall, it is important to clarify the idea that mutation bias is a possible factor explaining "genetic hotspots of evolution" (or genetic parallelism sensu 10.1038/nrg3483), but it is also possible that many genetic hotspots are repeated mutational targets because of their "optimal pleiotropy" (e.g. hub position in GRNs, such as mamo might be), or because of particularly modular CRE region that allow fine-tuning. Thus, I find the "fragility" argument misleading here. In fact the finding that "bd" and "bdf" alleles are different in nature is against the idea of a fragility bias (unless the authors can show increased mutation rates at this locus in a wild silkmoth species?). These alleles are also artificially-selected ie. they increased in frequency by breeding rather than natural selection in the wild, so while interesting for our understand of the genotype-phenotype map, they are not necessarily representative of the mutations that may underlie evolution in the wild.<br /> - Curiously, the last paragraph ("Some research suggests that common fragile sites...") elaborate on the idea that some sites of the genome are prone to mutation. The connection with mamo and the current article are extremely thin. There is here an attempt to connect meiotic and mitotic breaks to Bm-mamo, but this is confusing : it seems to propose Bm-mamo as a recruiter of epigenetic modulators that may drive higher mutation rates elsewhere. Not only I am not convinced by this argument without actual data, but this would not explain how the mutations at the Bm-mamo itself evolved.

On a more positive note, I find it fascinating that the authors identified a TF that clearly articulates or orchestrate larval pattern development, and that when it is deleted, can generate healthy individuals. In other words, while it is a TF with many targets, it is not too pleiotropic. This idea, that the genetically causal modulators of developmental evolution are regulatory genes, has been described elsewhere (e.g. Fig 4c in 10.1038/s41576-020-0234-z, and associated refs). To me, the beautiful findings about Bm-mamo make sense in the general, existing framework that developmental processes and regulatory networks "shape" the evolutionary potential and trajectories of organisms. There is a degree of "programmability" in the genomes, because some loci are particularly prone to modulate a given type of trait. Here, Bm-mamo, as a potentially regulator of both CPs and melanin pathway genes, appear to be a potent modulator of epithelial traits. Claiming that there are inherent mutational biases behind this is unwarranted.

Reviewer #1 (Public Review):

In 2019, Wilkinson and colleagues (PMID: 31142833) managed to break the veil on a 20-year open question on how to properly culture and expand Hematopoietic Stem Cells (HSCs). Although this study is revolutionizing the HSC biology field, several questions regarding the mechanisms of expansion remain open. Leveraging on this gap, Zhang et al.; embarked on a much-needed investigation regarding HSC self-renewal in this particular culturing setting.

The authors firstly tacked the known caveat that some HSC membrane markers are altered during in vitro cultures by functionally establishing EPCR (CD201) as a reliable and stable HSC marker (Figure 1), demonstrating that this compartment is also responsible for long-term hematopoietic reconstitution (Figure 3). Next in Figure 2, the authors performed single-cell omics to shed light into the potential mechanisms involved into HSC maintenance, and interestingly it was shown that several hematopoietic populations like monocytes and neutrophils are also present in this culture conditions, which has not been reported. The study goes on to functionally characterize these cultured HSCs (cHSC). The authors elegantly demonstrate using state-of-the-art barcoding strategies that these culturing conditions provoke heterogeneity in the expanding HSC pool (Figure 4). In the last experiment (Figure 5), it was demonstrated that cHSC not only retain their high EPCR expression levels but upon transplantation these cells remain more quiescent than freshly-isolated controls.

Taken together, this study independently validates that the proposed culturing system works and provide new insights into the mechanisms whereby HSC expansion takes place.

Following a first round of comments, the authors provided a comprehensive point-by-point response to the different points raised by reviewers, which significantly helps on better understanding some of the decisions taken by the authors. However, it is surprising that the current manuscript is practically unchanged compared to the previous version. Effectively, all major comments raised by reviewers are address in the response letter rather than incorporated into a truly updated version, which would be of great benefit for readers.

Further comments:<br /> 1. It is highly appreciated that the authors provide a comprehensive and cohesive explanations on i) the rationale for employing SAILERX for single-cell RNA and ATAC-seq, ii) data on HSC signature projected on independent scRNA-seq datasets and iii) further context on the Fgd5 expression limitations. These are important snippets of information which do not only further validate this manuscript's data but also provide context within the HSC biology field.<br /> However, I do not fully agree with the author statement "our primary objective in this study was to highlight the relatively low content of HSCs in cultures" (page 1, response to Reviewers) justifying why single-cell genome-wise approaches were used. As the authors are aware HSCs are defined by functional characterization rather than transcriptional/chromatin accessibility profiles, so it seems odd that this was the rationale to perform omics for this purpose. More importantly, the authors had gone through the lengths of already performing this costly and time-consuming experiment, but miss out on the opportunity to take a deeper dive into the molecular characteristics that could explain divergent behavior between freshly-isolated and cultured HSCs. It would be extremely relevant to the HSC biology community to understand, for example, if these two HSC populations have differences in enhancer accessibility (if the data quality allows), which could provide an upstream explanation for differences in transcription (is also not explored in this manuscript version).

2. It intriguing that the authors acknowledge that there are already more recent versions of this expansion protocol (page 2, response to Reviewers) and provided a convoluted explanation on why these were not included in the original manuscript. Both papers (PMID: 36809781 and PMID: 37385251) have now been published in respected peer-reviewed journals and provide insights which are pertinent for this work. Yet, the authors decided not to discuss these findings. It is understandable that repeating experiments with these updated conditions is outside of the scope of this manuscript, but it would be relevant to discuss how these recent advances in the protocol impact the work presented in this manuscript.

3. Regarding the previous comment on how cultured HSC are related to HSC aging, I highly appreciate both data on serial transplantation and also on scRNA-seq.

Reviewer #1 (Public Review):

Summary:<br /> TRAP transporters are an unusual class of secondary active transporters that utilize periplasmic binding proteins to deliver their substrates. This paper contributes a new 3 Å structure of the Haemophilus influenzae TRAP transporter. The structure joins two other recent cryo-EM structures of TRAP transporters, including a lower resolution structure of the same H. influenzae protein (overall 4.7 Å), and a ~3 Å structure of a homologue from P. profundum. In addition to reporting a higher resolution cryo-EM structure, the authors also recapitulate protein activity in a reconstituted system, investigate protein oligomerization using analytic ultracentrifugation, and evaluate interactions and function in "mix and match" configurations with periplasmic subunits from other homologues.

Strengths:<br /> The strength of the paper is that the better resolution cryo-EM data permits sidechain assignment, the identification of bound lipids, and the identification of sodium ions. It is important to get this structure out there, since the resolution passes an important threshold for model building accuracy. The current structure nicely explains a lot of prior mutagenesis data on the H. influenzae TRAP. This is also the first structure of a TRAP protein to be solved without a fiducial, although the overall structure is not very different than those solved with fiducials.

Weaknesses:<br /> The experiments examining the monomer/dimer equilibrium appear somewhat preliminary. The biological or mechanistic importance of oligomerization is not established, so these experiments are inherently of limited scope. Moreover, cryo-EM datasets exhibit both parallel and antiparallel dimers, the latter of which are clearly not biologically relevant. It is probably impossible to distinguish these in the AUC experiments, which makes interpretation of these experiments more difficult.

Similarly, the importance of the lipid binding sites observed in cryo-EM aren't experimentally established (for example by mutating the binding site) and it is thus unknown whether they are important for function (as the authors acknowledge).

Reviewer #1 (Public Review):

Summary:

The manuscript by Xia et al. investigated the mechanisms underlying Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). The authors observed that abnormal osteogenesis and adipogenesis is associated with decreased β-catenin in the necrotic femoral head of GONFH patients and inhibition of β-catenin signaling leads to abnormal osteogenesis and adipogenesis in GONFH rats. Of interest, deletion of β-catenin in Col2-expressing cells rather than in osx-expressing cells leads to a GONFH-like phenotype in femoral head of mice.

Strengths:

A strength of the study is that it sets up a Col2-expressing cell-specific β-catenin knockout mouse model that mimics full spectrum of osteonecrosis phenotype of GONFH. This is interesting and provides new insights into the understanding of GONFH. Overall, the data are solid and support their conclusions.

Reviewer #1 (Public Review):

Summary:<br /> This paper investigates the neural mechanisms underlying the change in perception when viewing ambiguous figures. Each possible percept is related to an attractor-like brain state and a perceptual switch corresponds to a transition between these states. The hypothesis is that these switches are promoted by bursts of noradrenaline that change the gain of neural circuits. The authors present several lines of evidence consistent with this view: pupil diameter changes during the time point of the perceptual change; a gain change in neural network models promotes a state transition; and large-scale fMRI dynamics in a different experiment suggests a lower barrier between brain states at the change point. However, some assumptions of the computational model seem not well justified and the theoretical analysis is incomplete. The paper would also benefit from a more in-depth analysis of the experimental data.

Strengths:<br /> The main strength of the paper is that it attempts to combine experimental measurements - from psychophysics, pupil measurements, and fMRI dynamics - and computational modeling to provide an emerging picture of how a perceptual switch emerges. This integrative approach is highly useful because the model has the potential to make the underlying mechanisms explicit and to make concrete predictions.

Weaknesses:<br /> A general weakness is that the link between the three parts of the paper is not very strong. Pupil and fMRI measurements come from different experiments and additional analysis showing that the two experiments are comparable should be included. Crucially, the assumptions underlying the RNN modeling are unclear and the conclusions drawn from the simulation may depend on those assumptions.

Main points:<br /> Perceptual tasks in pupil and fMRI experiments: how comparable are these two tasks? It seems that the timing is very different, with long stimulus presentations and breaks in the fMRI task and a rapid sequence in the pupil task. Detailed information about the task timing in the pupil task is missing. What evidence is there that the same mechanisms underlie perceptual switches at these different timescales? Quantification of the distributions of switching times/switching points in both tasks is missing. Do the subjects in the fMRI task show the same overall behavior as in the pupil task? More information is needed to clarify these points.

Computational model:<br /> 1. Modeling noradrenalin effects in the RNN: The pupil data suggests phasic bursts of NA would promote perceptual switches. But as I understand, in the RNN neuromodulation is modeled as different levels of gain throughout the trial. Making the neural gain time-dependent would allow investigation of whether a phasic gain change can explain the experimentally observed distribution of switching times.

2. Modeling perceptual switches: in the results, it is described that the networks were trained to output a categorical response, but the firing rates in Fig 2B do not seem categorical but rather seem to follow the input stimulus. The output signals of the network are not shown. If I understand correctly, a trivial network that would just represent the two input signals without any internal computation and relay them to the output would do the task correctly (because "the network's choice at each time point was the maximum of the two-dimensional output", p. 22). This seems like cheating: the very operation that the model should perform is to signal the change, in a categorical manner, not to represent the gradually changing input signals.

3. The mechanism of how increased gain leads to faster switches remains unclear to me. My first intuition was that increasing the gain of excitatory populations (the situation shown in Fig. 2E) in discrete attractor models would lead to deeper attractor wells and this would make it more difficult to switch. That is, a higher gain should lead to slower decisions in this case. However, here the switching time remains constant for a gain between 1 and 1.5. Lowering the gain, on the other hand, leads to slower switching. It is, of course, possible that the RNN behaves differently than classical point attractor models or that my intuition is incorrect (though I believe it is consistent with previous literature, e.g. Niyogi & Wong-Lin 2013 (doi:10.1371/journal.pcbi.1003099) who show higher firing rates - more stable attractors - for increased excitatory gain).

4. From the RNN model it is not clear how changes in excitatory and inhibitory gain lead to slower/faster switching. In order to better understand the role of inhibitory and excitatory gain on switching, I would suggest studying a simple discrete attractor model (a rate model, for example as in Wong and Wang 2006 or Roxin and Ledberg, Plos Comp. Bio 2008) which will allow to study these effects in terms of a very few model parameters. The Roxin paper also shows how to map rate models onto simplified one-dimensional systems such as the one in Fig S3. Setting up the model using this framework would allow for making much stronger, principled statements about how gain changes affect the energy landscape, and under which conditions increased inhibitory gain leads to faster switching.

One possibility is that increasing the excitatory gain in the RNN leads to saturated firing rates. If this is the reason for the different effects of excitatory and inhibitory gain changes, it should be properly explained. Moreover, the biological relevance of this effect should be discussed (assuming that saturation is indeed the explanation).

Alternative mechanisms:<br /> It is mentioned in the introduction that changes in attention could drive perceptual switches. A priori, attention signals originating in the frontal cortex may be plausible mechanisms for perceptual switches, as an alternative to LC-controlled gain modulation. Does the observed fMRI dynamics allow us to distinguish these two hypotheses? In any case, I would suggest including alternative scenarios that may be compatible with the observed findings in the discussion.

Reviewer #1 (Public Review):

In this manuscript, the authors explore the effects of DNA methylation on the strength of regulatory activity using massively parallel reporter assays in cell lines on a genome-wide level. This is a follow-up of their first paper from 2018 that describes this method for the first time. In addition to adding more in depth information on sequences that are explored by many researchers using two main methods, reduced bisulfite sequencing and sites represented on the Illumina EPIC array, they now show also that DNA methylation can influence changes in regulatory activity following a specific stimulation, even in absence of baseline effects of DNA methylation on activity. In this manuscript, the authors explore the effects of DNA methylation on the response to Interferon alpha (INFA) and a glucocorticoid receptor agonist (dexamethasone). The author validate their baseline findings using additional datasets, including RNAseq data and show convergences across two cell lines. The authors then map the methylation x environmental challenge (IFNA and dex) sequences identified in vitro to explore whether their methylation status is also predictive of regulatory activity in vivo. This is very convincingly shown for INFA response sequences, where baseline methylation is predictive of the transcriptional response to flu infection in human macrophages, an infection that triggers the INF pathways. The extension of the functional validity of the dex-response altering sequences is less convincing. Sequences altering the response to glucocorticoids, however, were not enriched in DNA methylation sites associated with exposure to early adversity which the authors interpret that "they are not links on the causal pathway between early life disadvantage and later life health outcomes, but rather passive biomarkers. However, this approach does not seem an optimal model to explore this relationship in vivo. This is because exposure to early adversity and its consequences is not directly correlated with glucocorticoid release and changes in DNA methylation levels following early adversity could be related to many physiological mechanisms, and overall, large datasets and meta-analyses do not show robust associations of exposure to early adversity and DNA methylation changes. Here other datasets, such as from Cushing patients maybe of more interest.<br /> ***<br /> After revision, the authors have now discussed this issue carefully, so that this point is addressed.<br /> ***<br /> Overall, the authors provide a great resource of DNA methylation sensitive enhancers that can now be used for functional interpretation of large scale datasets (that are widely generated in the research community), given the focus on sites included in RBSS and the Illumina EPIC array. In addition, their data lends support that difference in DNA methylation can alter responses to environmental stimuli and thus of the possibility that environmental exposures that alter DNS methylation can also alter subsequent response to this exposure, in line with the theory of epigenetic embedding of prior stimuli/experiences. The conclusions related to the early adversity data should be reconsidered in light of the comments above.

