Reviewer #2 (Public review):

Summary:

In this manuscript, the authors follow up on their previous work showing that in the absence of the Sir2 deacetylase the MCM replicative helicase at the rDNA spacer region is repositioned to a region of low nucleosome occupancy. Here they show that the repositioned displaced MCMs have increased firing propensity relative to non-displaced MCMs. In addition, they show that activation of the repositioned MCMs and low nucleosome occupancy in the adjacent region depend on the chromatin remodeling activity of Fun30.

Strengths:

The paper provides new information on the role of a conserved chromatin remodeling protein in regulation of origin firing and in addition provides evidence that not all loaded MCMs fire and that origin firing is regulated at a step downstream of MCM loading.

Comments on revisions:

The authors have addressed my concerns with the addition of new experiments and analysis.

Reviewer #2 (Public review):

Summary:

The study explores the functional consequence of CDK12 loss in prostate cancer. While CDK12 loss has been shown to confer homologous recombination (HR) deficiency through premature intronic polyadenylation of HR genes, the response of PARPi monotherapy has failed. This study therefore performed an in-depth analysis of genomic sequencing data from mCRPC patient tumors, and showed that tumors with CDK12 loss lack pertinent HR signatures and scars. Furthermore, functional exploration in human prostate cancer cell lines showed that while the acute inhibition of CDK12 resulted in aberrant polyadenylation of HR genes like BRCA1/2, HR-specific effects were overall modest or absent in cell lines or xenografts adapted to chronic CDK12 loss. Instead, vulnerability to genetically targeting CDK13 resulted in a synthetic lethality in tumors with CDK12 loss, as shown in vivo with SR4825, a CDK12/13 inhibitor - thus serving as a potential therapeutic avenue.

The evidence supporting this study is based on in-depth genomic analyses of human patients, acute knockdown studies of CDK12 using a CDK12/13 inhibitors SR4835, adaptive knockout of CDK12 using LuCaP 189.4_CL and inducible re-expression of CDK12, CDK12 single clones in 22Rv1 (KO2 and KO5) and Skov3 (KO1), Tet-inducible knockdown of BRCA2 or CDK12 followed by ionizing radiation and measurement of RAD51 foci, lack of sensitivity generally to PARPi and platinum chemotherapy in cells adapted to CDK12 loss, loss of viability of CDK13 knockout in CDK12 knockout cells, and in vivo testing of SE4825 in LuCaP xenografts with intact and CDK12 loss.

Strengths:

Overall, this study is robust and of interest to the broader homologous recombination and CDK field. First, the topic is clinically relevant given the lack of PARPi response in CDK12 loss tumors. Second, the strength of the genomic analysis in CDK12 lost PCa tumors is robust with clear delineation that BRCA1/2 genes and maintenance of most genes regulating HR are intact. Specifically, the authors find that there is no mutational signature or genomic features suggestive of HR, such as those found in BRCA1/2 tumors. Lastly, novel lines are generated in this study, including de novo LuCaP 189.4_CL with CDK12 loss that can be profound for potential synthetic lethalities.

Weakness:

One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?

While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12-K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout.

Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.

The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.

Reviewer #3 (Public review):

The manuscript presents interesting findings on the role of gut microbiota in gout, focusing on the interplay between age-related changes, inflammation, and microbiota-derived metabolites, particularly butyrate. The study provides valuable insights into the therapeutic potential of microbiota interventions and metabolites for managing hyperuricemia and gout.

The manuscript has improved with the revisions made, particularly regarding clarifications on experimental design and the inclusion of supplementary data.

Comments on latest version:

The authors have addressed many previous concerns; however, some areas still require clarification and improvement to support more definitive conclusions.

(1) This study suggests that microbiota interventions, particularly butyrate, show promising therapeutic potential for hyperuricemia and gout. While the authors discuss the functions of certain butyrate-producing bacteria, I recommend further validating the gut microbiota-butyrate pathway by supplementing germ-free animal models with a single butyrate-producing strain, such as Clostridium butyricum. To strengthen the manuscript, I suggest the authors make further revisions to address these key issues.

(2) Additionally, I was unable to locate the full-length, uncropped Western blot images in the manuscript or supplementary materials. Could the authors please provide these?

Reviewer #3 (Public review):

Previous work (Chouhan et al., 2022) from the Sehgal group investigated the relationship between sleep and long-term memory formation by dissecting the role of mushroom body intrinsic neurons, extrinsic neurons, and output neurons during sleep-dependent and sleep-independent memory consolidation. In this manuscript, Li et al., profiled transcriptome in the anterior-posterior (ap) α'/β' neurons and identified genes that are differentially expressed after training in fed condition, which supports sleep-dependent memory formation. By knocking down candidate genes systematically, the authors identified Polr1F and Regnase-1 as two important hits that play potential roles in sleep and memory formation. What is the function of sleep and how to create a memory are two long-standing questions in science. The present study used a new approach to identify novel components that may link sleep and memory consolidation in a specific type of neuron. Importantly, these components implicated that RNA processing may play a role in these processes.

I am enthusiastic about the innovative approach employed to identify RNA processing genes involved in sleep regulation and memory consolidation. During the revision process, the authors fully addressed major concerns raised by reviewers. First, the author used the Gal80ts to restrict the knockdown of Regnase-1 in adult animals and concluded that Regnase-1 RNAi appears to affect sleep through development. Second, the author showed that Regnase-1 knockdown produced robust phenotypes for both sleep-dependent and sleep-independent memory, as well as a severe short-term memory phenotype. The author cautiously concluded that flies with constitutive Regnase-1 knockdown could be poor learners, thereby exhibiting a memory phenotype. Although we don't yet have a strong link between sleep and long-term memory consolidation, the interpretation presented in the manuscript is sufficiently justified by the data. This work presents a novel strategy to explore the link between sleep and memory consolidation.

Reviewer #2 (Public review):

This study utilizes an extensive series of neurulation human embryos to address several open questions about the similarities and differences between human primary and secondary neurulation in the tail. Results are compared to other model systems, such as the chicken and rodent. Histology, in situ hybridization, and apoptosis analysis provide molecular data about how the tail regresses in the human embryo. The number of embryos utilized for the analysis and the quality of the histological analysis provide robustness to the findings.

Comments on revised version:

The authors have meticulously addressed all the concerns raised by the reviewers, using new data and modifications to the text to further strengthen the quality of the manuscript.

This is a fabulous manuscript. I have nothing more scientifically to critique.

Reviewer #2 (Public review):

Summary:

The current work investigates the neural signature of category representation in infancy. Neural responses during steady-state visually-evoked potentials (ssVEPs) were recorded in four age groups of infants between 3 and 15 months. Stimuli (i.e., faces, limbs, corridors, characters, and cars) were presented at 4.286 Hz with category changes occurring at a frequency of 0.857 Hz. Results of the category frequency analyses showed that reliable responses to faces emerge around 4-6 months, whereas response to libs, corridors, and characters emerge around 6-8 months. Additionally, the authors trained a classifier for each category to assess how consistent the responses were across participants (leave-one-out approach). Spatiotemporal responses to faces were more consistent than the responses to the remaining categories and increased with increasing age. Faces showed an advantage over other categories in two additional measures (i.e., representation similarity and distinctiveness). Together, these results suggest a different developmental timing of category representation.

Strengths:

The study design is well organized. The authors described and performed analyses on several measures of neural categorization, including innovative approaches to assess the organization of neural responses. Results are in support of one of the two main hypotheses on the development of category representation described in the introduction. Specifically, the results suggest a different timing in the formation of category representations, with earlier and more robust responses emerging for faces over the remaining categories. Graphic representations and figures are very useful when reading the results. The inclusion of the adult sample and results further validate the approach utilized with infants.

Comments on revised submission:

The revised manuscript satisfactorily addressed all my previous comments.

Reviewer #2 (Public review):

Summary:

This is an outstanding piece of work on the potential of FLO as a viable analgesic biologic for the treatment of postsurgical pain. The authors purified the HC-HA/PTX3 from FLO and demonstrated its potential as an effective non-opioid therapy for postsurgical pain. They further unraveled the mechanisms of action of the compound at cellular and molecular levels.

Strengths:

Prominent strengths include the incorporation of behavioral assessment, electrophysiological and imaging recordings, the use of knockout and knockdown animals, and the use of antagonist agents to verify biological effects. The integrated use of these techniques, combined with the hypothesis-driven approach and logical reasoning, provides compelling evidence and novel insight into the mechanisms of the significant findings of this work.

Weaknesses:

I did not find any significant weaknesses even with a critical set of mind. The only minor suggestion is that the Results section may focus on the results from this study and minimize the discussions of background information.

Comments on revisions:

The authors have adequately addressed all the points raised in the last round of review. Thanks!

Reviewer #2 (Public review):

Summary:

The goal of this work is to define the functions of T-box transcription factors Tbx3 and Tbx5 in the adult mouse ventricular cardiac conduction system (VCS) using a novel conditional mouse allele in which both genes are targeted in cis. A series of studies over the past 2 decades by this group and others have shown that Tbx3 is a transcriptional repressor that patterns the conduction system by repressing genes associated with working myocardium, while Tbx5 is a potent transcriptional activator of "fast" conduction system genes in the VCS. In a previous work, the authors of the present study further demonstrated that Tbx3 and Tbx5 exhibit an epistatic relationship whereby the relief of Tbx3-mediated repression through VCS conditional haploinsufficiency allows better toleration of Tbx5 VCS haploinsufficiency. Conversely, excess Tbx3-mediated repression through overexpression results in disruption of the fast-conduction gene network despite normal levels of Tbx5. Based on these data the authors proposed a model in which repressive functions of Tbx3 drive the adoption of conduction system fate, followed by segregation into a fast-conducting VCS and slow-conduction AVN through modulation of the Tbx5/Tbx3 ratio in these respective tissue compartments.

The question motivating the present work is: If Tbx5/Tbx3 ratio is important for slow versus fast VCS identity, what happens when both genes are completely deleted from the VCS? Is conduction system identity completely lost without both factors and if so, does the VCS network transform into a working myocardium-like state? To address this question, the authors have generated a novel mouse line in which both Tbx5 and Tbx3 are floxed on the same allele, allowing complete conditional deletion of both factors using the VCS-specific MinK-CreERT2 line, convincingly validated in previous work. The goal is to use these double conditional knockout mice to further explore the model of Tbx3/Tbx5 co-dependent gene networks and VCS patterning. First, the authors demonstrate that the double conditional knockout allele results in the expected loss of Tbx3 and Tbx5 specifically in the VCS when crossed with Mink-CreERT2 and induced with tamoxifen. The double conditional knockout also results in premature mortality. Detailed electrophysiological phenotyping demonstrated prolonged PR and QRS intervals, inducible ventricular tachycardia, and evidence of abnormal impulse propagation along the septal aspect of the right ventricle. In addition, the mutants exhibit downregulation of VCS genes responsible for both fast conduction AND slow conduction phenotypes with upregulation of 2 working myocardial genes including connexin-43. The authors conclude that loss of both Tbx3 and Tbx5 results in "reversion" or "transformation" of the VCS network to a working myocardial phenotype, which they further claim is a prediction of their model and establishes that Tbx3 and Tbx5 "coordinate" transcriptional control of VCS identity.

Overall Appraisal:

As noted above, the present study does not further explore the Tbx5/Tbx3 ratio concept since both genes are completely knocked out in the VCS. Instead, the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function. However, only limited data are presented to support the claim of transcriptional reprogramming since the knockout cells are not directly compared to working myocardial cells at the transcriptional level and only a small number of key genes are assessed (versus genome-wide assessment). In addition, the optical mapping dataset is incomplete and has alternative interpretations that are not excluded or thoroughly discussed.

In sum, while this study adds an elegantly constructed genetic model to the field, the data presented fit well within the existing paradigm of established functions of Tbx3 and Tbx5 in the VCS and in that sense do not decisively advance the field. Moreover, the authors' claims about the implications of the data are not always strongly supported by the data presented and do not fully explore alternative possibilities.

Strengths:

(1) Successful generation of a novel Tbx3-Tbx5 double conditional mouse model.

(2) Successful VCS-specific deletion of Tbx3 and Tbx5 using a VCS-specific inducible Cre driver line.

(3) Well-powered and convincing assessments of mortality and physiological phenotypes.

(4) Isolation of genetically modified VCS cells using flow.

Weaknesses:

(1) In general, the data is consistent with a long-standing and well-supported model in which Tbx3 represses working myocardial genes and Tbx5 activates the expression of VCS genes, which seem like distinct roles in VCS patterning. However, the authors move between different descriptions of the functional relationship and epistatic relationship between these factors, including terms like "cooperative", "coordinated", and "distinct" at various points. In a similar vein, sometimes terms like "reversion" are used to describe how VCS cells change after Tbx3/Tbx5 conditional knockout, and other times "transcriptional shift" and at other times "reprogramming". But these are all different concepts. The lack of a clear and consistent terminology for describing the phenomena observed makes the overarching claims of the manuscript more difficult to evaluate.

(2) A more direct quantitative comparison of Tbx5 Adult VCS KO with Tbx5/Tbx3 Adult VCS double KO would be helpful to ascertain whether deletion of Tbx3 on top of Tbx5 deletion changes the underlying phenotype in some discernable way beyond mRNA expression of a few genes. Superficially, the phenotypes look quite similar at the EKG and arrhythmia inducibility level and no optical mapping data from a single Tbx5 KO is presented for comparison to the double KO.

(3) The authors claim that double knockout VCS cells transform to working myocardial fate, but there is no comparison of gene expression levels between actual working myocardial cells and the Tbx3/Tbx5 DKO VCS cells so it's hard to know if the data reflect an actual cell state change or a more non-specific phenomenon with global dysregulation of gene expression or perhaps dedifferentiation. I understand that the upregulation of Gja1 and Smpx is intended to address this, but it's only two genes and it seems relevant to understand their degree of expression relative to actual working myocardium. In addition, the gene panel is somewhat limited and does not include other key transcriptional regulators in the VCS such as Irx3 and Nkx2-5. RNA-seq in these populations would provide a clearer comparison among the groups.

(4) From the optical mapping data, it is difficult to distinguish between the presence of (a) a focal proximal right bundle branch block due to dysregulation of gene expression in the VCS but overall preservation of the right bundle and its distal ramifications; from (b) actual loss of the VCS with reversion of VCS cells to a working myocardial fate. Related to this, the authors claim that this experiment allows for direct visualization of His bundle activation, but can the authors confirm or provide evidence that the tissue penetration of their imaging modality allows for imaging of a deep structure like the AV bundle as opposed to the right bundle branch which is more superficial? Does the timing of the separation of the sharp deflection from the subsequent local activation suggest visualization of more distal components of the VCS rather than the AV bundle itself? Additional clarification would be helpful.

Impact:

The present study contributes a novel and elegantly constructed mouse model to the field. The data presented generally corroborate existing models of transcriptional regulation in the VCS but do not, as presented, constitute a decisive advance.

Reviewer #2 (Public review):

Summary:

This study by Sanchez-Vasquez is a very innovative approach to inducing aneuploidy and then studying the contribution of treated cells to different lineages, including post-implantation. It connects well to the authors' previous work to induce mosaic aneuploidies. The authors identify sensitivity to HIF1a loss in treated embryos with likely aneuploidy. This work is part of an important line of work with evaluates the consequences of aneuploidy in the mammalian embryo.

Weaknesses:

Given that this is a study on the induction of aneuploidy, it would be meaningful to assess aneuploidy immediately after induction, and then again before implantation. This is also applicable to the competition experiments on page 7/8. What is shown is the competitiveness of treated cells. Because the publication centers around aneuploidy, the inclusion of such data in the main figure at all relevant points would strengthen it. There is some evaluation of karyotypes only in the supplemental - why? It would be good not to rely on a single assay that the authors appear to not give much importance.

Reviewer #2 (Public review):

Gaertner and colleagues present a study examining the transcriptomic diversity and spatial location of dopaminergic neurons from mice and examine the changes in gene expression resulting from knock-in of the Parkinson's LRRK G2019S risk variant. Overall, I found the manuscript presented their study very clearly, well written with very clear figures for the most part. I am not an expert on mouse neuroanatomy but found their classification reasonably well justified and the spatial orientation of dopaminergic neurons within the mouse brain informative and clear. While trends were clear and well presented, the apparent spatial heterogeneity suggests that knowledge of the functional connections and roles of these neurons will be required to better interpret the results presented, but nonetheless their findings exposed significant detail that is required for further understanding.

The study of the transcriptional effects of the LRRK2 KI was also informative and clearly framed in terms of a focused analysis on the effects of the KI only on dopaminergic neurons. However, I think there are issues here in both methodology, narrative, and clarity.

(1) In the GO pathway analyses (both GSEA and DEG GO), I did not see a correction applied to the gene background considered. The study focusses on dopaminergic neurons and thus the gene background should be restricted to genes expressed in dopaminergic neurons, rather than all genes in the mouse genome. The problem arises that if we randomly sample genes from dopaminergic neurons instead of the whole genome, we are predisposed to sampling genes enriched in relevant cell-type-specific roles (and their relevant GO terms) and correspondingly depleted in genes enriched in functions not associated with this cell type. Thus, I am unsure whether the results presented in Figures 8 and 9 may be more likely to be obtained just by randomly sampling genes from a dopaminergic neuron. The background should be limited and these functional analyses rerun.

(2) In the scRDS results, I am unsure what is significant and what isn't. The authors refer to relative measures in the text ("highest") but I do not know whether these differences are significant nor whether any associations are significantly unexpected. Can the x-axis of scRDS results presented in Figure 9 H and I be replaced with a corrected p-value instead of the scRDS score?

(3) The results discussed at the bottom of page 13 state that 48.82% of the proteins encoded by the Calb1 DEGs have pre-synaptic localisations as opposed to 45.83% of the SOX6 DEGs, which does not support the statement that "greater proportions of DEGs are associated with presynaptic locations in cells from vulnerable DA neurons (Sox6 family, [and in particular,Sox6^tafa1]), compared to less vulnerable ones (Calb1 family)".

(4) While an interest in the Sox6^tafa1 subtype is explained through their expression of Anxa1 denoting a previously identified subtype associated with locomotory behaviours, it was unclear to me how to interpret the functional associations made to DEGs in this subtype taken out of context of other subtypes. Given all the other subtypes, it is not possible to ascertain how specific and thus how interesting these results are unless other subtypes are analysed in the same way and this Sox6^tafa1 subtype is demonstrated as unusual given results from other subtypes.

(5) On p12, the authors highlight Mir124a-1hg that encodes miR-124. This is upregulated in Figure 8D but the authors note this has been to be downregulated in PD patients and some PD mouse models. Can the authors comment on the directional difference?

(6) Lastly, can the authors comment on the selection of a LogFC cut-off of 0.15 for their DEG selection? I couldn't see this explained (apologies if I missed it).

Reviewer #2 (Public review):

Summary:

The goal of this study was to develop a model for CDHR1-based Con-rod dystrophy and study the role of this cadherin in cone photoreceptors. Using genetic manipulation, a cell binding assay, and high-resolution microscopy the authors find that like rods, cones localize CDHR1 to the lateral edge of outer segment (OS) discs and closely oppose PCDH15b which is known to localize to calyceal processes (CPs). Ectopic expression of CDHR1 and PCDH15b in K652 cells indicates these cadherins promote cell aggregation as heterophilic interactants, but not through homophilic binding. This data suggests a model where CDHR1 and PCDH15b link OS and CPs and potentially stabilize cone photoreceptor structure. Mutation analysis of each cadherin results in cone structural defects at late larval stages. While pcdh15b homozygous mutants are lethal, cdhr1 mutants are viable and subsequently show photoreceptor degeneration by 3-6 months.

Strengths:

A major strength of this research is the development of an animal model to study the cone-specific phenotypes associated with CDHR1-based CRD. The data supporting CDHR1 (OS) and PCDH15 (CP) binding is also a strength, although this interaction could be better characterized in future studies. The quality of the high-resolution imaging (at the light and EM levels) is outstanding. In general, the results support the conclusions of the authors.

Weaknesses:

While the cellular phenotyping is strong, the functional consequences of CDHR1 disruption are not addressed. While this is not the focus of the investigation, such analysis would raise the impact of the study overall. This is particularly important given some of the small changes observed in OS and CP structure. While statistically significant, are the subtle changes biologically significant? Examples include cone OS length (Figures 4F, 6E) as well as other morphometric data (Figure 7I in particular). Related, for quantitative data and analysis throughout the manuscript, more information regarding the number of fish/eyes analyzed as well as cells per sample would provide confidence in the rigor. The authors should also note whether the analysis was done in an automated and/or masked manner.

Reviewer #2 (Public review):

Summary:

Identifying an important role for the Integrator complex in repressing HIV transcription and suggesting that by targeting subunits of this complex specifically, INTS12, reversal of latency with and without latency reversal agents can be enhanced.

Strengths:

The strengths of the paper include the general strategy for screening targets that may activate HIV latency and the rigor of exploring the mechanism of INTS12 repression of HIV transcriptional elongation. I found the mechanism of INTS12 interesting and maybe even the most impactful part of the findings.

Weaknesses:

I have two minor comments:

There was an opportunity to examine a larger panel of latency reversal agents that reactivate by different mechanisms to determine whether INTS12 and transcriptional elongation are limiting for a broad spectrum of latency reversal agents.

I felt the authors could have extended their discussion of how exquisitely sensitive HIV transcription is to pausing and transcriptional elongation and the insights this provides about general HIV transcriptional regulation.

Reviewer #2 (Public review):

Summary:<br /> In their manuscript, Fang and colleagues make a notable contribution to the field of oncology, particularly in advancing our understanding of triple-negative breast cancer (TNBC). The research delineates the role of STAMBPL1 in promoting angiogenesis in TNBC through its interaction with FOXO1 and the subsequent activation of the GRHL3/HIF1A/VEGFA axis. The evidence presented is robust, with a combination of in vitro experiments, RNA sequencing, and in vivo studies providing a comprehensive view of the molecular mechanisms at play. The strength of the evidence is anchored in the systematic approach and the utilization of multiple methodologies to substantiate the findings.

Strengths:<br /> The manuscript presents a methodologically robust framework, incorporating RNA-sequencing, chromatin immunoprecipitation (ChIP) assays, and a suite of in vitro and in vivo model systems, which collectively substantiate the claims regarding the pro-angiogenic role of STAMBPL1 in TNBC. The employment of multiple cellular models, conditioned media to assess HUVEC functional responses, and xenograft tumor models in murine hosts offers a comprehensive evaluation of STAMBPL1's impact on angiogenic processes.A salient strength of this work is the identification of GRHL3 as a transcriptional target of STAMBPL1 and the demonstration of a physical interaction between STAMBPL1 and FOXO1, which modulates GRHL3-driven HIF1A transcription. The study further suggests a potential therapeutic strategy by revealing the synergistic inhibitory effects of combined VEGFR and FOXO1 inhibitor treatment on TNBC tumor growth.

Weaknesses:<br /> A potential limitation of the study is the reliance on specific cellular and animal models, which may constrain the extrapolation of these findings to the broader spectrum of human TNBC biology. Furthermore, while the study provides evidence for a novel regulatory axis involving STAMBPL1, FOXO1, and GRHL3, the multifaceted nature of angiogenesis may implicate additional regulatory factors not exhaustively addressed in this research.

Appraisal of Achievement and Conclusion Support:<br /> The authors have successfully demonstrated that STAMBPL1 promotes HIF1A transcription and activates the HIF1α/VEGFA axis in a non-enzymatic manner, leading to increased angiogenesis in TNBC. The results are generally supportive of their conclusions, with clear evidence that STAMBPL1 upregulates HIF1α expression and enhances the activity of HUVECs. The study also shows that STAMBPL1 interacts with FOXO1 to promote GRHL3 transcription, which in turn activates HIF1A.

Impact on the Field and Utility:<br /> This research is poised to exert a substantial impact on the oncological research community by uncovering the role of STAMBPL1 in TNBC angiogenesis and by identifying the STAMBPL1/FOXO1/GRHL3/HIF1α/VEGFA axis as a potential therapeutic target. The findings could pave the way for the development of novel therapeutic strategies for TNBC, a subtype characterized by a paucity of effective treatment options. The methodologies utilized in this study are likely to be valuable to the research community, offering a paradigm for investigating the role of deubiquitinating enzymes in oncogenic processes.

Additional Context:<br /> It would be beneficial for readers to understand the broader context of TNBC research and the current challenges in treating this aggressive cancer subtype. The significance of this work is heightened by the lack of effective treatments for TNBC, making the identification of new therapeutic targets particularly important. Furthermore, understanding the specific mechanisms by which STAMBPL1 regulates HIF1α expression could provide insights into hypoxia signaling in other cancer types as well.

Reviewer #2 (Public review):

Summary:

Chronic inflammation of the bone microenvironment conferred by T2DM and obesity may inhibit bone formation and bone strength by decreasing the ratio of Wnt ligands/Wnt inhibitors.

The authors studied 63 postmenopausal women (age >65 years) undergoing hip replacement for osteoarthritis. These were grouped into T2DM and obesity, obesity only, and normal subjects. A set of inflammatory markers was measured in the serum and gene expression of members of the Wnt system in the bone tissue. Bone samples were assessed by micro-CT.

While TNF-α serum levels were higher in T2DM, IL-6 levels were higher in obesity as compared to control. In the bone compartment the most consistent finding was decreased mRNA levels for WNt10b and increased sclerostin mRNA levels, translating into a suppressed Wnt-to-Wnt inhibitor ratio, which was associated with low bone strength.

Strengths:

The study includes clinically well-characterized subjects of three defined subgroups. The analyses were comprehensive.

Weaknesses:

Including data or information on the Wnt inhibitor Dkk1 would be instructive. Analysis were limited to mRNA studies. Validation of protein levels would be supportive (although technically challenging).

Reviewer #2 (Public review):

Summary:

In this study, the authors investigated the chemotaxis of E. coli swimming close to the bottom surface in gradients of attractant in channels of increasingly smaller width but fixed height = 30 µm and length ~160 µm. In relatively large channels, they find that on average the cells drift in response to the gradient, despite cells close to the surface away from the walls being known to not be chemotactic because they swim in circles.

They find that this average drift is due to the cell localization close to the side walls, where they slide along the wall. Whereas the bacteria away from the walls have no chemotaxis (as shown before), the ones on the left side wall go down-gradient on average, but the ones on the right side wall go up-gradient faster, hence the average drift. They then study the effect of reducing channel width. They find that chemotaxis is higher in channels with a width of about 8 µm, which approximately corresponds to the radius of the circular swimming R. This higher chemotactic drift is concomitant to an increased density of cells on the RSW. They do simulations and modeling to suggest that the disruption of circular swimming upon collision with the wall increases the density of cells on the RSW, with a maximal effect at w = ~ 2/3 R, which is a good match for their experiments.

Strengths:

The overall result that confinement at the edge stabilises bacterial motion and allows chemotaxis is very interesting although not entirely unexpected. It is also important for understanding bacterial motility and chemotaxis under ecologically relevant conditions, where bacteria frequently swim under confinement (although its relevance for controlling infections could be questioned). The experimental part of the study is nicely supported by the model.

