Reviewer #2 (Public review):

Summary:

This paper presents a very interesting use of a causal graph framework to identify the "root genes" of a disease phenotype. Root genes are the genes that cause a cascade of events that ultimately leads to the disease phenotype, assuming the disease progression is linear.

Strengths:

- The methodology has a solid theoretical background.<br /> - This is a novel use of the causal graph framework to infer root causes in a graph

Weaknesses:

(1) General Comments<br /> First, I have some general comments. I would argue that the main premise of the study might be inaccurate or incomplete. There are three major attributes of real biological systems, which are not considered in this work.

One is that the process from health-to-disease is not linear most of the time with many checks along the way that aim to prevent the disease phenotype. This leads to a non-deterministic nature of the path from health-to-disease. In other words, with the same root gene perturbations, and depending on other factors outside of gene expression, someone may develop a phenotype in a year, another in 10 years and someone else never. Claiming that this information is included in the error terms might not be sufficient to address this issue. The authors should discuss this limitation.

Two, the paper assumes that the network connectivity will remain the same after perturbation. This is not always true due to backup mechanisms in the cells. For example, suppose that a cell wants to create product P and it can do it through two alternative paths:<br /> Path #1: A -> B -> P Path #2: A -> C -> P<br /> Now suppose that path #1 is more efficient, so when B can be produced, path #2 is inactive. Once the perturbation blocks element B from being produced, the graph connectivity changes by activation of path #2. I did not see the authors taking this into consideration, which seems to be a major limitation in using perturb-seq results to infer connectivities.

Three, there is substantial system heterogeneity that may cause the same phenotype. This goes beyond the authors claim that although the initial gene causes of a disease may differ from person to person, at some point they will all converge to changes in the same set of "root genes". This is not true for many diseases, which are defined based on symptoms and lab tests at the patient level. You may have two completely different molecular pathologies that lead to the development of the same symptoms and test results. Breast cancer with its subtypes is a prime example of that. In theory, this issue could be addressed if there is infinite sample size. However, this assumption is largely violated in all existing biological datasets.

All the above limit the usefulness of this method for most chronic diseases, although it might still lead to interesting discoveries in cancer (in which the association between genes' dysregulation and development of cancer is more direct and occurs in less amount of time).

With these in mind, the theoretical and algorithmic advances this paper offers are interesting. And the theoretical proofs are solid.

(2) Specific comments.<br /> I am curious how the simulated data were generated and processed. Specifically, were the values of the synthetic variables Z-scored? If not, then I would expect that the variance of every variable will increase from the roots of the graph to the leaves. That will give an advantage in any algorithm aiming to identify causal relations based on error terms. For fairness and completeness, the authors should Z-score the values in the synthetic data and compare the results.

The algorithm seems to require both RNA-seq and Perturb-seq data (Algorithm 1, page 14). Can it function with RNA-seq data only? What will be different in this case?

(3) Additional comments:<br /> Although the manuscript is generally written clearly, some parts are not clear and others have missing details that make the narrative difficult to follow up. Some specific examples:<br /> - Synthetic data generation: how many different graphs (SEMs) did they start from? (30?) How many samples per graph? Did they test different sample sizes?<br /> - The presentation of comparative results (Suppl fig 4 and 7) is not clear. No details are given on how these results were generated. (what does it mean "The first column denotes the standard deviation of the outputs for each algorithm"?) Why all other methods have higher SD differences than RCSP? Is it a matter of scaling? Shouldn't they have at least some values near zero since the authors "added the minimum value so that all histograms begin at zero"? also, why RCSP results are more like a negative binomial distribution and every other is kind of normal?<br /> - What is the significance of genes changing expression "from left to right" in a UMAP plot? (eg Fig. 3h and 3g)

The authors somewhat overstate the novelty of their algorithm. Representation of GRNs as causal graphs dates back in 2000 with the work of Nir Friedman in yeast. Other methods were developed more recently that look on regulatory network changes at the single sample level which the authors do not seem to be aware (e.g., Ellington et al, NeurIPS 2023 workshop GenBio and Bushur et al, 2019, Bioinformatics are two such examples). The methods they mention are for single cell data and they are not designed to connect single sample-level changes to a person's phenotype. The RCS method needs to be put in the right background context in order to bring up what is really novel about it.

Reviewer #3 (Public review):

Summary:

In this manuscript, the Authors propose that astrocytic water channel AQP4 represents the dominant pathway for tonic water efflux without which astrocytes undergo cell swelling. The authors measure changes in astrocytic sulforhodamine B fluorescence as the proxy for cell volume dynamics. Using this approach, they have performed a technically elegant series of ex vivo and in vivo experiments exploring changes in astrocytic volume "signal" in response to the AQP4 inhibitor TGN-020 and/or neuronal stimulation. The key findings are that TGN-020 produces an apparent swelling of astrocytes and modifies astrocytic cell volume dynamics after spreading depolarizations. This study is perceived as potentially highly significant. However, several technical caveats could be considered better and perhaps addressed through additional experiments.

Strengths:

(1) This is a technically sound study, in which the Authors employed a number of complementary ex vivo and in vivo techniques. The presented results are of interest to the field and potentially highly significant.

(2) The innovative use of sulforhodamine B for in situ measurements of astrocyte cell volume dynamics is thoroughly validated in brain slices by quantifying changes in sulforhodamine fluorescence in response to hypoosmotic and hyperosmotic media.

(3) The combination of cell volume measurements with registering functional outcomes in both astrocytes and neurons (cell-specific GCaMP6 signaling) is appropriate and adds to the significance of the work.

(4) The use of ChR2 optogenetics for producing spreading depolarization allows to avoid many complications of chemical manipulations and much appreciated.

Remaining limitations:

(1) In the opinion of this reviewer, the effects of TGN-020 are not entirely consistent with the current knowledge on water permeability in astrocytes and the relative contribution of AQP4 to this process.

Specifically, genetic deletion of AQP4 reduces plasmalemmal water permeability in astrocytes by ~two-three-fold (when measured at 37oC, E. Solenov et al., AJP-Cell, 2004). This difference is significant but thought to have limited impact on steady-state water distribution. To the best of this reviewer's knowledge, cultured AQP4-null astrocytes do not show changes in degree of hypoosmotic swelling or hyperosmotic shrinkage. Thus, the findings of Solenov et al. are not (entirely) congruent with the conclusions of the current manuscript.

Also, as noted by the Authors, the AQP4 knockout does not modify astrocytes swelling induced by hypoosmotic solution in brain slices (T.R. Murphy et al., Front Neurosci., 2017), further suggesting that AQP4 is not a significant rate-limiting factor for water movement across astrocyte membranes.

The Authors do discuss the above-mentioned discrepancies and explain them by the context-dependent changes in water fluxes. Nevertheless, with these caveats in mind, it would be highly desirable to utilize an independent method measuring astrocytic volume and extracellular volume fraction.

(2) As noted by this reviewer and now discussed by the Authors, changes in ADC signal (presented in in Fig. 5) may be confounded by the previously reported TGN-020-induced hyphemia (e.g., H. Igarashi et al., NeuroReport, 2013) and/or changes water fluxes across pia matter which is highly enriched in AQP4. If this is the case, the proposed brain water accumulation may be explained by factors other than astrocytic water homeostasis. This caveat certainly deserves further experimental exploration.

Reviewer #2 (Public review):

Summary:

The authors have utilised deep profiling methods to generate deeper insights into the features of the TME that drive responsiveness to PD-1 therapy in NSCLC.

Strengths:

The main strengths of this work lie in the methodology of integrating single cell sequencing, genetic data and TCRseq data to generate hypotheses regarding determinants of IO responsiveness.

Some of the findings in this study are not surprising and well precedented eg. association of Treg, STAT3 and NFkB with ICI resistance and CD8+ activation in ICI responders and thus act as an additional dataset to add weight to this prior body of evidence. Whilst the role of Th17 in PD-1 resistance has been previously reported (eg. Cancer Immunol Immunother 2023 Apr;72(4):1047-1058, Cancer Immunol Immunother 2024 Feb 13;73(3):47, Nat Commun. 2021; 12: 2606 ) these studies have used non-clinical models or peripheral blood readouts. Here the authors have supplemented current knowledge by characterization of the TME of the tumor itself.

Weaknesses:

Unfortunately, the study is hampered by the small sample size and heterogeneous population and whilst the authors have attempted to bring in an additional dataset to demonstrate robustness of their approach, the small sample size has limited their ability to draw statistically supported conclusions. There is also limited validation of signatures/methods in independent cohorts and no functional characterisation of the findings. Because of these factors, this work (as it stands) does have value to the field but will likely have a relatively low overall impact.

Reviewer #2 (Public review):

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

Review for second submission:

In this revised manuscript, the authors provided new data to further support that suspension culture with Wnt/PKC inhibitors can be used for long-term hiPSC maintenance across multiple cell lines, as well as comparison with current benchmark culture system. New discussion sections were also added to put the findings into perspective of current development and the need for hiPSC maintenance culture system, and the figures were updated to improve readability. Overall, the authors have addressed all my concerns in this revised manuscript. Congratulations to the authors on this very interesting study.

Reviewer #2 (Public review):

Summary

The authors present multiple machine-learning methodologies to predict post-stroke epilepsy (PSE) from admission clinical data.

Strengths

The Statistical Approach section is very well written. The approaches used in this section are very sensible for the data in question.

Typos have now been addressed and improved interpretability has been added to the paper, which is appreciated.

Weaknesses

The authors have clarified that the first features available for each patient have been used. However, they have not shown that these features did not occur before the time of post-stroke epilepsy. Explicit clarification of this should be performed.

The likely impact of the work on the field

If this model works as claimed, it will be useful for predicting PSE. This has some direct clinical utility.

Analysis of features contributing to PSE may provide clinical researchers with ideas for further research on the underlying aetiology of PSE.

Reviewer #2 (Public review):

Summary:

Watanabe et al. introduce a novel approach for activity-dependent labeling of neural circuits in Drosophila at single-cell resolution, based on detecting the expression of the immediate early gene Hr38 using in situ hybridization. While activity mapping of neurons during specific behaviors is well-established in rodent models, its application in Drosophila has been limited, primarily due to technical constraints. By overcoming these challenges, this study tackles an important and timely issue, providing a foundational tool that will serve as a key reference in the field of circuit neuroscience.

Strengths:

The principal strength of this method lies in its versatility and high sensitivity. It can be applied to a broad range of biological questions and enables the investigation of dynamic transcriptional regulation across an unlimited number of genes with a strong signal-to-noise ratio. As such, it holds great potential for widespread use across research labs.

Weaknesses:

No major weaknesses; all concerns have been adequately addressed.

Reviewer #2 (Public Review):

Adjuvants boost antigen-specific immune responses to vaccines. However, whether adjuvants modulate the epitope immunodominance and the mechanisms involved in adjuvant's effect on antigen processing and presentation are not fully characterized. In this manuscript, Li et al report that immunodominant epitopes recognized by antigen-specific T cells are altered by adjuvants.

Using MPLA, CpG, and MDP adjuvants and H. pylori antigens, the authors screened the dominant epitopes of Th1 responses in mice post-vaccination with different adjuvants and found that adjuvants altered antigen-specific CD4+ T cell immunodominant epitope hierarchy. They show that adjuvants, MPLA and CpG especially, modulate the peptide repertoires presented on the surface of APCs. Surprisingly, adjuvant favored the presentation of low-stability peptides rather than high-stability peptides by APCs. As a result, the low stability peptide presented in adjuvant groups elicits T cell response effectively.

Reviewer #2 (Public review):

Summary:

This important study uses convincing evidence to compare how different operationalizations of adverse childhood experience exposure related to patterns of skin conductance response during a fear conditioning task in a large sample of adults. Specifically, the authors compared the following operationalizations: dichotomization of the sample into "exposed" and "non-exposed" categories, cumulative adversity exposure, specificity of adversity exposure, and dimensional (threat versus deprivation) adversity exposure. The paper is thoughtfully framed and provides clear descriptions and rationale for procedures, as well as package version information and code. The authors' overall aim of translating theoretical models of adversity into statistical models, and comparing the explanatory power of each model, respectively, is an important and helpful addition to the literature.

Several outstanding strengths of this paper are the large sample size and its primary aim of statistically comparing leading theoretical models of adversity exposure in the context of skin conductance response. This paper also helpfully reports Cohen's d effect sizes, which aid in interpreting the magnitude of the findings. The methods and results are thorough and well-described.

Reviewer #2 (Public review):

Shah et al. investigate the role of an understudied neural circuitry, specifically the dLS -> LHA -> RVM pathway, in mediating stress-induced analgesia. The authors use a combination of advanced techniques to provide convincing evidence for the involvement of this circuit in modulating pain under stress.

The study begins by mapping the neural circuitry through a series of intersectional tracings. Following this, the authors use behavioral tests along with optogenetic and chemogenetic manipulations to confirm the pathway's role in promoting analgesia. Additionally, fiber photometry is employed to monitor the activity of each brain region in response to stress and pain.

While the study is comprehensive and the findings are convincing, a key concern arises regarding the overarching hypothesis that restraint-induced stress promotes analgesia. A more straightforward interpretation could be that intense struggling, rather than stress itself, might drive the observed analgesic responses.

Reviewer #2 (Public review):

Summary:

This study investigates cold induced states in C. elegans, using polysome profiling and RNA seq to identify genes that are differentially regulated and concluding that cold-specific gene regulation occurs at the transcriptional level. This study also includes analysis of one gene from the differentially regulated set, lips-11 (a lipase), and finds that it is regulated in response to a specific set of ER stress factors.

Strengths:

(1) Understanding how environmental conditions are linked to stress pathways is generally interesting.

(2) The study used well-established genetic tools to analyze ER stress pathways.

Weaknesses:

(1) The conclusions regarding a general transcriptional response are based on one gene, lips-11, which does not affect survival in response to cold. We would suggest altering the title, to replace "Reprograming gene expression: with" Regulation of the lipase lips-11".

(2) There is no gene ontology with the gene expression data.

(3) Definitive conclusions regarding transcription vs translational effects would require use of blockers such as alpha amanatin or cyclohexamide.

(4) Conclusions regarding the role of lipids are based on supplementation with oleic acid or choline, yet there is no lipid analysis of the cold animals, or after lips-1 knockdown. Although choline is important for PC production, adding choline in normal PC could have many other metabolic impacts and doesn't necessarily implicate PC with out lipidomic or genetic evidence.

Reviewer #2 (Public review):

Summary:

In the present work, the authors present an engineering solution to sample preparation in 96-well plates for high-throughput super resolution microscopy via Expansion Microscopy. This is not a trivial problem, as the well cannot be filled with the gel, which would prohibit expansion of the gel. They thus engineered a device that can spot a small droplet of hydrogel solution and keep it in place as it polymerises. It occupies only a small portion space at the center of each well, the gel can expand into all directions and imaging and staining can proceed by liquid handling robots and an automated microscope.

Strengths:

In contrast to Reference 8, the authors system is compatible with standard 96 well imaging plates for high-throughput automated microscopy and automated liquid handling for most parts of the protocol. They thus provide a clear path towards high throughput exM and high throughout super resolution microscopy, which is a timely and important goal.

Addition upon revision:

The authors addressed this reviewer's suggestions.

Reviewer #2 (Public review):

This study advances the model that the first canonical amino acids to emerge in life bound the earliest cofactors and led to the first proteins. The focus is on organic/organometallic cofactors, building on previous work on metals - ie. those in the groups of Bromberg, Dupont and others as well cited in the manuscript. Studies of this type are limited both by data availability and confounding chemical effects that are exacerbated by the timescale of evolutionary inference tackled here. However, the analysis provides a solid addition to the field and complements existing metal-focused studies as well as those Longo, Russell and others (also well cited).

Reviewer #2 (Public Review):

Summary:

The authors propose a new method for self-supervised learning of 3d semantic segmentation for fluorescence microscopy. It is based on a WNet architecture (Encoder / Decoder using a UNet for each of these components) that reconstructs the image data after binarization in the bottleneck with a soft n-cuts clustering. They annotate a new dataset for nucleus segmentation in mesoSPIM imaging and train their model on this dataset. They create a napari plugin that provides access to this model and provides additional functionality for training of own models (both supervised and self-supervised), data labeling, and instance segmentation via post-processing of the semantic model predictions. This plugin also provides access to models trained on the contributed dataset in a supervised fashion.

Strengths:

(1) The idea behind the self-supervised learning loss is interesting.

(2) The paper addresses an important challenge. Data annotation is very time-consuming for 3d microscopy data, so a self-supervised method that yields similar results to supervised segmentation would provide massive benefits.

Weaknesses:

The experiments presented by the authors do not adequately support the claims made in the paper. There are several shortcomings in the design of the experiment and presentation of the results. Further, it is unclear if results of similar quality as reported can be achieved within the GUI by non-expert users.

Major weaknesses:

(1) The main experiments are conducted on the new mesoSPIM dataset, which contains quite small and well separated nuclei. It is unclear if the good performance of the novel self-supervised learning method compared to CellPose and StarDist would hold for dataset with other characteristics, such as larger nuclei with a more complex morphology or crowded nuclei. Further, additional preprocessing of the mesoSPIM images may improve results for StarDist and CellPose (see the first point in minor weaknesses). Note: having a method that works better for small nuclei would be an important contribution. But I am uncertain the claims hold for larger and/or more crowded nuclei as the current version of the paper implies. The contribution of the paper would be stronger if a comparison with StarDist / CellPose was also done on the additional datasets from Figure 2.

(2) The experimental setup for the additional datasets seems to be unrealistic. In general, the description of these experiments is quite short and so the exact strategy is unclear from the text. However, you write the following: "The channel containing the foreground was then thresholded and the Voronoi-Otsu algorithm used to generate instance labels (for Platynereis data), with hyperparameters based on the Dice metric with the ground truth." I.e., the hyperparameters for the post-processing are found based on the ground truth. From the description it is unclear whether this is done a) on the part of the data that is then also used to compute metrics or b) on a separate validation split that is not used to compute metrics. If a): this is not a valid experimental setup and amounts to training on your test set. If b): this is ok from an experimental point of view, but likely still significantly overestimates the quality of predictions that can be achieved by manual tuning of these hyperparameters by a user that is not themselves a developer of this plugin or an absolute expert in classical image analysis, see also 3. Note that the paper provides notebooks to reproduce the experimental results. This is very laudable, but I believe that a more extended description of the experiments in the text would still be very helpful to understand the set-up for the reader. Further, from inspection of these notebooks it becomes clear that hyper-parameters where indeed found on the testset (a), so the results are not valid in the current form.