Reviewer #1 (Public Review):

Summary:<br /> Alonso-Calleja and colleagues explore the role of TGR5 in adult hematopoiesis at both steady state and post-transplantation. The authors utilize two different mouse models including a TGR5-GFP reporter mouse to analyze the expression of TGR5 in various hematopoietic cell subsets. Using germline Tgr5-/- mice it's reported that loss of Tgr5 has no significant impact on steady-state hematopoiesis, with a small decrease in trabecular bone fraction, associated with a reduction in proximal tibia adipose tissue, and an increase in marrow phenotypic adipocytic precursors. The authors further explored the role of stroma TGR5 expression in the hematopoietic recovery upon bone marrow transplantation of wild-type cells, although the studies supporting this claim are weak. Overall, while most of the hematopoietic phenotypes have negative results or small effects, the role of TGR5 in adipose tissue regulation is interesting to the field.

Strengths:<br /> • This is the first time the role of TGR5 has been examined in the bone marrow.<br /> • This paper supports further exploration of the role of bile acids in bone marrow transplantation and possible therapeutic strategies.

Weaknesses:<br /> • The authors fail to describe whether niche stroma cells or adipocyte progenitor cells (APCs) express TGR5.<br /> • Although the authors note a significant reduction in bone marrow adipose tissue in Tgr5-/- mice, they do not address whether this is white or brown adipose tissue especially since BA-TGR5 signaling has been shown to play a role in beiging.<br /> • In Figure 1, the authors explore different progenitor subsets but stop short of describing whether TGR5 is expressed in hematopoietic stem cells (HSCs).<br /> • Are there more CD45+ cells in the BM because hematopoietic cells are proliferating more due to a direct effect of the loss of Tgr5 or is it because there is just more space due to less trabecular bone?<br /> • In Figure 4 no absolute cell counts are provided to support the increase in immunophenotypic APCs (CD45-Ter119-CD31-Sca1+CD24-) in the stroma of Tgr5-/- mice. Accordingly, the absolute number of total stromal cells and other stroma niche cells such as MSCs, ECs are missing.<br /> • There are issues with the reciprocal transplantation design in Fig 4. Why did the authors choose such a low dose (250 000) of BM cells to transplant? If the effect is true and relevant, the early recovery would be observed independently of the setup and a more robust engraftment dataset would be observed without having lethality post-transplant. On the same note, it's surprising that the authors report ~70% lethality post-transplant from wild-type control mice (Fig 4E), according to the literature 200 000 BM cells should ensure the survival of the recipient post-TBI. Overall, the results even in such a stringent setup still show minimal differences and the study lacks further in-depth analyses to support the main claim.<br /> • Mechanistically, how does the loss of Tgr5 impact hematopoietic regeneration following sublethal irradiation?<br /> • Only male mice were used throughout this study. It would be beneficial to know whether female mice show similar results.

Reviewer #1 (Public Review):

Here, Muronova et al., demonstrate the physiological importance of a centriole and microtubule-associated protein, CCDC146, in sperm flagellar formation and male reproduction. This study identifies novel causal variants to cause male infertility and resolves the pathogenicity by the mutation with characterizing mouse models. Furthermore, the authors' claims are well supported by the biochemical and imaging approaches used in this study.

Reviewer #1 (Public Review):

Summary:<br /> The study by He et al. investigates the relationship of an increased susceptibility of diabetes patients to COVID-19. The paper raises the possibility that hyperglycemia-induced cathepsin L maturation could be one of the driving forces in this pathology, suggesting that an increased activity of CTSL leads to accelerated virus infection rates due to an elevated processing of the SARS-CoV-2 spike protein.

In a clinical case-control study, the team found that the severity of corona infections was higher in diabetic patients, and their CTSL levels correlated well with the progression of the disease. They further showed an increase in CTSL activity in the long term as well as acute hyperglycemia. SARS-CoV-2 increasingly infected cells that were cultured in serum from diabetic patients, the same was observed using high glucose medium. No effect was observed in the medium with increased concentrations of insulin. CTSL knockout abolished the glucose-dependent increase in infection.

Increased glucose levels did not correlate with an increase in CTSL transcription. Rather He et al. could show that high glucose levels led to CTSL translocation from the ER into the lysosome. It was the glucose-dependent processing of the protease to its active form which promoted infection.

Strengths:<br /> It is a complete study starting from a clinical observation and ending on the molecular mechanism. A strength is certainly the wide selection of experiments. The clinical study to investigate the effect of glucose on CTSL concentrations in healthy individuals sets the stage for experiments in cell culture, animal models, and human tissue. The effect of CTSL knockout cell lines on glucose-induced SARS-CoV2 infection rates is convincing. Finally, the team used a combination of Western blots and confocal microscopy to identify the underlying molecular mechanisms. The authors manage to keep the diabetic condition at the center of their study and therefore extend on previous knowledge of glucose-induced CTSL activation and their consequences for COVID-19 infections. By doing so, they create a novel connection between CTSL involvement in SARS-CoV2 infections and diabetes.

Weaknesses:<br /> The authors suggest that hyperglycemia as a symptom of diabetes leads to an increased infection rate in those patients. Throughout their study, the team focuses on two select symptoms of a diabetic condition, hyperglycemia and hyperinsulinemia. The team acknowledges in the discussion that there could be various other reasons. Hyperglycemia can lead to metabolic acidosis and a shift in blood pH. As CTSL activity is highly dependent on pH, it would have been crucial to include this parameter in the study.

The study rarely differentiates between cellular and extracellular CTSL activity. A more detailed explanation for the connection between the intracellular CTSL and serum CTSL in diabetic individuals, presumably via lysosomal exocytosis, could be helpful with regard to the final model to give a more complete picture.

In the early result section, an effect of hyperglycemia on total CTSL concentrations is described, but the data is not very convincing. Over the course of the manuscript, the hypothesis shifts increasingly towards an increase in protease trans-localization and processing to the active form rather than a change in total protease amounts. The overall importance of CTSL concentrations remains questionable.

Reviewer #1 (Public Review):

Summary:<br /> This important study provides a comprehensive evaluation of skeletal muscle mitochondrial function and remodeling in a genetically engineered mouse model of pancreatic cancer cachexia. The study builds upon and extends previous findings that implicate mitochondrial defects in the pathophysiology of cancer cachexia. The authors demonstrate that while the total quantity of mitochondria from skeletal muscles of mice with pancreatic cancer cachexia is similar to controls, mitochondria were elongated with disorganized cristae, and had reduced oxidative capacity. The mitochondrial dysfunction was not associated with exercise-induced metabolic stress (insufficient ATP production), suggesting compensation by glycolysis or other metabolic pathways. However, mitochondrial dysfunction can lead to increased production of ROS/oxidative stress and would be expected to interfere with carbohydrate and lipid metabolism, events that are linked to cancer-induced muscle loss. The data are convincing and were collected and analyzed using state-of-the-art techniques, with unbiased proteomics and transcriptomics analyses supporting most of their conclusions.

Additional Strengths:<br /> The authors utilize a genetically engineered mouse model of pancreatic cancer which recapitulates key aspects of human PDAC including the development of cachexia, making the model highly appropriate and translational.

The authors perform transcriptomic and proteomics analyses on the same tissue, providing a comprehensive analysis of the transcriptional networks and protein networks changed in the context of PDAC cachexia.

Weaknesses:<br /> The authors refer to skeletal muscle wasting induced by PDAC as sarcopenia. However, the term sarcopenia is typically reserved for the loss of skeletal muscle mass associated with aging.

In Figure 2, the MuRF1 IHC staining appears localized to the extracellular space surrounding blood vessels and myofibers-which causes concern as to the specificity of the antibody staining. MuRF1, as a muscle-specific E3 ubiquitin ligase that degrades myofibrillar proteins, would be expected to be expressed in the cytosol of muscle fibers.

Disruptions to skeletal muscle metabolism in PDAC mice are predicted based on mitochondrial dysfunction and the transcriptomic and proteomics data. The manuscript could therefore be strengthened by additional measures looking at skeletal muscle metabolites, or linking the findings to previous work that has looked at the skeletal muscle metabolome in related models of PDAC cachexia (Neyroud et al., 2023).

Reviewer #1 (Public Review):

The study is highly interesting and the applied methods are target-oriented. The biophysical characterization of viable N-protein species and several representative N-protein mutants is supported by the data, including polarity, hydrophobicity, thermodynamic stability, CD spectra, particle size, and especially protein self-association. The physicochemical parameters for viable N-protein and related coronavirus are described for comparison in detail. However, the conclusion becomes less convincing that the interaction of peptides or motifs was judged by different biophysical results, with no more direct data about peptide interaction. Additionally, the manuscript could benefit from more results involving peptide interaction to support the author's opinions or make expression more accurate when concerning the interaction of motifs. Although the authors put a lot of effort into the study, there are still some questions to answer.

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.

However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts.

Joint Public Review

The present study focuses on the structure and function of human PURA, a regulator of gene transcription and mRNA transport and translation whose mutation causes the neurodevelopmental PURA syndrome, characterized by developmental delay, intellectual disability, hypotonia, epileptic seizures, a.o. deficits. The authors combined structural biology, molecular dynamics simulation, and various cell biological assays to study the effects of disease-causing PURA mutations on protein structure and function. The corresponding data reveal a highly dynamic PURA structure and show that disease-related mutations in PURA cause complex defects in folding, DNA-unwinding activity, RNA binding, dimerization, and partitioning into processing bodies. These findings provide first insights into how very diverse PURA mutations can cause penetrant molecular, cellular, and clinical defects. This will be of substantial interest to cell biologists, neurogeneticists, and neurologists alike.

A particular strength of the present study is the structural characterization of human PURA, which is a challenging target for structural biology approaches. The molecular dynamics simulations are state-of-the-art, allowing a statistically meaningful assessment of the differences between wild-type and mutant proteins. The functional consequences of PURA mutations at the cellular level are fascinating, particularly the differential compartmentalization of wild-type and mutant PURA variants into certain subcellular condensates.

Weaknesses that warrant rectification relate to (i) the interpretation of statistically non-significant effects seen in the molecular dynamics simulations, (ii) the statistical analysis of the differential compartmentalization of PURA variants into processing bodies vs. stress granules, and (iii) the documentation of protein expression levels and knock-down efficiencies.

Reviewer #1 (Public Review):

Summary:<br /> Tian et al. describe how TIPE regulates melanoma progression, stemness, and glycolysis. The authors link high TIPE expression to increased melanoma cell proliferation and tumor growth. TIPE causes dimerization of PKM2, as well as translocation of PKM2 to the nucleus, thereby activating HIF-1alpha. TIPE promotes the phosphorylation of S37 on PKM2 in an ERK-dependent manner. TIPE is shown to increase stem-like phenotype markers. The expression of TIPE is positively correlated with the levels of PKM2 Ser37 phosphorylation in murine and clinical tissue samples. Taken together, the authors demonstrate how TIPE impacts melanoma progression, stemness, and glycolysis through dimeric PKM2 and HIF-1alpha crosstalk.

Strengths:<br /> The authors manipulated TIPE expression using both shRNA and overexpression approaches throughout the manuscript. Using these models, they provide strong evidence of the involvement of TIPE in mediating PKM2 Ser37 phosphorylation and dimerization. The authors also used mutants of PKM2 at S37A to block its interaction with TIPE and HIF-1alpha. In addition, an ERK inhibitor (U0126) was used to block the phosphorylation of Ser37 on PKM2. The authors show how dimerization of PKM2 by TIPE causes nuclear import of PKM2 and activation of HIF-1alpha and target genes. Pyridoxine was used to induce PKM2 dimer formation, while TEPP-46 was used to suppress PKM2 dimer formation. TIPE maintains stem cell phenotypes by increasing the expression of stem-like markers. Furthermore, the relationship between TIPE and Ser37 PKM2 was demonstrated in murine and clinical tissue samples.

Weaknesses:<br /> The evaluation of how TIPE causes metabolic reprogramming can be better assessed using isotope tracing experiments and improved bioenergetic analysis.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript ``Self-inhibiting percolation and viral spreading in epithelial tissue' describes a model based on 5-state cellular automata of development of an infection. The model is motivated and qualitatively justified by time-resolved measurements of expression levels of viral, interferon-producing, and antiviral genes. The model is set up in such a way that the crucial difference in outcomes (infection spreading vs. confinement) depends on the initial fraction of special virus-sensing cells. Those cells (denoted as 'type a') cannot be infected and do not support the propagation of infection, but rather inhibit it in a somewhat autocatalytic way. Presumably, such feedback makes the transition between two outcomes very sharp: a minor variation in concentration of ``a' cells results in qualitative change from one outcome to another. As in any percolation-like system, the transition between propagation and inhibition of infection goes through a critical state with all its attributes. A power-law distribution of the cluster size (corresponding to the fraction of infected cells) with a fairly universal exponent and a cutoff at the upper limit of this distribution.

Strengths:<br /> The proposed model suggests an explanation for the apparent diversity of outcomes of viral infections such as COVID.

Weaknesses:<br /> Those are not real points of weakness, though I think addressing them would substantially improve the manuscript.

The key point in the manuscript is the reduction of actual biochemical processes to the NOVAa rules. I think more could be said about it, be it referring to a set of well-known connections between expression states of cells and their reaction to infection or justifying it as an educated guess.

Another aspect where the manuscript could be improved would be to look a little beyond the strange and 'not-so-relevant for a biomedical audience' focus on the percolation critical state. While the presented calculation of the precise percolation threshold and the critical exponent confirm the numerical skills of the authors, the probability that an actual infected tissue is right at the threshold is negligible. So in addition to the critical properties, it would be interesting to learn about the system not exactly at the threshold: For example, how the speed of propagation of infection depends on subcritical p_a and what is the cluster size distribution for supercritical p_a.

Reviewer #1 (Public Review):

This manuscript from Zaman et al., investigates the role of cKit and Kit ligand in inhibitory synapse function at molecular layer interneuron (MLI) synapses onto cerebellar Purkinje cells (PC). cKit is a receptor tyrosine kinase expressed in multiple tissues, including select populations of neurons in the CNS. cKIt is activated by Kit ligand, a transmembrane protein typically expressed at the membrane of connected cells. A strength of this paper is the use of cell-specific knockouts of cKit and Kit ligand, in MLIs and PCs, respectively. In both cases, the frequency of spontaneous or miniature (in the presence of TTX) IPSCs was reduced. This suggests either a reduction in the number of functional inhibitory release sites or reduced release probability. IPSCs evoked by electrical stimulation in the molecular layer showed no change in paired-pulse ratio, indicating release probability is not changed in the cKit KO, and favoring a reduction in the number of release sites. Changes in IPSC amplitude were more subtle, with some analyses showing a decrease and others not. These data suggest that disruption of the cKit-Kit ligand complex reduces the number of functional synapses with only minor changes in synapse strength.