Weaknesses:

Several points of this study, in particular the interpretation of the width effect, need better clarification:

(1) Context:

There are a number of highly relevant previous publications that should have been acknowledged and discussed in relation to the current work:<br /> https://pubs.rsc.org/en/content/articlehtml/2023/sm/d3sm00286a<br /> https://link.springer.com/article/10.1140/epje/s10189-024-00450-7<br /> https://doi.org/10.1016/j.bpj.2022.04.008<br /> https://doi.org/10.1073/pnas.1816315116<br /> https://www.pnas.org/doi/full/10.1073/pnas.0907542106<br /> https://doi.org/10.1038/s41467-020-15711-0<br /> http://doi.org/10.1038/s41467-020-15711-0<br /> http://doi.org/10.1039/c5sm00939a

(2) Experimental setup:

a) The channels are built with asymmetric entrances (Figure 1), which could trigger a ratchet effect (because bacteria swim in circle) that could bias the rate at which cells enter into the channel, and which side they follow preferentially, especially for the narrow channel. Since the channel is short (160 µm), that would reflect on the statistics of cell distribution. Controls with straight entrances or with a reversed symmetry of the channel need to be performed to ensure that the reported results are not affected by this asymmetry.

b) The authors say the motile bacteria accumulate mostly at the bottom surface. This is strange, for a small height of 30 µm, the bacteria should be more-or-less evenly spread between the top and bottom surface. How can this be explained?

c) At the edge, some of the bacteria could escape up in the third dimension (http://doi.org/10.1039/c5sm00939a). What is the magnitude of this phenomenon in the current setup? Does it have an effect?

d) What is the cell density in the device? Should we expect cell-cell interactions to play a role here? If not, I would suggest to de-emphasize the connection to chemotaxis in the swarming paper in the introduction and discussion, which doesn't feel very relevant here, and rather focus on the other papers mentioned in point 1.

e) We are not entirely convinced by the interpretation of the results in narrow channels. What is the causal relationship between the increased density on the RSW and the higher chemotactic drift? The authors seem to attribute higher drift to this increased RSW density, which emerges due to the geometric reasons. But if there is no initial bias, the same geometric argument would induce the same increased density of down-gradient swimmers on the LSW, and so, no imbalance between RSW and LSW density. Could it be the opposite that the increased RSW density results from chemotaxis (and maybe reinforces it), not the other way around? Confinement could then deplete one wall due to the proximity of the other, and/or modify the swimming pattern - 8 µm is very close to the size of the body + flagellum. To clarify this point, we suggest measuring the bacterial distributions in the absence of a gradient for all channel widths as a control.

(3) Simulations:

The simulations treat the wall interaction very crudely. We would suggest treating it as a mechanical object that exerts elastic or "hard sphere" forces and torques on the bacteria for more realistic modeling. Notably, the simulations have a constant (chemotaxis independent) rate of wall escape by tumbling. We would expect that reduced tumbling due to up-gradient motility induces a longer dwell time at the wall.

Reviewer #2 (Public review):

Summary:

In this work, the authors use a theoretical model to study the potential impact of Horizontal Gene Transfer on the number of alternative stable states of microbial communities. For this, they use a modified version of the competitive Lotka Volterra model-which accounts for the effects of pairwise, competitive interactions on species growth-that incorporates terms for the effects of both an added death (dilution) rate acting on all species and the rates of horizontal transfer of mobile genetic elements-which can in turn affect species growth rates. The authors analyze the impact of horizontal gene transfer in different scenarios: bistability between pairs of species, multistability in communities, and a modular structure in the interaction matrix to simulate multiple niches. They also incorporate additional elements to the model, such as spatial structure to simulate metacommunities and modification of pairwise interactions by mobile genetic elements. In almost all these cases, the authors report an increase in either the number of alternative stable states or the parameter region (e.g. growth rate values) in which they occur.

In my opinion, understanding the role of horizontal gene transfer in community multistability is a very important subject. This manuscript is a useful approach to the subject, but I'm afraid that a thorough analysis of the role of different parameters under different scenarios is missing in order to support the general claims of the authors. The authors have extended their analysis to increase their biological relevance, but I believe that the analysis still lacks comprehensiveness.

Understanding the origin of alternative stable states in microbial communities and how often they may occur is an important challenge in microbial ecology and evolution. Shifts between these alternative stable states can drive transitions between e.g. a healthy microbiome and dysbiosis. A better understanding of how horizontal gene transfer can drive multistability could help predict alternative stable states in microbial communities, as well as inspire novel treatments to steer communities towards the most desired (e.g. healthy) stable states.

Strengths:

(1) Generality of the model: the work is based on a phenomenological model that has been extensively used to predict the dynamics of ecological communities in many different scenarios.

(2) The question of how horizontal gene transfer can drive alternative stable states in microbial communities is important and there are very few studies addressing it.

Weaknesses:

(1) There is a need for a more comprehensive analysis of the relative importance of the different model parameters in driving multistability. For example, there is no analysis of the effects of the added death rate in multistability. This parameter has been shown to determine whether a given pair of interacting species exhibits bistability or not (see e.g. Abreu et al 2019 Nature Communications 10:2120). Similarly, each scenario is analyzed for a unique value of species interspecies interaction strength-with the exception of the case for mobile genetic elements affecting interaction strength, which considers three specific values. Considering heterogeneous interaction strengths (e.g. sampling from a random distribution) could also lead to more realistic scenarios - the authors generally considered that all species pairs interact with the same strength. Analyzing a larger range of growth rates effects of mobile genetic elements would also help generalize the results. In order to achieve a more generic assessment of the impact of horizontal gene transfer in driving multistability, its role should be systematically compared to the effects of the rest of the parameters of the model.

(2) The authors previously developed this theoretical model to study the impact of horizontal gene transfer on species coexistence. In this sense, it seems that the authors are exploring a different (stronger interspecies competition) range of parameter values of the same model, which could potentially limit novelty and generality.

(3) The authors analyze several scenarios that, in my opinion, naturally follow from the results and parameter value choices in the first sections, making their analysis not very informative. For example, after showing that horizontal gene transfer can increase multistability both between pairs of species and in a community context, the way they model different niches does not bring significantly new results. Given that the authors showed previously in the manuscript that horizontal gene transfer can impact multistability in a community in which all species interact with each other, one might expect that it will also impact multistability in a larger community made of (sub)communities that are independent of (not interacting with) each-which is the proposed way for modelling niches. A similar argument can be made regarding the analysis of (spatially structured) metacommunities. It is known that, for smaller enough dispersal rates, space can promote regional diversity by enabling each local community to remain in a different stable state. Therefore, in conditions in which the impact of horizontal gene transfer drives multistability, it will also drive regional diversity in a metacommunity.

(4) In some cases, the authors consider that mobile genetic elements can lead to ~50% growth rate differences. In the presence of an added death rate, this can be a relatively strong advantage that makes the fastest grower easily take over their competitors. It would be important to discuss biologically relevant examples in which such growth advantages driven by mobile genetic elements could be expected, and how common such scenarios might be.

Reviewer #2 (Public review):

Summary:

Weiler et al use retrograde tracers, two-photon tomography, and automatic cell detection to provide a detailed quantitative description of the laminar and area sources of ipsi- and contralateral cortico-cortical inputs to two primary sensory areas and a primary motor area. They found considerable bilateral symmetry in the areas providing cortico-cortical inputs. However, although the same regions in both hemispheres tended to supply inputs, a larger proportion of inputs from contralateral areas originated from deeper layers (L5 and L6).

Strengths:

The study applies state-of-the-art anatomical methods, and the data is very effectively presented and carefully analyzed. The results provide many novel insights into the similarities and differences of inputs from the two hemispheres. While over the past decade there have been many studies quantitively and comprehensively describing cortico-cortical connections, by directly comparing inputs from the ipsi and contralateral hemispheres, this study fills in an important gap in the field. It should be of great utility and an important reference for future studies on inter-hemispheric interactions.

Weaknesses:

Overall, I do not find any major weakness in the analyses or their interpretation. However, one must keep in mind that the study only analyses inputs projecting to three areas. This is not an inherent flaw of the study; however, it warrants caution when extrapolating the results to callosal projections terminating in other areas. As inputs to two primary sensory areas and one is the primary motor cortex are studied, some of the conclusions could potentially be different for inputs terminating in high-order sensory and motor areas. Given that primary areas were injected, there are few instances of feedforward connections sampled in the ipsilateral hemisphere. The study finds that while ipsi-projections from the visual cortex to the barrel cortex are feedforward given its fILN values, those from the contralateral visual cortex are feedback instead. One is left to wonder whether this is due to the cross-modal nature of these particular inputs and whether the same rule (that contralateral inputs consistently exhibit feedback characteristics regardless of the hierarchical relationship of their ipsilateral counterparts with the target area,) would also apply to feedforward inputs within the same sensory cortices.

Another issue that is left unexplored is that, in the current analyses the barrel and primary visual cortex are analyzed as a uniform structure. It is well established that both the laminar sources of callosal inputs and their terminations differ in the monocular and binocular areas of the visual cortex (border with V2L). Similarly, callosal projections differ when terminating the border of S1 (a row of whiskers), and then in other parts of S1. Thus, some of the conclusions regarding the laminar sources of callosal inputs might depend on whether one is analyzing inputs terminating or originating in these border regions.

Finally, while the paper emphasizes that projections from L6 "dominate" intra and contralateral cortico-cortical inputs, the data shows a more nuanced scenario. While it is true that the areas for which L6 neurons are the most common source of cortico-cortical projections are the most abundant, the picture becomes less clear when considering the number of neurons sending these connections. In fact, inputs from L2/3 and L5 combined are more abundant than those from L6 (Figure 3B), challenging the view that projections from L6 dominate ipsi- and contralateral projecting cortico-cortical inputs.

Reviewer #2 (Public review):

Summary:

The manuscript 'Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease' from Angel D'Oliviera et al., uncovers that TRMT1 can be cleaved by SARS-CoV-2 main protease (Mpro) and defines the structural basis of TRMT1 recognition by Mpro. They use both recombinant TRMT1 and Mpro as well as endogenous TRMT1 from HEK293T cell lysates to convincingly show cleavage of TRMT1 by the SARS-CoV-2 protease. Using in vitro assays, the authors demonstrate that TRMT1 cleavage by Mpro blocks its enzymatic activity leading to hypomodification of RNA. To understand how Mpro recognizes TRMT1, they solved a co-crystal structure of Mpro bound to a peptide derived from the predicted cleavage site of TRMT1. This structure revealed important protein-protein interfaces and highlights the importance of the conserved Q530 for cleavage by Mpro. They then compare their structure with previous X-ray crystal structures of Mpro bound to substrate peptides derived from the viral polyprotein and propose the concept of two distinct binding conformations to Mpro: P3´-out and P3´-in conformations (here P3´ stands for the third residue downstream of the cleavage site). It remains unknown what is the physiological role of these two binding conformations on Mpro function, but the authors established that Mpro has dramatically different cleavage efficiencies for three distinct substrates. In an effort to rationalize this observation, a series of mutations in Mpro's active site and the substrate peptide were tested but unexpectedly had no significant impact on cleavage efficiency. While molecular dynamic simulations further confirmed the propensity of certain substrates to adopt the P3´-out or P3´-in conformation, it did not provide additional insights into the dramatic differences in cleavage efficiencies between substrates. This led the authors to propose that the discrimination of Mpro for preferred substrates might occur at a later stage of catalysis after binding of the peptide. Overall, this work will be of interest to biologists studying proteases and substrate recognition by enzymes and RNA modifications as well as help efforts to target Mpro with peptide-like drugs.

Strengths:

• The authors' statements are well supported by their data, and they used relevant controls when needed. Indeed, they used the Mpro C145A inactive variant to unambiguously show that the TRMT1 cleavage detected in vitro is solely due to Mpro's activity. Moreover, they used two distinct polyclonal antibodies to probe TRMT1 cleavage.<br /> • They demonstrate the impact of TRMT1 cleavage on RNA modification by quantifying both its activity and binding to RNA.<br /> • Their 1.9 Å crystal structure is of high quality and increases the confidence in the reported protein-protein contacts seen between TRMT1-derived peptide and Mpro.<br /> • Their extensive in vitro kinetic assay was performed in ideal conditions although it is sometimes unclear how many replicates were performed.<br /> • They convincingly show how Mpro cleavage is conserved among most but not all mammalian TRMT1 bringing an interesting evolutionary perspective on virus-host interactions.<br /> • The authors test multiple hypotheses to rationalize the preference of Mpro for certain substrates.<br /> • While this reviewer is not able to comment on the rigor of the MD simulations, the interpretations made by the authors seem reasonable and convincing.<br /> • The concept of two binding conformations (P3´-out or P3´-in) for the substrate in the active site of Mpro is significant and can guide drug design.

Weaknesses:

• The two polyclonal antibodies used by the authors seem to have strong non-specific binding to proteins other than TRMT1 but did not impact the author's conclusions or statements. This is a limitation of the commercially available antibodies for TRMT1.<br /> • Despite the reasonable efforts of the authors, it remains unknown why Mpro shows higher cleavage efficiency for the nsp4/5 sequence compared to TRMT1 or nsp8/9 sequences. This is a challenging problem that will take substantially more effort by several labs to decipher mechanistically.<br /> • The peptide cleavage kinetic assay used by the authors relies on a peptide labelled with a fluorophore (MCA) on the N-terminus and a quencher (Dpn) on the C-terminus. This design allows high-throughput measurements compatible with plate readers and is a robust and convenient tool. Nevertheless, the authors did not control for the impact of the labels (MCA and Dpn) on the activity of Mpro. While in most cases the introduced fluorophore/quencher do not impact activity, sometimes it can.<br /> • An unanswered question not addressed by the authors is if the peptides undergo conformational changes upon Mpro binding or if they are pre-organized to adopt the P3´-out and P3´-in conformations. This might require substantially more work outside the scope of this immediate article.

Reviewer #3 (Public review):

This study provides valuable insights into the cellular responses to complex aneuploidy in human preimplantation embryos. The authors have significantly expanded their sample size and conducted additional analysis and experiments to address previous concerns. The revised manuscript presents stronger evidence for gene dosage-dependent effects of aneuploidy on stress responses and lineage segregation. Overall, the findings contribute important knowledge to our understanding of how human embryos respond to chromosomal abnormalities.

Overall, the revision has substantially improved the manuscript and addressed the major concerns raised in the initial review.

Reviewer #2 (Public review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. This finding highlights the potentially complex role of PdeI in regulation of c-di-GMP levels and persister formation in microbial biofilms.

Comments on revised version:

The authors edited the manuscript thoroughly in response to the comments, including both performing new experiments and showing more data and information. Most of the major points raised between both reviewers were addressed. The authors explained the seeming contradiction between c-di-GMP levels and PdeI expression.

Reviewer #2 (Public review):

FOXP3 has been known to form diverse complexes with different transcription factors and enzymes responsible for epigenetic modifications, but how extracellular signals timely regulate FOXP3 complex dynamics remains to be fully understood. Histone H3K4 tri-methylation (H3K4me3) and CXXC finger protein 1 (CXXC1), which is required to regulate H3K4me3, also remain to be fully investigated in Treg cells. Here, Meng et al. performed a comprehensive analysis of H3K4me3 CUT&Tag assay on Treg cells and a comparison of the dataset with the FOXP3 ChIP-seq dataset revealed that FOXP3 could facilitate the regulation of target genes by promoting H3K4me3 deposition.

Moreover, CXXC1-FOXP3 interaction is required for this regulation. They found that specific knockdown of Cxxc1 in Treg leads to spontaneous severe multi-organ inflammation in mice and that Cxxc1-deficient Treg exhibits enhanced activation and impaired suppression activity. In addition, they have also found that CXXC1 shares several binding sites with FOXP3 especially on Treg signature gene loci, which are necessary for maintaining homeostasis and identity of Treg cells.

The findings of the current study are pretty intriguing, and it would be great if the authors could fully address the following comments to support these interesting findings.

Major points:

(1) There is insufficient evidence in the first part of the Results to support the conclusion that "FOXP3 functions as an activator by promoting H3K4Me3 deposition in Treg cells". The authors should compare the results for H3K4Me3 in FOXP3-negative conventional T cells to demonstrate that at these promoter loci, FOXP3 promotes H3K4Me3 deposition.

(2) In Figure 3 F&G, the activation status and IFNγ production should be analyzed in Treg cells and Tconv cells separately rather than in total CD4+ T cells. Moreover, are there changes in autoantibodies and IgG and IgE levels in the serum of cKO mice?

(3) Why did Cxxc1-deficient Treg cells not show impaired suppression than WT Treg during in vitro suppression assay, despite the reduced expression of Treg cell suppression assay -associated markers at the transcriptional level demonstrated in both scRNA-seq and bulk RNA-seq?

(4) Is there a disease in which Cxxc1 is expressed at low levels or absent in Treg cells? Is the same immunodeficiency phenotype present in patients as in mice?

Reviewer #2 (Public review):

Summary:

The authors comprehensively assess differences in the TCRB and TCRA repertoires in the fetal and adult mouse thymus by deep sequencing of sorted cell populations. For TCRB and TCRA they observed biased gene segment usage, less diversity, and greater repertoire sharing among individuals in fetal thymocytes. The TCRB repertoire was less evenly distributed and displayed more evidence of clonal expansions in fetal thymocytes. Both fetal and adult thymocytes demonstrated repertoire skewing in CD4 and CD8 as compared to DP thymocytes, which was attributed to MHC-I- vs MHC-II-restriction during positive selection. Effects of MHC-restriction were notably weaker in fetal thymocytes. The authors conclude that in multiple respects fetal repertoires are distinct from adult repertoires.

Strengths:

The analyses of the F18.5 and adult thymic repertoires are comprehensive with respect to the cell populations analyzed and the diversity of statistical approaches used to characterize the repertoires. Because repertoires were analyzed in pre- and post-selection thymocyte subsets, the data allowed assessment of repertoire selection at different developmental stages. Intriguing differences between fetus and adult are identified.

Weaknesses:

Some of the repertoire characteristics reported are already fairly well documented in the literature. Moreover, an unaddressed limitation of the study is that fetal thymocytes were analyzed at single time-point in their development. As a result, at least some of the conclusions about the fetal repertoire may be viewed not as general conclusions, but rather, due to the synchronous development of fetal thymocytes, as pertaining to the one day of fetal/early neonatal development assayed. Statements suggesting that (1) "progressive TCRa rearrangements occur less frequently in foetal DP cells" (Abstract), (2) "One possible explanation for this bias is that in the foetus progressive rounds of TCRa rearrangement are less common than in young adult" (Discussion), and (3) "Overall, the differences between the foetal and adult thymus TCR repertoires are consistent with the foetal thymus producing abT-cells ... with preference for particular gene segment usage" (Discussion), are oversimplified and potentially misleading.

Reviewer #2 (Public Review):

In this manuscript, the authors revealed that genetic deficiencies of ACK1 and BRK are associated with human SLE. First, the authors found that compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in one multiplex family and PTK6/BRK in another family. Then, by an experimental blockade of ACK1 or BRK in a mouse SLE model, they found an increase in glomerular IgG deposits and circulating autoantibodies. Furthermore, they reported that ACK and BRK variants from the SLE patients impaired the MERTK-mediated anti-inflammatory response to apoptotic cells in human induced pluripotent stem cells (hiPSC)-derived macrophages. This work identified new SLE-associated ACK and BRK variants and a role for the NRTK TNK2/ACK1 and PTK6/BRK in efferocytosis, providing a new molecular and cellular mechanism of SLE pathogenesis.

Reviewer #3 (Public Review):

Heparan Sulphate is a general association factor in the extracellular matrix which assists in host cell entry for a multitude of viral and bacterial pathogens by concentrating them in the vicinity of cellular membranes. The neurotropic picornavirus, EV-71 utilizes a protein receptor SCARB-2, in conjunction with Heparan Sulfate, in order to enter cells through the endo-lysosomal pathway. The uncoating and release of viral genome requires both receptor binding and late endosomal pH conditions. The authors have attempted to address a seeming contradiction in the in vitro and in vivo infectivity of strain MP4 variants of EV-71. One of the cell culture adapted strains MP4-L97R/E167G has stronger association with HS, which translates to higher infectivity in cell culture models; however, viral virulence is significantly lower in animal models.

Using an elegant and methodical set of experiments, the authors have probed the steps in the cellular entry pathway of MP4 and its L97R/E167G variant. Their experiments strongly suggest a difference in capsid uncoating mechanisms in the variant, with the L97R/E167G variant being significantly less robust and prone to destabilize earlier in the pathway. While this confers an advantage in terms of cell culture based infectivity, it is posited that the particles will not survive the gastric pH intact, which compromises virulence in the animal model. While the cell culture based uncoating experiments somewhat support this hypothesis, the main weakness of this work is a lack of explanation for the mechanism(s) of capsid destabilization conferred by overall increased positive charge. The structural bioinformatics study in the supplementary section does not explain how receptor binding, pocket factor expulsion, subunit interactions and low pH based capsid dynamics may be influenced by the mutations. Capsid destabilization could be an outcome in alteration of any or all of these processes. It is also unclear whether it is suggested that all mutations enhancing the net positive charge of VP1, or any other structural protein, will cause capsid destabilization by similar pathways. A clearer analysis of the influence of overall charge alterations, or individual mutations, on subunit interaction or particle conformation is needed. The enhancement in cell culture infectivity of the L97R/E167G variant under elevated endosomal pH is also unclear and requires further experimentation.

It has been suggested earlier that increased HS binding in vivo results in virus "trapping" and decreased infectivity. This may still be a major reason for reduced infectivity in vivo, in addition to the capsid destabilization as proposed in this work.

Reviewer #2 (Public Review):

Many naturally occurring networks are assumed to have a power-law (PL) degree distribution. This assumption has certainly been widely held in the field of protein interactomes (PPIs), although important studies around 2010 have conclusively shown that many of these PL distributions are either the result of data mis-handling or of sloppy statistical procedures (see e.g. Porter and Stumpf in Science around 2014, which I would advise the authors to cite). The value of the present study is to introduce a new mechanism, experiment bias, to explain the appearance of such distributions in the PPI case, and in particular to show how correcting empirically for this mechanism can lead to a reappraisal of which proteins are genuine hubs in these networks. The claims are well supported by empirical evidence and some theoretical analysis. Overall, this is a worthwhile contribution although its significance is somewhat dented by the fact that the PL enthusiasm of many had already been tempered by the studies mentioned above.

Reviewer #2 (Public review):

Summary:

This was a well-executed and well-written paper. The authors have provided important new datasets that expand on previous investigations substantially. The discovery that changes in diet are not so closely correlated with the presence of alkaloids (based on the expanded sampling of non-defended species) is important, in my opinion.

Strengths:

Provision of several new expanded datasets using cutting edge technology and sampling a wide range of species that had not been sampled previously. A conceptually important paper that provides evidence for the importance of intermediate stages in the evolution of chemical defense and aposematism.

Weaknesses:

There were some aspects of the paper that I thought could be revised. One thing I was struck by is lack of discussion of the potentially negative effects of toxin accumulation, and how this might play out in terms of different levels of toxicity in different species. Further, are there aspects of ecology or evolutionary history that might make some species less vulnerable to the accumulation of toxins than others? This could be another factor that strongly influences the ultimate trajectory of a species in terms of being well-defended. I think the authors did a good job in terms of describing mechanistic factors that could affect toxicity (e.g. potential molecular mechanisms), but did not make much of an attempt to describe potential ecological factors that could impact trajectories of the evolution of toxicity. This may have been done on purpose (to avoid being too speculative), but I think it would be worth some consideration.

In the discussion, the authors make the claim that poison frogs don't (seem to) suffer from eating alkaloids. I don't think this claim has been properly tested (the cited references don't adequately address it). To do so would require an experimental approach, ideally obtained data on both lifespan and lifetime reproductive success.

Update: Revised version: The authors carefully addressed the comments and suggestions on the first draft of the manuscript. In my opinion, these revisions were sufficient and the authors have adequately addressed the previously noted weaknesses in the manuscript.

Reviewer #2 (Public review):

This study builds upon the team's recent discovery that antibiotic treatment and other disturbances favours the persistence of bacteria with genomes that encode complete modules for the synthesis of essential metabolites (Watson et al. 2023). Veseli and collaborators now provide an in-depth analysis of metabolic pathway completeness within microbiomes, finding strong evidence for an enrichment of bacteria with high metabolic independence in the microbiomes associated with IBD and other gastrointestinal disorders. Importantly, this study provides a new open-source software to facilitate the reconstruction of metabolic pathways, estimate their completeness and normalize their results according to species diversity. Finally, this study also shows that metabolic independence of microbial communities can be used as a marker of dysbiosis. The function-based health index proposed here is more robust to individual's lifestyles and geographic origin than previously proposed methods based on bacterial taxonomy.

The implications of this study have the potential to spur a paradigm shift in the field. It shows that certain bacterial taxa that have been consistently associated with disease might not be harmful to their host as previously thought. These bacteria seem to be the only species that are able to survive in a stressed gut environment. They might even be important to rebuild a healthy microbiome (although the authors are careful in not making this speculation).

This paper provides an in-depth discussion of the results, and limitations are clearly addressed throughout the manuscript (see also the supplementary files for an in-depth assessment of the robustness of the methods). Some of the potential limitations relate to the use of large publicly available datasets, where sample processing and the definition of healthy status varies between studies. The authors have recognised these issues and their results were robust to analyses performed at a per-cohort basis. The potential limitations therefore are unlikely to have affected the conclusions of this study.

Overall, this is manuscript is a magnificent contribution to the field, likely to inspire many other studies to come.

Comments on revisions:

The authors have performed a detailed assessment of the accuracy and robustness of their new methods, and included an informative session comparing their new approach with existing ones. The new analyses have strengthened the manuscript, and the results support the biological interpretations of the study.<br /> I commend the authors for the effort and the excellent research.

Reviewer #2 (Public Review):

In this manuscript, Senn, Lipinski, and colleagues report on the structure and function of the conserved spliceosomal protein Fyv6. Pre-mRNA splicing is a critical gene expression step that occurs in two steps, branching and exon ligation. Fyv6 had been recently identified by the Hoskins' lab as a factor that aids exon ligation (Lipinski et al., 2023), yet the mechanistic basis for Fyv6 function was less clear. Here, the authors combine yeast genetics, transcriptomics, biochemical assays, and structural biology to reveal the function of Fyv6. Specifically, they describe that Fyv6 promotes the usage of distal 3'SSs by stabilizing a network of interactions that include the RNA helicase PRP22 and the spliceosome subunit SYF1. They discuss a generalizible mechanism for splice site proofreading by spliceosomsal RNA helicases that could be modulated by other, regulatory splicing factors.

This is a very high quality study, which expertly combines various approaches to provide new insights into the regulation of 3'SS choice, docking, and undocking. The cryo-EM data is also of excellent quality, which substantially extends on previous yeast P complex structures. This is also supported by the authors use of the latest data analysis tools (Relion-5, AlphaFold2 multimer predictions, Modelangelo). The authors re-evaluate published EM densities of yeast spliceosome complexes (B*, C,C*,P) for the presence or absence of Fyv6, substantiate Fyv6 as a 2nd step specific factor, confirm it as the homolog of the human protein FAM192A, and provide a model for how Fyv6 may fit into the splicing pathway. The biochemical experiments on probing the splicing effects of BP to 3'SS distances after Fyv6 KO, genetic experiments to probe Fyv6 and Syf1 domains, and the suppressor screening add substantially to the study and are well executed. The manuscript is clearly written and we particularly appreciated the nuanced discussions, for example for an alternative model by which Prp22 influences 3'SS undocking. The research findings will be of great interest to the pre-mRNA splicing community.

Comments on revisions:

I'm satisfied with the changes.

Reviewer #2 (Public review):

HIV infection is characterized by viral integration into permissive host cells - an event that occurs very early in viral-host encounter. This constitutes the HIV proviral reservoir and is a feature of HIV infection that provides the greatest challenge for eradicating HIV-1 infection once an individual is infected.

This study looks at how starting HIV treatment very early after infection, which substantially reduces the peak viral load detectable (compared to untreated infection), affects the amount and characteristics of the viral reservoir. The authors studied 35 women in South Africa who were at high risk of getting HIV. Some of these women started HIV treatment very soon after getting infected, while others started later. This study is well-designed and has as its focus a very well characterized cohort. Comparison groups are appropriately selected to address proviral DNA characterization and dynamics in the context of acute and chronic treated HIV-1. The amount of HIV and various characteristics of the genetic makeup of the virus (intact/defective proviral genome) was evaluated over one year of treatment. Methods employed for proviral DNA characterization are state-of-the-art and provide in-depth insights into the reservoir in peripheral blood.

While starting treatment early didn't reduce the amount of HIV DNA at the outset, it did lead to a gradual decrease in total HIV DNA quantity over time. In contrast, those who started treatment later didn't see much change in this parameter. Starting treatment early led to a faster decrease in intact provirus (a measure of replication-competence), compared to starting treatment later. Additionally, early treatment reduced genetic diversity of the viral DNA and resulted in fewer immune escape variants within intact genomes. This suggests that collectively having a smaller intact replication-competent reservoir, less viral variability, and less opportunity for virus to evade the immune system - are all features that are likely to facilitate more effective clearance of viral reservoir, especially when combined with other intervention strategies.