(3) I cannot obtain similar results to the ones reported in the manuscript using the plugin. I tried to obtain some of the results from the paper qualitatively: First I downloaded one of the volumes from the mesoSPIM dataset (c5image) and applied the WNet3D to it. The prediction looks ok, however the value range is quite narrow (Average BG intensity ~0.4, FG intensity 0.6-0.7). I try to apply the instance segmentation using "Convert to instance labels" from "Utilities". Using "Voronoi-Otsu" does not work due to an error in pyClesperanto ("clGetPlatformIDs failed: PLATFORM_NOT_FOUND_KHR"). Segmentation via "Connected Components" and "Watershed" requires extensive manual tuning to get a somewhat decent result, which is still far from perfect.

Then I tried to obtain the results for the Mouse Skull Nuclei Dataset from EmbedSeg. The results look like a denoised version of the input image, not a semantic segmentation. I was skeptical from the beginning that the method would transfer without retraining, due to the very different morphology of nuclei (much larger and elongated). None of the available segmentation methods yield a good result, the best I can achieve is a strong over-segmentation with watersheds.

Minor weaknesses:

(1) CellPose can work better if images are resized so that the median object size in new images matches the training data. For CellPose the cyto2 model should do this automatically. It would be important to report if this was done, and if not would be advisable to check if this can improve results.

(2) It is a bit confusing that F1-Score and Dice Score are used interchangeably to evaluate results. The dice score only evaluates semantic predictions, whereas F1-Score evaluates the actual instance segmentation results. I would advise to only use F1-Score, which is the more appropriate metric. For Figure 1f either the mean F1 score over thresholds or F1 @ 0.5 could be reported. Furthermore, I would advise adopting the recommendations on metric reporting from https://www.nature.com/articles/s41592-023-01942-8.

(3) A more conceptual limitation is that the (self-supervised) method is limited to intensity-based segmentation, and so will not be able to work for cases where structures cannot be distinguished based on intensity only. It is further unclear how well it can separate crowded nuclei. While some object separation can be achieved by morphological operations this is generally limited for crowded segmentation tasks and the main motivation behind the segmentation objective used in StarDist, CellPose, and other instance segmentation methods. This limitation is only superficially acknowledged in "Note that WNet3D uses brightness to detect objects [...]" but should be discussed in more depth.

Note: this limitation does not mean at all that the underlying contribution is not significant, but I think it is important to address this in more detail so that potential users know where the method is applicable and where it isn't.

Reviewer #2 (Public review):

Summary:

The authors have worked up a ``virtual thymus' using EPISIM, which has already been published. Attractive features of the computational model are stochasticity, cell-to-cell variability, and spatial heterogeneiety. They seek to explore the role of TECs, that release IL-7 which is important in the process of thymocyte division.

In the model, ordinary clones have IL7R levels chosen from a distribution, while `lesioned' clones have an IL7R value set to the maximum. The observation is that the lesioned clones are larger families, but the difference is not dramatic. This might be called a cell-intrinsic mechanism. One promising cell-extrinsic mechanism is mentioned: if a lesioned clone happens to be near a source of IL-7 and begins to proliferate, the progeny can crowd out cells of other clones and monopolise the IL-7 source. The effect will be more noticeable if sources are rare, so is seen when the TEC network is sparse.

Strengths:

Thymic disfunctions are of interest, not least because of T-ALL. New cells are added, one at a time, to simulate the conveyor belt of thymocytes on a background of stationary cells. They are thus able to follow cell lineages, which is interesting because one progenitor can give rise to many progeny.

There are some experimental results in Figures 4,5 and 6. For example, il7 crispant embryos have fewer thymocytes and smaller thymii; but increasing IL-7 availability produces large thymii.

Weaknesses:

On the negative side, like most agent-based models, there are dozens of parameters and assumptions whose values and validity are hard to ascertain.

The stated aim is to mimic a 2.5-to-11 day-old medaka thymus, but the constructed model is a geometrical subset that holds about 100 cells at a time in a steady state. The manuscript contains very many figures and lengthy descriptions of simulations run with different parameters values and assumptions. The abstract and conclusion did not help me understand what exactly has been done and learned. No attempt to synthesise observations in any mathematical formula is made.

Reviewer #2 (Public review):

Summary:

The study by Huang and colleagues focuses on GLP-1 producing enteroendocrine (EEC) L-cells and their regulation of GLP-1 production by a mechanogated ion channel Piezo1. The study describes Piezo1 expression by L-cells and using an exciting intersectional mouse model (villin to target epithelium and Gcg to target GLP-1 producing cells and others like glucagon producing pancreatic endocrine cells), which allows L-cell specific Piezo1 knockout. Using this model, they find an impairment of glucose tolerance, increased body weight, reduced GLP-1 content, and changes to the CaMKKbeta-CaMKIV-mTORC1 signaling pathway using normal diet and then high fat diet. Piezo1 chemical agonist and intestinal bead implantation reversed these changes and improved the disrupted phenotype. Using primary sorted L-cells and cell model STC-1, they found that stretch and Piezo1 activation increased GLP-1 and altered the molecular changes described above.

Strengths:

This is an interesting study testing a novel hypothesis that may have important mechanistic and translational implications. The authors generated an important intersectional genetics mouse model that allowed them to target Piezo1 L-cells specifically, and the surprising result of impaired metabolism is intriguing.

Weaknesses:

However, there are several critical limitations that require resolution before making the conclusions that the authors make. (1) A potential explanation for the data, and one that is consistent with existing literature [see for example, PMC5334365, PMC4593481], is that epithelial Piezo1, which is broadly expressed by the GI epithelium, impacts epithelial cell density and survival, and as such, if Piezo1 is involved in L-cell physiology, it may be through regulation of cell density. Thus, it is critical to determine L-cell densities and epithelial integrity in controls and Piezo1 knockouts systematically across the length of the gut, since the authors do not make it clear which gut region contributes to the phenotype they see. Current immunohistochemistry data are not convincing. (2) Calcium signaling in L-cells is implicated in their typical role of being gut chemosensors, and Piezo1 is a calcium channel, so it is not clear whether any calcium-related signaling mechanism would phenocopy these results. (3) Intestinal bead implantation, while intriguing, does not have clear mechanisms - and is likely to provide a point of intestinal obstruction and dysmotility. (4) previous studies, some that are very important, but not cited, contradict the presented results (e.g., epithelial Piezo1 role in insulin secretion) and require reconciliation.<br /> Overall, this study makes an interesting observation but the data are not currently strong enough to support the conclusions.

- There needs to be data localizing Piezo1 to L-cells and importantly, this needs to be quantified - are all L-cells (small bowel and colon) Piezo1 positive? This is because several studies show Piezo1 affecting epithelial cell densities. If there are changes in L-cell or other EEC densities in Piezo1 knockout, that shift can potentially explain the changes that the authors see in glucose metabolism and weight.<br /> - The intersectional model for L-cell transduction needs a deeper validation. Images in Fig 1e are not convincing for transduction of GFP in L-cells. The co-localization studies are not convincing, especially because Piezo1 labeling is very broad. There needs to be stronger validation of the intersectional Gcg-Villin-Piezo1 KO model. It is important to determine whether L-cell Piezo1 localization epithelium in small bowel and colon is present (above) and affected specifically in the knockout.<br /> - The authors state that "Villin-1 (encoded by Vill1 gene) is expressed in the gastrointestinal epithelium, including L cells, but not in pancreatic α cells" (line 378-379). However, Villin is highly expressed in whole mouse islets (https://doi.org/10.1016/j.molmet.2016.05.015, Figure 1A).<br /> - There needs to be quantification of L-cells in Piezo1 knockout. This is because several studies show Piezo1 affecting epithelial cell densities. If there are changes in L-cell or other EEC densities in Piezo1 knockout, that shift can potentially explain the changes that the authors see in glucose metabolism and weight.<br /> - L-cells are classically considered to be chemosensors. Do nutritive signals, which presumably also increase calcium compete or complement or dominate L-cell GLP1 synthesis regulation?<br /> - The mechanism of Glp1 synthesis vs release downstream of Piezo1 is not clear. The authors hypothesize that "Piezo1 might regulate GLP-1 synthesis through the CaMKKβ/CaMKIV-mTOR signaling pathway". However, references cited suggest that Ca2+ or cAMP lead to GLP-1-release, while mTOR primarily acts on the regulation of gene expression by promoting Gcg gene expression. These pathways do not clearly link to Piezo1  GLP-1 production. These mechanisms need to be reconciled.<br /> - Previous study PMID 32640190 (not cited here) found that Villin-driven Piezo1 knockout, which knocks out Piezo1 from all epithelial intestinal cells (including L-cells), showed no significant alterations in blood glucose or body weight. This is opposite of the presented findings and therefore the current results require reconciliation.

Comments on revised version:

The authors have addressed several comments that were common to the reviewers - specificity and validity of the intersectional model, mechanism of signaling downstream of Piezo1 and reconciliation of the results with previous studies. The authors have provided extensive experiments and revisions which have made the manuscript stronger. However, many important questions remain, and unfortunately, the intersectional mouse model and mechanisms remain unclear.

- I appreciate the authors quantifying the density of L cells in the intersectional Piezo knockout. There is a very clear >50% drop-off in GLP-1+ cells with the Piezo1 knockout (Supp fig 7c, d). Interestingly, there was not a decrease in PYY+ cells, which is curious because GLP1 and PYY are co-expressed in L cells. The mechanism of regulation of one hormone but not the other in the same cell requires clarification and would be relevant for this work. To begin with, co-labeling PYY and GLP1 and showing that one hormone can be found without the other would be useful.<br /> - Piezo1 immunofluorescence has very high background and overall poor specificity (Fig supp 5 and Fig supp 6B are good examples of poor Piezo1 immunofluorescence). Another method for labeling Piezo1 (e.g. via RNAscope) is required - and where tried (e.g., Fig 1L), the results are not convincing.<br /> - The intersectional mouse model requires further validation. The data presented in Fig 1E do not help - the GFP positive cells do not look like L-cells and there appear to be GFP positive cells in the muscle and submucosa.<br /> - Since Piezo1 is known to affect epithelial cell life span, barrier function maybe compromised. While I appreciate that the authors have obtain some images and measured zonular and occluded, this is unfortunately a suboptimal evaluation of barrier function.<br /> - The mechanisms of calcium signaling that will presumably lead to GLP1 release due to Piezo1 activation and mTOR which authors link to GLP1 synthesis remain unreconciled.<br /> - Intestinal bead implantation may provide an important area of obstruction, in addition to potential mechanical stimulation. Unfortunately whole gut transit time and fecal weight do not assay these functions well.<br /> - I believe that the explanation regarding lack of previous findings connecting Piezo1 in the epithelium and glucose tolerance remain poorly reconciled with the current findings.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the role of Hox genes in the specification of forelimb position. The central conclusions are that Hox paralogy group (PG) 6/7 genes are both necessary and sufficient to induce forelimb buds. In addition, the authors argue that HoxPG4/5 genes are necessary, but, by contrast to Hox PG6/7 genes, Hox PG4/5 genes are not sufficient to induce forelimb budding. To test the roles of Hox4-7 genes in limb development, the authors use both gain-of-function (GOF) and loss-of-function (LOF) approaches in chick embryos.

In LOF experiments, they produced dominant negative forms of Hoxa4, Hoxa5, Hoxa6, and Hoxa7, which lack the DNA-binding domain, and they electroporated these constructs into the prospective wing field of the lateral plate mesoderm (LPM) in pre-limb bud stage (HH12) chick embryos. All 4 constructs resulted in down-regulation of Tbx5 (an early marker of forelimb development), and of its target gene, Fgf10, which is required for the initiation of limb budding, in the lateral plate mesoderm. The dominant negative experiments also caused down-regulation of Fgf8 in the overlying limb ectoderm and a marked reduction in the size of the early wing bud. Based on the LOF results, the authors conclude that each of the Hoxa4-7 genes is required for the specification of the forelimb field and for the establishment of the Fgf10-Fgf8 feedback loop in wing bud mesenchyme and overlying epithelium.

The authors then use a GOF strategy to investigate whether the same genes are sufficient to induce forelimb budding. They test this hypothesis using the neck, a region that is known to be incompetent to form limbs in response to Fgf signaling. Overexpression of full-length Hoxa6 and Hoxa7 in the neck region caused ectopic expression of Tbx5 in the neck region, which fits with "posteriorization" of cells at neck level, as Tbx5 typically marks the forelimb and flank (interlimb) region of the lateral plate mesoderm. Consistent with a posterior transformation of positional identity (neck to forelimb), overexpression of Hoxa6 or Hoxa7 leads to activation of Fgf10 expression and development of an ectopic forelimb bud from (or extension of the normal forelimb bud into) the neck region). By contrast, overexpression of either Hoxa4 or Hoxa5 in the neck region is not sufficient to induce ectopic forelimb budding. Curiously, the ectopic forelimb buds do not express Fgf8 in the overlying ectoderm or develop beyond the bud stage. The latter finding is consistent with previous work showing that neck ectoderm is not competent to support outgrowth of transplanted limb bud mesenchyme. The authors investigate the mechanistic basis of this early arrest of outgrowth by comparing the transcriptomes of ectopic limb buds, normal forelimb buds, and normal neck cells.

The RNA sequencing analysis shows that while some limb development genes (e.g., Lmx1b, Hoxa9, Hoxd9, Hoxa10, Hoxd10) are activated in the ectopic limb bud, other key components of the circuit (e.g., Shh, Fgf8, Hox12/13 paralogs) are not established, leading them to conclude that failure of neck ectoderm to form an AER underlies the arrested outgrowth of ectopic limb buds.

Strengths:

This study provides the first evidence that altering the Hox code in neck lateral plate mesoderm (LPM) is sufficient to induce ectopic development of forelimb buds at the neck level. For more than 30 years, developmental biologists have speculated and provided indirect evidence that Hox genes are involved in the specification of forelimb position, but to my knowledge, no study has shown that altering Hox gene expression alone can induce limb development outside of the normal limb field. The finding that Hox6/7 paralogs are sufficient for forelimb bud development, whereas Hox4/5 paralogs are not, suggests that specification of forelimb identity requires instructive signaling that is a specific property of Hox6/7 paralogs. The GOF experiments significantly extend the knowledge of limb specification beyond that which has come from Hox gene manipulations in mice.

Weaknesses:

(1) By contrast to the GOF experiments that induce ectopic limb budding, the LOF experiments, which use dominant negative forms of Hoxa4, Hoxa5, Hoxa6, and Hoxa7, are more challenging to interpret due to the absence of data on the specificity of the dominant negative constructs. Absent such controls, one cannot be certain that effects on limb development are due to disruption of the specific Hox proteins that are being targeted.

(2) A test of their central hypothesis regarding the necessity and sufficiency of the Hox genes under investigation would be to co-transfect the neck with full-length Hoxa6/a7 AND the dnHoxA4/a5. If their hypothesis is correct, then the dn constructs should block the limb-inducing ability of Hoxa6/a7 overexpression (again, validation of specificity of the DN constructs is important here).

(3) The paper could be strengthened by providing some additional data, which should already exist in their RNA-Seq dataset, such as supplementary material that shows the actual gene expression data that are represented in the Venn diagram, heatmap, and GO analysis in Figure 3.

(4) The results of these experiments in chick embryos are rather unexpected based on previous knockout experiments in mice, and this needs to be discussed.

Reviewer #2 (Public review):

Summary:

This study explores how maternal behaviors influence vocal learning in the greater sac-winged bat (Saccopteryx bilineata). Over two field seasons, researchers tracked 19 bat pups from six wild colonies, examining vocal development aspects such as vocal practice duration, syllable repertoire size, and song syllable acquisition. The findings show that maternal behaviors significantly impact the length of daily babbling sessions and the overall babbling phase, while the presence of adult male tutors does not.

The researchers conducted detailed acoustic analyses, categorizing syllables and evaluating the variety and presence of learned song syllables. They discovered that maternal interactions enhance both the number and diversity of learned syllables and the production of mature syllables in the pups' vocalizations. A notable correlation was found between the extent of acoustic changes in the most common learned syllable type and maternal activity, highlighting the key role of maternal feedback in shaping pups' vocal development.

In summary, this study emphasizes the crucial role of maternal social feedback in the vocal development of S. bilineata. Maternal behaviors not only increase vocal practice but also aid in acquiring and refining a complex vocal repertoire. These insights enhance our understanding of social interactions in mammalian vocal learning and draw interesting parallels between bat and human vocal development.

Strengths:

This paper makes significant contributions to the field of vocal learning by looking at the role of maternal behaviors in shaping the vocal learning phenotype of Saccopteryx bilineata. The paper uses a longitudinal approach, tracking the vocal ontogeny of bat pups from birth to weaning across six colonies and two field seasons, allowing the authors to assess how maternal interactions influence various aspects of vocal practice and learning, providing strong empirical evidence for the critical role of social feedback in non-human mammalian vocal learners. This kind of evidence highlights the complexity of the vocal learning phenotype and shows that it goes beyond the right auditory experience and having the right circuitry.

The paper offers a nuanced understanding of how specific maternal behaviors impact the acquisition and refinement of the vocal repertoire, while showing the number of male tutors - the source of adult song - did not have much of an effect. The correlation between maternal activity and acoustic changes in learned syllable types is a novel finding that underscores the importance of non-vocal social interactions in vocal learning. In vocal learning research, with some notable exceptions, experience is often understood as auditory experience. This paper highlights how, even though that is one important piece of the puzzle, other kinds of experience directly affect the development of vocal behavior. This is of particular importance in the case of a mammalian species such as Saccopteryx bilineata, as this kind of result is perhaps more often associated with avian species.