Reviewer #1 (Public Review):

Summary:

The main goal of the authors was to study the testis-specific role of the protein FBXO24 in the formation and function of the ribonucleoprotein granules (membraneless electron-dense structures rich in RNAs and proteins).

Strengths:

The wide variety of methods used to support their conclusions (including transgenic models)

Weaknesses:

The lack of specific antibodies against FBXO24. Some of the experiments showing a specific phenotype are descriptive and lack of logical explanation about the possible mechanism (i.e. AR or the tail structure).

Questions:

The paper is excellent and employs a wide variety of methods to substantiate the conclusions. I have very few questions to ask:

1) KO mice cannot undergo acrosome reaction (AR) even spontaneously. How do you account for this, given that no visible defects were observed in the acrosome?

2) KO sperm are unable to migrate in the female tract, and, more intriguingly, they do not pass through the utero-tubal junction (UTJ). The levels of ADAM3 are normal, suggesting that the phenotype is influenced by other factors. The authors should investigate the levels of Ly6K since mice also exhibit the same phenotype but with normal levels of ADAM3.

3) In Figure 4A, the authors assert that "RBGS Tg mice revealed that mitochondria were abnormally segmented in Fbxo24 KO spermatozoa." I am unable to discern this from the picture shown in that panel. Could you please provide a more detailed explanation or display the information more explicitly?

Reviewer #1 (Public Review):

Summary:<br /> This study used a unique acute HIV-1 infection cohort, RV217, to study the evolution of transmitted founder viral Envelope sequences under nascent immune pressure. The striking feature of the RV217 cohort is the ability to detect viremia in the first week of infection, which can be followed at discrete Fiebig stages over long time intervals. This study evaluated Env sequences at 1 week, 4 weeks, and 24 weeks to provide a picture of viral and immunological co-evolution from Fiebig Stage I (1 week), Fiebig Stages IV (4 weeks), and Fiebig Stage VI (>24 weeks). This study design enabled lineage tracing of viral variants from a single transmitted founder (T/F) over the Fiebig Stages I, IV, and VI under nascent immune pressure generated in response to the T/F virus and its subsequent mutants.

Strengths:<br /> As expected, there were temporal differences in the appearance of virus quasispecies among the individuals, which were located predominantly in solvent-exposed residues of Env, which is consistent with prior literature. Interestingly, two waves of antibody reactivity were observed for variants with mutations in the V2 region that harbors V2i and V2p epitopes correlated with protection in the RV144 clinical trial. Two waves of antibody response, detected by SPR, were observed, with the first wave being predominated by antibodies specific for the T/F07 V2 epitope associated with H173 located on the C β-strand in the V2 region. The second wave was dominated by antibodies specific for an H to Y mutation at 173 that emerged due to antibody-mediated pressure to the original H173 virus. This is a remarkable finding in three ways.

First, the mutation is in the C β-strand, an unlikely paratope contact residue, as this region of the V2 loop is shielded by glycans in Env trimer structures with full glycan representation (see PDB:5t3x). The structure used for modeling in the current study was an earlier structure, PDB:4TVP, that had many truncated glycans. This does not detract from the finding that the H173Y mutation likely causes a conformational shift from a more rigid helical/coil conformation to a more dynamic conformation with a β-stranded and β-sheet core preference as indicated by the literature and by the MD simulations carried out by the authors. This observation suggests that using V2 scaffolds with both the H173 and H173Y variants will increase the breadth of potentially protective antibody responses to HIV-1, as indicated in reference 42, cited by the authors. Interestingly, the H173Y mutation abrogates reactivity to mAb CH58 and attenuates reactivity to mAb CH59 isolated from RV144 volunteers. These mAbs recognize conformationally distinct V2 epitopes, adding further credence to the conclusion that the H173Y mutation results in a conformational switch of the V2 region.

Second, the H173Y mutation affects the conformation of V2 epitopes recognized by mAbs that do not neutralize T/F HIV-1 while mediating potent ADCC. The ADCC data in the manuscript provides strong support for this hypothesis and augers for further exploration of the V2 epitopes as HIV-1 vaccine targets.<br /> Third, it is striking that immunogens based on the H173Y mutation successfully recapitulated the observed human antibody responses in wild-type Balb/c mice. The investigators used their extensive knowledge of V2 structures and scaffold-immunogens to create the library of constructs used for this study. In this case, the ΔDSV mutation increased the breadth and magnitude of the murine antibody responses.

Weaknesses:<br /> 1. V2 epitopes exhibit properties of CD4i epitopes in that they are largely absent from the native Env surface, probably by glycan-occlusion, but become more exposed upon CD4 binding. Although the V2-scaffolds were produced in GnTi- cells to produce high-mannose proteins, it appears that no systematic analysis of glycan content or structure was carried out save for enzymatic deglycosylation of the constructs to sharpen bands on SDS-PAGE gels. It would be helpful if the authors could comment on how the lack of this information might impact their conclusions.

2. Similarly, the MD simulations appear to be performed without taking glycan structure/occupancy.

Reviewer #1 (Public Review):

Mignerot et al. performed a Herculean effort to measure and describe natural variation in C. elegans egg-laying behavior and egg retention. The paper is well written and organized. The authors show wild strains vary in egg retention with some extremes that appear phenotypically similar to species with viviparity (or live birth / internal hatching of offspring). They previously published a rare variant in the gene kcnl-1 that plays a role in egg retention but identify common variants in this study. They classify wild strains based on egg-retention to separate out the extremely different isolates. Egg laying has been extensively studied in the laboratory strain N2, but rarely addressed in natural strains. The authors investigate egg-laying behaviors using standard assays and find that their classified egg-laying groups have differences in sub-behaviors suggesting diverse roles in the ultimate egg-laying output. Then, they turn to the egg-laying circuit using both exogenous serotonin (5-HT), 5-HT modulatory drugs (e.g. SSRIs), and even genome editing to test epistasis with the mod-5 5-HT reuptake. The effects of 5-HT modulation and mutants are not predictive based on the basal behaviors and egg-retention phenotypes with the most extreme egg-retention strains differing in their responses. Interestingly, strains with more egg retention have decreased fitness (in their laboratory) measures but also provide a protective environment for offspring when exposed to common "natural" stressors. Their final conclusion that egg retention could be a trade-off between antagonistic effects of maternal vs. offspring fitness is supported well and sets the stage for future mechanistic studies across Caenorhabditis.

Reviewer #1 (Public Review):

Koumoundourou et al., identify a pathway downstream of Bcl11b that controls synapse morphology and plasticity of hippocampal mossy fiber synapses. Using an elegant combination of in vivo, ex vivo, and in vitro approaches, the authors build on their previous work that indicated C1ql2 as a functional target of Bcl11b (De Bruyckere et al., 2018). Here, they examine the functional implications of C1ql2 at MF synapses in Bcl11b cKO mice and following C1ql2 shRNA. The authors find that Bcl11b KO and shRNA against C1ql2 significantly reduces the recruitment of synaptic vesicles and impairs LTP at MF synapses. Importantly, the authors test a role for the previously identified C1ql2 binding partner, exon 25b-containing Nrxn3 (Matsuda et al., 2016), as relevant at MF synapses to maintain synaptic vesicle recruitment. To test this, the authors developed a K262E C1ql2 mutant that disrupts binding to Nrxn3. Curiously, while Bcl11b KO and C1ql2 KD largely phenocopy (reduced vesicle recruitment and impaired LTP), only vesicle recruitment is dependent on C1ql2-Nrxn3 interactions. These findings provide new insight into the functional role of C1ql2 at MF synapses. The authors utilize a multidisciplinary approach to convincingly demonstrate a role for C1ql2-Nrxn3(25b+) interactions for vesicle recruitment and a Nrxn3(25b+)-independent role for C1ql2 in LTP, The authors establish an important signaling pathway that offers insight into how disruptions of Bcl11b contribute to synapse dysfunction and provide a much needed advance toward understanding the functional consequences of neurexin alternative splicing.

Reviewer #1 (Public Review):

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

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

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

Revision: I appreciate the authors revising the manuscript with additional data that have clarified my prior comments. They now show that TH levels in the striatum decrease with SNpc neurons. Further, while there is some disagreement regarding the expression LRRK1 in SNpc, the authors provide a convincing case that LRRK1 function is important in SNpc DA neurons.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript by Liu et al explores the role of the UPR and immune regulators in the evaluation of nutritional quality in C. elegans. They identify neuronal UPR activation and the MAPK PMK-1 as key responders to low food quality. In particular, the data suggest that these pathways are activated by low levels of vitamin C synthesis that result from the low sugar levels present in heat-killed E. coli.

Strengths:<br /> The results are intriguing and expand our understanding both of physiological food evaluation systems, and of the known roles of stress response pathways in organismal physiology. The authors use a range of techniques, encompassing imaging, metabolomic analysis, gene expression analysis, and behavioural assays, to support their claims.

Weaknesses:<br /> There is limited mechanistic analysis in the study. In particular, how does low vitamin C trigger UPR activation? This is an intriguing finding that, if followed up, could potentially reveal a novel mechanism of UPR activation. In addition, how is the activation of the PMK-1 pathway driven by/coordinated with UPR activation? The data in some figures is not as convincing as it could be: the magnitude of the effect size is small in the supplementation experiments, and the statistical tests used are not always appropriate to enable multiple comparisons.

Reviewer #1 (Public Review):

Summary:<br /> The authors introduce two preparations for observing large-scale cortical activity in mice during behavior. Alongside this, they present intriguing preliminary findings utilizing these methods. This paper is poised to be an invaluable resource for researchers engaged in extensive cortical recording in behaving mice.

Strengths:<br /> -Comprehensive methodological detailing:<br /> The paper excels in providing an exceptionally detailed description of the methods used. This meticulous documentation includes a step-by-step workflow, complemented by thorough workflow, protocols, and a list of materials in the supplementary materials.

-Minimal movement artifacts:<br /> A notable strength of this study is the remarkably low movement artifacts. To further underscore this achievement, a more robust quantification across all subjects, coupled with benchmarking against established tools (such as those from suite2p), would be beneficial.

Insightful preliminary data and analysis:<br /> The preliminary data unveiled in the study reveal interesting heterogeneity in the relationships between neural activity and detailed behavioral features, particularly notable in the lateral cortex. This aspect of the findings is intriguing and suggests avenues for further exploration.

Weaknesses:<br /> -Clarification about the extent of the method in the title and text:<br /> The title of the paper, using the term "pan-cortical," along with certain phrases in the text, may inadvertently suggest that both the top and lateral view preparations are utilized in the same set of mice. To avoid confusion, it should be explicitly stated that the authors employ either the dorsal view (which offers limited access to the lateral ventral regions) or the lateral view (which restricts access to the opposite side of the cortex). For instance, in line 545, the phrase "lateral cortex with our dorsal and side mount preparations" should be revised to "lateral cortex with our dorsal or side mount preparations" for greater clarity.

-Comparison with existing methods:<br /> A more detailed contrast between this method and other published techniques would add value to the paper. Specifically, the lateral view appears somewhat narrower than that described in Esmaeili et al., 2021; a discussion of this comparison would be useful. Furthermore, the number of neurons analyzed seems modest compared to recent papers (50k) - elaborating on this aspect could provide important context for the readers.

-Discussion of methodological limitations:<br /> The limitations inherent to the method, such as the potential behavioral effects of tilting the mouse's head, are not thoroughly examined. A more comprehensive discussion of these limitations would enhance the paper's balance and depth.

-Preliminary nature of results:<br /> The results are at a preliminary stage; for example, the B-soid analysis is based on a single mouse, and the validation data are derived from the training data set. The discrepancy between the maps in Figures 5e and 6e might indicate that a significant portion of the map represents noise. An analysis of variability across mice and a method to assign significance to these maps would be beneficial.

-Analysis details:<br /> More comprehensive details on the analysis would be beneficial for replicability and deeper understanding. For instance, the statement "Rigid and non-rigid motion correction were performed in Suite2p" could be expanded with a brief explanation of the underlying principles, such as phase correlation, to provide readers with a better grasp of the methodologies employed.

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and documented the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attach if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes the crystal structures of Streptococcus pneumoniae NOXs. Crystals were obtained for the wild-type and mutant dehydrogenase domain, as well as for the full-length protein comprising the membrane domain. The manuscript further carefully studies the enzyme's kinetics and substrate-specificity properties. Streptococcus pneumoniae NOX is a non-regulated enzyme, and therefore, its structure should provide a view of the NOX active conformation. The structural and biochemical data are discussed on this ground.

Strengths:<br /> This is very solid work. The protein chemistry and biochemical analysis are well executed and carefully described. Similarly, the crystallography must be appreciated given the difficulty of obtaining good enzyme preparations and the flexibility of the protein. Even if solved at medium resolution, the crystal structure of the full-length protein conveys relevant information. The manuscript nicely shows that the domain rotations are unlikely to be the main mechanistic element of NOX regulation. It rather appears that the NADPH-binding conformation is pivotal to enzyme activation. The paper extensively refers to the previous literature and analyses the structures comprehensively with a comparison to previously reported structures of eukaryotic and prokaryotic NOXs.

Weaknesses:<br /> The manuscript is not always very clear with regard to the analysis of NADPH binding. The last section describes a "crevice" featured by the NADPH-binding sites in NOXs. It remains unclear whether this element corresponds to the different conformations of the protein C-terminal residues or more extensive structural differences. This point must be clarified.<br /> A second less convincing point concerns the nature of the electron acceptor. The manuscript states that this NOX might not physiologically act as a ROS producer. A question then immediately arises: Is this protein an iron reductase? Can the authors better discuss or provide more data about this point?

Reviewer #1 (Public Review):

This paper performed a functional analysis of the poorly characterized pseudo-phosphatase Styxl2, one of the targets of the Jak/Stat pathway in muscle cells. The authors propose that Styxl2 is essential for de novo sarcomere assembly by regulating autophagic degradation of non-muscle myosin IIs (NM IIs). Although a previous study by Fero et al. (2014) has already reported that Styxl2 is essential for the integrity of sarcomeres, this study provides new mechanistic insights into the phenomenon. In vivo studies in this manuscript are compelling; however, I feel the contribution of autophagy in the degradation of NM IIs is still unclear.

Reviewer #1 (Public Review):

Summary:<br /> This paper presents evidence that a relatively common genetic variant tied to several disease phenotypes affects the interaction between the mRNA of CCL2 and the RNA binding protein HuR. CCL2 is an immune cell chemoattractant protein.

