Reviewer #1 (Public review):

Summary:

In this study, the authors used a chronic murine dietary restriction model to study the effects of chronic malnutrition on controls of bacterial infection and overall immunity, including cellularity and functions of different immune cell types. They further attempted to determine whether refeeding can revert the infection susceptibility and immunodeficiency. Although refeeding here improves anthropometric deficits, the authors of this study show that this is insufficient to recover the impairments across the immune cell compartments.

Strengths:

The manuscript is well-written and conceived around a valid scientific question. The data supports the idea that malnutrition contributes to infection susceptibility and causes some immunological changes. The malnourished mouse model also displayed growth and development delays. The work's significance is well justified. Immunological studies in the malnourished cohort (human and mice) are scarce, so this could add valuable information.

Weaknesses:

The assays on myeloid cells are limited, and the study is descriptive and overstated. The authors claim that "this work identifies a novel cellular link between prior nutritional state and immunocompetency, highlighting dysregulated myelopoiesis as a major." However, after reviewing the entire manuscript, I found no cellular mechanism defining the link between nutritional state and immunocompetency.

Reviewer #2 (Public review):

Summary:

Sukhina et al. use a chronic murine dietary restriction model to investigate the cellular mechanisms underlying nutritionally acquired immunodeficiency as well as the consequences of a refeeding intervention. The authors report a substantial impact of undernutrition on the myeloid compartment, which is not rescued by refeeding despite rescue of other phenotypes including lymphocyte levels, and which is associated with maintained partial susceptibility to bacterial infection.

Strengths:

Overall, this is a nicely executed study with appropriate numbers of mice, robust phenotypes, and interesting conclusions, and the text is very well-written. The authors' conclusions are generally well-supported by their data.

Weaknesses:

There is little evaluation of known critical drivers of myelopoiesis (e.g. PMID 20535209, 26072330, 29218601) over the course of the 40% diet, which would be of interest with regard to comparing this chronic model to other more short-term models of undernutrition.

Further, the microbiota, which is well-established to be regulated by undernutrition (e.g. PMID 22674549, 27339978, etc.), and also well-established to be a critical regulator of hematopoiesis/myelopoiesis (e.g. PMID 27879260, 27799160, etc.), is completely ignored here.

Reviewer #3 (Public review):

Summary:

Sukhina et al are trying to understand the impacts of malnutrition on immunity. They model malnutrition with a diet switch from ad libitum to 40% caloric restriction (CR) in post-weaned mice. They test impacts on immune function with listeriosis. They then test whether re-feeding corrects these defects and find aspects of emergency myelopoiesis that remain defective after a precedent period of 40% CR. Overall, this is a very interesting observational study on the impacts of sudden prolonged exposure to less caloric intake.

Strengths:

The study is rigorously done. The observation of lasting defects after a bout of 40% CR is quite interesting. Overall, I think the topic and findings are of interest.

Weaknesses:

While the observations are interesting, in this reviewer's opinion, there is both a lack of mechanistic understanding of the phenomena and also some lack of resolution/detail about the phenomena itself. Addressing the following major issues would be helpful towards aspects of both:

(1) Is it calories, per se, or macro/micronutrients that drive these phenotypes observed with 40% CR. At the least, I would want to see isocaloric diets (primarily protein, fat, or carbs) and then some of the same readouts after 40% CR. Ie does low energy with relatively more eg protein prevent immunosuppression (as is commonly suggested)? Micronutrients would be harder to test experimentally and may be out of the scope of this study. However, it is worth noting that many of the malnutrition-associated diseases are micronutrient deficiencies.

(2) Is immunosuppression a function of a certain weight loss threshold? Or something else? Some idea of either the tempo of immunosuppression (happens at 1, in which weight loss is detected; vs 2-3, when body length and condition appear to diverge; or 5 weeks), or grade of CR (40% vs 60% vs 80%) would be helpful since the mechanism of immunosuppression overall is unclear (but nailing it may be beyond the scope of this communication).

(3) Does an obese mouse that gets 40% CR also become immunodeficient? As it stands, this ad libitum --> 40% CR model perhaps best models problems in the industrial world (as opposed to always being 40% CR from weaning, as might be more common in the developing world), and so modeling an obese person losing a lot of weight from CR (like would be achieved with GLP-1 drugs now) would be valuable to understanding generalizability.

(4) Generalizing this phenomenon as "bacterial" with listeriosis, which is more like a virus in many ways (intracellular phase, requires type I IFN, etc.) and cannot be given by the natural route of infection in mice, may not be most accurate. I would want to see an experiment with E.Coli, or some other bacteria, to test the statement of generalizability (ie is it bacteria, or type I IFN-pathway dominant infections, like viruses). If this is unique listeriosis, it doesn't undermine the story as it is at all, but it would just require some word-smithing.

(5) Previous reports (which the authors cite) implicate Leptin, the levels of which scale with fat mass, as "permissive" of a larger immune compartment (immune compartment as "luxury function" idea). Is their phenotype also leptin-mediated (ie leptin AAV)?

(6) The inability of re-feeding to "rescue" the myeloid compartment is really interesting. Can the authors do a bone marrow transplantation (CR-->ad libitum) to test if this effect is intrinsic to the CR-experienced bone marrow?

(7) Is the defect in emergency myelopoiesis a defect in G-CSF? Ie if the authors injected G-CSF in CR animals, do they equivalently mobilize neutrophils? Does G-CSF supplementation (as one does in humans) rescue host defense against Listeria in the CR or re-feeding paradigms?

Reviewer #1 (Public review):

Summary:

The authors aim to assess the effect of salt stress on root:shoot ratio, identify the underlying genetic mechanisms, and evaluate their contribution to salt tolerance. To this end, the authors systematically quantified natural variations in salt-induced changes in root:shoot ratio. This innovative approach considers the coordination of root and shoot growth rather than exploring biomass and the development of each organ separately. Using this approach, the authors identified a gene cluster encoding eight paralog genes with a domain-of-unknown-function 247 (DUF247), with the majority of SNPs clustering into SR3G (At3g50160). In the manuscript, the authors utilized an integrative approach that includes genomic, genetic, evolutionary, histological, and physiological assays to functionally assess the contribution of their genes of interest to salt tolerance and root development.

Comments on revisions:

As the authors correctly noted, variations across samples, genotypes, or experiments make achieving statistical significance challenging. Should the authors choose to emphasize trends across experiments to draw biological conclusions, careful revisions of the text, including titles and figure legends, will be necessary to address some of the inconsistencies between figures (see examples below). However, I would caution that this approach may dilute the overall impact of the work on SR3G function and regulation. Therefore, I strongly recommend pursuing additional experimental evidence wherever possible to strengthen the conclusions.

(1) Given the phenotypic differences shown in Figures S17A-B, 10A-C, and 6A, the statement that "SR3G does not play a role in plant development under non-stress conditions" (lines 680-681) requires revision to better reflect the observed data.<br /> (2) I agree with the authors that detecting expression differences in lowly expressed genes can be challenging. However, as demonstrated in the reference provided (Lu et al., 2023), a significant reduction in WRKY75 expression is observed in T-DNA insertion mutant alleles of WRKY75. In contrast, Fig. 9B in the current manuscript shows no reduction in WRKY75 expression in the two mutant alleles selected by the authors, which suggests that these alleles cannot be classified as loss-of-function mutants (line 745). Additionally, the authors note that the wrky75 mutant exhibits reduced main root length under salt stress, consistent with the phenotype reported by Lu et al. (2023). However, other phenotypic discrepancies exist between the two studies. For example, 1) Lu et al. (2023) report that w¬rky75 root length is comparable to WT under control conditions, whereas the current manuscript shows that wrky75 root growth is significantly lower than WT; 2) under salt stress, Lu et al. (2023) show that wrky75 accumulates higher levels of Na+, whereas the current study finds Na+ levels in wrky75 indistinguishable from WT. To confirm the loss of WRKY75 function in these T-DNA insertion alleles the authors should provide additional evidence (e.g., Western blot analysis).

Reviewer #1 (Public review):

Syngnathid fishes (seahorses, pipefishes, and seadragons) present very particular and elaborated features among teleosts and a major challenge is to understand the cellular and molecular mechanisms that permitted such innovations and adaptations. The study provides a valuable new resource to investigate the morphogenetic basis of four main traits characterizing syngnathids, including the elongated snout, toothlessness, dermal armor and male pregnancy. More particularly, the authors have focused on a late stage of pipefish organogenesis to perform single-cell RNA-sequencing (scRNA-seq) completed by in situ hybridization analyses to identify molecular pathways implicated in the formation of the different specific traits.<br /> The first set of data explores the scRNA-seq atlas composed of 35,785 cells from two samples of gulf pipefish embryos that authors have been able to classify into major cell types characterizing vertebrate organogenesis, including epithelial, connective, neural and muscle progenitors. To affirm identities and discover potential properties of clusters, authors primarily use KEGG analysis that reveals enriched genetic pathways in each cell types. After revisions, the authors have provided extended supplementary files to well interpret the dataset and some statements have been clarified. I thank the authors for the revisions/completions of ISH results compared to initial submission.

To conclude, the scRNA-seq dataset in this unconventional model organism will be useful for the community and will provide clues for future research to understand the extraordinary evolution of the Syngnathidae family.

Reviewer #2 (Public review):

Summary:

The authors present the first single-cell atlas for syngathid fishes, providing a resource for future evolution & development studies in this group.

Strengths:

The concept here is simple and I find the manuscript to be well written. I like the in situ hybridization of marker genes >> this is really nice. I also appreciate the gene co-expression analysis to identify modules of expression. There are no explicit hypotheses tested in the manuscript, but the discovery of these cell types should have value in this organism and in the determination of morphological novelties in seahorses and their relatives.

Weaknesses:

I think there are a few computational analyses that might improve the generality of the results.

(1) The cell types: The authors use marker gene analysis and KEGG pathways to identify cell types. I'd suggest a tool like SAMap (https://elifesciences.org/articles/66747) which compares single cell data sets from distinct organisms to identify 'homologous' cell types -- I imagine the zebrafish developmental atlases could serve as a reasonable comparative reference.

(2) Trajectory analyses: Authors suggest that their analyses might identify progenitor cell states and perhaps related differentiated states. They might explore cytoTRACE and/or pseudotime-based trajectory analyses to more fully delineate these ideas.

(3) Cell-cell communication: I think it's very difficult to identify 'tooth primordium' cell types, because cell types won't be defined by organ in this way. for instance dental glia will cluster with other glia, dental mesenchyme will likely cluster with other mesenchymal cell types. so the histology and ISH in most convincing in this regard. having said this, given the known signaling interactions in the developing tooth (and in development generally) the authors might explore cell-cell communication analysis (e.g., CellChat) to identify cell types that may be interacting.

Comments on revisions:

I feel essentially the same about this manuscript. it's a useful resource for future experimental forays into this unique system. The team made improvements to deal with comments from other reviewers related to quality of confirmatory in situ hybridization. This is good.

Regarding their response that one can't use CellChat if you're not working in mice or human, this is inaccurate. the assumption one makes is that ligand-receptor pairs and signaling pathways have conserved functions across animals (vertebrates). It's the same assumption the authors make when using the KEGG pathway to score enrichment of pathways in clusters. CellChat used in fishes in Johnson et al 2023 Nature Communications | ( 2023) 14:4891.

Reviewer #3 (Public review):

Summary:

This study established a single-cell RNA sequencing atlas of pipefish embryos. The results obtained identified unique gene expression patterns for pipefish-specific characteristics, such as fgf22 in the tip of the palatoquadrate and Meckel's cartilage, broadly informing the genetic mechanisms underlying morphological novelty in teleost fishes. The data obtained are unique and novel, potentially important in understanding fish diversity. Thus, I would enthusiastically support this manuscript if the authors improve it to generate stronger and more convincing conclusions than the current forms.

Weakness:

Regarding the expression of sfrp1a and bmp4 dorsal to the elongating ethmoid plate and surrounding the ceratohyal: Are their expression patterns spatially extended or broader compared to the pipefish ancestor? Is there a much closer species available to compare gene expression patterns with pipefish? Did the authors consider using other species closely related to pipefish for ISH? Sfrp1a and bmp4 may be expressed in the same regions of much more closely related species without face elongation. I understand that embryos of such species are not always accessible, but it is also hard to argue responsible genes for a specific phenotype by only comparing gene expression patterns between distantly related species (e.g., pipefish vs. zebrafish). Due to the same reason, I would not directly compare/argue gene expression patterns between pipefish and mice, although I should admit that mice gene expression patterns are sometimes helpful to make a hypothesis of fish evolution. Alternatively, can the authors conduct ISH in other species of pipefish? If the expression patterns of sfrp1a and bmp4 are common among fishes with face elongation, the conclusion would become more solid. If these embryos are not available, is it possible to reduce the amount of Wnt and BMP signal using Crispr/Cas, MO, or chemical inhibitor? I do think that there are several ways to test the Wnt and/or BMP hypothesis in face elongation.

Reviewer #1 (Public review):

Summary:

This study investigates an intriguing question in cognitive control from a temporal dynamics perspective: why does concurrent verbal working memory load eliminate the color-word Stroop effect? Through a series of thorough data analyses, the authors propose that verbal working memory load occupies the stimulus-response mapping resources represented by theta-band activity, thereby disrupting the mapping process for task-irrelevant distractors. This reduces the response tendency to the distractors, ultimately leading to the elimination of the Stroop effect.

Strengths:

The behavioral and neural evidence presented in the manuscript is solid, and the findings have valuable theoretical implications for research on Stroop conflict processing.

Weaknesses:

There are several areas where the manuscript could be improved.

Major Comments:

(1) In the Results section, the rationale behind selecting the beta band for the central (C3, CP3, Cz, CP4, C4) regions and the theta band for the fronto-central (Fz, FCz, Cz) regions is not clearly explained in the main text. This information is only mentioned in the figure captions. Additionally, why was the beta band chosen for the S-ROI fronto-central region and the theta band for the S-ROI central region? Was this choice influenced by the MVPA results?

(2) In the Data Analysis section, line 424 states: "Only trials that were correct in both the memory task and the Stroop task were included in all subsequent analyses. In addition, trials in which response times (RTs) deviated by more than three standard deviations from the condition mean were excluded from behavioral analyses." The percentage of excluded trials should be reported. Also, for the EEG-related analyses, were the same trials excluded, or were different criteria applied?

(3) In the Methods section, line 493 mentions: "A 400-200 ms pre-stimulus time window was selected as the baseline time window." What is the justification in the literature for choosing the 400-200 ms pre-stimulus window as the baseline? Why was the 200-0 ms pre-stimulus period not considered?

(4) Is the primary innovation of this study limited to the methodology, such as employing MVPA and RSA to establish the relationship between late theta activity and behavior?

(5) On page 14, lines 280-287, the authors discuss a specific pattern observed in the alpha band. However, the manuscript does not provide the corresponding results to substantiate this discussion. It is recommended to include these results as supplementary material.

(6) On page 16, lines 323-328, the authors provide a generalized explanation of the findings. According to load theory, stimuli compete for resources only when represented in the same form. Since the pre-memorized Chinese characters are represented semantically in working memory, this explanation lacks a critical premise: that semantic-response mapping is also represented semantically during processing.

(7) The classic Stroop task includes both a manual and a vocal version. Since stimulus-response mapping in the vocal version is more automatic than in the manual version, it is unclear whether the findings of this study would generalize to the impact of working memory load on the Stroop effect in the vocal version.

(8) While the discussion section provides a comprehensive analysis of the study's results, the authors could further elaborate on the theoretical and practical contributions of this work.

Reviewer #2 (Public review):

Summary:

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strengths:

(1) The experiment is well-designed.

(2) The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Weaknesses:

(1) As the researchers mentioned, a previous study reported a diminished Stroop effect with concurrent working memory tasks to memorize meaningless visual shapes rather than memorize Chinese characters as in the study. My main concern is that lower-level graphic processing when memorizing visual shapes also influences the Stroop effect. The stage of Stroop conflict processing affected by the working memory load may depend on the specific content of the concurrent working memory task. If that's the case, I sense that the generalization of this finding may be limited.

(2) The P1 and N450 components are sensitive to congruency in previous studies as mentioned by the researchers, but the results in the present study did not replicate them. This raised concerns about data quality and needs to be explained.

Reviewer #1 (Public review):

The revision by Ruan et al clarifies several aspects of the original manuscript that were difficult to understand, and I think it presents some useful and interesting ideas. I understand that the authors are distinguishing their model from the standard Wright-Fisher model in that the population size is not imposed externally, but is instead a consequence of the stochastic reproduction scheme. Here, the authors chose a branching process but in principle any Markov chain can probably be used. Within this framework, the authors are particularly interested in cases where the variance in reproductive success changes through time, as explored by the DDH model, for example. They argue with some experimental results that there is a reason to believe that the variance in reproductive success does change over time.

One of the key aspects of the original manuscript that I want to engage with is the DDH model. As the authors point out, their equations 5 and 6 are assumptions, and not derived from any principles. In essence, the authors are positing that that the variance in reproductive success, given by 6, changes as a function of the current population size. There is nothing "inherent" to a negative binomial branching mechanism that results in this: in fact, the the variance in offspring number could in principle be the same for all time. As relates to models that exist in the literature, I believe that this is the key difference: unlike Cannings models, the authors allow for a changing variance in reproduction through time.

This is, of course, an interesting thing to consider, and I think that the situation the authors point out, in which drift is lower at small population sizes and larger at large population sizes, is not appreciated in the literature. However, I am not so sure that there is anything that needs to be resolved in Paradox 1. A very strong prediction of that model is that Ne and N could be inversely related, as shown by the blue line in Fig 3b. This suggests that you could see something very strange if you, for example, infer a population size history using a Wright-Fisher framework, because you would infer a population *decline* when there is in fact a population *expansion*. However, as far as I know there are very few "surprising population declines" found in empirical data. An obvious case where we know there is very rapid population growth is human populations; I don't think I've ever seen an inference of recent human demographic history from genetic data that suggests anything other than a massive population expansion. While I appreciate the authors empirical data supporting their claim of Paradox 1 (more on the empirical data later), it's not clear to me that there's a "paradox" in the literature that needs explaining so much as this is a "words of caution about interpreting inferred effective population sizes". To be clear, I think those words of caution are important, and I had never considered that you might be so fundamentally misled as to infer decline when there is growth, but calling it a "paradox" seems to suggest that this is an outstanding problem in the literature, when in fact I think the authors are raising a *new* and important problem. Perhaps an interesting thing for the authors to do to raise the salience of this point would be to perform simulations under this model and then infer effective population sizes using e.g. dadi or psmc and show that you could identify a situation in which the true history is one of growth, but the best fit would be one of decline

The authors also highlight that their approach reflects a case where the population size is determined by the population dynamics themselves, as opposed to being imposed externally as is typical in Cannings models. I agree with the authors that this aspect of population regulation is understudied. Nonetheless, several manuscripts have dealt with the case of population genetic dynamics in populations of stochastically fluctuating size. For example, Kaj and Krone (2003) show that under pretty general conditions you get something very much like a standard coalescent; for example, combining their theorem 1 with their arguments on page 36 and 37, they find that exchangeable populations with stochastic population dynamics where the variance does not change with time still converge to exactly the coalescent you would expect from Cannings models. This is strongly suggestive that the authors key result isn't about stochastic population dynamics per se, but instead related to arguing that variance in reproductive success could change through time. In fact, I believe that the result of Kaj and Krone (2003) is substantially more general than the models considered in this manuscript. That being said, I believe that the authors of this manuscript do a much better job of making the implications for evolutionary processes clear than Kaj and Krone, which is important---it's very difficult to understand from Kaj and Krone the conditions under which effective population sizes will be substantially impacted by stochastic population dynamics.

I also find the authors exposition on Paradox 3 to be somewhat strange. First of all, I'm not sure there's a paradox there at all? The authors claim that the lack of dependence of the fixation probability on Ne is a paradox, but this is ultimately not surprising---fixation of a positively selected allele depends mostly on escaping the boundary layer, which doesn't really depend on the population size (see Gillespie's book "The Causes of Molecular Evolution" for great exposition on boundary layer effects). Moreover, the authors *use a Cannings-style argument* to get gain a good approximation of how the fixation probability changes when there is non-Poisson reproduction. So it's not clear that the WFH model is really doing a lot of work here. I suppose they raise the interesting point that the particularly simple form of p(fix) = 2s is due to the assumption that variance in offspring is equal to 1.

In addition, I raised some concerns about the analysis of empirical results on reproductive variance in my original review, and I don't believe that the authors responded to it at all. I'm not super worried about that analysis, but I think that the authors should probably respond to me.

Overall, I feel like I now have a better understanding of this manuscript. However, I think it still presents its results too strongly: Paradox 1 contains important words of caution that reflect what I am confident is an under appreciated possibility, and Paradox 3 is, as far as I'm concerned, not a paradox at all. I have not addressed Paradox 2 very much because I think that another reviewer had solid and interesting comments on that front and I am leaving it to them. That being said, I do think Paradox 2 actually presents a deep problem in the literature and that the authors' argument may actually represent a path toward a solution.

This manuscript can be a useful contribution to the literature, but as it's presented at the moment, I think most of it is worded too strongly and it continues to not engage appropriately with the literature. Theoretical advances are undoubtedly important, and I think the manuscript presents some interesting things to think about but ultimately needs to be better situated and several of the claims strongly toned down.

References:<br /> Kaj, I., & Krone, S. M. (2003). The coalescent process in a population with stochastically varying size. Journal of Applied Probability, 40(1), 33-48.

Reviewer #2 (Public review):

Summary:

This theoretical paper examines genetic drift in scenarios deviating from the standard Wright-Fisher model. The authors discuss Haldane's branching process model, highlighting that the variance in reproductive success equates to genetic drift. By integrating the Wright-Fisher model with the Haldane model, the authors derive theoretical results that resolve paradoxes related to effective population size.

Strengths:

The most significant and compelling result from this paper is perhaps that the probability of fixing a new beneficial mutation is 2s/V(K). This is an intriguing and potentially generalizable discovery that could be applied to many different study systems.

The authors also made a lot of effort to connect theory with various real-world examples, such as genetic diversity in sex chromosomes and reproductive variance across different species.

Comments on revisions:

The author has addressed some of the concerns in my review, and I think the revised manuscript is more clear. I like the discussion about the caveats of the WFH model.

I hope the authors could also discuss the conditions needed for V(K)/Ne to be a reasonable approximation. It is currently unclear how the framework should be adopted in general.

The idea about estimating male-female V(K) ratios from population genetic data is interesting. Unfortunately, the results fell short. The accuracy of their estimators (derived using approximation Ne/V(K) approximation, and certain choice of theta, and then theta estimated with Watterson's estimator) should be tested with simulated results before applying to real data. The reliability of their estimator and their results from real data are unclear.

Arguments made in this paper sometimes lack precision (perhaps the authors want to emphasize intuition, but it seems more confusing than otherwise). For example: The authors stated that "This independence from N seems intuitively obvious: when an advantageous mutation increases to say, 100 copies in determining a population (depending mainly on s), its fixation would be almost certain, regardless of N.". Assuming large Ne, and with approximation, one could assume the probability of loss is e^(-2sn), but the writing about "100 copies" and "almost certain" is very imprecise, in fact, a mutation with s=0.001 segregating at 100 copies in a large Ne population is most probably lost. Whereas in a small population, it will be fixed. Yet the following sentence states "regardless of N. This may be a most direct argument against equating genetic drift, certainly no less important than 1/ N . with N, or Ne (which is supposed to be a function of N's)." I find this new paragraph misleading.

Some of the statements/wordings in this paper still seem too strong to me.

Reviewer #3 (Public review):

Summary:

Ruan and colleagues consider a branching process model (in their terminology the "Haldane model") and the most basic Wright-Fisher model. They convincingly show that offspring distributions are usually non-Poissonian (as opposed to what's assumed in the Wright-Fisher model), and can depend on short-term ecological dynamics (e.g., variance in offspring number may be smaller during exponential growth). The authors discuss branching processes and the Wright-Fisher model in the context of 3 "paradoxes" --- 1) how Ne depends on N might depend on population dynamics; 2) how Ne is different on the X chromosome, the Y chromosome, and the autosomes, and these differences do match the expectations base on simple counts of the number of chromosomes in the populations; 3) how genetic drift interacts with selection. The authors provide some theoretical explanations for the role of variance in the offspring distribution in each of these three paradoxes. They also perform some experiments to directly measure the variance in offspring number, as well as perform some analyses of published data.

Strengths:

- The theoretical results are well-described and easy to follow.<br /> - The analyses of different variances in offspring number (both experimentally and analyzing public data) are convincing that non-Poissonian offspring distributions are the norm.<br /> - The point that this variance can change as the population size (or population dynamics) change is also very interesting and important to keep in mind.<br /> - I enjoyed the Density-Dependent Haldane model. It was a nice example of the decoupling of census size and effective size.<br /> - Equation (10) is a nice result (but see below)

Weaknesses:

- I am not convinced that these types of effects cannot just be absorbed into some time-varying Ne and still be well-modeled by the Wright-Fisher process. As a concrete example, Mohle and Sagitov 2001 show that a "coalescent Ne" for the WF model should be (N-1)/Var(K). This resolves the exponentially growing bacteria "paradox" raised in the present paper --- when the bacteria are growing Var(K) ~ 0, and hence there should be very little drift. This exactly resolves the "paradox" raised by the authors. Instead, it merely underscores that Ne does not need to be equal to (or even positively correlated!) with N. I absolutely do not see this as a failure of the WF model. Whether one finds branching processes or the WF model more biologically intuitive is a matter of taste, but to say that WF models cannot explain this "paradox" is false, when a well-known paper from more than 20 years ago does just that.<br /> - Along these lines, the result that Ne in the Wright-Fisher process might not be related to N in any straightforward (or even positively correlated) way are well-known (e.g., Neher and Hallatschek 2012; Spence, Kamm, and Song 2016; Matuszewski, Hildebrandt, Achaz, and Jensen 2018; Rice, Novembre, and Desai 2018; the work of Lounès Chikhi on how Ne can be affected by population structure; etc...)<br /> - I was also missing some discussion of the relationship between the branching process and the Wright-Fisher model (or more generally Cannings' Exchangeable Models) when conditioning on the total population size. In particular, if the offspring distribution is Poisson, then conditioned on the total population size, the branching process is identical to the Wright-Fisher model.<br /> - Given that Cannings' exchangeable models decouple N and Ne, it would not surprise me if something like equation (10) could be derived under such a model. I have not seen such a derivation, and the authors' result is nice, but I do not see it as proof that WF-type models (i.e., Cannings' models) are irreparably broken.

Reviewer #1 (Public review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group

Weaknesses:

Too few cells included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more a collection of preliminary transcriptomic data than a true scientific report to help advance the field.

Comments on revisions:

Thank you for the revision that helps clarify some outstanding questions.

(1) I still have questions relating to the relevance of the spatial maps generated and shown in fig 3C. They are supposedly generated using a 'molecular finger print' specific to each sub-cluster of LECs. However, given that at early stages postMI most populations are exceedingly rare in your analyses, could you please explain or comment on the relevance of the spatial maps?

(2) Fig 3 s1 would indicate that the population CaII is the majoritarian one in healthy hearts, while quantifications in Fig 3A show that rather the LEC Co subpopulation is majoritarian. Further, in mouse hearts histological analyses have demonstrated that cardiac lymphatics are restricted to the outer layers of the heart. This is not seen in your spatial maps. This seems to be the case only for the LEc Co population in healthy hearts, but not for other subpopulation signatures. Please explain.

(3) Further, the population of CaI, with 1 cell analysed in d3, but appears very prevalent in the spatial maps at d3. Please explain.

(4) In your list of 12 genes used as matrix anchors to identify LEC subpopulations in your screens, it is not apparent how LEC CaI, II and III differ so much as to allow selective detection of subpopulations. This similitude of profiles is supported by Fig 2F, and further explanations are needed to explain how the spatial maps of LEC ca subpopulations appear as distinct as shown in fig 3 S1 and Fig 3C.

Reviewer #2 (Public review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Editors' comments (Public review):

Weaknesses:

(1) Figure 7C, 7E, 7I, and "Figure7-figure supplement 1 ": All data in these data panels are based on only n=3, which is insufficient. Sample sizes of n=3 are too low to correctly assess normality of distribution and, as a consequence, do not allow to select the appropriate parametric/non-parametric tests. Accordingly, no statistical comparison can be performed and all p values and symbols currently indicating statistically significant differences between groups must be removed.

(2) Figure 3A, 3B, or 3C: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(3) Figure 4 E and 4F: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(4) Figure 5: No information about n numbers per group is provided. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

Reviewer #1 (Public review):

Summary:

In the work Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly-developed in vitro KO model, with live imaging experiments, electrophysiological recordings and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with higher number of docked vesicles and shorter AZ. The evaluation of presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted, and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that results in chronic deletion of SNX4 in neurons. A convincing sequence of experimental techniques are applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at presynaptic site is relevant and of high interest in the field of synaptic physiology and on the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow to address the physiological roles of SNX4 at synaptic site and speculate on the underlying molecular mechanisms. The novel experiments on autophagy progression as well as spontaneous neurotransmission are well conducted, although do not assist for the explanation of the molecular mechanism underneath.

Comments on revisions:

Other implementations in the revised version are quite limited and would benefit from a more detailed presentation and description. i.e.: Sholl analysis in the new figure 1h, is presented with no definition of number of cells employed and standard deviations of the replication. The "simil" Sholl analysis performed on VAMP2 is still puzzling and some explanations on the reason for the constant value of VAMP2 fluorescent signal from less than 0 to 160 µm from the cell body is to be added. How is the increased number of active synapses explained? How is this related to shorter AZ and higher number of docked vesicles?

Reviewer #2 (Public review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

Comments on revisions:

I am fine with revision in principle. the authors have addressed my concerns.

Reviewer #3 (Public review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4.

Comments on revisions:

The authors have addressed a couple of the more major concerns with the manuscript, however many of the original weaknesses remain. The primary weakness being the lack of mechanism. It is disappointing that real-time VAMP2 trafficking was not investigated, and the authors justification as to why the experiment was not performed was not convincing (especially since this is the approach that all other groups employ to examine SV cargo trafficking). In a number of instances "contractual constraints" are referred to as an explanation for not performing additional experiments. It was unclear whether this refers to licencing issues with the mouse line or the lack of personnel to perform the work. Regardless it still leaves this work as somewhat incomplete.

Reviewer #1 (Public review):

Summary:

Arafi et al. present results of studies designed to better understand the effects of mutations in the presenilin-1 (PSEN1) gene on proteolytic processing of the amyloid precursor protein (APP). This is important because APP processing can result in the production of the amyloid β-protein (Aβ), a key pathologic protein in Alzheimer's disease (AD). Aβ exists in various forms that differ in amino acid sequence and assembly state. The predominant forms of Aβ are Aβ40 and Aβ42, which are 40 and 42 amino acids in length, respectively. Shorter and longer forms derive from processive proteolysis of the Aβ region of APP by the heterotetramer β-secretase, within which presenilin 1 possesses the active site of the enzyme. Each form may become toxic if it assembles into non-natively folded, oligomeric, or fibrillar structures. A deep mechanistic understanding of enzyme-substrate interactions is a first step toward the design and successful use of small-molecule therapeutics for AD.

The key finding of Arafi et al. is that three PSEN mutations display unusual profiles of effects on Aβ production that have novel implications for the stalled E-S complex hypothesis. PSEN1 F386S is unique in that initial ε cleavage is not reduced compared with WT PSEN1; only certain trimming steps are deficient, results consistent with FLIM experiments that reveal stabilized E-S complexes only in Aβ-rich regions in the cell. In contrast, PSEN1 A431E and A434T display very little ε cleavage and therefore very little overall Aβ production, suggesting a limited role of Aβ in the pathogenesis of these two mutants and pointing to stalled E-S complexes as the common factor. For the biochemist, this may not be surprising, but in the context of understanding and treating AD, it is immense because it shifts the paradigm from targeting the results of γ-secretase action, viz., Aβ oligomers and fibrils, to targeting initial Aβ production at the molecular level. It is the equivalent of taking cancer treatment from simple removal of tumorous tissue to prevention of tumor formation and growth. Arafi et al. have provided us with a blueprint for the design of small-molecule inhibitors of γ-secretase. The significance of this achievement cannot be overstated.

Strengths and weaknesses:

The comprehensiveness and rigor of the study are notable. Rarely have I reviewed a manuscript reporting the results of so many orthogonal experiments, all of which support the authors' hypotheses, and of so many excellent controls. In addition, as found in clinical trial reports, the limitations of the study were discussed explicitly. None of these significantly affected the conclusions of the study.

Reviewer #2 (Public review):

Summary:

The work by Arafi et al. shows the effect of Familial Alzheimer's Disease presenilin-1 mutants on endoproteinase and carboxylase activity. They have elegantly demonstrated how some mutants alter each step of processing. Together with FLIM experiments, this study provides additional evidence to support their 'stalled complex hypotheses'.

Strengths:

This is a beautiful biochemical work. The approach is comprehensive.

Weaknesses:

(1) It appears that the purified g-secretase complex generates the same amount of Ab40 and Ab42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

(2) It has been reported the Ab production lines from Ab49 and Ab48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Ab species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

(4) It is interesting that both Ab40 and Ab42 Elisa kits detect Ab43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

Reviewer #1 (Public review):

In this study, Rosenblum et al introduce a novel and automatic way of calculating sleep cycles from human EEG. Previous results have shown that the slope of the non-oscillatory component of the power spectrum (called the aperiodic or fractal component) changes with sleep stage. Building on this, the authors present an algorithm that extracts the continuous-time fluctuations in the fractal slope and propose that peaks in this variable can be used to identify sleep cycle limits. Cycles defined in this way are termed "fractal cycles". The main focus of the article is a comparison of "fractal" and "classical" (ie defined manually based on the hypnogram) sleep cycles in numerous datasets.