Moreover, the study's findings have broader implications for our understanding of vocal learning across species. By drawing parallels between bat and human vocal development (and in some ways to bird vocal development), the paper highlights common mechanisms that may underlie vocal practice and learning in both humans and other mammals. This interdisciplinary perspective enriches the field and encourages further comparative studies, ultimately advancing our knowledge of the evolutionary and developmental processes that shape vocal productive learning in all its dimensions.

Weaknesses:

Some weaknesses can be pointed out, but in fairness, the authors acknowledge them in one way or another. As such, these are not flaws per se, but gaps that can be filled with further research.

Experimental manipulations, such as controlled playback experiments or controlled environments, could strengthen the causal claims by directly testing the effects of specific maternal behaviors on vocal development. Certainly, the strengths of the paper will be consolidated after such work is performed.

The reliance on the number of singing males as a proxy for social acoustic input. This measure does not account for the variability in the quality, frequency, or duration of the male songs to which the pups are exposed. A more detailed analysis of the acoustic environment, including direct measurements of song exposure and its impact on vocal learning, would provide a clearer understanding of the role of male tutors.

Finally, and although it would be unlikely that these results are unique to Saccopteryx bilineata, the study's focus on a single species limits at present the generalizability of some of its findings to other vocal learning mammals. While the parallels drawn between bat and human vocal development are intriguing, the conclusions will be more robust when supported by comparative studies involving multiple species of vocal learners. This will help to identify whether the observed maternal influences on vocal development reported here are unique to Saccopteryx bilineata or represent a broader phenomenon in chiropteran, mammalian, or general vocal learning. Expanding the scope of research to include a wider range of species and incorporating cross-species comparisons will significantly enhance the contribution of this study to the field of vocal learning.

Reviewer #2 (Public review):

Summary

Schubert et al. recorded MEG and eye-tracking activity while participants were listening to stories in single-speaker or multi-speaker speech. In a separate task, MEG was recorded while the same participants were listening to four types of pure tones in either structured (75% predictable) or random (25%) sequences. The MEG data from this task was used to quantify individual 'prediction tendency': the amount by which the neural signal is modulated by whether or not a repeated tone was (un)predictable, given the context. In a replication of earlier work, this prediction tendency was found to correlate with 'neural speech tracking' during the main task. Neural speech tracking is quantified as the multivariate relationship between MEG activity and speech amplitude envelope. Prediction tendency did not correlate with 'ocular speech tracking' during the main task. Neural speech tracking was further modulated by local semantic violations in the speech material, and by whether or not a distracting speaker was present. The authors suggest that part of the neural speech tracking is mediated by ocular speech tracking. Story comprehension was negatively related to ocular speech tracking.

Strengths

This is an ambitious study, and the authors' attempt to integrate the many reported findings related to prediction and attention in one framework is laudable. The data acquisition and analyses appear to be done with great attention to methodological detail (perhaps even with too much focus on detail-see below). Furthermore, the experimental paradigm used is more naturalistic than was previously done in similar setups (i.e. stories instead of sentences).

Weaknesses

For many of the key variables and analysis choices (e.g. neural/ocular speech tracking, prediction tendency, mediation) it is not directly clear how these relate to the theoretical entities under study, and why they were quantified in this particular way. Relatedly, while the analysis pipeline is outlined in much detail, an overarching rationale and important intermediate results are often missing, which makes it difficult to judge the strength of the evidence presented. Furthermore, some analysis choices appear rather ad-hoc and should be made uniform and/or better motivated.

Reviewer #2 (Public review):

Summary:

This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

Strengths:

(1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.

(2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with the thalamus, as well as migration of cINs from CGE and MGE to the cortex.

Weaknesses:

(1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

(2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same mice. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

(3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

(4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell-intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

Reviewer #2 (Public review):

Summary:

This work used multiple approaches to show that CCK is critical for long-term potentiation (LTP) in the auditory thalamocortical pathway. They also showed that the CCK mediation of LTP is age-dependent and supports frequency discrimination. This work is important because it opens up a new avenue of investigation of the roles of neuropeptides in sensory plasticity.

Strengths:

The main strength is the multiple approaches used to comprehensively examine the role of CCK in auditory thalamocortical LTP. Thus, the authors do provide a compelling set of data that CCK mediates thalamocortical LTP in an age-dependent manner.

Weaknesses:

The behavioral assessment is relatively limited but may be fleshed out in future work.

Reviewer #2 (Public review):

The authors of this article investigated the impact of the host enzyme AOAH on the progression of MASLD in mice. To achieve this, they utilized whole-body Aoah-/- mice. The authors demonstrated that AOAH reduced LPS-induced lipid accumulation in the liver, probably by decreasing the expression and activation of SREBP1. In addition, AOAH reduced hepatic inflammation and minimized tissue damage.

However, this paper is descriptive without a clear mechanistic study. Another major limitation is the use of who-body KO mice so the cellular source of the enzyme remains undefined. Moreover, since LPS-mediated SREBP1 regulation or LPS-mediated MASLD progression is already documented, the role of AOAH in SREBP1-dependent lipid accumulation and MASLD progression is largely expected.

Specific comments:

(1) The overall human relevance of the current study remains unclear.

(2) Is AOAH secreted from macrophages or other immune cells? Are there any other functions of AOAH within the cells?

(3) Due to using whole-body KO mice, the role of AOAH in specific cell types was unclear in this study, which is one of the major limitations of this study. The authors should at least conduct in vitro experiments using a co-culture system of hepatocytes and Kupffer cells (or other immune cells) isolated from WT or Aoah-/- mice.

(4) It has been well-known that intestinal tight junction permeability is increased by LPS or inflammatory cytokines. However, in Figure 3E, intestinal permeability is comparable between the groups in both diet groups. The authors should discuss more about this result. In addition, intestinal junctional protein should be determined by Western blot and IHC (or IF) to further confirm this finding.

(5) In Figure 6, LPS i.g. Aoah-/- group is missing. This group should be included to better interpret the results.

(6) The term NAFLD has been suggested to be changed to MASLD as the novel nomenclature according to the guidelines of AASLD and EASL.

Reviewer #2 (Public review):

Summary:

The authors present the results of molecular phylogenetic analysis with very comprehensive samplings including 471 specimens belonging to 250 species, trying to give a holistic reconstruction of the evolutionary history of freshwater fishes (Nemacheilidae) across Eurasia since the early Eocene. This is of great interest to general readers.

Strengths:

They provide very vast data and conduct comprehensive analyses. They suggested that Nemacheilidae contain 6 major clades, and the earliest differentiation can be dated to the early Eocene.

Weaknesses:

The analysis is incomplete, and the manuscript discussion is not well organized. The authors did not discuss the systematic problems that widely exist. They also did not use the conventional way to discuss the evolutionary process of branches or clades, but just chronologically described the overall history.

Reviewer #2 (Public review):

Summary:

Tanaka et al. investigated the role of CCR4 in early atherosclerosis, focusing on the immune modulation elicited by this chemokine receptor under hypercholesterolemia. The study found that Ccr4 deficiency led to qualitative changes in atherosclerotic plaques, characterized by an increased inflammatory phenotype. The authors further analyzed the CD4 T cell immune response in para-aortic lymph nodes and atherosclerotic aorta, showing an increase mainly in Th1 cells and the Th1/Treg ratio in Ccr4-/-Apoe-/- mice compared to Apoe-/- mice. They then focused on Tregs, demonstrating that Ccr4 deficiency impaired their immunosuppressive function in in-vitro assays and elegantly showed that Ccr4-deficient Tregs had, as expected, impaired migration to the atherosclerotic aorta. Adoptive cell transfer of Ccr4-/- Tregs to Apoe-/- mice mimicked early atherosclerosis development in Ccr4-/-Apoe-/- mice. Therefore, this work shows that CCR4 plays an important role in early atherosclerosis but not in advanced stages.

Strengths:

Several in vivo and in vitro approaches were used to address the role of CCR4 in early atherosclerosis. Particularly, through the adoptive cell transfer of CCR4+ or CCR4- Tregs, the authors aimed to directly demonstrate the role of CCR4 in Tregs' protection against early atherosclerosis.

Weaknesses:

The isolation of Tregs was inadequately controlled; they were isolated based solely on CD4 and CD25 expression. CD25 is also expressed by activated effector T cells, meaning the analyzed cells could be a pool of mainly Tregs but also include effector T cells.

The study primarily focused on Th1 and Tregs without thoroughly investigating other CD4 T cell subsets. Th17 cells are known to play an important role in atherosclerosis; non-pathogenic Th17 cells express CCR4, while pathogenic Th17 cells do not. Considering that Figure 3 shows an increased frequency of IL17-expressing CD4 T cells compared to Apoe-/- mice, and given the imprecise Treg isolation, differences in non-pathogenic Th17 cells could be contributing to the observed effects.

Furthermore, the clinical relevance of these findings is not discussed. As an initial approach, the authors could analyze public datasets to determine if certain Ccr4 single nucleotide polymorphisms correlate with a higher incidence of atherosclerosis.

Reviewer #2 (Public review):

I appreciate the authors' thorough revision of the manuscript, which has significantly improved its quality. I have no additional comments or requests for further changes.

However, I remain in slight disagreement regarding the characterization of the neutral condition. My perspective is that it resembles more of a "medium" condition, making it challenging to understand what would be common to "high-medium" and "low-medium" contrasts. I suspect that the neutral condition might represent a state of high uncertainty since participants are informed that the algorithm cannot provide a prediction. From this viewpoint, the observed similarities in effects for both positive and negative expectations may actually reflect differences between certainty and uncertainty rather than the specific expectations themselves.

Nevertheless, the authors have addressed alternative interpretations of their discussion section, and I have no further requests. The paper is well-executed and demonstrates several strengths: the procedure effectively induced varying levels of expectations with clear impacts on pain ratings. Additionally, the integration of fMRI with EEG is commendable for tracking the transition from anticipatory to pain periods. Overall, the manuscript is strong and contributes valuable insights to the field.

Reviewer #2 (Public review):

The present study aims to investigate brain white matter predictors of back pain chronicity. To this end, a discovery cohort of 28 patients with subacute back pain (SBP) was studied using white matter diffusion imaging. The cohort was investigated at baseline and one-year follow-up when 16 patients had recovered (SBPr) and 12 had persistent back pain (SBPp). A comparison of baseline scans revealed that SBPr patients had higher fractional anisotropy values in the right superior longitudinal fasciculus SLF) than SBPp patients and that FA values predicted changes in pain severity. Moreover, the FA values of SBPr patients were larger than those of healthy participants, suggesting a role of FA of the SLF in resilience to chronic pain. These findings were replicated in two other independent datasets. The authors conclude that the right SLF might be a robust predictive biomarker of CBP development with the potential for clinical translation.<br /> Developing predictive biomarkers for pain chronicity is an interesting, timely, and potentially clinically relevant topic. The paradigm and the analysis are sound, the results are convincing, and the interpretation is adequate. A particular strength of the study is the discovery-replication approach with replications of the findings in two independent datasets.

Reviewer #2 (Public review):

Summary:

The main aim of this research was to explore whether and how self-associations (as opposed to other associations) bias early attentional selection, and whether this can explain well-known self-prioritization phenomena, such as the self-advantage in perceptual matching tasks. The authors adopted the Visual Attention Theory (VAT) by estimating VAT parameters using a hierarchical Bayesian model from the field of attention and applied it to investigate the mechanisms underlying self-prioritization. They also discussed the constraints on the self-prioritization effect in attentional selection. The key conclusions reported were:

(1) Self-association enhances both attentional weights and processing capacity

(2) Self-prioritization in attentional selection occurs automatically but diminishes when active social decoding is required, and

(3) Social and perceptual salience capture attention through distinct mechanisms.

Strengths:

Transferring the Theory of Visual Attention parameters estimated by a hierarchical Bayesian model to investigate self-prioritization in attentional selection was a smart approach. This method provides a valuable tool for accessing the very early stages of self-processing, i.e., attention selection. The authors conclude that self-associations can bias visual attention by enhancing both attentional weights and processing capacity and that this process occurs automatically. These findings offer new insights into self-prioritization from the perspective of the early stage of attentional selection.

Weaknesses:

(1) The results are not convincing enough to definitively support their conclusions. This is due to inconsistent findings (e.g., the model selection suggested condition-specific c parameters, but the increase in processing capacity was only slight; the correlations between attentional selection bias and SPE were inconsistent across experiments), unexpected results (e.g., when examining the impact of social association on processing rates, the other-associated stimuli were processed faster after social association, while the self-associated stimuli were processed more slowly), and weak correlations between attentional bias and behavioral SPE, which were reported without any p-value corrections. Additionally, the reasons why the attentional bias of self-association occurs automatically but disappears during active social decoding remain difficult to explain. It is also possible that the self-association with shapes was not strong enough to demonstrate attention bias, rather than the automatic processes as the authors suggest. Although these inconsistencies and unexpected results were discussed, all were post hoc explanations. To convince readers, empirical evidence is needed to support these unexpected findings.

(2) The generalization of the findings needs further examination. The current results seem to rely heavily on the perceptual matching task. Whether this attentional selection mechanism of self-prioritization can be generalized to other stimuli, such as self-name, self-face, or other domains of self-association advantages, remains to be tested. In other words, more converging evidence is needed.

(3) The comparison between the "social" and "perceptual" tasks remains debatable, as it is challenging to equate the levels of social salience and perceptual salience. In addition, these two tasks differ not only in terms of social decoding processes but also in other aspects such as task difficulty. Whether the observed differences between the tasks can definitively suggest the specificity of social decoding, as the authors claim, needs further confirmation.

Reviewer #2 (Public review):

Summary:

Liu et al investigated the performance of a novel imaging technique called RIM-Deep to enhance the imaging depth for cleared samples. Usually, the imaging depth using the classical confocal microscopy sample chamber is limited due to optical aberrations, resulting in loss of resolution and image quality. To overcome this limitation and increase depth, they generated a special imaging chamber, that is affixed to the objective and filled with a solution matching the refractive indices to reduce aberrations. Importantly, the study was conducted using a standard confocal microscope, that has not been modified apart from exchanging the standard sample chamber with the RIM-Deep sample holder. Upon analysing the imaging depth, the authors claim that the RIM-Deep method increased the depth from 2 mm to 5 mm. In summary, RIM-Deep has the potential to significantly enhance imaging quality of thick samples on a low budget, making in-depth measurements possible for a wide range of researchers that have access to an inverted confocal microscope.

Strengths:

The authors used different clearing methods to demonstrate the suitability of RIM-Deep for various sample preparation protocols with clearing solutions of different refractive indices. They clearly demonstrate that the RIM-Deep chamber is compatible with all 3 methods. Brain samples are characterized by complex networks of cells and are often hard to visualize. Despite the dense, complex structure of brain tissue, the RIM-Deep method generated high quality images of all 3 samples given. As the authors already stated, increasing imaging depth often goes hand in hand with purchasing expensive new equipment, exchanging several microscopy parts or purchasing a new microscopy set-up. Innovations, such as the RIM-Deep chamber, hence, might pave the way for cost-effective imaging and expand the applicability of an inverted confocal microscope.

Weaknesses:

(1) However, since this study introduces a novel imaging technique, and therefore, aims to revolutionize the way of imaging large samples, additional control experiments would strengthen the data. From the 3 clearing protocol used (CUBIC, MACS and iDISCO), only the brain section from Macaca fascicularis cleared with iDISCO was imaged with the standard chamber and the RIM-Deep method. This comparison indeed shows that the imaging depth thereby increases more than 2-fold, which is a significant enhancement in terms of microscopy. However, it would have been important to evaluate and show the difference of the imaging depth also on the other two samples, since they were cleared with different protocols and, thus, treated with clearing solutions of different refractive indices compared to iDCISCO.

(2) The description of the figures and figure panels should be improved for a better understanding of the experiments performed and the thus resulting images/data.

(3) While the authors used a Nikon AX inverted laser scanning confocal microscope, the study would highly benefit from evaluating the performance of the RIM-Deep method using other inverted confocal microscopes or even wide-field microscopes.

Reviewer #2 (Public review):

Summary:

The paper reconsiders the formation of Hebbian-type assemblies, with their spontaneous reactivation representing the statistics of the sensory inputs, in the light of predictive synaptic plasticity. It convincingly shows that not all plasticity rules can be predictive in the narrow sense. While plasticity for the excitatory synapses (the forward projecting and recurrent ones) are predictive, two types of plasticity in the recurrent inhibition is required: a homeostatic and competitive one.

Details:

Besides the excitatory forward and recurrent connections that are learned based on predictive synaptic plasticity, two types of inhibitory plasticity are considered. A first type of inhibition is homeostatic and roughly balances excitation within the cell assemblies. Plasticity in this type 1 inhibition is also predictive, analogous to the plasticity of the excitatory synapses. However, plasticity in type 2 inhibition is competitive and has a switched sign. Both types of inhibitory plasticity, the predictive (homeostatic) and the anti-predictive (competitive) one, work together with the predictive excitatory plasticity to form cell assemblies representing sensory stimuli. Only if the two types of homeostatic and competitive inhibitory plasticity are present, will the spontaneous replay of the assemblies reflect the statistics of the stimulus presentation.

Critical review:

The simulations include Dale's law, making them more biologically realistic. The paper emphasizes predictive plasticity and introduces type 1 inhibitory plasticity that, by construction, tries to fully explain away the excitatory input. In the absence of external inputs, however, due to the symmetry between the excitatory and inhibitory-type-1 plasticity rules, excitation and inhibition tend to fully cancel each other. Multiple options may solve the dilemma:

(1) As other predictive dendritic plasticity models assume, the presynaptic source for recurrent inhibition is typically less informative than the presynaptic source of excitation, so that inhibition is not able to fully explain away excitation.

(2) Beside the inhibitory predictive plasticity that mirrors the analogous excitatory predictive plasticity, and additional competitive plasticity can be introduced.

The paper chooses solution (2) and suggests and additional inhibitory recurrent pathway that is not predictive, but instead anti-predictive with a reversed sign. The combination of the two types of inhibitory plasticities lead to a stable formation of cell assemblies. The stable target activity of the plasticity rules in a memory recall is not anymore 0, as it would be with only type-1-inhibitory plasticity.<br /> Instead, the target activity of plasticity is now enhanced within a winning assembly, and also positive but reduced in the loosing assemblies.