Strengths:<br /> The study is well conducted with relevant controls. The techniques are appropriate, and several approaches provided concordant results that were generally supportive of the conclusions reached. The impact of this work, identifying a genetic variant that works by altering the binding of an RNA-regulatory protein, has important implications given that the HuR protein could be a drug target to improve its function and override this genetic change. This could have important implications for a number of diseases where this genetic variant contributes to disease risk.

Weaknesses:<br /> The authors need to do a better job of citing prior work. Certain details of the experimental protocols need to be further elaborated or clarified to contextualize the significance of the findings, Some of the findings need to be better described.

Reviewer #1 (Public Review):

The authors examine the fascinating question of how T lymphocytes regulate proteome expression during the dramatic cell state change that accompanies the transition from the resting quiescent state to the activated, dividing state. Orthogonal, complementary assays for translation (RPM/RTA, metabolic labeling) are combined with polyribosome profiling and quantitative, biochemical determinations of protein and ribosome content to explore this question, primarily in the OT-I T lymphocyte model system. The authors conclude that the ratio of protein levels to ribosomes/protein synthesis capacity is insufficient to support activation-coupled T cell division and cell size expansion. The authors hint at cellular mechanisms to explain this apparent paradox, focusing on protein acquisition strategies, including emperipolesis and entosis, though these remain topic areas for future study.

The strengths of the paper include the focus on a fundamental biological question - the transcriptional/translational control mechanisms that support the rapid, dramatic cell state change that accompanies lymphocyte activation from the quiescent to activated state, the use of orthogonal approaches to validate the primary findings, and the creative proposal for how this state change is achieved.

The weakness of the work is that several cellular regulatory processes that could explain the apparent paradox are not explored, though they are accessible to experimental analysis. In the accounting narrative that the authors highlight, a thorough accounting of the cellular process inventory that could support the cell state change should be further explored before committing to the proposal, provocative as it is, that protein acquisition provides a principal mechanism for supporting lymphocyte activation cell state change.

Appraisal and Discussion:

1) Relating to the points raised above, two recent review articles explore this topic area and highlight important areas of study in RNA biology and translational control that likely contribute to the paradox noted by the authors: Choi et al. 2022,<br /> doi.org/10.4110/in.2022.22.e39 ("RNA metabolism in T lymphocytes") and Turner 2023, DOI: 10.1002/bies.202200236 ("Regulation and function of poised mRNAs in lymphocytes"). These should be cited, and the broader areas of RNA biology discussed by these authors integrated into the current manuscript.

2) The authors cite the Wolf et al. study from the Geiger lab (doi.org/10.1038/s41590-020-0714-5, ref. 41) though largely to compare determined values for ribosome number. Many other elements of the Wolf paper seem quite relevant, for example, the very high abundance of glycolytic enzymes (and whose mRNAs are quite abundant as well), where (and as others have reported) there is a dramatic activation of glycolytic flux upon T cell activation that is largely independent of transcription and translation, the evidence for "pre-existing, idle ribosomes", the changes in mRNA copy number and protein synthesis rate Spearman correlation that accompanies activation, and that the efficiencies of mRNA translation are heterogeneous. These data suggest that more accounting needs to be done to establish that there is a paradox.

As one example, what if glycolytic enzyme protein levels in the resting cell are in substantial excess of what's need to support glycolysis (likely true) and so translational upregulation can be directed to other mRNAs whose products are necessary for function of the activated cell? In this scenario the dilution of glycolytic enzyme concentration that would come with cell division would not necessarily have a functional consequence. And the idle ribosomes could be recruited to key subsets of mRNAs (transcriptionally or post-transcriptionally upregulated) and with that a substantial remodeling of the proteome (authors ref. 44). The study of Ricciardi et al. 2018 (The translational machinery of human CD4+ T cells is poised for activation and controls the switch from quiescence to metabolic remodeling (doi.org/10.1016/j.cmet.2018.08.009) is consistent with this possibility. That study, and the short reviews noted above, are useful in highlighting the contributions of selective translational remodeling and the signaling pathways that contribute to the cell state change of T cell activation. From this perspective an alternative view can be posited, where the quiescent state is biologically poised to support activation, where subsets of proteins and mRNAs are present in far higher levels than that necessary to support basal function of the quiescent lymphocyte. In such a model, the early stages of lymphocyte activation and cell division are supported by this surplus inventory, with transcriptional activation, including ribosomal genes, primarily contributing at later stages of the activation process. An obvious analogy is the developing Drosophila embryo where maternal inheritance supports early-stage development and zygotic transcriptional contributions subsequently assuming primary control (e.g. DOI 10.1002/1873-3468.13183 , DOI: 10.1126/science.abq4835). To pursue that biological logic would require quantifying individual mRNAs and their ribosome loading states, mRNA-specific elongation rates, existing individual protein levels, turnover rates of both mRNAs and proteins, ribosome levels, mean ribosome occupancy state, and how each of these parameters are altered in response to activation. Such accounting could go far to unveil the paradox. This is a considerable undertaking, though, and outside the scope of the current paper.

Regarding the revised manuscript:

I am largely satisfied with the authors responses to the review and have but a few remaining thoughts, some mirrored in the comments from the other reviewers and some that came to mind upon reading the revision.

1) In the Introduction, it would be (have been) helpful if in paragraph two, it was stated that the current study was designed to test that assumption made in prior reports that the fold-increase in protein synthesis in response to mitogen activation was sufficient to endow the daughter cells with "the same protein content as their progenitor".

2) The primary conclusion, that "...protein synthesis activity or capacity of in vivo activated T cells does not support their doubling times" remains, to my eye, insufficiently supported by the data, though I agree it is a rational interpretation. My concern is that the devil is deeper in the details and without knowing the mRNA transcriptome composition pre- and post-activation, mean CDS length, 5' UTR structural features, perhaps codon optimality, etc., etc., the broader conclusion could be premature. As a first check, it would be useful to determine poly(A) mRNA and ribosome concentrations/cell, pre- and post-activation, and subsequently to compare mRNA transcriptome compositions in greater detail. Do mRNA:ribosome levels and ratios diverge as a consequence of activation? Poly(A) mRNA compositions? Does protein half-life change pre- and post-activation? mRNA half-life? My view is that additional molecular accounting is likely necessary to be confident in the primary conclusion.

3) I did not provide a clear description of the alternative interpretation I was imagining, which is that in the resting, unstimulated state, mRNA:ribosome and/or protein levels may be much higher than that necessary for lymphocyte viability. As in early development, this could be a mechanism to then provide sufficient protein synthesis capacity and/or proteins to daughter cells following activation of cell division and cell growth. In other words, it's a dynamic range question; the daughter cells exploit "unused" protein synthesis capacity to sustain their growth and division. Quantification and analysis of the additional variables noted in point 2) could reconcile the different interpretations.

Reviewer #1 (Public Review):

The association of vitamin D supplementation in reducing Asthma risk is well studied, although the mechanistic basis for this remains unanswered. In the presented study, Kilic and co-authors aim to dissect the pathway of Vitamin D-mediated amelioration of allergic airway inflammation. They use initial leads from bioinformatic approaches, which they then associate with results from a clinical trial (VDAART) and then validate them using experimental approaches in murine models. The authors identify a role of VDR in inducing the expression of the key regulator Ikzf3, which possibly suppresses the IL-2/STAT5 axis, consequently blunting the Th2 response and mitigating allergic airway inflammation.

The major strength of the paper lies in its interdisciplinary approach, right from hypothesis generation, and linkage with clinical data, as well as in the use of extensive ex vivo experiments and in vivo approaches using knock-out mice. The study presents some interesting findings including an inducible baseline absence/minimal expression of VDR in lymphocytes, which could have physiological implications and needs to be explored in future studies.<br /> The study presents a potential for further dissection of relevant pathophysiological pathways to explain certain seemingly associative results, and allow for a more effective translation.

Several results in the study suggest multiple factors and pathways influencing the phenotype seen, which could be explored in the future. The inferences of this study also need to be read in the context of the different sub-phenotypes and endotypes of Asthma, where the Th2 response may not be predominant. While this does not undermine the importance of this elegant study, it is essential to emphasise a holistic picture while interpreting the results.

Reviewer #1 (Public Review):

The manuscript investigates the role of membrane contact sites (MCSs) and sphingolipid metabolism in regulating vacuolar morphology in the yeast Saccharomyces cerevisiae. The authors show that tricalbin (1-3) deletion leads to vacuolar fragmentation and the accumulation of the sphingolipid phytosphingosine (PHS). They propose that PHS triggers vacuole division through MCSs and the nuclear-vacuolar junction (NVJ). The study presents some solid data and proposes potential mechanisms underlying vacuolar fragmentation driven by this pathway. Although the manuscript is clear in what the data indicates and what is more hypothetical, the story would benefit from providing more conclusive evidence to support these hypothesis. Overall, the study provides valuable insights into the connection between MCSs, lipid metabolism, and vacuole dynamics.

Reviewer #1 (Public Review):

Summary:<br /> This paper addresses the mechanisms positioning microtubule asters in Drosophila explants. Taking advantage of a genetic mutant, blocking the cell cycle in early embryos, the authors generate embryos with centrosomes detached from nuclei and then study the positioning mechanisms of such asters in explants. They conclude that asters interact via pushing forces. While this is an artificial system, understanding the mechanics of asters positioning, in particular, whether forces are pushing or pulling is an important one.

Strengths:<br /> The major strength of this paper is the series of laser cutting experiments supporting that asters position via pushing forces acting both on the boundary (see below for a relevant comment) and between asters. The combination of imaging, data analysis and mathematical modeling is also powerful.

Weaknesses:<br /> This paper has overlap in the conclusions with a previous paper from the same authors, so its impact is reduced. In Figure 2, the tracking of fluid flows is hard to see and better quantifications/analyses would lead to stronger conclusions. In Figure 4, it is not clear that the acceleration is significant and no statistical test is provided or described, as far as I can tell.

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.

At the top of the results, the authors should say what species they're working with and give some background about the nature of the reduced genome. It is important to know what the changes were and especially how much of the genome was deleted. Some insights into the genes that were deleted would also be useful context for understanding the evolution experiment. This could be included in the introduction or results.

Reviewer #1 (Public Review):

Summary:

The manuscript by Hann et. al examines the role of survival motor neuron protein (SMN) in lateral plate mesoderm-derived cells using the Prrx1Cre to elucidate how changing cell-specific SMN levels coordinate aspects of the spinal muscular atrophy (SMA) pathology. SMN has generally been studied in neuronal cells, and this is one of the first insights into non-neuronal cells that may contribute to SMA disease. The authors generated 3 mouse lines: a Prrx1;Smnf/f conditional null mouse, as well as, single and double copy Prrx1;Smnf/f;SMN2 mice carrying either one or two copies of a human SMN2 transgene. First, the bone development and growth of all three were assessed; the conditional null Smn mutation was lethal shortly after birth, while the SMN2 2-copy mutant did not exhibit bone growth phenotypes. Meanwhile, single-copy SMN2 mutant mice showed reduced size and shorter limbs with shorter proliferative and hypertrophic chondrocyte zones. The authors suggested that this was cell autonomous by assessing the expression of extrinsic factors known to modulate proliferation/differentiation of growth plate chondrocytes. After assessing bone phenotypes, the authors transitioned to the assessments of neuromuscular junction (NMJ) phenotypes, since there are documented neuromuscular impairments in SMA and the Prrx1Cre transgene is expressed in muscle-associated fibro-adipogenic progenitors (FAPs). Neonatal NMJ development was unchanged in mutant mice with two copies of SMN2 , but adult single-copy SMN2 mutant mice had abnormal NMJ morphology, altered presynaptic neurotransmission, and problematic nerve terminal structure. Finally, the authors sought to assess the ability to rescue NMJ phenotypes via FAP cell transplantation and showed wild-type FAPs were able to reduce pre/postsynaptic fragmentation and neurofilament varicosities.

Strengths:

The conditional genetic approaches are novel and interestingly demonstrate the potential for chondrocyte and fibro-adipogenic progenitor-specific contributions to the SMA pathology.

The characterizations of the neuromuscular and NMJ phenotypes are relatively strong.

The data strongly suggest a non-neuronal contribution to SMA, which indicates a need for further mechanistic (cellular and molecular) studies to better understand SMA.

Weaknesses:

The skeletal analyses are not rigorous and likely do not get to the core of how SMN regulates bone development.

The overall work is descriptive and lacks convincing mechanisms.

Additional experimentation is likely needed to fully justify the conclusions.

Reviewer #1 (Public Review):

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths<br /> 1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty.

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding. These conflict measures use several best-practice approaches towards estimating representational similarity.

3. The authors quantify several salient alternative hypothesis, and systematically distinguish their core results from these alternatives.

4. The question that the authors tackle is important to cognitive control, and they make a solid contribution.

Concerns<br /> 1. The framing of 'infinite possible types of conflict' feels like a strawman. While they might be true across stimuli (which may motivate a feature-based account of control), the authors explore the interpolation between two stimuli. Instead, this work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with literatures like n-back, multiple object tracking, and random dot motion). This parametric encoding is standard in feature-based attention, and it's not clear what the cognitive map framing is contributing.

2. The representations within DLPFC appear to treat 100% Stoop and (to a lesser extent) 100% Simon differently than mixed trials. Within mixed trials, the RDM within this region don't strongly match the predictions of the conflict similarity model. It appears that there may be a more complex relationship encoded in this region.

3. To orthogonalized their variables, the authors need to employ a complex linear mixed effects analysis, with a potential influence of implementation details (e.g., high-level interactions and inflated degrees of freedom).

Reviewer #1 (Public Review):

Summary:

Kinase inhibitors represent a highly valuable class of drugs as evidenced by their continued clinical success. The target landscape of kinase targeting small molecules can be leveraged to alter multiple phenotypes with increasing complexity that broadly aligns with increasing target promiscuity. This 'tools and resources' contribution provides a starting point for researchers interested in aligning kinase inhibitor activity with cytokine/chemokine stimulated signal transduction networks.

Strengths:

KinCytE is a forward-thinking database that yields hypothesis-generating options for researchers interested in pharmacologically modulating cytokine/chemokine signaling.

Weaknesses:

As a 'tools and resources' contribution, the primary (potential) weakness will be the authors' willingness to update and improve the tool. KinCytE will require frequent updating to better inform users in terms of contextual cytokine/chemokine stimulated signaling and the target landscape of those agents that are included as options.