Major strengths of the study include the cohort of very early treated persons with HIV and the depth of study. These are important findings, particularly as the study was conducted in HIV-1 subtype C infected women (more cure studies have focussed on men and with subtype B infection)- and in populations most affected by HIV and in need of HIV cure interventions. This is highly relevant because it cannot be assumed that any interventions employed for reducing/clearing the HIV reservoir would perform similarly in men and women or across different populations. Other factors also deserve consideration and include age, and environment (e.g. other comorbidities and coinfections).

Reviewer #2 (Public review):

Summary:

In this manuscript by Torelli et al., the authors propose that the major function of MYR1 and MYR1-dependent secreted proteins is to contribute to parasite survival in a paracrine manner rather than to protect parasites from cell-autonomous immune response. The authors conclude that these paracrine effects rescue ∆MYR1 or knockouts of MYR1-dependent effectors within pooled in vivo CRISPR screens.

Strengths:

The authors raised a more general concern that pooled CRISPR screens (not only in Toxoplasma but also other microbes or cancers) would miss important genes by "paracrine masking effect". Although there is no doubt that pooled CRISPR screens (especially in vivo CRISPR screens) are powerful techniques, I think this topic could be of interest to those fields and researchers.

Weaknesses:

In this version, the reviewer is not entirely convinced of the 'paracrine masking effect' because the in vivo experiments should include appropriate controls (see major point 2) in the first submission.

After the revision, although no experiments were added, this reviewer considered that the points have been sufficiently discussed and commented on.

Reviewer #2 (Public review):

Summary:

The manuscript by Isotani et al characterizes the hyperproliferation of intestinal stem cells (ISCs) induced by nicotine treatment in vivo. Employing a range of small molecule inhibitors, the authors systematically investigated potential receptors and downstream pathways associated with nicotine-induced phenotypes through in vitro organoid experiments. Notably, the study specifically highlights a signaling cascade involving α7-nAChR/PKC/YAP/TAZ/Notch as a key driver of nicotine-induced stem cell hyperproliferation. Utilizing a Lgr5CreER Apcfl/fl mouse model, the authors extend their findings to propose a potential role of nicotine in stem cell tumorgenesis. The study posits that Notch signaling is essential during this process.

Strengths and Weaknesses:

One noteworthy research highlight in this study is the indication, as shown in Figure 2 and S2, that the trophic effect of nicotine on ISC expansion is independent of Paneth cells. In the Discussion section, the authors propose that this independence may be attributed to distinct expression patterns of nAChRs in different cell types. To further substantiate these findings, the authors provided qPCR analysis of nAchRs in ISCs and Paneth cells from isolated whole small intestine, indicating that α7-nAChR uniquely responds to nicotine treatment among various nAChRs. The authors further strengthen the clinical relevance of the study by exploring human scRNA-seq dataset, in which α7-nAChR is indeed also expressed in human ISCs and Paneth cells.

As shown in the same result section, the effect of nicotine on ISC organoid formation appears to be independent of CHIR99021, a Wnt activator. In the Lgr5CreER Apcfl/fl mouse model, it is known that APC loss results in a constitutive stabilization of β-catenin, thus the hyperproliferation of ISCs by nicotine treatment in this mouse model is likely beyond Wnt activation. The authors have included such discussion.

In Figure 4, the authors investigate ISC organoid formation with a pan-PKC inhibitor, revealing that PKC inhibition blocks nicotine-induced ISC expansion. It's noteworthy that PKC inhibitors have historically been used successfully to isolate and maintain stem cells by promoting self-renewal. Therefore, it is surprising to observe no or reversal effect on ISCs in this context. The authors have now included an additional PKC inhibitor Sotrastaurin to confirm the role of PKC in nicotine-induced ISC expansion.

Overall, the manuscript has provided sufficient experimental evidence to address my concerns and also significantly enhanced its quality.

Reviewer #3 (Public review):

Summary:

The manuscript coins a term "the census population size" which they define from the diversity of malaria parasites observed in the human community. They use it to explore changes in parasite diversity in more than 2000 people in Ghana following different control interventions.

Strengths:

This is a good demonstration of how genetic information can be used to augment routinely recorded epidemiological and entomological data to understand the dynamics of malaria and how it is controlled. The genetic information does add to our understanding, though by how much is currently unclear (in this setting it says the same thing as age stratified parasite prevalence), and its relevance moving forward will depend on the practicalities and cost of the data collection and analysis. Nevertheless, this is a great dataset with good analysis and a good attempt to understand more about what is going on in the parasite population.

Weaknesses:

None

Reviewer #2 (Public review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The use of multiscale MD simulations allows for a detailed exploration of the system at different time and length scales. The combination of MD simulations and structural bioinformatics provides a comprehensive approach to validate the identified binding sites. Finally, the steered MD simulations offer quantitative insights into the binding strength between the membrane and bnAbs.

While the simulations and analyses provide qualitative insights into the binding interactions, they do not offer a quantitative assessment of energetics. The coarse-grained simulations exhibit artifacts and thus require careful analysis.

This study contributes to a deeper understanding of the molecular mechanisms underlying bnAb recognition of the HIV-1 envelope. The insights gained from this work could inform the design of more potent and broadly neutralizing antibodies.

Reviewer #2 (Public review):

Summary:

Intrinsic primary afferent neurons are an interesting population of enteric neurons that transduce stimuli from the mucosa, initiate reflexive neurocircuitry involved in motor and secretory functions, and modulate gut immune responses. The morphology, neurochemical coding, and electrophysiological properties of these cells have been relatively well described in a long literature dating back to the late 1800's but questions remain regarding their roles in enteric neurocircuitry, potential subsets with unique functions, and contributions to disease. Here, the authors provide RNAscope, immunolabeling, electrophysiological, and organ function data characterizing IPANs in mice and suggest that Cdh6 is an additional marker of these cells.

Strengths:

This paper would likely be of interest to a focused enteric neuroscience audience and increase information regarding the properties of IPANs in mice. These data are useful and suggest that prior data from studies of IPANs in other species are likely translatable to mice.

Weaknesses:

The advance presented here beyond what is already known is minimal. Some of the core conclusions are overstated and there are multiple other major issues that limit enthusiasm. Key control experiments are lacking and data do not specifically address the properties of the proposed Cdh6+ population.

Major weaknesses:

(1) The novelty of this study is relatively low. The main point of novelty suggests an additional marker of IPANs (Cdh6) that would add to the known list of markers for these cells. How useful this would be is unclear. Other main findings basically confirm that IPANs in mice display the same classical characteristics that have been known for many years from studies in guinea pigs, rats, mice and humans.

(2) Some of the main conclusions of this study are overstated and claims of priority are made that are not true. For example, the authors state in lines 27-28 of the abstract that their findings provide the "first demonstration of selective activation of a single neurochemical and functional class of enteric neurons". This is certainly not true since Gould et al (AJP-GIL 2019) expressed ChR2 in nitrergic enteric neurons and showed that activating those cells disrupted CMC activity. In fact, prior work by the authors themselves (Hibberd et al Gastro 2018) showed that activating calretinin neurons with ChR2 evoked motor responses. Work by other groups has used chemogenetics and optogenetics to show the effects of activating multiple other classes of neurons in the gut.

(3) Critical controls are needed to support the optogenetic experiments. Control experiments are needed to show that ChR2 expression a) does not change the baseline properties of the neurons, b) that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons, and c) that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions focused on here.

(4) The electrophysiological characterization of mouse IPANs is useful but this is a basic characterization of any IPAN and really says nothing specifically about Cdh6+ neurons. The electrophysiological characterization was also only done in a small fraction of colonic IPANs, and it is not clear if these represent cell properties in the distal colon or proximal colon, and whether these properties might be extrapolated to IPANs in the different regions. Similarly, blocking IH with ZD7288 affects all IPANs and does not add specific information regarding the role of the proposed Cdh6+ subtype.

(5) Why SMP IPANs were not included in the analysis of Cdh6 expression is a little puzzling. IPANs are present in the SMP of the small intestine and colon, and it would be useful to know if this proposed marker is also present in these cells.

(6) The emphasis on IH being a rhythmicity indicator seems a bit premature. There is no evidence to suggest that IH and IT are rhythm-generating currents in the ENS.

(7) As the authors point out in the introduction and discuss later on, Type II Cadherins such as Cdh6 bind homophillically to the same cadherin at both pre- and post-synapse. The apparent enrichment of Cdh6 in IPANs would suggest extensive expression in synaptic terminals that would also suggest extensive IPAN-IPAN connections unless other subtypes of neurons express this protein. Such synaptic connections are not typical of IPANs and raise the question of whether or not IPANs actually express the functional protein and if so, what might be its role. Not having this information limits the usefulness of this as a proposed marker.

(8) Experiments shown in Figures 6J and K use a tethered pellet to drive motor responses. By definition, these are not CMCs as stated by the authors.

(9) The data from the optogenetic experiments are difficult to understand. How would stimulating IPANs in the distal colon generate retrograde CMCs and stimulating IPANs in the proximal colon do nothing? Additional characterization of the Cdh6+ population of cells is needed to understand the mechanisms underlying these effects.

Reviewer #2 (Public review):

This manuscript explores the molecular mechanisms that are involved in substrate recognition by the PP1 phosphatase. The authors previously showed that the PP1 interacting protein (PPI), PhactrI, conferred substrate specificity by remodelling the PP1 hydrophobic substrate groove. In this work, the authors aimed to understand the key determinant of how other PIPs, Neurabin and Spinophilin, mediate substrate recognition.

The authors generated a few PP1-PIP fusion constructs, undertook TMT phosphoproteomics and validated their method using PP1-Phactr1/2/3/4 fusion constructs. Using this method, the authors identified phsophorylation sites controlled by PP1-Neurabin and focussed their work on 4E-BP1, thereby linking PP1-Neurabin to mTORC1 signalling. Upon validating that PP1-Neurabin dephosphorylates 4E-BP1, they determined that 4E-BP1 PBM binds to the PDZ domain of Neurabin with an affinity that was greater than 30-fold as compared to other substrates. PP1-Neurabin dephosphorylated 4E-BP1WT and IRSp53WT with a catalytic efficiency much greater than PP1 alone. However, PP1-Neurabin bound to 4E-BP1 and IRSp53 mutants lacking the Neurabin PDZ domain with a catalytic efficiency lesser than that observed with 4E-BP1WT. These results indicate the involvement of the PDZ domain in facilitating substrate recruitment by PP1-Neurabin. Interestingly, PP1-Phactr1 dephosphorylation of 4E-BP1 phenocopies PP1 alone, while PP1-Phactr1 dephosphorylates IRSp53 to a much higher extent than PP1 alone. These results highlight the importance of the PDZ domain and also shed light on how different PP1-PIP holoenzymes mediate substrate recognition using distinct mechanisms. The authors also show that the remodelling of the hydrophobic PP1 substrate groove which is essential for substrate recognition by PP1-Phactr1, was not required by PP1-Neurabin. Additionally, the authors also resolved the structure of a PP1-4E-BP1 fusion with the PDZ-containing C-terminal of Neurabin and observed that the Neurabin/PP1-4E-BP1 complex structure was oriented at 21{degree sign} to that in the unliganded Spinophilin/PP1 complex (resolved by Ragusa et al., 2010) owing to a slight bend in the C-terminal section that connects it to the RVxF-ΦΦ-R-W string. Since no interaction was observed with the remodelled PP1-Neurabin hydrophobic groove, the authors utilised AlphaFold3 to further answer this. They observed a high confidence of interaction between the groove and phosphorylated substrate and a low confidence of interaction between the groove and unphosphorylated substrate, thereby suggesting that the hydrophobic groove remodelling is not involved in PP1-Neurabin recognition and dephosphorylation of 4E-BP1.

In this work, the authors provide novel insights into how Neurabin depends on the interaction between its PDZ domain and PBM domains of potential substrates to mediate its recruitment by PP1. Additionally, they uncover a novel PP1-Neurabin substrate, 4E-BP1. They systematically employ phosphoproteomics, biochemical, and structural methods to investigate substrate specificity in a robust fashion. Furthermore, the authors also compare the interactions between PP1-Neurabin to 4E-BP1 and IRSp53 (PP1-Phactr1 substrate) with PP1-Phactr1, to showcase the specificity of the mode of action employed by these complexes in mediating substrate specificity. The authors employ an innovative PP1-PIP fusion strategy previously explored by Oberoi et al., 2016 and the authors themselves in Fedoryshchak et al., 2020. Although this method, allows for a more controlled investigation of the interactions between PP1-PIPs and its substrates, this methodology may not fully recapitulate the interactions that may occur in a physiological setting. This could potentially be overcome by studying the interactions of the full proteins using classical biochemical approaches in cell lines. Furthermore, the authors have substantially characterised the importance of the PDZ domain using their fusion constructs, however, I believe that further exploration into either structural or AlphaFold3 modelling of PBM domain substrate mutants, or a Neurabin PDZ-domain mutant might further strengthen this claim. Overall, the paper makes a substantial contribution to understanding substrate recognition and specificity in PP1-PIP complexes. The study's innovative methods, biological relevance, and mechanistic insights are strengths, but whether this mechanism occurs in a physiological context is unclear.

Reviewer #2 (Public review):

Summary:

This study develops a new artificial intelligence method for high-throughput analysis of skull bone marrow from MRI data, which may be useful for large-scale biological analyses. Using this method, the authors then attempt to estimate skull bone marrow adiposity (BMA) using T1-weighted signal intensity from MRI scans of ~33,000 people, followed by genome-wide association analysis; however, the approach is inadequate because T1-weighted signal intensity is not validated for measurement of bone marrow adiposity. If it could be validated, the study would be an important advance in understanding of bone marrow adiposity and skeletal biology.

Strengths:

This paper is well-written, and the figures are nicely presented. The neural network method used for analysing skull bone marrow is innovative, and the authors validate this through several approaches. Therefore, the authors have achieved the aim of developing a method for large-scale analysis of skull bone marrow from MRI data.

The GWAS is reasonably well-powered and addresses potential ethnicity differences, with one GWAS done across white males and females, and a separate GWAS in non-white participants. The methodology also conforms to common GWAS standards, including for mapping genetic variants to candidate genes. Moreover, the study further investigates the biological roles of these genes by analysing their expression in single-cell RNA sequencing data.

Weaknesses:

The fundamental weakness is that T1-weighted MRI signal intensity (T1W) is used as an estimate of BMA, but it has never been validated for this. The authors show that this T1W parameter measures something that is heritable and can be compared between subjects, but they don't show that it actually measures (or even estimates) calvarial BMA. There is an attempt to do so by comparing the T1W parameter with data from quantitative T1 images: the authors show a reasonable correlation with some of the quantitative T1 image data. However, this still does not show that the parameter is measuring BMA; it could be measuring some other biological characteristic, but this remains unclear. So, there is a need to validate the T1W parameter against an established measure of BMA, such as the bone marrow fat-fraction or proton density fat fraction measured from multi-echo MRI analysis.

Without validating this BMA measurement method, it is not possible to interpret the GWAS or other findings reported in the study.

A less critical weakness is that the GWAS has been done only on a single cohort, without replicating the findings in a follow-up cohort. For example, the authors could repeat their analysis on the remaining ~50,000 UK Biobank imaging participants for whom MRI data is now available. However, this would be pointless without knowing what biological characteristic(s) the T1W parameter is actually reflecting.

Reviewer #2 (Public review):

Summary:

Salmonella exploits host- and bacteria-derived β-alanine to efficiently replicate in host macrophages and cause systemic disease. β-alanine executes this by increasing the expression of zinc transporter genes and therefore the uptake of zinc by intracellular Salmonella.

Strengths:

The experiments designed are thorough and the claims made are directly related to the outcome of the experiments. No overreaching claims were made.

Weaknesses:

A little deeper insight was expected, particularly towards the mechanistic aspects. For example, zinc transport was found to be the cause of the b-alanine-mediated effect on Salmonella intracellular replication. It would have been very interesting to see which are the governing factors that may get activated or inhibited due to Zn accumulation that supports such intracellular replication.

Reviewer #2 (Public review):

Summary:

In this manuscript, Tolossa et al. analyze Inter-spike intervals from various freely available datasets from the Allen Institute and from a dataset from Steinmetz et al. They show that they can modestly decode between gross brain regions (Visual vs. Hippocampus vs. Thalamus), and modestly separate sub-areas within brain regions (DG vs. CA1 or various visual brain areas).

Strengths:

The paper is reasonably well written, and the definitions are quite well done. For example, the authors clearly explained transductive vs. inductive inference in their decoders. E.g., transductive learning allows the decoder to learn features from each animal, whereas inductive inference focuses on withheld animals and prioritizes the learning of generalizable features.

Weaknesses:

However, even with some of these positive aspects, I still found the manuscript to be a laundry list of results, where some results are overly explained and not particularly compelling or interesting, whereas interesting results are not strongly described or emphasized. The overall problem is that the study is not cohesive, and the authors need to either come up with a tool or demonstrate a scientific finding. The current version attempts to split the middle and thus is not as impactful as it could be.

Reviewer #2 (Public review):

Summary:

Previous work in the field highlighted the role of the kinesin-10 motor protein Kid (KIF22) in the polar ejection force during prometaphase. However, the biochemical and biophysical properties of Kid that enabled it to serve in this role were unclear. The authors demonstrate that human and xenopus Kid proteins are processive kinesins that function as homodimeric molecules. The data are solid and support the findings although the text could use some editing to improve clarity.

Strengths:

A highlight of the work is the reconstitution of DNA transport in vitro.

A second highlight is the demonstration that the monomer vs dimer state is dependent on protein concentration.

Weaknesses:

The authors make several assumptions of the monomer vs dimer state of various Kid constructs without verifying the protein state using e.g. size exclusion chromatography and/or nanophotometry. They also make statements about monomer-to-dimer transitions on the microtubule without showing or quantifying the data.

The discussion needs to better put the work into context regarding the ability of non-processive motors to work in teams (formerly thought to be the case for Kid) and how their findings on Kid change this prevailing view in the case of polar ejection force.

The authors also do not mention previous work on kinesins with non-conventional neck linker/neck coil regions that have been shown to move processively. Their work on Kid needs to be put into this context.

Reviewer #2 (Public review):

Summary:

The authors aim to provide a comprehensive description of the neurosecretory network in the adult Drosophila brain. They sought to assign and verify the types of 80 neurosecretory cells (NSCs) found in the publicly available FlyWire female brain connectome. They then describe the organization of synaptic inputs and outputs across NSC types and outline circuits by which olfaction may regulate NSCs, and by which Corazon-producing NSCs may regulate flight behavior. Leveraging existing transcriptomic data, they also describe the hormone and receptor expressions in the NSCs and suggest putative paracrine signaling between NSCs. Taken together, these analyses provide a framework for future experiments, which may demonstrate whether and how NSCs, and the circuits to which they belong, may shape physiological function or animal behavior.

Strengths:

This study uses the FlyWire female brain connectome (Dorkenwald et al. 2023) to assign putative cell types to the 80 neurosecretory cells (NSCs) based on clustering of synaptic connectivity and morphological features. The authors then verify type assignments for selected populations by matching cluster sizes to anatomical localization and cell counts using immunohistochemistry of neuropeptide expression and markers with known co-expression.

The authors compare their findings to previous work describing the synaptic connectivity of the neurosecretory network in larval Drosophila (Huckesfeld et al., 2021), finding that there are some differences between these developmental stages. Direct comparisons between adults and larvae are made possible through direct comparison in Table 1, as well as the authors' choice to adopt similar (or equivalent) analyses and data visualizations in the present paper's figures.

The authors extract core themes in NSC synaptic connectivity that speak to their function: different NSC types are downstream of shared presynaptic outputs, suggesting the possibility of joint or coordinated activation, depending on upstream activity. NSCs receive some but not all modalities of sensory input. NSCs have more synaptic inputs than outputs, suggesting they predominantly influence neuronal and whole-body physiology through paracrine and endocrine signaling.

The authors outline synaptic pathways by which olfactory inputs may influence NSC activity and by which Corazon-releasing NSCs may regulate flight. These analyses provide a basis for future experiments, which may demonstrate whether and how such circuits shape physiological function or animal behavior.

The authors extract expression patterns of neuropeptides and receptors across NSC cell types from existing transcriptomic data (Davie et al., 2018) and present the hypothesis that NSCs could be interconnected via paracrine signaling. The authors also catalog hormone receptor expression across tissues, drawing from the Fly Cell Atlas (Li et al., 2022).

Weaknesses:

The clustering of NSCs by their presynaptic inputs and morphological features, along with corroboration with their anatomical locations, distinguished some, but not all cell types. The authors attempt to distinguish cell types using additional methodologies: immunohistochemistry (Figure 2), retrograde trans-synaptic labeling, and characterization of dense core vesicle characteristics in the FlyWire dataset (Figure 1, Supplement 1). However, these corroborating experiments often lacked experimental replicates, were not rigorously quantified, and/or were presented as singular images from individual animals or even individual cells of interest. The assignments of DH44 and DMS types remain particularly unconvincing.

The authors present connectivity diagrams for visualization of putative paracrine signaling between NSCs based on their peptide and receptor expression patterns. These transcriptomic data alone are inadequate for drawing these conclusions, and these connectivity diagrams are untested hypotheses rather than results. The authors do discuss this in the Discussion section.

Reviewer #2 (Public review):

Summary:

Adhikari and colleagues developed a new technique, rapamycin-induced proximity assay (RiPA), to identify E3-ubiquitin (ub) ligases of a protein target, aiming at identifying additional E3 ligases that could be targeted for PROTAC generation or ligases that may degrade a protein target. The study is timely, as expanding the landscape of E3-ub ligases for developing targeted degraders is a primary direction in the field.

Strengths:

(1) The study's strength lies in its practical application of the FRB:FKBP12 system. This system is used to identify E3-ub ligases that would degrade a target of interest, as evidenced by the reduction in luminescence upon the addition of rapamycin. This approach effectively mimics the potential action of a PROTAC.

Weaknesses:

(1) While the technique shows promise, its application in a discovery setting, particularly for high-throughput or unbiased E3-ub ligase identification, may pose challenges. The authors now discuss these potential difficulties providing a more comprehensive understanding of RiPA's limitations.

(2) While RiPA will help identify E3 ligases, PROTAC design would still be empirical. The authors provide some discussion of this limitation.

Comments on revisions:

I thank the authors for addressing my prior concerns. I would recommend that individual replicate values are plotted in all the mean -/+ s.d or sem graphs.

Reviewer #2 (Public review):

Summary:

This well-written manuscript addresses an important but recalcitrant problem - the molecular mechanism of protein misfolding in Ig light chain (LC) amyloidosis (AL), a major life-threatening form of systemic human amyloidosis. The authors use expertly recorded and analyzed small-angle X-ray scattering (SAXS) data as a restraint for molecular dynamics simulations (called M&M) and to explore six patient-based LC proteins. The authors report that a highly populated "H-state" determined computationally, wherein the two domains in an LC molecule acquire a straight rather than bent conformation, is what distinguishes AL from non-AL LCs. They then use H-D exchange mass spectrometry to verify this conclusion. If confirmed, this is a novel and interesting finding with potentially important translational implications.

Strengths:

Expertly recorded and analyzed SAXS data combined with clever M&M simulations lead to a novel and interesting conclusion.

Regardless of whether or not the CL-CL domain interface is destabilized in AL LCs explored in this (Figure 6) and other studies, stabilization of this interface is an excellent idea that may help protect at least a subset of AL LCs from misfolding in amyloid. This idea increases the potential impact of this interesting study.

Weaknesses:

The HDX analysis could be strengthened.

Reviewer #2 (Public review):

Summary:

These studies investigate the phenotypic variability and roles of neutrophils in tuberculosis (TB) susceptibility by using a diverse collection of wild-derived inbred mouse lines. The authors aimed to identify new phenotypes during Mycobacterium tuberculosis infection by developing, infecting, and phenotyping 19 genetically diverse wild-derived inbred mouse lines originating from different geographic regions in North America and South America. The investigators achieved their main goals, which were to show that increasing genetic diversity increases the phenotypic spectrum observed in response to aerosolized M. tuberculosis, and further to provide insights into immune and/or inflammatory correlates of pulmonary TB. Briefly, investigators infected wild-derived mice with aerosolized M. tuberculosis and assessed early infection control at 21 days post-infection. The time point was specifically selected to correspond to the period after infection when acquired immunity and antigen-specific responses manifest strongly, and also early susceptibility (morbidity and mortality) due to M. tuberculosis infection has been observed in other highly susceptible wild-derived mouse strains, some Collaborative Cross inbred strains, and approximately 30% of individuals in the Diversity Outbred mouse population. Here, the investigators normalized bacterial burden across mice based on inoculum dose and determined the percent of immune cells using flow cytometry, primarily focused on macrophages, neutrophils, CD4 T cells, CD8 T cells, and B cells in the lungs. They also used single-cell RNA sequencing to identify neutrophil subpopulations and immune phenotypes, elegantly supplemented with in vitro macrophage infections and antibody depletion assays to confirm immune cell contributions to susceptibility. The main results from this study confirm that mouse strains show considerable variability to M. tuberculosis susceptibility. Authors observed that enhanced infection control correlated with higher percentages of CD4 and CD8 T cells, and B cells, but not necessarily with the percentage of interferon-gamma (IFN-γ) producing cells. High levels of neutrophils and immature neutrophils (band cells) were associated with increased susceptibility, and the mouse strain with the most neutrophils, the MANC line, exhibited a transcriptional signature indicative of a highly activated state, and containing potentially tissue-destructive, mediators that could contribute to the strain's increased susceptibility and be leveraged to understand how neutrophils drive lung tissue damage, cavitation, and granuloma necrosis in pulmonary TB.

Strengths:

The strengths are addressing a critically important consideration in the tuberculosis field - mouse model(s) of the human disease, and taking advantage of the novel phenotypes observed to determine potential mechanisms. Notable strengths include,

(1) Innovative generation and use of mouse models: Developing wild-derived inbred mice from diverse geographic locations is innovative, and this approach expands the range of phenotypic responses observed during M. tuberculosis infection. Additionally, the authors have deposited strains at The Jackson Laboratory making these valuable resources available to the scientific community.

(2) Potential for translational research: The findings have implications for human pulmonary TB, particularly the discovery of neutrophil-associated susceptibility in primary infection and/or neutrophil-mediated disease progression that could both inform the development of therapeutic targets and also be used to test the effectiveness of such therapies.

(3) Comprehensive experimental design: The investigators use many complementary approaches including in vivo M. tuberculosis infection, in vitro macrophage studies, neutrophil depletion experiments, flow cytometry, and a number of data mining, machine learning, and imaging to produce robust and comprehensive analyses of the wild-derives d strains and neutrophil subpopulations in 3 weeks after M. tuberculosis infection.

Weaknesses:

The manuscript and studies have considerable strengths and very few weaknesses. One minor consideration is that phenotyping is limited to a single limited-time point; however, this time point was carefully selected and has a strong biological rationale provided by investigators. This potential weakness does not diminish the overall findings, exciting results, or conclusions.

Reviewer #2 (Public review):

Summary:

This work is towards the development of nonantibiotic treatment for C. difficile. The authors screened a chemical library for activity against the C. difficile toxin TcdB, and found a group of compounds with antitoxin activity. Caffeic acid derivatives were highly represented within this group of antitoxin compounds, and the remaining portion of this work involves defining the mechanism of action of caffeic acid phenethyl ester (CAPE) and testing CAPE in mouse C. difficile infection model. The authors conclude CAPE attenuates C. difficile disease by limiting toxin activity and increasing microbial diversity during C. difficile infection.

Strengths/ Weaknesses:

The strategy employed by the authors is sound although not necessarily novel. A compound that can target multiple steps in the pathogenies of C. difficile would be an exciting finding. However, the data presented does not convincingly demonstrate that CAPE attenuates C. difficile disease and the mechanism of action of CAPE is not convincingly defined. The following points highlight the rationale for my evaluation.

(1) The toxin exposure in tissue culture seems brief (Figure 1). Do longer incubation times between the toxin and cells still show CAPE prevents toxin activity?

(2) The conclusion that CAPE has antitoxin activity during infection would be strengthened if the mouse was pretreated with CAPE before toxin injections (Figure 1D).

(3) CAPE does not bind to TcdB with high affinity as shown by SPR (Figure 4). A higher affinity may be necessary to inhibit TcdB during infection. The GTD binds with millimolar affinity and does not show saturable binding. Is the GTD the binding site for CAPE? Autoprocessing is also affected by CAPE indicating CAPE is binding non-GTD sites on TcdB.