The manuscript amply illustrates through examples the strong overlap between fractal and classical cycle identification. Accordingly, a high percentage (81%) can be matched one-to-one between methods and sleep cycle duration is well correlated (around R = 0.5). Moreover, the methods track certain global changes in sleep structure in different populations: shorter cycles in children and longer cycles in patients medicated with REM-suppressing anti-depressants. Finally, a major strength of the results is that they show similar agreement between fractal and classical sleep cycle length in 5 different data sets, showing that it is robust to changes in recording settings and methods.

The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method.<br /> The difference between the fractal and classical methods appear to be linked to the uncertain definition of sleep cycles since they are tied to when exactly the cycle begins/ends and whether or not to count cycles during fractured sleep architecture at sleep onset. Moreover, the discrepancies between the two are on the order of that found between classical cycles defined manually or via an automatic algorithm.

Overall the fractal cycle is an attractive method to study sleep architecture since it dispenses with time-consuming and potentially subjective manual identification of sleep cycles. However, given its difference with the classical method, it is unlikely that fractal scoring will be able to replace classical scoring directly. By providing a complementary quantification, it will likely contribute to refining the definition of sleep cycles that is currently ambiguous in certain cases. Moreover, it has the potential to be applied on animal studies which rarely deal with sleep cycle structure.

Reviewer #2 (Public review):

Summary:

This study focused on using strictly the slope of the power spectral density (PSD) to perform automated sleep scoring and evaluation of the durations of sleep cycles. The method appears to work well because the slope of the PSD is highest during slow-wave sleep, and lowest during waking and REM sleep. Therefore, when smoothed and analyzed across time, there are cyclical variations in the slope of the PSD, fit using an IRASA (Irregularly resampled auto-spectral analysis) algorithm proposed by Wen & Liu (2016).

Strengths:

The main novelty of the study is that the non-fractal (oscillatory) components of the PSD that are more typically used during sleep scoring can be essentially ignored because the key information is already contained within the fractal (slope) component. The authors show that for the most part, results are fairly consistent between this and conventional sleep scoring, but in some cases show disagreements that may be scientifically interesting.

Weaknesses:

The previous weaknesses were well-addressed by the authors in the revised manuscript. I will note that from the fractal cycle perspective, waking and REM sleep are not very dissimilar. Combining these states underlies some of the key results of this study.

Reviewer #1 (Public review):

Summary:

This study investigates an intriguing question in cognitive control from a temporal dynamics perspective: why does concurrent verbal working memory load eliminate the color-word Stroop effect? Through a series of thorough data analyses, the authors propose that verbal working memory load occupies the stimulus-response mapping resources represented by theta-band activity, thereby disrupting the mapping process for task-irrelevant distractors. This reduces the response tendency to the distractors, ultimately leading to the elimination of the Stroop effect.

Strengths:

The behavioral and neural evidence presented in the manuscript is solid, and the findings have valuable theoretical implications for research on Stroop conflict processing.

Comments on revisions:

The authors have addressed all concerns

Reviewer #2 (Public review):

Summary

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strength

The experiment is well designed.<br /> The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

The authors define a new metric for visual displays, derived from psychophysical response times, called visual homogeneity (VH). They attempt to show that VH is explanatory of response times across multiple visual tasks. They use fMRI to find visual cortex regions with VH-correlated activity. On this basis, they declare a new visual region in human brain, area VH, whose purpose is to represent VH for the purpose of visual search and symmetry tasks.

Link to original review: https://elifesciences.org/reviewed-preprints/93033v2/reviews#peer-review-0

Comments on latest version:

Authors rebuttal: We agree that visual homogeneity is similar to existing concepts such as target saliency, memorability etc. We have proposed it as a separate concept because visual homogeneity has an independent empirical measure (the reciprocal of target-absent search time in oddball search, or the reciprocal of same response time in a same-different task, etc) that may or may not be the same as other empirical measures such as saliency and memorability. Investigating these possibilities is beyond the scope of our study but would be interesting for future work. We have now clarified this in the revised manuscript (Discussion, p. 42).

Reviewer response to rebuttal: Neither the original ms nor the comments on that ms pretended that "visual homogeneity" was entirely separate from target saliency etc. So this is a response to a criticism that was never made. What the authors do claim, and what the comments question, is that they have successfully subsumed long-recognized psychophysical concepts like target saliency etc. under a new, uber-concept, "visual homogeneity" that explains psychophysical experimental results in a more unified and satisfying way. This subsumption of several well-established psychophysical concepts under a new, unified category is what reviewers objected to.

Authors rebuttal: However, we'd like to emphasize that the question of whether visual homogeneity is novel or related to existing concepts misses entirely the key contribution of our study.

Reviewer response to rebuttal: Sorry, but the claim of a new uber-concept in psychophysics, "visual homogeneity", is a major claim of the paper. The fact that it is not the only claim made does not absolve the authors from having to prove it satisfactorily.

"Authors rebuttal: "In addition, the large regions of VH correlations identified in Experiments 1 and 2 vs. Experiments 3 and 4 are barely overlapping. This undermines the claim that VH is a universal quantity, represented in a newly discovered area of visual cortex, that underlies a wide variety of visual tasks and functions."<br /> • We respectfully disagree with your assertion. First of all, there is partial overlap between the VH regions, for which there are several other obvious explanations that must be considered first before dismissing VH outright as a flawed construct. We acknowledge these alternatives in the Results (p. 27), and the relevant text is reproduced below.

"We note that it is not straightforward to interpret the overlap between the VH regions identified in Experiments 2 & 4. The lack of overlap could be due to stimulus differences (natural images in Experiment 2 vs silhouettes in Experiment 4), visual field differences (items in the periphery in Experiment 2 vs items at the fovea in Experiment 4) and even due to different participants in the two experiments. There is evidence supporting all these possibilities: stimulus differences (Yue et al., 2014), visual field differences (Kravitz et al., 2013) as well as individual differences can all change the locus of neural activations in object-selective cortex (Weiner and Grill-Spector, 2012a; Glezer and Riesenhuber, 2013). We speculate that testing the same participants on search and symmetry tasks using similar stimuli and display properties would reveal even larger overlap in the VH regions that drive behavior."

Reviewer response to rebuttal: The authors are saying that their results merely look unconvincing (weak overlap between VH regions defined in different experiments) because there were confounding differences between their experiments, in subject population, stimuli, etc. That is possible, but in that case it is up to the authors to show that their definition of a new "area VH" is convincing when the confounding differences are resolved, e.g. by using the same stimuli in the different experiments they attempt to agglomerate here. That would require new experiments, and none are offered in this revision.

Authors rebuttal: • Thank you for carefully thinking through our logic. We agree that a distance-to-centre calculation is entirely unnecessary as an explanation for target-present visual search. The similarity between target and distractor, so there is nothing new to explain here. However, this is a narrow and selective interpretation of our findings because you are focusing only on our results on target-present searches, which are only half of all our data. The other half is the target-absent responses which previously have had no clear explanation. You are also missing the fact that we are explaining same-different and symmetry tasks as well using the same visual homogeneity computation. We urge you to think more deeply about the problem of how to decide whether an oddball is present or not in the first place. How do we actually solve this task?

Reviewer response to rebuttal: It is the role of the authors to think deeply about their paper and on that basis present a clear and compelling case that readers can understand quickly and agree with. That is not done here.

Authors rebuttal: There must be some underlying representation and decision process. Our study shows that a distance-to-centre computation can actually serve as a decision variable to solve disparate property-based visual tasks. These tasks pose a major challenge to standard models of decision-making because the underlying representation and decision variable have been unclear. Our study resolves this challenge by proposing a novel computation that can be used by the brain to solve all these disparate tasks, and bring these tasks into the ambit of standard theories of decision-making.

Reviewer response to rebuttal: There is only a "challenge" if you accept the authors' a priori assumption that all of these tasks must have a common explanation and rely on a single neural mechanism. I do not accept that assumption, and I don't think the authors provide evidence to support the assumption. There is nothing "unclear" about how search, oddball, etc. have been thoroughly explained, separately, in the psychophysical literature that spans more than a century.

Authors rebuttal: • You are indeed correct in noting that both Experiment 1 & 2 involve oddball search, and so at the superficial level, it looks circular that the oddball search data of Experiment 1 is being used to explain the oddball search data of Experiment 2.<br /> However a deeper scrutiny reveals more fundamental differences: Experiment 1 consisted of only oddball search with the target appearing on the left or right, whereas Experiment 2 consisted of oddball search with the target either present or completely absent. In fact, we were merely using the search dissimilarities from Experiment 1 to reconstruct the underlying object representation, because it is well-known that neural dissimilarities are predicted well by search dissimilarities (Sripati & Olson, 2009; Zhivago et al, 2014).

Reviewer response to rebuttal: Here again the authors cite differences between their multiple experiments as a virtue that supports their conclusions. Instead, the experiments should have been designed for maximum similarity if the authors intended to explain them with the same theory.

Authors rebuttal: To thoroughly refute any lingering concern about circularity, we reasoned that the model predictions for Experiment 2 could have been obtained by a distance-to-center computation on any brain like object representation. To this end, we used object representations from deep neural networks pretrained on object categorization, whose representations are known to match well with the brain, and asked if a distance-to-centre computation on these representations could predict the search data in Experiment 2. This was indeed the case, and these results are now included an additional section in Supplementary Material (Section S1).

Reviewer response to rebuttal: The authors' claims are about human performance and how it is based on the human brain. Their claims are not well supported by the human experiments that they performed. It serves no purpose to redo the same experiments in silico, which cannot provide stronger evidence that compensates for what was lacking in the human data.

Authors rebuttal: "Confirming the generality of visual homogeneity<br /> We performed several additional analyses to confirm the generality of our results, and to reject alternate explanations.

First, it could be argued that our results are circular because they involve taking oddball search times from Experiment 1 and using them to explain search response times in Experiment 2. This is a superficial concern since we are using the search dissimilarities from Experiment 1 only as a proxy for the underlying neural representation, based on previous reports that neural dissimilarities closely match oddball search dissimilarities (Sripati and Olson, 2010; Zhivago and Arun, 2014). Nonetheless, to thoroughly refute this possibility, we reasoned that we would get similar predictions of the target present/absent responses in Experiment using any other brain-like object representation. To confirm this, we replaced the object representations derived from Experiment 1 with object representations derived from deep neural networks pretrained for object categorization, and asked if distance-to-center computations could predict the target present/absent responses in Experiment 2. This was indeed the case (Section S1).

Second, we wondered whether the nonlinear optimization process of finding the best-fitting center could be yielding disparate optimal centres each time. To investigate this, we repeated the optimization procedure with many randomly initialized starting points, and obtained the same best-fitting center each time (see Methods).

Third, to confirm that the above model fits are not due to overfitting, we performed a leave-one-out cross validation analysis. We left out all target-present and target-absent searches involving a particular image, and then predicted these searches by calculating visual homogeneity estimated from all other images. This too yielded similar positive and negative correlations (r = 0.63, p < 0.0001 for target-present, r = -0.63, p < 0.001 for target-absent).

Fourth, if heterogeneous displays indeed elicit similar neural responses due to mixing, then their average distance to other objects must be related to their visual homogeneity. We confirmed that this was indeed the case, suggesting that the average distance of an object from all other objects in visual search can predict visual homogeneity (Section S1).

Fifth, the above results are based on taking the neural response to oddball arrays to be the average of the target and distractor responses. To confirm that averaging was indeed the optimal choice, we repeated the above analysis by assuming a range of relative weights between the target and distractor. The best correlation was obtained for almost equal weights in the lateral occipital (LO) region, consistent with averaging and its role in the underlying perceptual representation (Section S1).

Finally, we performed several additional experiments on a larger set of natural objects as well as on silhouette shapes. In all cases, present/absent responses were explained using visual homogeneity (Section S2)."

Reviewer response to rebuttal: The authors can experiment on side questions for as long as they please, but none of the results described above answer the concern about how center-fitting undercuts the evidentiary value of their main results.

Authors rebuttal: • While it is true that the optimal center needs to be found by fitting to the data, there no particular mystery to the algorithm: we are simply performing a standard gradient-descent to maximize the fit to the data. We have described the algorithm clearly and are making our codes public. We find the algorithm to yield stable optimal centers despite many randomly initialized starting points. We find the optimal center to be able to predict responses to entirely novel images that were excluded during model training. We are making no assumption about the location of centre with respect to individual points. Therefore, we see no cause for concern regarding the center-finding algorithm.

Reviewer response to rebuttal: The point of the original comment was that center-fitting should not be done in the first place because it introduces unknowable effects.

•Authors rebuttal: Most visual tasks, such as finding an animal, are thought to involve building a decision boundary on some underlying neural representation. Even visual search has been portrayed as a signal-detection problem where a particular target is to be discriminated from a distractor. However none of these formulations work in the case of property-based visual tasks, where there is no unique feature to look for.<br /> We are proposing that, when we view a search array, the neural response to the search array can be deduced from the neural responses to the individual elements using well-known rules, and that decisions about an oddball target being present or absent can be made by computing the distance of this neural response from some canonical mean firing rate of a population of neurons. This distance to center computation is what we denote as visual homogeneity. We have revised our manuscript throughout to make this clearer and we hope that this helps you understand the logic better.<br /> • You are absolutely correct that the stimulus complexity should matter, but there are no good empirically derived measures for stimulus complexity, other than subjective ratings which are complex on their own and could be based on any number of other cognitive and semantic factors. But considering what factors are correlated with target-absent response times is entirely different from asking what decision variable or template is being used by participants to solve the task.

Reviewer response to rebuttal: If stimulus complexity is what matters, as the authors agree here, then it is incumbent on them to measure stimulus complexity. The difficulty of measuring stimulus complexity does not justify avoiding the problem with an analysis that ignores complexity.

Authors rebuttal: • We have provided empirical proof for our claims, by showing that target-present response times in a visual search task are correlated with "different" responses in the same-different task, and that target-absent response times in the visual search task are correlated with "same" responses in the same-different task (Section S4).

Reviewer response to rebuttal: Sorry, but there is still no reason to think that same-different judgments are based on a mythical boundary halfway between the two. If there is a boundary, it will be close to the same end of the continuum, where subjects might conceivably miss some tiny difference between two stimuli. The vast majority of "different" stimuli will be entirely different from the same stimulus, producing no confusability, and certainly not a decision boundary halfway between two extremes.

Authors rebuttal: • Again, the opposite correlations between target present/absent search times with VH are the crucial empirical validation of our claims that a distance-to-center calculation explain how we perform these property-based tasks. The VH predictions do not fully explain the data. We have explicitly acknowledged this shortcoming, so we are hardly dismissing it as a problem.

Reviewer response to rebuttal: The authors' acknowledgement of flaws in the ms does not argue in favor of publication, but rather just the opposite.

Authors rebuttal: • Finding an oddball, deciding if two items are same or different and symmetry tasks are disparate visual tasks that do not fit neatly into standard models of decision-making. The key conceptual advance of our study is that we propose a plausible neural representation and decision variable that allows all three property-based visual tasks to be reconciled with standard models of decision-making.

Reviewer response to rebuttal: The original comment stands as written. Same/different will have a boundary very close to the "same" end of the continuum. The boundary is only halfway between two choices if the stimulus design forces the boundary to be there, as in the motion and cat/dog experiments.

Authors rebuttal: "There is no inherent middle point boundary between target present and target absent. Instead, in both types of trial, maximum information is present when target and distractors are most dissimilar, and minimum information is present when target and distractors are most similar. The point of greatest similarity occurs at then limit of any metric for similarity. Correspondingly, there is no middle point dip in information that would produce greater difficulty and higher response times. Instead, task difficulty and response times increase monotonically with similarity between targets and distractors, for both target present and target absent decisions. Thus, in Figs. 2F and 2G, response times appear to be highest for animals, which share the largest numbers of closely similar distractors."<br /> • Your alternative explanation rests on vague factors like "maximum information" which cannot be quantified. By contrast we are proposing a concrete, falsifiable model for three property-based tasks - same/different, oddball present/absent and object symmetry. Any argument based solely on item similarity to explain visual search or symmetry responses cannot explain systematic variations observed for target-absent arrays and for symmetric objects, for the reasons explained earlier.

Reviewer response to rebuttal: There is nothing vague about this comment. The authors use an analysis that assumes a decision boundary at the centerpoint of their arbitrarily defined stimulus space. This assumption is not supported, and it is unlikely, considering that subjects are likely to notice all but the smallest variations between same and different stimuli, putting the boundary nearly at the same end of the continuum, not the very middle.

Authors rebuttal: "(1) The area VH boundaries from different experiments are nearly completely non-overlapping.

In line with their theory that VH is a single continuum with a decision boundary somewhere in the middle, the authors use fMRI searchlight to find an area whose responses positively correlate with homogeneity, as calculated across all of their target present and target absent arrays. They report VH-correlated activity in regions anterior to LO. However, the VH defined by symmetry Experiments 3 and 4 (VHsymmetry) is substantially anterior to LO, while the VH defined by target detection Experiments 1 and 2 (VHdetection) is almost immediately adjacent to LO. Fig. S13 shows that VHsymmetry and VHdetection are nearly non-overlapping. This is a fundamental problem with the claim of discovering a new area that represents a new quantity that explains response times across multiple visual tasks. In addition, it is hard to understand why VHsymmetry does not show up in a straightforward subtraction between symmetric and asymmetric objects, which should show a clear difference in homogeneity."

• We respectfully disagree. The partial overlap between the VH regions identified in Experiments 1 & 2 can hardly be taken as evidence against the quantity VH itself, because there are several other obvious alternate explanations for this partial overlap, as summarized earlier as well. The VH region does show up in a straightforward subtraction between symmetric and asymmetric objects (Section S7), so we are not sure what the Reviewer is referring to here.

Reviewer response to rebuttal: In disagreeing with the comment quoted above, the authors are maintaining that a new functional area of cerebral cortex can be declared even if that area changes location on the cortical map from one experiment to another. That position is patently absurd.

Authors rebuttal: "(3) Definition of the boundaries and purpose of a new visual area in the brain requires circumspection, abundant and convergent evidence, and careful controls.

Even if the VH metric, as defined and calculated by the authors here, is a meaningful quantity, it is a bold claim that a large cortical area just anterior to LO is devoted to calculating this metric as its major task. Vision involves much more than target detection and symmetry detection. Cortex anterior to LO is bound to perform a much wider range of visual functionalities. If the reported correlations can be clarified and supported, it would be more circumspect to treat them as one byproduct of unknown visual processing in cortex anterior to LO, rather than treating them as the defining purpose for a large area of visual cortex."

• We totally agree with you that reporting a new brain region would require careful interpretation and abundant and converging evidence. However, this requires many studies worth of work, and historically category-selective regions like the FFA have achieved consensus only after they were replicated and confirmed across many studies. We believe our proposal for the computation of a quantity like visual homogeneity is conceptually novel, and our study represents a first step that provides some converging evidence (through replicable results across different experiments) for such a region. We have reworked our manuscript to make this point clearer (Discussion, p 32).

Reviewer response to rebuttal: Indeed, declaring a new brain area depends on much more work than is done here. Thus, the appropriate course here is to wait before claiming to have identified a new cortical area.

Reviewer #2 (Public review):

Summary:

This study proposes visual homogeneity as a novel visual property that enables observers perform to several seemingly disparate visual tasks, such as finding an odd item, deciding if two items are same, or judging if an object is symmetric. In Exp 1, the reaction times on several objects were measured in human subjects. In Exp 2, visual homogeneity of each object was calculated based on the reaction time data. The visual homogeneity scores predicted reaction times. This value was also correlated with the BOLD signals in a specific region anterior to LO. Similar methods were used to analyze reaction time and fMRI data in a symmetry detection task. It is concluded that visual homogeneity is an important feature that enables observers to solve these two tasks.

Strengths:

(1) The writing is very clear. The presentation of the study is informative.

(2) This study includes several behavioral and fMRI experiments. I appreciate the scientific rigor of the authors.

Weaknesses:

Before addressing the manuscript itself, I would like to comment the review process first. Having read the lasted revised manuscript, I shared many of the concerns raised by the two reviewers in the last two rounds of review. It appears that the authors have disagreed with the majority of comments made by the two reviewers. If so, I strongly recommend that the authors proceed to make this revision as a Version of Record and conclude this review process. According to eLife's policy that the authors have the right to make a Version of Record at any time during the review process, and I fully respect that right. However, I also ask that the authors respect the reviewer's right to retain the comments regarding this paper.

Beside that, I still have several further questions about this study.

(1) My main concern with this paper is the way visual homogeneity is computed. On page 10, lines 188-192, it says: "we then asked if there is any point in this multidimensional representation such that distances from this point to the target-present and target-absent response vectors can accurately predict the target-present and target-absent response times with a positive and negative correlation respectively (see Methods)". This is also true for the symmetry detection task. If I understand correctly, the reference point in this perceptual space was found by deliberating satisfying the negative and positive correlations in response times. And then on page 10, lines 200-205, it shows that the positive and negative correlations actually exist. This logic is confusing. The positive and negative correlations emerge only because this method is optimized to do so. It seems more reasonable to identify the reference point of this perceptual space independently, without using the reaction time data. Otherwise, the inference process sounds circular. A simple way is to just use the mean point of all objects in Exp 1, without any optimization towards reaction time data.<br /> I raised this question in my initial review. However, the authors did not address whether the positive and negative correlations still hold if the mean point is defined as the reference point without any optimization. The authors also argue that it is similar to a case of fitting a straight line. It is fine that the authors insist on the straight line (e.g., correlation). However, I would not call "straight line correlations" a "quantitative model" as a high-profile journals like eLife. Please remove all related arguments of a novel quantitative model.

(2) Visual homogeneity (at least given the current form) is an unnecessary term. It is similar to distractor heterogeneity/distractor variability/distractor saliency in literature. However, the authors attempt to claim it as a novel concept. Both R1 and me raised this question in the very first review. However, the authors refused to revise the manuscript. In the last review, I mentioned this and provided some example sentences claiming novelty. The authors only revised the last sentence of the abstract, and even did not bother to revise the last sentence of significance: "we show that these tasks can be solved using a simple property WE DEFINE as visual homogeneity". Also, lines 851 still shows "we have defined a NOVEL image property, visual homogeneity...". I am confused about whether the authors agree or disagree that "visual homogeneity is an unnecessary term". If the authors agree, they should completely remove the related phrase throughout the paper. If not, they should keep all these and state the reasons. I don't think this is a correct approach to revising a manuscript.

(3) If the authors agree that visual homogeneity is not new, I suggest a complete rewrite of the title, abstract, significance, and introduction. Let me ask a simple question, can we remove "visual homogeneity" and use some more well-established term like "image feature similarity"? If yes, visual homogeneity is unnecessary.

(4) If I understand it correctly, one of the key findings of this paper is "the response times for target-present searches were positively correlated with visual homogeneity. By contrast, the response times for target-absent searches were negatively correlated with visual homogeneity" (lines 204-207). I think the authors have already acknowledged that this positive correlation is not surprising at all because it reflects the classic target-distractor similarity effect. If this is the case, please completely remove the positive correlation as a novel prediction and finding.

(5) In my last review, I mentioned the seminal paper by Duncan and Humphreys (1989) has clearly stated that "difficulty increases with increased similarity of targets to nontargets and decreased similarity between nontargets" (the sentence in their abstract). Here, "similarity between nontargets" is the same as the visual homogeneity defined here. Similar effects have been shown in Duncan (1989) and Nagy, Neriani, and Young (2005). See also the inconsistent results in Nagy& Thomas, 2003, Vicent, Baddeley, Troscianko&Gilchrist, 2009. More recently, Wei Ji Ma has systematically investigated the effects of heterogeneous distractors in visual search. I think the introduction part of Wei Ji Ma's paper (2020) provides a nice summary of this line of research.

Thanks to the authors' revision, I now better understand the negative correlation. The between-distrator similarity mentioned above describes the heterogeneity of distractors WITHIN an image. However, if I understand it correctly, this study aims to address the negative correlation of reaction time and target-absent stimuli ACROSS images. In other words, why do humans show a shorter reaction time to an image of four pigeons than to an image of four dogs (as shown in Figure 2C), simply because the later image is closer to the reference point of the image space. In this sense, this negative correlation is indeed not the same as distractor heterogeneity. However, this is known as the saliency effect or oddball effects. For example, it seems quite natural to me that humans respond faster to a fish image if the image set contains many images of four-leg dogs that look very different from fish. If this is indeed a saliency effect, why should we define a new term "visual homogeneity"?

(6) The section "key predictions" is quite straightforward. I understand the logic of positive and negative correlations. However, what is the physical meaning of "decision boundary" (Fig. 1G) here? How does the "decision boundary" map on the image space?

(7) In my opinion, one of the advantages of this study is the fMRI dataset, which is valuable because previous studies did not collect fMRI data. The key contribution may be the novel brain region associated with display heterogeneity. If this is the case, I would suggest using a more parametric way to measure this region. For example, one can use Gabor stimuli and systematically manipulate the variations of multiple Gabor stimuli, the same logic also applies to motion direction. If this study uses static Gabor, random dot motion, object images that span from low-level to high-level visual stimuli, and consistently shows that the stimulus heterogeneity is encoded in one brain region, I would say this finding is valuable. But this sounds another experiment. In other words, it is insufficient to claim a new brain region given the current form of the manuscript.

References:

* Duncan, J., & Humphreys, G. W. (1989). Visual search and stimulus similarity. Psychological Review, 96(3), 433-458. doi: 10.1037/0033-295x.96.3.433<br /> * Duncan, J. (1989). Boundary conditions on parallel processing in human vision. Perception, 18(4), 457-469. doi: 10.1068/p180457<br /> * Nagy, A. L., Neriani, K. E., & Young, T. L. (2005). Effects of target and distractor heterogeneity on search for a color target. Vision Research, 45(14), 1885-1899. doi: 10.1016/j.visres.2005.01.007<br /> * Nagy, A. L., & Thomas, G. (2003). Distractor heterogeneity, attention, and color in visual search. Vision Research, 43(14), 1541-1552. doi: 10.1016/s0042-6989(03)00234-7<br /> * Vincent, B., Baddeley, R., Troscianko, T., & Gilchrist, I. (2009). Optimal feature integration in visual search. Journal of Vision, 9(5), 15-15. doi: 10.1167/9.5.15<br /> * Singh, A., Mihali, A., Chou, W. C., & Ma, W. J. (2023). A Computational Approach to Search in Visual Working Memory.<br /> * Mihali, A., & Ma, W. J. (2020). The psychophysics of visual search with heterogeneous distractors. BioRxiv, 2020-08.<br /> * Calder-Travis, J., & Ma, W. J. (2020). Explaining the effects of distractor statistics in visual search. Journal of Vision, 20(13), 11-11.

Reviewer #3 (Public review):

Summary of the review process from the Reviewing Editor:

The authors and the reviewers did not agree on several important points made in this paper. The reviewers were critical of the operationalisation of the concept of visual homogeneity (VH), and questioned its validity. For instance, they found it unsatisfying that VH was not calculated on the basis of images themselves, but on the basis of reaction times instead. The authors responded by providing further explanation and argumentation for the importance of this novel concept, but the reviewers were not persuaded. The reviewers also pointed out some data features that did not fit the theory (e.g., overlapping VH between present and absent stimuli), which the authors acknowledge as a point that needs further refining. Finally, the reviewers pointed out that the new so-called visual homogeneity brain region does not overlap very much in the two studies, to which the authors have responded that it is remarkable that there is even partial overlap, given the many confounding differences between the two studies. Altogether, the authors have greatly elaborated their case for VH as an important concept, but the reviewers were not persuaded, and we conclude that the current evidence does not yet meet the high bar for declaring that a novel image property, visual homogeneity, is computed in a localised brain region.

Reviewer #1 (Public review):

The authors explain that an action potential that reach an axon terminal emits a small electrical field as it "annihilates". This happens even though there is no gap junction, at chemical synapses. The generated electrical field is simulated to show that it can affect a nearby, disconnected target membrane by tens of microvolts for tenths of a microsecond. Longer effects are simulated for target locations a few microns away.

To simulate action potentials (APs), the paper does not use the standard Hodgkin-Huxley formalism because it fails to explain AP collision. Instead it uses the Tasaki and Matsumoto (TM) model which is simplified to only models APs with three parameters and as a membrane transition between two states of resting versus excited. The authors expand the strictly binary, discrete TM method to a Relaxing Tasaki Model (RTM) that models the relaxation of the membrane potential after an AP. They find that the membrane leak can be neglected in determining AP propagation and that the capacitive currents dominate the process.

The strength of the work is that authors identified an important interaction between neurons that is neglected by the standard models. A weakness of the proposed approach is the assumptions that it makes. For instance, the external medium is modeled as a homogeneous conductive medium, which may be further explored to properly account for biological processes. To the authors' credit, the external medium can be largely varying and could be left out from the general model, only to be modeled specific instances.

The authors provide convincing evidence by performing experiments to record action potential propagation and collision properties and then developing a theoretical framework to simulate effect of their annihilation on nearby membranes. They provide both experimental evidence and rigorous mathematical and computer simulation findings to support their claims. The work has a potential of explaining significant electrical interaction between nerve centers that are connected via a large number of parallel fibers.

Comments on revisions:

The authors responded to all of my previous concerns and significantly improved the manuscript.

Reviewer #2 (Public review):

In this study, the authors measured extracellular electrical features of colliding APs travelling in different directions down an isolated earthworm axon. They then used these features to build a model of the potential ephaptic effects of AP annihilation, i.e. the electrical signals produced by colliding/annihilating APs that may influence neighbouring tissue. The model was then applied to some different hypothetical scenarios involving synaptic connections. In a revised version of the manuscript, it was also applied, with success, to published experimental data on the cerebellar basket cell-to-Purkinje cell pinceau connection. The conclusion is that an annihilating AP at a presynaptic terminal can emphatically influence the voltage of a postsynaptic cell (this is, presumably, the 'electrical coupling between neurons' of the title), and that the nature of this influence depends on the physical configuration of the synapse.

As an experimental neuroscientist who has never used computational approaches, I am unable to comment on the rigour of the analytical approaches that form the bulk of this paper. The experimental approaches appear very well carried out, and the data showing equal conduction velocity of anti- and orthodromically propagating APs in every preparation is now convincing.

The conclusions drawn from the synaptic modelling have been considerably strengthened by the new Figure 5. Here, the authors' model - including AP annihilation at a synaptic terminal - is used to predict the amplitude and direction of experimentally observed effects at the cerebellar basket cell-to-Purkinje cell synapse (Blot & Barbour 2014). One particular form of the model (RTM with tau=0.5ms and realistic non-excitability of the terminal) matches the experimental data extremely well. This is a much more convincing demonstration that the authors' model of ephaptic effects can quantitatively explain key features of experimental data pertaining to synaptic function. As such, the implications for the relevance of ephaptic coupling at different synaptic contacts may be widespread and important.

However, it appears that all of the models in the new Fig5 involve annihilating APs, yet only one fits the data closely. A key question, which should be addressed if at all possible, is what happens to the predictive power of the best-fitting model in Fig5 if the annihilation, and only the annihilation, is removed? In other words, can the authors show that it is specifically the ephaptic effects of AP annihilation, rather than other ephaptic effects of, say AP waveform/amplitude/propagation, that explain the synaptic effects measured in Blot & Barbour (2014)? This would appear to be a necessary demonstration to fully support the claims of the title.

Reviewer #1 (Public review):

Summary:

This paper contains what could be described as a "classic" approach towards evaluating a novel taste stimuli in an animal model, including standard behavioral tests (some with nerve transections), taste nerve physiology, and immunocytochemistry of the tongue. The stimulus being tested is ornithine, from a class of stimuli called "kokumi", which are stimuli that enhance other canonical tastes, increasing essentially the hedonic attributes of these other stimuli; the mechanism for ornithine detection is thought to be GPRC6A receptors expressed in taste cells. The authors showed evidence for this in an earlier paper with mice; this paper evaluates ornithine taste in a rat model.

Strengths:

The data show the effects of ornithine on taste: in two-bottle and briefer intake tests, adding ornithine results in a higher intake of most, but not all, stimuli tests. Bilateral nerve cuts or the addition of GPRC6A antagonists decrease this effect. Small effects of ornithine are shown in whole-nerve recordings.

Weaknesses:

The conclusion seems to be that the authors have found evidence for ornithine acting as a taste modifier through the GPRC6A receptor expressed on the anterior tongue. It is hard to separate their conclusions from the possibility that any effects are additive rather than modulatory. Animals did prefer ornithine to water when presented by itself. Additionally, the authors refer to evidence that ornithine is activating the T1R1-T1R3 amino acid taste receptor, possibly at higher concentrations than they use for most of the study, although this seems speculative. It is striking that the largest effects on taste are found with the other amino acid (umami) stimuli, leading to the possibility that these are largely synergistic effects taking place at the tas1r receptor heterodimer.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors examined a new and exciting taste enhancer (ornithine). They used a variety of experimental approaches in rats to document the impact of ornithine on taste preference and peripheral taste nerve recordings. Further, they provided evidence pointing to a potential receptor for ornithine.

Weaknesses:

The authors have not established that the rat is an appropriate model system for studying kokumi. Their measurements do not provide insight into any of the established effects of kokumi on human flavor perception. The small study on humans is difficult to compare to the rat study because the authors made completely different types of measurements. Thus, I think that the authors need to substantially scale back the scope of their interpretations. These weaknesses diminish the likely impact of the work on the field of flavor perception.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein-coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste.

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants, including inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl); citric acid and quinine hydrochloride. Robust effects of ornithine were observed in the cases of IMP, MSG, MPG, and sucrose, and little or no effects were observed in the cases of sodium chloride, citric acid, and quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. The inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify the role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally, they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

Weaknesses:

The researchers undertook what turned out to be largely confirmatory studies in rats with respect to their previously published work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9). They miss an opportunity to outline the experimental results from the study that favor their preferred interpretation that ornithine is a taste enhancer rather than a tastant.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). While the experimental results as a whole favor the authors' interpretation that C6A mediates the Ornithine responses, they do not make clear either the nature of the 'receptor identification problem' in the Introduction or the way in which they approached that problem in the Results and Discussion sections. It would be helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response. In addition, while they showed that C6A-positive cells were clearly distinct from gustducin-positive, and thus T1R-positive cells, they missed an opportunity to clearly differentiate C6A-expressing taste cells and CaSR-expressing taste cells in the rat tongue sections.