Reviewer #1 (Public Review):

Summary:<br /> Tuberculous meningitis (TBM) is one of the most severe forms 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:<br /> 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 a test that can be used in the clinical with a minimally invasive sample. Furthermore, the data from this study has also revealed important insights into 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:<br /> The data on blood neutrophil count is really intriguing and seems to provide a very powerful yet easy-to-measure method to differentiate survival vs. death in TBM patients. It would be quite useful in this case to perform predictive analysis to see if neutrophil count alone, or in combination with gene signature, can predict (or better predict) mortality, as it would be far easier for clinical implementation than the RNA-based method. Moreover, genes associated with increased neutrophil activation and decreased T cell activation both have significantly higher enrichment scores in TBM (Figure 9) and in morality (Figure 8). While I understand the basis of selecting hub genes in the significant modules, they often do not represent these biological pathways (at least not directly associated in most cases). If genes were selected based on these biologically relevant pathways, would they have better predictive values?

Reviewer #1 (Public Review):

Summary:<br /> The investigators have performed a state-of-the art systematic review and meta-analysis of studies that may help to answer the research question: if administration of multiple antibiotics simultaneously prevents antibiotic resistance development in individuals. The amount of studies eligible for analysis is very low, and within that low number, there is huge variability in bug-drug combinations studied and most studies had a high risk of bias, further limiting the capability of meta-analysis to answer the research question. In addition, based on I2 values there is also huge statistical heterogeneity between outcomes of studies compared, further limiting the predictive value of meta-analysis. In fact, the only 2 studies meeting all eligibility criteria addressed the treatment of mycobacterium tuberculosis, for which the research question is hardly applicable. The authors, therefore, conclude that "our analysis could not identify any benefit or harm of using a higher or a lower number of antibiotics regarding within-patient resistance development." Apart from articulating this knowledge gap, the findings will not have consequences for patient care, but may stimulate the scientific community to better address this research question in future studies.

Strengths:<br /> The systematic and rigorous approach for the review and meta-analysis.

Weaknesses:<br /> None identified.

Reviewer #1 (Public Review):

Summary:

This manuscript by Vuong and colleagues reports a study that pooled data from 3 separate longitudinal studies that collectively spanned an observation period of over 15 years. The authors examined for correlation between viraemia measured at various days from illness onset with thrombocytopaenia and severe dengue, according to the WHO 2009 classification scheme. The motivation for this study is both to support the use of viraemia measurement as a prognostic indicator of dengue and also when an antiviral drug becomes licensed for use, to guide the selection of patients for antiviral therapy. They found that the four DENVs show differences in peak and duration of viraemia and that viraemia levels before day 5 but not those after from illness onset correlated with platelet count and plasma leakage at day 7 onwards. They concluded that the viraemia kinetics call for early measurement of viraemia levels in the early febrile phase of illness.

Strengths:

This is a unique study due to the large sample size and longitudinal viraemia measurements in the study subjects. The data addresses a gap in information in the literature, where although it has been widely indicated that viraemia levels are useful when collected early in the course of illness, this is the first time anyone has systematically examined this notion.

Weaknesses:

The study only analysed data from dengue patients in Vietnam. Moreover, the majority of these patients had DENV-1 infection; few had DENV-4 infection. The data could thus be skewed by the imbalance in the prevalence of the different types of DENV during the period of observation. The use of patient-reported time of symptom onset as a reference point for viraemia measurement is pragmatic although there is subjectivity and thus noise in the data.

Reviewer #1 (Public Review):

Summary:

The authors presented here a novel 3D fibroblast culture and transdifferentiation approach for potential meat production with GelMA hydrogel.

Strengths:

1. Reduced serum concentration for 3D chicken fibroblast culture and transdifferentiation is optimized.<br /> 2. Efficient myogenic transdifferentiation and lipogenesis as well as controlled fat deposition are achieved in the 3D GelMA.

Weaknesses:

1. While the authors stated the rationale of using fibroblasts instead of myogenic/adipogenic stem cells for meat production, the authors did not comment on the drawbacks/disadvantages of genetic engineering (e.g., forced expression of MyoD) in meat production.<br /> 2. While the authors cited one paper to state the properties and applications of GelMA hydrogel in tissue engineering and food processing, concerns/examples of the food safety with GelMA hydrogel are not discussed thoroughly.<br /> 3. In Fig. 4C, there seems no significant difference in the Vimentin expression between Fibroblast_MyoD and Myofibroblast. The conclusion of "greatly reduced in the myogenic transdifferentiated cells" is overstated.<br /> 4. The presented cell culture platform is only applied to chicken fibroblasts and should be tested in other species such as pigs and fish.

Reviewer #1 (Public Review):

In this manuscript, Manessero and colleagues argue that the prefrontal cortex (PFC), given its exquisite capability to down-regulate down-stream regions central in driving emotional responses to threat, maybe a promising target to stimulate in order to reduce aberrant fear memory responses. They aim to differ from previous studies that tested the strengthening of extinction learning, by merely focusing on the expression of threat memory without extinction learning. Given that other studies have often focused on the dorsolateral prefrontal cortex as promising target to regulate fear responses, they also ran experiments to directly compare effectiveness of targeting the mPFC and dlPFC in reducing fear memory responses. These aims are all focused on what the authors describe as "implicit memory", but they also test the effects of the interventions on "explicit memory" of the presented cues. However, in the introduction, the authors do not explicitly describe what their aim or theoretical rationale to implement these tests was. Likewise, the authors implemented generalisation stimuli (i.e., cues similar to the original CS) in the implicit memory tests, but the aim of these tests is also not explained.

In order to test their hypotheses, the authors adopt a single-cue fear conditioning paradigm where participants learned to associate an auditory cue with the occurrence of short electrical stimulation across 15 repetitions of the CS-US pairing (80% reinforcement rate). One week later, for the second session, this cue was again presented 4 times, along with 2 types of generalization stimuli, that were each also presented four times. This test session took place in another environment. Conditioned skin conductance responses were measured as index of defensive responding. In the critical condition, during 10 minutes prior to these cue presentations, repetitive Transcranial Magnetic Stimulation (rTMS) was applied to specifically target the medial PFC. Another independent group of participants completed a two-alternative forced-choice (2AFC) explicit recognition test, to inquire to what extent they could recognize whether a given tone was presented during the conditioning phase (basically a source memory task). Finally, a two-alternative forced-choice (2AFC) perceptual discrimination test was presented, to ascertain that participants could discern the different tones presented. The second session was repeated yet another week later, but without any rTMS and in the original conditioning context again, to test whether any potential fear dampening effects were retained.

The observations are quite straightforward: compared to sham and an active control group, mPFC stimulation prior to fear memory retrieval resulted in an immediate reduction of conditioned responses, a difference that was consistent across all 4 test trials. Also conditioned responses to the generalization cues were reduced upon mPFC stimulation. These effects seemed to be specific for memories, since responding to novel unconditioned cues (loud female scream) were not affected by prior mPFC stimulation. Likewise, measures of explicit memory were unaffected. In separate experiments, stimulation of the mPFC also outperformed stimulation of the dlPFC. This pattern of results was again observed during the tests a week later.

The authors conclude that, since these outcomes were observed in the absence of extinction training, the rTMS procedure directly modulated the defensive responses activated by the threat memory trace. The fact that defensive responses to novel unconditioned stimuli were not affected are in line with earlier observations that the mPFC seems critical for the expression of conditioned but not innate fear. Given that dlPFC stimulation seemed less effective, the mPFC may be the most suitable candidate for future therapeutic interventions.

Major strengths:<br /> - Earlier work delving into the involvement of the prefrontal cortex in fear regulation has not only revealed a central role for the mPFC, but also for the dlPFC. An important strength of this study is that the authors therefore also directly compare groups that are targeted in either one of these regions, thereby revealing that even though stimulating the dlPFC results in some fear reduction, the effect is much stronger for mPFC. Another nice consequence of this extra group is that the earlier observations when targeting the mPFC are being replicated.

- It is important to test novel avenues to achieve enduring fear dampening effects of interventions. An intervention that only exerts immediate but transient effects does not bring much clinical value. So the fact that this study incorporates a follow-up test and then shows that the acute fear dampening effects are retained in the absence of any TMS stimulation certainly is important.

- It is only natural to show defensive responses to cues that previously have been paired with something aversive, like a shock. For this reason, generalized fear responses to cues that are similar to fear cues but in fact innocuous is considered maladaptive, and at the core of anxiety disorders. A strength of the paper is that the authors have added generalization tests in addition to (adaptive) fear retention, to ascertain that their intervention in fact also targets maladaptive responding.

Major weaknesses<br /> - There are two major weaknesses in this paper, that can have a potentially detrimental consequence for the robustness of the results and conclusions. First of all, even though comparing the effect of mPFC stimulation with other groups that have been stimulated in other brain regions is important, another comparison - perhaps an even more essential one - is lacking: is there a significant reduction in conditioned fear responses after targeting the mPFC as compared to that group's own fear acquisition (or at least the final phase of acquisition)? Instead, the authors compare fear responses with responses PRIOR to conditioning, which is not meaningful. The same goes for the long-term follow-up: also here, a comparison with fear responding prior to the intervention is lacking. Such a reduction in conditioned fear responding should be larger than any reduction (e.g., due to habituation or forgetting) in fear responding in the (sham) control groups (i.e., an overall interaction between group and fear responding should be present). Whether this is the case is unfortunately unknown, since the fear acquisition data (neither raw, nor pre-processed) are not to be found in the manuscript, and are therefore also not included in any of the analyses. Since there is also no safe control stimulus, the crucial comparison is made entirely between-subjects, and for such a comparison groups of n~20 are quite modest.

- Second, against commons practice, the authors commence by square root transforming all SCR data to normalize the data (while this should only be done in the final phase or preprocessing, if the variables entered in the statistical tests require so), only to then again normalize these obtained values by dividing by the unconditioned responses of the participants, that then are used to calculate differences scores with preconditioning. In these descriptions it is unclear which unconditioned stimulus it was (the original one, from conditioning?) and whether it was standardised to the highest response or an average of all the responses. Decisions that are taken in these early pre-processing steps can have a gigantic impact on the outcomes and conclusions, so this is not trivial. One may say that this cannot explain the group effects that have been observed, given the fact that all groups have been pre-processed in the same way. However, the mPFC group of interest seems to display relatively high unconditioned responses - standardising with these measures may result in relatively low conditioned responses in this particular group. This shortcoming is therefore closely related to the point made above: given that conditioning data would be standardised in the same vein, a test that included the within-subject comparison between acquisition and post-intervention is absolutely crucial to ascertain that the effects observed are not merely due to coincidences in pre-processing values and pre-existing group differences.

- In addition to the above-described main analyses, some other potential weaknesses concern the analysis strategy applied to the generalization tests. Several ANOVAS are being run, one to test for the pattern of generalization responses within-subjects (i.e., the CS, NS1 and NS2), and several ones to compare each of these between the three groups. But such analyses are not warranted in the absence of an overall interaction between the within subject factors and group factor. Such overall omnibus tests however are lacking, and the high number of separate anovas risks false positives (i.e., these comparisons should have been made with planned contrasts). The fact that the included factors and levels are not being described, makes it generally hard to gauge what variables exactly have been entered in every analysis.

Further remarks:<br /> - There is a possibility that a re-analysis of the data using properly preprocessed SCR data along with analyses that include comparisons with the conditioned responses during acquisition reveal a different pattern of results. Therefore, whether the authors truly achieved their aims and whether the results support their conclusions is as of yet undecided.

- Even if the pattern of results holds, then the claim that the long-term follow-up reductions of fear were achieved in the absence of any extinction cannot be made with confidence: after all, upon mPFC stimulation during the second session, the CS was presented four times, and so were each of the two generalization stimuli. So perhaps extinction was not complete, but almost certainly some extinction has taken place: it is well-known that the strongest extinction-learning typically takes place in the first trials (e.g., due to higher prediction errors). The authors do not give any alternative theoretical explanation for the enduring reduction of fear reduction, which would be interesting to learn their thoughts on.

- If the results hold and satisfiable reasons are provided as to why the effects remain visible in the follow-up, this study could be a valuable contribution to the field: it may refocus future studies to the mPFC as major target to not only promote acute fear regulation, but perhaps even more importantly form a clinical perspective, a route for enduring fear reductions.

Reviewer #1 (Public Review):

In this manuscript, the Authors implement a delayed feedback control method and use it for the first time in biological neuronal networks. They extend a well-established computational theory and expand it into the biological realm. With this, they obtain novel evidence, never considered before, that showcases the difference between simulated neuronal networks and biological ones. Furthermore, they optimize the DFC method to achieve optimal results in the control of cell excitability in the content of biological neuronal networks, taking advantage of a closed-loop stimulation setup that, by itself, is not trivial to build and operate and that will certainly have a positive impact the fields of cellular and network electrophysiology.

Regarding the results, it would be very constructive if the Authors could share the code for the quasi-real-time interface with the Multichannel Systems software (current and older hardware versions), as this represents likely a bottleneck preventing more researchers to implement such an experimental paradigm.

On the data focusing on the effects of the DFC algorithms on neuronal behavior, the evidence is very compelling, although more care should be devoted to the statistical analyses, since some of the applied statistical tests are not appropriate. In a more biological sense, further discussion and clarification of the experimental details would improve this manuscript, making it more accessible and clearer for researchers across disciplines (i.e., ranging from computational to experimental Neuroscience) and increasing the impact of this research.

In summary, this work represents a necessary bridge between recent advances in computational neuroscience and the biological implementation of neuronal control mechanisms.

Reviewer #1 (Public Review):

The manuscript by Grove and colleagues analyzes the role of TEAD1 transcription factors in all events regulating PNS myelin formation and maintenance and regeneration. Throughout the manuscript, the authors compare the results obtained to those they previously described in YAP/TAZ double knockout mice. Strengths of the manuscript are combined in vivo analyses by generating mutants constitutively lacking TEAD1 expression in myelinating Schwann cells (P0Cre//TEAD1f/f mice: cKO) and mutants in which TEAD1 expression can be ablated after tamoxifen-mediated recombination is myelinating Schwann cells (PlpCreER//TEAD1f/f mice: iKO). Using this approach the authors were able to assess the role of TEAD1 in all aspects related to PNS myelin: formation as well as maintenance and remyelination after injury. By exploiting these models, they were able to define the role of TEAD1 in regulating Schwann cell proliferation as well as in the cholesterol biosynthetic pathway.

Collectively, their data indicate that TEAD 1 has a composite role in PNS myelination being required for developmental myelination, but dispensable for myelin maintenance. Further, they also describe a role for TEAD1 in promoting PNS remyelination after an injury event.

Despite these strengths, there are some weaknesses that should be addressed by the authors:

1. The manuscript would benefit from better and more detailed analysis of the role of the other TEAD transcription factors, as they are likely redundant in function to TEAD1. For example, since in cKO mice some fibers can escape the sorting defect and eventually myelinate, albeit at a lower level, could they determine whether TEAD2-4 transcription factors might compensate for TEAD1 absence in this setting?