(4) In the infection model, CAPE does not statistically significantly attenuate weight loss during C. difficile infection (Figure 6). I recognize that weight loss is an indirect measure of C. difficile disease but histopathology also does not show substantial disease alleviation (see below).

(5) In the infection model (Figure 6), the histopathology analysis shows substantial improvement in edema but limited improvement in cellular infiltration and epithelial damage. Histopathology is probably the most critical parameter in this model and a compound with disease-modifying effects should provide substantial improvements.

(6) The reduction in C. difficile colonization is interesting. It is unclear if this is due to antitoxin activity and/or due to CAPE modifying the gut microbiota and metabolites (Figure 6). To interpret these data, a control is needed that has CAPE treatment without C. difficile infection or infection with an atoxicogenic strain.

(7) Similar to the CAPE data, the melatonin data does not display potent antitoxin activity and the mouse model experiment shows marginal improvement in the histopathological analysis (Figure 9). Using 100 µg/ml of melatonin (~ 400 micromolar) to inactivate TcdB in cell culture seems high. Can that level be achieved in the gut?

(8) The following parameters should be considered and would aid in the interpretation of this work. Does CAPE directly affect the growth of C. difficile? Does CAPE affect the secretion of TcdB from C. difficile? Does CAPE alter the sporulation and germination of C. diffcile?

Reviewer #2 (Public review):

Summary:

The authors present an interesting paper where they test the antagonistic pleiotropy theory. Based on this theory they hypothesize that genetic variants associated with later onset of age at menarche and age at first birth have a positive causal effect on a multitude of health outcomes later in life, such as epigenetic aging and prevalence of chronic diseases. Using a mendelian randomization and colocalization approach, the authors show that SNPs associated with later age at menarche are associated with delayed aging measurements, such as slower epigenetic aging and reduced facial aging, and a lower risk of chronic diseases, such as type 2 diabetes and hypertension. Moreover, they identified 128 fertility-related SNPs that are associated with age-related outcomes and they identified BMI as a mediating factor for disease risk, discussing this finding in the context of evolutionary theory.

Strengths:

The major strength of this manuscript is that it addresses the antagonistic pleiotropy theory in aging. Aging theories are not frequently empirically tested although this is highly necessary. The work is therefore relevant for the aging field as well as beyond this field, as the antagonistic pleiotropy theory addresses the link between fitness (early life health and reproduction) and aging.

Points that have to be clarified/addressed:

(1) The antagonistic pleiotropy is an evolutionary theory pointing to the possibility that mutations that are beneficial for fitness (early life health and reproduction) may be detrimental later in life. As it concerns an evolutionary process and the authors focus on contemporary data from a single generation, more context is necessary on how this theory is accurately testable. For example, why and how much natural variation is there for fitness outcomes in humans? How do genetic risk score distributions of the exposure data look like? Also, how can the authors distinguish in their data between the antagonistic pleiotropy theory and the disposable soma theory, which considers a trade-off between investment in reproduction and somatic maintenance and can be used to derive similar hypotheses? There is just a very brief mention of the disposable soma theory in lines 196-198.

(2) The antagonistic pleiotropy theory, used to derive the hypothesis, does not necessarily distinguish between male and female fitness. Would the authors expect that their results extrapolate to males as well? And can they test that?

(3) There is no statistical analyses section providing the exact equations that are tested. Hence it's not clear how many tests were performed and if correction for multiple testing is necessary. It is also not clear what type of analyses have been done and why they have been done. For example in the section starting at line 47, Odds Ratios are presented, indicating that logistic regression analyses have been performed. As it's not clear how the outcomes are defined (genotype or phenotype, cross-sectional or longitudinal, etc.) it's also not clear why logistic regression analysis was used for the analyses.

(4) Mendelian Randomization is an important part of the analyses done in the manuscript. It is not clear to what extent the MR assumptions are met, how the assumptions were tested, and if/what sensitivity analyses are performed; e.g. reverse MR, biological knowledge of the studied traits, etc. Can the authors explain to what extent the genetic instruments represent their targets (applicable expression/protein levels) well?

(5) It is not clear what reference genome is used and if or what imputation panel is used. It is also not clear what QC steps are applied to the genotype data in order to construct the genetic instruments of MR.

(6) A code availability statement is missing. It is understandable that data cannot always be shared, but code should be openly accessible.

Reviewer #2 (Public review):

Summary:

The authors provided further evidence that menstrual fluid (MF) can be used as a non-invasive source of endometrial tissue for studying its normal physiological state and when it is abnormal such as in endometriosis. Single-cell RNA sequencing confirmed the presence of the major cell types -blood and tissue immune cells and endometrial stromal, epithelial, and vascular cells. The major new finding was that interindividual variation for the blood immune cells was minimal between multiple MF samples from an individual. A comparison between the ex vivo MF gene profile and cultured MF showed the expected attachment and culture of stromal (and a small number of epithelial) cells, but the immune cells failed to attach. Several differentially expressed genes between controls and endometriosis were suggested as potential biomarkers of the disease, however, these were a mitochondrial pseudogene and a hemoglobin subunit, both very unlikely related to endometriosis pathogenesis.

Strengths:

The Spearman correlation analysis between the control MF gene profiles of multiple samples from the same individual and its graphic presentation provided strong evidence that there is little variation between MF samples. Together with another study which showed similar findings for endometrial stem cells and a number of proteins in MF supernatant, this important data shows MF as a promising biofluid for pathology testing.

The bioinformatic analyses conducted by bioinformatic and computational experts are a major strength of the manuscript and in particular the comparison between MF and endometrial biopsy data obtained from published scRNAseq studies. This is an important finding, particularly if comparisons included late secretory and early proliferative stage biopsy tissue which would be most similar to shedding menstrual endometrium.

The inclusion of workflows in the Figures for the various studies and the use of symbols in the various panels is very helpful for the reader.

MF cell suspensions were enriched for stromal and epithelial cells to enable a detailed bioinformatic analysis of their respective gene profiles

Weaknesses:

Two patient cohorts from different institutions were used in the study and somewhat different methods were used to extract the cellular fraction from these cohorts for further study: (1) sample dilution and differential filtration to separate blood-derived immune cells from endometrial tissue then dissociated into single cells and separated into CD45+, CD45-EpCAM+ and CD45-EpCAM- cells, and (2) gradient density separation to generate unsorted, CD45+, CD45- and putative mesenchymal stem cells (MSC) CD45-CD105+ which were also cultured. In addition, questions on pelvic pain and proven fertility would have addressed the 2 key symptoms of endometriosis.

The use of CD105 to purify MSC from MF rather than well-characterised markers of clonogenic, self-renewing, and mesodermal differentiating endometrial MSC such as CD146+PDGFRB+ or SUSD2 (both mentioned in references 22 and 23) is a weakness. The ISCT markers are not specific and are also found on stromal fibroblasts of many tissues (Phinney and Sensebe Cytotherapy 2013; Demu et al Acta Haematologica 2016).<br /> The UMAPs generated from the scRNAseq were at low resolution and more individual immune and endometrial cell types have previously been identified and reported in MF. More comparisons with these studies would also have enhanced the Discussion.

It was not always possible to work out how the data was reported in the gene expression tables (Supplementary Tables 2, 4-10) as they were not in adjusted P value order and sometimes positive log2 fold change values appeared amongst the negative log2FC. In some comparisons described, the adj P values were not significant but were described as up or down-regulated in the text.

The 2 DEGs highlighted in the endometriosis and control arm of the study appear as poor choices from many others that could have been chosen as MTRNR2L1 is a mitochondrial pseudogene and HBG2 is a hemoglobin subunit. Neither are likely indicators of endometriosis pathogenesis.

The manuscript format and organisation could be improved by reducing the discussion in the Results section and providing a more in-depth Discussion. More references need to be included in the Discussion and other work in the MF analysis field that supports - or not - the authors' findings or at least puts them into context, and should be included and referenced.

The potential to use MF as a non-invasive source of endometrial tissue for potential diagnosis is a very important avenue of research that is currently in its infancy and could have a major impact in the endometriosis research arena.

Reviewer #2 (Public review):

Summary:

The present study sets out to examine the impact of counterconditioning (CC) and extinction on conditioned threat responses in humans, particularly looking at neural mechanisms involved in threat memory suppression. By combining behavioral, physiological, and neuroimaging (fMRI) data, the authors aim to provide a clear picture of how CC might engage unique neural circuits and coding dynamics, potentially offering a more robust reduction in threat responses compared to traditional extinction.

Strengths:

One major strength of this work lies in its thoughtful and unique design - integrating subjective, physiological, and neuroimaging measures to capture the variouse aspects of counterconditioning (CC) in humans. Additionally, the study is centered on a well-motivated hypothesis and the findings have the potential to improve the current understanding of pathways associated with emotional and cognitive control.

The data presentation is systematic, and the results on behavioral and physiological measures fit well with the hypothesized outcomes. The neuroimaging results also provide strong support for distinct neural mechanisms underlying CC versus extinction.

Weaknesses:

Overall, this study is a well-conducted and thought-provoking investigation into counterconditioning, with strong potential to advance our understanding of threat modulation mechanisms. Two main weaknesses concern the scope and decisions regarding analysis choices. First, while the findings are solid, the topic of counterconditioning is relatively niche and may have limited appeal to a broader audience. Expanding the discussion to connect counterconditioning more explicitly to widely studied frameworks in emotional regulation or cognitive control would enhance the paper's accessibility and relevance to a wider range of readers. This broader framing could also underscore the generalizability and broader significance of the results. In addition, detailed steps in the statistical procedures and analysis parameters seem to be missing. This makes it challenging for readers to interpret the results in light of potential limitations given the data modality and/or analysis choices.

Reviewer #2 (Public review):

This study found that D1-MSNs and D2-MSNs have opposing dynamics during interval timing in a mouse-optimized interval timing task. Further optogenetic and pharmacologic inhibition of either D1 or D2 MSNs increased response time. This study provides useful experimental evidence in the coding of time in striatum. However, there are some major weaknesses in this study.

(1) Regarding the data in Figure S3, The variance within each mouse was too big, the authors need to figure out and explain what caused the large variance within the same mouse, or the authors need to increase the sample size.<br /> (2) Regarding the results in Figure 3 C and D, Figure 6 H and Figure 7 D, what is the sample size? From the single data points in the figures, it seems that the authors were using the number of cells to do statistical tests and plot the figures. For example, Figure 3 C, if the authors use n= 32 D2 MSNs and n= 41D1 MSNs to do the statistical test, it could make small difference to be statistically significant. The authors should use the number of mice to do the statistical tests.<br /> (3) Regarding the results in Figure 5, what is the reason for the increase in the response times? The authors should plot the position track during intervals (0-6 s) with or without optogenetic or pharmacologic inhibition. The authors can check Figure 3, 5, and 6 in paper https://doi.org/10.1016/j.cell.2016.06.032 for reference to analyze the data.

Reviewer #2 (Public review):

Summary:

Mitochondria import hundreds of precursor proteins from the cytosol. The TOM and TIM23 complexes facilitate the import on the matrix-targeting pathway of mitochondria. In yeast, Tim50 is a critical and essential subunit of the TIM23 complex that mediates the transition of precursors from the outer to the inner membrane. The human Tim50 homolog TIMM50 is highly similar in structure and a comparable function of Tim50 and TIMM50 was proven by several biochemical and genetic studies in the past.

In this study, the authors characterize human cells which express lower levels or mutated versions of TIMM50. They found that in these TIMM50-depletion cells, the levels of other TIM23 core subunits are also diminished but many mitochondrial proteins are unaffected. Moreover, they observed alterations in the electrical activity and the levels of potassium channels in neuronal cells of TIMM50-deficient mice. They propose that these changes explain the pathology of patients who often suffer from epilepsy.

Strengths:

The paper is written by experts in the field, and it is very clear. The experiments are of high quality and sufficiently well-controlled. The study is interesting for a broad readership.

Weaknesses:

The authors show that even upon low levels of Tim50, mitochondrial proteins are not considerably depleted. However, it remains somewhat unclear why this is. TIMM50 and the TIM23 complex might not be rate-limiting for the biogenesis of mitochondrial proteins. Alternatively, the import defect is compensated indirectly, for example by a reduced growth of cells. It will be interesting to study the physiological consequences of TIMM50-depletion in more depth in the future.

Reviewer #3 (Public review):

Summary:

The authors aimed to determine the mechanism by which seizures emerge in Developmental and Epileptic Encephalopathies caused by variants in the gene FGF13. Loss of FGF13 in excitatory neurons had no effect on seizure phenotype as compared to loss of FGF13 in GABAergic interneurons, which in contrast caused a dramatic proseizure phenotype and early death in these animals. They were able to show that Fgf13 ablation and consequent loss of FGF13-S and FGF13-VY reduced overall inhibitory input from Fgf13-expressing interneurons onto hippocampal pyramidal neurons. This was shown to occur not via disruption to voltage gated sodium channels but rather by reducing potassium currents and action potential repolarisation in these interneurons.

Strengths:

The authors employed multiple well validated, novel mouse lines with FGF13 knocked out in specific cell types including all neurons, all excitatory cells, all GABAergic interneurons, or a subset of MGE-derived interneurons, including axo-axonic chandelier cells. The phenotypes of each of these four mouse lines were carefully characterised to reveal clear differences with the most fundamental being that Interneuron-targeted deletion of FGF13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while deletion of FGF13 in excitatory neurons caused no detectable seizures and no survival deficits.<br /> The authors made excellent use of western blotting and in situ hybridisation of the different FGF13 isoforms to determine which isoforms are expressed in which cell types, with FGF3-S predominantly in excitatory neurons and FGF13-VY and FGF13-V predominantly in GABAergic neurons.

The authors performed highly detailed electrophysiological analysis of excitatory neurons and GABAergic interneurons with FGF13 deficits using whole-cell patch clamp. This enabled them to show that FGF13 removal did not affect voltage-gated sodium channels in interneurons, but rather reduced the action of potassium channels, with the resultant effect of making it more likely that interneurons enter depolarisation block. These findings were strengthened by the demonstration that viral re-expression of different Fgf13 splice isoforms could partially rescue deficits in interneuron action potential output and restore K+ channel current size.

Additionally, the discussion was nuanced, and demonstrated how the current findings resolved previous apparent contradictions in the field involving the function of FGF13.

These findings will have a significant impact on our understanding of how FGF13 causes seizures and death in DEEs, and the action of different FGF13 isoforms within different neuronal cell types, particularly GABAergic interneurons.

Comments on revisions:

I appreciate the author's responses to the previous round of reviews. All my comments have been addressed. Congratulations on an excellent body of work.

Reviewer #2 (Public review):

In this manuscript, Li and collaborators set out to investigate the neuronal mechanisms underlying "subjective time estimation" in rats. For this purpose, they conducted calcium imaging in the prefrontal cortex of water-restricted rats that were required to perform an action (nose-poking) for a short duration to obtain drops of water. The authors provided evidence that animals progressively improved in performing their task. They subsequently analyzed the calcium imaging activity of neurons and identify start, duration, and stop cells associated with the nose poke. Specifically, they focused on duration cells and demonstrated that these cells served as a good proxy for timing on a trial-by-trial basis, scaling their pattern of actvity in accordance with changes in behavioral performance. In summary, as stated in the title, the authors claim to provide mechanistic insights into subjective time estimation in rats, a function they deem important for various cognitive conditions.

This study aligns with a wide range of studies in system neuroscience that presume that rodents solve timing tasks through an explicit internal estimation of duration, underpinned by neuronal representations of time. Within this framework, the authors performed complex and challenging experiments, along with advanced data analysis, which undoubtedly merits acknowledgement. However, the question of time perception is a challenging one, and caution should be exercised when applying abstract ideas derived from human cognition to animals. Studying so-called time perception in rats has significant shortcomings because, whether acknowledged or not, rats do not passively estimate time in their heads. They are constantly in motion. Moreover, rats do not perform the task for the sake of estimating time but to obtain their rewards are they water restricted. Their behavior will therefore reflect their motivation and urgency to obtain rewards. Unfortunately, it appears that the authors are not aware of these shortcomings. These alternative processes (motivation, sensorimotor dynamics) that occur during task performance are likely to influence neuronal activity. Consequently, my review will be rather critical. It is not however intended to be dismissive. I acknowledge that the authors may have been influenced by numerous published studies that already draw similar conclusions. Unfortunately, all the data presented in this study can be explained without invoking the concept of time estimation. Therefore, I hope the authors will find my comments constructive and understand that as scientists, we cannot ignore alternative interpretations, even if they conflict with our a priori philosophical stance (e.g., duration can be explicitly estimated by reading neuronal representation of time) and anthropomorphic assumptions (e.g., rats estimate time as humans do). While space is limited in a review, if the authors are interested, they can refer to a lengthy review I recently published on this topic, which demonstrates that my criticism is supported by a wide range of timing experiments across species (Robbe, 2023). In addition to this major conceptual issue that casts doubt on most of the conclusions of the study, there are also several major statistical issues.

Main Concerns

(1) The authors used a task in which rats must poke for a minimal amount of time (300 ms and then 1500 ms) to be able to obtain a drop of water delivered a few centimeters right below the nosepoke. They claim that their task is a time estimation task. However, they forget that they work with thirsty rats that are eager to get water sooner than later (there is a reason why they start by a short duration!). This task is mainly probing the animals ability to wait (that is impulse control) rather than time estimation per se. Second, the task does not require to estimate precise time because there appear to be no penalties when the nosepokes are too short or when they exceed. So it will be unclear if the variation in nosepoke reflects motivational changes rather than time estimation changes. The fact that this behavioral task is a poor assay for time estimation and rather reflects impulse control is shown by the tendency of animals to perform nose-pokes that are too short, the very slow improvement in their performance (Figure 1, with most of the mice making short responses), and the huge variability. Not only do the behavioral data not support the claim of the authors in terms of what the animals are actually doing (estimating time), but this also completely annihilates the interpretation of the Ca++ imaging data, which can be explained by motivational factors (changes in neuronal activity occurring while the animals nose poke may reflect a growing sens of urgency to check if water is available).

(2) A second issue is that the authors seem to assume that rats are perfectly immobile and perform like some kind of robots that would initiate nose pokes, maintain them, and remove them in a very discretized manner. However, in this kind of task, rats are constantly moving from the reward magazine to the nose poke. They also move while nose-poking (either their body or their mouth), and when they come out of the nose poke, they immediately move toward the reward spout. Thus, there is a continuous stream of movements, including fidgeting, that will covary with timing. Numerous studies have shown that sensorimotor dynamics influence neural activity, even in the prefrontal cortex. Therefore, the authors cannot rule out that what the records reflect are movements (and the scaling of movement) rather than underlying processes of time estimation (some kind of timer). Concretely, start cells could represent the ending of the movement going from the water spout to the nosepoke, and end cells could be neurons that initiate (if one can really isolate any initiation, which I doubt) the movement from the nosepoke to the water spout. Duration cells could reflect fidgeting or orofacial movements combined with an increasing urgency to leave the nose pokes.

(3) The statistics should be rethought for both the behavioral and neuronal data. They should be conducted separately for all the rats, as there is likely interindividual variability in the impulsivity of the animals.

(4) The fact that neuronal activity reflects an integration of movement and motivational factors rather than some abstract timing appears to be well compatible with the analysis conducted on the error trials (Figure 4), considering that the sensorimotor and motivational dynamics will rescale with the durations of the nose poke.

(5) The authors should mention upfront in the main text (result section) the temporal resolution allowed by their Ca+ probe and discuss whether it is fast enough in regard of behavioral dynamics occurring in the task.

Comments on the revised version

I have read the revised version of the manuscript and the rebuttal letter. My major concern was that the task used is not a time estimation task but primarily taps into impulse control and that animals are not immobile during the nose-poking epoch. I provided factual evidence for this (the animal's timing performance is poor and, on average, animals struggle to wait long enough), and I pointed to a review that discusses the results of many studies congruent with the importance of movement/motivation, not only in constraining the timing of reward-oriented actions during so-called time estimation tasks but also in powerfully modulating neuronal activity.

The authors' responses to my comments are puzzling and unconvincing. First, on the one hand, they acknowledge in their rebuttal letter the difficulty of demonstrating a neuronal representation of explicit internal estimation of time. Then, they seem to imply that this issue is beyond the scope of their study and focus in the rebuttal on whether the neuronal activity they report shows signs of being sensitive to movement and motivation, which they claim is independent of movement and motivation. This leads the authors to make no major changes in their manuscript. Their title, abstract, introduction, and discussion are largely unchanged and do not reflect the possibility that there are major confounding factors in so-called time estimation (rodents are not disembodied passive information processors) that may well explain some of the neuronal patterns. Evidently, the dismissive treatment by the authors is not satisfying. I will briefly restate my comments and reply to their responses and their new figure, which not only is unconvincing but raises new questions.

My comments were primarily focused on the behavioral task. The authors replied: "Studying the neural representation of any internal state may suffer from the same ambiguity [by ambiguity they meant that it is difficult to know if animals are explicitly estimating time]. With all due respect, however, we would like to limit our response to the scope of our results. According to the reviewer, two alternative interpretations of the task-related sequential activity exist." The authors imply that my comments are beyond the scope of their study. That is not true. My comments were targeted at the behavior of the animals, behavior they rely on to title their study: "Stable sequential dynamics in prefrontal cortex represents a subjective estimation of time." When I question whether the task and behavioral data presented are congruent with "subjective estimation of time," my comments are not beyond the scope of the study-they directly tackle the main point of the authors. Other researchers will read the title and abstract of this manuscript and conclude: "Here is a paper that provides evidence of a mechanism for animals estimating duration internally (because subjective time perception is assumed to be different from using clocks)." Still, there is a large body of literature showing that the behavior of animals in such tasks can be entirely explained without invoking subjective time perception and internal representation. How can the authors acknowledge that they can't be sure that mice are estimating time and then have such an affirmative title and abstract?

In my opinion, science is not just about forcing ideas (often reflecting philosophical preconceptions) on data and dismissing those who disagree. It is about discussing alternative possibilities fairly and being humble. In their revised version, I see no effort by the authors to investigate the importance of movement and motivation during their task or seriously engage with this idea. It's much easier to dismiss my comments as being beyond the scope of their results. According to the authors, it seems that movements and motivations play no role in the task. Still, the animals are water-restricted, and during the task, they will display decreased motivation (due to increased satiety), and their history of rewarded vs. non-rewarded trials will affect their behavior. This is one of the most robust effects seen across all behavioral studies. Moreover, the animals are constantly moving. Maybe the authors used a special breed of mice that behave like some kind of robots? I acknowledge that this is not easy to investigate, but if the authors did not use high-quality video recording or an experimental paradigm that allows disentangling motivational confounds, then they should refrain from using big words such as subjective time estimation and discuss alternative representations by acknowledging the studies that do find that movement and motivation are present during reward-based timing tasks and do in fact modulate neuronal activity, even in associative brain regions.

To sustain their claim that what they reported is movement-independent, the authors provided a supplementary figure in which they correlated neuronal activity and head movement tracked using DeepLabCut. I have to say that I was particularly surprised by this figure. First, in the original manuscript, there was absolutely no mention of video recording. Now it appears in the methods section, but the description is very short. There is no information on how these video recordings were made. The quality of the images provided in Figure S2 is far from reassuring. It is unclear whether the temporal and spatial resolution would be good enough to make meaningful correlations. Fast head/orofacial movements that occur during nose-poking can be on the order of 20 Hz. To be tracked, this would require at least a 40 Hz sampling rate. But no sampling information is provided. The authors should explain how they synchronized behavioral and neuronal data acquisition. Could the authors share behavioral videos of the 5 sessions shown in Figure S2 so we can judge the behavior of the animals, the quality of the video, and the possibility of making correlations?

Figure S2A-F: I am not sure why the authors correlated nose-poking duration (time estimation) and the duration between upper and lower nose-pokes (reward-oriented movement). It is not relevant to the issue I raised. Without any information about video acquisition frame rate, the y-axis legend (frame) is not very informative. Still, in Figure S2A-F, Rat 5 shows a clear increase in nose-poke duration, which is congruent with decreased impulsivity. Is the time coding different in this rat compared to other rats? There are some similar trends in other animals (Rat 1 and maybe Rat 3), but what is surprising is the huge variability (big downward deflections in the nose-poke duration). I would not be surprised if those deflections occurred after a long pause in activity. Could the authors plot trial time instead of trial number? How do the authors explain such a huge deflection if the animals are estimating time?

Regarding Figure S2H: I don't see how it addresses my concern. My concern is that some of the Ca activity recorded during nose-poking reflects head movements. The authors need to show if they can detect head movement during nose-poking. Aligning the Ca data relative to head movement should give the same result as when aligning the data relative to the time at which the animals pull out of the upper nose-poke.

Minor comments:

In their introduction, the authors wrote: "While these findings [correlates of time perception] provide strong evidence for a neural mechanism of time coding in the brain, true causal evidence at single-cell resolution remains beyond reach due to technical limitations. Although inhibiting certain brain regions (such as medial prefrontal cortex, mPFC,22) led to disruption in the performance of the timing task, it is difficult to attribute the effect specifically to the ramping or sequential activity patterns seen in those regions as other processes may be involved. Lacking direct experimental evidence, one potential way of testing the causal involvement of 'time codes' in time estimation function is to examine their correlation at a finer resolution."<br /> This statement is inaccurate at two levels. First, very good causal evidence has been obtained on this topic (see Monteiro et al., 2023, Nature Neuroscience), and see my News & Views on the strengths and weaknesses of this paper. Second, their proposal is inaccurate. Looking at a finer correlation will still be a correlative approach, and the authors will not be able to disentangle motor/motivation confounds.

Reviewer #3 (Public Review):

Summary:

The study aims to elucidate the spatial dynamics of subcellular astrocytic calcium signaling. Specifically, they elucidate how subdomain activity above a certain spatial threshold (~23% of domains being active) heralds a calcium surge that also affects the astrocytic soma. Moreover, they demonstrate that processes on average are included earlier than the soma and that IP3R2 is necessary for calcium surges to occur. Finally, they associate calcium surges with slow inward currents.

The revised manuscript is improved compared to the first iteration. While some concerns have been addressed, my main critique pertaining to ROI approach/sampled area, statistical analyses and anesthesia are in my view still important caveats of the study that I think should have been even more clearly addressed in the manuscript.

Strengths:<br /> The study addresses an interesting topic that is only partially understood. The study uses multiple methods including in vivo two-photon microscopy, acute brain slices, electrophysiology, pharmacology, and knockout models. The conclusions are strengthened by the same findings in both in vivo anesthetized mice and in brain slices.

Weaknesses:

The method that has been used to quantify astrocytic calcium signals only analyzes what seems to be a small proportion of the total astrocytic domain on the example micrographs, where a structure is visible in the SR101 channel (see for instance Reeves et al. J. Neurosci. 2011, demonstrating to what extent SR101 outlines an astrocyte). This would potentially heavily bias the results: from the example illustrations presented it is clear that the calcium increases in what is putatively the same astrocyte goes well beyond what is outlined with automatically placed small ROIs. The smallest astrocytic processes are an order of magnitude smaller than the resolution of optical imaging and would not be outlined by either SR101 or with the segmentation method judged by the ROIs presented in the figures. Completely ignoring these very large parts of the spatial domain of an astrocyte, in particular when making claims about a spatial threshold, seems inappropriate. Several recent methods published use pixel-by-pixel event-based approaches to define calcium signals. The data should have been analyzed using such a method within a complete astrocyte spatial domain in addition to the analyses presented. Also, the authors do not discuss how two-dimensional sampling of calcium signals from an astrocyte that has processes in three dimensions (see Bindocci et al, Science 2017) may affect the results: if subdomain activation is not homogeneously distributed in the three-dimensional space within the astrocyte territory, the assumptions and findings between a correlation between subdomain activation and somatic activation may be affected.

Authors reply: In order to reduce noise from individual pixels, we chose to segment astrocyte arborizations into domains of several pixels. As pointed out previously, including pixels outside of the SR101-positive territory runs the risk of including a pixel that may be from a neighboring cell or mostly comprised of extracellular space, and we chose the conservative approach to avoid this source of error. We agree that the results have limitations from being acquired in 2D instead of 3D, but it is likely to assume the 3D astrocyte is homogeneously distributed and that the 2D plane is representative of the whole astrocyte. Indeed, no dimensional effects were reported in Bindocci et al, Science 2017. We have included a paragraph in the discussion to address this limitation in our study on P15, L23-27:<br /> "The investigation of the spatial threshold could be improved in the future in a number of ways. One being the use of state-of-the-art imaging in 3D(Bindocci et al., 2017). While the original publication using 3D imaging to study astrocyte physiology does not necessarily imply that there would be different calcium dynamics in one axis over another, the three-dimensional examination of the spatial threshold could refine the findings we present here.