It would have been helpful to include a positive control kokumi substance in the two-bottle preference experiment (e.g., one of the known gamma-glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

The results demonstrate that enhancement of the chorda tympani nerve response to MSG occurs at substantially greater Ornithine concentrations (10 and 30 mM) than were required to observe differences in the two bottle preference experiments (1.0 mM; Figure 2). The discrepancy requires careful discussion and if necessary further experiments using the two-bottle preference format.

Reviewer #1 (Public review):

Summary:

This work proposes a new approach to analyse cell-count data from multiple brain regions. Collecting such data can be expensive and time-intensive, so, more often than not, the dimensionality of the data is larger than the number of samples. The authors argue that Bayesian methods are much better suited to correctly analyse such data compared to classical (frequentist) statistical methods. They define a hierarchical structure, partial pooling, in which each observation contributes to the population estimate to more accurately explain the variance in the data. They present two case studies in which their method proves more sensitive in identifying regions where there are significant differences between conditions, which otherwise would be hidden.

Strengths:

The model is presented clearly, and the advantages of the hierarchical structure are strongly justified. Two alternative ways are presented to account for the presence of zero counts. The first involves the use of a horseshoe prior, which is the more flexible option, while the second involves a modified Poisson likelihood, which is better suited to datasets with a large number of zero counts, perhaps due to experimental artifacts. The results show a clear advantage of the Bayesian method for both case studies.

The code is freely available, and it does not require a high-performance cluster to execute for smaller datasets. As Bayesian statistical methods become more accessible in various scientific fields, the whole scientific community will benefit from the transition away from p-values. Hierarchical Bayesian models are an especially useful tool that can be applied to many different experimental designs. However, while conceptually intuitive, their implementation can be difficult. The authors provide a good framework with room for improvement.

Weaknesses:

Alternative possibilities are discussed regarding the prior and likelihood of the model. Given that the second case study inspired the introduction of the zero-inflation likelihood, it is not clear how applicable the general methodology is to various datasets. If every unique dataset requires a tailored prior or likelihood to produce the best results, the methodology will not easily replace more traditional statistical analyses that can be applied in a straightforward manner. Furthermore, the differences between the results produced by the two Bayesian models in case study 2 are not discussed. In specific regions, the models provide conflicting results (e.g., regions MH, VPMpc, RCH, SCH, etc.), which are not addressed by the authors. A third case study would have provided further evidence for the generalizability of the methodology.

Reviewer #2 (Public review):

Summary:

This is a well-written methodology paper applying a Bayesian framework to the statistics of cell counts in brain slices. A sharpening of the bounds on measured quantities is demonstrated over existing frequentist methods and therefore the work is a contribution to the field.

Strengths:

As well as a mathematical description of the approach, the code used is provided in a linked repository.

Weaknesses:

A clearer link between the experimental data and model-structure terminology would be a benefit to the non-expert reader.

Reviewer #1 (Public review):

Summary:

Loh and colleagues investigate valence encoding in the mesolimbic dopamine system. Using an elegant approach, they show that sucrose, which normally evokes strong dopamine neuron activity and release in the nucleus accumbens, is made aversive via conditioned taste aversion, the same sucrose stimulus later evokes much less dopamine neuron activity and release. Thus, dopamine activity can dynamically track the changing valence of an unconditioned stimulus. These results are important for helping clarify valence and value related questions that are the matter of ongoing debate regarding dopamine functions in the field.

Strengths:

This is an elegant way to ask this question, the within subject's design and the continuity of the stimulus is a strong way to remove a lot of the common confounds that make it difficult to interpret valence-related questions. I think these are valuable studies that help tie up questions in the field while also setting up a number of interesting future directions. There are number of control experiments and tweaks to the design that help eliminate a number of competing hypotheses regarding the results. The data are clearly presented and contextualized.

Weaknesses for consideration:

The focus on one relatively understudied region of the rat striatum for dopamine recordings could potentially limit generalization of the findings. While this can be determined in future studies, the implications should be further discussed in the current manuscript.

Reviewer #2 (Public review):

Summary:

Koh et al. report an interesting manuscript studying dopamine binding in the lateral accumbens shell of rats across the course of conditioned taste aversion. The question being asked here is how does the dopamine system respond to aversion? The authors take advantage of unique properties of taste aversion learning (notably, within-subjects remapping of valence to the same physical stimulus) to address this.

They combine a well controlled behavioural design (including key, unpaired controls) with fibre photometry of dopamine binding via GrabDA and of dopamine neuron activity by gCaMP, careful analyses of behaviour (e.g., head movements; home cage ingestion), the authors show that, 1) conditioned taste aversion of sucrose suppresses the activity of VTA dopamine neurons and lateral shell dopamine binding to subsequent presentations of the sucrose tastant; 2) this pattern of activity was similar to the innately aversive tastant quinine; 3) dopamine responses were negatively correlated with behavioural (inferred taste reactivity) reactivity; and 4) dopamine responses tracked the contingency of between sucrose and illness because these responses recovered across extinction of the conditioned taste aversion.

Strengths:

There are important strengths here. The use of a well-controlled design, the measurement of both dopamine binding and VTA dopamine neuron activity, the inclusion of an extinction manipulation; and the thorough reporting of the data. I was not especially surprised by these results, but these data are a potentially important piece of the dopamine puzzle (e.g., as the authors note, salience-based argument struggles to explain these data).

Weaknesses for consideration:

(1) The focus here is on the lateral shell. This is a poorly investigated region in the context of the questions being asked here. Indeed, I suspect many readers might expect a focus on the medial shell. So, I think this focus is important. But, I think it does warrant greater attention in both the introduction and discussion. We do know from past work that there can be extensive compartmentalisation of dopamine responses to appetitive and aversive events and many of the inconsistent findings in the literature can be reconciled by careful examination of where dopamine is assessed. I do think readers would benefit from acknowledgement this - for example it is entirely reasonable to suppose that the findings here may be specific to the lateral shell.

(2) Relatedly, I think readers would benefit from an explicit rationale for studying the lateral shell as well as consideration of this in the discussion. We know that there are anatomical (PMID: 17574681), functional (PMID: 10357457), and cellular (PMID: 7906426) differences between the lateral shell and the rest of the ventral striatum. Critically, we know that profiles of dopamine binding during ingestive behaviours there can be highly dissimilar to the rest of ventral striatum (PMID: 32669355). I do think these points are worth considering.

(3) I found the data to be very thoughtfully analysed. But in places I was somewhat unsure:<br /> (a) Please indicate clearly in the text when photometry data show averages across trials versus when they show averages across animals.<br /> (b) I did struggle with the correlation analyses, for two reasons.<br /> (i) First, the key finding here is that the dopamine response to intraoral sucrose is suppressed by taste aversion. So, this will significantly restrict the range of dopamine transients, making interpretation of the correlations difficult.

(ii) Second, the authors report correlations by combining data across groups/conditions. I understand why the authors have done this, but it does risk obscuring differences between the groups. So, my question is: what happens to this trend when the correlations are computed separately for each group? I suspect other readers will share the same question. I think reporting these separate correlations would be very helpful for the field - regardless of the outcome.

(4) Figure 1A is not as helpful as it might be. I do think readers would expect a more precise reporting of GCaMP expression in TH+ and TH- neurons. I also note that many of the nuances in terms of compartmentalisation of dopamine signalling discussed above apply to ventral tegmental area dopamine neurons (e.g. medial v lateral) and this is worth acknowledging when interpreting.

Reviewer #3 (Public review):

Summary:

This study helps to clarify the mixed literature on dopamine responses to aversive stimuli. While it is well accepted that dopamine in the ventral striatum increases in response to various rewarding and appetitive stimuli, aversive stimuli have been shown to evoke phasic increases or decreasing depending on the exact aversive stimuli, behavioral paradigm, and/or dopamine recording method and location examined. Here the authors use a well-designed set of experiments to show differential responses to an appetitive primary reward (sucrose) that later becomes a conditioned aversive stimulus (sucrose previously paired with lithium chloride in a conditioned taste aversion paradigm). The results are interesting and add valuable data to the question of how the mesolimbic dopamine system encodes aversive stimuli, however, the conclusions are strongly stated given that the current data do not necessarily align with prior conflicting data in terms of recording location, and it is not clear exactly how to interpret the generally biphasic dopamine response to the CTA-sucrose which also evolves over exposures within a single session.

Strengths:

• The authors nicely demonstrate that their two aversive stimuli examined, quinine and sucrose following CTA, evoked aversive facial expressions and paw movements that differed from those following rewarding sucrose to support that the stimuli experienced by the rats differ in valence.

• Examined dopamine responses to the exact same sensory stimuli conditioned to have opposing valences, avoiding standard confounds of appetitive and aversive stimuli being sensed by different sensory modalities (i.e., sweet taste vs. electric shock).

• The authors examined multiple measurements of dopamine activity - cell body calcium (GCaMP6f) in midbrain and release in NAc (Grab-DA2h), which is useful as the prior mixed literature on aversive dopamine responses comes from a variety of recording methods.

• Correlations between sucrose preference and dopamine signals demonstrate behavioral relevance of the differential dopamine signals.

• The delayed testing experiment in Figure 7 nicely controls for the effect of time to demonstrate that the "rewarding" dopamine response to sucrose only recovers after multiple extinction sucrose exposures to extinguish the CTA.

Weaknesses for consideration:

• Regional differences in dopamine signaling to aversive stimuli are mentioned in the introduction and discussion. For instance, the idea that dopamine encodes salience is strongly argued against in the discussion, but the paper cited as arguing for that (Kutlu et al. 2021) is recording from the medial core in mice. Given other papers cited in the text about the regional differences in dopamine signaling in the NAc and from different populations of dopamine neurons in midbrain, it's important to mention this distinction wrt to salience signaling. Relatedly, the text says that the lateral NAc shell was targeted for accumbens recordings, but the histology figure looks like the majority of fibers were in the anterior lateral core of NAc. For the current paper to be a convincing last word on the issue, it would be extremely helpful to have similar recordings done in other parts of the NAc to do a more thorough comparison against other studies.

• Dopamine release in the NAc never dips below baseline for the conditioned sucrose. Is it possible to really consider this as a signal for valence per se, as opposed to it being a weaker response relative to the original sucrose response?

• Related to this, the main measure of the dopamine signal here, "mean z-score," obscures the temporal dynamics of the aversive dopamine response across a trial. This measure is used to claim that sucrose after CTA is "suppressing" dopamine neuron activity and release, which is true relative to the positive valence sucrose response. However, both GRAB-DA and cell-body GCaMP measurements show clear increases after onset of sucrose infusion before dipping back to baseline or slightly below in the average of all example experiments displayed. One could point to these data to argue either that aversive stimuli cause phasic increases in dopamine (due to the initial increase) or decreases (due to the delayed dip below baseline) depending on the measurement window. Some discussion of the dynamics of the response and how it relates to the prior literature would be useful.<br /> - Would this delayed below-baseline dip be visible with a shorter infusion time?<br /> - Does the max of the increase or the dip of the decrease better correlate with the behavioral measures of aversion (orofacial, paw movements) or sucrose preference than "mean z-score" measure used here?<br /> - The authors argue strongly in the discussion against the idea that dopamine is encoding "salience." Could this initial peak (also seen in the first few trials of quinine delivery, fig 1c color plot) be a "salience" response?

• Related to this, the color plots showing individual trials show a reduction in the increases to positive valence sucrose across conditioning day trials and a flip from infusion-onset increase to delayed increases across test day trials. This evolution across days makes it appear that the last few conditioning day trials would be impossible to discriminate from the first few test day trials in the CTA-paired. Presumably, from strength of CTA as a paradigm, the sucrose is already aversive to the animals at the first trial of test day. Why do the authors think the response evolves across this session?

• Given that most of the work is using a conditioned aversive stimulus, the comparison to a primary aversive tastant quinine is useful. However, the authors saw basically no dopamine response to a primary aversive tastant quinine (measured only with GRAB-DA) and saw less noticeable decreases following CTA for NAc recordings with GRAB-DA2h than with cell body GCaMP. Given that they are using the high-affinity version of the GRAB sensor, this calls into question whether this is a true difference in release vs. soma activity or issue of high affinity release sensor making decreases in dopamine levels more difficult to observe.

Reviewer #1 (Public review):

Summary:

The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.

Strengths:

(1) The insight into how cone and rod transcripts are mixed together at first is important and clarifies a long-standing notion in the field.

(2) The discovery of distinct active vs inactive mRNA isoforms for rod and cone determinants is crucial to understanding how cells make the decision to form one or the other cell type. This is only really possible with full-length scRNAseq analysis.

(3) New markers of subpopulations are also uncovered, such as CHRNA1 in rod/cone hybrids that seem to give rise to either rods or cones.

(4) Regulon analyses provide insight into key transcription factor programs linked to rod or cone fates.

(5) The gradient of lncRNAs in maturing cones is novel, and while the functional significance is unclear, it opens up a new line of questioning around photoreceptor maturation.

(6) The finding that SYK mRNA is naturally expressed in cone precursors is novel, as previously it was assumed that SYK expression required epigenetic rewiring in tumors.

Weaknesses:

(1) The writing is very difficult to follow. The nomenclature is confusing and there are contradictory statements that need to be clarified.

(2) The drug data is not enough to conclude that SYK inhibition is sufficient to prevent the division of RB1 null cone precursors. Drugs are never completely specific so validation is critical to make the conclusion drawn in the paper.

Reviewer #2 (Public review):

Summary:

The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.

Strengths:

This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, long-read RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.

Weaknesses:

The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on the developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.

Reviewer #3 (Public review):

Summary:

The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.

Strengths:

The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.

Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.

Weaknesses:

The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.

Reviewer #1 (Public review):

The authors aim to investigate the relationship between low estrogen levels, postmenopausal hypertension, and the potential role of the molecule L-AABA as a biomarker for hypertension. By employing metabolomic analysis and various statistical methods, the study seeks to understand how estrogen deficiency affects blood pressure and identify key metabolites involved in this process, with a particular focus on L-AABA.

Strengths:

The study addresses a relevant and understudied area: the role of estrogen and metabolites in postmenopausal hypertension. It presents a novel hypothesis that L-AABA may serve as a protective factor against hypertension, which could have significant clinical implications if proven.

Weaknesses:

The evidence linking L-AABA to hypertension is largely correlative, lacking experimental validation or mechanistic proof. Key limitations, such as the inadequacy of the ovariectomy model in replicating human menopause, are acknowledged but not addressed with alternative approaches. In summary, while the study offers an intriguing hypothesis, its conclusions are premature and require further experimental validation and human data to substantiate the claims.

Reviewer #2 (Public review):

Summary:

In this manuscript, Dr. Yao Li et al. documented the metabolomic profile of the aorta from OVX rats and that from OVX plus E2. These conditions mimic post-menopause hypertension and hormonal replacement therapy.

Strengths:

The authors state that this is probably the first study to examine the metabolic changes in the aorta of post-menopause hypertension.

Weaknesses:

There are several weaknesses, and a few of them are quite serious.

(1) The aorta is not a resistant artery and has little to do with hypertension. The authors should have used resistant arteries for this study. The expression of several adrenergic receptors and cholinergic receptors in the aorta and resistant arteries are different. It is unknown whether the aorta metabolomic profile has any relevance to BP and whether they are similar to that of the resistant arteries. I understand the logistics issue of obtaining enough tissues from resistant arteries. At least, once some leads are discovered in the aorta, the authors should validate it in resistant arteries. This should be feasible.

(2) The aorta and all the arteries have three layers. It is critically important to know whether the metabolic changes occur in the intima or in the media, while the adventitia probably has little to do with vasoconstriction and hypertension. If the authors want to use the aorta to conduct the preliminary study, they should completely remove the adventitia and then use samples with and without their endothelium stripped and then assess their metabolomic profiles. After the leads are obtained from this preliminary profiling, they should be validated in endothelium and smooth muscles of the resistant artery. The current experiments are not appropriately designed.

(3) The tail-cuff BP measurement is a technique of the last century. The current gold standard of BP measurement is by telemetry. The tail-cuff method is particularly problematic in this study because the 1-2 h restraining of the rats for more than 10 times BP measurement will cause significant stress in the animal, and their stress hormone secretion might cause biased metabolomic profiles in the OVX versus shames operated mice. The problem can be totally avoided by using telemetry.

(4) Although the L-AABA showed a high p-value (10^-4) of a decrease in the OVX rats, the fold change is small (2-3 folds). Such a small change should be validated using a different method to be convincing.

(5) The authors claim (or hypothesize) that the reduced AABA level in OVX can cause vascular remodeling. This can be easily validated by the histology of the OVX-resistant artery, and they should do that during the revision. The authors should also examine the M1 macrophage function from the OVX mice to validate their claimed link of AABA to M1.

(6) As mentioned above, the authors need to pinpoint the changes of AABA to target cells, i.e., endothelial cells, SMC, or M1, and then use in vitro or in vivo cell biology approaches to assess whether these cells in the OVX rat indeed have an abnormality in function and, indeed, such functional changes are responsible for the BP phenotype.

(7) The results of the current study can be condensed into 1 or 2 figures that can serve as a base or a starting point for a deeper scientific study.

Summary

The experimental design of this manuscript is inappropriate, and the methods are not up to the current standards. The whole study is descriptive and rudimentary. It lacks validation and mechanism. The data from this manuscript might be of some value and can serve as the first step for more investigation of the mechanism of post-menopause hypertension.

Reviewer #3 (Public review):

Summary:

The decrease in estrogen levels is strongly associated with postmenopausal hypertension. Dr. Yao Li and colleagues aimed to investigate the metabolomic mechanisms of underlying postmenopausal hypertension using OVX and OVX+E2 rat models. They successfully established a correlation between reduced estrogen levels and the development of hypertension in rats. They identified L-alpha-aminobutyric acid (AABA) as a potential marker for postmenopausal hypertension. The research explored the metabolic alterations in aortic tissues and proposed several potential mechanisms contributing to postmenopausal hypertension.

Strengths:

The group performed a comprehensive enrichment analysis and various statistical analyses of the metabolomics data.

Weaknesses:

(1) The manuscript is descriptive in nature, although they mentioned their primary objective is to explore the potential mechanisms linking low estrogen levels with postmenopausal hypertension. No mechanism insights have been interrogated in this study, which has been mentioned by the authors in the discussion. The connection between E2, AABA, and macrophage needs to be validated in endothelial cells, vascular smooth muscle cells, and other aortic tissue cells. Without such verification, the manuscript predominantly raises hypotheses only based on metabolomic data.

(2) The serum contains three forms of estrogen: Estradiol, Estrone, and Estriol. The authors used the Rat E2 ELISA kit. Ideally, all three forms of estrogen should be measured.

Reviewer #1 (Public review):

Summary:

This manuscript reports the investigation of PriC activity during DNA replication initiation in Escherichia coli. It is reported that PriC is necessary for the growth and control of DNA replication initiation under diverse conditions where helicase loading is perturbed at the chromosome origin oriC. A model is proposed where PriC loads helicase onto ssDNA at the open complex formed by DnaA at oriC. Reconstituted helicase loading assays in vitro support the model. The manuscript is well-written and has a logical narrative.

Major Questions/Comments:

An important observation here is that a ΔpriC mutant alone displays under-replication, suggesting that this helicase loading pathway is physiologically relevant. Has this PriC phenotype been reported previously? If not, would it be possible to confirm this result using an independent experimental approach (e.g. marker frequency analysis or fluorescent reporter-operator systems)?

Is PriA necessary for the observed PriC activity at oriC? Is there evidence that PriC functions independently of PriA in vivo?

Is PriC helicase loading activity in vivo at the origin direct (the genetic analysis leaves other possibilities tenable)? Could PriC enrichment at oriC be detected using chromatin immunoprecipitation?

Reviewer #2 (Public review):

This is a great paper. Yoshida et al. convincingly show that DnaA does not exclusively do loading of the replicative helicase at the E. coli oriC, but that PriC can also perform this function. Importantly, PriC seems to contribute to helicase loading even in wt cells albeit to a much lesser degree than DnaA. On the other hand, PriC takes a larger role in helicase loading during aberrant initiation, i.e. when the origin sequence is truncated or when the properties of initiation proteins are suboptimal. Here highlighted by mutations in dnaA or dnaC.

This is a major finding because it clearly demonstrates that the two roles of DnaA in the initiation process can be separated into initially forming an open complex at the DUE region by binding/nucleation onto DnaA-boxes and second by loading of the helicase. Whereas these two functions are normally assumed to be coupled, the present data clearly show that they can be separated and that PriC can perform at least part of the helicase loading provided that an area of duplex opening is formed by DnaA.

This puts into question the interpretation of a large body of previous work on mutagenesis of oriC and dnaA to find a minimal oriC/DnaA complex in many bacteria. In other words, mutants in which oriC is truncated/mutated may support the initiation of replication and cell viability only in the presence of PriC. Such mutants are capable of generating single-strand openings but may fail to load the helicase in the absence of PriC. Similarly, dnaA mutants may generate an aberrant complex on oriC that trigger strand opening but are incapable of loading DnaB unless PriC is present.

In the present work, the sequence of experiments presented is logical and the manuscript is clearly written and easy to follow. The very last part regarding PriC in cSDR replication does not add much to the story and may be omitted.

Reviewer #3 (Public review):

Summary:

At the abandoned replication fork, loading of DnaB helicase requires assistance from PriABC, repA, and other protein partners, but it does not require replication initiator protein, DnaA. In contrast, nucleotide-dependent DnaA binding at the specific functional elements is fundamental for helicase loading, leading to the DUE region's opening. However, the authors questioned in this study that in case of impeding replication at the bacterial chromosomal origins, oriC, a strategy similar to an abandoned replication fork for loading DnaB via bypassing the DnaA interaction step could be functional. The study by Yoshida et al. suggests that PriC could promote DnaB helicase loading on the chromosomal oriC ssDNA without interacting with the DnaA protein. However, the conclusions drawn from the primarily qualitative data presented in the study could be slightly overwhelming and need supportive evidence.

Strengths:

Understanding the mechanism of how DNA replication restarts via reloading the replisomes onto abandoned DNA replication forks is crucial. Notably, this knowledge becomes crucial to understanding how bacterial cells maintain DNA replication from a stalled replication fork when challenging or non-permissive conditions prevail. This critical study combines experiments to address a fundamental question of how DnaB helicase loading could occur when replication initiation impedes at the chromosomal origin, leading to replication restart.

Weaknesses:

The term colony formation used for a spotting assay could be misleading for apparent reasons. Both assess cell viability and growth; while colony formation is quantitative, spotting is qualitative. Particularly in this study, where differences appear minor but draw significant conclusions, the colony formation assays representing growth versus moderate or severe inhibition are a more precise measure of viability.

Figure 2<br /> The reduced number of two oriC copies per cell in the dnaA46priC-deficient strain was considered moderate inhibition. When combined with the data suggested by the dnaAC2priC-deficient strain containing two origins in cells with or without PriC (indicating no inhibition)-the conclusion was drawn that PriC rescue blocked replication via assisting DnaC-dependent DnaB loading step at oriC ssDNA.

The results provided by Saifi B, Ferat JL. PLoS One. 2012;7(3):e33613 suggests the idea that in an asynchronous DnaA46 ts culture, the rate by which dividing cells start accumulating arrested replication forks might differ (indicated by the two subpopulations, one with single oriC and the other with two oriC). DnaA46 protein has significantly reduced ATP binding at 42C, and growing the strain at 42C for 40-80 minutes before releasing them at 30 C for 5 minutes has the probability that the two subpopulations may have differences in the active ATP-DnaA. The above could be why only 50% of cells contain two oriC. Releasing cells for more time before adding rifampicin and cephalexin could increase the number of cells with two oriCs. In contrast, DnaC2 cells have inactive helicase loader at 42 C but intact DnaA-ATP population (WT-DnaA at 42 or 30 C should not differ in ATP-binding). Once released at 30 C, the reduced but active DnaC population could assist in loading DnaB to DnaA, engaged in normal replication initiation, and thus should appear with two oriC in a PriC-independent manner.

Broadly, the evidence provided by the authors may support the primary hypothesis. Still, it could call for an alternative hypothesis: PriC involvement in stabilizing the DnaA-DnaB complex (this possibility could exist here). To prove that the conclusions made from the set of experiments in Figures 2 and 3, which laid the foundations for supporting the primary hypothesis, require insights using on/off rates of DnaB loading onto DnaA and the stability of the complexes in the presence or absence of PriC, I have a few other reasons to consider the latter arguments.

Figure 3<br /> One should consider the fact that dnA46 is present in these cells. Overexpressing pdnaAFH could produce mixed multimers containing subunits of DnaA46 (reduced ATP binding) and DnaAFH (reduced DnaB binding). Both have intact DnaA-DnaA oligomerization ability. The cooperativity between the two functions by a subpopulation of two DnaA variants may compensate for the individual deficiencies, making a population of an active protein, which in the presence of PriC could lead to the promotion of the stable DnaA: DnaBC complexes, able to initiate replication. In the light of results presented in Hayashi et al. and J Biol Chem. 2020 Aug 7;295(32):11131-11143, where mutant DnaBL160A identified was shown to be impaired in DnaA binding but contained an active helicase function and still inhibited for growth; how one could explain the hypothesis presented in this manuscript. If PriC-assisted helicase loading could bypass DnaA interaction, then how growth inhibition in a strain carrying DnaBL160A should be described. However, seeing the results in light of the alternative possibility that PriC assists in stabilizing the DnaA: DnaBC complex is more compatible with the previously published data.

Figure 4<br /> Overexpression of DiaA could contribute to removing a higher number of DnaA populations. This could be more aggravated in the absence of PriC (DiaA could titrate out more DnaA)- the complex formed between DnaA: DnaBC is not stable, therefore reduced DUE opening and replication initiation leading to growth inhibition (Fig. 4A ∆priC-pNA135). Figure 7C: Again, in the absence of PriC, the reduced stability of DnaA: DnaBC complex leaves more DnaA to titrate out by DiaA, and thus less Form I*. However, adding PriC stabilizes the DnaA: DnaBC hetero-complexes, with reduced DnaA titration by DiaA, producing additional Form I*. Adding a panel with DnaBL160A that does not interact with DnaA but contains helicase activity could be helpful. Would the inclusion of PriC increase the ability of mutant helicase to produce additional Form I*?

Figure 5<br /> The interpretation is that colony formation of the Left-oriC ∆priC double mutant was markedly compromised at 37˚C (Figure 5B), and 256 the growth defects of the Left-oriC mutant at 25{degree sign}C and 30{degree sign}C were aggravated. However, prima facia, the relative differences in the growth of cells containing and lacking PriC are similar. Quantitative colony-forming data is required to claim these results. Otherwise, it is slightly confusing.

A minor suggestion is to include cells expressing PriC using plasmid DNA to show that adding PriC should reverse the growth defect of dnaA46 and dnaC2 strains at non-permissive temperatures. The same should be added at other appropriate places.

Reviewer #1 (Public review):

The authors present an important work where they model some of the complex interactions between immune cells, fibroblasts and cancer cells. The model takes into account the increased ECM production of cancer-associated fibroblasts. These fibres trap the cancer but also protect it from immune system cells. In this way, these fibroblasts' actions both promote and hinder cancer growth. By exploring different scenarios, the authors can model different cancer fates depending on the parameters regulating cancer cells, immune system cells and fibroblasts. In this way, the model explores non-trivial scenarios. An important weakness of this study is that, though it is inspired by NSCLC tumors, it is restricted to modelling circular tumor lesions and does not explore the formation of ramified tumors, as in NSCLC. In this way, is only a general model and it is not clear how it can be adapted to simulate more realistic tumor morphologies.

Reviewer #2 (Public review):

Summary:

The authors develop a computational model (and a simplified version thereof) to treat an extremely important issue regarding tumor growth. Specifically, it has been argued that fibroblasts have the ability to support tumor growth by creating physical conditions in the tumor microenvironment that prevent the relevant immune cells from entering into contact with, and ultimately killing, the cancer cells. This inhibition is referred to as immune exclusion. The computational approach follows standard procedures in the formulation of models for mixtures of different material species, adapted to the problem at hand by making a variety of assumptions as to the activity of different types of fibroblasts, namely "normal" versus "cancer-associated". The model itself is relatively complex, but the authors do a convincing job of analyzing possible behaviors and attempting to relate these to experimental observations.

Strengths:

As mentioned, the authors do an excellent job of analyzing the behavior of their model both in its full form (which includes spatial variation of the concentrations of the different cellular species) and in its simplified mean field form. The model itself is formulated based on established physical principles, although the extent to which some of these principles apply to active biological systems is not clear (see Weaknesses). The results of the model do offer some significant insights into the critical factors which determine how fibroblasts might affect tumor growth; these insights could lead to new experimental ways of unraveling these complex sets of issues and enhancing immunotherapy.

Weaknesses:

Models of the form being studied here rely on a large number of assumptions regarding cellular behavior. Some of these seemed questionable, based on what we have learned about active systems. The problem of T cell infiltration as well as the patterning of the extracellular matrix (ECM) by fibroblasts necessarily involve understanding cell motion and cell interactions due e.g. to cell signaling. Adopting an approach based purely on physical systems driven by free energies alone does not consider the special role that active processes can play, both in motility itself and in the type of self-organization that can occur due to these cell-cell interactions. This to me is the primary weakness of this paper.

A separate weakness concerns the assumption that fibroblasts affect T cell behavior primarily by just making a more dense ECM. There are a number of papers in the cancer literature (see, for some examples, Carstens, J., Correa de Sampaio, P., Yang, D. et al. Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun 8, 15095 (2017); Sun, Xiujie, Bogang Wu, Huai-Chin Chiang, Hui Deng, Xiaowen Zhang, Wei Xiong, Junquan Liu et al. "Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion." Nature 599, no. 7886 (2021): 673-678) that seem to indicate that density alone is not a sufficient indicator of T cell behavior. Instead, the organization of the ECM (for example, its anisotropy) could be playing a much more essential role than is given credit for here. This possibility is hinted at in the Discussion section but deserves much more emphasis.

Finally, the mixed version of the model is, from a general perspective, not very different from many other published models treating the ecology of the tumor microenvironment (for a survey, see Arabameri A, Asemani D, Hadjati J (2018), A structural methodology for modeling immune-tumor interactions including pro-and anti-tumor factors for clinical applications. Math Biosci 304:48-61). There are even papers in this literature that specifically investigate effects due to allowing cancer cells to instigate changes in other cells from being tumor-inhibiting to tumor-promoting. This feature occurs not only for fibroblasts but also for example for macrophages which can change their polarization from M1 to M2. There needed to be some more detailed comparison with this existing literature.

Reviewer #1 (Public review):

Summary:

In this manuscript by Obray et al., the authors show that adolescent ethanol exposure increases mechanical allodynia in adulthood. Additionally, they show that BLA-mediated inhibition of the prelimbic cortex is reduced, resulting in increased excitability in neurons that then project to vlPAG. This effect was mediated by BLA inputs onto PV interneurons. The primary finding of the manuscript is that these AIE-induced changes further impact acute pain processing in the BLA-PrL-vlPAG circuit, albeit behavioral readouts after inducing acute pain were not different between AIE rats and controls. These results provide novel insights into how AIE can have long-lasting effects on pain-related behaviors and neurophysiology. In this manuscript by Obray et al., the authors show that adolescent ethanol exposure increases mechanical allodynia in adulthood. Additionally, they show that BLA-mediated inhibition of the prelimbic cortex is reduced, resulting in increased excitability in neurons that then project to vlPAG. This effect was mediated by BLA inputs onto PV interneurons. The primary finding of the manuscript is that these AIE-induced changes further impact acute pain processing in the BLA-PrL-vlPAG circuit, albeit behavioral readouts after inducing acute pain were not different between AIE rats and controls. These results provide novel insights into how AIE can have long-lasting effects on pain-related behaviors and neurophysiology.

Strengths:

The manuscript was very well written and the experiments were rigorously conducted. The inclusion of both behavioral and neurophysiological circuit recordings was appropriate and compelling. The attention to SABV and appropriate controls was well thought out. The Discussion provided novel ideas for how to think about AIE and chronic pain and proposed several interesting mechanisms. This was a very well-executed set of experiments.

Weaknesses:

There is a mild disconnect between behavioral readout (reflexive pain) and neural circuits of interest (emotional). Considering that this circuit is likely engaged in the aversiveness of pain, it would have been interesting to see how carrageenan and/or AIE impacted non-reflexive pain measures. Perhaps this would reveal a potentiated or dysregulated phenotype that matches the neurophysiological changes reported. However, this critique does not take away from the value of the paper or its conclusions.

Reviewer #2 (Public review):

Summary:

The study by Obray et al. entitled "Adolescent alcohol exposure promotes mechanical allodynia and alters synaptic function at inputs from the basolateral amygdala to the prelimbic cortex" investigated how adolescent intermittent ethanol exposure (AIE) affects the BLA -> PL circuit, with an emphasis on PAG projecting PL neurons, and how AIE changes mechanical and thermal nociception. The authors found that AIE increased mechanical, but not thermal nociception, and an injection of an inflammatory agent did not produce changes in an ethanol-dependent manner. Physiologically, a variety of AIE-specific effects were found in PL neuron firing at BLA synapses, suggestive of AIE-induced alterations in neurotransmission at BLA-PVIN synapses.

Strengths:

This was a comprehensive examination of the effects of AIE on this neural circuit, with an in-depth dissection of the various neuronal connections within the PL.

Sex was included as a biological variable, yet there were little to no sex differences in AIE's effects, suggestive of similar adaptations in males and females.