2. A striking result of the study is the morphological defects observed in the process of axonal sorting and in the Remak fibers formation of TEAD1 cKO mice. To explain the sorting defect, the authors correctly analyze Schwann cell proliferation. However, since axonal sorting is mediated by the interaction between the extracellular matrix and intracellular cytoskeleton rearrangement they should address also these two aspects. As per the Remak bundles and the poly-axonal myelination they observe, it is difficult to reconcile this "abnormal" myelination with the fact that TEAD 1 cKO mice have a very severe myelinating phenotype, which is persistent in adulthood.

3. In the analyses of the cholesterol biosynthetic pathway, TEAD1 seems to be only partly involved. Again, which is the role of any of the other TEADs?

4. Why do cKO mice die before P60?

Reviewer #2 (Public Review):

In this paper, the authors discover that postsynaptic mitochondria in C. elegans govern glutamate receptor trafficking dynamics. The core results are two-fold. For one, they find that loss or inhibition of mcu-1 - the C. elegans mitochondrial calcium uniporter - increases GLR-1 glutamate receptor accumulation at the postsynaptic dendritic sites and enhances its trafficking dynamics. The authors hypothesize that this effect on glutamate receptors may have something to do with mitochondrial ROS production. This is because ROS is a by-product of normal oxidative phosphorylation, downstream of calcium import. Indeed, the generation of artificially high amounts of mitochondrial ROS has the opposite effect of mcu-1 loss: decreased glutamate receptor subunit accumulation. Collectively, the results support the idea that mitochondrial function can control receptor dynamics at synaptic sites. This is interesting because tight control of synaptic function likely combines several mitochondrial functions: energy production, calcium buffering, and (here) ROS signaling.

STRENGTHS

• The C. elegans genetic model is a strength because the authors are able to make refined conclusions by classical loss-of-function mutants (e.g., mcu-1) along with an impressive cytological toolkit to examine GLR-1 dynamics.

• The use of pharmacology as a second means to test those genetic conclusions is a strength.

• The authors' careful reagent verification of reporters (Ca2+, ROS, etc.) is a strength.

• The ability to link fundamental mitochondrial processes to GLR-1 exocytosis will expand how the field thinks about mitochondrial synapse function.

WEAKNESSES

For the most part, the data in the paper support the conclusions, and the authors were careful to try experiments in multiple ways. But please see below:

• (Main Point) The data are good, but they fall short of mechanism (e.g., Line 322). Figure 6 is accurate as drawn. But calcium and ROS are not abstract signals. They are likely exerting affirmative actions on specific targets. The Discussion does acknowledge this in terms of ROS and it speculates on possible targets.

The general idea seems to be that mitochondria import calcium through MCU-1 (and interacting factors). As a result, oxidative phosphorylation successfully occurs and mitochondrial ROS is a signaling by-product that signals glutamate receptors not to undergo exocytosis. But there are other interpretations of what might happen in between. In fact, if OXPHOS is disrupted, it is known that this can generate a lot more mitochondrial ROS than the normal by-product levels.

This reviewer wonders if excess ROS would cause an extreme response. Or alternatively, if scavenging ROS via pharmacological scavengers or SOD expression would reverse the effects.

Small Points

• 33.3 mHz - just making sure, do the authors mean once every 30 seconds? That would be more straightforward.

• Figure 2 is confusing. The text says that the mcu-1 mutants have a GLR-1::GFP FRAP rate that is comparable to controls (Lines 165-167). But Figure 2E suggests that it is markedly less, which is the opposite result of the slight increase in rate resulting from Ru360 treatment. And is the explanation why the GLR-1::GFP results differ from the SEP::GLR-1 results a difference between total GFP vs. surface GFP?

• I could not watch Video 2 (not sure if it is the file or just the copy I downloaded).

• It is good that the authors tried both optical stimulation and mechanical stimulation (dropping culture plates to stimulate the worms, Figure 3). Why was the mechanical stimulation set aside for further tests in the paper?

• Does this process affect all kinds of transport, or is it just the glutamate receptors? Was anything else examined?

Reviewer #1 (Public Review):

Payne et al. have investigated the neural basis of VOR adaptation with the goal of constraining sites and mechanisms of plasticity supporting cerebellar learning. This has been an area of intense debate for decades; previous competing models have argued extensively about the sites of plasticity and the strength of eye velocity feedback/ efference copy signals to Purkinje cells has been central to the debate. This paper nicely explores the consequences of varying the strength of this feedback and in so doing, provides a potential explanation for why Purkinje cell responses during VOR cancellation could exhibit stronger responses following learning, despite net depression of the strength of their vestibular inputs. In that sense it provides some reconciliation of existing models. The work appears to be well done and the paper is well written. The manuscript could be improved and the significance of the work clarified and enhanced by contextualizing the work more appropriately within the existing literature in this area.

Reviewer #1 (Public Review):

This interesting study by Miyano combines slice electrophysiology and superresolution microscopy to address the role of RBP2 in Ca2+ channel clustering and neurotransmitter release at hippocampal mossy fiber terminals. While a number of studies demonstrated a critical role for RBPs in clustering Ca2+ channels at other synapses and some provided evidence for a role of the protein in molecular coupling of Ca2+ channels and release sites, the present study targets another key synapse that is an important model for presynaptic studies and offers access to a microdomain controlled synaptic vesicle (SV) release mechanism with low initial release probability.

Summarizing a large body of high quality work, the authors demonstrate reduced Ca2+ currents and a reduced release probability. They attribute the latter to the reduced Ca2+ influx and can restore release by increasing Ca2+ influx. Moreover they propose an altered fusion competence of the SVs, which is not so strongly supported by the data in my view.

The effects are relatively small, but I think the careful analysis of the RBP role at the mossy fiber synapse is an important contribution.

Share an example of a technology you frequently use and share some of its advantages and disadvantages.

Joint Public Review:

In this manuscript, the authors examined the role of transcription readout and intron retention in increasing transcription of transposable elements during aging in mammals. It is assumed that most transposable elements have lost the regulatory elements necessary for transcription activation. Using available RNA-seq datasets, the authors showed that an increase in intron retention and readthrough transcription during aging contributes to an increase in the number of transcripts containing transposable elements.

Previously, it was assumed that the activation of transposable elements during aging is a consequence of a gradual imbalance of transcriptional repression and a decrease in the functionality of heterochromatin (de repression of transcription in heterochromatin). Therefore, this is an interesting study with important novel conclusion.

The authors revised the manuscript in accordance with the comments. Overall, the manuscript is useful because it shows that there is no direct connection between increased levels of transposon RNA and aging, and further demonstrates the disorganization of the transcriptional apparatus during aging.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #1 (Public Review):

The authors present a study focused on addressing the key challenge in drug discovery, which is the optimization of absorption and affinity properties of small molecules through in silico methods. They propose active learning as a strategy for optimizing these properties and describe the development of two novel active learning batch selection methods. The methods are tested on various public datasets with different optimization goals and sizes, and new affinity datasets are curated to provide up-to-date experimental information. The authors claim that their active learning methods outperform existing batch selection methods, potentially reducing the number of experiments required to achieve the same model performance. They also emphasize the general applicability of their methods, including compatibility with popular packages like DeepChem.

Strengths:

Relevance and Importance: The study addresses a significant challenge in the field of drug discovery, highlighting the importance of optimizing absorption and affinity properties of small molecules through in silico methods. This topic is of great interest to researchers and pharmaceutical industries.

Novelty: The development of two novel active learning batch selection methods is a commendable contribution. The study also adds value by curating new affinity datasets that provide chronological information on state-of-the-art experimental strategies.<br /> Comprehensive Evaluation: Testing the proposed methods on multiple public datasets with varying optimization goals and sizes enhances the credibility and generalizability of the findings. The focus on comparing the performance of the new methods against existing batch selection methods further strengthens the evaluation.

Weaknesses:

Lack of Technical Details: The feedback lacks specific technical details regarding the developed active learning batch selection methods. Information such as the underlying algorithms, implementation specifics, and key design choices should be provided to enable readers to understand and evaluate the methods thoroughly.

Evaluation Metrics: The feedback does not mention the specific evaluation metrics used to assess the performance of the proposed methods. The authors should clarify the criteria employed to compare their methods against existing batch selection methods and demonstrate the statistical significance of the observed improvements.

Reproducibility: While the authors claim that their methods can be used with any package, including DeepChem, no mention is made of providing the necessary code or resources to reproduce the experiments. Including code repositories or detailed instructions would enhance the reproducibility and practical utility of the study.

Suggestions for Improvement:

Elaborate on the Methodology: Provide an in-depth explanation of the two active learning batch selection methods, including algorithmic details, implementation considerations, and any specific assumptions made. This will enable readers to better comprehend and evaluate the proposed techniques.

Clarify Evaluation Metrics: Clearly specify the evaluation metrics employed in the study to measure the performance of the active learning methods. Additionally, conduct statistical tests to establish the significance of the improvements observed over existing batch selection methods.

Enhance Reproducibility: To facilitate the reproducibility of the study, consider sharing the code, data, and resources necessary for readers to replicate the experiments. This will allow researchers in the field to validate and build upon your work more effectively.

Conclusion:<br /> The authors' study on active learning methods for optimizing drug discovery presents an important and relevant contribution to the field. The proposed batch selection methods and curated affinity datasets hold promise for improving the efficiency of drug discovery processes. However, to strengthen the study, it is crucial to provide more technical details, clarify evaluation metrics, and enhance reproducibility by sharing code and resources. Addressing these limitations will further enhance the value and impact of the research.

Reviewer #2 (Public Review):

Summary:

This paper tests the idea that schooling can provide an energetic advantage over solitary swimming. The present study measures oxygen consumption over a wide range of speeds, to determine the differences in aerobic and anaerobic cost of swimming, providing a potentially valuable addition to the literature related to the advantages of group living.

Strengths:<br /> The strength of this paper is related to providing direct measurements of the energetics (oxygen consumption) of fish while swimming in a group vs solitary. The energetic advantages of schooling has been claimed to be one of the major advantages of schooling and therefore a direct energetic assessment is a useful result.

Weaknesses:

1) Regarding the fish to water volume ratio, the arguments raised by the authors are valid. However, the ratio used is still quite high (as high as >2000 in solitary fish), much higher than that recommended by Svendsen et al (2006). Hence this point needs to be discussed in the ms (summarising the points raised in the authors' response)

2) Wall effects: Fish in a school may have been swimming closer to the wall. The fact that the convex hull volume of the fish school did not change as speed increased is not a demonstration that fish were not closer to the wall, nor is it a demonstration that wall effect were not present. Therefore the issue of potential wall effects is a weakness of this paper.

3) The authors stated "Because we took high-speed videos simultaneously with the respirometry measurements, we can state unequivocally that individual fish within the school did not swim closer to the walls than solitary fish over the testing period". This is however not quantified.

4) Statistical analysis. The authors have dealt satisfactorily with most of the comments.<br /> However :<br /> (a) the following comment has not been dealt with directly in the ms "One can see from the graphs that schooling MO2 tends to have a smaller SD than solitary data. This may well be due to the fact that schooling data are based on 5 points (five schools) and each point is the result of the MO2 of five fish, thereby reducing the variability compared to solitary fish."<br /> (b) Different sizes were used for solitary and schooling fishes. The authors justify using larger fish as solitary to provide a better ratio of respirometer volume to fish volume in the tests on individual fish. However, mass scaling for tail beat frequency was not provided. Although (1) this is because of lack of data for this species and (2) using scaling exponent of distant species would introduce errors of unknown magnitude, this is still a weakness of the paper that needs to be acknowledged here and in the ms.

Reviewer #1 (Public Review):

Summary:<br /> The authors describe their work on finding optimal ways of infusing organoids into mice. They describe five delivery methods and compare organoid survival two weeks after delivery. This work is concluded with the use of a vascularized chamber being the most optimal for organoid viability.

Strengths:<br /> The aim is to have a preclinical, translational model to test methods of organoid infusion. This is important and timely to the field.

Weaknesses:<br /> - A clear aim seems to be missing, although I can extract this from the manuscript. The approach is described a bit cryptically. The manuscript could use a bit more explanation here and there.<br /> - Although the authors themselves argue in the introduction that the use of mice is not optimal, they show a mouse study in which human-derived iPSC organoids are infused in mice.<br /> - As far as I can extract from the Methods section, only one iPSC line was used. Given the huge donor variance, it is essential to repeat the work with multiple iPSC lines.<br /> - I am missing the right control groups, especially for the surgical groups. And the group size is very variable (3 to 7 mice per group). Three per group is then somewhat small in size.

Reviewer #1 (Public Review):

This is a very well-written and performed study describing a TOPBP1 separation of function mutation, resulting in defective MSCI maintenance but normal sex body formation. The phenotype differs from that of a previous TOPBP1 null allele, in which both MSCI and sex body formation were defective. Additional defects in CHK phosphorylation and SETX localization are also described.

Strengths:

The study is very rigorous, with a remarkably large number spectrum of techniques deployed to support the conclusions

Weaknesses

The study claims that MSCI is initiated but not maintained in the mutant. I think alternative hypotheses are possible.

Reviewer #1 (Public Review):

Summary:

In this study, the authors developed a mathematical model to predict human biological ages using physiological traits. This model provides a way to identify environmental and genetic factors that impact aging and lifespan.

Strengths:

1. The topic addressed by the authors - human age predication using physiological traits - is an extremely interesting, important, and challenging question in the aging field. One of the biggest challenges is the lack of well-controlled data from a large number of humans. However, the authors took this challenge and tried their best to extract useful information from available data.

2. Some of the findings can provide valuable guidelines for future experimental design for human and animal studies. For example, it was found that this mathematical model can best predict age when all different organ and physiological systems are sampled. This finding makes sense in general but can be, and has been, neglected when people use molecular markers to predict age. Most of those studies have used only one molecular trait or different traits from one tissue.

Weaknesses:

1. As I mentioned above, the Biobank data used here are not designed for this current study, so there are many limitations for model development using these data, e.g., missing data points and irrelevant measurements for aging. This is a common caveat for human studies and has been discussed by the authors.

2. There is no validation dataset to verify the proposed model. The authors suggested that human biological age can be predicted with high accuracy using 12 simple physiological measurements. It will be super useful and convincing if another biobank dataset containing those 12 traits can be applied to the current model.

Reviewer #1 (Public Review):

The manuscript by Kadkova et al. describes an electrophysiological analysis of 3 neurodevelopmental disease-causing SNAP-25 mutations in hippocampal neuron autaptic cultures. The work expands on a prior study of these 3 mutations, along with several others in SNAP-25, that was performed in acutely dissociated hippocampal cultures by another group (Alten et al, 2021). Most of the physiology defects found are pretty similar for the 3 mutations the two research groups characterized, with differences largely found in the effects on the size of the readily releasable pool (RRP) of SVs. These differences could be due to technical differences in the approach but are also likely to reflect in part differences in autapses as a model that have been previously described. In addition to the physiological analysis in cultured neurons, the current work extends the analysis beyond the prior study by analyzing the effects of these SNAP-25 mutations in in vitro liposome fusion assays with purified proteins, and some modeling of the effects on energy landscapes during priming and fusion.