Reviewer #2 (Public review):

Summary:

This study primarily aims to examine the relationship between collective performance and group identification. Additionally, the authors propose that inter-brain synchronization (IBS) underlies collective performance and that changes in intra-brain functional connectivity or single-brain activation may, in turn, underlie IBS. The topic addressed in this paper is of great importance in the field using hyperscanning. However, the details of the experiments and analysis described in the paper are unclear, and the hypothesis as to why IBS is thought to underlie collective performance is not clearly presented. In addition, some of the analysis seems to be inappropriate.

Strengths:

I find the model presented in Figure 7 to be intriguing. Understanding why inter-brain synchronization occurs and how it is supported by specific single-brain activations or intra-brain functional connectivity is indeed a critical area for researchers conducting hyperscanning studies to explore.

Understanding triadic-interaction is really important, while almost all hyperscanning neuroimaging focuses on the dyadic interaction. The exploring neural/behavioral/psychological basis behind triadic interaction is a promising method for understanding collective behavior and decision-making.

Weaknesses:

The authors need to clearly articulate their hypothesis regarding why neural synchronization occurs during social interaction. For example, in line 284, it is stated that "It is plausible that neural synchronization is closely associated with group identification and collective performance...", but this is far from self-evident. Neural synchronization can occur even when people are merely watching a movie (Hasson et al., 2004), and movie-watchers are not engaged in collective behavior. There is no direct link between the IBS and collective behavior. The authors should explain why they believe inter-brain synchronization occurs in interactive settings and why they think it is related to collective behavior/performance.

The authors state that "GNS in the OFC was a reliable neuromarker, indicating the influence of group identification on collective performance," but this claim is too strong. Please refer to Figure 4B. Do the authors really believe that collective performance can be predicted given the correlation with the large variance shown? There is a significant discrepancy between observing a correlation between two variables and asserting that one variable is a predictive biomarker for the other.

Why are the individual answers being analyzed as collective performance (See, L-184)? Although these are performances that emerge after the group discussion, they seem to be individual performances rather than collective ones. Typically, wouldn't the result of a consensus be considered a collective performance? The authors should clarify why the individual's answer is being treated as the measure of collective performance.

Performing SPM-based mapping followed by conducting a t-test on the channels within statistically significant regions constitutes double dipping, which is not an acceptable method (Kriegeskorte et al., 2011). This issue is evident in, for example, Figures 3A and 4A.

Please refer to the following source:<br /> https://www.nature.com/articles/nn.2303

In several key analyses within this study (e.g., single-brain activation in the paragraph starting from L398, neural synchronization in the paragraph starting from L393), the TPJ is mentioned alongside the DLPFC. However, in subsequent detailed analyses, the TPJ is entirely ignored.

The method for analyzing single-brain activation is unclear. Although it is mentioned that GLM (generalized linear model) was used, it is not specified what regressors were prepared, nor which regressor's β-values are reported as brain activity. Without this information, it is difficult to assess the validity of the reported results.

While the model illustrated in Figure 7 seems to be interesting, for me, it seems not to be based on the results of this study. This is because the study did not investigate the causal relationships among the three metrics. I guess, Figure 5D might be intended to explain this, but the details of the analysis are not provided, making it unclear what is being presented.

The details of the experiment are not described at all. While I can somewhat grasp what was done abstractly, the lack of specific information makes it impossible to replicate the study.

Reviewer #2 (Public review):

Summary:

Nishi et al, investigate the well-known and previously described phenomenon of age-associated myeloid-biased hematopoiesis. Using a previously established HoxB5mCherry mouse model, they used HoxB5+ and HoxB5- HSCs to discriminate cells with long-term (LT-HSCs) and short-term (ST-HSCs) reconstitution potential and compared these populations to immunophenotypically defined 'bulk HSCs' that consists of a mixture of LT-HSC and ST-HSCs. They then isolated these HSC populations from young and aged mice to test their function and myeloid bias in non-competitive and competitive transplants into young and aged recipients. Based on quantification of hematopoietic cell frequencies in the bone marrow, peripheral blood, and in some experiments the spleen and thymus, the authors argue against the currently held belief that myeloid-biased HSCs expand with age.

While aspects of their work are fascinating and might have merit, several issues weaken the overall strength of the arguments and interpretation. Multiple experiments were done with a very low number of recipient mice, showed very large standard deviations, and had no statistically detectable difference between experimental groups. While the authors conclude that these experimental groups are not different, the displayed results seem too variable to conclude anything with certainty. The sensitivity of the performed experiments (e.g. Fig 3; Fig 6C, D) is too low to detect even reasonably strong differences between experimental groups and is thus inadequate to support the author's claims. This weakness of the study is not acknowledged in the text and is also not discussed. To support their conclusions the authors need to provide higher n-numbers and provide a detailed power analysis of the transplants in the methods section.

As the authors attempt to challenge the current model of the age-associated expansion of myeloid-biased HSCs (which has been observed and reproduced by many different groups), ideally additional strong evidence in the form of single-cell transplants is provided.

It is also unclear why the authors believe that the observed reduction of ST-HSCs relative to LT-HSCs explains the myeloid-biased phenotype observed in the peripheral blood. This point seems counterintuitive and requires further explanation.

Based on my understanding of the presented data, the authors argue that myeloid-biased HSCs do not exist, as<br /> a) they detect no difference between young/aged HSCs after transplant (mind low n-numbers and large std!!!); b) myeloid progenitors downstream of HSCs only show minor or no changes in frequency and c) aged LT-HSCs do not outperform young LT-HSC in myeloid output LT-HScs in competitive transplants (mind low n-numbers and large std!!!).<br /> However, given the low n-numbers and high variance of the results, the argument seems weak and the presented data does not support the claims sufficiently. That the number of downstream progenitors does not change could be explained by other mechanisms, for instance, the frequently reported differentiation short-cuts of HSCs and/or changes in the microenvironment.

Strengths:

The authors present an interesting observation and offer an alternative explanation of the origins of aged-associated myeloid-biased hematopoiesis. Their data regarding the role of the microenvironment in the spleen and thymus appears to be convincing.

Weaknesses:

"Then, we found that the myeloid lineage proportions from young and aged LT-HSCs were nearly comparable during the observation period after transplantation (Fig. 3, B and C)."<br /> [Comment to the authors]: Given the large standard deviation and low n-numbers, the power of the analysis to detect differences between experimental groups is very low. Experimental groups with too large standard deviations (as displayed here) are difficult to interpret and might be inconclusive. The absence of clearly detectable differences between young and aged transplanted HSCs could thus simply be a false-negative result. The shown experimental results hence do not provide strong evidence for the author's interpretation of the data. The authors should add additional transplants and include a detailed power analysis to be able to detect differences between experimental groups with reasonable sensitivity.

Line 293: "Based on these findings, we concluded that myeloid-biased hematopoiesis observed following transplantation of aged HSCs was caused by a relative decrease in ST-HSC in the bulk-HSC compartment in aged mice rather than the selective expansion of myeloid-biased HSC clones."<br /> Couldn't that also be explained by an increase in myeloid-biased HSCs, as repeatedly reported and seen in the expansion of CD150+ HSCs? It is not intuitively clear why a reduction of ST-HSCs clones would lead to a myeloid bias. The author should try to explain more clearly where they believe the increased number of myeloid cells comes from. What is the source of myeloid cells if the authors believe they are not derived from the expanded population of myeloid-biased HSCs?

Reviewer #2 (Public review):

This paper shows and analyzes an interesting phenomenon. It shows that when people are exposed to sequences of moving dots (that is moving dots in one direction, followed by another direction, etc.), showing either the starting movement direction or ending movement direction causes a coarse-grained brain response that is similar to that elicited by the complete sequence of 4 directions. However, they show by decoding the sensor responses that this brain activity actually does not carry information about the actual sequence and the motion directions, at least not on the time scale of the initial sequence. They also show a reverse reply on a highly compressed time scale, which is elicited during the period of elevated activity, and activated by the first and last elements of the sequence, but not others. Additionally, these replays seem to occur during periods of cortical ripples, similar to what is found in animal studies.

These results are intriguing. They are based on MEG recordings in humans, and finding such replays in humans is novel. Also, this is based on what seems to be sophisticated statistical analysis. However, this is the main problem with this paper. The statistical analysis is not explained well at all, and therefore its validity is hard to evaluate. I am not at all saying it is incorrect; what I am saying is that given how it is explained, it cannot be evaluated.

Reviewer #2 (Public review):

Summary:

Using in vivo fiber-photometry the authors first establish that DA release when contacting their partner mouse increases with days of cohabitation while this increase is not observed when contacting a stranger mouse. Similar effects are found in D1-MSNs and D2-MSNs with the D1-MSN responses increasing and D2-MSN responses decreasing with days of cohabitation. They then use slice physiology to identify underlying plasticity/adaptation mechanisms that could contribute to the changes in D1/D2-MSN responses. Last, to address causality the authors use chemogenetic tools to selectively inhibit or activate NAc shell D1 or D2 neurons that project to the ventral pallidum. They found that D2 inhibition facilitates bond formation while D2 excitation inhibits bond formation. In contrast, both D1-MSN activation and inhibition inhibit bond formation.

Strengths:

The strength of the manuscript lies in combining in vivo physiology to demonstrate circuit engagement and chemogenetic manipulation studies to address circuit involvement in pair bond formation in a monogamous vole.

Weaknesses:

Weaknesses include that a large set of experiments within the manuscript are dependent on using short promoters for D1 and D2 receptors in viral vectors. As the authors acknowledge this approach can lead to ectopic expression and the presented immunohistochemistry supports this notion. It seems to me that the presented quantification underestimates the degree of ectopic expression that is observed by eye when looking at the presented immunohistochemistry. However, given that Cre transgenic animals are not available for Microtus mandarinus and given the distinct physiological and behavioral outcomes when imaging and manipulating both viral-targeted populations this concern is minor.

The slice physiology experiments provide some interesting outcomes but it is unclear how they can be linked to the in vivo physiological outcomes and some of the outcomes don't match intuitively (e.g. cohabitation enhances excitatory/inhibitory balance in D2-MSNs but the degree of contact-induced inhibition is enhanced in D2-MSN).

One interesting finding is that the relationship between D2-MSN and pair bond formation is quite clear (inhibition facilitates while excitation inhibits pair bond formation). In contrast, the role of D1-MSNs is more complicated since both excitation and inhibition disrupt pair bond formation. This is not convincingly discussed.

It seemed a missed opportunity that physiological readout is limited to males. I understand though that adding females may be beyond the scope of this manuscript.

In what ways was Russian slavery similar to and different from American slavery?

Cite your sources.

Reviewer #2 (Public review):

Summary:

This manuscript reports that a combination of two small molecules, 2C (CHIR99027 and A-485) enabled to induce the dedifferentiation of hESC-derived cardiomyocytes (CMs) into regenerative cardiac cells (RCC). These RCCs had disassembled sarcomeric structures and elevated expression of embryonic cardiogenic genes such as ISL1, which exhibited proliferative potential and were able to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Lineage tracing further suggested that RCCs originated from TNNT2+ cells, not pre-existing ISL1+ cells. Furthermore, 2C treatment increased the numbers of RCC cells in neonatal rat and adult mouse hearts, and improves cardiac function post-MI in adult mice. Mechanistically, bulk RNA-seq analysis revealed that 2C led to elevated expression of embryonic cardiogenic genes while down-regulation of CM-specific genes. Single-cell RNA-seq data showed that 2C promoted cardiomyocyte transition into an intermediate state that are marked with ACTA2 and COL1A1, which subsequently transform into RCCs. Finally, ChIP-seq analysis demonstrated that CHIR99027 enhanced H3K9Ac and H3K27Ac modifications in embryonic cardiac genes, while A-485 inhibited these modifications in cardiac-specific genes. These combined alterations effectively induced the dedifferentiation of cardiomyocytes into RCCs. Overall, this is an important work, presenting a putative cardiac regenerative cell types that may represent endogenous cardiac regeneration in regenerative animals. With that said, here are suggestions for the authors:

Strengths:

Overall, this work is quite comprehensive and is logically and rigorously designed. The phenotypic and functional data on 2C are strong.

Weaknesses or suggestions:

(1) In Figure 4, the authors should perform additional experiments on analyzing 2C effect on cardiomyocytes, endothelial cells, and fibroblasts in adult mouse hearts after myocardial infarction.<br /> (2) In Figures 5-7, the mechanistic insights of 2C are primarily derived from transcriptomic and genomic datasets without experimental verification.<br /> (3) The authors should compare transcriptomic profiling of the RCCs with other putative cardiac progenitors from public databases.

Reviewer #2 (Public review):

Summary:

This study by Mendes et al provides novel key insights in the role of chemotaxis and immune cell recruitment into the hypothalamus in the development of diet-induced obesity. Specifically, the authors first revealed that although transcriptional changes in hypothalamic resident microglia following exposure to high-fat feeding are minor, there are compelling transcriptomic differences between resident microglia and microglia recruited to the hypothalamus, and these are sexually dimorphic. Using independent loss-of-function studies, the authors also demonstrate an important role of CXCR3 and hypothalamic CXCL10 in the hypothalamic recruitment of CCR2+ positive cells on metabolism following exposure to high-fat diet-feeding in mice. This manuscript puts forth conceptually novel evidence that inhibition of chemotaxis-mediated immune cell recruitment accelerates body weight gain in high-fat diet-feeding, suggesting that a subset of microglia which express CXCR3 may confer protective, anti-obesogenic effects.

Strengths:

The work is exciting and relevant given the prevalence of obesity and the consequences of inflammation in the brain on perturbations of energy metabolism and ensuant metabolic diseases. Hypothalamic inflammation is associated with disrupted energy balance, and activated microglia within the hypothalamus resulting from excessive caloric intake and saturated fatty acids are often thought to be mediators of impairment of hypothalamic regulation of metabolism. The present work reports a novel notion in which immune cells recruited into the hypothalamus which express chemokine receptor CXCR3 may have a protective role against diet-induced obesity. In vivo studies reported herein demonstrate that inhibition of CXCR3 exacerbates high-fat diet-induced body weight gain, increases circulating triglycerides and fasting glucose levels, worsens glucose tolerance, and increases the expression of orexigenic neuropeptides, at least in female mice.

This work provides a highly interesting and needed overview of preclinical and clinical brain inflammation, which is relevant to readers with an interest in metabolism and immunometabolism in the context of obesity.

Using flow cytometry, cell sorting, and transcriptomics including RNA-sequencing, the manuscript provides novel insights on transcriptional landscapes of resident and recruited microglia in the hypothalamus. Importantly, sex differences are investigated.

Overall, the manuscript is perceived to be highly interesting, relevant, and timely. The discussion is thoughtful, well-articulated, and a pleasure to read and felt to be of interest to a broad audience.

Weaknesses:

There were no major weaknesses perceived. Some comments for potential textual additions to the results/discussion are provided below.

Could the authors comment on the choice of peripheral administration of CXCR3 antagonist as opposed to central (e.g. icv) administration? Indeed, systemic inhibition of CXCR3 produced significant alterations in body weight gain and glucose tolerance in female mice given high-fat diet and reduced CCR2 and CXCR3 immunostaining in the hypothalamus. Could changes to peripheral (e.g. WAT, liver) immune responses to the diet underlie the metabolic changes observed?

Besides hypothalamic mRNA levels of chemokines and chemokine receptors, does systemic CXCR3 antagonism affect other aspects linked to diet-induced impairments of hypothalamic regulation of energy homeostasis, like inflammation, ER stress and/or mitochondrial dynamics/function? It would be interesting to reveal the consequence of reduced CCR2+ microglial migration to the hypothalamus with chronic high-fat diet exposure.

Reviewer #2 (Public review):

Summary:

This paper reports the structures of two human biotin-dependent carboxylases. The authors used endogenously purified proteins and solved the structures in high resolutions. Based on the structures, they defined the binding site for acyl-CoA and biotin and reported the potential conformational changes in biotin position.

Strengths:

The authors effectively utilized the biotin of the two proteins and obtained homogeneous proteins from human cells. They determined the high-resolution structures of the two enzymes in apo and substrate-bound states.

Comments and questions to the manuscripts:

(1) I'm quite impressed with the protein purification and structure determination, but I think some functional characterization of the purified proteins should be included in the manuscript. The activity of enzymes should be the foundation of all structures and other speculations based on structures.

(2) In Figure 1B, the structure of MCC is shown as two layers of beta units and two layers of alpha units, while there is only one layer of alpha units resolved in the density maps. I suggest the authors show the structures resolved based on the density maps and show the complete structure with the docked layer in the supplementary figure.

(3) In the introduction, I suggest the author provide more information about the previous studies about the structure and reaction mechanisms of BDCs, what is the knowledge gap, and what problem you will resolve with a higher resolution structure. For example, you mentioned in line 52 that G437 and A438 are catalytic residues, are these residues reported as catalytic residues or this is based on your structures? Has the catalytic mechanism been reported before? Has the role of biotin in catalytic reactions revealed in previous studies?

(4) In the discussion, the authors indicate that the movement of biotin could be related to the recognition of acyl-CoA in BDCs, however, they didn't observe a change in the propionyl-CoA bound MCC structure, which is contradictory to their speculation. What could be the explanation for the exception in the MCC structure?

(5) In the discussion, the authors indicate that the selectivity of PCC to different acyl-CoA is determined by the recognition of the acyl chain. However, there are no figures or descriptions about the recognition of the acyl chain by PCC and MCC. It will be more informative if they can show more details about substrate recognition in Figures 3 and 4.

(6) How are the solved structures compared with the latest Alphafold3 prediction?