Reviewer #3 (Public review):

Summary:

Obray et al. investigate the long-lasting effects of adolescent intermittent ethanol (AIE) in rats, a model of alcohol dependence, on a neural circuit within the prefrontal cortex. The studies are focused on inputs from the basolateral amygdala (BLA) onto parvalbumin (PV) interneurons and pyramidal cells that project to the periaqueductal gray (PAG). The authors found that AIE increased BLA excitatory drive onto parvalbumin interneurons and increased BLA feedforward inhibition onto PAG-projecting neurons.

Strengths:

Fully powered cohorts of male and female rodents are used, and the design incorporates both AIE and an acute pain model. The authors used several electrophysiological techniques to assess synaptic strength and excitability from a few complimentary angles. The design and statistical analysis are sound, and the strength of evidence supporting synaptic changes following AIE results is solid.

Weaknesses:

(1) There is incomplete evidence supporting some of the conclusions drawn in this manuscript. The authors claim that the changes in feedforward inhibition onto pyramidal cells are due to the changes in parvalbumin interneurons, but evidence is not provided to support that idea. PV cells do not spontaneously fire action potentials spontaneously in slices (nor do they receive high levels of BLA activity while at rest in slices). It is possible that spontaneous GABA release from PV cells is increased after AIE but the authors did not report sIPSC frequency. Second, the authors did not determine that PV cells mediate the feedforward BLA op-IPSCs and changes following AIE (this would require manipulation to reduce/block PV-IN activity). This limitation in results and interpretation is important because prior work shows BLA-PFC feedforward IPSCs can be driven by somatostatin cells. Cholecystokinin cells are also abundant basket cells in PFC and have been recently shown to mediate feedforward inhibition from the thalamus and ventral hippocampus, so it's also possible that CCK cells are involved in the effects observed here.

(2) The authors conclude that the changes in this circuit likely mediate long-lasting hyperalgesia, but this is not addressed experimentally. In some ways, the focused nature of the study is a benefit in this regard, as there is extensive prior literature linking this circuit with pain behaviors in alternative models (e.g., SNI), but it should be noted that these studies have not assessed hyperalgesia stemming from prior alcohol exposure. While the current studies do not include a causative behavioral manipulation, the strength of the association between BLA-PL-PAG function and hyperalgesia could be bolstered by current data if there were relationships detected between electrophysiological properties and hyperalgesia. Have the authors assessed this? In addition, this study is limited by not addressing the specificity of synaptic adaptations to the BLA-PL-PAG circuit. For instance, PL neurons send reciprocal projections to BLA and send direct projections to the locus coeruleus (which the authors note is an important downstream node of the PAG for regulating pain).

(3) I have some concerns about methodology. First, 5-ms is a long light pulse for optogenetics and might induce action-potential independent release. Does TTX alone block op-EPSCs under these conditions? Second, PV cells express a high degree of calcium-permeable AMPA receptors, which display inward rectification at positive holding potentials due to blockade from intracellular polyamines. Typically, this is controlled/promoted by including spermine in the internal solution, but I do not believe the authors did that. Nonetheless, the relatively low A/N ratios for this cell type suggest that CP-AMPA receptors were not sampled with the +40/+40 design of this experiment, raising concerns that the majority of AMPA receptors in these cells were not sampled during this experiment. Finally, it should be noted that asEPSC frequency can also reflect changes in a number of functional/detectable synapses. This measurement is also fairly susceptible to differences in inter-animal differences in ChR2 expression. There are other techniques for assessing presynaptic release probability (e.g., PPR, MK-801 sensitivity) that would improve the interpretation of these studies if that is intended to be a point of emphasis.

(4) In a few places in the manuscript, results following voluntary drinking experiments (especially Salling et al. and Sicher et al.) are discussed without clear distinction from prior work in vapor models of dependence

(5) Discussion (lines 416-420). The authors describe some differing results with the literature and mention that the maximum current injection might be a factor. To me, this does not seem like the most important factor and potentially undercuts the relevance of the findings. Are the cells undergoing a depolarization block? Did the authors observe any changes in the rheobase or AP threshold? On the other hand, a more likely difference between this and previous work is that the proportion of PAG-projecting cells is relatively low, so previous work in L5 likely sampled many types of pyramidal cells that project to other areas. This is a key example where additional studies by the current group assessing a distinct or parallel set of pyramidal cells would aid in the interpretation of these results and help to place them within the existing literature. Along these lines, PAG-projecting neurons are Type A cells with significant hyperpolarization sag. Previous studies showed that adolescent binge drinking stunts the development of HCN channel function and ensuing hyperpolarization sag. Have the authors observed this in PAG-projecting cells? Another interesting membrane property worth exploring with the existing data set is the afterhyperpolarization / SK channel function.

Reviewer #1 (Public Review):

Otero-Coronel et al. address an important question for neuroscience - how does a premotor neuron capable of directly controlling behavior integrate multiple sources of sensory inputs to inform action selection? For this, they focused on the teleost Mauthner cell, long known to be at the core of a fast escape circuit. What is particularly interesting in this work is the naturalistic approach they took. Classically, the M-cell was characterized, both behaviorally and physiologically, using an unimodal sensory space. Here the authors make the effort (substantial!) to study the physiology of the M-cell taking into account both the visual and auditory inputs. They performed well-informed electrophysiological approaches to decipher how the M-cell integrates the information of two sensory modalities depending on the strength and temporal relation between them.

The empirical results are convincing and well-supported. The manuscript is well-written and organized. The experimental approaches and the selection of stimulus parameters are clear and informed by the bibliography. The major finding is that multisensory integration increases the certainty of environmental information in an inherently noisy environment.

Reviewer #2 (Public Review):

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well-written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength that MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Reviewer #1 (Public Review):

This work addresses how to quantify functional compensation throughout the aging process and identifies brain regions that engage in compensatory mechanisms during the Cattell task, a measure of fluid cognition. The authors find that regions of the frontal cortex and cuneus showed unique effects of both age and performance. Interestingly, these two regions demonstrated differential activation patterns taking into account both age and performance. Specifically, the researchers found that the relationship between performance and activation in the cuneal ROI was strongest in older adults, however, this was not found in younger adults. These findings suggest that specifically within the cuneus, greater activation is needed by older adults to maintain performance, suggestive of functional compensation.

The conclusions derived from the study are well supported by the data. The authors validated the use of the in-scanner Cattell task by demonstrating high reliability in the same sample with the standard out-of-scanner version. Some strengths of the study include the large sample size and wide age range of participants. The authors use a stringent Bayes factor of 20 to assess the strength of evidence. The authors used a whole-brain approach to define regions of interest (ROIs) based on activation patterns that were jointly related to age and performance. Overall, the methods are technically sound and support the authors' conclusions.

Comment from Reviewing Editor: In the revised manuscript, the authors have addressed the weaknesses previously identified by reviewer 1.

Reviewer #2 (Public Review):

This work by Knights et al., makes use of the Cam-CAN dataset to investigate functional compensation during a fluid processing task in older adults, in a fairly large sample of approximately 200 healthy adults ranging from 19 to 87. Using univariate methods, the authors identify two brain regions in which activity increases as a function of both age and performance and conduct further investigations to assess whether the activity of these regions provides information regarding task difficulty. The authors conclude that the cuneal cortex - a region of the brain previously implicated in visual attention - shows evidence of compensation in older adults.

The conclusions of the paper are well supported by the data, and the authors use appropriate statistical analyses. The use of multivariate methods over the last 20 years has demonstrated many effects that would have been missed using more traditional univariate analysis techniques. The data set is also of an appropriate size, and as the authors note, fluid processing is an extremely important domain in the field of cognition in aging, due to its steep decline over aging.

Comment from Reviewing Editor: It would have been nice to see an analysis of a more crystallised intelligence task included too, as a contrast since this is an area that does not demonstrate such a decline (and perhaps continues to improve over aging). This comment does not take away the important contributions of the manuscript.

Reviewer #3 (Public Review):

This neuroimaging study investigated how brain activity related to visual pattern-based reasoning changes over the adult lifespan, addressing the topic of functional compensation in older age. To this end, the authors employed a version of the Cattell task, which probes visual pattern recognition for identifying commonalities and differences within sets of abstract objects in order to infer the odd object among a given set. Using a state-of-the-art univariate analysis approach on fMRI data from a large lifespan sample, the authors identified brain regions in which the activation contrast between hard and easy Cattell task conditions was modulated by both age and performance. Regions identified comprised prefrontal areas and bilateral cuneus. Applying a multivariate decoding approach to activity in these regions, the authors went on to show that only in older adults, the cuneus, but not the prefrontal regions, carried information about the task condition (hard vs. easy) beyond that already provided by activity patterns of voxels that showed a univariate main effect of task difficulty. This was taken as compelling evidence for task-specific compensatory activity in the cuneus in advanced age.

The study is well-motivated and well-written. The authors used appropriate, rigorous methods that allowed them to control for a range of possible confounds or alternative explanations. Laudable aspects include the large sample with a wide and even age distribution, the validation of the in-scanner task performance against previous results obtained with a more standard version outside the scanner, and the control for vascular age-related differences in hemodynamic activity via a BOLD signal amplitude measure obtained from a separate resting-state fMRI scan. Overall, the conclusions are well-supported by the data.

Comment from Reviewing Editor: The revised manuscript has addressed the points raised during the review of the original submission.

Reviewer #1 (Public review):

Summary:

The authors sought to identify unknown factors involved in the repair of uracil in DNA through a CRISPR knockout screen.

Strengths:

The screen identified both known and unknown proteins involved in DNA repair resulting from uracil or modified uracil base incorporation into DNA. The conclusion is that the protein activity of METTL3, which converts A nucleotides to 6mA nucleotides, plays a role in the DNA damage/repair response. The importance of METTL3 in DNA repair, and its colocalization with a known DNA repair enzyme, UNG2, is well characterized.

Weaknesses:

This reviewer identified no major weaknesses in this study. The manuscript could be improved by tightening the text throughout, and more accurate and consistent word choice around the origin of U and 6mA in DNA. The dUTP nucleotide is misincorporated into DNA, and 6mA is formed by methylation of the A base present in DNA. Using words like 6mA "deposition in DNA" seems to imply it results from incorporation of a methylated dATP nucleotide during DNA synthesis.

Reviewer #2 (Public review):

Summary:

In this work, the authors performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA, and identified unexpected factors, such as the RNA m6A methyltransferase METTL3, as required to overcome floxuridine-driven cytotoxicity in mammalian cells. Interestingly, the observed N6-methyladenosine was embedded in DNA, which has been reported as DNA 6mA in mammalian genomes and is currently confirmed with mass spectrometry in this model. Therefore, this work consolidated the functional role of mammalian genomic DNA 6mA, and supported with solid evidence to uncover the METTL3-6mA-UNG2 axis in response to DNA base damage.

Strengths:

In this work, the authors took an unbiased, genome-wide CRISPR approach to identify novel factors involved in uracil repair with potential clinical interest.

The authors designed elegant experiments to confirm the METTL3 works through genomic DNA, adding the methylation into DNA (6mA) but not the RNA (m6A), in this base damage repair context. The authors employ different enzymes, such as RNase A, RNase H, DNase, and liquid chromatography coupled to tandem mass spectrometry to validate that METTL3 deposits 6mA in DNA in response to agents that increase genomic uracil.

They also have the Mettl3-KO and the METTL3 inhibition results to support their conclusion.

Weaknesses:

Although this study demonstrates that METTL3-dependent 6mA deposition in DNA is functionally relevant to DNA damage repair in mammalian cells, there are still several concerns and issues that need to be improved to strengthen this research.

First, in the whole paper, the authors never claim or mention the mammalian cell lines contamination testing result, which is the fundamental assay that has to be done for the mammalian cell lines DNA 6mA study.

Second, in the whole work, the authors have not supplied any genomic sequencing data to support their conclusions. Although the sequencing of DNA 6mA in mammalian models is challenging, recent breakthroughs in sequencing techniques, such as DR-Seq or NT/NAME-seq, have lowered the bar and improved a lot in the 6mA sequencing assay. Therefore, the authors should consider employing the sequencing methods to further confirm the functional role of 6mA in base repair.

Third, the authors used the METTL3 inhibitor and Mettl3-KO to validate the METTL3-6mA-UNG2 functional roles. However, the catalytic mutant and rescue of Mettl3 may be the further experiments to confirm the conclusion.

Reviewer #3 (Public review):

Summary:

The authors are showing evidence that they claim establishes the controversial epigenetic mark, DNA 6mA, as promoting genome stability.

Strengths:

The identification of a poorly understood protein, METTL3, and its subsequent characterization in DDR is of high quality and interesting.

Weaknesses:

(1) The very presence of 6mA (DNA) in mammalian DNA is still highly controversial and numerous studies have been conclusively shown to have reported the presence of 6mA due to technical artifacts and bacterial contamination. Thus, to my knowledge there is no clear evidence for 6mA as an epigenetic mark in mammals, and consequently, no evidence of writers and readers of 6mA. None of this is mentioned in the introduction. Much of the introduction can be reduced, but a paragraph clearly stating the controversy and lack of evidence for 6mA in mammals needs to be added, otherwise, the reader is given an entirely distorted view of the field.

These concerns must also be clearly in the limitations section and even in the results section which fails to nuance the authors' findings.

(2) What is the motivation for using HT-29 cells? Moreover, the materials and methods do not state how the authors controlled for bacterial contamination, which has been the most common cause of erroneous 6mA signals to date. Did the authors routinely check for mycoplasma?

(3) The single cell imaging of 6mA in various cells is nice. The results are confirmed by mass spec as an orthogonal approach. Another orthogonal and quantitative approach to assessing 6mA levels would be PacBio. Similarly, it is unclear why the authors have not performed dot-blots of 6mA for genomic DNA from the given cell lines.

(4) The results of Figure 3 need further investigation and validation. If the results are correct the authors are suggesting that the majority of 6mA in their cell lines is present in the DNA, and not the RNA, which is completely contrary to every other study of 6mA in mammalian cells that I am aware of. This could suggest that the antibody is not, in fact, binding to 6mA, but to unmodified adenine, which would explain why the signal disappears after DNAse treatment. Indeed, binding of 6mA to unmethylated DNA is a commonly known problem with most 6mA antibodies and is well described elsewhere.

(5) Given the lack of orthologous validation of the observed DNA 6mA and the lack of evidence supporting the presence of 6mA in mammalian DNA and consequently any functional role for 6mA in mammalian biology, the manuscript's conclusions need to be toned down significantly, and the inherent difficulty in assessing 6mA accurately in mammals acknowledged throughout.

Reviewer #1 (Public review):

Summary:

In this study, the authors describe the construction of an extremely large-scale anatomical model of juvenile rat somatosensory cortex (excluding the barrel region), which extends earlier iterations of these models by expanding across multiple interconnected cortical areas. The models are constructed in a way to maintain biological detail from a granular scale - for example, individual cell morphologies are maintained, and synaptic connectivity is founded on anatomical contacts. The authors use this model to investigate a variety of properties, from cell-type specific targeting (where the model results are compared to findings from recent large-scale electron microscopy studies) to network metrics. The model is also intended to serve as a platform and resource for the community by being a foundation for simulations of neuronal circuit activity and for additional anatomical studies that rely on the detailed knowledge of cellular identity and connectivity.

Strengths:

As the authors point out, the combination of scale and granularity of their model are what make this study valuable and unique. The comparisons with recent electron microscopy findings are some of the most compelling results presented in the study, showing that certain connectivity patterns can arise directly from the anatomical configuration, while other discrepancies highlight where more selective targeting rules (perhaps based on molecular cues) are likely employed. They also describe intriguing effects of cortical thickness and curvature on circuit connectivity and characterize the magnitude of those effects on different cortical layers.

The detailed construction of the model is drawn on wide range of data sources (cellular and synaptic density measures, neuronal morphologies, cellular composition measures, brain geometry, etc.) that are integrated together; other data sources are used for comparison and validation. This consolidation and comparison also represents a valuable contribution to the overall understanding of the modeled system.

Weaknesses:

The scale of the model, which is a primary strength, also can carry some drawbacks. In order to integrate all the diverse data sources together, many specific decisions must be made about, for example, translating findings from different species or regions to the modeled system, or deciding which aspects of the system can be assumed to be same and which should vary. All these decisions will have effects on the predicted results from the model, which could limit the types of conclusions that can be made (both by the others and by others in the community who may wish to use the model for their own work). However, the public release of the models and most of the associated tools does provide others a somewhat easier path to modify and evaluate this iteration of the model for their own studies.

Overall, the model presented in this study represents an enormous amount of work and stands as the basis for other work by the same group as well as a unique resource for the community, even while acknowledging that it may be somewhat unwieldy for the community to employ due to the weight of its manifold specific construction decisions, size, and complexity.

Reviewer #2 (Public review):

Summary:

The authors build a colossal anatomical model of juvenile rat non-barrel primary somatosensory cortex, including inputs from the thalamus. This enhances past models by incorporating information on the shape of the cortex and estimated densities of various types of excitatory and inhibitory neuron across layers. This is intended to enable analysis of the micro- and mesoscopic organisation of cortical connectivity and to be a base anatomical model for large-scale simulations of physiology.

Strengths:

• The authors incorporate many diverse data sources on morphology and connectivity.<br /> • This paper takes on the challenging task of linking micro- and meso-scale connectivity<br /> • By building in the shape of the cortex, the authors were able to link cortical geometry to connectivity. In particular they make an unexpected prediction that cortical conicality affects the modularity of local connectivity, which should be testable.<br /> • The author's analysis of the model led to the interesting prediction that layer 5 neurons' connect local modules, which may be testable in the future, and provide a basis to link from detailed anatomy to functional computations.<br /> • The visualisation of the anatomy in various forms is excellent<br /> • The model is openly shared

Weaknesses:

• There is no effort to determine how specific or generalisable the findings here are to other parts of cortex.<br /> • Although there is a link to physiological modelling in another paper, there is no clear pathway to go from this type of model to understanding how the specific function of the modelled areas may emerge here (and not in other cortical areas).<br /> • Some of the decisions seem a little ad-hoc, and the means to assess those decisions is not always easily available to the reader<br /> • The shape of the juvenile cortex - a key novelty of this work - was based on merely a scalar reduction of the adult cortex. This is very surprising, and surely an oversimplification. Huge efforts have gone into modelling the complex nonlinear development of cortex, by teams including the developing Human Connectome Project. For such a fundamental aspect of this work, why isn't it possible to reconstruct the shape of this relatively small part of juvenile rat cortex?<br /> • The same relative laminar depths are used for all subregions. This will have a large impact on the model. However, relative laminar depths can change drastically across the cortex (see e.g. many papers by Palomero-Gallagher, Zilles and colleagues). The authors should incorporate the real laminar depths, or, failing that, show evidence to show that the laminar depth differences across the subregions included in the model are negligible.<br /> • The authors perform an affine mapping between mouse and rat cortex. This is again surprising. In human imaging, affine mappings are insufficient to map between two individual brains of the same species, and nonlinear transformations are instead used. That an affine transformation should be considered sufficient to map between two different species is then very surprising. For some models, this may be fine, but there is a supposed emphasis here on biological precision in terms of anatomical location.<br /> o Live nature of the model. This is such a colossal model, and effort, that I worry that it may be quite difficult to update in light of new data. For example, how much person and compute time would it take to update the model to account for different layer sizes across subregions? Or to more precisely account for the shape of juvenile rat cortex?

Reviewer #3 (Public review):

This manuscript reports a detailed model of the rat non-barrel somatosensory cortex, consisting of 4.2 million morphologically and biophysically detailed neuron models, arranged in space and connected according to highly sophisticated rules informed by diverse experimental data. Due to its breadth and sophistication the model will undoubtedly be of interest to the community, and the reporting of anatomical details of modeling in this paper is important for understanding all the assumptions and procedures involved in constructing the model. While a useful contribution to this field, the model and the manuscript could be improved by employing data more directly and comparing simple features of the model's connectivity - in particular, connection probabilities - with relevant experimental data.

The manuscript is overall well-written, but contains a substantial number of confusing or unclear statements, and some important information is not provided.

Comments on revisions:

The authors mostly addressed all my points and improved the paper substantially. I do not have further extensive comments except one general point below.

Regarding section 2.3 and metrics of connectivity like pairwise connection probabilities, it is great that the authors rewrote that section and added comparisons with experimental data in Figs. 4 and S9. Unfortunately, what one finds when direct comparisons are made is that the modeled pairwise connectivity is quite different from the data. Fig. S9 shows that the model's results do not agree with data in about half of the cases (purple and red arrows). Similarly large discrepancies can be seen for some other metrics, like in Fig. S10B and S10C1,C2. (And similar concerns apply to thalamocortical connections in section 2.5, where it looks like little to no data are available to verify the pairwise connectivity between the thalamic and cortical neurons via a direct comparison.)

This is concerning since this model forms the basis for multiple other studies of cortical dynamics and function by the same group and potentially others in the community, with multiple papers relying on it, whereas basic properties of connectivity are apparently not captured well.

On the other hand, this is also a "glass half full" situation, showing that the sophisticated algorithms for establishing connections, developed by the authors, are working well in at least half of the connection types explored. It is therefore imperative that the authors continue refining these algorithms to capture the remaining half in future iterations and producing improved models that the community can better rely on.

Please also note that Fig. S11 does not have a caption.

Reviewer #1 (Public review):

Summary:

The manuscript "Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation" by Cho and colleagues investigates de novo tight junction formation during the differentiation of immortalized human HaCaT keratinocytes to granular-like cells, as well as during epithelial remodeling that occurs upon the apoptotic of individual cells in confluent monolayers of the representative epithelial cell line EpH4. The authors demonstrate the involvement of Rho-ROCK with well-conducted experiments and convincing images. Moreover, they unravel the underlying molecular mechanism, with Rho-ROCK activity activating the transmembrane serine protease Matriptase, which in turn leads to the cleavage of EpCAM and TROP2, respectively, releasing Claudins from EpCAM/TROP2/Claudin complexes at the cell membrane to become available for polymerization and de novo tight junction formation. These functional studies in the two different cell culture systems are complemented by localization studies of the according proteins in the stratified mouse epidermis in vivo.

In total, these are new and very intriguing and interesting findings that add important new insights into the molecular mechanisms of tight junction formation, identifying Matriptase as the "missing link" in the cascade of formerly described regulators. The involvement of TROP2/EpCAM/Claudin has been reported recently (Szabo et al., Biol. Open 2022; Bugge lab), and Matriptase had been formerly described to be required for in tight junction formation as well, again from the Bugge lab. Yet, the functional correlation/epistasis between them, and their relation to Rho signaling, had not been known thus far.

However, experiments addressing the role of Matriptase require a little more work.

Strengths:

Convincing functional studies in two different cell culture systems, complemented by supporting protein localization studies in vivo. The manuscript is clearly written and most data are convincingly demonstrated, with beautiful images and movies.

Weaknesses:

The central finding that Rho signaling leads to increased Matriptase activity needs to be more rigorously demonstrated (e.g. western blot specifically detecting the activated version or distinguishing between the full-length/inactive and processed/active version).

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate how epithelia maintain intercellular barrier function despite and during cellular rearrangements upon e.g. apoptotic extrusion in simple epithelia or regenerative turnover in stratified epithelia like this epidermis. A fundamental question in epithelial biology. Previous literature has shown that Rho-mediated local regulation of actomyosin is essential not only for cellular rearrangement itself but also for directly controlling tight junction barrier function. The molecular mechanics however remained unclear. Here the authors use extensive fluorescent imaging of fixed and live cells together with genetic and drug-mediated interference to show that Rho activation is required and sufficient to form novo tight junctional strands at intercellular contacts in epidermal keratinocytes (HaCat) and mammary epithelial cells. After having confirmed previous literature they then show that Rho activation activates the transmembrane protease Matriptase which cleaves EpCAM and TROP2, two claudin-binding transmembrane proteins, to release claudins and enable claudin strand formation and therefore tight junction barrier function.

Strengths:

The presented mechanism is shown to be relevant for epithelial barriers being conserved in simple and stratifying epithelial cells and mainly differs due to tissue-specific expression of EpCAM and TROP2. The authors present careful state-of-the-art imaging and logical experiments that convincingly support the statements and conclusion. The manuscript is well-written and easy to follow.

Weaknesses:

Whereas the in vitro evidence of the presented mechanism is strongly supported by the data, the in vivo confirmation is mostly based on the predicted distribution of TROP2. Whereas the causality of Rho-mediated Matriptase activation has been nicely demonstrated it remains unclear how Rho activates Matriptase.

Reviewer #1 (Public review):

Summary:

The ingenious design in this study achieved the observation of 3D cell spheroids from an additional lateral view and gained more comprehensive information than the traditional one angle of imaging, which extensively extended the methods to investigate cell behaviors in the growth or migration of tumor organoids in the present study. I believe that this study opens an avenue and provides an opportunity to characterize the spheroid formation dynamics from different angles, in particular side-view with high resolution, in other organoids study in the future.

Reviewer #2 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Strengths:

The system allows the in situ observation of the 3D structures along the 3 axes (x,y and z) without disrupting the integrity of the spheroids; in a time-lapse manner it is possible to follow the formation of the 3D structure and the spheroids fusion from multiple angles, allowing a better understanding of the cell aggregation/growth and kinetic of the cells.

Interestingly the system allows the analysis of cell migration/ escape from the 3D structure analysing not only the morphological changes in the periphery of the spheroids but also from the inner region demonstrating that the proliferating cells in the periphery of the structure are more involved in the migration and dissemination process. The application of the system in the study of the effects of doxorubicin and NK cells would give new insights in the description of the response of tumor 3D structure to killing agents.

Reviewer #1 (Public review):

Epigenetic regulation complex (PRC2) is essential for neural crest specification, and its misregulation has been shown to cause severe craniofacial defects. This study shows that Eed, a core PRC2 component, is critical for craniofacial osteoblast differentiation and mesenchymal proliferation after neural crest induction. Using mouse genetics and single-cell RNA sequencing, the researcher found that conditional knockout of Eed leads to significant craniofacial hypoplasia, impaired osteogenesis, and reduced proliferation of mesenchymal cells in post-migratory neural crest populations.

Overall, the study is superficial and descriptive. No in-depth mechanism was analyzed and the phenotype analysis is not comprehensive.

Reviewer #2 (Public review):

Summary:

The role of PRC2 in post-neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.

Strengths:

(1) Elegant genetic system to ablate EED post neural crest induction.

(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type-specific gene expression changes in developmental systems.

Weaknesses:

(1) Although this study is well designed and contains state-of-the-art single-cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.

(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novel in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.

(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.

(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Figure 4e).

Reviewer #1 (Public review):

One of the roadblocks in PfEMP1 research has been the challenges in manipulating var genes to incorporate markers to allow the transport of this protein to be tracked and to investigate the interactions taking place within the infected erythrocyte. In addition, the ability of Plasmodium falciparum to switch to different PfEMP1 variants during in vitro culture has complicated studies due to parasite populations drifting from the original (manipulated) var gene expression. Cronshagen et al have provided a useful system with which they demonstrate the ability to integrate a selectable drug marker into several different var genes that allows the PfEMP1 variant expression to be 'fixed'. This on its own represents a useful addition to the molecular toolbox and the range of var genes that have been modified suggests that the system will have broad application. As well as incorporating a selectable marker, the authors have also used selective linked integration (SLI) to introduce markers to track the transport of PfEMP1, investigate the route of transport, and probe interactions with PfEMP1 proteins in the infected host cell.

What I particularly like about this paper is that the authors have not only put together what appears to be a largely robust system for further functional studies, but they have used it to produce a range of interesting findings including:

- Co-activation of rif and var genes when in a head-to-head orientation.

- The reduced control of expression of var genes in the 3D7-MEED parasite line.

- More support for the PTEX transport route for PfEMP1.

- Identification of new proteins involved in PfEMP1 interactions in the infected erythrocyte, including some required for cytoadherence.

In most cases the experimental evidence is straightforward, and the data support the conclusions strongly. The authors have been very careful in the depth of their investigation, and where unexpected results have been obtained, they have looked carefully at why these have occurred.

(1) In terms of incorporating a drug marker to drive mono-variant expression, the authors show that they can manipulate a range of var genes in two parasite lines (3D7 and IT4), producing around 90% expression of the targeted PfEMP1. Removal of drug selection produces the expected 'drift' in variant types being expressed. The exceptions to this are the 3D7-MEED line, which looks to be an interesting starting point to understand why this variant appears to have impaired mutually exclusive var gene expression and the EPCR-binding IT4var19 line. This latter finding was unexpected and the modified construct required several rounds of panning to produce parasites expressing the targeted PfEMP1 and bind to EPCR. The authors identified a PTP3 deficiency as the cause of the lack of PfEMP1 expression, which is an interesting finding in itself but potentially worrying for future studies. What was not clear was whether the selected IT4var19 line retained specific PfEMP1 expression once receptor panning was removed.

(2) The transport studies using the mDHFR constructs were quite complicated to understand but were explained very clearly in the text with good logical reasoning.

(3) By introducing a second SLI system, the authors have been able to alter other genes thought to be involved in PfEMP1 biology, particularly transport. An example of this is the inactivation of PTP1, which causes a loss of binding to CD36 and ICAM-1. It would have been helpful to have more insight into the interpretation of the IFAs as the anti-SBP1 staining in Figure 5D (PTP-TGD) looks similar to that shown in Figure 1C, which has PTP intact. The anti-EXP2 results are clearly different.

(4) It is good to see the validation of PfEMP1 expression includes binding to several relevant receptors. The data presented use CHO-GFP as a negative control, which is relevant, but it would have been good to also see the use of receptor mAbs to indicate specific adhesion patterns. The CHO system if fine for expression validation studies, but due to the high levels of receptor expression on these cells, moving to the use of microvascular endothelial cells would be advisable. This may explain the unexpected ICAM-1 binding seen with the panned IT4var19 line.

(5) The proxiome work is very interesting and has identified new leads for proteins interacting with PfEMP1, as well as suggesting that KAHRP is not one of these. The reduced expression seen with BirA* in position 3 is a little concerning but there appears to be sufficient expression to allow interactions to be identified with this construct. The quantitative impact of reduced expression for proxiome experiments will clearly require further work to define it.

(6) The reduced receptor binding results from the TryThrA and EMPIC3 knockouts were very interesting, particularly as both still display PfEMP1 on the surface of the infected erythrocyte. While care needs to be taken in cross-referencing adhesion work in P. berghei and whether the machinery truly is functionally orthologous, it is a fair point to make in the discussion. The suggestion that interacting proteins may influence the "correct presentation of PfEMP1" is intriguing and I look forward to further work on this.<br /> Overall, the authors have produced a useful and reasonably robust system to support functional studies on PfEMP1, which may provide a platform for future studies manipulating the domain content in the exon 1 portion of var genes. They have used this system to produce a range of interesting findings and to support its use by the research community.<br /> Finally, a small concern. Being able to select specific var gene switches using drug markers could provide some useful starting points to understand how switching happens in P. falciparum. However, our trypanosome colleagues might remind us that forcing switches may show us some mechanisms but perhaps not all.

Reviewer #2 (Public review):

Summary

Croshagen et al develop a range of tools based on selection-linked integration (SLI) to study PfEMP1 function in P. falciparum. PfEMP1 is encoded by a family of ~60 var genes subject to mutually exclusive expression. Switching expression between different family members can modify the binding properties of the infected erythrocyte while avoiding the adaptive immune response. Although critical to parasite survival and Malaria disease pathology, PfEMP1 proteins are difficult to study owing to their large size and variable expression between parasites within the same population. The SLI approach previously developed by this group for genetic modification of P. falciparum is employed here to selectively and stably activate the expression of target var genes at the population level. Using this strategy, the binding properties of specific PfEMP1 variants were measured for several distinct var genes with a novel semi-automated pipeline to increase throughput and reduce bias. Activation of similar var genes in both the common lab strain 3D7 and the cytoadhesion competent FCR3/IT4 strain revealed higher binding for several PfEMP1 IT4 variants with distinct receptors, indicating this strain provides a superior background for studying PfEMP1 binding. SLI also enables modifications to target var gene products to study PfEMP1 trafficking and identify interacting partners by proximity-labeling proteomics, revealing two novel exported proteins required for cytoadherence. Overall, the data demonstrate a range of SLI-based approaches for studying PfEMP1 that will be broadly useful for understanding the basis for cytoadhesion and parasite virulence.

Comments

(1) While the capability of SLI to actively select var gene expression was initially reported by Omelianczyk et al., the present study greatly expands the utility of this approach. Several distinct var genes are activated in two different P. falciparum strains and shown to modify the binding properties of infected RBCs to distinct endothelial receptors; development of SLI2 enables multiple SLI modifications in the same parasite line; SLI is used to modify target var genes to study PfEMP1 trafficking and determine PfEMP1 interactomes with BioID. Curiously, Omelianczyk et al activated a single var (Pf3D7_0421300) and observed elevated expression of an adjacent var arranged in a head-to-tail manner, possibly resulting from local chromatin modifications enabling expression of the neighboring gene. In contrast, the present study observed activation of neighboring genes with head-to-head but not head-to-tail arrangement, which may be the result of shared promoter regions. The reason for these differing results is unclear although it should be noted that the two studies examined different var loci.

(2) The IT4var19 panned line that became binding-competent showed increased expression of both paralogs of ptp3 (as well as a phista and gbp), suggesting that overexpression of PTP3 may improve PfEMP1 display and binding. Interestingly, IT4 appears to be the only known P. falciparum strain (only available in PlasmoDB) that encodes more than one ptp3 gene (PfIT_140083100 and PfIT_140084700). PfIT_140084700 is almost identical to the 3D7 PTP3 (except for a ~120 residue insertion in 3D7 beginning at residue 400). In contrast, while the C-terminal region of PfIT_140083100 shows near-perfect conservation with 3D7 PTP3 beginning at residue 450, the N-terminal regions between the PEXEL and residue 450 are quite different. This may indicate the generally stronger receptor binding observed in IT4 relative to 3D7 results from increased PTP3 activity due to multiple isoforms or that specialized trafficking machinery exists for some PfEMP1 proteins.