The authors use lentiviral expression of wildtype or one of the 3 mutants in SNAP-25 autaptic neurons and assay neuronal survival and synaptic output. The authors also combine wildtype with each of the 3 mutants as well, given these diseases manifest as spontaneous mutations in only 1 of the SNAP-25 alleles, suggesting a dominant effect. The authors observe that the V48F and D166Y alleles (that are suggested to disrupt the Syt1-SNAP-25/SNARE interface) result in a very large increase in spontaneous release that exceeds the Syt1 null mutant alone, suggesting an effect on spontaneous SV release beyond a lack of Syt1 regulation of SNARE-mediated fusion. In contrast, Syt1 nulls have a much more severe loss of evoked release, through both V48F and D166Y also have modest decreases in release. They find both mutants also cause a decrease in the RRP. Applying some modeling for these results, the authors suggest V48F and D166Y lowers the energy barrier for fusion, creating the enhanced spontaneous release rates and causing a decrease of the RRP. They also find evidence for reduced SV priming. In contrast, a SNAP-25 I167N disease mutation in the SNARE assembly interaction layer causes dramatic decreases in both evoked and spontaneous release, consistent with a disruption to SNARE assembly/stability. In vitro fusion assays with these mutant SNAP-25 alleles was also done and provided supportive evidence for these interpretations for all 3 alleles. The ability to control calcium, Syt1, PIP2 and Complexin levels in the in vitro assays provided additional information on defining the precise steps of the fusion process these mutations disrupt. Together, the study indicates the I167N mutation acts as a dominant-negative allele to block fusion, while the other two alleles have both loss- and gain-of function properties that cause more complex disruptions that decrease evoked release while dramatically enhancing spontaneous fusion.

Overall, these results build on prior work and shed light on how disruptions to the SNAP-25 t-SNARE alter the process of SV priming and fusion.

Reviewer #1 (Public Review):

Summary:<br /> Moon et al analyse ECoG data obtained during speech listening and focus on the relationship of two aspects: 1) delays between voltage signals at individual electrodes to other electrodes in the vicinity and 2) the power of those signals in a range of spectral bands. They find that high power in frequencies below 30 Hz is correlated with longer delays. They further look for this pattern of results in an oscillator model.

Strengths:<br /> The manuscript examines whether a finding made in cats in the late 90s generalises to intracranial recordings from humans. Specifically, the amplitude of low-frequency oscillations should be related to the delay of cross-correlation between areas. The authors find evidence for such a relationship and show this in individual participants. After inspecting this phenomenon from many different angles, they also added an oscillator model and claimed that they found a similar pattern there. As such, the manuscript reports an extensive body of work carried out on high-quality data.

Weaknesses:<br /> The manuscript's readability and flow could be optimised: terms are used that aren't explained, and the structure seems somewhat convoluted. Showing single-subject results is laudable, however, the authors could consider adding group results that integrate across participants, and perhaps relaying single-participant plots to the supplemental material. The manuscript would benefit if analyses were motivated more clearly. Sometimes, I am unsure why a given analysis was carried out, why it was carried out in a specific way, and what question it was intended to answer.

Reviewer #1 (Public Review):

Summary:<br /> In this important work, the authors propose and test a model for the control of murine ultrasonic vocalizations (USV) in which two independent mechanisms, involving changes in laryngeal opening or airflow, control vocal tone. They present compelling experimental evidence for this dual control model by demonstrating the ability of freely behaving adult mice to generate vocalizations with various intonations by modulating both the breathing pattern and the laryngeal muscles. They also present novel evidence that these mechanisms are encoded in the brainstem vocalization central neural pattern generator, particularly in the component in the medulla called the intermediate reticular oscillator (iRO). The results presented clearly advance understanding of the developmental nature of the iRO, its ability to intrinsically generate and control many of the dynamic features of USV including those related to intonation, and its coordination with/control of expiratory airflow patterns. This work will interest neuroscientists investigating the neural generation and control of vocalization, breathing, and more generally, neuromotor control mechanisms.

Strengths:<br /> Important features and novelty of this work include:

1) The study employs an effective combination of anatomical, molecular, and functional/ behavioral approaches to examine the hypothesis and provide novel data indicating that variations in expiratory airflow can change the pitch patterns of adult murine USV.

2) The results significantly extend the authors' previous work that identified the iRO in neonatal mice by now presenting data that functionally demonstrates the existence of the critical Penk+Vglut2+ iRO neurons in adult mice, indicating that the iRO neurons maintain their function in generating vocalization throughout development.

3) The results convincingly demonstrate that the iRO neurons encode and can generate vocalizations by modulating both breathing and the laryngeal muscles.

4) The anatomical mapping and tracing results establish an important set of input and output circuit connections to the iRO, including input from the vocalization-promoting subregions of the midbrain periaqueductal gray (PAG), as well as output axonal projections to laryngeal motoneurons, and to the respiratory rhythm generator in the preBötzinger complex.

5) These studies advance the important concept that the brainstem vocalization pattern generator integrates with the medullary respiratory pattern generator to control expiratory airflow as a key mechanism to produce various USV types characterized by different pitch patterns.

Weaknesses:<br /> A limitation is that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow have not been fully worked out, requiring future studies.

Reviewer #1 (Public Review):

Summary:<br /> This work studies spatiotemporal patterns of structure-function coupling in developing brains, using a large set of imaging data acquired from children and young adults aged 5-22. Magnetic resonance imaging data of brain structure and function were obtained from a publicly available database, from which structural and functional features and measures were derived. The authors examined the spatial patterns of structure-function coupling and how they evolve with brain development. This work further examined correlations between brain structure-function coupling and behaviour, and explored evolutionary, microarchitectural and genetic bases that could potentially account for the observed patterns.

Strengths:<br /> The strength of this work is the use of currently available state-of-the-art analysis methods, along with a large set of high-quality imaging data, and comprehensive examination of structure-function coupling in developing brains. The results are comprehensive and illuminative.

Weakness:<br /> As in most other studies, transcriptomic and cellular architectures of structure-function coupling were characterized only on the basis of a common atlas in this work.

The authors have achieved their aims in this study, and the findings provide mechanistic insights into brain development, which could inspire further basic and clinical studies along this line.

Reviewer #1 (Public Review):

He et al. investigate the requirement and function of Blimp1 (encoded by Prdm1) in murine NK cells and ILC1. Employing a conditional knockout mouse model (Prdm1flox x Ncr1cre), the authors describe impaired abundance and maturation of Prdm1-deficient NK cells and ILC1 in different tissues. Blimp1-deficient NK cells have reduced expression of cytotoxic molecules (Gzmb, Prf1) and, in some instances, Ifng production, and Prdm1flox x Ncr1cre mice show impaired tumor control in experimental metastasis models. Using single-cell RNA sequencing analysis, the authors propose that Prdm1 regulates JunB expression and NK cell maturation. Based on in silico analyses, the authors suggest manifold intercellular communication between NK/ILC1 and macrophages. Without following up on any of these potentially interesting suggestions, the authors conclude their study reiterating that Prdm1 regulates IFNg-production of tumor-infiltrating NK cells and ILC1.

Many of the reported functions of Blimp1 in NK cells have previously been identified using a mixed-chimera strategy comparing Prdm1 WT and KO NK cells (Kallies et al., Blood 2011). Here, the authors expand on these findings using a conditional model to delete Prdm1 in NK/ILC1 and single-cell sequencing and provide a more refined analysis of the functions of Blimp1 in these cells. Cell-chat analysis suggests close interactions of Blimp-dependent NK/ILC1 subsets with hepatic macrophages, but these suggestions are not followed up by experiments. Potentially interesting differences in the macrophage compartment of Ncr1-Cre x Prdm1-fl/fl mice are suggested by the scc-RNA-Seq data but are not validated e.g. by FACS. The study falls short in providing new mechanistic insights. Nevertheless, it is an interesting confirmation of "old" suggestions in a more refined setting, and the provided single-cell mRNA-Seq data represents a potentially valuable resource for the community. There are some control analyses that are required to support the conclusions of the authors, and I have a few suggestions that would help to improve the manuscript.

Major comments:

- The authors do not control for the potential effects of Cre expression. Expression of Cre from within the Ncr1 locus (using the mouse model established by Narni-Mancinelli et al.) has significant effects on NK cells and especially ILC1s (reducing their frequency and absolute numbers and altering their functionality. The authors should characterize the Ncr1cre mice used here (developed by Shanghai Model Organism Center) in this regard and should use proper controls (Ncr1Cre+ Prdm1wt/wt as control for Ncr1Cre+ Prdm1fl/fl, instead of WT littermates) for all of their key data, e.g. those depicted in Fig 1FG, 2ADFH, 7D, S2,3,4.

- Several of the phenotypic findings on NK cells have been described before by Kallies et al. in 2011 (Ref 29), although using a different genetic Prdm1-ablation model (Prdm1-GFP/GFP knockin/knockout model). This study reported impaired NK cell maturation, reduced Gzmb expression, impaired in vivo cytotoxicity against subcutaneous RMA-S cells, impaired in vitro proliferation, comparable in vitro killing, increase in BM NK cell numbers. The authors should discuss/mention this more prominently in their manuscript, and highlight where they confirm or refine these previous findings, and where they actually provide new information.

- What is the reason to refer to the enriched cluster in Blimp1-deficient NK cells as "Junbhi"? There is no follow-up for a function of Junb, and there are many other genes upregulated in these cells. Most critically, these cells seem to represent exactly the c-Kithi cells that Kallies et al. already showed and discussed in their paper. The authors should stain for Kit, and also refer to this. Also, MacKay et al. performed Blimp1-Chip-Seq (in T cells), maybe it would be interesting to check to which of the identified DEGs Blimp1 can bind.

- cNK cells are considered circulating cells, that transiently pass through the liver. Previous studies have suggested almost identical gene expression patterns in hepatic and splenic NK cells. In functional tests, they often "perform" identically. I am therefore a bit surprised that the authors find a differential dependency of Blimp1 for the IFNg production of splenic (no role of Blimp1) versus hepatic (Blimp1 regulating IFNg production) NK cells (Fig S3). Do the authors have any suggestions on that? The analyses are performed by 12+4h stimulations with IL12/18, which could involve the effects of altered bystander cells (as suggested by Figure 6). Therefore, these analyses should be provided upon standard 4h stimulations with IL12/18 and also with PMA/I under BFA. Note: liver and splenic cNK cells look quite different in the chosen histograms in Figures 7 A, B, C, yet there is massive variability in these analyses - is there any systematic/technical problem?

- Figure 4 H/I - In contrast to NK cells in Fig 4E, F, the KO and WT ILC1s seem to co-cluster largely. Authors should validate differentially expressed genes. How strong is the effect of Blimp1 in ILC1s? Or is Blimp1 a critical TF driving effector differentiation in NK cells, while it has only subtle effects in ILC1 (these may be regulated by Hobit?)? This seems an interesting finding that should at least be discussed. For these types of small differences in ILC1, FACS confirmation analyses should be performed and findings be reevaluated using Cre-expressing controls (see above).

The authors describe and discuss some of Figure 1 and 2 data as if Blimp1 would be involved in alternative NK versus ILC1 fates, but there is no evidence for this.

- There are several recent studies suggesting a role for Hobit, homologue of Blimp1, in NK cells and in ILC1, and in the control of liver metastases. The authors should discuss similar and unique functions of Hobit and Blimp1, also in the regulation of gene expression patterns, and should refer to these studies.

- Figure 4: The authors should discuss (and cross-validate) their liver gene expression analyses in the context of published datasets of NK and ILC1, such as the ones by Lopez et al, Friedrich et al, Ducimetiere et al and Yomogida et al.

Reviewer #1 (Public Review):

Summary:<br /> Functionally important alternative isoforms are gold nuggets found in a swamp of errors produced by the splicing machinery.

The architecture of eukaryotic genomes, when compared with prokaryotes, is characterised by a preponderance of introns. These elements, which are still present within transcripts, are rapidly removed during the splicing of messenger RNA (mRNA), thus not contributing to the final protein. The extreme rarity of introns in prokaryotes, and the elimination of these introns from mRNAs before translation into protein, raises questions about the function of introns in genomes. One explanation comes from functional biology: introns are thought to be involved in post-transcriptional regulation and in the production of translational variants. The latter function is possible when the positions of the edges of the spliced intron vary. While some light has been shed on specific examples of the functional role of alternative splicing, to what extent are they representative of all introns in metazoans?

In this study, the hypothesis of a functional role for alternative splicing, and therefore to a certain extent for introns, is evaluated against another explanation coming from evolutionary biology: isoforms are above all errors of imprecision by the molecular machinery at work during splicing. This hypothesis is based on a principle established by Motoo Mikura, which has become central to population genetics, explaining that the evolutionary trajectory of a mutation with a given effect is intimately linked to the effective population size (Ne) where this mutation emerges. Thus, the probability of fixation of a weakly deleterious mutation increases when Ne decreases, and the probability of fixation of a weakly advantageous mutation increases when Ne increases. The genomes of populations with low Ne are therefore expected to accumulate more weakly deleterious mutations and fewer weakly advantageous mutations than populations with high Ne. In this framework, if splicing errors have only small effects on the fitness of individuals, then natural selection cannot increase the precision of the splicing machinery, allowing tolerance for the production of alternative isoforms.

In the past, the debate opposed one-off observations of effectively functional isoforms on the one hand, to global genomic quantities describing patterns without the possibility of interpreting them in detail. The authors here propose an elegant quantitative approach in line with the expected continuous variation in the effectiveness of selection, both between species and within genomes. The result describing the inter-specific pattern on a large scale confirms what was already known (there is a negative relationship between effective size and average alternative splicing rate). The essential novelty of this study lies in 1) the quantification, for each intron studied, of the relative abundance of each isoform, and 2) the analysis of a relationship between this abundance and the evolutionary constraints acting on these isoforms.

What is striking is the light shed on the general very low abundance of alternative isoforms. Depending on the species, 60% to 96% of cases of alternatively spliced introns lead to an isoform whose abundance is less than 5% of the total variants for a given intron.

In addition to the fact that 60%-96% of the total isoforms are more than 20 times less abundant than their majority form, this large proportion of alternative isoforms exhibit coding-phase shift at rates similar to what would be expected by chance, i.e. for a third of them, which reinforces the idea that there is no particular constraint on these isoforms.

The remaining 4%-40% of isoforms see their coding-phase shift rate decrease as their relative abundance increases. This result represents a major step forward in our understanding of alternative splicing and makes it possible to establish a quantitative model directly linking the relative abundance of an isoform with a putative functional role concerning only those isoforms produced in abundance. Only the (rare) isoforms which are abundantly produced are thought to be involved in a biological function.