Comments on revisions: It is good that 3D imaging aspects are mentioned as a limitation, and I agree that Bindocci et al. do not necessarily suggest that results in this manuscript would have been different if also the third spatial dimension was included in the analyses. However, the way I see it, the added analyses and text changes throughtout still do not adequately address my concern pertaining to basing a spatial threshold on a fraction of the astrocyte territory.

The study uses a heaviside step function to define a spatial 'threshold' for somata either being included or not in a calcium signal. However, Fig 4E and 5D showing how the method separates the signal provide little understanding for the reader. The most informative figure that could support the main finding of the study, namely a ~23% spatial threshold for astrocyte calcium surges reaching the soma, is Fig. 4G, showing the relationship between the percentage of arborizations active and the soma calcium signal. A similar plot should have been presented in Fig 5 as well. Looking at this distribution, though, it is not clear why ~23% would be a clear threshold to separate soma involvement, one can only speculate how the threshold for a soma event would influence this number. Even if the analyses in Fig. 4H and the fact that the same threshold appears in two experimental paradigms strengthen the case, the results would have been more convincing if several types of statistical modeling describing the continuous distribution of values presented in Fig. 4E (in addition to the heaviside step function) were presented.

Authors reply: We agree with the reviewer and have added to the paper a discussion for our justification on the use of the Heaviside step function, and have included this in the methods section. We chose the Heaviside step function to represent the on/off situation that we observed in the data that suggested a threshold in the biology. We agree with the reviewer that Fig. 4G is informative and demonstrates that under 23% most of the soma fluorescence values are clustered at baseline. We agree that a different statistical model describing the data would be more convincing and confirmed the spatial threshold with the use of a confidence interval in the text and supported the use of percent domains active for this threshold over other properties such as spatial or temporal clustering using a general linear model. P18-19, L34-2:<br /> "Heaviside step function<br /> The Heaviside step function below in equation 4 is used to mathematically model the transition from one state to the next and has been used in simple integrate and fire models (Bueno-Orovio et al., 2008; Gerstner, 2000).<br /> 𝐻(𝑎) ∶=<br /> 0, 𝑎 < 𝑎T<br /> {<br /> 1, 𝑎 {greater than or equal to} 𝑎T<br /> (4)<br /> The Heaviside step function 𝐻(𝑎) is zero everywhere before the threshold area (𝑎T) and one everywhere afterwards. From the data shown in Figure 4E where each point (𝑆(𝑎)) is an individual astrocyte response with its percent area (𝑎) domains active and if the soma was active or not denoted by a 1 or 0 respectively. To determine 𝑎T in our data we iteratively subtracted 𝐻(𝑎) from 𝑆(𝑎) for all possible values of 𝑎T to create an error term over 𝑎. The area of the minimum of that error term was denoted the threshold area.

Comments on revisions: Even with the added explanations, I am still not sure that the data show a specific threshold, or that the statistical model enforce a threshold onto the data. The data in Fig. 4G does not in my view clearly show a clear threshold as suggested. The analyses are strengthened with an added statistical modeling, however, the details of the modeling is not presented in the manuscript as far as I can see. As a bare minimum the statistical packages/tools used, the model details and goodness of fit as residual plots must be shown/commented.

The description of methods should have been considerably more thorough throughout. For instance which temperature the acute slice experiments were performed at, and whether slices were prepared in ice-cold solution, are crucial to know as these parameters heavily influence both astrocyte morphology and signaling. Moreover, no monitoring of physiological parameters (oxygen level, CO2, arterial blood gas analyses, temperature etc) of the in vivo anesthetized mice is mentioned. These aspects are critical to control for when working with acute in vivo two-photon microscopy of mice; the physiological parameters rapidly decay within a few hours with anesthesia and following surgery.

Authors reply: We have increased the thoroughness of our methods section. Especially including that body temperature and respiration were indeed monitored throughout anesthesia.

Comments on revisions: Bath temperature for slice experiments, or cutting conditions are still not reported. For the in vivo experiments, it must be commented that this level of physiological monitoring for acute in vivo brain physiology experiments (self breathing, no control of O2/CO2) is barely adequate and could represent a considerable caveat of the study.

Reviewer #2 (Public review):

Zylberberg and colleagues show that food choice outcomes and BOLD signal in the vmPFC are better explained by algorithms that update subjective values during the sequence of choices compared to algorithms based on static values acquired before the decision phase. This study presents a valuable means of reducing the apparent stochasticity of choices in common laboratory experiment designs. The evidence supporting the claims of the authors is solid, although currently limited to choices between food items because no other goods were examined. The work will be of interest to researchers examining decision making across various social and biological sciences.

Comments on revisions:

We thank the authors for carefully addressing our concerns about the first version of the manuscript. The manuscript text and contributions are now much more clear and convincing.

Reviewer #2 (Public review):

The authors of this work set out to test ideas about how observers learn to ignore irrelevant visual information. Specifically, they used fMRI to scan participants who performed a visual search task. The task was designed in such a way that highly salient but irrelevant search items were more likely to appear at a given spatial location. With a region-of-interest approach, the authors found that activity in visual cortex that selectively responds to that location was generally suppressed, in response to all stimuli (search targets, salient distractors, or neutral items), as well as in the absence of an anticipated stimulus.

Strengths of the study include: A well-written and well-argued manuscript; clever application of a region of interest approach to fMRI design, which allows articulating clear tests of different hypotheses; careful application of follow-up analyses to rule out alternative, strategy-based accounts of the findings; tests of the robustness of the findings to detailed analysis parameters such as ROI size; and exclusion of the role of regional baseline differences in BOLD responses.

The report might be enhanced by analyses (perhaps in a surface space) that distinguish amongst the multiple "early" retinotopic visual areas that are analysed in the aggregate here. Furthermore, the study could benefit from an analysis that tests the correlation over observers between the magnitude of their behavioural effects and their neural responses.

The study provides an advance over previous studies, which identified enhancement or suppression in visual cortex as a function of search target/distractor predictability, but in less spatially-specific way. It also speaks to open questions about whether such suppression/enhancement is observed only in response to the arrival of visual information, or instead is preparatory, favouring the latter view. The theoretical advance is moderate, in that it is largely congruent with previous frameworks, rather than strongly excluding an opposing view or providing a major step change in our understanding of how distractor suppression unfolds.

Reviewer #2 (Public review):

Summary:

The authors try to show the importance of CHMP5 for skeletal development.

Strengths:

The findings of this manuscript are interesting. The mouse phenotypes are well done and are of interest to a broader (bone) field.

Weaknesses:

The mechanistic insights are mediocre, and the cellular senescence aspect poor.

In total, it has not been shown that there are actual senescent cells that are reduced after D+Q-treatment. These statements need to be scaled back substantially.

Reviewer #2 (Public review):

Summary:

The authors aimed to explore how a key protein in the circadian clock of plants, ELF3, responds to temperature changes by forming molecular condensates. They focused on understanding the role of a specific region of the protein, a polyQ tract, in promoting temperature-sensitive structural changes and regulating the formation of condensates. Through a series of computational simulations, they sought to uncover the molecular basis for ELF3's temperature responsiveness and its broader implications for plant growth and adaptation to environmental conditions.

Strengths:

The study's strength lies in its focus on an important biological question: how plants sense and respond to temperature changes at the molecular level. The authors employed a variety of computational techniques, including coarse-grained simulations, to explore the role of specific molecular features in this process. These methods provide a multi-scale view of protein behavior and offer valuable insights into how molecular structures may influence biological function.

Weaknesses:

However, there are notable weaknesses in the evidence provided. While the authors present trends in molecular changes, such as shifts in helical propensity and the formation of condensates, these results seem subtle and are not strongly substantiated by statistical analysis. The lack of error bars in the figures makes it difficult to distinguish between meaningful signals and potential noise in the data. Furthermore, the temperature-sensitive behavior appears to be influenced more by chain length than by sequence-specific effects of the polyQ region, raising questions about whether the findings truly capture the molecular mechanisms responsible for temperature sensing. Additionally, some simulations, particularly those related to the formation of condensates, do not appear fully converged, which casts further doubt on the robustness of the results.

Additional Context for Readers:

Readers should interpret the results with caution, especially regarding the molecular mechanisms proposed for temperature sensing. While the study presents interesting trends, the evidence is not definitive, and the findings may be more reflective of general protein behavior (such as the effect of chain length on condensate formation) than specific sequence-driven responses to temperature. Further experimental studies and more converged simulations will be necessary to fully understand the role of ELF3 in temperature regulation.

Reviewer #2 (Public review):

Summary:

The authors report several interesting species and sex differences in cell type expression that may relate to species differences in behavior. The differential cell type abundance findings build on previously observed species/sex differences in behavior and brain anatomy. These data will be a valuable resource for behavioral neuroscientists. These findings are important but the manuscript goes too far in attributing causal influences to differences in behavior. A second important problem is that dissections used for the sequencing data include other neuropeptide-rich areas of the hypothalamus like the PVN. Although histology is included, the results in the main manuscript often do not include the mPOA making it hard to know if species/sex differences are consistent across different hypothalamic regions. The manuscript would benefit from more precise language.

Strengths:

The data are novel because cell-type atlases are available for only a few species.

The authors have clearly defined appropriate steps taken to obtain trustworthy estimations of cell type abundance. Furthermore, the criteria for each cell type assignment were described in a way for readers to easily replicate. The rigor in comparing cell abundance provides convincing evidence that these species have differences in MPOA cellular composition.

The authors have a good explanation for why 19 of the 53 neuron clusters were not classified (possible Mus/Peromyscus anatomical differences, some cell types don't have well-defined transcriptional profiles).

Validated findings with histology

Weaknesses:

Some methodology could be further explained, like the decision of a 15% cutoff value for cell type assignment per cluster, or the necessity of a multi-step analysis pipeline for gene enrichment studies.

The authors should exercise strong caution in making inferences about these differences being the basis of parental behavior. It is possible, given connections to relevant research, but without direct intervention, direct claims should be avoided. There should be clear distinctions of what to conclude and what to propose as possibilities for future research.

Histology is not performed on all regions included in the sequencing analysis.

Reviewer #2 (Public review):

Summary:

Haupt and colleagues performed a well-designed study to test the spatial and temporal gradient of perceiving braille letters in blind individuals. Using cross-hand decoding of the read letters, and comparing it to the decoding of the read letter for each hand, they defined perceptual and sensory responses. Then they compared where (using fMRI) and when (using EEG) these were decodable. Using fMRI, they showed that low-level tactile responses specific to each hand are decodable from the primary and secondary somatosensory cortex as well as from IPS subregions, the insula and LOC. In contrast, more abstract representations of the braille letter independent from the reading hand were decodable from several visual ROIs, LOC, VWFA and surprisingly also EVC. Using a parallel EEG design, they showed that sensory hand-specific responses emerge in time before perceptual braille letter representations. Last, they used RSA to show that the behavioral similarity of the letter pairs correlates to the neural signal of both fMRI (for the perceptual decoding, in visual and ventral ROIs) and EEG (for both sensory and perceptual decoding).

Strengths:

This is a very well-designed study and it is analyzed well. The writing clearly describes the analyses and results. Overall, the study provides convincing evidence from EEG and fMRI that the decoding of letter identity across the reading hand occurs in the visual cortex in blindness. Further, it addresses important questions about the visual cortex hierarchy in blindness (whether it parallels that of the sighted brain or is inverted) and its link to braille reading.

Reviewer #2 (Public review):

Summary:

This study aims to explore the ferroptosis-related immune landscape of TNBC through the integration of single-cell and bulk RNA sequencing data, followed by the development of a risk prediction model for prognosis and drug response. The authors identified key subpopulations of immune cells within the TME, particularly focusing on T cells and macrophages. Using machine learning algorithms, the authors constructed a ferroptosis-related gene risk score that accurately predicts survival and the potential response to specific drugs in TNBC patients.

Strengths:

The study identifies distinct subpopulations of T cells and macrophages with differential expression of ferroptosis-related genes. The clustering of these subpopulations and their correlation with patient prognosis is highly insightful, especially the identification of the TREM2+ and FOLR2+ macrophage subtypes, which are linked to either favorable or poor prognoses. The risk model thus holds potential not only for prognosis but also for guiding treatment selection in personalized oncology.

Weaknesses:

The study has a relatively small sample size, with only 9 samples analyzed by scRNA-seq. Given the typically high heterogeneity of the tumor microenvironment (TME) in cancer patients, this may affect the accuracy of the conclusions. The scRNA-seq analysis focuses on the expression of ferroptosis-related genes in various cells within the TME. In contrast, bulk RNA sequencing uses data from tumor samples, and the results between the two analyses are not consistent. The bulk RNA sequencing results may not accurately capture the changes happening in the microenvironment.

Reviewer #2 (Public review):

Avrillon et al. provides a comprehensive assessment of firing rate parameters from a large percentage of the motor unit pool, in two muscles, during voluntary isometric contractions. The authors have used new quantitative methods to extract more unique motor units across contractions than prior studies. This was achieved by recording muscle fibre action potentials from four high density surface electromyogram (HDsEMG) arrays, quantifying residual EMG comparing the recorded and data-based simulation (Fig. 1A-B), and developing a metric to compare the spatial identification for each motor unit (Fig. 1D-E). From identified motor units, the authors have provided a detailed characterization of recruitment and firing rate responses during slow voluntary isometric contractions in the vastus lateralis and tibialis anterior muscles up to 75-80% of maximum intensity. In the lower limb it is interesting how lower threshold motor units have firing rate responses that saturate, whereas higher threshold units that presumably produce higher muscle contractile forces continue to increase their firing rate. Conceptually, the authors rightly focus on the literature of intrinsic motoneurone properties, but in vivo, other possibilities (that are difficult to measure in awake human participants) are that the form of descending supraspinal drive, spinal network dynamics and afferent inputs may have different effects across motor unit sizes, muscles and types of contractions. These results from single trail contractions and with a larger sample of motor units, supports the summary rate coding profiles of motor units in the extensor digitorum communis muscle (Monster and Chan, 1977).

Reviewer #2 (Public review):

In this study, Solyga and Keller use multimodal closed-loop paradigms in conjunction with multiphoton imaging of cortical responses to assess whether and how sensorimotor prediction errors in one modality influence the computation of prediction errors in another modality. Their work addresses an important open question pertaining to the relevance of non-hierarchical (lateral cortico-cortical) interactions in predictive processing within the neocortex.

Specifically, they monitor GCaMP6f responses of layer 2/3 neurons in the auditory cortex of head-fixed mice engaged in VR paradigms where running is coupled to auditory, visual, or audio-visual sensory feedback. The authors find strong auditory and motor responses in the auditory cortex, as well as weak responses to visual stimuli. Further, in agreement with previous work, they find that the auditory cortex responds to audiomotor mismatches in a manner similar to that observed in visual cortex for visuomotor mismatches. Most importantly, while visuomotor mismatches by themselves do not trigger significant responses in the auditory cortex, simultaneous coupling of audio-visual inputs to movement non-linearly enhances mismatch responses in the auditory cortex.

Their results thus suggest that prediction errors within a given sensory modality are non-trivially influenced by prediction errors from another modality. These findings are novel, interesting, and important, especially in the context of understanding the role of lateral cortico-cortical interactions and in outlining predictive processing as a general theory of cortical function.

Comments on revisions:

The authors thoroughly addressed the concerns raised. In my opinion, this has substantially strengthened the manuscript, enabling much clearer interpretation of the results reported. I commend the authors for the response to review. Overall, I find the experiments elegantly designed, and the results robust, providing compelling evidence for non-hierarchical interactions across neocortical areas and more specifically for the exchange of sensorimotor prediction error signals across modalities.

Reviewer #2 (Public review):

Summary:

In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

Strengths:

(1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.<br /> (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

Reviewer #2 (Public review):

Summary:

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

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

Reviewer #2 (Public review):

Summary:

In this paper, the authors use AlphaFold2 to identify potential binding partners of nuage localizing proteins.

Strengths:

The main strength of the paper is that the authors experimentally verify a subset of the predicted interactions.

Many studies have been performed to predict protein-protein interactions in various subsets of proteins. The interesting story here is that the authors (i) focus on an organelle that contains quite some intrinsically disordered proteins and (ii) experimentally verify some (but not all) predictions.

Weaknesses:

Identification of pairwise interactions is only a first step towards understanding complex interactions. It is pretty clear from the predictions that some (but certainly not all) of the pairs could be used to build larger complexes. AlphaFold easily handles proteins up to 4-5000 residues, so this should be possible. I suggest that the authors do this to provide more biological insights.

Another weakness is the use of a non-standard name for "ranking confidence" - the author calls it the pcScore - while the name used in AlphaFold (and many other publications) is ranking confidence.

Reviewer #2 (Public review):

Summary:

Lam et al., present a very intriguing whole genome CRISPR screen in Syrian Hamster cells as well as K562 cells to identify key genes involved in hypothermia-rewarming tolerance. Survival screens were performed by exposing cells to 4C in a cooled CO2 incubator followed by a rewarming period of 30 minutes prior to survival analysis. In this paradigm, Syrian hamster-derived cell lines exhibit more robust survival than human cell lines (BHK-21 and HaK vs HT1080, HeLa, RPE1, and K562). A genome-wide Syrian hamster CRISPR library was created targeting all annotated genes with 10 guides/gene. LV transduction of the library was performed in BHK-21 cells and the survival screen procedures involved 3 cycles of 4C cold exposure x4 days followed by 2 days of re-warming.

When compared to controls maintained at 37C, 9 genes were required for BHK-21 survival of cold cycling conditions and 5 of these 9 are known components of the GPX4 antioxidant pathway. GPX4 KO BHK-21 cells had reduced cell growth at 37C and profoundly worse cold tolerance which could be reduced by GPX4 expression. GPX4 inhibitors also reduced survival in cold. CRISPR KO screens and GPX4 KO in K562 cells revealed comparable results (though intriguingly glutathione biosynthesis genes were more critical to K562 cells than BHK-21 cells). Human or Syrian hamster GPX4 overexpression improved cold tolerance.

Strengths:

This is a very nicely written paper that clearly communicates in figures and text complicated experimental manipulations and in vitro genetic screening and cell survival data. The focus on GPX4 is interesting and relatively novel. The converging pharmacologic, loss-of-function, and gain-of-function experiments are also a strength.

Weaknesses:

A recently published article (Reference 43, Sone et al.) also independently explored the role of GPX4 in Syrian hamster cold tolerance through gain-of-function screening. Further exploration of the GPX4 species-specific mechanisms would be of great interest, but this is considered a minor weakness given the already very comprehensive and compelling data presented.

Reviewer #2 (Public review):

Summary:

The manuscript by Thakur et. al seeks to establish a novel ASO-based approach to treat 22q11.2 deletion syndrome. Central to this thesis is that an ER membrane complex member called EMC10 is significantly increased in the disorder, which is largely attributed to the loss of miRNA-mediated repression. The authors generated three new iPSC cell lines for the disorder and showed that deletion of EMC10 rescues morphology and Ca-flux deficits. They go on to show that post-symptomatic deletion of Emc10 in mice using a conditional-off tamoxifen allele reverses social memory phenotypes. Finally, in collaboration with Ionis, they developed two new ASOs to knock down EMC10 and show that social and spatial memory phenotypes are rescued, even two months after injection.

Strengths:

In general, this represents a substantial undertaking and an impressive body of work. The experiments follow a logical progression and in most cases are well-controlled. The isolation of EMC10 effects relative to the broader miRNA disruption is viewed as impactful. The use of both genetic and ASO approaches to validate the therapeutic strategy is also viewed as highly positive. The authors' contention that EMC10 can be targeted at post-symptomatic time points to reverse 22q11.2 deletion syndrome is supported by the data. Further, they have provided a therapeutic mechanism to do so. These findings are likely to be impactful and lead to further development efforts.

Weaknesses:

The primary weaknesses of the manuscript lie in incomplete or inappropriate data analysis, as well as a failure to validate key experiments. For example, both genetic and ASO-mediated EMC10-mediated reductions are assessed at the level of mRNA, but only one experiment, in one brain region, is validated at the protein level. This brain region is the PFC, which is problematic when many of the phenotypes used have a strong hippocampal component. Likewise, the iPSC experiments make the case that excitatory neurons are central to the phenotype, but no effort is made to show that the ASOs are entering that type of neuron, or even any quantification of what percentage of cells in the target brain regions (HPC, PFC, etc.) are positive for the ASO. There is only a single image provided of staining with a phosphorothioate antibody and a claim of robust uptake, which cannot be assumed. The iPSC transcriptomics work would also benefit from a more comprehensive comparison between the EMC10 knockout lines and their parent 22q11 deletion lines. Further, there are other examples where the statistics used are either wrong (Figure 3 t-test vs ANOVA) or missing (Figure S2). These technical and analytical shortcomings make it challenging to fully interpret the data and detract from an otherwise exciting manuscript.

Reviewer #2 (Public review):

Summary

In this study, rats were trained to discriminate auditory frequency and visual form/orientation for both unisensory and coherently presented AV stimuli. Recordings were made in the auditory cortex during behaviour and compared to those obtained in various control animals/conditions. The central finding is that AC neurons preferentially represent the contralateral-conditioned stimulus - for the main animal cohort this was a 10k tone and a vertically oriented bar. Over 1/3rd of neurons in AC were either AV/V/A+V and while a variety of multisensory neurons were recorded, the dominant response was excitation by the correctly oriented visual stimulus (interestingly this preference was absent in the visual-only neurons). Animals performing a simple version of the task in which responses were contingent on the presence of a stimulus rather than its identity showed a smaller proportion of AV stimuli and did not exhibit a preference for contralateral conditioned stimuli. The contralateral conditioned dominance was substantially less under anesthesia in the trained animals and was present in a cohort of animals trained with the reverse left/right contingency. Population decoding showed that visual cues did not increase the performance of the decoder but accelerated the rate at which it saturated. Rats trained on auditory and then visual stimuli (rather than simultaneously with A/V/AV) showed many fewer integrative neurons.

Strengths

There is a lot that I like about this paper - the study is well-powered with multiple groups (free choice, reversed contingency, unisensory trained, anesthesia) which provides a lot of strength to their conclusions and there are many interesting details within the paper itself. Surprisingly few studies have attempted to address whether multisensory responses in the unisensory cortex contribute to behaviour - and the main one that attempted to address this question (Lemus et al., 2010, uncited by this study) showed that while present in AC, somatosensory responses did not appear to contribute to perception. The present manuscript suggests otherwise and critically does so in the context of a task in which animals exhibit a multisensory advantage (this was lacking in Lemus et al.,). The behaviour is robust, with AV stimuli eliciting superior performance to either auditory or visual unisensory stimuli (visual were slightly worse than auditory but both were well above chance).

Weaknesses

I have a number of points that in my opinion require clarification and I have suggestions for ways in which the paper could be strengthened. In addition to these points, I admit to being slightly baffled by the response latencies; while I am not an expert in the rat, usually in the early sensory cortex auditory responses are significantly faster than visual ones (mirroring the relative first spike latencies of A1 and V1 and the different transduction mechanisms in the cochlea and retina). Yet here, the latencies look identical - if I draw a line down the pdf on the population level responses the peak of the visual and auditory is indistinguishable. This makes me wonder whether these are not sensory responses - yet, they look sensory (very tightly stimulus-locked). Are these latencies a consequence of this being AuD and not A1, or ... ? Have the authors performed movement-triggered analysis to illustrate that these responses are not related to movement out of the central port, or is it possible that both sounds and visual stimuli elicit characteristic whisking movements? Lastly, has the latency of the signals been measured (i.e. you generate and play them out synchronously, but is it possible that there is a delay on the audio channel introduced by the amp, which in turn makes it appear as if the neural signals are synchronous? If the latter were the case I wouldn't see it as a problem as many studies use a temporal offset in order to give the best chance of aligning signals in the brain, but this is such an obvious difference from what we would expect in other species that it requires some sort of explanation.

Reaction times were faster in the AV condition - it would be of interest to know whether this acceleration is sufficient to violate a race model, given the arbitrary pairing of these stimuli. This would give some insight into whether the animals are really integrating the sensory information. It would also be good to clarify whether the reaction time is the time taken to leave the center port or respond at the peripheral one.

The manuscript is very vague about the origin or responses - are these in AuD, A1, AuV... ? Some attempts to separate out responses if possible by laminar depth and certainly by field are necessary. It is known from other species that multisensory responses are more numerous, and show greater behavioural modulation in non-primary areas (e.g. Atilgan et al., 2018).

Reviewer #2 (Public Review):

Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing wave-like electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that should have a great impact on the field.

The authors provide convincing evidence that chirps may function in homeoactive sensing. In particular, the evidence showing increased chirping in more cluttered environments and a relationship between chirping and movement are especially strong and suggestive. Their evidence arguing against a role for chirps in communication is not as strong. However, based on an extensive review of the literature, the authors conclude, I think fairly, that the evidence arguing in favor of a communication function is limited and inconclusive. Thus, the real strength of this study is not that it conclusively refutes the communication hypothesis, but that it calls this hypothesis into question while also providing compelling evidence in favor of an alternative function.

In summary, although the evidence against a role for chirps in communication is not as strong as the evidence for a role in active sensing, this study presents very interesting data that is sure to stimulate discussion and follow-up studies. The authors acknowledge that chirps could function as both a communication and homeactive sensing signal, and the language arguing against a communication function is appropriately measured. A given electrical behavior could serve both communication and homeoactive sensing. I suspect this is quite common in electric fish (not just in gymnotiforms such as the species studied here, but also in the distantly related mormyrids), and perhaps in other actively sensing species such as echolocating animals.

Reviewer #2 (Public review):

Summary:

The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.

Strengths:

They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria and the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.

Weaknesses:

Many data sets are shown in figures that cannot be understood without more descriptions either in the text or the legend, e.g., Fig. 1A. Similarly, many abbreviations are not defined.

The revision addressed these issues.

Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Fig. 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Fig. 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Fig. 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.

The revision addressed these problems.

The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation, and analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.

The revision corrected this method.

To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and also show whether a mutation that disrupt the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Fig. 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Fig. 1G is a quantification of a Western blot data that should be shown.

The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Fig. 1I, more Flag-C1QBP 1-167 was pull-down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?

The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect.

The revision added AlphaFold models for the protein interaction. However, the models were not analyzed and potential mutations that would disrupt the interact were not predicted, made and tested. The revision did not addressed the request for the protease inhibitor.

Reviewer #2 (Public review):

Summary:

This manuscript by Poltavski and colleagues explores the relative contributions of Pax2- and Wnt1- lineage derived cells in the enteric nervous system (ENS) and how they are each affected by disruptions in Ret and Endrb signaling. The current understanding of ENS development in mice is that vagal neural crest progenitors derived from a Wnt1+ lineage migrate into and colonize the developing gut. The sacral neural crest was thought to make a small contribution to the hindgut in addition but recent work has questioned that contribution and shown that the ENS is entirely populated by vagal crest (PMID: 38452824). GDNF-Ret and Endothelin3-Ednrb signaling are both known to be essential for normal ENS development and loss of function mutations are associated with a congenital disorder called Hirschsprung's disease. The transcription factor Pax2 has been studied in CNS and cranial placode development but has not been previously implicated in ENS development. In this work, the authors begin with the unexpected observation that conditional knockout of Ednrb in Pax2-expressing cells causes a similar aganglionosis, growth retardation, and obstructed defecation as conditional knockout of Ednrb in Wnt1-expressing cells. The investigators then use the Pax2 and Wnt1 Cre transgenic lines to lineage-trace ENS derivatives and assess the effects of loss of Ret or Ednrb during embryonic development in these lineages. Finally, they use explants from the corresponding embryos to examine the effects of GDNF on progenitor outgrowth and differentiation.

Strengths:

- The manuscript is overall very well illustrated with high resolution images and figures. Extensive data are presented.