Reviewer #3 (Public review):

Summary:

The submission from Cronshagen and colleagues describes the application of a previously described method (selection linked integration) to the systematic study of PfEMP1 trafficking in the human malaria parasite Plasmodium falciparum. PfEMP1 is the primary virulence factor and surface antigen of infected red blood cells and is therefore a major focus of research into malaria pathogenesis. Since the discovery of the var gene family that encodes PfEMP1 in the late 1990s, there have been multiple hypotheses for how the protein is trafficked to the infected cell surface, crossing multiple membranes along the way. One difficulty in studying this process is the large size of the var gene family and the propensity of the parasites to switch which var gene is expressed, thus preventing straightforward gene modification-based strategies for tagging the expressed PfEMP1. Here the authors solve this problem by forcing the expression of a targeted var gene by fusing the PfEMP1 coding region with a drug-selectable marker separated by a skip peptide. This enabled them to generate relatively homogenous populations of parasites all expressing tagged (or otherwise modified) forms of PfEMP1 suitable for study. They then applied this method to study various aspects of PfEMP1 trafficking.

Strengths:

The study is very thorough, and the data are well presented. The authors used SLI to target multiple var genes, thus demonstrating the robustness of their strategy. They then perform experiments to investigate possible trafficking through PTEX, they knock out proteins thought to be involved in PfEMP1 trafficking and observe defects in cytoadherence, and they perform proximity labeling to further identify proteins potentially involved in PfEMP1 export. These are independent and complimentary approaches that together tell a very compelling story.

Weaknesses:

(1) When the authors targeted IT4var19, they were successful in transcriptionally activating the gene, however, they did not initially obtain cytoadherent parasites. To observe binding to ICAM-1 and EPCR, they had to perform selection using panning. This is an interesting observation and potentially provides insights into PfEMP1 surface display, folding, etc. However, it also raises questions about other instances in which cytoadherence was not observed. Would panning of these other lines have been successfully selected for cytoadherent infected cells? Did the authors attempt panning of their 3D7 lines? Given that these parasites do export PfEMP1 to the infected cell surface (Figure 1D), it is possible that panning would similarly rescue binding. Likewise, the authors knocked out PTP1, TryThrA, and EMPIC3 and detected a loss of cytoadhesion, but they did not attempt panning to see if this could rescue binding. To ensure that the lack of cytoadhesion in these cases is not serendipitous (as it was when they activated IT4var19), they should demonstrate that panning cannot rescue binding.

(2) The authors perform a series of trafficking experiments to help discern whether PfEMP1 is trafficked through PTEX. While the results were not entirely definitive, they make a strong case for PTEX in PfEMP1 export. The authors then used BioID to obtain a proxiome for PfEMP1 and identified proteins they suggest are involved in PfEMP1 trafficking. However, it seemed that components of PTEX were missing from the list of interacting proteins. Is this surprising and does this observation shed any additional light on the possibility of PfEMP1 trafficking through PTEX? This warrants a comment or discussion.

Reviewer #1 (Public review):

The results of this manuscript look at the interplay between pleiotropy, standing genetic variation, and parallelism (i.e. predictability of evolution) in gene expression. Ultimately, their results suggest that (a) pleiotropic genes typically have a smaller range in variation/expression, and (b) adaptation to similar environments tends to favor changes in pleiotropic genes, which leads to parallelism in mechanisms (though not dramatically). However, it is still uncertain how much parallelism is directly due to pleiotropy, instead of a complex interplay between them and ancestral variation.

I have a few things that I was uncertain about. It may be these things are easily answered but require more discussion or clarity in the manuscript.

(1) The variation being talked about in this manuscript is expression levels, and not SNPs within coding regions (or elsewhere). The cause of any specific gene having a change in expression can obviously be varied - transcription factors, repressors, promoter region variation, etc. Is this taken into account within the "network connectivity" measurement? I understand the network connectivity is a proxy for pleiotropy - what I'm asking is, conceptually, what can be said about how/why those highly pleiotropic genes have a change (or not) in expression. This might be a question for another project/paper, but it feels like a next step worth mentioning somewhere.

(2) The authors do have a passing statement in line 361 about cis-regulatory regions. Is the assumption that genetic variation in promoter regions is the ultimate "mechanism" driving any change in expression? In the same vein, the authors bring up a potential confounding factor, though they dismiss it based on a specific citation (lines 476-481; citation 65). I'm of the mindset that in order to more confidently disregard this "issue" based on previous evidence, it requires more than one citation. Especially since the one citation is a plant. That specific point jumps out to me as needing a more careful rebuttal.

(3) I feel like there isn't enough exploration of tissue specificity versus network connectivity. Tissue specificity was best explained by a model in which pleiotropy had both direct and indirect effects on parallelism; while network connectivity was best explained (by a small margin) via the model which was mostly pleiotropy having a direct effect on ancestral variation, that then had a direct effect on parallelism. When the strengths of either direct/indirect effects were quantified, tissue specificity showed a stronger direct effect, while network connectivity had none (i.e. not significant). My confusion is with the last point - if network connectivity is explained by a direct effect in the best-supported model, how does this work, since the direct effect isn't significant? Perhaps I am misunderstanding something.

Also, network connectivity might favor the most pleiotropic genes being transcription factor hubs (or master regulators for various homeostasis pathways); while the tissue specificity metric perhaps is a kind of a space/time element. I get that a gene having expression across multiple tissues does fit the definition of pleiotropy in the broad sense, but I'm wondering if some important details are getting lost - I'm just thinking about the relative importance of what tissue specificity measurements say versus the network connectivity measurement.

Reviewer #2 (Public review):

Summary:

Lai and collaborators use a previously published RNAseq dataset derived from an experimental evolution set up to compare the pleiotropic properties of genes whose expression evolved in response to fluctuating temperature for over 100 generations. The authors correlate gene pleiotropy with the degree of parallelisms in the experimental evolution set up to ask: are genes that evolved in multiple replicates more or less pleiotropic?

They find that, maybe counter to expectation, highly pleiotropic genes show more replicated evolution. Such an effect seems to be driven by direct effects (which the authors can only speculate on) and indirect effects through low variance in pleiotropic genes (which the authors indirectly link to genetic variation underlying gene expression variance).

Weaknesses:

The results offer new insights into the evolution of gene expression and into the parameters that constrain such evolution, i.e., pleiotropy. Although the conclusions are supported by the data, I find the interpretation of the results a little bit complicated.

Major comment:

The major point I ask the authors to address is whether the connection between polygenic adaptation and parallelism can indeed be used to interpret gene expression parallelism. If the answer is not, please rephrase the introduction and discussion, if the answer is yes, please make it explicit in the text why it is so.

The authors' argument: parallelism in gene expression is the same as parallelism in SNP allele frequency (AFC) (see L389-383 here they don't mention that this explanation is derived from SNP parallelism and not trait parallelism, and see Figure 1 b). In previous publications, the authors have explained the low level of AFC parallelism using a polygenic argument. Polygenic traits can reach a new trait optimum via multiple SNPs and therefore although the trait is parallel across replicates, the SNPs are not necessarily so.

In the current paper, they seem to be exchanging SNP AFC by gene expression, and to me, those are two levels that cannot be interchanged. Gene expression is a trait, not an SNP, and therefore the fact that a gene expression doesn't replicate cannot be explained by a polygenic basis, because again the trait is gene expression itself. And, actually, the results of the simulations show that high polygenicity = less trait parallelism (Figure 4).

Now, if the authors focus on high parallel genes (present in e.g. 7 or more replicates) and they show that the eQTLs for those genes are many (highly polygenic) and the AFC of those eQTLs are not parallel, then I would agree with the interpretation. But, given that here they just assess gene expression and not eQTL AFC, I do not think they can use the 'highly polygenic = low parallelism' explanation.

The interpretation of the results to me, should be limited to: genes with low variance and high pleiotropy tend to be more parallel, and the explanation might be synergistic pleiotropy.

Reviewer #3 (Public review):

The authors aim to understand how gene pleiotropy affects parallel evolutionary changes among independent replicates of adaptation to a new hot environment of a set of experimental lines of Drosophila simulans using experimental evolution. The flies were RNAsequenced after more than 100 generations of lab adaptation and the changes in average gene expression were obtained relative to ancestral expression levels from reconstructed ancestral lines. Parallelism of gene expression change among lines is evaluated as variance in differential gene expression among lines relative to error variance. Similarly, the authors ask how the standing variation in gene expression estimated from a handful of flies from a reconstructed outbred line affects parallelism. The main findings are that parallelism in gene expression responses is positively associated with pleiotropy and negatively associated with expression variation. Those results are in contradiction with theoretical predictions and empirical findings. To explain those seemingly contradictory results the authors invoke the role of synergistic pleiotropy and correlated selection, although they do not attempt to measure either.

Strengths:

(1) The study uses highly replicated outbred laboratory lines of Drosophila simulans evolved in the lab under a constant hot regime for over 100 generations. This allows for robust comparisons of evolutionary responses among lines.

(2) The manuscript is well written and the hypotheses are clearly delineated at the onset.

(3) The authors have run a causal analysis to understand the causal dependencies between pleiotropy and expression variation on parallelism.

(4) The use of whole-body RNA extraction to study gene expression variation is well justified.

Weaknesses:

(1) It is unclear how well phenotypic variation in gene expression of the evolved lines has been estimated by the sample of 20 males from a reconstructed outbred line not directly linked to the evolved lines under study. I see this as a general weakness of the experimental design.

(2) There are no estimates of standing genetic variation of expression levels of the genes under study, only phenotypic variation. I wished the authors had been clear about that limitation and had discussed the consequences of the analysis. This also constitutes a weakness of the study.

(3) Moreover, since the phenotype studied is gene expression, its genetic basis extends beyond expressed sequences. The phenotypic variation of a gene's expression may thus likely misrepresent the genetic variation available for its evolution. The genetic variation of gene expression phenotypes could be estimated from a cross or pedigree information but since individuals were pool-sequenced (by batches of 50 males), this type of analysis is not possible in this study.

(4) The authors have not attempted to estimate synergistic pleiotropy among genes, nor how selection acts on gene expression modules. It makes any conclusion regarding the role of synergistic pleiotropy highly speculative.

I don't understand the reason why the analysis would be restricted to significantly differentially expressed genes only. It is then unclear whether pleiotropy, parallelism, and expression variation do play a role in adaptation because the two groups of adaptive and non-adaptive genes have not been compared. I recommend performing those comparisons to help us better understand how "adaptive" genes differentially contribute to adaptation relative to "non-adaptive" genes relative to their difference in population and genetic properties.

There is a lack of theoretical groundings on the role of so-called synergistic pleiotropy for parallel genetic evolution. The Discussion does not address this particular prediction. It could be removed from the Introduction.

Reviewer #1 (Public review):

Liang et al. have conducted a small-scale pilot study focusing on the feasibility and tolerability of Low-dose chemotherapy combined with delayed immunotherapy in the neoadjuvant treatment of non-small cell lung cancer. The design of delayed immunotherapy after chemotherapy is relatively novel, while the reduced chemotherapy, although somewhat lacking in innovation, still serves as an early clue for exploring future feasible strategies. Also, the dynamic ctDNA and TCR profiles could give some important hints of intrinsic tumor reaction.

However, as the author mentioned in the limitation part, due to the small sample size and lack of a control group, we cannot fully understand the advantages and disadvantages of this approach compared to standard treatment. Compared to standard immunotherapy, the treatment group in this study has three differences: (1) reduced chemotherapy, (2) the use of cisplatin instead of the commonly used carboplatin in neoadjuvant therapy trials, and (3) delayed immunotherapy. Generally, in the exploration of updated treatment strategies, the design should follow the principle of "controlling variables." If there are too many differences at once, it becomes difficult to determine which variable is responsible for the effects, leading to confusion in the interpretation of the results. Moreover, the therapeutic strategy may lack practical clinical operability due to the long treatment duration.

Furthermore, in the exploration of biomarkers, the authors emphasized the procedure of whole RNA sequencing in tumor tissues in the method section, and this was also noted in the flowchart in Figure 1. However, I didn't find any mention of RNA-related analyses in the Results section, which raises some concerns about the quality of this paper for me. If the authors have inadvertently omitted some results, they should supplement the RNA-related analyses so that I can re-evaluate the paper.

To sum up, this article exhibited a certain degree of innovation to some extent, However, due to its intrinsic design defects and data omissions, the quality of the research warranted further improvement.

Reviewer #2 (Public review):

Summary:

In this single center, single arm, open label non-randomised study the authors tested the use of paclitaxel at 180-220 mg/m2 and cisplatin at 60mg/m2 in patients with squamous NSCLC and pemetrexed at 500mg/m2 and cisplatin at 60mg/m2 in adenocarcinoma of lung origin in the neoadjuvant setting. The chemotherapy appears to have been given at a relatively standard dose; though the platin dose at 60mg/m2 is somewhat lower than has been used in the checkmate 816 trial (75mg/m2/dose), this is a well-established dose for NSCLC.

Key differences to currently approved neoadjuvant chemo-ICI treatment is that anti-PD1 antibody sintilimab (at 200mg/dose) was given on day 5 and that only 2 cycles of chemotherapy were given pre surgery, but then repeated on two occasions post surgery. Between May/2020 and Nov/2023 50 patients were screened, 38 went on to have this schedule of tx, 31 (~82%) went on to have surgery and 27 had the adjuvant treatment. The rate of surgery is entirely consistent with the checkmate 816 data.

Question to the authors:

It would be very helpful to understand why 7 (~18% of the population) patients did not make it to surgery and whether this is related to disease progression, toxicity or other reasons for withdrawal.

The key clinical endpoints were pCR and mPR rates. 2/38 patients are reported to have achieved a radiological pCR but only 31 patients underwent surgery with histological verification. Supp table2 suggests that 10/31 patients achieved a pCR, 6/31 additional patients achieved a major pathological response and that 13/31 did not achieve a major pathological response

It would be really helpful for understanding the clinical outcome to present the histopathological findings in the text in a bit more detail and to refer the outcome to the radiological findings. I note that the reference for pathological responses incorrectly is 38 patients as only 31 patients underwent surgery and were evaluated histologically.

The treatment was very well tolerated with only 1 grade 3 AE reported. The longer term outcome will need to be assessed over time as the cohort is very 'young'. It is not clear what the adjuvant chemo-ICI treatment would add and how this extra treatment would be evaluated for benefit - if all the benefit is in the neoadjuvant treatment then the extra post-operative tx would only add toxicity

Please consider what the two post-operative chemo-ICI cycles might add to the outcome and how the value of these cycles would be assessed. Would there be a case for a randomised assessment in the patients who have NOT achieved a mPR histologically?

While the clinical dataset identifies that the proposed reduced chemo-ICI therapy has clinical merit and should be assessed in a randomized study, the translational work is less informative.

The authors suggest that the treatment has a positive impact on T lymphocytes. Blood sampling was done at day 0 and day 5 of each of the four cycle of chemotherapy with an additional sample post cycle 4. The authors state that data were analysed at each stage.

The data in Figure 3B are reported for three sets of pairs: baseline to pre day 5 in cycle 1, day 5 to day 21 in cycle 1, baseline of cycle to to day 5. It remains unclear whether the datasets contain the same top 20 clones and it would be very helpful to show kinetic change for the individual 'top 20 clones' throughout the events in individual patients; as it stands the 'top20 clones' may vary widely from timepoint to timepoint. Of note, the figures do not demonstrate that the top 20 TCR clones were 'continuously increased'.

Instead, the data suggest that there are fluctuations in the relative distributions over time but that may simply be a reflection of shifts in T cell populations following chemotherapy rather than of immunological effects in the cancer tissue.<br /> Consistent with this the authors conclude (line 304/5): "No significant difference was observed in the diversity, evenness, and clonality of TCR clones across the whole treatment procedure" and this seems to be a more persuasive conclusion than the statement 'that a positive effect on T lymphocytes was observed' - where it is also not clear what 'positive' means.

The text needs a more balanced representation of the data: only a small subset of four patients appear to have been evaluated to generate the data for figure 3B and only three patients (P5, P6, P7) can have contributed to figure 3C if the sample collection is represented accurately in Figure 3A.

The text refers to flow cytometric results in SF3. However, no information is given on the flow cytometry in M&M, markers or gating strategy.

Please consider changing the terminology of the 'phases' into something that is easier to understand. One option would be to use a reference to a more standard unit (cycle 1-4 of chemotherapy and then d0/d5/d21).

Please make it explicit in the text that molecular analyses were undertaken for some patients only, and how many patients contribute to the data in figures 3B-F. Figure 3A suggests paired mRNA data were obtained in 2 patients (P2 and P5) but I cannot find the results on these analyses; four individual blood samples to assess TCR changes int PH1/PH2/PH3and PH4 were only available in four patients (P4,P5,P7,P9). Only three patients seem to have the right samples collected to allow the analysis for 'C3' in figure 3C.

Please display for each of the 'top 20 clones' at any one timepoint how these clones evolve throughout the study; I expect that a clone that is 'top 20' at a given timepoint may not be among the 'top twenty' at all timepoints.

Please also assess if the expanded clonotypes are present (and expanded) in the cancer tissue at resection, to link the effect in blood to the tumour. Given that tissue was collected for 31 patients, mRNA sequencing to generate TCR data should be possible to add to the blood analyses in the 12 patients in Figure 3A. Without this data no clear link can be made to events in the cancer.

Please provide in M&M the missing information on the flow cytometry methodology (instrument, antibody clones, gating strategy) and what markers were used to define T cell subsets (naïve, memory, central memory, effector memory).

The authors also describe that ctDNA reduces after chemo-ICI treatment. This is well documented in their data but ultimately irrelevant: if the cancer volume is reduced to the degree of a radiological or pathological response /complete response then the quantity of circulating DNA from the cancer cells must reduce. More interesting would be the question whether early changes predict clinical outcome and whether recurrent ct DNA elevations herald recurrence.

Please probe whether the molecular data identify good radiological or pathological outcomes before cycle 2 is started and whether the ctDNA levels identify patients who will have a poor response and/or who relapse early.

This is such a clever way to create a container that otherwise might not have existed for that work. I wonder if this would be a good way to highlight glue work?

Reviewer #1 (Public review):

This paper presents a mechanistic study of rDNA origin regulation in yeast by SIR2. Each of the ~180 tandemly repeated rDNA gene copies contains a potential replication origin. Early-efficient initiation of these origins is suppressed by Sir2, reducing competition with origins distributed throughout the genome for rate-limiting initiation factors. Previous studies by these authors showed that SIR2 deletion advances replication timing of rDNA origins by a complex mechanism of transcriptional de-repression of a local PolII promoter causing licensed origin proteins (MCMcomplexes) to re-localize (slide along the DNA) to a different (and altered) chromatin environment. In this study, they identify a chromatin remodeler, FUN30, that suppresses the sir2∆ effect, and remarkably, results in a contraction of the rDNA to about one-quarter it's normal length/number of repeats, implicating replication defects of the rDNA. Through examination of replication timing, MCM occupancy and nucleosome occupancy on the chromatin in sir2, fun30, and double mutants, they propose a model where nucleosome position relative to the licensed origin (MCM complexes) intrinsically determines origin timing/efficiency. While their interpretations of the data are largely reasonable and can be interpreted to support their model, a key weakness is the connection between Mcm ChEC signal disappearance and origin firing. While the cyclical chromatin association-dissociation of MCM proteins with potential origin sequences may be generally interpreted as licensing followed by firing, dissociation may also result from passive replication and as shown here, displacement by transcription and/or chromatin remodeling. Moreover, linking its disappearance from chromatin in the ChEC method with such precise resolution needs to be validated against an independent method to determine the initiation site(s). Differences in rDNA copy number and relative transcription levels also are not directly accounted for, obscuring a clearer interpretation of the results. Nevertheless, this paper makes a valuable advance with the finding of Fun30 involvement, which substantially reduces rDNA repeat number in sir2∆ background. The model they develop is compelling and I am inclined to agree, but I think the evidence on this specific point is purely correlative and a better method is needed to address the initiation site question. The authors deserve credit for their efforts to elucidate our obscure understanding of the intricacies of chromatin regulation.

Overall, the paper is improved by providing additional data and improved analysis. The paper nicely characterizes the effect of Fun30. The model is reasonable but remains lacking in precise details of mechanism.

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 #3 (Public review):

Summary:

Heterochromatin is characterized by low transcription activity and late replication timing, both dependent on the NAD-dependent protein deacetylase Sir2, the founding member of the sirtuins. This manuscript addresses the mechanism by which Sir2 delays replication timing at the rDNA in budding yeast. Previous work from the same laboratory (Foss et al. PLoS Genetics 15, e1008138) showed that Sir2 represses transcription-dependent displacement of the Mcm helicase in the rDNA. In this manuscript, the authors show convincingly that the repositioned Mcms fire earlier and that this early firing partly depends on the ATPase activity of the nucleosome remodeler Fun30. Using read-depth analysis of sorted G1/S cells, fun30 was the only chromatin remodeler mutant that somewhat delayed replication timing in sir2 mutants, while nhp10, chd1, isw1, htl1, swr1, isw2, and irc5 had no effect. The conclusion was corroborated with orthogonal assays including two-dimensional gel electrophoresis and analysis of EdU incorporation at early origins. Using an insightful analysis with an Mcm-MNase fusion (Mcm-ChEC), the authors show that the repositioned Mcms in sir2 mutants fire earlier than the Mcm at the normal position in wild type. This early firing at the repositioned Mcms is partially suppressed by Fun30. In addition, the authors show Fun30 affects nucleosome occupancy at the sites of the repositioned Mcm, providing a plausible mechanism for the effect of Fun30 on Mcm firing at that position. However, the results from the MNAse-seq and ChEC-seq assays are not fully congruent for the fun30 single mutant. Overall, the results support the conclusions providing a much better mechanistic understanding how Sir2 affects replication timing at rDNA,

Strengths:

(1) The data clearly show that the repositioned Mcm helicase fires earlier than the Mcm in the wild type position.

(2) The study identifies a specific role for Fun30 in replication timing and an effect on nucleosome occupancy around the newly positioned Mcm helicase in sir2 cells.

Comments on revisions:

In the previous revision the authors addressed my concerns and improved the manuscript and the presentation of the data. All my recommendations were implemented.

Reviewer #1 (Public review):

Summary:

The authors were attempting to identify the molecular and cellular basis for why modulators of the HR pathway, specifically PARPi, are not effective in CDK12 deleted or mutant prostate cancers and they seek to identify new therapeutic agents to treat this subset of metastatic prostate cancer patients. Overall, this is an outstanding manuscript with a number of strengths and in my opinion represents a significant advance in the field of prostate cancer biology and experimental therapeutics.

Strengths:

The patient data cohort size and clinical annotation from Figure 1 are compelling and comprehensive in scope. The associations between tandem duplications and amplifications of oncogenes that have been well-credentialed to be drivers of cancer development and progression are fascinating and the authors identify that in those that have AR amplification for example, there is evidence for AR pathway activation. The association between CDK12 inactivation and various specific gene/pathway perturbations is fascinating and is consistent with previously published studies - it would be interesting to correlate these changes with cell line-based studies in which CDK12 is specifically deleted or inhibited with small molecules to see how many pathways/gene perturbations are shared between the clinical samples and cell and mouse models with CDK12 perturbation. The short-term inhibitor studies related to changes in HRD genes and protein expression with CDK12/13 inhibition are fascinating and suggest differential pathway effects between short inhibition of CDK12/13 and long-term loss of CDK12. The in vivo studies with the inhibitor of CDK12/13 are intriguing but not definitive

Weaknesses:

Given that there are different mutations identified at different CDK12 sites as illustrated in Figure 1B it would be nice to know which ones have been functionally classified as pathogenic and for which ones that the pathogenicity has not been determined. This would be especially interesting to perform in light of the differences in the LOH scores and WES data presented - specifically, are the pathogenic mutations vs the mutations for which true pathogenicity is unknown more likely to display LOH or TD? For the cell inhibition studies with the CDK12/13 inhibitor, more details characterizing the specificity of this molecule to these targets would be useful. Additionally, could the authors perform short-term depletion studies with a PROTAC to the target or short shRNA or non-selected pool CRISPR deletion studies of CDK12 in these same cell lines to complement their pharmacological studies with genetic depletion studies? Also perhaps performing these same inhibitor studies in CDK12/13 deleted cells to test the specificity of the molecule would be useful. Additionally, expanding these studies to additional prostate cancer cell lines or organdies models would strengthen the conclusions being made. More information should be provided about the dose and schedule chosen and the rationale for choosing those doses and schedules for the in vivo studies proposed should be presented and discussed. Was there evidence for maximal evidence of inhibition of the target CDK12/13 at the dose tested given the very modest tumor growth inhibition noted in these studies?

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):

Significance:

About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.

In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.

Strengths:

Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.

Weaknesses:

(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

Reviewer #2 (Public review):

Summary:

In their manuscript, the authors report that fecal transplantation from young mice into old mice alleviates susceptibility to gout. The gut microbiota in young mice is found to inhibit activation of the NLRP3 inflammasome pathway and reduce uric acid levels in the blood in the gout model.

Strengths:

The authors focused on the butanoate metabolism pathway based on the results of metabolomics analysis after fecal transplantation and identified butyrate as the key factor in mitigating gout susceptibility. In general, this is a well-performed study.

Weaknesses:

The discussion on the current results and previous studies regarding the effect of butyrate on gout symptoms is insufficient.

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 #2 (Public review):

Sleep and memory are intertwined processes, with sleep-deprivation having a negative impact on long-term memory in many species. Recently, the authors showed that fruit flies form sleep-dependent long-term appetitive memory only when fed. They showed that this context-dependent memory trace maps to the anterior posterior (ap) α'β' mushroom body neurons (MBNs) (Chouhan et al., (2021) Nature). However, the molecular cascades induced by during training that promote sleep and memory have remained enigmatic.

Here the authors investigate this issue by combining cell-specific transcriptomics, genetic perturbations, and measurements of sleep and memory. They identify an array of genes altered in expression following appetitive training. These genes are mainly downregulated, and predominantly encode regulators of transcription and RNA biosynthesis. This is a conceptually attractive finding given that long-term memory requires de novo protein translation.

The authors then screen these genes for novel regulators of sleep and memory. They show that one of these genes (Polr1F) acts in ap α'β' MBNs to promote wakefulness, while another (Regnase-1) promotes sleep. They also identify a specific role for Regnase-1 in ap α'β' MBNs in regulating short- and long-term memory formation - likely through effects on the development of ap α'β' MBNs - and demonstrate that Pol1rF inhibits translation throughout the fly brain.

The analyses of molecular alterations in ap α'β' MBNs are interesting and impressive. However, as noted by the authors, further experiments are required to clarify the precise contribution of reductions in Polr1F and Regnase-1 to training-induced changes in memory and sleep. Nonetheless, this study provides a useful platform for such studies, and provides a conceptual advance in linking acute changes in RNA processing pathways to the interconnected processes of sleep, memory, and protein translation.

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 #4 (Public review):

Summary:

Li and Chouhan et al. follow up on a previous publication describing the role of anterior-posterior (ap) and medial (m) ɑ′/β′ Kenyon cells in mediating sleep-dependent and sleep-independent memory consolidation, respectively, based on feeding state in Drosophila melanogaster. The authors sequenced bulk RNA of ap ɑ′/β′ Kenyon cells 1h after flies were either trained-fed, trained-starved or untrained-fed and find a small number of genes (59) differentially expressed (3 upregulated, 56 downregulated) between trained-fed and trained-starved conditions. Many of these genes encode proteins involved in the regulation of gene expression. The authors then screened these differentially expressed genes for sleep phenotypes by expressing RNAi hairpins constitutively in ap ɑ′/β′ Kenyon cells and measuring sleep patterns. Two hits were selected for further analysis: Polr1F, which promoted sleep, and Regnase-1, which reduced sleep. The pan-neuronal expression of Polr1F and Regnase-1 RNAi constructs was then temporally restricted to adult flies using the GeneSwitch system. Polr1F sleep phenotypes were still observed, while Regnase-1 sleep phenotypes were not, indicating developmental defects. Appetitive memory was then assessed in flies with constitutive knockdown of Polr1F and Regnase-1 in ap ɑ′/β′ Kenyon cells. Polr1F knockdown did not affect sleep-dependent or sleep-independent memory, while Regnase-1 knockdown disrupted sleep-dependent memory, sleep-independent memory, as well as learning. Polr1F knockdown increased pre-ribosomal RNA transcripts in the brain, as measured by qPCR, in line with its predicted role as part of the RNA polymerase I complex. A puromycin incorporation assay to fluorescently label newly synthesized proteins also indicated higher levels of bulk translation upon Polr1F knockdown. Regnase-1 knockdown did not lead to observable changes in measurements of bulk translation.

Strengths:

The proposed involvement of RNA processing genes in regulating sleep and memory processes is interesting, and relatively unexplored. The methods are satisfactory.

Weaknesses:

The main weakness of previous versions of the paper was the over-interpretation of results, particularly relating to the proposed link between sleep and memory consolidation. This has now been appropriately addressed, as reflected in the change of title and incorporation of alternative interpretations of the data in the text.

Reviewer #1 (Public review):

Summary:

The authors analyzed 108 human embryos in order to address outstanding questions about human lower spinal development and secondary neural tube formation. Through whole embryo imaging and histologic analysis, they have provided exceptional quantification of the timing of posterior neuropore closure, rate of lower spinal somite formation, and formation and regression of the human tail. Their analysis has also provided convincing qualitative evidence of the cellular and molecular mechanisms at play during lower spinal development, by identifying the presence of caspase-dependent programmed cell death and the dynamic expression of FGF8/WNT3A within the elongating embryo. Interestingly, they identified multiple polarized lumens within the site of secondary neural tube formation, and added a solid argument for the mode of formation of this structure; however, the evidence for a conclusive morphogenetic mechanism remains elusive. Finally, the authors provided a substantial review of the existing publications related to human lower spinal development, creating an excellent reference and demonstrating the importance of continuing to use each of these precious samples to further advance our understanding of human development.

Strengths:

This manuscript provides an excellent window into the key morphogenetic events of human caudal neural tube formation. Figures 1 and 2 provide beautiful images and quantification of the developmental events, enabling comparison to models that are currently in use, including model organisms and the developing spinal organoid field. The characterization of somite development and later regression is particularly important.

In Figures 3 and 4, the authors use immunohistochemistry to examine the cellular death mechanisms and spatiotemporal organization of tissue regression within the tail. They demonstrate a proximal to distal tapering of the overall tail and neural tube areas that is not present for the notochord and reveal a proximal to distal degeneration of the tailgut, similar to what is observed in rodents. The identification of caspase-dependent cell death within the human tail provides an explanation for the mechanism of this regression, especially given the notable lack of presence of any gross necrosis.

Next, the authors have addressed current questions regarding the molecular pathways present during elongation of the embryo and later regression of the tail structure. The in situ hybridization experiments in Figure 5 show important evidence for a maintained neuromesodermal progenitor pool of stem cells that promote axial elongation. Additionally, the authors have conducted serial transverse sections of the tail to better understand the formation of the secondary neural tube in humans. They found a rodent-like formation involving a singular rosette caudally at the tailbud tip, and that multiple lumens, if present, were located more rostrally. This clearly differs from chick secondary neurulation. Finally, as mentioned above, the non-trivial collection and review of the existing human secondary neural tube and body formation literature is an important tool that organizes and synthesizes ~ 100 years of observations from precious human samples.

Weaknesses:

(1) The non-pathologic presence of multiple polarizations in human tails compared to the rodent pathogenic counterpart is interesting given that rodents obviously maintain this appendage that is lost in humans. A clear mechanism for how the secondary tube becomes continuous with the primary tube and how this relates to the presence of multiple polarizations in humans remains elusive.

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 #1 (Public review):

Summary:

In the paper, Yan and her colleagues investigate at which stage of development different categorical signals can be detected with EEG using a Steady-state visual evoked potential paradigm. The study reports the development trajectory of selective responses to five categories (i.e., faces, limbs, corridors, characters, and cars) over the first 1.5 years of life. It reveals that while responses to faces show significant early development, responses to other categories (i.e., characters and limbs) develop more gradually and emerge later in infancy. The insights the study provides are important. The paper is well-written and enjoyable, and the content is well-motivated and solid.

Strengths:

(1) This study contains a rich dataset with a good amount of effort. It covers a large sample of infants across ages (N=45) asking an interesting question about when we can robustly detect visual category representations during the first year of life of human infants.

(2) The chosen category stimuli are appropriate and well-controlled. These categories are classic and important for situating the study in the field within a well-established theoretical framework.

(3) The brain measurements are solid. Visual periodicity allows for the dissociation of selective responses to image categories within the same rapid image stream, which appears at different intervals. This is important for the infant field, where brain measures often lack sensitivity due to the developing brain's low signal-to-noise ratio and short recording time. Considering the significant changes in the brain during infancy, this robust measure of ERPs has good interpretability.

Weaknesses:

(1) There is limited data available for each category per infant, with an average of only 5 trials/epochs per category per participant. This insufficient data for each individual weakens the study, as it limits the power of analysis and constrains our understanding of the research question. If more data were available for each tested category per individual, the findings would be more robust and our ability to answer the questions more effectively would be enhanced.

(2) The study would benefit from a more detailed explanation of analysis choices, limitations, and broader interpretations of the findings. This should include: a) improving the treatment of bias from specific categories (e.g., faces) towards others; b) justifying the specific experimental and data analysis choices; and c) expanding the interpretation and discussion of the results. I believe that giving more attention to these aspects would improve the study and contribute positively to the field.

Comments on revised submission:

The authors thoroughly addressed my concerns, and I have no further issues with their response.

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 #3 (Public review):

Yan et al. ("When do visual category representations emerge in infant brains?") present an EEG study of category-specific visual responses in infancy from 3 to 15 months of age. In their experiment, infants viewed visually controlled images of faces and several non-face categories in a steady state evoked potential paradigm. The authors find visual responses at all ages, but face responses only at 4-6 months and older, and other category-selective responses at later ages. They find that spatiotemporal patterns of response can discriminate faces from other categories at later ages.

Overall, I found the study well-executed and a useful contribution to the literature. The study advances prior work by using well-controlled stimuli, subgroups at different ages, and new analytic approaches. The data and analyses support their conclusions regarding developmental change in neural responses to high-level visual stimuli.