Within the same genome, the authors show that only highly expressed genes, i.e. those that tend to be more constrained on average, are also the genes with the lowest alternative splicing rates on average.

The comparison between species in this study reveals that the smaller the effective size of a species, the more its genome produces isoforms that are low in abundance and low in constraint. Conversely, species with a large effective size relatively reduce rare isoforms, and increase stress on abundant isoforms.

To sum up:<br /> • the higher the effective size of a species, the fewer introns are spliced.<br /> • highly expressed genes are spliced less.<br /> • when splicing occurs, it is mainly to produce low-abundance isoforms.<br /> • low-abundance isoforms are also less constrained.

Taken together, these results reinforce a quantitative view of the evolution of alternative splicing as being mainly the product of imprecision in the splicing machinery, generating a great deal of molecular noise. Then, out of all this noise, a few functional gold nuggets can sometimes emerge. From the point of view of the reviewer, the evolutionary dynamics of genomes are depressing. The small effective population sizes are responsible for the accumulation of multiple slightly deleterious introns. Admittedly, metazoan genomes try to get rid of these introns during RNA maturation, but this mechanism is itself rendered imprecise by population sizes.

Strengths:<br /> • The authors simultaneously study the effects of effective population size, isoform abundance, and gene expression levels on the evolutionary constraints acting on isoforms. Within this framework, they clearly show that an isoform becomes functionally important only under certain rare conditions.<br /> • The authors rule out an effect putatively linked to variations in expression between different organs which could have biased comparisons between different species.

Weaknesses:<br /> • While the longevity of organisms as a measure of effective size seems to work overall, it may not be relevant for discriminating within a clade. For example, within Hymenoptera, we might expect them to have the same overall longevity, but that effective size would be influenced more by the degree of sociality: solitary bees/ants/wasps versus eusocial. I am therefore certain that the relationship shown in Figure 4D is currently not significant because the measure of effective size is not relevant for Hymenoptera. The article would have been even more convincing by contrasting the rates of alternative splicing between solitary versus social hymenopterans.<br /> • When functionalist biologists emphasise the role of the complexity of living things, I'm not sure they're thinking of the comparison between "drosophila" and "homo sapiens", but rather of a broader evolutionary scale. Which gives the impression of an exaggeration of the debate in the introduction.

Reviewer #1 (Public Review):

Summary:

This important work advances our understanding of sperm motility regulation during fertilization by uncovering the midpiece/mitochondria contraction associated with motility cessation and structural changes in the midpiece actin network as its mode of action. The evidence supporting the conclusion is solid, with rigorous live cell imaging using state-of-the-art microscopy, although more functional analysis of the midpiece/mitochondria contraction would have further strengthened the study. The work will be of broad interest to cell biologists working on the cytoskeleton, mitochondria, cell fusion, and fertilization.

Strengths:

The authors demonstrate that structural changes in the flagellar midpiece F-actin network are concomitant to midpiece/mitochondrial contraction and motility arrest during sperm-egg fusion by rigorous live cell imaging using state-of-art microscopy.

Weaknesses:

Many interesting observations are listed as correlated or in time series but do not necessarily demonstrate the causality and it remains to be further tested whether the sperm undergoing midpiece contraction are those that fertilize or those that are not selected. Further elaboration of the function of the midpiece contraction associated with motility cessation (a major key discovery of the manuscript) would benefit from a more mechanistic study.

Reviewer #1 (Public Review):

Summary:

Numerous pathways have been proposed to elucidate the nongenomic actions of progesterone within both male and female reproductive tissues. The authors employed the Xenopus oocyte system to investigate the PLA2 activity of ABHD2 and the downstream lipid mediators in conjunction with mPRb and P4, on their significance in meiosis. The research has been conducted extensively and is presented clearly.

Strengths:

While the interaction between membranous PR and ABHD2 is not a novel concept, this present study exhibits several strengths:

1. mPRbeta, a member of the PAQR family, has been elusive in terms of detailed signal transduction. Through mutation studies involving the Zn binding domain, the authors discovered that the hydrolase activity of mPRbeta is not essential for meiosis and oocyte maturation. Instead, they suggest that ABHD2, acting as a coreceptor of mPRbeta, demonstrates phospholipase activity, indicating that downstream lipid mediators may play a dominant role when stimulated by progesterone.

2. Extensive exploration of downstream signaling pathways and the identification of several potential meiotic activity-related lipid mediators make this aspect of the study novel and potentially significant.

Weaknesses:

However, there are some weaknesses and areas that need further clarification:

1. The mechanism governing the molecular assembly of mPRbeta and ABHD2 remains unclear. Are they constitutively associated or is their association ligand-dependent? Does P4 bind not only to mPRbeta but also to ABHD2, as indicated in Figure 6J? In the latter case, the reviewer suggests that the authors conduct a binding experiment using labeled P4 with ABHD2 to confirm this interaction and assess any potential positive or negative cooperativity with a partner receptor.

2. The authors have diligently determined the metabolite profile using numerous egg cells. However, the interpretation of the results appears incomplete, and inconsistencies were noted between Figure 2B and Supplementary Figure 2C. Furthermore, PGE2 and D2 serve distinct roles and have different elution patterns by LC-MS/MS, thus requiring separate measurements. In addition, the extremely short half-life of PGI2 necessitates the measurement of its stable metabolite, 6-keto-PGF1a, instead. The authors also need to clarify why they measured PGF1a but not PGF2a.

3. Although they propose PGs, LPA, and S1P are important downstream mediators, the exact roles of the identified lipid mediators have not been clearly demonstrated, as receptor expression and activation were not demonstrated. While the authors showed S1PR3 expression and its importance by genetic manipulation, there was no observed change in S1P levels following P4 treatment (Supplementary Figure 2D). It is essential to identify which receptors (subtypes) are expressed and how downstream signaling pathways (PKA, Ca, MAPK, etc.) relate to oocyte phenotypes.

These clarifications and further experiments would enhance the overall impact and comprehensiveness of the study.

Reviewer #1 (Public Review):

Summary:

In this study, Yue et al. re-processed publicly available DNA methylation data (published in 2012 and 2017 from the Meissner lab) from pre- and post-implantation mouse embryos. Against the global wave of genome-wide reduction of DNA methylation occurring during pre-implantation development, they detected a slight increase (~1% on average) of DNA methylation at gene promoter regions during the transition from 8-cell to blastocyst stage. They claim that many such promoters are located in the X chromosome. Subsequently, they knocked down Dnmt3b (presumably because of its upregulation during the transition from the 8-cell to blastocyst stage) and detected the aberrant patterning of H3K27me3 in the mutant female embryos. Based on this observation, they claim that imprinted X-chromosome inactivation is impaired in the Dnmt3b-Kd pre-implantation embryos. Finally, they propose a model where such an increase of DNA methylation together with H3K27me3 regulates imprinted X-chromosome inactivation in the pre-implantation embryos. While their observation is of potential interest, the current version of the work fails to provide enough evidence to support their conclusions. Below are suggestions and comments on the manuscript.

Major issues:

1. Sex of the embryos of the genome-wide bisulfite-sequencing data<br /> The authors re-analyzed publicly available genome-wide DNA methylation data from the Meissner lab published in 2012 and 2017. The former used reduced representation bisulfite sequencing (RRBS) and the latter used whole-genome bisulfite sequencing (WGBS). Based mainly on the RRBS data, Yue et al. detected de novo DNA methylated promoters during the transition from 8-cell to blastocyst against the global wave of genome-wide DNA demethylation. They claim that such promoter regions are enriched at the "inactive" X chromosome. However, it would be difficult to discuss DNA methylation at inactive X-chromosomes as the RRBS data were derived from a mixture of male and female embryos. It would also be notable that the increase of DNA methylation at these promoter regions is ~1% on average. Such a slight increase in DNA methylation during pre-implantation development could also be due to the developmental variations between the embryos or between the sexes of embryos.

2. Imprinted X-chromosome inactivation and evaluation of H3K27me3 (related to Figures 2C, D; 3F; Figure2-supplement 2 F, G; Figure3-supplement 3G)<br /> Based on the slight change in the H3K27me3 signals in the Dnmt3b-Kd blastocysts, the authors claim that imprinted X-chromosome inactivation is impaired in the mutant embryo. It would be not easy to reach this conclusion from such a rough analysis of H3K27me3 presented in Figure 2C, D. Rigorous quantification/evaluation of the H3K27me3 signals in the Dnmt3b-Kd embryos should be considered. Additional evidence for the impairment of H3K27me3 in the mutant embryos should also be provided (expression of a subset of X-linked genes by RNA-FISH or RT-PCR etc.). Though technically challenging, high-resolution genome-wide approach such as ChIP-seq of H3K27me3 in the Dnmt3b-kd female embryos (with traceable SNPs between maternal and paternal X chromosome to distinguish inactive and active X-chromosome) could more precisely evaluate regions that lose H3K27me3 in the X-chromosome (de novo DNA methylated promoters from 8-cell to blastocyst, for example).

3. Analysis of the developmental potential of Dnmt3b-kd embryos<br /> While the authors claim that Dnmt3b-mediated de novo DNA methylation plays an important role in imprinted X-chromosome inactivation, it remains unclear whether the analysis presented in Figure 4 is derived from "female" embryos. This analysis seemed confusing as the authors claim that de novo DNA methylation in the promoter regions during the transition from 8-cell to blastocyst regulates imprinted X-chromosome inactivation, but this should not happen in the male embryos. Was the impairment of embryonic proliferation and differentiation observed in both male and female embryos? Or is this specific to the female embryos? We think that the sex of the embryos would be critical for the analysis presented in Figure 4.

Reviewer #1 (Public Review):

Summary:

This manuscript reports experiments designed to dissect the function of N-cadherin during mammalian folliculogenesis, using the mouse as a model system. Prior studies have shown that this is the principal cadherin expressed by the follicular granulosa cells. Two main strategies are used - small-molecule inhibitors that target N-cadherin and a conditional knockout where the gene encoding N-cad is deleted in granulosa cells. The authors also take advantage of the ability to reproduce key events of folliculogenesis, such as oocyte meiotic maturation, in vitro. Four main conclusions are drawn from the studies: (i) cadherin-based cell contact is required to maintain cadherin (N-cad in the granulosa cells; E-cad in the oocyte) at the plasma membrane; (ii) N-cad is required for cumulus layer expansion; (iii) N-cad is required for meiotic maturation of the oocyte; (iv) N-cad is required for ovulation.

Strengths:

The experiments are logically conceived, clearly described and presented, and carefully interpreted. A key strength of the paper is that multiple approaches have been used (drugs, knockouts, immunofluorescence, PLA, in vitro and in vivo studies). Taken together, they clearly establish essential roles for N-cadherin during folliculogenesis.

It is intriguing that, when cadherin activity is impaired, the cadherins are lost from the plasma membrane. This suggests that, in a multicellular context, interactions with other cadherins, either in cis within the same cell or in trans with a neighboring cell, are required to maintain cadherins at the membrane. Hence, beyond their significance for understanding female reproductive biology, these experiments have broader implications for cell biology.

Weaknesses:

A few points could be considered or clarified by the authors:

The YAP experiments were confusing to the reviewer. CRS-066 increased YAP activity, as indicated by increased expression of target genes. Since CRS-066 prevents expansion, this result suggests that YAP antagonizes expansion. Therefore, blocking YAP should favor expansion. Yet, the YAP inhibitor impaired expansion. In the reviewer's eyes, these results seem to be contradictory.

It is intriguing that the inhibitors were able to efficiently block oocyte maturation. Oocytes from which the cumulus granulosa cells have been removed (denuded) will mature in vitro in the absence of LH or EGF. Since the effect of the inhibitors is to break the contact between the cumulus cells and oocyte, one might have predicted that this would not impair the ability of the oocytes to mature. Perhaps the authors could comment on this.

Regarding the experiments where the inhibitors were administered intra-peritoneally, the authors might comment on the rationale for choosing the doses that were used. An additional point to consider is that, since N-cadherin is expressed in a variety of tissues, an effect of interfering with N-cadherin at these non-ovarian sites could indirectly influence ovarian function.

Reviewer #1 (Public Review):

Weinberger et al. use different fate-mapping models, the FIRE model and PLX-diet to follow and target different macrophage populations and combine them with single-cell data to understand their contribution to heart regeneration after I/R injury. This question has already been addressed by other groups in the field using different models. However, the major strength of this manuscript is the usage of the FIRE mouse model that, for the first time, allows specific targeting of only fetal-derived macrophages.

The data show that the absence of resident macrophages is not influencing infarct size but instead is altering the immune cell crosstalk in response to injury, which is in line with the current idea in the field that macrophages of different origins have distinct functions in tissues, especially after an injury.

To fully support the claims of the study, specific targeting of monocyte-derived macrophages or the inhibition of their influx at different stages after injury would be of high interest.

In summary, the study is well done and important for the field of cardiac injury. But it also provides a novel model (FIRE mice + RANK-Cre fate-mapping) for other tissues to study the function of fetal-derived macrophages while monocyte-derived macrophages remain intact.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Xiong L et al., the authors have uncovered an important link between innate immune signaling and hair regeneration. The authors provide convincing evidence supporting the critical roles of TLR2 in sensing CEP levels in hair follicles, counteracting the action of BMP signaling, and facilitating the activation of HFSCs during the hair cycle and wound repair. Importantly, the authors also propose that decreased CEP production and TLR2 expression might be factors contributing to the decreased hair regeneration associated with aging.

Strengths:

The experiments in this manuscript are well-designed and presented. The authors provided extensive evidence supporting the roles of TLR2 signaling in regulating hair follicle stem cell functions. Importantly, the findings from this paper could have sustained impacts on our understanding of the roles of innate immunity in regulating tissue regeneration in the absence of inflammation.

Weaknesses:

1. The central conclusion of this study is that the activation of TLR2 can suppress BMP signaling. However, the molecular link between TLR2 and BMP signaling is still missing. Given the importance of this finding, it would be intriguing to further investigate how TLR2 activation suppresses BMP signaling. A better characterization of the molecular-level interaction between TLR2 and BMP signaling can further enhance the impact of this study.

2. The authors imply that the decreased CEP level in aged mice could lead to deficient TLR2 signaling, which could further cause aging-associated hair regeneration defects. But this has not been demonstrated. What are the BMPs and pSmad1/5 levels in aged skin? Another important experiment to confirm the importance of this link during aging would be to inject CEP into the aged skin and examine whether this could restore hair regeneration in aged mice.

3. The impacts of CEP/TLR2 on proliferation of keratinocytes is still weak. How much of this effect is a result of NFkB activation, and how much is simply due to inhibiting BMP signaling?

Updated comments on the revised manuscript:<br /> The authors have addressed my previous questions.