- The identification of Pax2 expression as a lineage marker that distinguishes a subset of cells in the ENS that may be distinct from cells derived from Wnt1+ progenitors is an interesting new observation that challenges current understanding of ENS development

- Pax2 has not been previously implicated in ENS development - this manuscript does not directly test that role but hints at the possibility

- Interrogation of two distinct signaling pathways involved in ENS development and their relative effects on the two purported lineages

Weaknesses:

- The major challenge with interpreting this work is the use of two transgenic lines, Wnt1-Cre and Pax2-Cre, which are not well characterized in terms of fidelity to native gene expression and recombination efficiency in the ENS. If 100% of cells that express Wnt1 do not express Cre or if the Pax2 transgene is expressed in cells that do not normally express Pax2, then these observations would have very different interpretations and would not support the conclusions made. The two lineages are never compared in the same embryo, which also makes it difficult to assess relative contributions and renders the evidence more circumstantial than definitive.

- Visualization of the Pax2-Cre and Wnt-1Cre induced recombination in cross-sections at postnatal ages would help with data interpretation. If there is recombination evident in the mesenchyme, this would particularly alter interpretation of Ednrb mutant experiments, since that pathway has been shown to alter gut mesenchyme and ECM, which could indirectly alter ENS colonization.

- The data on distinct lineages in Fig 3 is somewhat weak and the description in the Results section tends to over-interpretation. For example, "A minimum number (approx. 3%) of CGRP+ neurons were labeled by Wnt1Cre ... which indicates that Wnt1Cre-derived cells have little or no commitment to a mechanosensory fate in the distal colon." The data panel in Fig 3f shows that most of the CGRP-IR cells in Wnt1-Cre-Tomato mice are tdTomato+ though their tdTomato fluorescence is less intense than in neighboring smaller, likely glial cells. This suggests that CGRP+/Tomato+ neurons were likely undercounted. IHC for tdTomato to ensure detection of low levels of Tomato expression and quantification of observations would strengthen the authors' claim. CGRP+ enteric neurons have been visualized and functionally described by several investigators in the field using Wnt1-Cre-GCaMP mice, which also challenges the authors' conclusions. Finally, quantification of CGRP+ enteric neurons by measuring CGRP mucosal fiber immunoreactivity is not accurate because it would reflect both ENS CGRP-expressing neurons and visceral afferents from DRG. Moreover, it is not known if all CGRP+ enteric neurons project to the mucosa or if all mucosal-projecting neurons are mechanosensory. Finally, most of the signal seems to be non-specific background staining in the mucosa and quantification of mucosal signal in this context does not seem meaningful.

- No consideration of glia - are these derived from both lineages?

- No discussion of how these observations may fit in with recent work that suggests a mesenchymal contribution of enteric neurons (PMID: 38108810)

- Phospho-RET staining in Figure 7 is difficult to discern and interpret with high background. Positive and negative controls would strengthen these data.

Comments on revised version:

The authors have responded to the weaknesses identified above. Based on my own assessment of the revised manuscript, my assessment is unchanged because the manuscript is largely unchanged.

Reviewer #2 (Public review):

Summary:

Rai and coworkers have studied the regulation of the MICAL-family of actin regulators by Rab 8 family GTPases. Their work uses a combination of structural biology, biochemistry, and modelling approaches to identify the regions and specific residues interacting with Rabs and understand the consequences of MICAL1 regulation. The study extends previous work on individual domains by incorporating analysis of the full-length MICAL1 protein and provides compelling evidence for allosteric regulation by Rab binding to two low and high-affinity regulatory sites.

Strengths:

Excellent biochemical and structural analysis.

Weaknesses:

Additional data to test the model for Rab regulation of MICAL1 in the actin-pelleting assay would enhance the study.

Reviewer #2 (Public review):

This study shows that Osx plays a pivotal role in the dendritic network and intercellular communication of Col1α1-positive osteocytes via targeting Connexin43 (Cx43). It provides solid evidence to broaden our understanding of Osx's roles during bone homeostasis. This work will be of interest to investigators studying bone diseases involving osteocytes, such as delayed fracture healing or osteoporosis.

Comments:

(1) In Figure 1, it appears that the Osx- and Col1α1-positive cells may not be exclusively expressed by osteocytes. Possibly periosteum cells and osteoblasts are also included. This could potentially impact the interpretation of results. The authors should provide a clearer analysis to distinguish the cell types precisely.

(2) Jialiang S. Wang et al. (Nat Commun. 2021 Nov 1;12(1):6274.) have previously reported on the direct role of Osx in osteocytes. In light of this prior research, it is essential for the authors to thoroughly discuss how this study differs from previous findings.

(3) In the methods section, it is crucial to provide detailed information about the manufacturer and country of origin of reagents, like ATRA.

(4) The morphology of osteocytes in cortical bone can vary between the metaphysis site and the middle shaft site of long bones. For SEM data of osteocytes in Figure 2, it is necessary to address this issue. The authors should clarify whether morphological difference was observed between these sites and, if so, how these differences might impact the interpretation of the data.

(5) In the bone research field, two different Col1α1 - CreER mice were used. The authors should specify which type of Col1α1 - CreER mice were utilized in this research.

(6) A more detailed description of the statistical method used in Figure 2G - I is required, particularly with regard to quantifying the number of osteocyte dendritic processes.

(7) In Figure 6C and Figure 6D, while the legend indicates N = 3, there are five data points presented in the statistical graph.

Reviewer #2 (Public review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. This finding highlights the potentially complex role of PdeI in regulation of c-di-GMP levels and persister formation in microbial biofilms.

Weaknesses:

Given many current methods that also introduce different techniques for ribosomal RNA depletion in bacterial single-cell RNA sequencing, it is unclear what is the place and role of RiboD-PETRI. The efficiency of rRNA depletion varies greatly between species for the majority of the available methods, so it is not easy to select the best fitting technique for a specific application.

Despite transcriptome-wide coverage, the authors focused on the role of a single heterogeneously expressed gene, PdeI. A more integrated analysis of multiple genes and\or interactions between them using these data could reveal more insights into the biofilm biology.

The authors should also present the UMIs capture metrics for RiboD-PETRI method for all cells passing initial quality filter (>=15 UMIs/cell) both in the text and in the figures. Selection of the top few cells with higher UMI count may introduce biological biases in the analysis (the top 5% of cells could represent a distinct subpopulation with very high gene expression due to a biological process). For single-cell RNA sequencing, showing the statistics for a 'top' group of cells creates confusion and inflates the perceived resolution, especially when used to compare to other methods (e.g. the parent method PETRI-seq itself).

Reviewer #2 (Public review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. This finding highlights the potentially complex role of PdeI in regulation of c-di-GMP levels and persister formation in microbial biofilms.

Weaknesses:

Given many current methods that also introduce different techniques for ribosomal RNA depletion in bacterial single-cell RNA sequencing, it is unclear what is the place and role of RiboD-PETRI. The efficiency of rRNA depletion varies greatly between species for the majority of the available methods, so it is not easy to select the best fitting technique for a specific application.

Despite transcriptome-wide coverage, the authors focused on the role of a single heterogeneously expressed gene, PdeI. A more integrated analysis of multiple genes and\or interactions between them using these data could reveal more insights into the biofilm biology.

The authors should also present the UMIs capture metrics for RiboD-PETRI method for all cells passing initial quality filter (>=15 UMIs/cell) both in the text and in the figures. Selection of the top few cells with higher UMI count may introduce biological biases in the analysis (the top 5% of cells could represent a distinct subpopulation with very high gene expression due to a biological process). For single-cell RNA sequencing, showing the statistics for a 'top' group of cells creates confusion and inflates the perceived resolution, especially when used to compare to other methods (e.g. the parent method PETRI-seq itself).

Reviewer #2 (Public review):

I appreciate the authors' efforts in addressing previous feedback by correcting typos, clarifying terms, and expanding the methodological descriptions. The revisions have notably improved the manuscript's clarity and readability. However, despite these positive changes, I still have several significant concerns, both conceptual and technical, that need to be addressed to strengthen the conclusions of the paper.

The key idea of this paper is the treatment of rDNA copies in an individual as a pseudo-population and model their sequence evolution with the WFH framework by introducing the parameter V*(K). With this modeling framework, the authors claim that the molecular evolution rate of rDNA relative to that of single-copy genes can be expressed as a simple function V*(K) and C (the copy number per individual). Moreover, when V*(K) is sufficiently large, the neutral molecular evolution of rDNA can be faster than expected under a naïve model without considering horizontal, homogenizing processes and thus be potentially compatible with empirical data. However, several issues persist in the definition, assumptions, and derivation of the model:

(1) Several terms in the model remain undefined. While Ne is clearly defined in the standard single-copy gene model as the reciprocal of genetic drift (i.e., the decay in heterozygosity), its meaning for multiple-copy genes is unclear. Based on the context, it appears that the authors define Ne as the parameter that fits the population polymorphism level (Hs) using the equation in line 165. This definition is reasonable, but it should be explicitly clarified in the text."<br /> (2) Another key parameter V*(K) was still not defined within the paper. In response 9, the authors explained that V*(K) refers to "the number of progeny to whom the gene copy of interest is transmitted (K) over a specific time interval". However, the meaning of "progeny" remains unclear. Are the authors referring to the descendent copies of a gene copy, or the offspring individuals (i.e., the living organisms)? For example, if a variant spreads horizontally through homogenizing processes and transmits vertically to multiple offspring individuals, the number of descent gene copies could differ substantially from the number of descendent individuals to whom a gene copy is transmitted to. This distinction needs to be clarified and clearly stated in the paper.<br /> (3) The authors state that V*(K)>=1 for rDNA genes because of the homogenizing processes (lines 139-141) without providing justification. It is unclear, at least to me, whether homogenizing processes are expected increase or decrease the variance in "reproductive success" across gene copies. Moreover, the authors claim that V*(K) "can potentially reach values in the hundreds and may even exceed C, resulting in C*=C/V*(K)<1" (Response 7). This claim is unlikely to be true, as the minimum value of K is bounded by zero and E(K) is assumed to be 1. Even in the extreme case that 1% gene copies leave large numbers of descends while the others leave none, V*(K) would still be less than 100. Such extreme case seems highly improbable, given realistic rates of the homogenizing processes.<br /> (4) Regardless of how the authors define V*(K), it is not immediately clear why Equation 1 (N*=NC/V*(K)) holds. Both sides of the equation have their independent meanings, so the authors need to provide a step-by-step derivation demonstrating that they are equal. Only by doing this will the implicit underlying assumptions become clearer. I also strongly recommend that the authors conduct forward-in-time simulations with fixed N, C, V*(K) (however they define it) and μ to confirm that the right side of Equation 1 actually predicts the N* as calculated from the polymorphism level using the equation in line 165.<br /> (5) Without providing justification, the authors assumed that a certain number N* exists for rRNA such that it fits both the polymorphism level (line 156) in recent timescales and divergence level in longer timescales (i.e., in the comparison between Tf and Td). However, if N, C or any other relevant parameters have varied substantially throughout evolution, N* is expected to vary with time, and the same value may not fit both polymorphism and divergence data simultaneously.

The authors also provided more detailed description of their data analysis methods, but some of my major concerns remain:<br /> (1) A significant issue with aligning reads to a single reference genome is reference bias, referring to the phenomenon that reads carrying the reference alleles tend to align more easily than those with one or more non-reference alleles, thus creating a bias in genotype calling or variant allele frequency quantification. As a result, there may be an underrepresentation of non-reference alleles in called variants or an underestimate of non-reference allele frequency, particularly in regions with high genetic diversity. Simply focusing on bi-allelic SNVs is insufficient to minimize reference bias. Given the fourfold increase in diversity within rDNA, the authors must either provide evidence that reference bias is not a significant concern or adopt graph-based reference genomes or more sophisticated alignment algorithms to address this issue.<br /> (2) The potential for reference bias also renders the analysis of divergence sites unreliable, as aligning reads from one species (e.g. chimpanzee) to the reference of another species (e.g., human) is likely to introduce biases in variant calling between the two. One commonly adopted approach to address this imbalance is to align reads from both species to a third reference genome that is expected to be equidistantly related to both.<br /> (3) Although it is somewhat reassuring that the estimated divergence rate of rDNA between human and macaque is comparable to that of the rest of the genome, there still remains concern of a under-estimation of divergence in rDNA regions due to reference bias issue. Note that while the "third genome" approach reduces imbalance between two genomes in comparison, it may still under-estimate overall divergence level due to under-calling of non-reference variants.<br /> (4) In response to my question about the similarity in rDNA substitution rates estimated with or without CpG sites, the authors suggest that this "may be due to strong homogenizing forces, which can rapidly fix or eliminate variants" (response17). However, this explanation is insufficient, because the observed substitution rate depends on the mutation rate multiplied by the fixation probability, and accelerated fixation or loss does not alter either. Unless the authors can provide more convincing explanation, technical errors in calling of fixed sites still remain a concern.

Minor points<br /> Line 157: The statement "where μ is the mutation rate of the entire gene" must be wrong, as the heterozygosity calculated with such μ would correspond to the chance of seeing two different haplotypes at gene level, which is incompatible with the empirical calculation specified in Equation 2. Instead, μ must represent the mutation rate per site averaged over the entire gene.

In response 22, the authors explained that the allele frequency spectrum shown in Fig 3 is folded, because the ancestral allele was not determined. However, this is inconsistent with x-axis Fig 3 ranging between 0 and 1. I suspect the x-axis represents the frequency of the alternative (i.e., non-reference) allele. If so, the reported correlation is inflated, as the reference allele is somewhat random, and a variant at joint ALT allele frequencies of (0.9, 0.9) is no different from a variant at (0.1, 0.1). The proper way of calculate this correlation is to first determine the minor allele frequency across individuals and then calculate the correlation between minor allele frequencies.

Similarly, in response 14, it is unclear what the x-axis represents. Is it the ALT allele frequency or derived allele frequency? If the former, why are only variants with AF>0.8 defined as fixed variants, while those with AF<0.2 excluded? If it is the latter, please describe how ancestral state is determined.

Reviewer #2 (Public Review):

Summary:

Blocking a weak base compound's protonation increased intracellular diffusion and fractional recovery in the cytoplasm, which may improve the intracellular availability and distribution of weakly basic, small molecule drugs and be impactful in future drug development.

Strengths:

(1) The intracellular distribution of drugs and the chemical properties that drive their distribution are much needed in the literature. Thus, the idea behind this paper is of relevance.

(2) The study used common compounds that were relevant to others.

(3) Altering a compound's pKa value and measuring cytosolic diffusion rates certainly is inciteful on how weak base drugs and their relatively high pKa values affect distribution and pharmacokinetics. This particular experiment demonstrated relevance to drug targeting and drug development.

(4) The manuscript was fairly well written.

Comments on revised version:

After reviewing the authors' responses to my questions and concerns, they have adequately corrected the errors, added new information and data based off the reviewers suggestions that improved the manuscript. The manuscript in its current form would add quality information to a part of the literature that is lacking much needed information.

Reviewer #2 (Public review):

Notably, the authors provide the first structure of human PIEZO1 (hPIEZO1), which will facilitate future studies in the field. They reveal that hPIEZO1 has a more flattened shape than mouse PIEZO1 (mPIEZO1) and has lipids that insert into the hydrophobic pore region. To understand how PIEZO1 GOF mutations might affect this structure and the underlying mechanistic changes, they solve structures of hPIEZO1 as well as two HX causing mild GOF mutations (A1988V and E756del) and a severe GOF mutation (R2456H). Unable to glean too much information due to poor resolution of the mutant channels, the authors also attempt to resolve MCFIC-bound structures of the mutants. These structures show that MDFIC inserts into the pore region of hPIEZO1, similar to its interaction with mPIEZO1, and results in a more curved and contracted state than hPIEZO1 on its own. The authors use these structures to hypothesize that differences in curvature and pore lipid position underlie the differences in inactivation kinetics between wild-type hPIEZO1, hPIEZO1 GOF mutations, and hPIEZO1 in complex with MDFIC.

Strengths:

This is the first human PIEZO1 structure. Thus, these studies become the steppingstone for future investigations to better understand how disease-causing mutations affect channel gating kinetics.

Comments on revisions:

The revised version of the manuscript is stronger and the authors have addressed most of our concerns. The only clarification that remains is data related to the electrophysiology experiments, Figure S2. In the response, the authors mention that they were referring to previously reported mPIEZO1 mutants. However, it is still missing quantification from the human mutant + MDFIC data. This data should be available to the authors and will be more informative than just the representative traces. In the text line 151-152 "Indeed, electrophysiological studies showed that co-expression of these channelopathy mutants with MDFIC resulted in significantly reduced mechanosensitivity and inactivation rate (Fig. S2)." However the updated version does not have any number or the statistics that were performed to indicate significance. I acknowledge that in the response they describe threshold but very descriptively.

Reviewer #2 (Public review):

Summary:

In this manuscript, Kaplan et al. study mesenchymal Meis2 in whisker formation and the links between whisker formation and sensory innervation. To this end, they used conditional deletion of Meis2 using the Wnt1 driver. Whisker development was arrested at the placode induction stage in Meis2 conditional knockouts leading to the absence of expression of placodal genes such as Edar, Lef1, and Shh. The authors also show that branching of trigeminal nerves innervating whisker follicles was severely affected but that whiskers did form in the complete absence of trigeminal nerves.

Strengths:

The analysis of Meis2 conditional knockouts convincingly shows a lack of whisker formation and all epithelial whisker/hair placode markers were analyzed. Using Neurog1 knockout mice, the authors show equally convincingly that whiskers and teeth develop in the complete absence of trigeminal nerves.

Weaknesses:

The manuscript does not provide much mechanistic insight as to why mesenchymal Meis2 leads to the absence of whisker placodes. Using a previously generated scRNA-seq dataset they show that two early markers of dermal condensates, Foxd1 and Sox2, are downregulated in Meis2 mutants. However, given that placodes and dermal condensates do not form in the mutants, this is not surprising and their absence in the mutants does not provide any direct link between Meis2 and Foxd1 or Sox2. (The absence of a structure evidently leads to the absence of its markers.)

Reviewer #2 (Public Review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. Given that PdeI is a phosphodiesterase, which is supposed to promote hydrolysis of c-di-GMP, this finding is unexpected.

Weaknesses:

With the descriptions and writing of the manuscript, it is hard to place the findings about the PdeI into existing context (i.e. it is well known that c-di-GMP is involved in biofilm development and is heterogeneously distributed in several species' biofilms; it is also known that E.coli diesterases regulate this second messenger, i.e. https://journals.asm.org/doi/full/10.1128/jb.00604-15).<br /> There is also no explanation for the apparently contradictory upregulation of c-di-GMP in cells expressing higher PdeI levels. Perhaps the examination of the rest of the genes in cluster 2 of the biofilm sample could be useful to explain the observed association.

Reviewer #2 (Public review):

In the present study, Boffi et al. investigate the manner in which the dorsal cortex of the of the inferior colliculus (DCIC), an auditory midbrain area, encodes sound location azimuth in awake, passively listening mice. By employing volumetric calcium imaging (scanned temporal focusing or s-TeFo), complemented with high-density electrode electrophysiological recordings (neuropixels probes), they show that sound-evoked responses are exquisitely noisy, with only a small portion of neurons (units) exhibiting spatial sensitivity. Nevertheless, a naïve Bayesian classifier was able to predict the presented azimuth based on the responses from small populations of these spatially sensitive units. A portion of the spatial information was provided by correlated trial-to-trial response variability between individual units (noise correlations). The study presents a novel characterization of spatial auditory coding in a non-canonical structure, representing a noteworthy contribution specifically to the auditory field and generally to systems neuroscience, due to its implementation of state-of-the-art techniques in an experimentally challenging brain region. However, nuances in the calcium imaging dataset and the naïve Bayesian classifier warrant caution when interpreting some of the results.

Strengths:

The primary strength of the study lies in its methodological achievements, which allowed the authors to collect a comprehensive and novel dataset. While the DCIC is a dorsal structure, it extends up to a millimetre in depth, making it optically challenging to access in its entirety. It is also more highly myelinated and vascularised compared to e.g., the cerebral cortex, compounding the problem. The authors successfully overcame these challenges and present an impressive volumetric calcium imaging dataset. Furthermore, they corroborated this dataset with electrophysiological recordings, which produced overlapping results. This methodological combination ameliorates the natural concerns that arise from inferring neuronal activity from calcium signals alone, which are in essence an indirect measurement thereof.

Another strength of the study is its interdisciplinary relevance. For the auditory field, it represents a significant contribution to the question of how auditory space is represented in the mammalian brain. "Space" per se is not mapped onto the basilar membrane of the cochlea and must be computed entirely within the brain. For azimuth, this requires the comparison between miniscule differences between the timing and intensity of sounds arriving at each ear. It is now generally thought that azimuth is initially encoded in two, opposing hemispheric channels, but the extent to which this initial arrangement is maintained throughout the auditory system remains an open question. The authors observe only a slight contralateral bias in their data, suggesting that sound source azimuth in the DCIC is encoded in a more nuanced manner compared to earlier processing stages of the auditory hindbrain. This is interesting because it is also known to be an auditory structure to receive more descending inputs from the cortex.

Systems neuroscience continues to strive for the perfection of imaging novel, less accessible brain regions. Volumetric calcium imaging is a promising emerging technique, allowing the simultaneous measurement of large populations of neurons in three dimensions. But this necessitates corroboration with other methods, such as electrophysiological recordings, which the authors achieve. The dataset moreover highlights the distinctive characteristics of neuronal auditory representations in the brain. Its signals can be exceptionally sparse and noisy, which provide an additional layer of complexity in the processing and analysis of such datasets. This will undoubtedly be useful for future studies of other less accessible structures with sparse responsiveness.

Weaknesses:

Although the primary finding that small populations of neurons carry enough spatial information for a naïve Bayesian classifier to reasonably decode the presented stimulus is not called into question, certain idiosyncrasies, in particular the calcium imaging dataset and model, complicate specific interpretations of the model output, and the readership is urged to interpret these aspects of the study's conclusions with caution.

I remain in favour of volumetric calcium imaging as a suitable technique for the study, but the presently constrained spatial resolution is insufficient to unequivocally identify regions of interest as cell bodies (and are instead referred to as "units" akin to those of electrophysiological recordings). It remains possible that the imaging set is inadvertently influenced by non-somatic structures (including neuropil), which could report neuronal activity differently than cell bodies. Due to the lack of a comprehensive ground-truth comparison in this regard (which to my knowledge is impossible to achieve with current technology), it is difficult to imagine how many informative such units might have been missed because their signals were influenced by spurious, non-somatic signals, which could have subsequently misled the models. The authors reference the original Nature Methods article (Prevedel et al., 2016) throughout the manuscript, presumably in order to avoid having to repeat previously published experimental metrics. But the DCIC is neither the cortex nor hippocampus (for which the method was originally developed) and may not have the same light scattering properties (not to mention neuronal noise levels). Although the corroborative electrophysiology data largely alleviates these concerns for this particular study, the readership should be cognisant of such caveats, in particular those who are interested in implementing the technique for their own research.

A related technical limitation of the calcium imaging dataset is the relatively low number of trials (14) given the inherently high level of noise (both neuronal and imaging). Volumetric calcium imaging, while offering a uniquely expansive field of view, requires relatively high average excitation laser power (in this case nearly 200 mW), a level of exposure the authors may have wanted to minimise by maintaining a low number of repetitions, but I yield to them to explain. Calcium imaging is also inherently slow, requiring relatively long inter-stimulus intervals (in this case 5 s). This unfortunately renders any model designed to predict a stimulus (in this case sound azimuth) from particularly noisy population neuronal data like these as highly prone to overfitting, to which the authors correctly admit after a model trained on the entire raw dataset failed to perform significantly above chance level. This prompted them to feed the model only with data from neurons with the highest spatial sensitivity. This ultimately produced reasonable performance (and was implemented throughout the rest of the study), but it remains possible that if the model was fed with more repetitions of imaging data, its performance would have been more stable across the number of units used to train it. (All models trained with imaging data eventually failed to converge.) However, I also see these limitations as an opportunity to improve the technology further, which I reiterate will be generally important for volume imaging of other sparse or noisy calcium signals in the brain.

Transitioning to the naïve Bayesian classifier itself, I first openly ask the authors to justify their choice of this specific model. There are countless types of classifiers for these data, each with their own pros and cons. Did they actually try other models (such as support vector machines), which ultimately failed? If so, these negative results (even if mentioned en passant) would be extremely valuable to the community, in my view. I ask this specifically because different methods assume correspondingly different statistical properties of the input data, and to my knowledge naïve Bayesian classifiers assume that predictors (neuronal responses) are assumed to be independent within a class (azimuth). As the authors show that noise correlations are informative in predicting azimuth, I wonder why they chose a model that doesn't take advantage of these statistical regularities. It could be because of technical considerations (they mention computing efficiency), but I am left generally uncertain about the specific logic that was used to guide the authors through their analytical journey.

In a revised version of the manuscript, the authors indeed justify their choice of the naïve Bayesian classifier as a conservative approach (not taking into account noise correlations), which could only improve with other models (that do). They even tested various other commonly used models, such as support vector machines and k-nearest neighbours, to name a few, but do not report these efforts in the main manuscript. Interestingly, these models, which I supposed would perform better in fact did not overall - a finding that I have no way of interpreting but nevertheless find interesting.

That aside, there remain other peculiarities in model performance that warrant further investigation. For example, what spurious features (or lack of informative features) in these additional units prevented the models of imaging data from converging? In an orthogonal question, did the most spatially sensitive units share any detectable tuning features? A different model trained with electrophysiology data in contrast did not collapse in the range of top-ranked units plotted. Did this model collapse at some point after adding enough units, and how well did that correlate with the model for the imaging data? How well did the form (and diversity) of the spatial tuning functions as recorded with electrophysiology resemble their calcium imaging counterparts? These fundamental questions could be addressed with more basic, but transparent analyses of the data (e.g., the diversity of spatial tuning functions of their recorded units across the population). Even if the model extracts features that are not obvious to the human eye in traditional visualisations, I would still find this interesting.

Although these questions were not specifically addressed in the revised version of the manuscript, I also admit that I did not indent do assert that these should necessarily fall within the scope of the present study. I rather posed them as hypothetical directions one could pursue in future studies. Finally, further concerns I had with statements regarding the physiological meaning of the findings have been ameliorated by nicely modified statements, thus bringing transparency to the readership, which I appreciate.

In summary, the present study represents a significant body of work that contributes substantially to the field of spatial auditory coding and systems neuroscience. However, limitations of the imaging dataset and model as applied in the study muddles concrete conclusions about how the DCIC precisely encodes sound source azimuth and even more so to sound localisation in a behaving animal. Nevertheless, it presents a novel and unique dataset, which, regardless of secondary interpretation, corroborates the general notion that auditory space is encoded in an extraordinarily complex manner in the mammalian brain.

Reviewer #2 (Public review):

Summary:

Cell intrinsic signaling pathways controlling the function of macrophages in inflammatory processes, including in response to infection, injury or in the resolution of inflammation are incompletely understood. In this study, Rosell et al. investigate the contribution of RAS-p110α signaling to macrophage activity. p110α is a ubiquitously expressed catalytic subunit of PI3K with previously described roles in multiple biological processes including in epithelial cell growth and survival, and carcinogenesis. While previous studies have already suggested a role for RAS-p110α signaling in macrophage function, the cell intrinsic impact of disrupting the interaction between RAS and p110α in this central myeloid cell subset is not known.

Strengths:

Exploiting a sound previously described genetically engineered mouse model that allows tamoxifen-inducible disruption of the RAS-p110α pathway and using different readouts of macrophage activity in vitro and in vivo, the authors provide data consistent with their conclusion that alteration in RAS-p110α signaling impairs various but selective aspects of macrophage function in a cell-intrinsic manner.

Weaknesses:

My main concern is that for various readouts, the difference between wild-type and mutant macrophages in vitro or between wild-type and Pik3caRBD mice in vivo is modest, even if statistically significant. To further substantiate the extent of macrophage function alteration upon disruption of RAS-p110α signaling and its impact on the initiation and resolution of inflammatory responses, the manuscript would benefit from a more extensive assessment of macrophage activity and inflammatory responses in vivo.

In the in vivo model, all cells have disrupted RAS-p100α signaling, not only macrophages. Given that other myeloid cells besides macrophages contribute to the orchestration of inflammatory responses, it remains unclear whether the phenotype described in vivo results from impaired RAS-p100α signaling within macrophages or from defects in other haematopoietic cells such as neutrophils, dendritic cells, etc.