Reviewer #1 (Public review):

Summary:

Opioids and related drugs are powerful analgesics that reduce suffering from pain. Unfortunately, their use often leads to addiction and there is an opioid-abuse epidemic that affects people worldwide. This study represents an ongoing effort to develop non-opioid analgesics for pain management. The findings point to an alternative approach to control post-surgical pain in lieu of opioid medications.

Strengths:

(1) The study responds to the urgent need for the development of non-opioid analgesics.<br /> (2) The study demonstrates the efficacy of Clarix Flo (FLO) and HC-HA/PTX3 from the human amniotic membrane (AM) in reducing pain in a mouse model without the adverse effects of opioids.<br /> (3) The study further explored the underlying mechanisms of how HC-HA/PTX3 produces its effects on neurons, suggesting the molecules/pathways involved in pain relief.<br /> (4) The potential use of naturally derived biologics from human birth tissues (AM) is safe and sustainable, compared to synthetic pharmaceuticals.<br /> (5) The study was conducted with scientific rigor, involving purification of active components, comparative analysis with multiple controls, and mechanistic explorations.

Weaknesses:

(1) It should be cautioned that while the preclinical findings are promising, these results still need to be translated into clinical settings that are complex and often unpredictable.<br /> (2) The study shows the efficacy of FLO and HC-HA/PTX3 in one preclinical model of post-surgical pain. The observed effect may be variable in other pain conditions.

Comments on revisions:

The authors have addressed my concerns in the revision. I don't have further comments on this manuscript.

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 #3 (Public review):

Summary:

Non-opioid analgesics derived from human amniotic membrane (AM) product represents a novel and unique approach to analgesia that may avoid the traditional harms associated with opioids. Here, the study investigators demonstrate that HC-HAPTX3 is the primary bioactive component of the AM product FLO responsible for anti-nociception in mouse-model and in-vitro dorsal root ganglion (DRG) cell culture experiments. The mechanism is demonstrated to be via CD44 with an acute cytoskeleton rearrangement that is induced that inhibits Na+ and Ca++ current through ion channels. Taken together, the studies reported in the manuscript provide supportive evidence clarifying the mechanisms and efficacy of HC-HAPTX3 antinociception and analgesia.

Strengths:

Extensive experiments including murine behavioral paw withdrawal latency and Catwalk test data demonstrating analgesic properties. Breadth and depth of experimental data are clearly supporting mechanisms and antinociceptive properties.

Weaknesses:

None. Only a few minor directed changes to the text of the manuscript.<br /> P4 last sentence - "Our findings highlight the potential of a naturally derived biologic from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms." - another sentence clause is required before "unravel"<br /> P7 second paragraph - please edit the following sentence for clarity: "Since HC-HA/PTX3 mimics FLO in producing pain inhibition, and it has high-purity and is more water-soluble than FLO, making it suitable for probing cellular mechanisms."

Reviewer #1 (Public review):

Summary:

In a heroic effort, Ozanna Burnicka-Turek et al. have made and investigated conduction system-specific Tbx3-Tbx5 deficient mice and investigated their cardiac phenotype. Perhaps according to expectations, given the body of literature on the function of the two T-box transcription factors in the heart/conduction system, the cardiomyocytes of the ventricular conduction system seemed to convert to "ordinary" ventricular working myocytes. As a consequence, loss of VCS-specific conduction system propagation was observed in the compound KO mice, associated with PR and QRS prolongation and elevated susceptibility to ventricular tachycardia.

Strengths:

Great genetic model. Phenotypic consequences at the organ and organismal levels are well investigated. The requirement of both Tbx3 and Tbx5 for maintaining VCS cell state has been demonstrated.

Weaknesses:

The actual cell state of the Tbx3/Tbx5 deficient conducting cells was not investigated in detail, and therefore, these cells could well only partially convert to working cardiomyocytes, and may, in reality, acquire a unique state.

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 #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterised the cardiac phenotype associated with the cardiac conduction system (CCS)-specific combined deletion of Tbx5 and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen-inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9-week-old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterising VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analysed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutants and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lies in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Lineage tracing analysis using the reporter lines included in the crosses described in the study will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot, will demonstrate that indeed the cells that were specified as VCS in the adult heart, become working myocardium in the absence of Tbx3 and Tbx5 function.

Reviewer #1 (Public review):

Summary:

This paper developed a model of chromosome mosaicism by using a new aneuploidy-inducing drug (AZ3146), and compared this to their previous work where they used reversine, to demonstrate the fate of aneuploid cells during murine preimplantation embryo development. They found that AZ3146 acts similarly to reversine in inducing aneuploidy in embryos, but interestingly showed that the developmental potential of embryos is higher in AZ3146-treated vs. reversine-treated embryos. This difference was associated with changes in HIF1A, p53 gene regulation, DNA damage, and fate of euploid and aneuploid cells when embryos were cultured in a hypoxic environment.

Strengths:

In the current study, the authors investigate the fate of aneuploid cells in the preimplantation murine embryo using a specific aneuploidy-inducing compound to generate embryos that were chimeras of euploid and aneuploid cells. The strength of the work is that they investigate the developmental potential and changes in gene expression profiles under normoxic and hypoxic culture conditions. Further, they also assessed how levels of DNA damage and DNA repair are altered in these culture conditions. They also assessed the allocation of aneuploid cells to the divergent cell lineages of the blastocyst stage embryo.

Weaknesses:

Inconsistent/missing description for sample size, biological/technical replicates, label orientation, the appropriate number of * for each figure panel, and statistical tests used.

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 #1 (Public review):

Summary:

Dopamine neurons contribute to motivated and motor behaviors in many ways, and ample recent evidence has suggested that distinct dopamine neuron subclasses support discrete behavioral and circuit functions. Prior studies have subdivided dopamine neurons by spatial localization, gene expression patterns, and physiological properties. However, many of these studies were bound by previous technical limitations that made comprehensive subclassification efforts difficult or impossible. The main goal of this manuscript was to characterize and further define dopamine neuron heterogeneity in the ventral midbrain. The study uses cutting-edge single nucleus RNA-seq (on the 10X Genomics platform) and spatial transcriptomics (on the MERFISH platform) to define dopamine neuron heterogeneity with unprecedented resolution. The result is a convincing and comprehensive subclassification of dopamine neurons into three main families, each with major branches and subtypes. In addition, the study reports comparisons between wild-type mice and mice that harbor a G2019S mutation in the Lrrk2 gene, which models a common cause of autosomally dominant Parkinson's Disease in humans. These results, while less robust due to the nature of the group comparisons, nevertheless identify vulnerability within specific dopamine neuron subpopulations. This vulnerability may contribute unique risk of dopamine neuron loss in the context of Parkinson's disease. Overall, the study is careful and rigorous and provides a critical resource for the rapidly evolving knowledge of dopamine neuron subtypes.

Strengths:

(1) The creation of a public-facing app where the snRNA-seq data can be investigated by anyone is a major strength.

(2) The manuscript includes careful comparisons to prior datasets that have sought to explore dopamine neuron heterogeneity. The result is a useful synthesis of new findings with previously published work, which is helpful for moving the field forward in this area.

(3) The integration of snRNA-seq with MERFISH results is particularly strong and enables insight not only into subclassification but also into how this relates to spatial localization. The careful neuroanatomy reveals important distinctions between Sox6, Calb1, and Gad2 positive dopamine neuron families, with some degree of spatial overlap.

Weaknesses:

(1) Important details about the nature of DEG comparisons between the wild type and the Lrrk2 G2019S model are missing.

(2) Some aspects of the integration between snRNA-seq and MERFISH data are not clear, and many MERFISH-identified cells do not appear to have a high-confidence cluster transfer into the snRNA-seq data space. Imputation is used to overcome some issues with the MERFISH dataset, but it is not clear that this is appropriate.

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 #1 (Public review):

Mutations in CDHR1, the human gene encoding an atypical cadherin-related protein expressed in photoreceptors, are thought to cause cone-rod dystrophy (CRD). However, the pathogenesis leading to this disease is unknown. Previous work has led to the hypothesis that CDHR1 is part of a cadherin-based junction that facilitates the development of new membranous discs at the base of the photoreceptor outer segments, without which photoreceptors malfunction and ultimately degenerate. CDHR1 is hypothesized to bind to a transmembrane partner to accomplish this function, but the putative partner protein has yet to be identified.

The manuscript by Patel et al. makes an important contribution toward improving our understanding of the cellular and molecular basis of CDHR1-associated CRD. Using gene editing, they generate a loss of function mutation in the zebrafish cdhr1a gene, an ortholog of human CDHR1, and show that this novel mutant model has a retinal dystrophy phenotype, specifically related to defective growth and organization of photoreceptor outer segments (OS) and calyceal processes (CP). This phenotype seems to be progressive with age. Importantly, Patel et al, present intriguing evidence that pcdh15b, also known for causing retinal dystrophy in previous Xenopus and zebrafish loss of function studies, is the putative cdhr1a partner protein mediating the function of the junctional complex that regulates photoreceptor OS growth and stability.

This research is significant in that it:

(1) provides evidence for a progressive, dystrophic photoreceptor phenotype in the cdhr1a mutant and, therefore, effectively models human CRD; and

(2) identifies pcdh15b as the putative, and long sought after, binding partner for cdhr1a, further supporting the theory of a cadherin-based junction complex that facilitates OS disc biogenesis.

Nonetheless, the study has several shortcomings in methodology, analysis, and conceptual insight, which limits its overall impact.

Below I outline several issues that the authors should address to strengthen their findings.

Major comments:

(1) Co-localization of cdhr1a and pcdh15b proteins

The model proposed by the authors is that the interaction of cdhr1a and pcdh15b occurs in trans as a heterodimer. In cochlear hair cells, PCDH15 and CDHR23 are proposed to interact first as dimers in cis and then as heteromeric complexes in trans. This was not shown here for cdhr1a and pcdh15b, but it is a plausible configuration, as are single heteromeric dimers or homodimers. Regardless, this model depends on the differential compartmental expression of the cdhr1a and pcdh15b proteins. Data in Figure 1 show convincing evidence that these two proteins can, at least in some cases, be distributed along the length of photoreceptor membranes that are juxtaposed, as would be the case for OS and CP. If pcdh15b is predominantly expressed in CPs, whereas cdhr1a is predominantly expressed in OS, then this should be confirmed with actin double labeling with cdhr1a and pcdh15b since the apicobasal oriented (vertical) CPs would express actin in this same orientation but not in the OS. This would help to clarify whether cdhr1a and pcdh15b can be trafficked to both OS and CP compartments or whether they are mutually exclusive.

Photoreceptor heterogeneity goes beyond the cone versus rod subtypes discussed here and it is known that in zebrafish, CP morphology is distinct in different cone subtypes as well as cone versus rod. It would be important to know which specific photoreceptor subtypes are shown in zebrafish (Figures 1A-C) and the non-fish species depicted in Figures 1E-L. Also, a larger field of view of the staining patterns for Figures 1E-L would be a helpful comparison (could be added as a supplementary figure).

(2) Cdhr1a function in cell culture

The authors should explain the multiple bands in the anti-FLAG blots. Also, it would be interesting to confirm that the cdhr1a D173 mutant prevents the IP interaction with pcdh15b as well as the additive effects in aggregate assays of Figure 2.

Is it possible that the cultured cells undergo proliferation in the aggregation assays shown in Figure 2? Cells might differentially proliferate as clusters form in rotating cultures. A simple assay for cell proliferation under the different transfection conditions showing no differences would address this issue and lend further support to the proposed specific changes to cell adhesion as a readout of this assay.

Also, the authors report that the number of clusters was normalized to the field of view, but this was not defined. Were the n values different fields of view from one transfection experiment, or were they different fields of view from separate transfection experiments? More details and clarification are needed.

(3) Methodological issues in quantification and statistical analyses

Were all the OS and CP lengths counted in the observation region or just a sample within the region? If the latter, what were the sampling criteria? For CPs, it seems that the length was an average estimate based on all CPs observed surrounding one cone or one-rod cell. Is this correct? Again, if sampled, how was this implemented? In Fig 4M', the cdhr1a-/- ROS mostly looks curvilinear. Did the measurements account for this, or were they straight linear dimension measurements from base to tip of the OS as depicted in Fig 5A-E? A clearer explanation of the OS and CP length quantification methodology is required.

How were cone and rod photoreceptor cell counts performed? The legend in Figure 4 states that they again counted cells in the observation region, but no details were provided. For example, were cones and rods counted as an absolute number of cells in the observation region (e.g., number of cones per defined area) or relative to total (DAPI+) cell nuclei in the region? Changes in cell density in the mutant (smaller eye or thinner ONL) might affect this quantification so it would be important to know how cell quantification was normalized.

In Figure 6I, K, measuring the length of the signal seems problematic. The dimension of staining is not always in the apicobasal (vertical) orientation. It might be more accurate to measure the cdhr1a expression domain relative to the OS (since the length of the OS is already reduced in the mutants). Another possible approach could be to measure the intensity of cdhr1 staining relative to the intensity within a Prph2 expression domain in each group. The authors should provide complementary evidence to support their conclusion.

A better description of the statistical methodology is required. For example, the authors state that "each of the data points has an n of 5+ individuals." This is confusing and could indicate that in Figure 4F alone there were ~5000 individuals assayed (~100 data points per treatment group x n=5 individuals per data point x 10 treatment groups). I don't think that is what the authors intended. It would be clearer if the authors stated how many OS, CP, or cells were counted in their observation region averaged per individual, and then provided the n value of individuals used per treatment group (controls and mutants), on which the statistical analyses should be based.

There are hundreds of data points in the separate treatment groups shown in several of the graphs. It would not be correct to perform the ANOVA on the separate OS or CP length measurements alone as this will bias the estimates since they are not all independent samples. For example, in Figure 6H, 5dpf pcdh15b+/- have shorter CPs compared to WT but pcdh15b-/- have longer compared to WT. This could be an artifact of the analysis. Moreover, the authors should clarify in the Methods section which ANOVA post hoc tests were used to control for multiple pairwise comparisons.

(4) Cdhr1a function in photoreceptors

The cdhr1a IHC staining in 5dpf WT larvae in Figure 3E appears different from the cdhr1a IHC staining in 5dpf WT larvae in Figure 1A or Figure 6I. Perhaps this is just the choice of image. Can the authors comment or provide a more representative image?

The authors show that pcdh15b localization after 5dpf mirrored the disorganization of the CP observed with actin staining. They also show in Figure 5O that at 180dpf, very little pcdh15b signal remains. They suggest based on this data that total degradation of CPs has occurred in the cdhr1a-/- photoreceptors by this time. However, although reduced in length, COS and cone CPs are still present at 180dpf (Figure 5E, E'). Thus, contrary to the authors' general conclusion, it is possible that the localization, trafficking, and/or turnover of pcdh15b is maintained through a cdhr1a-dependent mechanism, irrespective of the degree to which CPs are maintained. The experiments presented here do not clearly distinguish between a requirement for maintenance of localization versus a secondary loss of localization due to defective CPs.

(5) Conceptual insights

The authors claim that cdhr1a and pcdh15b double mutants have synergistic OS and CP phenotypes. I think this interpretation should be revisited.

First, assuming the model of cdhr1a-pcdh15b interaction in trans is correct, the authors have not adequately explained the logic of why disrupting one side of this interaction in a single mutant would not give the same severity of phenotype as disrupting both sides of this interaction in a double mutant.

Second, and perhaps more critically, at 10dpf the OS and CP lengths in cdhr1a-/- mutants (Figure 7J, T) are significantly increased compared to WT. In contrast, there are no significant differences in these measurements in the pcdh15b-/- mutants. Yet in double homozygous mutants, there is a significant reduction of ~50% in these measurements compared to WT. A synergistic phenotype would imply that each mutant causes a change in the same direction and that the magnitude of this change is beyond additive in the double mutants (but still in the same direction). Instead, I would argue that the data presented in Figure 7 suggest that there might be a functionally antagonistic interaction between cdhr1a and pcdh15b with respect to OS and CP growth at 10dpf.

If these proteins physically interacted in vivo, it would appear that the interaction is complex and that this interaction underlies both OS growth-promoting and growth-restraining (stabilizing) mechanisms working in concert. Perhaps separate homodimers or heterodimers subserve distinct CP-OS functional interactions. This might explain the age-dependent differences in mutant CP and OS length phenotypes if these mechanisms are temporally dynamic or exhibit distinct OS growth versus maintenance phases. Regardless of my speculations, the model presented by the authors appears to be too simplistic to explain the data.

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 #3 (Public review):

Summary:

The manuscript by Patel et al investigates the hypothesis that CDHR1a on photoreceptor outer segments is the binding partner for PCDH15 on the calyceal processes, and the absence of either adhesion molecule results in separation between the two structures, eventually leading to degeneration. PCDH15 mutations cause Usher syndrome, a disease of combined hearing and vision loss. In the ear, PCDH15 binds CDH23 to form tip links between stereocilia. The vision loss is less understood. Previous work suggested PCDH15 is localized to the calyceal processes, but the expression of CDH23 is inconsistent between species. Patel et al suggest that CDHR1a (formerly PCDH21) fulfills the role of CDH23 in the retina.

The experiments are mainly performed using the zebrafish model system. Expression of Pcdh15b and Cdhr1a protein is shown in the photoreceptor layer through standard confocal and structured illumination microscopy. The two proteins co-IP and can induce aggregation in vitro. Loss of either Cdhr1a or Pcdh15, or both, results in degeneration of photoreceptor outer segments over time, with cones affected primarily.

The idea of the study is logical given the photoreceptor diseases caused by mutations in either gene, the comparisons to stereocilia tip links, and the protein localization near the outer segments. The work here demonstrates that the two proteins interact in vitro and are both required for ongoing outer segment maintenance. The major novelty of this paper would be the demonstration that Pcdh15 localized to calyceal processes interacts with Cdhr1a on the outer segment, thereby connecting the two structures. Unfortunately, the data presented are inadequate proof of this model.

Strengths:

The in vitro data to support the ability of Pcdh15b and Cdhr1a to bind is well done. The use of pcdh15b and cdhr1a single and double mutants is also a strength of the study, especially being that this would be the first characterization of a zebrafish cdhr1a mutant.

Weaknesses:

(1) The imaging data in Figure 1 is insufficient to show the specific localization of Pcdh15 to calyceal processes or Cdhr1a to the outer segment membrane. The addition of actin co-labelling with Pcdh15/Cdhr1a would be a good start, as would axial sections. The division into rod and cone-specific imaging panels is confusing because the two cell types are in close physical proximity at 5 dpf, but the cone Cdhr1a expression is somehow missing in the rod images. The SIM data appear to be disrupted by chromatic aberration but also have no context. In the zebrafish image, the lines of Pcdh15/Cdhr1a expression would be 40-50 um in length if the scale bar is correct, which is much longer than the outer segments at this stage and therefore hard to explain.

(2) Figure 3E staining of Cdhr1a looks very different from the staining in Figure 1. It is unclear what the authors are proposing as to the localization of Cdhr1a. In the lab's previous paper, they describe Cdhr1a as being associated with the connecting cilium and nascent OS discs, and fail to address how that reconciles with the new model of mediating CP-OS interaction. And whether Cdhr1a localizes to discrete domains on the disc edges, where it interacts with Pcdh15 on individual calyceal processes.

(3) The authors state "In PRCs, Pcdh15 has been unequivocally shown to be localized in the CPs". However, the immunostaining here does not match the pattern seen in the Miles et al 2021 paper, which used a different antibody. Both showed loss of staining in pcdh15b mutants so unclear how to reconcile the two patterns.

(4) The explanation for the CRISPR targets for cdhr1a and the diagram in Figure 3 does not fit with crRNA sequences or the mutation as shown. The mutation spans from the latter part of exon 5 to the initial portion of exon 6, removing intron 5-6. It should nevertheless be a frameshift mutation but requires proper documentation.

(5) There are complications with the quantification of data. First, the number of fish analyzed for each experiment is not provided, nor is the justification for performing statistics on individual cell measurements rather than using averages for individual fish. Second, all cone subtypes are lumped together for analysis despite their variable sizes. Third, t-tests are inappropriately used for post-hoc analysis of ANOVA calculations.

(6) Unclear how calyceal process length is being measured. The cone measurements are shown as starting at the external limiting membrane, which is not equivalent to the origin of calyceal processes, and it is uncertain what defines the apical limit given the multiple subtypes of cones. In Figure 5, the lines demonstrating the measurements seem inconsistently placed.

(7) The number of fish analyzed by TEM and the prevalence of the phenotype across cells are not provided. A lower magnification view would provide context. Also, the authors should explain whether or not overgrowth of basal discs was observed, as seen previously in cdhr1-null frogs (Carr et al., 2021).

(8) The statement describing the separation between calyceal processes and the outer segment in the mutants is not backed up by the data. TEM or co-labelling of the structures in SIM could be done to provide evidence.

(9) "Based on work in the murine model and our own observations of rod CPs, we hypothesize that zebrafish rod CPs only extend along the newly forming OS discs and do not provide structural support to the ROS." Unclear how murine work would support that conclusion given the lack of CPs in mice, or what data in the manuscript supports this conclusion.

(10) The authors state "from the fact that rod CPs are inherently much smaller than cone CPs" without providing a reference. In the manuscript, the measurements do show rod CPs to be shorter, but there are errors in the cone measurements, and it is possible that the RPE pigment is interfering with the rod measurements.

(11) The discussion should include a better comparison of the results with ocular phenotypes in previously generated pcdh15 and cdhr1 mutant animals.

(12) The images in panels B-F of the Supplemental Figure are uncannily similar, possibly even of the same fish at different focal planes.

Reviewer #1 (Public review):

Gray and colleagues describe the identification of Integrator complex subunit 12 (INTS12) as a contributor to HIV latency in two different cell lines and in cells isolated from the blood of people living with HIV. The authors employed a high-throughput CRISPR screening strategy to knock down genes and assess their relevance in maintaining HIV latency. They had used a similar approach in two previous studies, finding genes required for latency reactivation or genes preventing it and whose knockdown could enhance the latency-reactivating effect of the NFκB activator AZD5582. This work builds on the latter approach by testing the ability of gene knockdowns to complement the latency-reactivating effects of AZD5582 in combination with the BET inhibitor I-BET151. This drug combination was selected because it has been previously shown to display synergistic effects on latency reactivation.

The finding that INTS12 may play a role in HIV latency is novel, and the effect of its knockdown in inducing HIV transcription in primary cells, albeit in only a subset of donors, is intriguing. However, there are some data and clarifications that would be important to include to complement the information provided in the current version of the manuscript.

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 #3 (Public review):

Summary:

Transcriptionally silent HIV-1 genomes integrated into the host`s genome represent the main obstacle to an HIV-1 cure. Therefore, agents aimed at promoting HIV transcription, the so-called latency reactivating agents (LRAs) might represent useful tools to render these hidden proviruses visible to the immune system. The authors successfully identified, through multiple techniques, INTS12, a component of the Integrator complex involved in 3' processing of small nuclear RNAs U1 and U2, as a factor promoting HIV-1 latency and hindering elongation of the HIV RNA transcripts. This factor synergizes with a previously identified combination of LRAs, one of which, AZD5582, has been validated in the macaque model for HIV persistence during therapy (https://pubmed.ncbi.nlm.nih.gov/37783968/). The other compound, I-BET151, is known to synergize with AZD5582, and is a inhibitor of BET, factors counteracting the elongation of RNA transcripts.

Strengths:

The findings were confirmed through multiple screens and multiple techniques. The authors successfully mapped the identified HIV silencing factor at the HIV promoter.

Weaknesses:

(1) Initial bias:<br /> In the choice of the genes comprised in the library, the authors readdress their previous paper (Hsieh et al.) where it is stated: "To specifically investigate host epigenetic regulators involved in the maintenance of HIV-1 latency, we generated a custom human epigenome specific sgRNA CRISPR library (HuEpi). This library contains sgRNAs targeting epigenome factors such as histones, histone binders (e.g., histone readers and chaperones), histone modifiers (e.g., histone writers and erasers), and general chromatin associated factors (e.g., RNA and DNA modifiers) (Fig 1B and 1C)".

From these figure panels, it clearly appears that the genes chosen are all belonging to the indicated pathways. While I have nothing to object to on the pertinence to HIV latency of the pathways selected, the authors should spend some words on the criteria followed to select these pathways. Other pathways involving epigenetic modifications and containing genes not represented in the indicated pathways may have been left apart.

(2) Dereplication:<br /> From Figure 1 it appears that INTS12 alone reactivates HIV -1 from latency alone without any drug intervention as shown by the MACGeCk score of DMSO-alone controls. If INTS12 knockdown alone shows antilatency effects, why, then were they unable to identify it in their previous article (Hsieh et al., 2023)? The authors should include some words on the comparison of the results using DMSO alone with those of the previous screen that they conducted.

(3) Translational potential:<br /> In order to propose a protein as a drug target, it is necessary to adhere to the "primum non nocere" principle in medicine. It is therefore fundamental to show the effects of INTS12 knockdown on cell viability/proliferation (and, advisably, T-cell activation). These data are not reported in the manuscript in its current form, and the authors are strongly encouraged to provide them.

Finally, as many readers may not be very familiar with the general principles behind CRISPR Cas9 screening techniques, I suggest addressing them in this excellent review: https://pmc.ncbi.nlm.nih.gov/articles/PMC7479249/.

Reviewer #1 (Public review):

Summary:<br /> In this study by Fang et al., the authors show how STAMBPL1 promotes TNBC angiogenesis via a feed-forward GRHL3/HIF1a/VEGFA axis. They demonstrate that STAMBPL1 interacts with FOXO1, define the required domains in each protein, and illustrate that this interaction facilitates FOXO1 transcriptional factor activity, which then activating GRHL3/HIF1a/VEGFA signaling. Lastly, they show that the combination of VEGFR and FOXO1 inhibitors can synergistically suppress STAMBPL1-overexpressing TNBC.

Strengths:

The manuscript is clearly written, and the results are well explained. The observation that STAMBPL1 mediates GRHL3 transcription through its interaction with FOXO1 is novel. The findings also have important translational potential.

Weaknesses:<br /> The mechanism by which STAMBPL1 mediates GRHL3 transcription through its interaction with FOXO1 is not sufficiently discussed, especially in relation to how STAMBPL1 regulates FOXO1. Some reported effects are modest.

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 #3 (Public review):

In this manuscript, Fang et al. describe a new oncogenic function of the STAMBPL1 protein in triple-negative breast cancer (TNBC). STAMBPL1 is a deubiquitinase that has been poorly studied in cancer. Previous reports identify it as a promoter of epithelial to mesenchymal transition or an inhibitor of cisplatin-induced cell death, but its participation to other cancer phenotypes has not been investigated. Fang et al. find that in cell line models of TNBC, STAMBPL1 promotes expression of the transcription factor HIF-1a and its downstream target VEGF, with the consequence of stimulating neo-angiogenesis in vitro and in vivo. Mechanistically, the authors find that this occurs via a non-enzymatic and indirect mechanism, that is by promoting the expression of GRHL3, a transcription factor that in turn binds to the HIF-1a promoter to stimulate its transcription. Interestingly, the way by which STAMPB1 promotes GRHL3 expression is by facilitating the transcriptional activity of FOXO1, a known regulator of GRHL3. Because the authors find that STAMBPL1 and FOXO1 interact, they suggest that STAMBPL1 may promote the formation of an active transcriptional complex containing FOXO1, perhaps by facilitating the recruitment of transcriptional coactivators.<br /> In conclusion, these data position for the first time the STAMBPL1 deubiquitinase in a FOXO-GRHL3 regulatory axis for the control of VEGF expression and tumor angiogenesis.<br /> The main weaknesses of this work are that the relevance of this molecular axis to the pathogenesis of TNBC is not clear, and it is not clearly established whether this is a regulatory pathway that occurs in hypoxic conditions or independently of oxygen levels.<br /> With respect to the first point, both FOXO1 and GRHL3 have been previously described as tumor suppressors, with reports of FOXO1 inhibiting tumor angiogenesis. Therefore, this works describes an apparently contradictory function of these proteins in TNBC. While it is not surprising that the same genes perform divergent functions in different tumor contexts, a stronger evidence in support of the oncogenic function of these two genes should be provided to make the data more convincing. As an example, the data in support of high STAMBPL1, FOXO and GRHL3 gene expression in TNBC TCGA specimens provided in Figure 8 is not very strong and it is not clear what the non-TNBC specimens are (whether other breast cancers or other tumors, perhaps those tumors whether these genes perform tumor suppressive functions). To strengthen the notion that STAMBPL1, FOXO and GRHL3 are overexpressed in TNCB, the authors could provide a comparison with normal tissue, as well as the analysis of other publicly available datasets (like the NCI Clinical Proteomic Tumor Analysis Consortium as an example). Finally, is it not clear what are the basal protein expression levels of STAMBPL1 in the cell lines used in this study, as based on the data presented in Figures 2D and F it appears that the protein is not expressed if not exogenously overexpressed. It would be helpful if the authors addressed this issue and provided further evidence of STAMBPL1 expression in TNBC cell lines.<br /> Linked to these considerations is the second major criticism, namely that it is not made clear if this new regulatory axis is proposed to act in normoxic or hypoxic conditions. The experiments presented in this paper are performed in both conditions but a clear explanation as to why cells are exposed to hypoxia is not given and would be necessary being that HIF-1a transcription and not protein stability is being analyzed. Also, different hypoxic conditions are sometimes used, resulting in different mRNA levels of HIF-1a and its downstream targets and quite significant fluctuations within the same cell line from one experimental setting to the next. The authors should provide an explanation as to why experimental conditions are changed and, more importantly, the experiments presented in Figure 2 should be performed also in normoxia.<br /> Another critical point is that necessary experimental controls are sometimes missing, and this is reducing the strength of some of the conclusions enunciated by the authors. As examples, experiments where overexpression of STAMBPL1 is coupled to silencing of FOXO1 to demonstrate dependency lack FOXO1silencing the absence of STAMBPL1 overexpression. Because diminishing FOXO1 expression affects HIF-1a/VEGF transcription even in the absence of STAMBPL1 (shown in Figure 7C, D), it is not clear if the data presented in Figure 7G are significant. The difference between HIF-1a expression upon FOXO1 silencing should be compared in the presence or absence of STAMBPL1 overexpression to understand if FOXO1 impacts HIF-1a transcription dependently or independently of STAMBPL1.

In addition, some minor comments to improve the quality of this manuscript are provided.<br /> (1) As a general statement, the manuscript is extremely synthetic. While this is not necessarily a negative feature, sometimes results are discussed in the figure legends and not in the main text (as an example, western blots showing HIF-1a expression) and this makes it hard to read thought the data in an easy and enjoyable manner.<br /> (2) The effect of STAMBPL1 overexpression on HIF-1a transcription is minor (Figure 2) The authors should explain why they think this is the case and whether hypoxia may provide a molecular environment that is more permissive to this type of regulation.<br /> (3) HIF-1a does not appear upregulated at the protein level protein by STAMBPL1 or GRLH3 overexpression, even though this is stated in the legends of Figures 2 and 6. The authors should show unsaturated western blots images and provide quantitative data of independent experiments to make this point.<br /> In summary, adding necessary controls and performing additional experiments to substantiate the oncogenic function of these genes in TNCB would strengthen the authors' conclusions.

Reviewer #1 (Public review):

This is a well-done clinical study which provides new information on the effects of metabolic disturbances in the human skeleton. 63 postmenopausal women undergoing hip arthroplasty, consisting of T2D with obesity; obesity alone; and neither T2D nor obesity were studied. Most of the findings relate to T2D. Increased serum TNF-α was found in T2D, as well as increased bone gene expression of TNF- α, which was associated with reduced expression of Wnt pathway genes. mRNA levels of certain of the cytokines correlated with Wnt signaling components. In addition, the increased serum TNF- α in T2D was associated with reduced Young's modulus, a measure of bone strength. A strength of this paper is that it provides information in an area that is not well-understood. However, there are a number of concerns that warrant direct addressing.

(1) Can the authors speculate why the changes in cytokines and Wnt expression do not impact bone microarchitecture?

(2) The authors state that they are showing an association between inflammation and bone strength via the regulation of Wnt signaling. However they have only shown here that serum cytokines correlate with bone strength. It is true that the authors have previously shown that Wnt signaling correlated with bone strength. But here it would be useful to show if bone strength is also correlated with inflammatory genes.

(3) AGEs increase inflammation (by binding to RAGE which triggers an inflammatory cascade). AGEs might also increase SOST. From their previous work, it seems that the authors have bone AGE measures on these patients and they have shown their relationship with SOST. Do the increased AGEs relate to inflammation as measured by serum and bone expression?

(4) Were bone turnover markers done to show how the inflammation and Wnt findings relate to bone resorption and formation?

(5) RNA integrity values should be reported to confirm that the RNA has not degraded.

(6) The discussion of adiponectin could be clearer (studies are cited that show both positive and negative effects). Please clarify that adiponectin effects on bone are complex and what they are.

(7) Were patients excluded for prior as well as current antiresorptive medication use?

(8) Fig 4A. correlation between SOST mRNA and TNF-a mRNA seems to be driven by 1 outlier. Does the relationship persist if it is removed?

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 #3 (Public review):

In this manuscript, the authors examine circulating and bone parameters in patients with T2DM or obesity vs control subjects. Based on their findings they conclude that increased inflammation in bone of subjects with T2DM and obesity is negatively correlated with Wnt pathway signaling and bone strength.

Overall, this is a well done clinical study that provides further insights into the pathogenesis of bone loss associated with T2DM. However, there are a number of issues that the authors should address:

(1) The major conceptual problem is that the alterations in circulating and bone factors they observed would predominantly affect bone turnover and thus, bone mass. But bone mass is preserved in T2DM (as their own data show). They postulate that their findings lead to impaired bone quality, but it is not clear how this would occur. For example, the impairment in bone quality could be due to the accumulation of AGEs in bone in T2DM, and the correlations observed be true but unrelated. Along these lines, were serum or bone AGEs measured - and if not, is it possible for the authors to do so? At the least, this issue should be fully addressed in the Discussion if the authors are unable to provide additional data to address this.