Reviewer #3 (Public review):

Summary:

In this study the authors aim to develop an experimental/computational pipeline to assess the modification status of an RNA following treatment with dimethylsulfate (DMS). Building upon the more common DMS Map method, which predominantly assesses the modification status of the Watson-Crick-Franklin face of A's and C's, the authors insert a chemical processing step in the workflow prior to deep sequencing that enables detection of methylation at the N7 position of guanosine residues. This approach, termed BASH MaP, provides a more complete assessment of the true modification status of an RNA following DMS treatment, and this new information provides a powerful set of constraints for assessing the secondary structure and conformational state of an RNA. In developing this work, the authors use Spinach as a model RNA. Spinach is a fluorogenic RNA that binds and activates the fluorescence of a small molecule ligand. Crystal structures of this RNA with ligand bound show that it contains a G-quadruplex motif. In applying BASH MaP to Spinach, the authors also perform the more standard DMS MaP for comparison. They show that the BASH MaP workflow appears to retain the information yielded by DMS MaP while providing new information about guanosine modifications. In Spinach, the G-quadruplex G's have the least reactive N7 positions, consistent with the engagement of N7 in hydrogen bonding interactions at G's involved in quadruplex formation. Moreover, because the inclusion of data corresponding to G increases the number of misincorporations per transcript, BASH MaP is more amenable to analysis of co-occurring misincorporations through statistical analysis, especially in combination with site-specific mutations. These co-occurring misincorporations provide information regarding what nucleotides are structurally coupled within an RNA conformation. By deploying a likelihood-ratio statistical test on BASH MaP data, the authors can identify Gs in G-quadruplexes, deconvolute G-G correlation networks, base-triple interactions and even stacking interactions. Further, the authors develop a pipeline to use the BASH MaP-derived G-modification data to assist in the prediction of RNA secondary structure and identify alternative conformations adopted by a particular RNA. This seems to help with the prediction of secondary structure for Spinach RNA.

Strengths:

The BASH Map procedure and downstream data analysis pipeline more fully identifies the complement of methylations to be identified from DMS treatment of RNA, thereby enriching the information content. This in turn allows for more robust computational/statistical analysis, which likely will lead to more accurate structure predictions. This seems to be the case for the Spinach RNA.

Weaknesses:

The authors demonstrate that their method can detect G-quadruplexes in Spinach and some other RNAs both in vitro and in cells. While application to other RNAs is beyond the scope of the current manuscript, the performance of BASH MaP and associated computational analysis in the context of other RNAs remains to be determined.

Reviewer #3 (Public review):

Summary:

The work by Graca et al. describes a GMC flavoprotein dehydrogenase (MftG) in the ethanol metabolism of mycobacteria and provides evidence that it shuttles electrons from the mycofactocin redox cofactor to the electron transport chain.

Strengths:

Overall, this study is compelling, exceptionally well designed and thoroughly conducted. An impressively diverse set of different experimental approaches is combined to pin down the role of this enzyme and scrutinize the effects of its presence or absence in mycobacteria cells growing on ethanol and other substrates. Other strengths of this work are the clear writing style and stellar data presentation in the figures, which makes it easy also for non-experts to follow the logic of the paper. Overall, this work therefore closes an important gap in our understanding of ethanol oxidation in mycobacteria, with possible implications for the future treatment of bacterial infections.

Weaknesses:

I see no major weaknesses of this work, which in my opinion leaves no doubt about the role of MftG.

Reviewer #2 (Public review):

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

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

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

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

Reviewer #2 (Public review):

Summary:

In this work, Vivian Salgueiro et al. have comprehensively investigated the role of VirR in the vesicle production process in Mtb using state-of-the-art omics, imaging, and several biochemical assays. From the present study, authors have drawn a positive correlation between cell membrane permeability and vasculogenesis and implicated VirR in affecting membrane permeability, thereby impacting vasculogenesis.

Strengths:

The authors have discovered a critical factor (i.e. membrane permeability) that affects vesicle production and release in Mycobacteria, which can broadly be applied to other bacteria and may be of significant interest to other scientists in the field. Through omics and multiple targeted assays such as targeted metabolomics, PG isolation, analysis of Diaminopimelic acid and glycosyl composition of the cell wall, and, importantly, molecular interactions with PG-AG ligating canonical LCP proteins, the authors have established that VirR is a central scaffold at the cell envelope remodelling process which is critical for MEV production.

Weaknesses:

Throughout the study, the authors have utilized a CRISPR knockout of VirR. VirR is a non-essential gene for the growth of Mtb; a null mutant of VirR would have been a better choice for the study.

Comments on the revised version:

Concerns flagged about using CRISPR -guide RNA mediated knockdown of viral has yet to be addressed entirely. I understand that the authors could not get knock out despite attempts and hence they have guide RNA mediated knockdown strategy. However, I wondered if the authors looked at the levels of the downstream genes in this knockdown.

Authors have used the virmut-Comp strain for some of the experiments. However, the materials and methods must describe how this strain was generated. Given the mutant is a CRISPR-guide RNA mediated knockdown. The CRISPR construct may have taken up the L5 loci. Did authors use episomal construct for complementation? If so, what is the expression level of virR in the complementation construct? What are the expression levels of downstream genes in mutant and complementation strains? This is important because the transcriptome analysis was redone by considering complementation strain. The complemented strain is written as virmut-C or virmut-Comp. This has to be consistent.

Reviewer #2 (Public review):

Summary:

The overall goal of Eleni et al. is to determine if the suppression of LH pulses during lactation is mediated by prolactin signaling at kisspeptin neurons. To address this, the authors used GCaMP fiber photometry and serial blood sampling to reveal that in vivo episodic arcuate kisspeptin neuron activity and LH pulses are suppressed throughout pregnancy and lactation. The authors further utilized knockout models to demonstrate that the loss of prolactin receptor signaling at kisspeptin cells prevents the suppression of kisspeptin cell activity and results in the early reestablishment of fertility during lactation. The work demonstrates exemplary design and technique, and the outcomes of these experiments are sophistically discussed.

Strengths:

This manuscript demonstrates exceptional skill with powerful techniques and reveals a key role for arcuate kisspeptin neurons in maintaining lactation-induced infertility in mice. In a difficult feat, the authors used fiber photometry to map the activity of arcuate kisspeptin cells into lactation and weaning without disrupting parturition, lactation, or maternal behavior. The authors used a knockout approach to identify if the inhibition of fertility by prolactin is mediated via direct signaling at arcuate kisspeptin cells. Although the model does not perfectly eliminate prolactin receptor expression in all kisspeptin neurons, results from the achieved knockdown support the conclusion that prolactin signaling at kisspeptin neurons is required to maintain lactational infertility. The methods are advanced and appropriate for the aims, the study is rigorously conducted, and the conclusions are thoughtfully discussed.

Comments on the latest version:

All comments and suggestions have been addressed by the authors in this revision.

Reviewer #2 (Public review):

This work by Pal et al. studied the relationship between protein expression noise and translational efficiency. They proposed a model based on ribosome demand to explain the positive correlation between them, which is new as far as I realize. Nevertheless, I found the evidence of the main idea that it is the ribosome demand generating this correlation is weak. Below are my major and minor comments.

Major comments:

(1) Besides a hypothetical numerical model, I did not find any direct experimental evidence supporting the ribosome demand model. Therefore, I think the main conclusions of this work are a bit overstated.

(2) I found that the enhancement of protein noise due to high translational efficiency is quite mild, as shown in Figure 6A-B, which makes the biological significance of this effect unclear.

(3) The captions for most of the figures are short and do not provide much explanation, making the figures difficult to read.

(4) It would be helpful if the authors could define the meanings of noise (e.g., coefficient of variation?) and translational efficiency in the very beginning to avoid any confusion. It is also unclear to me whether the noise from the experimental data is defined according to protein numbers or concentrations, which is presumably important since budding yeasts are growing cells.

(5) The conclusions from Figures 1D and 1E are not new. For example, the constant protein noise as a function of mean protein expression is a known result of the two-state model of gene expression, e.g., see Equation (4) in Paulsson, Physics of Life Reviews 2005.

(6) In Figure 4C-D, it is unclear to me how the authors changed the mean protein expression if the translation initiation rate is a function of variation in mRNA number and other random variables.

(7) If I understand correctly, the authors somehow changed the translation initiation rate to change the mean protein expression in Figures 4C-D. However, the authors changed the protein sequences in the experimental data of Figure 6. I am not sure if the comparison between simulations and experimental data is appropriate.

Reviewer #2 (Public review):

In this manuscript, Hou et al. investigate the interplay between OCT4 and SOX2 in driving the pluripotent state during early embryonic lineage development. Using knockout (KO) embryos, the authors specifically analyze the transcriptome and chromatin state within the ICM-to-EPI developmental trajectory. They emphasize the critical role of OCT4 and the supportive function of SOX2, along with other factors, in promoting embryonic fate. Although the paper presents high-quality data, several key claims are not well-supported, and direct evidence is generally lacking.

Major Points:

(1) Although the authors claim that both maternal KO and maternal KO/zygotic hetero KO mice develop normally, the molecular changes in these groups appear overestimated. A wildtype control is recommended for a more robust comparison.

(2) The authors assert that OCT4 and SOX2 activate the pluripotent network via the OCT-SOX enhancer. However, the definition of this enhancer is based solely on proximity to TSSs, which is a rough approximation. Canonical enhancers are typically located in intronic and intergenic regions and marked by H3K4me1 or H3K27ac. Re-analyzing enhancer regions with these standards could be beneficial. Additionally, the definitions of "close to" or "near" in lines 183-184 are unclear and not defined in the legends or methods.

(3) There is no evidence that the decreased peaks/enhancers could be the direct targets of Oct4 and Sox2 throughout this manuscript. Figures 2 and 4 show only minimal peak annotations related to OCT and SOX motifs, and there is a lack of chromatin IP data. Therefore, claims about direct targets are not substantiated and should be appropriately revised.

(4) Lines 143-146 lack direct data to support the claim. Actually, the main difference in cluster I, 11 and 3, 8, 14 is whether the peak contains OCT-SOX motif. However, the reviewer cannot get any information of peaks activated by OCT4 rather than SOX2 in cluster I, 11.

Minor Points:

(1) Lines 153-159: The figure panel does not show obvious enrichment of SOX2 signals or significant differences in H3K27ac signals across clusters, thus not supporting the claim.

(2) Lines 189-190: The term "identify" is overstated for the integrative analysis of RNA-seq and ATAC-seq, which typically helps infer TF targets rather than definitively identifying them.

(3) The Discussion is lengthy and should be condensed.

Reviewer #2 (Public review):

Summary:

This article investigates the distribution of synapses on the dendritic arbors of descending neurons in the looming circuit of the fly visual system. The authors use publicly available EM reconstruction data of the adult fly brain to identify the positions of synapses from several types of visual projection neuron (VPN) to descending neuron (DN) connections. VPN dendrites are retinotopically organized, and axons from different VPN populations innervate distinct optic glomeruli. Yet the authors did not find any retinotopic organization of the synapses in the VPN-DN pairs they analyzed. They then constructed passive electrical models of the DNs with their structures extracted from the EM reconstructions. They focused on two specific DNs and parameterized their models by conducting whole-cell recordings within a voltage range below spiking threshold. Simulation of these passive models showed that irrespective of the location of a synapse, EPSPs became very similar at the spike initiation zone. This is consistent with the idea of synaptic democracy where EPSPs at far away synapses have higher amplitude compared to those nearer to the spike initiation zone so that they all attenuate to similar amplitudes while reaching there. The authors found that activating synapses from individual VPNs have the same effect as activating a random set of synapses. They conclude that despite some clustering of VPN synapses at small scale, they are distributed randomly over the dendritic arbor of DNs so that their EPSP amplitude encode the number of activated synapses, avoiding sublinearity from shunting effect.

Strengths:

- Experimental confirmation of the location of the spike initiation zone in the DN arbors is interesting and may provide better understanding of signal processing in these neurons.<br /> - Passive parameters obtained through electrophysiological recordings are useful.<br /> - These morphologically detailed single neuron models, if made available publicly, will be beneficial for building more complete models to understand the fly visual circuit.<br /> - The authors have complemented the work of Dombrovski et al by analyzing the distribution of synapses in more detail from EM data for a different set of neurons.

Weaknesses:

DNs are upstream of motorneurons, and one would expect, as demonstrated by Dombrovski et al, that specific DNs being activated by input from specific regions of the visual field will activate motoneurons so that the fly moves away from a looming object.

The current work analyzed the synapse distribution on two DNs that do not seem to have such role, and emphasize the lack of retinotopy. However, it is not clear why one would expect retinotopy in synapse location on the dendritic arbor. The comparison with mammalian visual circuits is not appropriate because those layers are extracting more and more complex visual features, whereas Drosophila DNs are supposed to drive motoneurons to generate suitable escape behavior.

- The authors do not suggest the functional roles of these DNs in controlling the movement of the fly. They argue that the synapse distribution and the passive electrotonic structure of these neurons are optimized to make the composite EPSP encode the number of activated synapses, but do not explain why this is important.

- Although DNs are spiking neurons, the authors limit their work to the subthreshold passive domain. If the EPSP at the spike initiation zone crosses spiking threshold, will encoding the number of synapses in EPSP amplitude still matter? Will it matter either if the composite EPSP remains subthreshold?

- The temporal aspect of the input has been ignored by the authors in their simulations. First, it is not clear all the synapses from a single VPN should get activated together. One would expect a spike in a VPN to arrive at different synapses with different time delays depending on their electrotonic distance from the spike initiation zone and the signal propagation speed in the neurites.

A looming stimulus should be expanding with time, but from the description of the simulations it does not seem that the authors have tried to incorporate this aspect in their design of the synaptic activation.

- The suggestion in the abstract that linear encoding of synapse number is default strategy which is then tuned by active properties and plasticity seems strange. Developmentally active properties do not get inserted into passive neurons.

- Much of the analysis (Figures 4, 5, 12) show relationships with physical distance along dendrite. In studying passive neurons it is more informative to use electrotonic distance which provides better insight.

Reviewer #2 (Public review):

Summary:

In their manuscript, Quian and colleagues identified a novel mechanism by which Pseudomonas control inflammatory responses upon inflammasome activation. They identified a caspase-11 substrate (VgrG2b) which, upon cleavage, binds and inhibits the NLRP3 to reduce the production of pro-inflammatory cytokines. This is a unique mechanism that allows for the tailoring of the innate immune response upon bacterial recognition.

Strengths:

The authors are presenting here a novel conceptual framework in host-pathogen interactions. Their work is supported by a range of approaches (biochemical, cellular immunology, microbiology, animal models), and their conclusions are supported by multiple independent evidences. The work is likely to have an important impact on the innate immunity field and host-pathogen interactions field and may guide the development of novel inhibitors.

Weaknesses:

Although quite exhaustive, a few of the authors' conclusions are not fully supported (e.g., caspase-11 directly cleaving VgrG2b, the unique affinity of VgrG2b-C for NLRP3) and would require complementary approaches to validate their findings fully. This is minimal.

Reviewer #2 (Public review):

Summary:

The authors decoupled several players that are thought to contribute to the establishment of epithelial polarity and determined their causal relationship. This provides a new picture of the respective roles of junctional proteins (Par3), the centrosome, and endomembrane compartments (Cdc42, Rab11, Gp135) from upstream to downstream.<br /> Their conclusions are based on live imaging of all players during the early steps of polarity establishment and on the knock-down of their expression in the simplest ever model of epithelial polarity: a cell doublet surrounded by ECM.

The position of the centrosome is often taken as a readout for the orientation of the cell polarity axis. There is a long-standing debate about the actual role of the centrosome in the establishment of this polarity axis. Here, using a minimal model of epithelial polarization, a doublet of daugthers MDCK cultured in Matrigel, the authors made several key observations that bring new light to our understanding of a mechanism that has been studied for many years without being fully explained:

(1) They showed that centriole can reach their polarized position without most of their microtubule-anchoring structures. These observations challenge the standard model according to which centrosomes are moved by the production and transmission of forces along microtubules.

(2) (However) they showed that epithelial polarity can be established in the absence of centriole.

(3) (Somehow more expectedly) they also showed that epithelial polarity can't be established in the absence of Par3.

(4) They found that most other polarity players that are transported through the cytoplasm in lipid vesicles, and finally fused to the basal or apical pole of epithelial cells, are moved along an axis which is defined by the position of centrosome and orientation of microtubules.

(5) Surprisingly, two non-daughters cells that were brought in contact (for 6h) could partially polarize by recruiting a few Par3 molecules but not the other polarity markers.

(6) Even more surprisingly, in the absence of ECM, Par 3 and centrosomes could move to their proper position close to the intercellular junction after cytokinesis but other polarity markers (at least GP135) localized to the opposite, non-adhesive, side. So the polarity of the centrosome-microtubule network could be dissociated from the localisation of GP135 (which was believed to be transported along this network).

Strengths:

(1) The simplicity and reproducibility of the system allow a very quantitative description of cell polarity and protein localisation.

(2) The experiments are quite straightforward, well-executed, and properly analyzed.

(3) The writing is clear and conclusions are convincing.

Weaknesses:

(1) The simplicity of the system may not capture some of the mechanisms involved in the establishment of cell polarity in more physiological conditions (fluid flow, electrical potential, ion gradients,...).

(2) The absence of centriole in centrinone-treated cells might not prevent the coalescence of centrosomal protein in a kind of MTOC which might still orient microtubules and intracellular traffic. How are microtubules organized in the absence of centriole? If they still form a radial array, the absence of a centriole at the center of it somehow does not conflict with classical views in the field.

(3) The mechanism is still far from clear and this study shines some light on our lack of understanding. Basic and key questions remain:<br /> a) How is the centrosome moved toward the Par3-rich pole? This is particularly difficult to answer if the mechanism does not imply the anchoring of MTs to the centriole or PCM.<br /> b) What happens during cytokinesis that organises Par3 and intercellular junction in a way that can't be achieved by simply bringing two cells together? In larger epithelia cells have neighbours that are not daughters, still, they can form tight junctions with Par3 which participates in the establishment of cell polarity as much as those that are closer to the cytokinetic bridge (as judged by the overall cell symmetry). Is the protocol of cell aggregation fully capturing the interaction mechanism of non-daughter cells?

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).

(1) It is convincing that co-infection of WT and ∆MYR1 parasites could rescue the growth of ∆MYR1 in mice shown by in vivo luciferase imaging. Also, this is consistent with ∆MYR1 parasites showing no in vivo fitness defect in the in vivo CRISPR screens conducted by several groups. Meanwhile, it has been reported previously and shown in this manuscript that ∆MYR1 parasites have an in vitro growth defect; however, ∆MYR1 parasites show no in vitro fitness defect the in vitro pooled CRISPR screen. The authors show that the competition defect of ∆MYR1 parasites cannot be rescued by co-infection with WT parasites in Figure 1c, which might indicate that no paracrine rescue occurred in an in vitro environment. The authors seem not to mention these discrepancies between in vitro CRISPR screens and in vitro competition assays. Why do ∆MYR1 parasites possess neutral in vitro fitness scores in in vitro CRISPR screens? Could the authors describe a reasonable hypothesis?