Inclusion of information on the absolute number of macrophages, and total immune cells (e.g. for the spleen analysis) would help determine if the reduced frequency of macrophages represents an actual difference in their total number or rather reflects a relative decrease due to an increase in the number of other/s immune cell/s.

Reviewer #2 (Public review):

In this article, Tian et al present a convincing analysis of the molecular mechanisms underpinning TIPE-mediated regulation of glycolysis and tumor growth in melanoma. The authors begin by confirming TIPE expression in melanoma cell lines and identify "high" and "low" expressing models for functional analysis. They show that TIPE depletion slows tumour growth in vivo, and using both knockdown and over expression approaches, show that this is associated with changes in glycolysis in vitro. Compelling data using multiple independent approaches is presented to support an interaction between TIPE and the glycolysis regulator PKM2, and over-expression of TIPE promoted nuclear translocation of PKM2 dimers. Mechanistically, the authors also demonstrate that PKM2 is required for TIPE-mediated activation of HIF1a transcriptional activity, as assessed using an HRE-promoter reporter assay, and that TIPE-mediated PKM2 dimerization is p-ERK dependent. Finally, the dependence of TIPE activity on PKM2 dimerization was demonstrated on tumor growth in vivo and in regulation of glycolysis in vitro, and ectopic expression of HIF1a could rescue inhibition of PKM2 dimerization in TIPE overexpressing cells and reduced induction of general cancer stem cell markers, showing a clear role for HIF1a in this pathway.

The detailed mechanistic analysis of TIPE mediated regulation of PKM2 to control aerobic glycolysis and tumor growth is a major strength of the study and provides new insights into the molecular mechanisms that underpin the Warburg effect in melanoma cells. The main conclusions of this paper are well supported by data, however further investigation of a potential oncogenic effect of TIPE in melanoma patients is warranted to support the tumor promoting role of TIPE identified in the experimental models. Analysis of patient samples showed a significant increase in TIPE protein levels in primary melanoma compared to benign skin tumours, and a further increase upon metastatic progression. Moreover, TIPE levels correlate with proliferation (Ki67) and hypoxia gene sets in the TCGA melanoma patient dataset. However, intriguingly, high TIPE expression associates with better survival outcomes in the TCGA melanoma patient cohort, therefore further investigation of how TIPE-mediated regulation of glycolysis contributes to melanoma progression is warranted to confirm the authors claims of a potential oncogenic function. Regardless, the new insights into the molecular mechanisms underpinning TIPE-mediated aerobic glycolysis in melanoma are convincing and will likely generate interest in the cancer metabolism field.

Reviewer #2 (Public Review):

Summary:

Previous NMR and HDX-MS studies on full-length (FL) BTK showed that the covalent BTKi, ibrutinib, causes long-range effects on the conformation of BTK consistent with disruption of the autoinhibited conformation, based on HDX deuterium uptake patterns and NMR chemical shift perturbations. This study extends the analyses to four new covalent BTKi, acalabrutinib, zanubrutinib, tirabrutinib/ONO4059, and a noncovalent ATP competitive BTKi, pirtobrutinib/LOXO405.

The results show distinct conformational changes that occur upon binding each BTKi. The findings show consistent NMR and HDX changes with covalent inhibitors, which move helix aC to an 'out' position and disrupt SH3-kinase interactions, in agreement with X-ray structures of the BTKi complexed with the BTK kinase domain. In contrast, the solution measurements show that pirtobrutinib maintains and even stabilizes the helix aC-in and autoinhibited conformation, even though the BTK:pritobrutinib crystallizes with helix aC-out. This and unexpected variations in NMR and HDX behavior between inhibitors highlight the need for solution measurements to understand drug interactions with the full-length BTK. Overall the findings present good evidence for allosteric effects by each BTKi that induce distal conformational changes which are sensitive to differences in inhibitor structure.

The study goes on to examine BTK mutants T474I and L528W, which are known to confer resistance to pirtobrutinib, zanubritinib, and tirabrutinib. T474I reduces and L528W eliminates BTK autophosphorylation at pY551, while both FL-BTK-WT and FL-BTK-L528W increase HCK autophosphorylation and PLCg phosphorylation. These show that mutants partially or completely inactivate BTK and that inactive FL-BTK can activate HCK, potentially by direct BTK-HCK interactions. But they do not explain drug resistance. However, HDX and NMR show that each mutant alters the effects of BTKi binding compared to WT. In particular, T474I alters the effects of all three inhibitors around W395 and the activation loop, while L528W alters interactions around W395 with tirabrutinib and pirtobrutinib, and does not appear to bind zanubrutinib at all. The study concludes that the mutations might block drug efficacy by reducing affinity or altering binding mode.

Strengths:

The work presents convincing evidence that BTK inhibitors alter the conformation of regions distal to their binding sites, including those involved in the SH3-kinase interface, the activation loop, and a substrate binding surface between helix aF and helix aG. The findings add to the growing understanding of allosteric effects of kinase inhibitors, and their potential regulation of interactions between kinase and binding proteins.

Comments on the revised version:

The authors have satisfactorily addressed my concerns in their revised manuscript.

Reviewer #2 (Public Review):

Summary:

The aim of this manuscript is to use molecular dynamics (MD) simulations to describe the conformational changes of the neurotransmitter binding site of a nicotinic receptor. The study uses a simplified model including the alpha-delta subunit interface of the extracellular domain of the channel and describes the binding of four agonists to observe conformational changes during the weak to strong affinity transition.

Strength:

The 200 ns-long simulations of this model suggest that the agonist rotates about its centre in a 'flip' motion, while loop C 'flops' to restructure the site. The changes appear to be reproduced across simulations and different ligands and are thus a strong point of the study.

Weaknesses:

After carrying out all-atom molecular dynamics, the authors revert to a model of binding using continuum Poisson-Boltzmann, surface area and vibrational entropy. The motivations for and limitations associated with this approximate model for the thermodynamics of binding, rather than using modern atomistic MD free energy methods (that would fully incorporate configurational sampling of the protein, ligand and solvent) could be provided. Despite this, the authors report correlation between their free energy estimates and those inferred from the experiment. This did, however, reveal shortcomings for two of the agonists. The authors mention their trouble getting correlation to experiment for Ebt and Ebx and refer to up to 130% errors in free energy. But this is far worse than a simple proportional error, because -24 Vs -10 kcal/mol is a massive overestimation of free energy, as would be evident if it the authors were to instead to express results in terms of KD values (which would have error exceeding a billion fold). The MD analysis could be improved with better measures of convergence, as well as a more careful discussion of free energy maps as function of identified principal components, as described below. Overall, however, the study has provided useful observations and interpretations of agonist binding that will help understand pentameric ligand-gated ion channel activation.

Reviewer #2 (Public review):

Summary:

This study reveals a novel role of TET2 in regulating gluconeogenesis. It shows that fasting and a high-fat diet increase TET2 expression in mice, and TET2 knockout reduces glucose production. The findings highlight that TET2 positively regulates FBP1, a key enzyme in gluconeogenesis, by interacting with HNF4α to demethylate the FBP1 promoter in response to glucagon. Additionally, metformin reduces FBP1 expression by preventing TET2-HNF4α interaction. This identifies an HNF4α-TET2-FBP1 axis as a potential target for T2D treatment.

Strengths:

The authors use several methods in vivo (PTT, GTT, and ITT in fasted and HFD mice; and KO mice) and in vitro (in HepG2 and primary hepatocytes) to support the existence of the HNF4alpha-TET-2-FBP-1 axis in the control of gluconeogenesis. These findings uncovered a previously unknown function of TET2 in gluconeogenesis.

Weaknesses:

Although the authors provide evidence of an HNF4α-TET2-FBP1 axis in the control of gluconeogenesis, which contributes to the therapeutic effect of metformin on T2D, its role in the pathogenesis of T2D is less clear. The mechanisms by which TET2 is up-regulated by glucagon should be more explored.

Reviewer #2 (Public review):

Summary:

Golov et al has performed the capture MChIP-C using H3K4me3 antibody. The new method significantly increases the resolution of Micro-C and can detect the clear interactions which is not well described in the previous HiChIP/PLAC-seq method. Overall, the paper represented a significant technological advance which can be valuable to the 3D genomic field in the future.

The authors have addressed all my concerns and comments.

Reviewer #2 (Public review):

5-methylcytosine (5mC) is a key epigenetic mark in DNA and plays a crucial role in regulating gene expression in many eukaryotes including humans. The DNA methyltransferases (DNMTs) that establish and maintain 5mC, are conserved in many species across eukaryotes, including animals, plants, and fungi, mainly in a CpG context. Interestingly, 5mC levels and distributions are quite variable across phylogenies with some species even appearing to have no such DNA methylation.

This interesting and well-written paper discusses the continuation of some of the authors' work published several years ago. In that previous paper, the laboratory demonstrated that DNA methylation pathways coevolved with DNA repair mechanisms, specifically with the alkylation repair system. Specifically, they discovered that DNMTs can introduce alkylation damage into DNA, specifically in the form of 3-methylcytosine (3mC). (This appears to be an error in the DNMT enzymatic mechanism where the generation 3mC as opposed to its preferred product 5-methylcytosine (5mC), is caused by the flipped target cytosine binding to the active site pocket of the DNMT in an inverted orientation.) The presence of 3mC is potentially toxic and can cause replication stress, which this paper suggests may explain the loss of DNA methylation in different species. They further showed that the ALKB2 enzyme plays a crucial role in repairing this alkylation damage, further emphasizing the link between DNA methylation and DNA repair.

The co-evolution of DNMTs with DNA repair mechanisms suggests there can be distinct advantages and disadvantages of DNA methylation to different species which might depend on their environmental niche. In environments that expose species to high levels of DNA damage, high levels of 5mC in their genome may be disadvantageous. This present paper sets out to examine the sensitivity of an organism to genotoxic stresses such as alkylation and oxidation agents as the consequence of DNMT activity. Since such a study in eukaryotes would be complicated by DNA methylation controlling gene regulation, these authors cleverly utilize Escherichia coli (E.coli) and incorporate into it the DNMTs from other bacteria that methylate the cytosines of DNA in a CpG context like that observed in eukaryotes; the active sites of these enzymes are very similar to eukaryotic DNMTs and basically utilize the same catalytic mechanism (also this strain of E.coli does not specifically degrade this methylated DNA) .

The experiments in this paper more than adequately show that E. coli expression of these DNMTs (comparing to the same strain without the DNMTS) do indeed show increased sensitivity to alkylating agents and this sensitivity was even greater than expected when a DNA repair mechanism was inactivated. Moreover, they show that this E. coli expressing this DNMT is more sensitive to oxidizing agents such as H2O2 and has exacerbated sensitivity when a DNA repair glycosylase is inactivated. Both propensities suggest that DNMT activity itself may generate additional genotoxic stress. Intrigued that DNMT expression itself might induce sensitivity to oxidative stress, the experimenters used a fluorescent sensor to show that H2O2 induced reactive oxygen species (ROS) are markedly enhanced with DNMT expression. Importantly, they show that DNMT expression alone gave rise to increased ROS amounts and both H2O2 addition and DNMT expression has greater effect that the linear combination of the two separately. They also carefully checked that the increased sensitivity to H2O2 was not potentially caused by some effect on gene expression of detoxification genes by DNMT expression and activity. Finally, by using mass spectroscopy, they show that DNMT expression led to production of the 5mC oxidation derivatives 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) in DNA. 5fC is a substrate for base excision repair while 5hmC is not; more 5fC was observed. Introduction of non-bacterial enzymes that produce 5hmC and 5fC into the DNMT expressing bacteria again showed a greater sensitivity than expected. Remarkedly, in their assay with addition of H2O2, bacteria showed no growth with this dual expression of DNMT and these enzymes.

Overall, the authors conduct well thought-out and simple experiments to show that a disadvantageous consequence of DNMT expression leading to 5mC in DNA is increased sensitivity to oxidative stress as well as alkylating agents.

Again, the paper is well-written and organized. The hypotheses are well-examined by simple experiments. The results are interesting and can impact many scientific areas such as our understanding of evolutionary pressures on an organism by environment to impacting our understanding about how environment of a malignant cell in the human body may lead to cancer.

Reviewer #2 (Public review):

Summary:

If synaptic input is functionally clustered on dendrites, nonlinear integration could increase the computational power of neural networks. But this requires the right synapses to be located in the right places. This paper aims to address the question of whether such synaptic arrangements could arise by chance (i.e. without special rules for axon guidance or structural plasticity), and could therefore be exploited even in randomly connected networks. This is important, particularly for the dendrites and biological computation communities, where there is a pressing need to integrate decades of work at the single-neuron level with contemporary ideas about network function.

Using an abstract model where ensembles of neurons project randomly to a postsynaptic population, back-of-envelope calculations are presented that predict the probability of finding clustered synapses and spatiotemporal sequences. Using data-constrained parameters, the authors conclude that clustering and sequences are indeed likely to occur by chance (for large enough ensembles), but require strong dendritic nonlinearities and low background noise to be useful.

Strengths:

- The back-of-envelope reasoning presented can provide fast and valuable intuition. The authors have also made the effort to connect the model parameters with measured values. Even an approximate understanding of cluster probability can direct theory and experiments towards promising directions, or away from lost causes.

- I found the general approach to be refreshingly transparent and objective. Assumptions are stated clearly about the model and statistics of different circuits. Along with some positive results, many of the computed cluster probabilities are vanishingly small, and noise is found to be quite detrimental in several cases. This is important to know, and I was happy to see the authors take a balanced look at conditions that help/hinder clustering, rather than just focus on a particular regime that works.

- This paper is also a timely reminder that synaptic clusters and sequences can exist on multiple spatial and temporal scales. The authors present results pertaining to the standard `electrical' regime (~50-100 µm, <50 ms), as well as two modes of chemical signaling (~10 µm, 100-1000 ms). The senior author is indeed an authority on the latter, and the simulations in Figure 5, extending those from Bhalla (2017), are unique in this area. In my view, the role of chemical signaling in neural computation is understudied theoretically, but research will be increasingly important as experimental technologies continue to develop.

Weaknesses:

- The paper is mostly let down by the presentation. In the current form, some patience is needed to grasp the main questions and results, and it is hard to keep track of the many abbreviations and definitions. A paper like this can be impactful, but the writing needs to be crisp, and the logic of the derivation accessible to non-experts. See, for instance, Stepanyants, Hof & Chklovskii (2002) for a relevant example.

It would be good to see a restructure that communicates the main points clearly and concisely, perhaps leaving other observations to an optional appendix. For the interested but time-pressed reader, I recommend starting with the last paragraph of the introduction, working through the main derivation on page 7, and writing out the full expression with key parameters exposed. Next, look at Table 1 and Figure 2J to see where different circuits and mechanisms fit in this scheme. Beyond this, the sequence derivation on page 17 and biophysical simulations in Figures 5 and 6 are also highlights.

- The analysis supporting the claim that strong nonlinearities are needed for cluster/sequence detection is unconvincing. In the analysis, different synapse distributions on a single long dendrite are convolved with a sigmoid function and then the sum is taken to reflect the somatic response. In reality, dendritic nonlinearities influence the soma in a complex and dynamic manner. It may be that the abstract approach the authors use captures some of this, but it needs to be validated with simulations to be trusted (in line with previous work, e.g. Poirazi, Brannon & Mel, (2003)).

- It is unclear whether some of the conclusions would hold in the presence of learning. In the signal-to-noise analysis, all synaptic strengths are assumed equal. But if synapses involved in salient clusters or sequences were potentiated, presumably detection would become easier? Similarly, if presynaptic tuning and/or timing was reorganized through learning, the conditions for synaptic arrangements to be useful could be relaxed. Answering these questions is beyond the scope of the study, but there is a caveat there nonetheless.

Reviewer #2 (Public review):

In this study, Wada et al. investigate the low potential ferredoxin from Bacillus thermoproteolyticus (BtFd) using a combination of neutron crystallography, x-ray crystallography, DFT and spectroscopy to determine the influence of hydrogen bonding networks on the redox potential of ferredoxin's 4Fe-4S cluster. The use of neutron diffraction allowed the authors to probe the precise location of hydrogens around the 4Fe-4S cluster, which was not possible from prior studies, even with the previously reported high-resolution (0.92 Å) structure of BtFd. This allowed the authors to revise prior models of the proposed H bonding network theorized from earlier x-ray crystallography studies ( for example, showing that there is not in fact a H bond formed between the Thr63-O𝛾1 and the [4Fe-4S]-S4 atoms). With this newly described H-bonding network established, the electronic structure of the 4Fe-4S cluster was then investigated using DFT methodology, revealing a startling role of the deprotonated surface residue Asp64, which bears substantial electronic density in the LUMO which is otherwise localized to the 4Fe-4S cluster. While aspartate is usually deprotonated at physiological pH, the authors provide compelling evidence that this aspartate has a much higher pKa than is usual, and is able to act as a protonation-dependent switch which controls the stability of the reduced state of the 4Fe-4S cluster, and thus the redox potential.

The findings of this study and the conclusions drawn from them are well supported by the data and computational work. Their findings have implications for similar control mechanisms in other, non-ferredoxin 4Fe-4S bearing electron transport proteins which have yet to be explored, providing great value to the metalloprotein community. One change that the authors may consider to enhance the clarity of the manuscript regards the nomenclature used for the varying models discussed (CM, CMNA, CMH and so forth). It would be beneficial to the reader if the nomenclature included the redox state (ox. vs red.) of the model in the model's name.

Reviewer #2 (Public review):

This manuscript by Yang et al. describes a variety of bilateral and segmented microfossils from the basal Cambrian (Fortunian Stage) Kuanchuanpu Formation, South China. During the Fortunian Stage, body fossils are scarce, and key evidence for the presence of different clades relies on exceptionally preserved microfossils of embryos and larvae. The authors interpret the described microfossils as segmented bilaterians, with anteroposterior and dorsoventral differentiation and paired appendages. The implication of this interpretation is that the microfossils represent important evidence for early bilaterian evolution.

The strength of the manuscript is the convincing presentation of the material's bilateral and segmented nature and its taphonomy. The combined use of scanning electron microscopy and X-ray computed tomography to illustrate the material convincingly supports the argument of a bilaterian affinity. Likewise, the visualization of the cemented vesicles composed of phosphate nanocrystals that make up the fossils' internal molds supports the proposed taphonomic pathway.

The weakness of the manuscript is the further biological interpretations. While the manuscript presents a convincing argument that the molds derive from overall segmented (metameric) body plans, it does not fully explore which cavities/organs are actually molded. Instead, it assumes without discussion that the molds reflect the cuticle with a loss of fine external structures (e.g., setae). While external sclerites and cuticles are convincingly displayed in one case (Figure Supplement 5), more options exist for the rest of the material. Here, molds could perhaps represent other cavities, such as guts (including diverticula) or perivisceral cavities, both consistent with a lack of fine external details as well as an endogenous taphonomic pathway. A proper exploration of what these molds actually represent is, therefore, crucial to interpreting the ecological and evolutionary implications of the fossils.

Despite its weakness, the manuscript demonstrates convincing evidence of bilaterian microfossils in the Fortunian Stage. This evidence, in itself, contributes valuable information on the Cambrian animal radiation.

Reviewer #2 (Public review):

Summary:

The authors utilize biochemical approaches to determine and validate NRL protein-protein interactions to further understand the mechanisms by which the NRL transcription factor controls rod photoreceptor gene regulatory networks. Observations that NRL displays numerous protein-protein interactions with RNA-binding proteins, many of which are involved in R-loop biology, led the authors to investigate the role of RNA and R-loops in mediating protein-protein interactions and profile the co-localization of R-loops with NRL genomic occupancy.

Strengths:

Overall, the manuscript is very well written, providing succinct explanations of the observed results and potential implications. Additionally, the authors use multiple orthogonal techniques and tissue samples to reproduce and validate that NRL interacts with DHX9 and DDX5. Experiments also utilize specific assays to understand the influence of RNA and R-loops on protein-protein interactions. The authors also use state-of-the-art techniques to profile R-loop localization within the retina and integrate multiple previously established datasets to correlate R-loop presence with transcription factor binding and chromatin marks in an attempt to understand the significance of R-loops in the retina.

Weaknesses:

In general, the authors provide superficial interpretations of the data that fit a narrative but fail to provide alternative explanations or address caveats of the results. Specifically, many bands are present in interaction studies either in control lanes (GST controls) of Westerns or large amounts of background in PLA experiments. Additionally, the lack of experiments testing the functional significance of Nrl interactions or R-loops within the developing retina fails to provide novel biological insights into the regulation of gene regulatory networks other than, 'This could be a potentially important new mechanism'. Additionally, the authors test the necessity of RNA for NRL/DHX9 interactions but don't show RNA binding of NRL or DHX9 or the sufficiency of RNA to interfere/mediate protein-protein interactions. Recent work has highlighted the prevalence of RNA binding by transcription factors through Arginine Rich Motifs that are located near the DNA binding domains of transcription factors.

Reviewer #2 (Public review):

Summary:

Madrigal et al identified Tom1, a E3 ubiquitin ligase previously known to be involved in ribosome biogenesis, as a protein that binds to terminal tandem Src-homology 2 (tSH2) domain of Spt6. They mapped this interaction to the acid region of Tom1, which is also known to interact with histones. Cells with tom1 mutants that cannot bind Spt6 did not show temperature sensitive phenotypes displaced for tom1 null mutant. Using ChIP assays, they showed that Tom1 is enriched at gene bodies of highly transcribed genes, and a loss of tom1 leads to reduced nucleosomal changes at gene promoters. Finally, they also solved the structures of Tom1 lacking the acidic region and found that Tom1p can adopt a compact a-solenoidal "basket" similar to the previously described structure of HUWE1. Overall, this is an interesting study and I have the following suggestions to improve the manuscript.

Major concerns.

(1) Promoter regions are in general nucleosome free. How does Tom1 mutant affect nucleosome-sized fragments at the promoter regions?<br /> (2) While Tom1 antibodies may not specific, could the author perform Tom1 ChIP-seq in wild type and tom 1 null cells? This dataset may be more informative than tagged Tom1 that may not be functional.

Reviewer #2 (Public review):

Summary:

Brooks et al. generate a gene expression atlas of the early embryonic cranial neural plate. They generate single-cell transcriptome data from early cranial neural plate cells at 6 consecutive stages between E7.5 to E9. Utilizing computational analysis they infer temporal gene expression dynamics and spatial gene expression patterns along the anterior-posterior and mediolateral axis of the neural plate. Subsequent comparison with known gene expression patterns revealed a good agreement with their inferred patterns, thus validating their approach. They then focus on Sonic Hedgehog (Shh) signalling, a key morphogen signal, whose activities partition the neural plate into distinct gene expression domains along the mediolateral axis. Single-cell transcriptome analysis of embryos in which the Shh pathway was pharmacologically activated throughout the neural plate revealed characteristic changes in gene expression along the mediolateral axis and the induction of distinct Shh-regulated gene expression programs in the developing fore-, mid-, and hindbrain.

Strengths:

This manuscript provides a comprehensive transcriptomic characterisation of the developing cranial neural plate, a part of the embryo that to my knowledge has not been extensively analysed by single-cell transcriptomic approaches. The single-cell sequencing data appears to be of high quality and will be a great resource for the wider scientific community. Moreover, the computational analysis is well executed and the validation of the sequencing data using published gene expression patterns is convincing. Taken together, this is a well-executed study that describes a relevant scientific resource for the wider scientific community.

Weaknesses:

Conceptually, the findings that gene expression patterns differ along the rostrocaudal, mediolateral, and temporal axes of the neural plate and that Shh signalling induces distinct target genes along the anterior-posterior axis of the nervous system are more expected than surprising. However, the strength of this manuscript is again the comprehensive characterization of the spatiotemporal gene expression patterns and how they change upon ectopic activation of the Shh pathway.

Reviewer #2 (Public review):

Summary:

Protein synthesis - translation - involves repeated recognition and incorporation of amino-acyl-tRNAs by the ribosome. This process is a trade-off between the rate and accuracy of selection (for review see (Johansson et al, 2008; Wohlgemuth et al, 2011)). The ribosome does not just maximise the rate or the accuracy, it balances the two. Therefore, it is possible to select mutants that translate faster than the wt (but are sloppy) or that are very accurate (more than the wt) but translate slower. Slow translation is detrimental as it limits the rate of protein synthesis (and, therefore, growth) and hyper-accurate mutants accumulate mis-translated proteins, which is detrimental for the cell.

Bi and colleagues employ genetics, MIC measurements, reporter assays, and structural biology to characterise the role of GidB rRNA methylase in translational accuracy in Mycobacterium smegmatis.

Strengths:

The genetics and phenotypic assays are convincing and establish the biological role of the methylase. The authors use a powerful set of complementary assays that convincingly demonstrate that the loss of GidB results in mistranslation.

Weaknesses:

(1) It would be essential to provide information regarding the growth rate and, ideally, translation rates in the gidB KO and the isogenic WT. As translation balances accuracy and speed, only characterising the speed is not sufficient to understand the phenomenon.

(2) Cryo-EM analysis of vacant 70S ribosomes is not sufficient for understanding the mechanisms underlying the accuracy defects in the gidB KO. One should assemble and solve structurally near-cognate and non-cognate complexes. I believe the authors are over-interpreting the scant structural data they have. Furthermore, current representation makes it impossible to assess the resolution of the structure, especially in the areas of interest.

References:

Johansson M, Lovmar M, Ehrenberg M (2008) Rate and accuracy of bacterial protein synthesis revisited. Curr Opin Microbiol 11: 141-147<br /> Wohlgemuth I, Pohl C, Mittelstaet J, Konevega AL, Rodnina MV (2011) Evolutionary optimization of speed and accuracy of decoding on the ribosome. Philos Trans R Soc Lond B Biol Sci 366: 2979-2986.

Reviewer #2 (Public review):

Summary:

In the manuscript "Mapping HIV-1 RNA Structure, Homodimers, Long-Range Interactions and 1 persistent domains by HiCapR" Zhang et al report results from an omics-type approach to mapping RNA crosslinks within the HIV RNA genome under different conditions i.e. in infected cells and in virions. Reportedly, they used a previously published method which, in the present case, was improved for application to RNAs of low abundance.

Their claims include the detection of numerous long-range interactions, some of which differ between cellular and virion RNA. Further claims concern the detection and analysis of homodimers.

Strengths:

(1) The method developed here works with extremely little viral RNA input and allows for the comparison of RNA from infected cells versus virions.

(2) The findings, if validated properly, are certainly interesting to the community.

Weaknesses:

(1) On the communication level, the present version of the manuscript suffers from a number of shortcomings. I may be insufficiently familiar with habits in this community, but for RNA afficionados just a little bit outside of the viral-RNA-X-link community, the original method (reference 22) and the presumed improvement here are far too little explained, namely in something like three lines (98-100). This is not at all conducive to further reading.

(2) Experimentally, the manuscript seems to be based on a single biological replicate, so there is strong concern about reproducibility.

(3) The authors perform an extensive computational analysis from a limited number of datasets, which are in thorough need of experimental validation.

Reviewer #3 (Public review):

Summary:

In this manuscript, Zhang et al. investigate the conductivity and inhibition mechanisms of the Kv2.1 channel, focusing on the distinct effects of TEA and RY785 on Kv2 potassium channels. The study employs microsecond-scale molecular dynamics simulations to characterize K+ ion permeation and compound binding inhibition in the central pore.

Strengths:

The findings reveal a unique inhibition mechanism for RY785, which binds to the channel walls in the open structure while allowing reduced K+ flow. The study also proposes a long-range allosteric coupling between RY785 binding in the central pore and its effects on voltage-sensing domain dynamics. Overall, this well-organized paper presents a high-quality study with robust simulation and analysis methods, offering novel insights into voltage-gated ion channel inhibition that could prove valuable for future drug design efforts.

Weaknesses:

(1) The study neglects to consider the possibility of multiple binding sites for RY785, particularly given its impact on voltage sensors and gating currents. Specifically, there is potential for allosteric binding sites in the voltage-sensing domain (VSD), as some allosteric modulators with thiazole moieties are known to bind VSD domains in multiple voltage-gated sodium channels (Ahuja et al., 2015; Li et al., 2022; McCormack et al., 2013; Mulcahy et al., 2019).