(2) The T2DM patients were extremely well controlled. This may have limited some of the differences between groups. Was it not possible to select a group of less well-controlled patients - that is more the norm? This may also explain why the biomechanical indices in Table 3 were only marginally different in the T2DM vs the other groups. This point should also be addressed.

(3) The authors found some interesting differences in bone sclerostin levels. Were circulating sclerostin levels measured? This data would be of interest and should be provided.

(4) Fig 4A - the correlation between TNFa and SOST seems to be driven by one highly influential point. What happens if this point is removed? Is this point a formal statistical outlier? Please check this.

Reviewer #1 (Public review):

This article deals with the chemotactic behavior of E coli bacteria in thin channels (a situation close to 2D). It combines experiments and simulations.

The authors show experimentally that, in 2D, bacteria swim up a chemotactic gradient much more effectively when they are in the presence of lateral walls. Systematic experiments identify an optimum for chemotaxis for a channel width of ~8µm, close to the average radius of the circle trajectories of the unconfined bacteria in 2D. It is known that these circles are chiral and impose that the bacteria swim preferentially along the right-side wall when there is no chemotactic gradient. In the presence of a chemotactic gradient, this larger proportion of bacteria swimming on the right wall yields chemotaxis. This effect is backed by numerical simulations and a geometrical analysis.

If the conclusions drawn from the experiments presented in this article seem clear and interesting, I find that the key elements of the mechanism of this wall-directed chemotaxis are not sufficiently emphasized. Moreover, the paper would be clearer with more details on the hypotheses and the essential ingredients of the analyses.

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 #3 (Public review):

This paper addresses through experiment and simulation the combined effects of bacterial circular swimming near no-slip surfaces and chemotaxis in simple linear gradients. The authors have constructed a microfluidic device in which a gradient of L-aspartate is established to which bacteria respond while swimming while confined in channels of different widths. There is a clear effect that the chemotactic drift velocity reaches a maximum in channel widths of about 8 microns, similar in size to the circular orbits that would prevail in the absence of side walls. Numerical studies of simplified models confirm this connection.

The experimental aspects of this study are well executed. The design of the microfluidic system is clever in that it allows a kind of "multiplexing" in which all the different channel widths are available to a given sample of bacteria.

While the data analysis is reasonably convincing, I think that the authors could make much better use of what must be voluminous data on the trajectories of cells by formulating the mathematical problem in terms of a suitable Fokker-Planck equation for the probability distribution of swimming directions. In particular, I would like to see much more analysis of how incipient circular trajectories are interrupted by collisions with the walls and how this relates to enhanced chemotaxis. In essence, there needs to be a much clearer control analysis of trajectories without sidewalls to understand the mechanism in their presence.

The authors argue that these findings may have relevance to a number of physiological and ecological contexts. Yet, each of these would be characterized by significant heterogeneity in pore sizes and geometries, and thus it is very unclear whether or how the findings in this work would carry over to those situations.

Reviewer #1 (Public review):

Summary:

The authors present a modelling study to test the hypothesis that horizontal gene transfer (HGT) can modulate the outcome of interspecies competition in microbiomes, and in particular promote bistability in systems across scales. The premise is a model developed by the same authors in a previous paper where bistability happens because of a balance between growth rates and competition for a mutual resource pool (common carrying capacity). They show that introducing a transferrable element that gives a "growth rate bonus" expands the region of parameter space where bistability happens. The authors then investigate how often (in terms of parameter space) this bistability occurs across different scales of complexity, and finally under selection for the mobile element (framed as ABR selection).

Strengths:

The authors tackle an important, yet complex, question: how do different evolutionary processes impact the ecology of microbial ecosystems? They do a nice job at increasing the scales of heterogeneity and asking how these impact their main observable: bistability.

Weaknesses:

The author's starting point is their interaction LV model and the manuscript then explores how this model behaves under different scenarios. Because the structure of the model and the underlying assumptions essentially dictate these outcomes, I would expect to see much more focus on how these two aspects relate to the specific scenarios that are discussed. For example:

A key assumption is that the mobile element conveys a multiplicative growth rate benefit (1+lambda). However, the competition between the species is modelled as a factor gamma that modulates the competition for overall resource and thus appears in the saturation term (1+ S1/Nm + gamma2*S2/Nm). This means that gamma changes the perceived abundance of the other species (if gamma > 1, then from the point of view of S1 it looks like there are more S2 than there really are). Most importantly, the relationship between these parameters dictates whether or not there will be bistability (as the authors state).

This decoupling between the transferred benefit and the competition can have different consequences. One of them is that - from the point of view of the mobile element - the mobile element competes at different strengths within the same population compared to between. To what degree introducing such a mobile element modifies the baseline bistability expectation thus strongly depends on how it modifies gamma and lambda.

Thus, this structural aspect needs to be much more carefully presented to help the reader follow how much of the results are just trivial given the model assumptions and which have more of an emergent flavour. From my point of view, this has an important impact on helping the reader understand how the model that the authors present can contribute to the understanding of the question "how microbes competing for a limited number of resources stably coexist". I do appreciate that this changes the focus of the manuscript from a presentation of simulation results to more of a discussion of mathematical modelling.

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 #3 (Public review):

Hong et al. used a model they previously developed to study the impact of horizontal gene transfer (HGT) on microbial multispecies communities. They investigated the effect of HGT on the existence of alternative stable states in a community. The model most closely resembles HGT through the conjugation of incompatible plasmids, where the transferred genes confer independent growth-related fitness effects. For this type of HGT, the authors find that increasing the rate of HGT leads to an increasing number of stable states. This effect of HGT persists when the model is extended to include multiple competitive niches (under a shared carrying capacity) or spatially distinct patches (that interact in a grid-like fashion). Instead, if the mobile gene is assumed to reduce between-species competition, increasing HGT leads to a smaller region of multistability and fewer stable states. Similarly, if the mobile gene is deleterious an increase in HGT reduces the parameter region that supports multistability.

This is an interesting and important topic, and I welcome the authors' efforts to explore these topics with mathematical modeling. The manuscript is well written and the analyses seem appropriate and well-carried out. However, I believe the model is not as general as the authors imply and more discussion of the assumptions would be helpful (both to readers + to promote future theoretical work on this topic). Also, given the model, it is not clear that the conclusions hold quite so generally as the authors claim and for biologically relevant parameters. To address this, I would recommend adding sensitivity analyses to the manuscript.

Specific points

(1) The model makes strong assumptions about the biology of HGT, that are not adequately spelled out in the main text or methods, and will not generally prove true in all biological systems. These include:<br /> a) The process of HGT can be described by mass action kinetics. This is a common assumption for plasmid conjugation, but for phage transduction and natural transformation, people use other models (e.g. with free phage that adsorp to all populations and transfer in bursts).<br /> b) A subpopulation will not acquire more than one mobile gene, subpopulations can not transfer multiple genes at a time, and populations do not lose their own mobilizable genes. [this may introduce bias, see below].<br /> c) The species internal inhibition is independent of the acquired MGE (i.e. for p1 the self-inhibition is by s1).<br /> These points are in addition to the assumptions explored in the supplementary materials, regarding epistasis, the independence of interspecies competition from the mobile genes, etc. I would appreciate it if the authors could be more explicit in the main text about the range of applicability of their model, and in the methods about the assumptions that are made.

(2) I am not surprised that a mechanism that creates diversity will lead to more alternative stable states. Specifically, the null model for the absence of HGT is to set gamma to zero, resulting in pij=0 for all subpopulations (line 454). This means that a model with N^2 classes is effectively reduced to N classes. It seems intuitive that an LV-model with many more species would also allow for more alternative stable states. For a fair comparison, one would really want to initialize these subpopulations in the model (with the same growth rates - e.g. mu1(1+lambda2)) but without gene mobility.

(3) I am worried that the absence of double gene acquisitions from the model may unintentionally promote bistability. This assumption is equivalent to an implicit assumption of incompatibility between the genes transferred from different species. A highly abundant species with high HGT rates could fill up the "MGE niche" in a species before any other species have reached appreciable size. This would lead to greater importance of initial conditions and could thus lead to increased multistability.

This concern also feels reminiscent of the "coexistence for free" literature (first described here http://dx.doi.org/10.1016/j.epidem.2008.07.001 ) which was recently discussed in the context of plasmid conjugation models in the supplementary material (section 3) of https://doi.org/10.1098/rstb.2020.0478 .

(4) The parameter values tested seem to focus on very large effects, which are unlikely to occur commonly in nature. If I understand the parameters in Figure 1b correctly for instance, lambda2 leads to a 60% increase in growth rate. Such huge effects of mobile genes (here also assumed independent from genetic background) seem unlikely except for rare cases. To make this figure easier to interpret and relate to real-world systems, it could be worthwhile to plot the axes in terms of the assumed cost/benefit of the mobile genes of each species.

Something similar holds for the HGT rate (eta): given that the population of E. coli or Klebsiella in the gut is probably closer to 10^9 than 10^12 (they make up only a fraction of all cells in the gut), the assumed rates for eta are definitely at the high end of measured plasmid transfer rates (e.g. F plasmid transfers at a rate of 10^-9 mL/CFU h-1, but it is derepressed and considered among the fastest - https://doi.org/10.1016/j.plasmid.2020.102489 ). To adequately assess the impact of the HGT rate on microbial community stability it would need to be scanned on a log (rather than a linear) scale. Considering the meta-analysis by Sheppard et al. it would make sense to scan it from 10^-7 to 1 for a community with a carrying capacity around 10^9.

(5) It is not clear how sensitive the results (e.g. Figure 2a on the effect of HGT) are to the assumption of the fitness effect distribution of the mobile genes. This is related to the previous point that these fitness effects seem quite large. I think some sensitivity analysis of the results to the other parameters of the simulation (also the assumed interspecies competition varies from figure to figure) would be helpful to put the results into perspective and relate them to real biological systems.

Reviewer #1 (Public review):

Weiler, Teichert, and Margrie systematically analyzed long-range cortical connectivity, using a retrograde viral tracing strategy to identify layer and region-specific cortical projections onto the primary visual, primary somatosensory, and primary motor cortices. Their analysis revealed several hundred thousand inputs into each region, with inputs originating from almost all cortical regions but dominated in number by connections within cortical sub-networks (e.g. anatomical modules). Generally, the relative areal distribution of contralateral inputs followed the distribution of corresponding ipsilateral inputs. The largest proportion of inputs originated from layer 6a cells, and this layer 6 dominance was more pronounced for contralateral than ipsilateral inputs, which suggests that these connections provide predominantly feedback inputs. The hierarchical organization of input regions was similar between ipsi- and contralateral regions, except for within-module connections, where ipsilateral connections were much more feed-forward than contralateral. These results contrast earlier studies which suggested that contralateral inputs only come from the same region (e.g. V1 to V1) and from L2/3 neurons. Thus, these results provide valuable data supporting a view of interhemispheric connectivity in which layer 6 neurons play an important role in providing modulatory feedback.

The conclusions of this paper are mostly well-supported by the data and analysis, but additional consideration of possible experimental biases is needed.

Further discussion or analysis is needed about possible biases in uptake efficiency for different cell types. Is it possible that the nuclear retro-AAV has a tropism for layer 6 axons? Quantitative comparisons with results obtained with alternative methods such as rabies virus (Yao et al., 2023) or anterograde tracing (Harris et al., 2019) may be helpful for this.

Quantitative analysis of the injection sites should be included to account for possible biases. For example, L6 neurons are known to be the main target of contralateral inputs into the visual cortex (Yao et al., 2023). Thus, if the injections are biased towards or against layer 6 neurons, this may change the layer distribution of retrogradely labeled input cells. Comparison across biological replicates may help reveal sensitivity to particular characteristics of the injections.

The possibility of labeling axons of passage within the white matter should be addressed. This could potentially lead to false positive connections, contributing to the broad connectivity from most cortical regions that were observed.

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 #1 (Public review):

D'Oliviera et al. have demonstrated cleavage of human TRMT1 by the SARS-CoV-2 main protease in vitro. Following, they solved the structure of Mpro (Nsp5)-C145A bound to TRMT1 substrate peptide, revealing binding conformation distinct from most viral substrates. Overall, this work enhances our understanding of substrate specificity for a key drug target of CoV2. The paper is well-written and the data is clearly presented. It complements the companion article by demonstrating interaction between Mpro and TRMT1, as well as TRMT1 cleavage under isolated conditions in vitro. They show that cleaved TRMT1 has reduced tRNA binding affinity, linking a functional consequence to TRMT1 cleavage by MPro. Importantly, the revelation for flexible substrate binding of Nsp5 is fundamental for understanding Nsp5 as a drug target. Trmt1 cleavage assays by Mpro revealed similar kinetics for TRMT1 cleavage as compared to nsp8/9 viral polyprotein cleavage site. They purify TRMT1-Q350K, in which there is a mutation in the predicted cleavage consensus sequence, and confirm that it is resistant to cleavage by recombinant Mpro. I am unable to comment critically on the structural analyses as it is outside of my expertise. Overall, I think that these findings are important for confirming TRMT1 as a substrate of Mpro, defining substrate binding and cleavage parameters for an important drug target of SARS-CoV-2, and may be of interest to researchers studying RNA modifications.

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):

Summary:

In this manuscript, the authors have used a combination of enzymatic, crystallographic, and in silico approaches to provide compelling evidence for substrate selectivity of SARS-CoV-2 Mpro for human TRMT1.

Strengths:

In my opinion, the authors came close to achieving their intended aim of demonstrating the structural and biochemical basis of Mpro catalysis and cleavage of human TRMT1 protein. The revised version of the manuscript has addressed most of the questions I had posed in my earlier review.

Weaknesses:

Although several new hypotheses are generated from the Mpro structural data, the manuscript falls a bit short of testing them in functional assays, which would have solidified the conclusions the authors have drawn.

Reviewer #2 (Public review):

A high fraction of cells in early embryos carry aneuploid karyotypes, yet even chromosomally mosaic human blastocysts can implant and lead to healthy newborns with diploid karyotypes. Previous studies in other models have shown that genotoxic and proteotoxic stresses arising from aneuploidy lead to the activation of the p53 pathway and autophagy, which helps eliminate cells with aberrant karyotypes. These observations have been here evaluated and confirmed in human blastocysts. The study also demonstrates that the second lineage and formation of primitive endoderm are particularly impaired by aneuploidy.

Comments on revisions:

The authors have addressed the critical issues sufficiently. In particular, they improved the data analysis and added additional data from embryonal samples.

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 #1 (Public review):

Summary:

In this manuscript, Yan and colleagues introduce a modification to the previously published PETRI-seq bacterial single cell protocol to include a ribosomal depletion step based on a DNA probe set that selectively hybridizes with ribosome-derived (rRNA) cDNA fragments. They show that their modification of the PETRI-seq protocol increases the fraction of informative non-rRNA reads from ~4-10% to 54-92%. The authors apply their protocol to investigating heterogeneity in a biofilm model of E. coli, and convincingly show how their technology can detect minority subpopulations within a complex community.

Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single cell RNA-seq.

Comments on revised version:

The reviewers have responded thoughtfully and comprehensively to all of my comments. I believe the details of the protocol are now much easier to understand, and the text and methods have been significantly clarified. I have no further comments.

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 #1 (Public review):

Summary:

This work investigated the role of CXXC-finger protein 1 (CXXC1) in regulatory T cells. CXXC1-bound genomic regions largely overlap with Foxp3-bound regions and regions with H3K4me3 histone modifications in Treg cells. CXXC1 and Foxp3 interact with each other, as shown by co-immunoprecipitation. Mice with Treg-specific CXXC1 knockout (KO) succumb to lymphoproliferative diseases between 3 to 4 weeks of age, similar to Foxp3 KO mice. Although the immune suppression function of CXXC1 KO Treg is comparable to WT Treg in an in vitro assay, these KO Tregs failed to suppress autoimmune diseases such as EAE and colitis in Treg transfer models in vivo. This is partly due to the diminished survival of the KO Tregs after transfer. CXXC1 KO Tregs do not have an altered DNA methylation pattern; instead, they display weakened H3K4me3 modifications within the broad H3K4me3 domains, which contain a set of Treg signature genes. These results suggest that CXXC1 and Foxp3 collaborate to regulate Treg homeostasis and function by promoting Treg signature gene expression through maintaining H3K4me3 modification.

Strengths:

Epigenetic regulation of Treg cells has been a constantly evolving area of research. The current study revealed CXXC1 as a previously unidentified epigenetic regulator of Tregs. The strong phenotype of the knockout mouse supports the critical role CXXC1 plays in Treg cells. Mechanistically, the link between CXXC1 and the maintenance of broad H3K4me3 domains is also a novel finding.

Weaknesses:

(1) It is not clear why the authors chose to compare H3K4me3 and H3K27me3 enriched genomic regions. There are other histone modifications associated with transcription activation or repression. Please provide justification.

(2) It is not clear what separates Clusters 1 and 3 in Figure 1C. It seems they share the same features.

(3) The claim, "These observations support the hypothesis that FOXP3 primarily functions as an activator by promoting H3K4me3 deposition in Treg cells." (line 344), seems to be a bit of an overstatement. Foxp3 certainly can promote transcription in ways other than promoting H3K3me3 deposition, and it also can repress gene transcription without affecting H3K27me3 deposition. Therefore, it is not justified to claim that promoting H3K4me3 deposition is Foxp3's primary function.

(4) For the in vitro suppression assay in Figure S4C, and the Treg transfer EAE and colitis experiments in Figure 4, the Tregs should be isolated from Cxxc1 fl/fl x Foxp3 cre/wt female heterozygous mice instead of Cxxc1 fl/fl x Foxp3 cre/cre (or cre/Y) mice. Tregs from the homozygous KO mice are already activated by the lymphoproliferative environment and could have vastly different gene expression patterns and homeostatic features compared to resting Tregs. Therefore, it's not a fair comparison between these activated KO Tregs and resting WT Tregs.

(5) The manuscript didn't provide a potential mechanism for how CXXC1 strengthens broad H3K4me3-modified genomic regions. The authors should perform Foxp3 ChIP-seq or Cut-n-Taq with WT and Cxxc1 cKO Tregs to determine whether CXXC1 deletion changes Foxp3's binding pattern in Treg cells.

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 #3 (Public review):

In the report entitled "CXXC-finger protein 1 associates with FOXP3 to stabilize homeostasis and suppressive functions of regulatory T cells", the authors demonstrated that Cxxc1-deletion in Treg cells leads to the development of severe inflammatory disease with impaired suppressive function. Mechanistically, CXXC1 interacts with Foxp3 and regulates the expression of key Treg signature genes by modulating H3K4me3 deposition. Their findings are interesting and significant. However, there are several concerns regarding their analysis and conclusions.

Major concerns:

(1) Despite cKO mice showing an increase in Treg cells in the lymph nodes and Cxxc1-deficient Treg cells having normal suppressive function, the majority of cKO mice died within a month. What causes cKO mice to die from severe inflammation?

Considering the results of Figures 4 and 5, a decrease in Treg cell population due to their reduced proliferative capacity may be one of the causes. It would be informative to analyze the population of tissue Treg cells.

(2) In Figure 5B, scRNA-seq analysis indicated that Mki67+ Treg subset are comparable between WT and Cxxc1-deficient Treg cells. On the other hand, FACS analysis demonstrated that Cxxc1-deficient Treg shows less Ki-67 expression compared to WT in Figure 5I. The authors should explain this discrepancy.

In addition, the authors concluded on line 441 that CXXC1 plays a crucial role in maintaining Treg cell stability. However, there appears to be no data on Treg stability. Which data represent the Treg stability?

(3) The authors found that Cxxc1-deficient Treg cells exhibit weaker H3K4me3 signals compared to WT in Figure 7. This result suggests that Cxxc1 regulates H3K4me3 modification via H3K4 methyltransferases in Treg cells. The authors should clarify which H3K4 methyltransferases contribute to the modulation of H3K4me3 deposition by Cxxc1 in Treg cells.

Furthermore, it would be important to investigate whether Cxxc1-deletion alters Foxp3 binding to target genes.

(4) In Figure 7, the authors concluded that CXXC1 promotes Treg cell homeostasis and function by preserving the H3K4me3 modification since Cxxc1-deficient Treg cells show lower H3K4me3 densities at the key Treg signature genes. Are these Cxxc1-deficient Treg cells derived from mosaic mice? If Cxxc1-deficient Treg cells are derived from cKO mice, the gene expression and H3K4me3 modification status are inconsistent because scRNA-seq analysis indicated that expression of these Treg signature genes was increased in Cxxc1-deficient Treg cells compared to WT (Figure 5F and G).

Reviewer #1 (Public review):

Summary:

The manuscript by Rowell et al aims to identify differences in TCR recombination and selection between foetal and adult thymus in mice. Authors sequenced the unpaired bulk TCR repertoire in foetal and adult mice thymi and studied both TCRB and TCRa characteristics in the double negative (DN, CD4-CD8-) and single positive (SP4 CD4+CD8- and SP8 CD4-CD8+) populations. They identified age-related differences in TCRa and TCRB segment usage, including a preferential bias toward 3'TRAV and 5' TRAJ rearrangements in foetal cells compared to adults who had a larger perveance for 5'TRAV segments. By depleting the thymocyte population in adult thymi using hydrocortisone, the authors demonstrated that the repertoire became more foetal like, they, therefore, argue that the preferential 5'TRAV rearrangements in adults may be resulting from prolonged/progressive TCRa rearrangements in the adult thymocytes. In line with previous studies, Authors demonstrate that the foetal TCR repertoire was less diverse, less evenly distributed and had fewer non-template insertions while containing more clonal expansions. In addition, the authors claim that changes in V-J usage and CDR1 and CDR2 in the DN vs SP repertoires indicated that positive selection of foetal thymocytes are less dependent on interactions with the MHC.

Strengths:

Overall, the manuscript provides an extensive analysis of the foetal and adult TCR repertoire in the thymus, resulting in new insights in T cell development in foetal and adult thymi.

Weaknesses:

Three major concerns arise:<br /> (1) the authors have analysed TCR repertoires of only 4 foetal and 4 adult mice, considering the high spread the study may have been underpowered.

- The sample size was increased in the revised version

(2) Gating strategies are missing and

- These have now been provided in the revised version

(3) The manuscript is very technical and clearly aimed for a highly specialised audience with expertise in both thymocyte development and TCR analysis. Considering eLife is a scientific journal with a broader readership, Authors are recommended to provide schematics of the TCR rearrangements/their findings and include a summary of conclusions/implications of their findings at the end of each results section rather than waiting till the discussion. This will help the reader to interpret their findings while reading the results.

- These have now been included in the revised version

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 #1 (Public Review):

The authors report compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in familial SLE. They suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis.

The experiments in this revision showing that a weekly injection of ACK1 or BRK inhibitors induced various kinds of lupus-related autoantibodies in BALB/c supported the pivotal role of ACK1/BRK in systemic autoimmunity, although treated mice failed to demonstrate the full picture of lupus.

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 #1 (Public Review):

This article is interesting because the phenotype of the virus with mutations that alter the affinity of HS has been associated with how the viral particle interacts with HS and, thus, with binding and entry. However, the data in this manuscript is compelling and strongly suggests that the mutation that increases the affinity of HS alters capsid stability. To my knowledge, this is the first evidence that such mutation causes capsid destabilization. Furthermore, the idea that this mutation increases infectivity in cell lines by also using a pH-independent route and that, in vivo, this mutation attenuates the virus is very novel. Last year Wa-Chu's lab proposed that encephalopathic Alphaviruses produce capsids with different sizes and that this helps to attenuate highly pathogenic viruses (which might not be the case for non encepahlopatic Alphavirsues). However, they did not demonstrate whether these alterations attenuate the virus and if the altered morphology affects capsid stability. Therefore, this manuscript is fundamental as it contributes to understanding how the assembly/disassembly mechanism can be used to attenuate a virus. Furthermore, it is possible this mechanism could not be restricted to viruses that belong to the Picornaviridae family and opens a new door to understanding viral attenuation in other icosahedral viruses.

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 #4 (Public Review):

In this work, Tee et al. study the implications of Heparan Sulfate (HS) binding mutations observed on the Enterovirus A71 (EV-A71) capsid. HS-binding mutations are observed for several virus infections and are often presumed to be a cell culture adaptation. However, in the case of EV-A71, the presence of HS-binding mutations in clinical samples and the contradictory findings in animal studies have made the clinical relevance of HS-binding a subject of debate. Therefore, to better understand the role of HS-binding in EV-A71, the authors use a mouse-adapted EV-A71 variant (MP4) and compare it to a cell-adapted strong HS-binder (MP4-97R/167G). Using these two variants, the authors show that the strong HS-binder does not require acidification for uncoating and genome release. Furthermore, it is demonstrated that the capsid stability of the HS-binding variant is compromised, resulting in pH-independent uncoating. Overall, this study provides new insights demonstrating that seemingly beneficial mutations increasing viral replication may be counterbalanced by other unintended consequences.

Strengths:

The thoroughness of the experiments performed to demonstrate that the HS-binding phenotype results in pH-independent entry and capsid destabilisation is worth highlighting. In this regard, the authors have explored viral entry using a range of approaches involving lysosomotropic drugs, viral binding assays, and neutral red-labelled viruses coupled with diverse techniques such as FISH, RNAscope, and transient expression of constitutively active molecules to inhibit parts of the viral cycle. In my opinion, this is necessary to rule out the other downstream effects of the lysomotropic drugs and to confirm the role of the HS-binding mutation in the entry phase. The use of in silico analysis coupled with negative staining electron microscopy and environmental challenge assays is notable. Finally, the demonstration of some of the work using a human-relevant strain is commendable.

Weaknesses:

A major weakness in this study is the focus on using a mouse-adapted EV-A71 strain (MP4). In the introduction, it is argued that HS-binding mutations are controversial due to their occurrence in cell culture. However, due to host limitations, mice are not the natural hosts for EV-A71 and thus, the same argument can be made for a mouse-adapted strain. It is not clear how different this strain is from circulating EV-A71 strains and the relevance of these findings to the human situation is questionable. This is particularly made evident in the discussion where it is highlighted that HS-binding variants (VP1-145G/Q mutants) have been associated with severe neurological cases while the same variants show attenuated phenotypes in mice and monkeys. This contrast between clinical data and animal studies should be highlighted in the introduction, rather than later in the discussion, as currently the in vivo animal studies are presented as the optimal situation and may lead to misconstrued conclusions from the results.

An important consideration is that the results are based primarily on image analysis. The inclusion of RT-qPCR and/or plaque assays as supplementary data will help strengthen the findings. Moreover, there are suggestions of an intermediate binder having a different phenotype. As this intermediate binder is the clinical phenotype, data on the entry of this intermediate binder will be valuable.

Another weakness in the study is the lack of contextualization of the results to current EV-A71 literature. For instance, SCARB2 is referred to as the internalization receptor but a recent study has shown that SCARB2 is not required for internalization (https://doi.org/10.1128%2Fjvi.02042-21). The findings from this study are consistent with the localization of SCARB2 in the lysosomal membranes. Furthermore, the same study has highlighted host sulfation as a key factor in EV-A71 entry. Post-translational sulfation introduces negatively charged residues on host proteins including HS and SCARB2. This increases the binding of HS-binding strains to these proteins. In this regard, the reduced infectivity upon soluble SCARB2 treatment may simply be due to enhanced binding rather than capsid opening as suggested in the results. Therefore, additional experiments (e.g. nSEM following soluble SCARB2 treatment) must be performed to support the conclusion of capsid opening, due to inherent instability, upon SCARB2 binding.

In addition to the above, other existing literature on EV-A71 pathogenesis using organoids contradicts some of the explanations of differential phenotype in clinical observations versus mice models. In the introduction, it is suggested that reduced neurovirulence of HS-binding strains is due to binding to the vascular endothelia. However, the correlation of clinical severity to viremia (https://doi.org/10.1186/1471-2334-14-417) and the association of HS-binding mutants to clinical disease counteract this suggestion. Similarly, viral infection in human organoids with EV-A71 results in as low as 0.4% of the cells being infected (https://doi.org/10.1038/s41564-023-01339-5). In this case, if viral binding to (ubiquitously expressed) HS results in viral trapping then the HS-binding mutants should show lowered infectivity in organoid models rather than the observed higher infectivity (https://doi.org/10.3389/fmicb.2023.1045587, https://doi.org/10.1038/s41426-018-0077-2). Finally, EV-A71 release has also been shown to occur in exosomes (https://doi.org/10.1093%2Finfdis%2Fjiaa174) which effectively provides a protective lipid membrane. These recent findings must be incorporated into the article and will help better contextualize their findings.

Overall, the authors present new findings with convincing methodology. The manuscript can be improved in the contextualization of the findings and highlighting the weakness in translating these findings to resolve the debate surrounding the relevance of HS-binding phenotype. The inclusion of additional experiments and data recommended to the authors will also help strengthen the manuscript.

Reviewer #1 (Public Review):

This manuscript was previously reviewed and this earlier evaluation resulted in two conflicting assessments. I fully endorse the favourable opinion of former Reviewer 1 and find most negative comments of former Reviewer 2 inappropriate.

This work is absolutely necessary. Even though the authors find it difficult to be fully assertive in the end, their ground work in trying to demonstrate the existence of bias in PPI data is undeniably valuable. Other authors have tried before to show the limitation of unequivocally assigning the degree distribution to a power law but these doubts have had a weak impact. This new study is a great opportunity to discuss further a concern for a simplistic view of PPI network topology. The recent contribution of Broido & Clauset was definitely one to bounce on. The approach of this new manuscript is compelling. Dividing the study in several parts, each reflecting an attempt to bring out commonly used shortcuts in PPI network analyses, makes sense.

Surprisingly, the authors do not refer to the endless controversy of labeling hubs as party or date, which is another manifestation of the interpretative bias of PPI data.

The only worthy point prompted by former Reviewer 2 is the effect of spoke expansion. In their response, the authors suggest that it would probably extend questioning and even if it is considered as future work, it could be mentioned in the main manuscript.

In the end, this submission is an invitation to constructively rethink the analysis of PPI networks and it feeds the discussion on modelling degree distributions that should not be considered as a solved issue.

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 #3 (Public Review):

I would like to congratulate the authors to an impressive piece of work highlighting important real and potential biases, which may lead to power-law distributed node degrees in protein-protein interaction networks.<br /> This manuscript is easy to follow and very well written manuscript.<br /> I truly enjoyed the concise and convincing scientific presentation.<br /> Even if some of the concerns have already been discussed or raised in the past, the manuscript assesses potential biases in PPIs in a rigorous manner.

I deem the following observations highly relevant to be communicated to the community again:<br /> (1) PL-like distributions emerge by aggregation of data sets alone.<br /> (2) Research interest in itself is PL-distributed and drives PL-like properties in PPI networks<br /> (3) Bait usage is a major driver of PL-like behaviour.<br /> (4) Accounting for biases changes the biological interpretation of the networks<br /> (5) Simulation studies further corroborate these findings.

Reviewer #1 (Public review):

This is a very relevant study, with the potential of having high impact on future research on the evolution of chemical defense mechanisms in animals. The authors present a substantial number of new and surprising experimental results, i.e., the presence in low quantities of alkaloids in amphibians previously deemed to lack these toxins. These data are then combined with literature data to weave the importance of passive accumulation mechanisms into a 4-phases scenario of the evolution of chemical defense in alkaloid-containing poison frogs.

In general, the new data presented in the manuscript are of high quality and high scientific interest, the suggested scenario compelling, and the discussion thorough. Also, the revised version of the manuscript has been carefully prepared with a high quality of illustrations. UI did not detect typos in the text

Understanding that the majority of dendrobatid frogs, including species considered undefended, can contain low quantities of alkaloids in their skin provides an entirely new perspective to our understanding of how the amazing specializations of poison frogs evolved. Although only few non-dendrobatids were included in the alkaloid screening, some of these also included minor quantities of alkaloids, and the capacity of passive alkaloid accumulation may therefore characterize numerous other frog clades, or even amphibians in general.

The overall quality of the work is exceptional. The authors also have done a fantastic job restructuring the manuscript in response to my initial comments, and it is now very clear which new hypotheses are presented and which testable predictions for future studies derive from these hypotheses. This study will be highly influential in informing and guiding future research on toxicity, alkaloid sequestration and resistance, and evolution of aposematism.

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 #1 (Public review):

Summary:

Utilizing transgenic lineage tracing techniques and tissue clearing-based advanced imaging and three-dimensional slices reconstruction, the authors comprehensively mapped the distribution atlas of NFATc1+ and PDGFR-α+ cells in dental and periodontal mesenchyme and tracked their in vivo trajectories. This important work expands our understanding of both single and double positive NFATc1 and PDGFR-α cells in maintaining dental and periodontal mesenchyme homeostasis, and will provide impact on clinical application and investigation. The strength of this work is convincing, as it employed CRISPR/Cas9-mediated gene editing to generate two dual recombination systems, and mapped gNFATc1+ and PDGFR-α+ cells residing in dental and periodontal mesenchyme, their capacity for progeny cell generation, and their inclusive, exclusive and hierarchical relations in homeostasis, generating a spatiotemporal atlas of these skeletal stem cell population.

This work has theoretical or practical implications in the periodontal field. The methods, data and analyses support the claims.

Comments on revised version:

The authors have addressed my main concerns.

Reviewer #1 (Public review):

In this work, Veseli et al. present a computational framework to infer the functional diversity of microbiomes in relation to microbial diversity directly from metagenomic data. The framework reconstructs metabolic modules form metagenomes and calculates the per-population copy number of each module, resulting in the proportion of microbes in the sample carrying certain genes. They applied this framework to a dataset of gut microbiomes from 109 inflammatory bowel disease (IBD) patients, 78 patients with other gastrointestinal conditions, and 229 healthy controls. The found that the microbiomes of IBD patients were enriched in a high fraction of metabolic pathways, including biosynthesis pathways such as those for amino acids, vitamins, nucleotides, and lipids. Hence, they had higher metabolic independence compared with healthy controls. To an extent, the authors also found a pathway enrichment suggesting higher metabolic independence in patients with gastrointestinal conditions other than IBD indicating this could be a signal for a general loss in host health. Finally, a machine learning classifier using high metabolic independence in microbiomes could predict IBD with good accuracy. Overall, this is an interesting and well-written article and presents a novel workflow that enables a comprehensive characterization of microbiome cohorts.