(2) The authors developed a mixed infection assay with an inoculum containing a 20:80 ratio of ΔMYR1-Luc parasites with either WT parasites or ΔMYR1 mutants not expressing luciferase, showing that the in vivo growth defect of ∆MYR1 parasites is rescued by the presence of WT parasites. Since this experiment lacks appropriate controls, interpretation could be difficult. Is this phenomenon specific to MYR1? If a co-inoculum of ∆GRA12-Luc with either WT parasites or GRA12 parasites not expressing luciferase is included, the data could be appropriately interpreted.

(3) In the Discussion part, the authors argue that the rescue phenotype of mixed infection is not due to co-infection of host cells (lines 307-310). This data is important to support the authors' paracrine hypothesis and should be shown in the main figure.

(4) In the Discussion part, the authors assume that the rescue phenotype is the result of multiple MYR1-dependent effectors. I admit that this hypothesis could be possible since a recently published paper described the concerted action of numerous MYR1-dependent or independent effectors contributing to the hypermigration of infected cells (Ten Hoeve et al., mBio, 2024). I think this paragraph would be kind of overstated since the authors did not test any of the candidate effectors. Since the authors possess ∆IST parasites, they can test whether IST is involved in the "paracrine masking effect" or not to support their claim.

Reviewer #2 (Public review):

In this paper, Hotinger et. al. propose an improved barcoded library system, called STAMPR, to study Salmonella population dynamics during infection. Using this system, the authors demonstrate significant diversity in the colonization of different Salmonella clones (defined by the presence of different barcodes) not only across different organs (liver, spleen, adipose tissues, pancreas, and gall bladder) but also within different compartments of the same gastrointestinal tissue. Additionally, this system revealed that microbiota competition is the major bottleneck in Salmonella intestinal colonization, which can be mitigated by streptomycin treatment. However, this has been demonstrated previously in numerous publications. They also show that there was minimal sharing between populations found in the intestine and those in the other organs. Upon IV and IP infection to bypass the intestinal bottleneck, they were able to demonstrate, using this library, that Salmonella can renter the intestine through two possible routes. One route is essentially the reverse path used to escape the gut, leading to a diverse intestinal population; while the other, through the bile, typically results in a clonal population. Although the authors showed that the STAMPR pipeline improved the ability to identify founder populations and their diversity within the same animal during infections, some of the conclusions appear speculative and not fully supported.

(1) It's particularly interesting how the authors, using this system, demonstrate the dominant role of the microbiota bottleneck in Salmonella colonization and how it is widened by antibiotic treatment (Figure 1). Additionally, the ability to track Salmonella reseeding of the gut from other organs starting with IV and IP injections of the pathogen provides a new tool to study population dynamics (Figure 5). However, I don't think it is possible to argue that the proximal and distal small intestine, Peyer's patches (PPs), cecum, colon, and feces have different founder populations for reasons other than stochastic variations. All the barcoded Salmonella clones have the same fitness and the fact that some are found or expanded in one region of the gastrointestinal tract rather than another likely results from random chance - such as being forced in a specific region of the gut for physical or spatial reasons-and subsequent expansion, rather than any inherent biological cause. For example, some bacteria may randomly adhere to the mucus, some may swim toward the epithelial layer, while others remain in the lumen; all will proliferate in those respective sites. In this way, different founder populations arise based on random localization during movement through the gastrointestinal tract, which is an observation, but it doesn't significantly contribute to understanding pathogen colonization dynamics or pathogenesis. Therefore, I would suggest placing less emphasis on describing these differences or better discussing this aspect, especially in the context of the gastrointestinal tract.

(2) I do think that STAMPR is useful for studying the dynamics of pathogen spread to organs where Salmonella likely resides intracellularly (Figure 3). The observation that the liver is colonized by an early intestinal population, which continues to proliferate at a steady rate throughout the infection, is very interesting and may be due to the unique nature of the organ compared to the mucosal environment. What is the biological relevance during infection? Do the authors observe the same pattern (Figures 3C and G) when normalizing the population data for the spleen and mesenteric lymph nodes (mLN)? If not, what do the authors think is driving this different distribution?

(3) Figure 6: Could the bile pathology be due to increased general bacterial translocation rather than Salmonella colonization specifically? Did the authors check for the presence of other bacteria (potentially also proliferating) in the bile? Do the authors know whether Salmonella's metabolic activity in the bile could be responsible for gallbladder pathology?

Reviewer #2 (Public review):

Summary:

The authors sequence 45 new samples of S. Gallinarum, a commensal Salmonella found in chickens, which can sometimes cause disease. They combine these sequences with around 500 from public databases, determine the population structure of the pathogen, and coarse relationships of lineages with geography. The authors further investigate known anti-microbial genes found in these genomes, how they associate with each other, whether they have been horizontally transferred, and date the emergence of clades.

Strengths:

(1) It doesn't seem that much is known about this serovar, so publicly available new sequences from a high-burden region are a valuable addition to the literature.

(2) Combining these sequences with publicly available sequences is a good way to better contextualise any findings.

Weaknesses:

There are many issues with the genomic analysis that undermine the conclusions, the major ones I identified being:

(1) Recombination removal using gubbins was not presented fully anywhere. In this diversity of species, it is usually impossible to remove recombination in this way. A phylogeny with genetic scale and the gubbins results is needed. Critically, results on timing the emergence (fig2) depend on this, and cannot be trusted given the data presented.

(2) The use of BEAST was also only briefly presented, but is the basis of a major conclusion of the paper. Plot S3 (root-to-tip regression) is unconvincing as a basis of this data fitting a molecular clock model. We would need more information on this analysis, including convergence and credible intervals.

(3) Using a distance of 100 SNPs for a transmission is completely arbitrary. This would at least need to be justified in terms of the evolutionary rate and serial interval.

(4) The HGT definition is non-standard, and phylogeny (vertical inheritance) is not controlled for.<br /> The cited method:<br /> 'In this study, potentially recently transferred ARGs were defined as those with perfect identity (more than 99% nucleotide identity and 100% coverage) in distinct plasmids in distinct host bacteria using BLASTn (E-value {less than or equal to}10−5)'<br /> This clearly does not apply here, as the application of distinct hosts and plasmids cannot be used. Subsequent analysis using this method is likely invalid, and some of it (e.g. Figure 6c) is statistically very poor.

(5) Associations between lineages, resistome, mobilome, etc do not control for the effect of genetic background/phylogeny. So e.g. the claim 'the resistome also demonstrated a lineage-preferential distribution' is not well-supported.

(6) The invasiveness index is not well described, and the difference in means is not biologically convincing as although it appears significant, it is very small.

(7) 'In more detail, both the resistome and mobilome exhibited a steady decline until the 1980s, followed by a consistent increase from the 1980s to the 2010s. However, after the 2010s, a subsequent decrease was identified.'<br /> Where is the data/plot to support this? Is it a significant change? Is this due to sampling or phylogenetics?

(8) It is not clear what the burden of disease this pathogen causes in the population, or how significant it is to agricultural policy. The article claims to 'provide valuable insights for targeted policy interventions.', but no such interventions are described.

(9) The abstract mentions stepwise evolution as a main aim, but no results refer to this.

(10) The authors attribute changes in population dynamics to normalisation in China-EU relations and hen fever. However, even if the date is correct, this is not a strongly supported causal claim, as many other reasons are also possible (for example other industrial processes which may have changed during this period).

(11) No acknowledgment of potential undersampling outside of China is made, for example, 'Notably, all bvSP isolates from Asia were exclusively found in China, which can be manually divided into three distinct regions (southern, eastern, and northern).'. Perhaps we just haven't looked in other places?

(12) Many of the conclusions are highly speculative and not supported by the data.

(13) The figures are not always the best presentation of the data:<br /> a. Stacked bar plots in Figure 1 are hard to interpret, the total numbers need to be shown. Panel C conveys little information.<br /> b. Figure 4B: stacked bars are hard to read and do not show totals.<br /> c. Figure 5 has no obvious interpretation or significance.

In summary, the quality of analysis is poor and likely flawed (although there is not always enough information on methods present to confidently assess this or provide recommendations for how it might be improved). So, the stated conclusions are not supported.

Reviewer #2 (Public review):

Temperature is a critical factor affecting the progression of viral diseases in vertebrates and invertebrates. In the current study, the authors investigate mechanisms by which high temperatures promote anti-viral resistance in shrimp. They show that high temperatures induce HSF1 expression, which in turn upregulates AMPs. The AMPs target viral envelope proteins and inhibit viral infection/replication. The authors confirm this process in drosophila and suggest that there may be a conserved mechanism of high-temperature mediated anti-viral response in arthropods. These findings will enhance our understanding of how high temperature improves resistance to viral infection in animals.

The conclusions of this paper are mostly well supported by data, but some aspects of data analysis need to be clarified and extended. Further investigation on how WSSV infection is affected by AMP would have strengthened the study.

https://www.liberatingstructures.com/1-1-2-4-all/

https://app.thebrain.com/brain/3d80058c-14d8-5361-0b61-a061f89baf87/fee4a5d8-6d7d-417a-8be2-2b2cc0e97966

Reviewer #2 (Public review):

Summary:

This study, based on their previous findings that TFH cells can be converted into TR1 cells, conducted a highly detailed and comprehensive epigenetic investigation to answer whether TR1 differentiation from TFH is driven by epigenetic changes. Their evidence indicated that the downregulation of TFH-related genes during the TFH to TR1 transition depends on chromatin closure, while the upregulation of TR1-related genes does not depend on epigenetic changes.

Strengths:

A significant advantage of their approach lies in its detailed and comprehensive assessment of epigenetics. Their analysis of epigenetics covers chromatin open regions, histone modifications, DNA methylation, and using both single-cell and bulk techniques to validate their findings. As for their results, observations from different epigenetic perspectives mutually supported each other, lending greater credibility to their conclusions. This study effectively demonstrates that 1. the TFH-to-TR1 differentiation process is associated with massive closure of OCRs, and 2. the TR1-poised epigenome of TFH cells is a key enabler of this transdifferentiation process. Considering the extensive changes in epigenetic patterns involved in other CD4+ T lineage commitment processes, the similarity between TFH and TR1 in their epigenetics is intriguing.

They performed correlation analysis to answer the association between "pMHC-NP-induced epigenetic change" and "gene expression change in TR1". Also, they have made their raw data publicly available, providing a comprehensive epigenomic database of pMHC-NP induced TR1 cells. This will serve as a valuable reference for future research.

Weaknesses:

A major limitation is that this study heavily relies on a premise from the previous studies performed by the same group on pMHC-NP-induced T cell responses. This significantly limits the relevance of their conclusion to a broader perspective. Specifically, differential OCRs between Tet+ and naïve T cells were limited to only 821, as compared to 10,919 differential OCRs between KLH-TFH and naïve T cells (Fig. 2A), indicating that the precursors and T cell clonotypes that responded to pMHC-NP were extremely limited. I acknowledge that this limitation has been added and discussed in the Discussion section of the revised manuscript.

Reviewer #2 (Public review):

It is challenging to study the biophysical properties of organelle channels using conventional electrophysiology. The conventional reconstitution methods requires multiple steps and can be contaminated by endogenous ionophores from the host cell lines after purification. To overcome this challenge, in this manuscript, Larmore et al. described a fully synthetic method to assay the functional properties of the TRPP channel family. The TRPP channels are an important organelle ion channel family that natively traffic to primary cilia and ER organelles. The authors utilized cell-free protein expression and reconstitution of the synthetic channel protein into giant unilamellar vesicles (GUV), the single channel properties can be measured using voltage-clamp electrophysiology. Using this innovative method, the authors characterized their membrane integration, orientation, and conductance, comparing the results to those of endogenous channels. The manuscript is well-written and may present broad interest to the ion channel community studying organelle ion channels. Particularly because of the challenges of patching native cilia cells, the functional characterization is highly concentrated in very few labs. This method may provide an alternative approach to investigate other channels resistant to biophysical analysis and pharmacological characterization.

Comments on revised version:

The authors have addressed my concerns. This excellent method manuscript would benefit the study of organelle channels.

Reviewer #2 (Public review):

The relationships between genes and phenotypes are complex and the impact of deleting or a gene can often have multifaceted and unforeseen consequences. This paper dissected the role of calcineurin, encoded by tax-6, in various phenotypes in C. elegans, including lifespan, pathogen susceptibility, the defecation motor program, and nutrient absorption or calorie restriction, through a series of genetic and behavioral analyses. Many genes in these pathways were tested yielding robust results. Classic epistasis analyses were used to distinguish between genes operating in the same or separate pathways. Researchers in the related fields will be very interested in looking through the data presented in this paper in great detail and benefit from it.

Overall, this paper supports a model in which the increased lifespan and heightened pathogen susceptibility observed following calcineurin inhibition result from the disruptions in the defecation motor program but through distinct pathways. A defective defecation motor program leads to intestine bloating and compromised nutrient absorption. Calorie restriction resulting from poor nutrient absorption affects lifespan, whereas increased colonization in the bloated intestine heightens pathogen susceptibility. The observation that knockdown of several other DMP-related genes also results in increased lifespan and pathogen susceptibility further reinforces the proposed model.

Reviewer #2 (Public Review):

Summary:

The authors inquire in particular whether the receptor Gpr156, which is necessary for hair cells to reverse their polarities in the zebrafish lateral line and mammalian otolith organs downstream of the differential expression of the transcription factor Emx2, also controls the mechanosensitive properties of hair cells and ultimately an animal's behavior. This study thoroughly addresses the issue by analyzing the morphology, electrophysiological responses, and afferent connections of hair cells found in different regions of the mammalian utricle and the Ca2+ responses of lateral line neuromasts in both wild-type animals and gpr156 mutants. Although many features of hair cell function are preserved in the mutants-such as development of the mechanosensory organs and the Emx2-dependent, polarity-specific afferent wiring and synaptic pairing-there are a few key changes. In the zebrafish neuromast, the magnitude of responses of all hair cells to water flow resembles that of the wild-type hair cells that respond to flow arriving from the tail. These responses are larger than those observed in hair cells that are sensitive to flow arriving from the head and resemble effects previously observed in Emx2 mutants. The authors note that this behavior suggests that the Emx2-GPR156 signaling axis also impinges on hair cell mechanotransduction. Although mutant mice exhibit normal posture and balance, they display defects in swimming behavior. Moreover, their vestibulo-ocular reflexes are perturbed. The authors note that the gpr156 mutant is a good model to study the role of opposing hair cell polarity in the vestibular system, for the wiring patterns follow the expression patterns of Emx2, even though hair cells are all of the same polarity. This paper excels at describing the effects of gpr156 perturbation in mouse and zebrafish models and will be of interest to those studying the vestibular system, hair cell polarity, and the role of inner-ear organs in animal behavior.

The study is exceptional in including, not only morphological and immunohistochemical indices of cellular identity but also electrophysiological properties. The mutant hair cells of murine maculæ display essentially normal mechanoelectrical transduction and adaptation-with two or even three kinetic components-as well as normal voltage-activated ionic currents.

Reviewer #2 (Public review):

Summary:

Here the authors describe a model for tracking time-varying coupling between neurons from multi-electrode spike recordings. Their approach extends a GLM with static coupling between neurons to include dynamic weights, learned by a long-short-term-memory (LSTM) model. Each connection has a corresponding LSTM embedding and is read-out by a multi-layer perceptron to predict the time-varying weight.

Strengths:

This is an interesting approach to an open problem in neural data analysis. I think, in general, the method would be interesting to computational neuroscientists.

Weaknesses:

It is somewhat difficult to interpret what the model is doing. I think it would be worthwhile to add some additional results that make it more clear what types of patterns are being described and how.

Major Issues:

Simulation for dynamic connectivity. It certainly seems doable to simulate a recurrent spiking network whose weights change over time, and I think this would be a worthwhile validation for this DyNetCP model. In particular, I think it would be valuable to understand how much the model overfits, and how accurately it can track known changes in coupling strength. If the only goal is "smoothing" time-varying CCGs, there are much easier statistical methods to do this (c.f. McKenzie et al. Neuron, 2021. Ren, Wei, Ghanbari, Stevenson. J Neurosci, 2022), and simulations could be useful to illustrate what the model adds beyond smoothing.

Stimulus vs noise correlations. For studying correlations between neurons in sensory systems that are strongly driven by stimuli, it's common to use shuffling over trials to distinguish between stimulus correlations and "noise" correlations or putative synaptic connections. This would be a valuable comparison for Fig 5 to show if these are dynamic stimulus correlations or noise correlations. I would also suggest just plotting the CCGs calculated with a moving window to better illustrate how (and if) the dynamic weights differ from the data.

Minor Issues:

Introduction - it may be useful to mention that there have been some previous attempts to describe time-varying connectivity from spikes both with probabilistic models: Stevenson and Kording, Neurips (2011), Linderman, Stock, and Adams, Neurips (2014), Robinson, Berger, and Song, Neural Computation (2016), Wei and Stevenson, Neural Comp (2021) ... and with descriptive statistics: Fujisawa et al. Nat Neuroscience (2008), English et al. Neuron (2017), McKenzie et al. Neuron (2021).

In the sections "Static DyNetCP ...reproduce". It may be useful to have some additional context to interpret the CCG-DyNetCP correlations and CCG-GLMCC correlations (for simulation). If I understand right, these are on training data (not cross-validated) and the DyNetCP model is using NM+1 parameters to predict ~100 data points (It would also be good to say what N and M are for the results here). The GLMCC model has 2 or 3 parameters (if I remember right?).

In the section "Static connectivity inferred by the DyNetCP from in-vivo recordings is biologically interpretable"... I may have missed it, but how is the "functional delay" calculated? And am I understanding right that for the DyNetCP you are just using [w_i\toj, w_j\toi] in place of the CCG?