(2) The study describes RY785 as a selective inhibitor of Kv2 channels and characterizes its binding residues through MD simulations. However, it is not clear whether the identified RY785-binding residues are indeed unique to Kv2 channels.

(3) The study does not clarify the details, rationale, and ramifications of a biasing potential to dihedral angles.

(4) The observation that the Kv2.1 central pore remains partially permeable to K+ ions when RY785 is bound is intriguing, yet it was not revealed whether polar groups of RY785 always interact with K+ ions.

Reviewer #2 (Public review):

Summary:

The work aims to further understand the role of macrophages in lung precancer/lung cancer evolution

Strengths:

(1) The use of single-cell RNA seq to provide comprehensive characterisation.

(2) Characterisation of cross-talk between macrophages and the lung precancerous cells.

(3) Functional validation of the effects of S100a4+ cells on lung precancerous cells using in vitro assays.

(4) Validation in human tissue samples of lung precancer / invasive lesions.

Weaknesses:

(1) The authors need to provide clarification of several points in the text.

(2) The authors need to carefully assess their assumptions regarding the role of macrophages in angiogenesis in precancerous lesions.

(3) The authors should discuss more broadly the current state of anti-macrophage therapies in the clinic.

Reviewer #3 (Public review):

Summary:

This study by Jiang et al. aims to establish the streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) mouse model in vivo and the STZ-induced pancreatic β cell MIN6 cell model in vitro to explore the protective effects of Eugenol (EUG) on T1DM. The authors tried to elucidate the potential mechanism by which EUG inhibits the NRF2-mediated anti-oxidative stress pathway. Overall, this study is well executed with solid data, offering an intriguing report from animal studies for a potential new treatment strategy for T1DM.

Strengths:

In vivo efficacy study is comprehensive and solid. Given STZ-induced T1DM is a devastating and harsh model, the in vivo efficacy from this compound is really impressive.

Reviewer #2 (Public review):

Summary:

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications.

Strengths:

The algorithm is almost as accurate as manual tracking and importantly saves time for researchers. The authors have discussed how their tool compares to other tracking methods.

Weaknesses:

Applicability can be questioned because the device used is 2D and physiological biology is in 3D. However, the authors have addressed this point in their revised manuscript.

Reviewer #2 (Public Review):

In the manuscript "Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress", Dar, Malla, and colleagues use direct RNA sequencing on nanopores to characterize the transcriptome after arsenite and oxidative stress. They observe a population of transcripts that are shortened during stress. The authors hypothesize that this shortening is mediated by the 5'-3' exonuclease XRN1, as XRN1 knockdown results in longer transcripts. Interestingly, the authors do not observe a polyA-tail shortening, which is typically thought to precede decapping and XRN1-mediated transcript decay. Finally, the authors use G3BP1 knockout cells to demonstrate that stress granule formation is required for the observed transcript shortening. The manuscript contains intriguing findings of interest to the mRNA decay community.

Reviewer #2 (Public review):

Lian et al. provide novel and exciting findings related to exercise-induced intestinal injury that have many implications for those engaging in any kind of training protocol. The authors continue to provide data demonstrating that different forms of exercise training impart a unique signature to the gut microbiota. The paper is well-written, easy to follow, and contains ample information in all sections. The figures are displayed in a clear and comprehensible format, with elegant images. I do have a few concerns regarding some aspects of the paper listed below, but otherwise, I feel that the authors clearly state their objectives, implement valid methods, and summarize their findings with the appropriate conclusions given their experimental constraints.

(1) The authors performed extensive experiments demonstrating the immediate effects of a bout of exercise on intestinal integrity throughout a 6-week training program. Additionally, the authors go as far as to show that successive exercise sessions appear to augment the observed damage. This is very important and noteworthy data. But I wonder, had the endpoint collections been taken 24 hours+ after the last exercise bout, would the findings be different? My concern is that the 1-hour time point is biased towards seeing more damage. I understand the acute effects of exercise occur and are important to report, but they can be transient, and adaptations ensue. My main concern is that the data shows the onset of the initial damage, but nothing addresses an adaptive or recovery response that could counter the observed exercise-induced intestinal injury. Even metrics such as stool consistency/ pellets per hour/ abnormal defecation measurements could indicate the function of the GI system after exercise and may offer more information related to damage vs recovery.

(2) An additional concern arises with the model of forced treadmill running. It was previously shown that forced treadmill running resulted in more gut damage compared to voluntary wheel running, with or without dextran sodium sulfate-induced colitis (PMID: 23707215). This type of training appears to be very important in initiating damage to the GI. Understanding how much of this is related to the chosen exercise protocol, forced treadmill running, will be very important for future experiments. Exercise intensity has been suggested to be a major factor in exercise-induced intestinal damage. Therefore, the group designated as MOD-EX in this paper may be over the intensity threshold that limits GI damage. The protocols used in this manuscript may be inherently biased towards enhancing exercise-induced GI damage, which is not necessarily negative, especially when a damaging protocol is needed. However, how much this relates to and can be translated to humans is not clear and needs further experimentation.

(3) I think the comparison between groups at the specified time point is important, but I believe additional comparisons should be included that show within-group differences across each time point. For example, in the Mod group, does FITC- dextran change between 4 and 6 weeks? Are there morphological change differences between 2, 4, and 6 weeks within each group? Essentially addressing a progression in damage as a function of the duration of exercise training. The authors clearly show exercise-induced damage to the GI, but we do not know how this damage is handled or if the continuation of exercise continues to reinforce the disruption in the epithelial cells.

(4) The authors describe the purpose of this study as being to identify key regulators of the destruction and reconstruction process of the GI after exercise (introduction lines 128-129). While the authors did sufficient work to describe certain contributing factors, I do not believe they have provided compelling data on the key regulators of exercise-induced intestinal injury, at least experimentally they did not perform exhaustive experiments to identify such. Nor did the authors include data showing any kind of reconstruction that occurs in the GI after exercise. I believe the authors need to revise this statement to reflect that they investigated certain or specific regulators of the damage response in the intestines after exercise training.

(5) Was water intake monitored and recorded per group? If so I think it would be important to include in the supplemental data. Fluid intake/proper hydration can also contribute to changes in the microbiome and if the data is available, it would complement the food intake. If for any reason the exercise groups were taking in less fluid it may be a confounding factor that should be considered.

(6) Methods section - Treadmill running exercise protocol, line 143, I think there is a typo with "exercise straining". Did the authors mean to write "exercise training"? If it is indeed a typo, the same appears in the supplemental material under the same section.

(7) The microbiome analysis is sufficient, and the authors speculate on the possible consequences of the observed changes to the microbiota. However, I believe Figures 5E-G are misleading. The positive correlation is present because of the increase in gut leakiness and the observed exercise-induced increase in microbes. However the same correlation could be made with any positive adaptation to exercise and the observed gut leakiness. I believe those correlations, as described now, postulate these microbes (members of the family Lachnospiraceae) are associated with increased gut leakiness. However, this correlation is not compelling as it is, and additional experiments are warranted to justify this. It cannot be ruled out that the microbes are increasing due to exercise itself. Additionally, reports have suggested species within the Lachnospiraceae family do increase in response to exercise in mice and are associated with positive adaptations to exercise (PMID: 28862530, PMID: 37940330, PMID: 36517598). With this, it should be noted that Lachnospiraceae was also found to be negatively associated with endurance performance (PMID: 35002754). Therefore, specific species or stains of Lachnospiraceae may be highly responsive to exercise while others are not. Without deeper sequencing it is impossible to tease this out and therefore, the authors should be careful with any interpretation beyond discussing what is observed. Additionally, these correlations between Lachnospiraceae and gut leakiness should be interpreted cautiously or more experiments should be included which demonstrate these microbes are connected to gut leakiness. Much more research is needed to determine exactly what strains are positively and negatively associated with exercise adaptations and performance.

Reviewer #2 (Public review):

Summary:

The authors utilize a new technique to measure mitochondrial respiration from frozen tissue extracts, which goes around the historical problem of purifying mitochondria prior to analysis, a process that requires a fair amount of time and cannot be easily scaled up.

Strengths:

A comprehensive analysis of mitochondrial respiration across tissues, sexes, and two different ages provides foundational knowledge needed in the field.

Weaknesses:

While many of the findings are mostly descriptive, this paper provides a large amount of data for the community and can be used as a reference for further studies. As the authors suggest, this is a new atlas of mitochondrial function in mouse. The inclusion of a middle aged time point and a slightly older young point (3-6 months) would be beneficial to the study.

Reviewer #2 (Public review):

Summary:

In this study the authors systematically explore mechanism(s) of impaired postnatal lung development with relevance to BPD (bronchopulmonary dysplasia) in two murine models of 'alveolar simplification', namely hyperoxia and epithelial loss of TGFb signaling. The work presented here is of great importance, given the limited treatment options for a clinical entity frequently encountered in newborns with high morbidity and mortality that is still poorly understood, and the unclear role of TGFb signaling, its signaling levels, and its cellular effects during secondary alveolar septum formation, a lung structure generating event heavily impacted by BPD. The authors show that hyperoxia and epithelial TGFb signaling loss have similar detrimental effects on lung structure and mechanical properties (emphysema-like phenotype) and are associated with significantly decreases numbers of PDGFRa-expressing cells, the major cell pool responsible for generation of postnatal myofibroblasts. They then use a single-cell transcriptomic approach combined with pathway enrichment analysis for both models to elucidate common factors that affect alveologenesis. Using cell communication analysis (NicheNet) between epithelial and myofibroblasts they confirm increased projected TGFb-TGFbR interactions and decreased projected interactions for PDGFA-PDGFRA, and other key pathways, such as SHH and WNT. Based on these results they go on to uncover in a sequela of experiments that surprisingly, increased TGFb appears reactive to postnatal lung injury and rather protective/homeostatic in nature, and the authors establish the requirement for alpha V integrins, but not the subtype alphaVbeta6, a known activator of TGFb signaling and implied in adult lung fibrosis. The authors then go beyond the TGFb axis evaluation to show that mere inhibition of proliferation by conditional KO of Ect2 in Pdgfra lineage results in alveolar simplification, pointing out the pivotal role of PDGFRa-expressing myofibroblasts for normal postnatal lung development.

Strengths:

(1) The approach including both pharmacologic and mechanistically-relevant transgenic interventions both of which produced consistent results provides robustness of the results presented here.

(2) Further adding to this robustness is the use of moderate levels of hyperoxia at 75% FiO2, which is less extreme than 100% FiO2 frequently used by others in the field, and therefore favors the null hypothesis.

(3) The prudent use of advancement single cell analysis tools, such as NicheNet to establish cell interactions through the pathways they tested and the validation of their scRNA-seq results by analysis of two external datasets. Delineation of the complexity of signals between different cell types during normal and perturbed lung development, such as attempted successfully in this study, will yield further insights into the underlying mechanism(s).

(4) The combined readout of lung morphometric (MLI) and lung physiologic parameters generates a clinically meaningful readout of lung structure and function.

(5) The systematic evaluation of TGFb signaling better determines the role in normal and postnatally-injured lung.

Weaknesses:

(1) While the study convincingly establishes the effect of lung injury on the proliferation of PDGFRa-expressing cells, differentiation is equally important. Characterization of PDGFRa expressing cells and tracking the changes in the injury models in the scRNA analysis, a key feature of this study, would benefit from expansion in this regard. PDGFRa lineage gives rise to several key fibroblast populations, including myofibroblasts, lipofibroblasts, and matrix-type fibroblasts (Collagen13a1, Collagen14a1). Lipofibroblasts constitute a significant fraction of PDGFRa+ cells, and expand in response to hyperoxic injury, as shown by others. Collagen13a1-expressing fibroblasts expand significantly under both conditions (Fig.3), and appear to contain a significant number of PDGFRa-expressing cells (Suppl Fig.1). Effects of the applied injuries on known differentiation markers for these populations should be documented. Another important aspect would be to evaluate whether the protective/homeostatic effect of TGFb signaling is by supporting differentiation of myofibroblasts. Postnatal Gli1 lineage gains expression of PDGFRa and differentiation markers, such as Acta2 (SMA) and Eln (Tropoelastin). Loss of PDGFRa expression was shown to alter Elastin and TGFb pathway related genes. TGFb signaling is tightly linked to the ECM via LTBPs, Fibrillins and Fibulins. An additional analysis in the aforementioned regards has great potential to more specifically identify the cell type(s) affected by the loss of TGFb signaling and allow analysis of their specific transcriptomic changes in response and underlying mechanism(s) to postnatal injury.

[The authors have added in detailed transcriptomic description of the fibroblast populations.]

(2) Of the three major lung abnormalities encountered in BPD, the authors focus on alveolarization impairment in great detail, to very limited extend on inflammation, and not on vascularization impairment. However, this would be important not only to better capture the established pathohistologic abnormalities of BPD, but also is needed since the authors alter TGFb signaling, and inflammatory and vascular phenotypes with developmental loss of TGFb signaling and its activators have been described. Since the authors make the point about absence of inflammation in their BPD model, it will be important to show the evidence.

[While this an important question, assessment of these components goes beyond the scope of this paper.]

(3) Conceptually it would be important that in the discussion the authors reconcile their findings in the experimental BPD models in light of human BPD and potential implications it might have on new ways to target key pathways and cell types for treatment. This allows the scientific community to formulate the next set of questions in a disease relevant manner.

[The authors have amended the discussion in this regard.]

Comments on latest version:

This reviewer would like to thank the authors for their efforts to address the concerns, in particular the better transcriptomic description of the fibroblast populations. The reviewer is well aware of the issues with PDGFRa antibodies that work on mouse tissue and also the problem with available reporters and lineage tracers in terms of haploinsufficiency.

There are no further concerns from this reviewer's side.

Reviewer #2 (Public review):

Summary:

Blackwell et al. investigated the structure, localization and physiological function of Plasmodium falciparum (Pf) heme oxygenase (HO). Pf and other malaria parasites scavenge and digest large amounts of hemoglobin from red cells for sustenance. To counter the potentially cytotoxic effects of heme, it is biomineralized into hemozoin and stored in the food vacuole. Another mechanism to counteract heme toxicity is through its enzymatic degradation via heme oxygenases. However, it was previously found by the authors that PfHO lacks the ability to catalyze heme degradation, raising the intriguing question of what the physiological function of PfHO is. In the current contribution, the authors determine that PfHO localizes to the apicoplast, determine its targeting sequence, establish the essentiality of PfHO for parasite viability, and determine that PfHO is required for proper maintenance of apicoplasts and apicoplast gene expression. In sum, the authors establish an essential physiological function for PfHO, thereby providing new insights into the role of PfHO in plasmodium metabolism.

Strengths:

The studies are rigorously conducted and the results of the experiments unambiguously support a role for PfHO as being an apicoplast targeted protein required for parasite viability and maintenance of apicoplasts.

Weaknesses:

While the studies conducted are rigorous and support the primary conclusions, the lack of experiments probing the molecular function of PfHO somewhat limits the impact of the work. Nevertheless, knowledge that PfHO is required for parasite viability and plays a role in the maintenance of apicoplasts is still an important advance.

Comments on revisions:

The authors thoughtfully addressed all the reviewer comments.

Reviewer #3 (Public review):

Mohseni and Elhaik challenge the widespread use of PCA as an analytical and interpretive tool in the study of geometric morphometrics. The standard approach in geometric morphometrics analysis involves Generalised Procrustes Analysis (GPA) followed by Principal Component Analysis (PCA). Recent research challenges PCA outcomes' accuracy, robustness, and reproducibility in morphometrics analysis. In this paper, the authors demonstrate that PCA is unreliable for such studies. Additionally, they test and compare several Machine-Learning methods and present MORPHIX, a Python package of their making that incorporates the tools necessary to perform morphometrics analysis using ML methods.

Mohseni and Elhaik conducted a set of thorough investigations to test PCA's accuracy, robustness, and reproducibility following renewed recent criticism and publications where this method was abused. Using a set of 2 and 3D morphometric benchmark data, the authors performed a traditional analysis using GPA and PCA, followed by a reanalysis of the data using alternative classifiers and rigorous testing of the different outcomes.

In the current paper, the authors evaluated eight ML methods and compared their classification accuracy to traditional PCA. Additionally, common occurrences in the attempted morphological classification of specimens, such as non-representative partial sampling, missing specimens, and missing landmarks, were simulated, and the performance of PCA vs ML methods was evaluated.

Comments on revisions:

I have gone over the revised manuscript and the detailed responses to the previous round of review. While there are places where I personally would have used slightly toned-down phrasing, the authors' get to set the tone of their manuscript, and I will not argue with that any further.

In general, the restructuring, addition of new paragraphs, minor revisions and new title make for a much better manuscript, which as stated in the previous review, will be a valuable resource for workers in the field.

Reviewer #2 (Public review):

Summary:

In this work, Walker and collaborators study the evolution of hepatitis C virus (HCV) in a cohort of 14 subjects with recent HCV infections. They focus in particular on the interplay between HCV and the immune system, including the accumulation of mutations in CD8+ T cell epitopes to evade immunity. Using a computational method to estimate the fitness effects of HCV mutations, they find that viral fitness declines as the virus mutates to escape T-cell responses. In long-term infections, they found that viral fitness can rebound later in infection as HCV accumulates additional mutations.

Strengths:

This work is especially interesting for several reasons. Individuals who developed chronic infections were followed over fairly long times and, in most cases, samples of the viral population were obtained frequently. At the same time, the authors also measured CD8+ T cell and antibody responses to infection. The analysis of HCV evolution focused not only on variation within particular CD8+ T cell epitopes but also on the surrounding proteins. Overall, this work is notable for integrating information about HCV sequence evolution, host immune responses, and computational metrics of fitness and sequence variation. The evidence presented by the authors supports the main conclusions of the paper described above.

Weaknesses:

One notable weakness of the present version of the manuscript is a lack of clarity in the description of the method of fitness estimation. In the previous studies of HIV and HCV cited by the authors, fitness models were derived by fitting the model (equation between lines 435 and 436) to viral sequence data collected from many different individuals. In the section "Estimating survival fitness of viral variants," it is not entirely clear if Walker and collaborators have used the same approach (i.e., fitting the model to viral sequences from many individuals), or whether they have used the sequence data from each individual to produce models that are specific to each subject. If it is the former, then the authors should describe where these sequences were obtained and the statistics of the data.

If the fitness models were inferred based on the data from each subject, then more explanation is needed. In prior work, the use of these models to estimate fitness was justified by arguing that sequence variants common to many individuals are likely to be well-tolerated by the virus, while ones that are rare are likely to have high fitness costs. This justification is less clear for sequence variation within a single individual, where the viral population has had much less time to "explore" the sequence landscape. Nonetheless, there is precedent for this kind of analysis (see, e.g., Asti et al., PLoS Comput Biol 2016). If the authors took this approach, then this point should be discussed clearly and contrasted with the prior HIV and HCV studies.

Another important point for clarification is the definition of fitness. In the abstract, the authors note that multiple studies have shown that viral escape variants can have reduced fitness, "diminishing the survival of the viral strain within the host, and the capacity of the variant to survive future transmission events." It would be helpful to distinguish between this notion of fitness, which has sometimes been referred to as "intrinsic fitness," and a definition of fitness that describes the success of different viral strains within a particular individual, including the potential benefits of immune escape. In many cases, escape variants displace variants without escape mutations, showing that their ability to survive and replicate within a specific host is actually improved relative to variants without escape mutations. However, escape mutations may harm the virus's ability to replicate in other contexts. Given the major role that fitness plays in this paper, it would be helpful for readers to clearly discuss how fitness is defined and to distinguish between fitness within and between hosts (potentially also mentioning relevant concepts such as "transmission fitness," i.e., the relative ability of a particular variant to establish new infections).

One concern about the analysis is in the test of Shannon entropy as a way to quantify the rate of escape. The authors describe computing the entropy at multiple time points preceding the time when escape mutations were observed to fix in a particular epitope. Which entropy values were used to compare with the escape rate? If just the time point directly preceding the fixation of escape mutations, could escape mutations have already been present in the population at that time, increasing the entropy and thus drawing an association with the rate of escape? It would also be helpful for readers to include a definition of entropy in the methods, in addition to a reference to prior work. For example, it is not clear what is being averaged when "average SE" is described.

Reviewer #2 (Public review):

Summary:

This work provides a comprehensive understanding of cellular immunity in bivalves. To precisely describe the hemocytes of the oyster C. gigas, the authors morphologically characterized seven distinct cell groups, which they then correlated with single-cell RNA sequencing analysis, also resulting in seven transcriptional profiles. They employed multiple strategies to establish relationships between each morphotype and the scRNAseq profile. The authors correlated the presence of marker genes from each cluster identified in scRNAseq with hemolymph fractions enriched for different hemocyte morphotypes. This approach allowed them to correlate three of the seven cell types, namely hyalinocytes (H), small granule cells (SGC), and vesicular cells (VC). A macrophage-like (ML) cell type was correlated through the expression of macrophage-specific genes and its capacity to produce reactive oxygen species. Three other cell types correspond to blast-like cells, including an immature blast cell type from which distinct hematopoietic lineages originate to give rise to H, SGC, VC, and ML cells. Additionally, ML cells and SGCs demonstrated phagocytic properties, with SGCs also involved in metal homeostasis. On the other hand, H cells, non-granular cells, and blast cells expressed antimicrobial peptides. This study thus provides a complete landscape of oyster hemocytes with functional validation linked to immune activities. This resource will be valuable for studying the impact of bacterial or viral infections in oysters.

Strengths:

The main strength of this study lies in its comprehensive and integrative approach, combining single-cell RNA sequencing, cytological analysis, cell fractionation, and functional assays to provide a robust characterization of hemocyte populations in Crassostrea gigas.

(1) The innovative use of marker genes, quantifying their expression within specific cell fractions, allows for precise annotation of different cellular clusters, bridging the gap between morphological observations and transcriptional profiles.

(2) The study provides detailed insights into the immune functions of different hemocyte types, including the identification of professional phagocytes, ROS-producing cells, and cells expressing antimicrobial peptides.

(3) The identification and analysis of transcription factors specific to different hemocyte types and lineages offer crucial insights into cell fate determination and differentiation processes in oyster immune cells.

(4) The authors significantly advance the understanding of oyster immune cell diversity by identifying and characterizing seven distinct hemocyte transcriptomic clusters and morphotypes.

These strengths collectively make this study a significant contribution to the field of invertebrate immunology, providing a comprehensive framework for understanding oyster hemocyte diversity and function.

Weaknesses:

(1) The authors performed scRNAseq/lineage analysis and cytological analysis on oysters from two different sources. The methodology of the study raises concerns about the consistency of the sample and the variability of the results. The specific post-processing of hemocytes for scRNAseq, such as cell filtering, might also affect cell populations or gene expression profiles. It's unclear if the seven hemocyte types and their proportions were consistent across both samples. This inconsistency may affect the correlation between morphological and transcriptomic data.

(2) The authors claim to use pathogen-free adult oysters (lines 95 and 119), but no supporting data is provided. It's unclear if the oysters were tested for bacterial and viral contaminations, particularly Vibrio and OsHV-1 μVar herpesvirus.

(3) The KEGG and Gene Ontology analyses, while informative, are very descriptive and lack interpretation. The use of heatmaps with dendrograms for grouping cell clusters and GO terms is not discussed in the results, missing an opportunity to explore cell-type relationships. The changing order of cell clusters across panels B, C, and D in Figure 2 makes it challenging to correlate with panel A and to compare across different GO term categories. The dendrograms suggest proximity between certain clusters (e.g., 4 and 1) across different GO term types, implying similarity in cell processes, but this is not discussed. Grouping GO terms as in Figure 2A, rather than by dendrogram, might provide a clearer visualization of main pathways. Lastly, a more integrated discussion linking GO term and KEGG pathway analyses could offer a more comprehensive view of cell type characteristics. The presentation of scRNAseq results lacks depth in interpretation, particularly regarding the potential roles of different cell types based on their transcriptional profiles and marker genes. Additionally, some figures (2B, C, D, and 7C to H) suffer from information overload and small size, further hampering readability and interpretation.

(4) The pseudotime analysis presented in the study provides modest additional information to what is already manifest from the clustering and UMAP visualization. The central and intermediate transcriptomic profile of cluster 4 relative to other clusters is apparent from the UMAP and the expression of shared marker genes across clusters (as shown in Figure 1D). The statement by the authors that 'the two types of professional phagocytes belong to the same granular cell lineage' (lines 594-596) should be formulated with more caution. While the pseudotime trajectory links macrophage-like (ML) and small granule-like (SGC) cells, this doesn't definitively establish a direct lineage relationship. Such trajectories can result from similarities in gene expression induced by factors other than lineage relationships, such as responses to environmental stimuli or cell cycle states. To conclusively establish this lineage relationship, additional experiments like cell lineage tracing would be necessary, if such tools are available for C. gigas.

(6) Given the mention of herpesvirus as a major oyster pathogen, the lack of discussion on genes associated with antiviral immunity is a notable omission. While KEGG pathway analysis associated herpesvirus with cluster 1, the specific genes involved are not elaborated upon.

(7) The discussion misses an opportunity for comparative analysis with related species. Specifically, a comparison of gene markers and cell populations with Crassostrea hongkongensis, could highlight similarities and differences across systems.

Conclusion:

The authors largely achieved their primary objective of providing a comprehensive characterization of oyster immune cells. They successfully integrated multiple approaches to identify and describe distinct hemocyte types. The correlation of these cell types with specific immune functions represents a significant advancement in understanding oyster immunity. However, certain aspects of their objectives have not been fully achieved. The lineage relationships proposed on the basis of pseudotime analysis, while interesting, require further experimental validation. The potential of antiviral defense mechanisms, an important aspect of oyster immunity, has not been discussed in depth.

This study is likely to have a significant impact on the field of invertebrate immunology, particularly in bivalve research. It provides a new standard for comprehensive immune cell characterization in invertebrates. The identification of specific markers for different hemocyte types will facilitate future research on oyster immunity. The proposed model of hemocyte lineages, while requiring further validation, offers a framework for studying hematopoiesis in bivalves.

Reviewer #2 (Public review):

Summary:

The authors aimed to understand the mechanisms underlying chronic kidney disease (CKD) induced by cisplatin treatment. Acute or chronic kidney diseases are major adverse effects of cisplatin chemotherapy for cancer, which limits the treatment's efficacy. Understanding the disease's genesis is fundamental to identifying targets for preventing or treating these conditions.

Strengths:

The authors employed an in vivo model of cisplatin-induced chronic kidney disease (CKD) in mice, which displayed similar adverse effects of the therapy as seen in humans. The model called repeated low-dose cisplatin (RLCD), caused similar tissue and functional damage in the kidneys, led to harmful effects on the intestines by altering the microbiota and epithelial cell barrier, and impaired systemic vascular blood flow.

The authors demonstrated that the detrimental effects on the intestinal barrier led to the release of bacterial compounds into the circulation, which, in association with reactive oxygen species formed by the inflammatory and oxidative action of cisplatin, activated blood, and kidney neutrophils to release neutrophil extracellular traps (NETs). In turn, they suggested circulating NETs migrated into kidney tissue, causing damage. Moreover, they showed NETs are capable of trapping coagulation factors responsible for impaired systemic blood flow.

These conclusions were primarily based on reduced CKD symptoms and vascular damage in genetically modified animals that do not form NETs, as well as the observation that a bacterial compound (lipopolysaccharide) associated with cisplatin induces NET formation in isolated neutrophils. Moreover, treating animals with an anti-inflammatory and antioxidant natural compound simultaneously with cisplatin administration abolished the harmful effects on the kidneys and intestines.

The authors conclude that the intestinal damage and inflammatory properties of cisplatin lead to NET release, which, in turn, is responsible for the kidney and vascular damage evoked by cisplatin treatment.

Hence, the manuscript employs a well-designed experimental model and covers several important manifestations of cisplatin toxicity. It also uses genetically deficient mice to demonstrate the involvement of NETs in the development of chronic kidney disease (CKD)

Weaknesses:

Overall, the work was well executed. However, a few aspects require additional experiments to confirm the conclusions. The involvement of NETs in the genesis of CKD is unquestionable; nonetheless, the roles of locally induced versus circulating NETs, as well as the translation of in vitro NET release to in vivo CKD genesis, need further evaluation. Additionally, the primary mechanism of the natural anti-inflammatory compound used appears to be antioxidative, which does not promote the formation of reactive oxygen species necessary for NET formation. It is not clear in the title.