Comments on revisions:

I believe that after the second round of revisions, the Reviewers sufficiently addressed the comments and improved the manuscript. Open questions have been answered. I have no further comments.

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 #3 (Public review):

The major strength of this manuscript is the "anvi-estimate-metabolism' tool, which is already accessible online, extensively documented, and potentially broadly useful to microbial ecologists. Inclusion of extensive benchmarking and validation on simulated metagenomes has further increased confidence in this approach. Further, the conceptual insights raise interesting hypotheses that could be pursued in follow-on experimental work.

Comments on revisions:

Thank you for the very thorough response and congratulations!

Joint Public Review:

The subject area will have general appeal to those interested in the study of Pavlovian conditioning. The paper is important, showcasing a rigorous experimental design, several different approaches to data analysis, careful consideration of prior literature, and a thorough introduction. The results indicate that the rate of Pavlovian learning is determined by the ratio of reward rate during cue to the overall reward rate, and that the asymptotic response rate is determined by the reward rate during cue. These findings provide context to many conflicting recent results on this topic and are supported by strong/convincing evidence.

It is additionally claimed that the parameter that governs the acquisition and asymptote of responding in rats is exactly the same as that which governs the acquisition and asymptote of responding in the Gibbon and Balsam (1981) study that used pigeons as experimental subjects; and that the rates of responding during the inter-trial interval and the cue are proportional to the corresponding reward rates with the same proportionality constant. In both of these respects, there are several points that stand in need of clarification - at present, the strength of the evidence in support of these claims is solid. More generally, there are some points that could clarify aspects of rate estimation theory and, thereby, increase the rating of the paper from important to fundamental. These points range from analytical to conceptual and are presented below.

ANALYTICAL

(1) A key claim made here is that the same relationship (including the same parameter) describes data from pigeons by Gibbon and Balsam (1981; Figure 1) and the rats in this study (Figure 3). The evidence for this claim, as presented here, is not as strong as it could be. This is because the measure used for identifying trials to criterion in Figure 1 appears to differ from any of the criteria used in Figure 3, and the exact measure used for identifying trials to criterion influences the interpretation of Figure 3***. To make the claim that the quantitative relationship is one and the same in the Gibbon-Balsam and present datasets, one would need to use the same measure of learning on both datasets and show that the resultant plots are statistically indistinguishable, rather than simply plotting the dots from both data sets and spotlighting their visual similarity. In terms of their visual characteristics, it is worth noting that the plots are in log-log axis and, as such, slight visual changes can mean a big difference in actual numbers. For instance, between Figure 3B and 3C, the highest information group moves up only "slightly" on the y-axis but the difference is a factor of 5 in the real numbers. Thus, in order to support the strong claim that the quantitative relationships obtained in the Gibbon-Balsam and present datasets are identical, a more rigorous approach is needed for the comparisons.

***The measure of acquisition in Figure 3A is based on a previously established metric, whereas the measure in Figure 3B employs the relatively novel nDKL measure that is argued to be a better and theoretically based metric. Surprisingly, when r and r2 values are converted to the same metric across analyses, it appears that this new metric (Figure 3B) does well but not as well as the approach in Figure 3A. This raises questions about why a theoretically derived measure might not be performing as well on this analysis, and whether the more effective measure is either more reliable or tapping into some aspect of the processes that underlie acquisition that is not accounted for by the nDKL metric.

(2) Another interesting claim here is that the rates of responding during ITI and the cue are proportional to the corresponding reward rates with the same proportionality constant. This too requires more quantification and conceptual explanation. For quantification, it would be more convincing to calculate the regression slope for the ITI data and the cue data separately and then show that the corresponding slopes are not statistically distinguishable from each other. Conceptually, it is not clear why the data used to test the ITI proportionality came from the last 5 conditioning sessions. What were the decision criteria used to decide on averaging the final 5 sessions as terminal responses for the analyses in Figure 5? Was this based on consistency with previous work, or based on the greatest number of sessions where stable data for all animals could be extracted?

If the model is that animals produce response rates during the ITI (a period with no possible rewards) based on the overall rate of rewards in the context, wouldn't it be better to test this before the cue learning has occurred? Before cue learning, the animals would presumably only have attributed rewards in the context to the context and thus, produce overall response rates in proportion to the contextual reward rate. After cue learning, the animals could technically know that the rate of rewards during ITI is zero. Why wouldn't it be better to test the plotted relationship for ITI before cue learning has occurred? Further, based on Figure 1, it seems that the overall ITI response rate reduces considerably with cue learning. What is the expected ITI response rate prior to learning based on the authors' conceptual model? Why does this rate differ from pre and post-cue learning? Finally, if the authors' conceptual framework predicts that ITI response rate after cue learning should be proportional to contextual reward rate, why should the cue response rate be proportional to the cue reward rate instead of the cue reward rate plus the contextual reward rate?

(3) There is a disconnect between the gradual nature of learning shown in Figures 7 and 8 and the information-theoretic model proposed by the authors. To the extent that we understand the model, the animals should simply learn the association once the evidence crosses a threshold (nDKL > threshold) and then produce behavior in proportion to the expected reward rate. If so, why should there be a gradual component of learning as shown in these figures? In terms of the proportional response rule to the rate of rewards, why is it changing as animals go from 10% to 90% of peak response? The manuscript would be greatly strengthened if these results were explained within the authors' conceptual framework. If these results are not anticipated by the authors' conceptual framework, this should be explicitly stated in the manuscript.

(4) Page 27, Procedure, final sentence: The magazine responding during the ITI is defined as the 20 s period immediately before CS onset. The range of ITI values (Table 1) always starts as low as 15 s in all 14 groups. Even in the case of an ITI on a trial that was exactly 20 s, this would also mean that the start of this period overlaps with the termination of the CS from the previous trial and delivery (and presumably consumption) of a pellet. It should be indicated whether the definition of the ITI period was modified on trials where the preceding ITI was < 20 s, and if any other criteria were used to define the ITI. Were the rats exposed to the reinforcers/pellets in their home cage prior to acquisition?

(5) For all the analyses, the exact models that were fit and the software used should be provided. For example, it is not necessarily clear to the reader (particularly in the absence of degrees of freedom) that the model discussed in Figure 3 fits on the individual subject data points or the group medians. Similarly, in Figure 6 there is no indication of whether a single regression model was fit to all the plotted data or whether tests of different slopes for each of the conditions were compared. With regards to the statistics in Figure 6, depending on how this was run, it is also a potential problem that the analyses do not correct for the potentially highly correlated multiple measurements from the same subjects, i.e. each rat provides 4 data points which are very unlikely to be independent observations.

CONCEPTUAL

(1) We take the point that where traditional theories (e.g., Rescorla-Wagner) and rate estimation theory (RET) both explain some phenomenon, the explanation in terms of RET may be preferred as it will be grounded in aspects of an animal's experience rather than a hypothetical construct. However, like traditional theories, RET does not explain a range of phenomena - notably, those that require some sort of expectancy/representation as part of their explanation. This being said, traditional theories have been incorporated within models that have the representational power to explain a broader array of phenomena, which makes me wonder: Can rate estimation be incorporated in models that have representational power; and, if so, what might this look like? Alternatively, do the authors intend to claim that expectancy and/or representation - which follow from probabilistic theories in the RW mould - are unnecessary for explanations of animal behaviour?***

***If the authors choose to reply to these points, they should consider taking advantage of an "Ideas and Speculation" subsection within the Discussion that is supported by eLife [ https://elifesciences.org/inside-elife/e3e52a93/elife-latest-including-ideas-and-speculation-in-elife-papers ].

(2) The discussion of Rescorla's (1967) and Kamin's (1968) findings needs some elaboration. These findings are already taken to mean that the target CS in each design is not informative about the occurrence of the US - hence, learning about this CS fails. In the case of blocking, we also know that changes in the rate of reinforcement across the shift from stage 1 to stage 2 of the protocol can produce unblocking. Perhaps more interesting from a rate estimation perspective, unblocking can also be achieved in a protocol that maintains the rate of reinforcement while varying the sensory properties of the US (Wagner). How does rate estimation theory account for these findings and/or the demonstrations of trans-reinforcer blocking (Pearce-Ganesan)? Are there other ways that the rate estimation account can be distinguished from traditional explanations of blocking and contingency effects? If so, these would be worth citing in the discussion. More generally, if one is going to highlight seminal findings (such as those by Rescorla and Kamin) that can be explained by rate estimation, it would be appropriate to acknowledge findings that challenge the theory - even if only to note that the theory, in its present form, is not all-encompassing. For example, it appears to me that the theory should not predict one-trial overshadowing or the overtraining reversal effect - both of which are amenable to discussion in terms of rates. I assume that the signature characteristics of latent inhibition and extinction would also pose a challenge to rate estimation theory, just as they pose a challenge to Rescorla-Wagner and other probability-based theories. Is this correct?

Reviewer #1 (Public Review):

Summary:

A key challenge at the second chemical step of splicing is the identification of the 3' splice site of an intron. This requires recruitment of factors dedicated to the second chemical step of splicing and exclusion of factors dedicated to the first chemical step of splicing. Through the highest resolution cyroEM structure of the spliceosome to-date, the authors show the binding site for Fyv6, a factor dedicated to the second chemical step of splicing, is mutually exclusive with the binding site for a distinct factor dedicated to the first chemical step of splicing, highlighting that splicing factors bind to the spliceosome at a specific stage not only by recognizing features specific to that stage but also by competing with factors that bind at other stages. The authors further reveal that Fyv6 functions at the second chemical step to promote selection of 3' splice sites distal to a branch point and thereby discriminate against proximal, suboptimal 3' splice site. Lastly, the authors show by cyroEM that Fyv6 physically interacts with the RNA helicase Prp22 and by genetics Fyv6 functionally interacts with this factor, implicating Fyv6 in 3'SS proofreading and mRNA release from the spliceosome. The evidence for this study is robust, with the inclusion of genomics, reporter assays, genetics, and cyroEM. Further, the data overall justify the conclusions, which will be of broad interest.

Strengths:

(1) The resolution of the cryoEM structure of Fyv6-bound spliceosomes at the second chemical step of splicing is exceptional (2.3 Angstroms at the catalytic core; 3.0-3.7 Angstroms at the periphery), providing the best view of this spliceosomal intermediate in particular and the core of the spliceosome in general.<br /> (2) The authors observe by cryoEM three distinct states of this spliceosome, each distinguished from the next by progressive loss of protein factors and/or RNA residues. The authors appropriately refrain from overinterpreting these states as reflecting distinct states in the splicing cycle, as too many cyroEM studies are prone to do, and instead interpret these observations to suggest interdependencies of binding. For example, when Fyv6, Slu7, and Prp18 are not observed, neither are the first and second residues of the intron, which otherwise interact, suggesting an interdependence between 3' splice site docking on the 5' splice site and binding of these second step factors to the spliceosome.<br /> (3) Conclusions are supported from multiple angles.<br /> (4) The interaction between Fyv6 and Syf1, revealed by the cyroEM structure, was shown to account for the temperature-sensitive phenotypes of a fyv6 deletion, through a truncation analysis.<br /> (5) Splicing changes were observed in vivo both by indirect copper reporter assays and directly by RT-PCR.<br /> (6) Changes observed by RNA-seq are validated by RT-PCR.<br /> (7) The authors go beyond simply observing a general shift to proximal 3'SS usage in the fyv6 deletion by RNA-seq by experimentally varying branch point to 3' splice site distance experimentally in a reporter and demonstrating in a controlled system that Fyv6 promotes distal 3' splice sites.<br /> (8) The importance of the Fyv6-Syf1 interaction for 3'SS recognition is demonstrated by truncations of both Fyv6 and of Syf1.<br /> (9) In general, the study was executed thoroughly and presented clearly.

Comments on revisions:

The authors have satisfactorily addressed the comments.

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 #3 (Public Review):

In this manuscript the authors expand their initial identification of Fyv6 as a protein involved in the second step of pre-mRNA splicing to investigate the transcriptome-wide impact of Fyv6 on splicing and gain a deeper understanding of the mechanism of Fyv6 action.

They first use deep sequencing of transcripts in cells depleted of Fyv6 together with Upf1 (to limit loss of mis-spliced transcripts) to identify broad changes in the transcriptome due to loss of Fyv6. This includes both changes in overall gene expression, that are not deeply discussed, as well as alterations in choice of 3' splice sites - which is the focus of the rest of the manuscript

They next provide the highest resolution structure of the post-catalytic spliceosome to date; providing unparalleled insight into details of the active site and peripheral components that haven't been well characterized previously.

Using this structure they identify functionally critical interactions of Fyv6 with Syf1 but not Prp22, Prp8 and Slu7. Finally, a suppressor screen additionally provides extensive new information regarding functional interactions between these second step factors.

Overall this manuscript reports new and essential information regarding molecular interactions within the spliceosome that determine the use of the 3' splice site. It would be helpful, especially to the non-expert, to summarize these in a table, figure or schematic in the discussion.

Comments on revisions:

I'm satisfied with the changes made in the revision.

Reviewer #1 (Public review):

Summary:

This study reports single-cell RNA sequencing results of lung adenocarcinoma, comparing 4 treatment-naive and 5 post-neoadjuvant chemotherapy tumor samples.<br /> The authors claim that there are metabolic reprogramming in tumor cells as well as stromal and immune cells after chemotherapy.<br /> The most significant findings are in the macrophages that there are more pro-tumorigenic cells after chemotherapy, i.e. CD45+CD11b+ARG+ cells. In the treatment-naive samples, more anti-tumorigenic CD45+CD11b+CD86+ macrophages are found. They sorted each population and performed functional analyses.

Strengths:

Comparison of the treatment-naive and post-chemotherapy samples of lung adenocarcinoma.

Weaknesses:

After the revision, issues remain with lengthy descriptive clustering type analysis, insufficient statistical support, and inefficient figure presentation.

Reviewer #1 (Public review):

Summary:

This study examined the associations of healthy lifestyles with comprehensive and organ-specific biological ages. It emphasized the importance of lifestyle factors in determining biological ages, which were using common blood biomarkers and body measures.

Strengths:

The data were from a large cohort study and defined comprehensive and six-specified BA.

Weaknesses highlighted previously:

(1) Since only 8.5% of participants from the CMEC were included in the study, has any section bias happened?

(2) The author should specify the efficiency of FFQ. How FFQ can genuinely reflect the actual intake? Moreover, how was the aMED calculated in your study?

(3) HLI (range) and HLI (category) should be clearly defined.

(4) The rationale of comprehensive and specific BA construction should be clearly defined and discussed. For example, can cardiopulmonary BA be reflected only by using cardiopulmonary status? I do not think so.

(5) The lifestyle index is defined based on an equal-weight approach, but this does not reflect reality and can not fully answer the research questions it raises.

Comments on the revised version:

The author answered most of the questions raised. However, since wine is the most important component of aMED, removing wine or alcohol may result in biased estimates. In addition, The authors acknowledge the limitations of this approach, namely that some biomarkers may not fully capture the complete aging process of the system; this weakness is particularly remarkable in organ-specific BA. The authors emphasize that it is cost-effective and easy to implement. However, the results associated with organ-specific BA may not be credible because they do not fully reflect the state of a particular organ. It is recommended that these shortcomings and the applicability of the results should be discussed in the text.

Reviewer #1 (Public review):

The authors sought to determine the impact of early antiretroviral treatment on the size, composition, and decay of the HIV latent reservoir. This reservoir represents the source of viral rebound upon treatment interruption and therefore constitutes the greatest challenge to achieving an HIV cure. A particular strength of this study is that it reports on reservoir characteristics in African women, a significantly understudied population, of whom some have initiated treatment within days of acute HIV diagnosis. With the use of highly sensitive and current technologies, including digital droplet PCR and near full-length genome next-generation sequencing, the authors generated a valuable dataset for investigation of proviral dynamics in women initiating early treatment compared to those initiating treatment in chronic infection. The authors confirm previous reports that early antiretroviral treatment restricts reservoir size, but further show that this restriction extends to defective viral genomes, where late treatment initiation was associated with a greater frequency of defective genomes. Furthermore, an additional strength of this study is the longitudinal comparison of viral dynamics post-treatment, wherein early treatment was shown to be associated with a more rapid rate of decay in proviral genomes, regardless of intactness, over a period of one year post-treatment. While it is indicated that intact genomes were not detected after one year following early treatment initiation, sampling depth is noted as a limitation of the study by the authors, and caution should thus be taken with interpretation where sequence numbers are low. Defective genomes are more abundant than intact genomes and are therefore more likely to be sampled. Early treatment was also associated with reduced proviral diversity and fewer instances of polymorphisms associated with cytotoxic T-lymphocyte immune selection. This is expected given that rapid evolution and extensive immune selection are synonymous with HIV infection in the absence of treatment, yet points to an additional benefit of early treatment in the context of immune therapies to restrict the reservoir.

This is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C. The data and findings from this study therefore represent a much-needed resource in furthering our understanding of HIV persistence and informing broadly impactful cure strategies. The analysis on clonal expansion of proviral genomes may be limited by higher sequence homogeneity in hyperacute infection i.e., cells with different proviral integration sites may have a higher likelihood of containing identical genomes compared to chronic infection.

Overall, these data demonstrate the distinct benefits of early treatment initiation at reducing the barrier to a functional cure for HIV, not only by restricting viral abundance and diversity but also potentially through the preservation of immune function and limiting immune escape. It therefore provides clues to curative strategies even in settings where early diagnosis and treatment may be unlikely.

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 #1 (Public review):

Previous studies have highlighted some of these paracrine activities of Toxoplasma - and Rasogi et al (mBio, 2020) used a single cell sequencing approach of cells infected in vitro with the WT or MYR KO parasites - and one of their conclusions was that MYR-1 dependent paracrine activities counteract ROP-dependent processes. Similarly, Chen et al (JEM 2020) highlighted that a particular rhoptry protein (ROP16) could be injected into uninfected macrophages and move them to an anti-inflammatory state that might benefit the parasite.

Caveats around immunity and as yet no insight into how this works. In Fig 2 there is a marked defect in the ability of the parasites to expand at day 2 and day 5. Together, these data sets suggest that this paracrine effect mediated by MYR-1 works early - well before the development of adaptive responses.

Comments on revisions:

The authors have provided their perspective on the original review. There were some previous comments that revolved around whether some of the early changes were masked by pooling data sets where they have reiterated that it is not statistically different. Would have been nice to have seen out addressed by having experiments that were appropriately powered. But it's their call.

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 #1 (Public review):

In their manuscript, authors Isotani et al used in vivo and ex vivo models to show that nicotine could promote stemness and tumorigenicity in murine model. The authors further provided data supporting that the effects of nicotine on stem cell proliferation and tumor initiation were mediated by the Hippo-YAP/TAZ and Notch signal pathway.

The major strength of this study is the using a set of tools, including Lgr5 reporter mice (Lgr5-EGFP-IRES-CreERT2 mice), stem cell-specific Apc knockout mice (Lgr5CreER Apcfl/fl mice), organoids derived from these mice and chemical compounds (agonists and antagonists) to demonstrate nicotine affects stem cells rather than Paneth cells, leading to increased intestinal stemness and tumorigenicity. Whereas, all models are restricted to mice, lacking analysis of human samples or human intestinal organoids to prove the human relevance of these findings.

Overall, the presented results support their conclusions. A previous study reported that nicotine acts through the α2β4 nAChR to enhance Wnt production by Paneth cells, which subsequently affects ISCs. In contrast, this manuscript demonstrated that nicotine directly promotes ISCs through α7-nAChR, independent of Paneth cells. Therefore, this manuscript offers novel insights into the mechanism of nicotine's effects on the mouse intestine.

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 #2 (Public review):

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

Strengths:

Overall, I found these results clear, convincing, and well-presented. There is growing interest in developing an expanded toolkit for genomic epidemiology in malaria, and detecting changes in transmission intensity is one major application. As the authors summarize, there is no one-size-fits-all approach, and the Bayesian MOIvar estimate developed here has the potential to complement currently used methods, particularly in regions with high diversity/transmission. I find its extension to a calculation of absolute parasite numbers appealing as this could serve as both a conceptually straightforward and biologically meaningful metric.

Weaknesses:

While I understand the conceptual importance of distinguishing among parasite prevalence, mean MOI, and absolute parasite number, I am not fully convinced by this manuscript's implementation of "census population size". The authors reference the population genetic literature, but within the context of that field, "census population size" refers to the total population size (which, if not formally counted, can be extrapolated) as opposed to "effective population" size, which accounts for a multitude of demographic factors. There is often interesting biology to be gleaned from the magnitude of difference between N and Ne. In this manuscript, however, "census population size" is used to describe the number of distinct parasites detected within a sample, not a population. As a result, the counts do not have an immediate population genetic interpretation and cannot be directly compared to Ne. This doesn't negate their usefulness but does complicate the use of a standard population genetic term. In contrast, I think that sample parasite count will be most useful in an epidemiological context, where the total number of sampled parasites can be contrasted with other metrics to help us better understand how parasites are divided across hosts, space and time. However, for this use, I find it problematic that the metric does not appear to correct for variations in participant number. For instance, in this study, participant numbers especially varied across time for 1-5 year-olds (N=356, 216, 405, and 354 in 2012, 2014, 2015, and 2017 respectively). This sample size variability is accounted for with other metrics like mean MOI. In sum, while the manuscript opens up an interesting discussion, I'm left with an incomplete understanding of the robustness and interpretability of the new proposed metric.

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 #1 (Public review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with two lipid compositions similar to native viral membranes. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region. The revised manuscript demonstrates that these lipid interactions are robust to alterations in membrane composition and rigidity. However, it does not address the reverse-that phospholipids known experimentally not to associate with these antibodies (if any such lipids exist) also fail to interact in MD simulations.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. These simulations recapitulate lipid binding interactions solved in published crystallographic studies but also lead to the discovery of a novel lipid binding site the authors term the "Loading Site", which could guide future experiments with this antibody.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. These CG simulations , which cannot resolve atomistic interactions, are nonetheless compelling because negative controls (ab 13h11, BSA) that should not associate with membrane indeed sample significantly less membrane.

Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive, creative, and biologically inspired. Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

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 #1 (Public review):

Summary:

In their manuscript, Gomez-Frittelli and colleagues characterize the expression of cadherin6 (and -8) in colonic IPANs of mice. Moreover, they found that these cdh6-expressing IPANs are capable of initiating colonic motor complexes in the distal colon, but not proximal and midcolon. They support their claim by morphological, electrophysiological, optogenetic, and pharmacological experiments.

Strengths:

The work is very impressive and involves several genetic models and state-of-the-art physiological setups including respective controls. It is a very well-written manuscript that truly contributes to our understanding of GI-motility and its anatomical and physiological basis. The authors were able to convincingly answer their research questions with a wide range of methods without overselling their results.

Weaknesses:

The authors put quite some emphasis on stating that cdh6 is a synaptic protein (in the title and throughout the text), which interacts in a homophilic fashion. They deduct that cdh6 might be involved in IPAN-IPAN synapses (line 247ff.). However, Cdh6 does not only interact in synapses and is expressed by non-neuronal cells as well (see e.g., expression in the proximal tubuli of the kidney). Moreover, cdh6 does not only build homodimers, but also heterodimers with Chd9 as well as Cdh7, -10, and -14 (see e.g., Shimoyama et al. 2000, DOI: 10.1042/0264-6021:3490159). It would therefore be interesting to assess the expression pattern of cdh6-proteins using immunostainings in combination with synaptic markers to substantiate the authors' claim or at least add the possibility of cell-cell-interactions other than synapses to the discussion. Additionally, an immunostaining of cdh6 would confirm if the expression of tdTomato in smooth muscle cells of the cdh6-creERT model is valid or a leaky expression (false positive).

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 #1 (Public review):

Summary:

In this manuscript the Treisman and colleagues address the question of how protein phosphatase 1 (PP1) regulatory subunits (or PP1-interacting protein (PIPs)) confer specificity on the PP1 catalytic subunit which by itself possesses little substrate specificity. In prior work the authors showed that the PIP Phactrs confers specificity by remodelling a hydrophobic groove immediately adjacent to the PP1 catalytic site through residues within the RVxF- ø ø -R-W string of Phactrs. Specifically, the residues proximal and including the 'W' of the RVxF- ø ø -R-W string remodel the hydrophobic groove. Other residues of the RVxF- ø ø -R-W string (i.e. the RVxF- ø ø -R) are not involved in this remodelling.

The authors suggest that the RVxF- ø ø -R-W string is a conserved feature of many PIPs including PNUTS, Neurabin/spinophilin and R15A. However, from a sequence and structural perspective, only the RVxF- ø ø -R- is conserved. The W is not conserved in most and in the R15A structure (PDB:7NZM) the Trp side chain points away from the hydrophobic channel - this could be a questionable interpretation due to model-building into the low-resolution cryo-EM map (4 A).

In this paper, the authors convincingly show that Neurabin confers substrate specificity through interactions of its PDZ domain with the PDZ domain-binding motif (PBM) of 4E-BP. They show the PBM motif is required for Neurabin to increase PP1 activity towards 4E-BP and a synthetic peptide modelled on 4E-BP and also a synthetic peptide based on IRSp53 with a PBM added. The PBM of 4E-BP1 confers high affinity binding to the Neurabin PDZ domain. A crystal structure of a PP1-4E-BP1 fusion with Neurabin shows that the PBM of 4E-BP interacts with the PDZ domain of Neurabin. No interactions of 4E-BP and the catalytic site of PP1 are observed. Cell biology work showed that Neurabin-PP1 regulates the TOR signalling pathway by dephosphorylating 4E-BPs.

Strengths:

This work demonstrates convincingly using a variety of cell biology, proteomics, biophysics and structural biology that the PP1 interacting protein Neurabin confers specificity on PP1 through an interaction of its PDZ domain with a PDZ-binding motif of 4E-BP1 proteins. Remodelling of the hydrophobic groove of the PP1 catalytic subunit is not involved in Neurabin-dependent substrate specificity, in contrast to how Phactrs confers specificity on PP1. The active site of the Neurabin/PP1 complex does not recognise residues in the vicinity of the phospho-residue, thus allowing for multiple phospho-sites on 4E-BP to be dephosphorylated by Neurabin/PP1. This contrasts with substrate specificity conferred by the Phactrs PIP that confers specificity of Phactrs/PP1 towards its substrates in a sequence-specific context by remodelling the hydrophobic groove immediately adjacent to the catalytic. The structural and biochemical insights are used to explore the role of Neurabin/PP1 in dephosphorylation 4E-BPs in vivo, showing that Neurabin/PP1 regulates the TOR signalling pathway, specifically mTORC1-dependent translational control.

Weaknesses:

The only weakness is the suggestion that a conserved RVxF- ø ø -R-W string exists in PIPs. The 'W' is not conserved in sequence and 3 dimensions in most of the PIPs discussed in this manuscript. The lack of conservation of the W would be consistent with the finding based on multiple PP1-PIP structures that apart from Phactrs, no other PIP appears to remodel the PP1 hydrophobic channel.

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 #3 (Public review):

Protein Phosphatase 1 (PP1), a vital member of the PPP superfamily, drives most cellular serine/threonine dephosphorylation. Despite PP1's low intrinsic sequence preference, its substrate specificity is finely tuned by over 200 PP1-interacting proteins (PIPs), which employ short linear motifs (SLIMs) to bind specific PP1 surface regions. By targeting PP1 to cellular sites, modifying substrate grooves, or altering surface electrostatics, PIPs influence substrate specificity. Although many PIP-PP1-substrate interactions remain uncharacterized, the Phactr family of PIPs uniquely imposes sequence specificity at dephosphorylation sites through a conserved "RVxF-ΦΦ-R-W" motif. In Phactr1-PP1, this motif forms a hydrophobic pocket that favors substrates with hydrophobic residues at +4/+5 in acidic contexts (the "LLD motif"), a specificity that endures even in PP1-Phactr1 fusions. Neurabin/Spinophilin remodel PP1's hydrophobic groove in distinct ways, creating unique holoenzyme surfaces, though the impact on substrate specificity remains underexplored. This study investigates Neurabin/Spinophilin specificity via PDZ domain-driven interactions, showing that Neurabin/PP1 specificity is governed more by PDZ domain interactions than by substrate sequence, unlike Phactr1/PP1.

A significant strength of this work is the use of PP1-PIP fusion proteins to effectively model intact PP1•PIP holoenzymes by replicating the interactions that remodel the PP1 interface and confer site-specific substrate specificity. When combined with proteomic analyses to assess phospho-site depletion in mammalian cells, these fusions offer critical insights into holoenzyme specificity, revealing new candidate substrates for Neurabin and Spinophilin. The studies present compelling evidence that the PDZ domain of PP1-Neurabin directs its specificity, with the remodelled PP1 hydrophobic groove interactions having minimal impact. This mechanism is supported by structural analysis of the PP1-4E-BP1 substrate fusion bound to a Neurabin construct, highlighting the 4E-BP1/PDZ interaction. This work delivers crucial insights into PP1-PIP holoenzyme function, combining biochemical, proteomic, and structural approaches. It validates the PP1-PIP fusion protein model as a powerful tool, suggesting it may extend to studying additional holoenzymes. While an extremely useful model, it must be considered unlikely the PP1-PIP fusions fully recapitulate the specificity and regulation of the holoenzyme.

Reviewer #1 (Public review):

The authors of this study developed a method to quantify calvarial bone marrow from MRI head scans, enabling the study of its composition in large datasets of adults, usually collected to study the brain. Bone marrow intensity can be semi-quantitatively measured in T1-weighted MRI scans due to the greater signal intensity of fat than watery red marrow. This is an ingenious use of the MRI-produced information for other important phenotypes, such as bone structure and marrow content. Different head types were tested for complying with the model, which is notable.

The model was also successfully validated using several publicly available MRI resources - real data - in (1) a dataset consisting of 30 individuals that were scanned 10 times each at 3-day intervals, and (2) the monozygotic (MZ) twin data from the Human Connectome Project cohort. Then the authors applied this validated method to head-MRI scans from the UK Biobank (n=33,042) to extract information on the spatial distribution of bone marrow adiposity (BMA) in the calvaria, allowing a GWAS to identify associated genes.

The authors revealed high heritability and identified 41 genetic loci significantly associated with the BMA trait, including six sex-specific loci. Of note, statistics estimate that 99% of BMA trait-influencing variants are shared with BMD (497 of 500 variants), which may mean these results demonstrate the biological relevance to bone health. Some of the BMA genes were found related to the Wnt pathway, including WNT16, WNT4, NXN; this is a "positive control", since the Wnt/β-catenin signaling pathway was suggested as an important determinant of BMA. Also, associations in genes (BMP4, DLX5, LGR4, LRP4, SFRP4) that are known to specifically influence adiposity, are encouraging. Integrating mapped genes with bone marrow single-cell RNA-seq data revealed patterns of adipogenic lineage differentiation and lipid loading.

The study also investigated the genetic overlap between BMA and twelve (or 13) "brain and body" traits and identified significant genetic correlations with BMI, cognitive ability, and Parkinson's disease.

In sum, since MRI head scans present a hitherto unexplored opportunity to address unresolved aspects of bone marrow biology, this study is both timely and innovative.

There are, however, some assumptions, findings, and their interpretation, which require more critical focus.

Sex-specificity is well described and studied here. Men have higher BMA than women, but post-menopausal women catch up in the BMA values. The authors believe that calvarial marrow has a number of features that make it particularly well-suited to the study of BMA process - which is clinically important in other bone sites. It has a simple "sandwiched" structure that they are able to model. This is true only to some extent: a condition called "Hyperostosis frontalis interna", of unknown etiology (described by Smith & Hemphill in 1956) - is characterized by irregular overgrowth of the inner table of the frontal bone (symmetric/bilateral). Although not of clinical significance, typically benign, studies report a prevalence of 12%; However, it's most common in postmenopausal women - where prevalences up to 49% in women over the age of 65 - have been reported. Thus, sexual dimorphism is obvious and the effect of estrogen is likely shared with whichever bone - and marrow - age-related pathology. So, for women not using HRT, this new layer of the bone might interfere with the calvarial BMA readings and in turn, affect the BMA-related analyses. The authors suspect that the effect of BMA on BMD may be biased in women; they should comment on those "with low BMD and high BMA" given that hyperostosis frontalis might be an issue. A strong effect of SNPs in the ESR1 chromosomal region might be akin to the above concern.

Then, there is a perfect overlap of the BMA SNPs that are shared with BMD (497 of 500 variants), which may prove a "face validity" of the MRI-derived BMA. However, the BMD in the study was heel-derived eBMD - which is a good proxy for osteoporosis and is mostly driven by trabecular bone. Thus, there might be a concern that the BMA metrics capture some trabecular BMD.

Next, integrating mapped genes with existing bone marrow single-cell RNA-sequencing data revealed patterns of adipogenic lineage differentiation and lipid loading. The problem here is that the scRNAseq studies of the Bone Marrow niche are overwhelmingly mouse. The authors might wish to justify why they are relevant to humans (in the absence of the human-specific scRNAseq).

For genetic correlation analysis, the authors selected 7 body and 6 brain traits. The latter traits reflect cognition (general cognitive ability and educational attainment) and brain-related disorders. This selection might seem arbitrary. The interpretation of genetic correlation with cognitive ability, education, and Parkinson's disease was attributed to the recently discovered vascular channels that link calvarial bone marrow to the meninges. This is a fascinating hypothesis, which requires functional proof. However, there might be simpler explanations. Thus, the diploe and the inner table of the calvarium are drained by the same veins as the dura. From the anatomy textbook, we know that diploic veins connect the pericranial and endocranial venous system through the skull.