Reviewer #2 (Public review):

Summary:

This paper tackles the problem of understanding when the dynamics of neural population activity do and do not align with some target output, such as an arm movement. The authors develop a theoretical framework based on RNNs showing that an alignment of neural dynamics to an output can be simply controlled by the magnitude of the read-out weight vector while the RNN is being trained: small magnitude vectors result in aligned dynamics, where low-dimensional neural activity recapitulates the target; large magnitude vectors result in "oblique" dynamics, where encoding is spread across many dimensions. The paper further explores how the aligned and oblique regimes differ, in particular that the oblique regime allows degenerate solutions for the same target output.

Strengths:

- A really interesting new idea that different dynamics of neural circuits can arise simply from the initial magnitude of the output weight vector: once written out (Eq 3) it becomes obvious, which I take as the mark of a genuinely insightful idea

- The offered framework potentially unifies a collection of separate experimental results and ideas, largely from studies of motor cortex in primate: the idea that much of the ongoing dynamics do not encode movement parameters; the existence of the "null space" of preparatory activity; and that ongoing dynamics of motor cortex can rotate in the same direction even when the arm movement is rotating in opposite directions.

- The main text is well written, with a wide-ranging set of key results synthesised and illustrated well and concisely

- Shows the occurrence of the aligned and oblique regimes generalises across a range of simulated behavioural tasks

- A deep analytical investigation of when the regimes occur and how they evolve over training

- Shows where the oblique regime may be advantageous: allows multiple solutions to the same problem; and differs in sensitivity to perturbation and noise

- An insightful corollary result that noise in training is needed to obtain the oblique regime

- Tests whether the aligned and oblique regimes can be seen in neural recordings from primate cortex in a range of motor control tasks

- The revised text offers greater clarity and precision about when the aligned and oblique regimes occur and in the interpretation of the analyses of neural data

Weaknesses:

- The depth of analytical treatment in the Methods is impressive; however, the paper and the Methods analyses are largely independent, with the numerous results in the latter not being mentioned in the Results or Discussion. It in effect operates as two papers.

Reviewer #2 (Public review):

In the revised manuscript, the authors investigated the role of a presynaptic protein, Rab3A, in the homeostatic synaptic plasticity in cultured cortical neurons. The study was motivated by their previous findings that Rab3A is required for expression of similar homeostatic mechanisms at the neuromuscular junction. The authors first show that untreated WT neurons express homeostatic synaptic plasticity in response to 48h of TTX treatment (upregulation of both mEPSC amplitude and frequency), whereas neurons lacking Rab3A or carrying a dominant negative mutated Rab3A (earlybird) do not. They also demonstrate that only neuronal, but not glial Rab3A is responsible for this defect. Furthermore, they confirm the increased mEPSC amplitudes in WT neurons reflect the addition of GluA2-containing AMPA receptors rather than calcium-permeable ones, as previously reported by multiple labs. However, the increase in mEPSC amplitude is not accompanied by a corresponding upregulation of GluA2 synaptic clusters according to their IHC data (cluster size and intensity trend slightly upwards but not reaching significance). They further show that this modest upward trend is absent in Rab3A KO neurons, and conclude that Rab3A is involved in postsynaptic GluA2 upregulation during homeostatic synaptic plasticity.

When compared to the original version, the authors have done an admirable job in switching to more established ways to assess homeostatic synaptic plasticity by comparing the mean mEPSC amplitude and frequency, which has greatly improved the legibility of the manuscript to the public. Their data in Figures 1,2, and 8 clearly demonstrate that functional Rab3A in cortical neurons is required for the homeostatic regulation of mEPSCs.

However, the authors still have not provided further investigation of the mechanisms behind the role of Rab3A in this form of plasticity, and the revision therefore has added little to the significance of the study. Moreover, the experimental design for the investigation of the mismatch between mEPSC amplitude and GluA2 cluster fluorescence remains questionable, making it difficult to draw any credible conclusions from groups of data that not only look similar to the eye but also show no significance statistically.

A major claim the authors want to make is that Rab3A, although a presynaptic protein, regulates postsynaptic GluA2, and they do this by showing in Figure 5 that the upward trend of GluA2 cluster size and intensity is absent in Rab3A KO neurons. First, it is difficult to convince readers that this upward trend is real in Figures 5B-D without getting more samples. Second, the authors pick GluA2 clusters on the primary dendrites, whereas mEPSC events come from a much larger synapse population (e.g., more distal), therefore it makes sense that these two forms of measurement do not show matching changes, and this caveat could be addressed by sampling more diverse dendritic locations. Without a convincing phenotype in WT neurons, the support for this claim is weak.

Another claim of the authors is that this mismatch between mEPSC amplitude and GluA2 cluster sizes with the same culture suggests there are other factors contributing to the mEPSC amplitude. They do this by comparing results from individual culture dissociations, which greatly suffer from undersampling (Figure 6). In particular, they point out that 2 out of 3 dissociations show "matching" upward trends in mEPSC and GluA2 cluster (figure 6A and 6B) while the third one shows opposite trends, and use this to support their claim. Anyone who has done culture preparation would know the high variability between dissociations, which is why culture data are always pooled for assessment of any population trend. Anything could have happened to this particular dissociation (culture #3, figure 6C), and drawing conclusion from this single incident does little to support this claim. At least, they should double the dissociation numbers, and their claim would be much more convincing if a similar phenomenon occurs again. Besides, as mentioned above, all these mismatching trends could just be due to sampling differences.

In summary, this study establishes that neuronal Rab3A plays a role in homeostatic synaptic plasticity, but so do a number of other molecules that have been implicated in homeostatic synaptic plasticity in the past two decades (only will grow with the new techniques such as RNAseq). Without going beyond this finding and demonstrating how exactly Rab3A participates in the induction and/or expression of this form of plasticity, or maybe the potential Rab3A-mediated functional and behavioral defects in vivo, the contribution of the current study to the field is limited. However, given the presynaptic location of Rab3A, this finding could serve as a starting point for researchers interested in pre-postsynaptic cross-talk during homeostatic plasticity in general.

Reviewer #2 (Public review):

Summary:

The present manuscript addresses a longstanding challenge in neuroscience: how the brain assigns credit for delayed outcomes, especially in real-world learning scenarios where decisions and outcomes are separated by time. The authors focus on the lateral orbitofrontal cortex and hippocampus, key regions involved in contingent learning. By integrating fMRI data and behavioral tasks, the authors examined how neural circuits maintain a causal link between past decisions and delayed outcomes. Their findings offer insights into mechanisms that could have critical implications for understanding human decision-making.

Strengths:

(1) The experimental designs were extremely well thought-out. The authors successfully coupled behavioral data and neural measures (through fMRI) to explore the neural mechanisms of contingent learning. This integration adds robustness to the findings and strengthens their relevance.

(2) The emphasis on the interaction between the lateral orbitofrontal cortex (lOFC) and hippocampus (HC) in this study is very well-targeted. The reported findings regarding their dynamic interactions provide valuable insights into contingent learning in humans.

(3) The use of an advanced modeling framework and analytical techniques allowed the authors to uncover new mechanistic insights regarding a complex case of the decision-making process. The methods developed will also benefit analyses of future neuroimaging data on a range of decision-making tasks as well.

Weaknesses:

Given the limited temporal resolution of fMRI and that the measured signal is an indirect measure of neural activity, it is unclear the extent to which the reported causality reflects the true relationship/interactions between neurons in different regions.

Reviewer #2 (Public review):

Summary:

Deshmukh and colleagues studied the evolution of mimetic morphs in the Papilio polytes species group. They investigate the timing of origin of haplotypes associated with different morphs, their dominance relationships, associations with different isoform expressions, and evidence for selection and recombination in the sequence data. P. polytes is a textbook example of a Batesian mimic, and this study provides important nuanced insights into its evolution, and will therefore be relevant to many evolutionary biologists. I find the results regarding dominance and the sequence of events generally convincing, but I have some concerns about the motivation and interpretation of some other analyses, particularly the tests for selection.

Strengths:

This study uses widespread sampling, large sample sizes from crossing experiments, and a wide range of data sources.

Weaknesses:

(1) Purpose and premise of selective sweep analysis

A major narrative of the paper is that new mimetic alleles have arisen and spread to high frequency, and their dominance over the pre-existing alleles is consistent with Haldane's sieve. It would therefore make sense to test for selective sweep signatures within each morph (and its corresponding dsx haplotype), rather than at the species level. This would allow a test of the prediction that those morphs that arose most recently would have the strongest sweep signatures.

Sweep signatures erode over time - see Figure 2 of Moest et al. 2020 (https://doi.org/10.1371/journal.pbio.3000597), and it is unclear whether we expect the signatures of the original sweeps of these haplotypes to still be detectable at all. Moest et al show that sweep signatures are completely eroded by 1N generations after the event, and probably not detectable much sooner than that, so assuming effective population sizes of these species of a few million, at what time scale can we expect to detect sweeps? If these putative sweeps are in fact more recent than the origin of the different morphs, perhaps they would more likely be associated with the refinement of mimicry, but not necessarily providing evidence for or against a Haldane's sieve process in the origin of the morphs.

(2) Selective sweep methods

A tool called RAiSD was used to detect signatures of selective sweeps, but this manuscript does not describe what signatures this tool considers (reduced diversity, skewed frequency spectrum, increased LD, all of the above?). Given the comment above, would this tool be sensitive to incomplete sweeps that affect only one morph in a species-level dataset? It is also not clear how RAiSD could identify signatures of selective sweeps at individual SNPs (line 206). Sweeps occur over tracts of the genome and it is often difficult to associate a sweep with a single gene.

(3) Episodic diversification

Very little information is provided about the Branch-site Unrestricted Statistical Test for Episodic Diversification (BUSTED) and Mixed Effects Model of Evolution (MEME), and what hypothesis the authors were testing by applying these methods. Although it is not mentioned in the manuscript, a quick search reveals that these are methods to study codon evolution along branches of a phylogeny. Without this information, it is difficult to understand the motivation for this analysis.

(4) GWAS for form romulus

The authors argue that the lack of SNP associations within dsx for form romulus is caused by poor read mapping in the inverted region itself (line 125). If this is true, we would expect strong association in the regions immediately outside the inversion. From Figure S3, there are four discrete peaks of association, and the location of dsx and the inversion are not indicated, so it is difficult to understand the authors' interpretation in light of this figure.

(5) Form theseus

Since there appears to be only one sequence available for form theseus (actually it is said to be "P. javanus f. polytes/theseus"), is it reasonable to conclude that "the dsx coding sequence of f. theseus was identical to that of f. polytes in both P. javanus and P. alphenor" (Line 151)? Looking at the Clarke and Sheppard (1972) paper cited in the statement that "f. polytes and f. theseus show equal dominance" (line 153), it seems to me that their definition of theseus is quite different from that here. Without addressing this discrepancy, the results are difficult to interpret.

Reviewer #2 (Public review):

Summary:

Pluripotent stem cells are powerful tools for understanding development, differentiation, and disease modeling. The capacity of stem cells to differentiate into various cell types holds great promise for therapeutic applications. However, ethical concerns restrict the use of human embryonic stem cells (hESCs). Consequently, induced human pluripotent stem cells (ihPSCs) offer an attractive alternative for modeling rare diseases, drug screening, and regenerative medicine. A comprehensive understanding of ihPSCs is crucial to establish their similarities and differences compared to hESCs. This work demonstrates systematic differences in the reprogramming of nuclear and non-nuclear proteomes in ihPSCs.

Strengths:

The authors employed quantitative mass spectrometry to compare protein expression differences between independently derived ihPSC and hESC cell lines. Qualitatively, protein expression profiles in ihPSC and hESC were found to be very similar. However, when comparing protein concentration at a cellular level, it became evident that ihPSCs express higher levels of proteins in the cytoplasm, mitochondria, and plasma membrane, while the expression of nuclear proteins is similar between ihPSCs and hESCs. A higher expression of proteins in ihPSCs was verified by an independent approach, and flow cytometry confirmed that ihPSCs had larger cell size than hESCs. The differences in protein expression were reflected in functional distinctions. For instance, the higher expression of mitochondrial metabolic enzymes, glutamine transporters, and lipid biosynthesis enzymes in ihPSCs was associated with enhanced mitochondrial potential, increased ability to uptake glutamine, and increased ability to form lipid droplets.

Weaknesses:

While this finding is intriguing and interesting, the study falls short of explaining the mechanistic reasons for the observed quantitative proteome differences. It remains unclear whether the increased expression of proteins in ihPSCs is due to enhanced transcription of the genes encoding this group of proteins or due to other reasons, for example, differences in mRNA translation efficiency. Another unresolved question pertains to how the cell type origin influences ihPSC proteomes. For instance, whether ihPSCs derived from fibroblasts, lymphocytes, and other cell types all exhibit differences in their cell size and increased expression of cytoplasmic and mitochondrial proteins. Analyzing ihPSCs derived from different cell types and by different investigators would be necessary to address these questions.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The methodology for generating and screening the anti-AG antibodies lacks pertinent details for recapitulating and interpreting the results.

The figure legends and methods associated with the microscopy assays lack sufficient details to appropriately interpret the experiments conducted.

The galectin-9 measured in the sera of TB patients does not approach the concentrations required for Mtb growth restriction in the in vitro assays performed by the authors. It remains difficult to envision how greater levels of galectin-9 release might contribute to Mtb control in severe forms of TB, since higher levels of serum Gal9 has been observed in other human studies and correlate with poorly controlled infection. The authors over-interpret the role of Gal9 in bacterial control during disease/infection without any evidence of impact on in vivo (animal model) control.

Reviewer #2 (Public review):

Summary:

In this work, Witten et al. assess visual acuity, cone density, and fixational behavior in the central foveal region in a large number of subjects.<br /> This work elegantly presents a number of important findings, and I can see this becoming a landmark work in the field. First, it shows that acuity is determined by the cone mosaic, hence, subjects characterized by higher cone densities show higher acuity in diffraction limited settings. Second, it shows that humans can achieve higher visual resolution than what is dictated by cone sampling, suggesting that this is likely the result of fixational drift, which constantly moves the stimuli over the cone mosaic. Third, the study reports a correlation between the amplitude of fixational motion and acuity, namely, subjects with smaller drifts have higher acuities and higher cone density. Fourth, it is shown that humans tend to move the fixated object toward the region of higher cone density in the retina, lending further support to the idea that drift is not a random process, but is likely controlled. This is a beautiful and unique work that furthers our understanding of the visuomotor system and the interplay of anatomy, oculomotor behavior, and visual acuity.

Strengths:

The work is rigorously conducted, it uses state-of-the-art technology to record fixational eye movements while imaging the central fovea at high resolution, and examines exactly where the viewed stimulus falls on individuals' foveal cone mosaic with respect to different anatomical landmarks in this region. Figures are clear and nicely packaged. It is important to emphasize that this study is a real tour-de-force in which the authors collected a massive amount of data on 20 subjects. This is particularly remarkable considering how challenging it is to run psychophysics experiments using this sophisticated technology. Most of the studies using psychophysics with AO are, indeed, limited to a few subjects. Therefore, this work shows a unique set of data, filling a gap in the literature.

Weaknesses:

Data analysis has been improved after the first round of review. The revised version of the manuscript is solid, and there are no weaknesses that should be addressed. The authors added more statistical tests and analyses, reported comparable effects even when different metrics are used (e.g., diffusion constant), and removed the confusing text on myopia. I think this work represents a significant scientific contribution to vision science.

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 #2 (Public review):

Liu et al. applied hidden Markov models (HMM) to fMRI data from 64 participants listening to audio stories. The authors identified three brain states, characterized by specific patterns of activity and connectivity, that the brain transitions between during story listening. Drawing on a theoretical framework proposed by Berwick et al. (TICS 2023), the authors interpret these states as corresponding to external sensory-motor processing (State 1), lexical processing (State 2), and internal mental representations (State 3). States 1 and 3 were more likely to transition to State 2 than between one another, suggesting that State 2 acts as a transition hub between states. Participants whose brain state trajectories closely matched those of an individual with high comprehension scores tended to have higher comprehension scores themselves, suggesting that optimal transitions between brain states facilitated narrative comprehension.

Overall, the conclusions of the paper are well-supported by the data. Several recent studies (e.g., Song, Shim, and Rosenberg, eLife, 2023) have found that the brain transitions between a small number of states; however, the functional role of these states remains under-explored. An important contribution of this paper is that it relates the expression of brain states to specific features of the stimulus in a manner that is consistent with theoretical predictions.

(1) It is worth noting, however, that the correlation between narrative features and brain state expression (as shown in Figure 3) is relatively low (~0.03). Additionally, it was unclear if the temporal correlation of the brain state expression was considered when generating the null distribution. It would be helpful to clarify whether the brain state expression time courses were circularly shifted when generating the null.

(2) A strength of the paper is that the authors repeated the HMM analyses across different tasks (Figure 5) and an independent dataset (Figure S3) and found that the data was consistently best fit by 3 brain states. However, it was not entirely clear to me how well the 3 states identified in these other analyses matched the brain states reported in the main analyses. In particular, the confusion matrices shown in Figure 5 and Figure S3 suggests that that states were confusable across studies (State 2 vs. State 3 in Fig. 5A and S3A, State 1 vs. State 2 in Figure 5B). I don't think this takes away from the main results, but it does call into question the generalizability of the brain states across tasks and populations.

(3) The three states identified in the manuscript correspond rather well to areas with short, medium, and long temporal timescales (see Hasson, Chen & Honey, TiCs, 2015). Given the relationship with behavior, where State 1 responds to acoustic properties, State 2 responds to word-level properties, and State 3 responds to clause-level properties, the authors may want to consider a "single-process" account where the states differ in terms of the temporal window for which one needs to integrate information over, rather than a multi-process account where the states correspond to distinct processes.

Reviewer #2 (Public review):

Summary:

The authors inspect the stability and compensatory plasticity in the retinotopic mapping in patients with congenital achromatopsia. They report an increased cortical thickness in central (eccentricities 0-2 deg) in V1 and the expansion of this effect to V2 (trend) and V3 in a cohort with an average age of adolescents.

In analyzing the receptive fields, they show that V1 had increased receptive field sizes in achromats, but there were no clear signs of reorganization filling in the rod-free area.<br /> In contrast, V3 showed an altered readout of V1 receptive fields. V3 of achromats oversampled the receptive fields bordering the rod-free zone, presumably to compensate and arrive at similar receptive fields as in the controls.

These findings support a retention of peripheral-V1 connectivity, but a reorganization of later hierarchical stages of the visual system to compensate for the loss, highlighting a balance between stability and compensation in different stages of the visual hierarchy.

Strengths:

The experiment is carefully analyzed, and the data convey a clear and interesting message about the capacities of plasticity.

Weaknesses:

The existence of unstable fixation and nystagmus in the patient group is alluded to, but not quantified or modeled out in the analyses. The authors may want to address this possible confound with a quantitative approach.

Reviewer #2 (Public review):

Summary:

Ziółkowska et al. characterize the synaptic mechanisms at the basis of the REdCA1 contribution to the consolidation of fear memory extinction. In particular, they describe a layer specific modulation of RE-dCA1 excitatory synapses modulation associated to contextual fear extinction which is impaired by transient chemogenetic inhibition of this pathway. These results indicate that RE activity-mediated modulation of synaptic morphology contributes to the consolidation of contextual fear extinction

Strengths:

The manuscript is well conceived, the statistical analysis is solid and methodology appropriate. The strength of this work is that it nicely builds up on existing literature and provides new molecular insight on a thalamo-hippocampal circuit previously known for its role in fear extinction. In addition, the quantification of pre- and post-synapses is particularly thorough.

Weaknesses:

The findings in this paper are well supported by the data more detailed description of the methods is needed.

(1) In the paragraph Analysis of dCA1 synapses after contextual fear extinction (CFE), more experimental and methodological data should be given in the text: -how was PSD95 used for the analysis, what was the difference between RE. Even if Thy1-GFP mice were used in Fig.2, it appears they were not used for bouton size analysis. To improve clarity, I suggest moving panel 2C to Figure 3. It is not clear whether all RE axons were indiscriminately analysed in Fig. 2 or if only the ones displaying colocalization with both PSD95 and GFP were analysed. If GFP was not taken into account here, analysed boutons could reflect synapses onto inhibitory neurons and this potential scenario should be discussed<br /> (2) in the methods: The volume of intra-hippocampal CNO injections should be indicated. The concentration of 3 uM seems pretty low in comparison with previous studies. More details of what software/algorithm was used to score freezing should be included. CNO source is missing. Antibody dilutions for IHC should be indicated. Secondary antibody incubation time should be indicated

No statement about code and data availability is present.

Reviewer #2 (Public review):

McDougal et al. describe the surprising finding that IFIT1 proteins from different mammalian species inhibit the replication of different viruses, indicating that the evolution of IFIT1 across mammals has resulted in host species-specific antiviral specificity. Before this work, research into the antiviral activity and specificity of IFIT1 had mostly focused on the human ortholog, which was described to inhibit viruses including vesicular stomatitis virus (VSV) and Venezuelan equine encephalitis virus (VEEV) but not other viruses including Sindbis virus (SINV) and parainfluenza virus type 3 (PIV3). In the current work, the authors first perform evolutionary analyses on IFIT1 genes across a wide range of mammalian species and reveal that IFIT1 genes have evolved under positive selection in primates, bats, carnivores, and ungulates. Based on these data, they hypothesize that IFIT1 proteins from these diverse mammalian groups may show distinct antiviral specificities against a panel of viruses. By generating human cells that express IFIT1 proteins from different mammalian species, the authors show a wide range of antiviral activities of mammalian IFIT1s. Most strikingly, they find several IFIT1 proteins that have completely different antiviral specificities relative to human IFIT1, including IFIT1s that fail to inhibit VSV or VEEV, but strongly inhibit PIV3 or SINV. These results indicate that there is potential for IFIT1 to inhibit a much wider range of viruses than human IFIT1 inhibits. Electrophoretic mobility shift assays (EMSAs) suggest that some of these changes in antiviral specificity can be ascribed to changes in the direct binding of viral RNAs. Interestingly, they also find that chimpanzee IFIT1, which is >98% identical to human IFIT1, fails to inhibit any tested virus. Replacing three residues from chimpanzee IFIT1 with those from human IFIT1, one of which has evolved under positive selection in primates, restores activity to chimpanzee IFIT1. Together, these data reveal a vast diversity of IFIT1 antiviral specificity encoded by mammals, consistent with an IFIT1-virus evolutionary "arms race".

Overall, this is a very interesting and well-written manuscript that combines evolutionary and functional approaches to provide new insight into IFIT1 antiviral activity and species-specific antiviral immunity. The conclusion that IFIT1 genes in several mammalian lineages are evolving under positive selection is supported by the data, although there are some important analyses that need to be done to remove any confounding effects from gene recombination that has previously been described between IFIT1 and its paralog IFIT1B. The virology results, which convincingly show that IFIT1s from different species have distinct antiviral specificity, are the most surprising and exciting part of the paper. As such, this paper will be interesting for researchers studying mechanisms of innate antiviral immunity, as well as those interested in species-specific antiviral immunity. Moreover, it may prompt others to test a wide range of orthologs of antiviral factors beyond those from humans or mice, which could further the concept of host-specific innate antiviral specificity. Additional areas for improvement, which are mostly to clarify the presentation of data and conclusions, are described below.

Strengths:

(1) This paper is a very strong demonstration of the concept that orthologous innate immune proteins can evolve distinct antiviral specificities. Specifically, the authors show that IFIT1 proteins from different mammalian species are able to inhibit the replication of distinct groups of viruses, which is most clearly illustrated in Figure 4G. This is an unexpected finding, as the mechanism by which IFIT1 inhibits viral replication was assumed to be similar across orthologs. While the molecular basis for these differences remains unresolved, this is a clear indication that IFIT1 evolution functionally impacts host-specific antiviral immunity and that IFIT1 has the potential to inhibit a much wider range of viruses than previously described.

(2) By revealing these differences in antiviral specificity across IFIT1 orthologs, the authors highlight the importance of sampling antiviral proteins from different mammalian species to understand what functions are conserved and what functions are lineage- or species-specific. These results might therefore prompt similar investigations with other antiviral proteins, which could reveal a previously undiscovered diversity of specificities for other antiviral immunity proteins.

(3) The authors also surprisingly reveal that chimpanzee IFIT1 shows no antiviral activity against any tested virus despite only differing from human IFIT1 by eight amino acids. By mapping this loss of function to three residues on one helix of the protein, the authors shed new light on a region of the protein with no previously known function.

(4) Combined with evolutionary analyses that indicate that IFIT1 genes are evolving under positive selection in several mammalian groups, these functional data indicate that IFIT1 is engaged in an evolutionary "arms race" with viruses, which results in distinct antiviral specificities of IFIT1 proteins from different species.

Weaknesses:

(1) The evolutionary analyses the authors perform appear to indicate that IFIT1 genes in several mammalian groups have evolved under positive selection. However, IFIT1 has previously been shown to have undergone recurrent instances of recombination with the paralogous IFIT1B, which can confound positive selection analyses such as the ones the authors perform. The authors should analyze their alignments for evidence of recombination using a tool such as GARD (in the same HyPhy package along with MEME and FUBAR). Detection of recombination in these alignments would invalidate their positive selection inferences, in which case the authors need to either analyze individual non-recombining domains or limit the number of species to those that are not undergoing recombination. While it is likely that these analyses will still reveal a signature of positive selection, this step is necessary to ensure that the signatures of selection and sites of positive selection are accurate.

(2) The choice of IFIT1 homologs chosen for study needs to be described in more detail. Many mammalian species encode IFIT1 and IFIT1B proteins, which have been shown to have different antiviral specificity, and the evolutionary relationship between IFIT1 and IFIT1B paralogs is complicated by recombination. As such, the assertion that the proteins studied in this manuscript are IFIT1 orthologs requires additional support than the percent identity plot shown in Figure 3B.

(3) Some of the results and discussion text could be more focused on the model of evolution-driven changes in IFIT1 specificity. In particular, the chimpanzee data are interesting, but it would appear that this protein has lost all antiviral function, rather than changing its antiviral specificity like some other examples in this paper. As such, the connection between the functional mapping of individual residues with the positive selection analysis is somewhat confusing. It would be more clear to discuss this as a natural loss of function of this IFIT1, which has occurred elsewhere repeatedly across the mammalian tree.

(4) In other places in the manuscript, the strength of the differences in antiviral specificity could be highlighted to a greater degree. Specifically, the text describes a number of interesting examples of differences in inhibition of VSV versus VEEV from Figure 3C and 3D, but it is difficult for a reader to assess this as most of the dots are unlabeled and the primary data are not uploaded. A few potential suggestions would be to have a table of each ortholog with % infection by VSV and % infection by VEEV. Another possibility would be to plot these data as an XY scatter plot. This would highlight any species that deviate from the expected linear relationship between the inhibition of these two viruses, which would provide a larger panel of interesting IFIT1 antiviral specificities than the smaller number of species shown in Figure 4.

Reviewer #2 (Public review):

Summary:

The authors try to demonstrate that PD-1 regulates not only the quantity but also the quality of the immune response determining the Th differentiation. The authors suggest that the ability of PD-1 agonists to dampen Th2 differentiation could be exploited in allergies or classical Th2-mediated disease as a therapeutical approach.

Strengths:<br /> The authors performed a series of elegant experiments using OVA-specific CD4 T cells from mice, showing a strong reduction of Th2 differentiation in vitro. They also perform some experiments with a model of allergies, showing an amelioration of the phenotype after administration of PD-1 agonist with a reduction of Th2 cells.

Weaknesses:

The authors perform all the experiments using DO11.10 mouse cells. Such cells have a TCR with very high affinity, it would be relevant to repeat at least some of the in vitro assays in a more physiological setting (you can immunise mice with ova to increase the pool of OVA-specific T cells, and then repeat the restimulation experiment). Also, a longer kinetic would be of interest to see the effect of the agonist on Th1 cells.

Another drawback is the lack of experiments with human cells. It would be really important to repeat the experiments with CD4 T cells from healthy donors (the antibody that the authors use as PD-1 agonist is human, so it would not be a complicated experiment).

It would be also interesting to show in the allergic disease model the effect of the agonism on the T cell response in general.

Reviewer #2 (Public review):

Summary:

The authors have set out to investigate and explain how early members of the Pterosauria were able to maintain stiffness in the vane of their tails. This stiffness, it is said, was crucial for flight in early members of this clade. Through the use Laser-Stimulated Fluorescence imaging, the authors have revealed that certain pterosaurs had a sophisticated dynamic tensioning system that has previously been unappreciated.

Strengths:

The choice of method of investigation for the key question is sound enough, and the execution of the same is excellent. Overall the paper is well written and well presented, and provides a very succinct, accessible and clear conclusion.

Weaknesses:

None

Reviewer #2 (Public review):

Summary:

This study evaluated the aperiodic component in the medial prefrontal cortex (mPFC) using resting-state EEG recordings from 149 individuals with chronic pain and 115 healthy participants. The findings showed no significant differences in the aperiodic component of the mPFC between the two groups, nor was there any correlation between the aperiodic component and pain intensity. These results were consistent across various chronic pain subtypes and were corroborated by whole-brain analyses. The study's robustness was further reinforced by preregistration and multiverse analyses, which accounted for a wide range of methodological choices.

Strengths:

This study was rigorously conducted, yielding clear and conclusive results. Furthermore, it adhered to stringent open and reproducible science practices, including preregistration, blinded data analysis, and Bayesian hypothesis testing. All data and code have been made openly available, underscoring the study's commitment to transparency and reproducibility.

Weaknesses:

The aperiodic exponent of the EEG power spectrum is often regarded as an indicator of the excitatory/inhibitory (E/I) balance. However, this measure may not be the most accurate or optimal for quantifying E/I balance, a limitation that the authors might consider addressing in the future.

Reviewer #2 (Public review):

This article presents an analysis of the chemical composition of head-space generated by fruit at differing stages of ripeness. The authors used gas chromatography-mass spectrometry (GC-MS) to record the chemical makeup of the respective head-space samples. The authors process the data and present it in a low dimensional space. They then draw conclusions from the geometry of that representation about the process of fermentation.

I have a number of major concerns with some of the stages in the argument advanced by the authors:

(1) As far as I understand, the authors restrict their analysis to 13 molecules which appear in samples of all three levels of ripeness. This choice causes the analysis to overlook the very likely (and meaningful) possibility that different molecules present at different levels of ripeness are informative and might support different results.

(2) It is unclear what was used as control? Empty bag? Please include the control results in your supplementary table, or indicate in the text if you eliminated compounds that were found in the control.

(3) It is not clear that Figure 2-H _looks_ like a spiral. The authors should provide a quantifiable measure of the quality of the fit of a spiral rather than other paths. Furthermore, in the section "collective spiral ..." the end of paragraph one, "the points were best fitted by a two parameter archemedian spiral" best out of what? best out of all two parameter spirals? Please explain

(4) In the section "estimating odor source phenotype ... " the authors write: "we first calculated the association of odorant compounds with different phenotypes in this dataset" how was that done?

(5) Even if hyperbolic space MDS is slightly better, an R^2 value for Euclidean MDS of 0.797 is very good and one could say that Euclidean MDS is also an option.

(6) In the section "collective spiral ..." near end of paragraph two: " we removed outlier samples for days 10 and 17 for two reasons...". Why does a smaller number of samples should make a certain day an outlier.

(7) In section titles "collective spiral progression of multiple..." the authors write: the hyperbolic t-sne embedding exhibited batch effects across runs that amounted to rotation of the data. To compensate for these effects and combine data across runs we performed Procrustes analysis to align data across runs".

Can we be sure that this process does itself not manufacture an alignment of data? The authors should apply the same process to random or shuffled data and see if the result is different from the actual data.

DOI: 10.1038/s41586-024-07972-2

Resource: RRID:IMSR_JAX:024858

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_JAX:024858

What is this?

Reviewer #3 (Public review):

The present study used an experimental procedure involving time-pressure for responding, in order to uncover how the control of saccades by exogenous and endogenous attention unfolds over time. The findings of the study indicate that saccade planning is influenced by the locus of endogenous attention, but that this influence was short-lasting and could be overcome quickly. Taken together, the present findings reveal new dynamics between endogenous attention and eye movement control and lead the way for studying them using experiments under time-pressure.

The results achieved by the present study advance our understanding of vision, eye movements, and their control by brain mechanisms for attention. In addition, they demonstrate how tasks involving time-pressure can be used to study the dynamics of cognitive processes. Therefore, the present study seems highly important not only for vision science, but also for psychology, (cognitive) neuroscience, and related research fields in general.

I think the authors' addressed all of the reviewers' points successfully and in detail, so that I don't have any further suggestions or comments.

Reviewer #2 (Public review):

Summary:

In this study, the authors aim to combine automated whole-cell patch clamp recording simultaneously from multiple cells. Using a 2-electrode approach, they are able to sample as many cells (and connections) from one slice, as would be achieved with a more technically demanding and materially expensive 8-electrode patch clamp system. They provide data to show that this approach is able to successfully record from 52% of attempted cells, which was able to detect 3 pairs in 71 screened neurons. The authors state that this is a step forward in our ability to record from randomly connected ensembles of neurons.

Strengths:

The conceptual approach of recording multiple partner cells from another in a step wise manner indeed increases the number of tested connections. An approach that is widely applicable to both automated and manual approaches. Such a method could be adopted for many connectivity studies using dual recording electrodes.

The implementation of automated robotic whole-cell patch-clamp techniques from multiple cells simultaneously is a useful addition to the multiple techniques available to ex vivo slice electrophysiologists.

The approach using 2 electrodes, which are washed between cells is economically favourable, as this reduces equipment costs for recording multiple cells, and limits the wastage of capillary glass that would otherwise be used once.

Weaknesses:

(1) Based on the revised manuscript - a discussion of the implementation of this approach to manual methods is still lacking,

(2) A comparison of measurements shown in Figure 2 to other methods has not been addressed adequately.

(3) The morphological identification of neurons is understandably outside the remit of this project - but should be discussed and/or addressed. It was not suggested to perform detailed anatomical analysis - but to highlight the importance of this, and it should still be discussed

(4) The revised manuscript does not clearly state which cells were included in the analysis as far as I can see - and indeed cells with Access Resistance >40 MOhm appear to still be included in the data.

Reviewer #2 (Public review):

Summary and strengths:

O'Brien et al. present a compelling strategy to both understand rare disease that could have a neuronal focus and discover drugs for repurposing that can affect rare disease phenotypes. Using C. elegans, they optimize the Brown lab worm tracker and Tierpsy analysis platform to look at movement behaviors of 25 knockout strains. These gene knockouts were chosen based on a process to identify human orthologs that could underlie rare diseases. I found the manuscript interesting and a powerful approach to make genotype-phenotype connections using C. elegans. Given the rate that rare Mendelian diseases are found and candidate genes suggested, human geneticists need to consider orthologous approaches to understand the disease and seek treatments on a rapid time scale. This approach is one such way. Overall, I have a few minor suggestions and some specific edits.

Weaknesses:

(1) Throughout the text on figures, labels are nearly impossible to read. I had to zoom into the PDF to determine what the figure was showing. Please make text in all figures a minimum of 10 point font. Similarly, Figure 2D point type is impossible to read. Points should be larger in all figures. Gene names should be in italics in all figures, following C. elegans convention.

(2) I have a strong bias against the second point in Figure 1A. Sequencing of trios, cohorts, or individuals NEVER identifies causal genes in the disease. This technique proposes a candidate gene. Future experiments (oftentimes in model organisms) are required to make those connections to causality. Please edit this figure and parts of the text.

(3) How were the high-confidence orthologs filtered from 767 to 543 (lines 128-131)? Also, the choice of the final list of 25 genes is not well justified. Please expand more about how these choices were made.

(4) Figures 3 and 4, why show all 8289 features? It might be easier to understand and read if only the 256 Tierpsy features were plotted in the heat maps.

(5) The unc-80 mutant screen is clever. In the feature space, it is likely better to focus on the 256 less-redundant Tierpsy features instead of just a number of features. It is unclear to me how many of these features are correlated and not providing more information. In other words, the "worsening" of less-redundant features is far more of a concern than "worsening" of 1000 correlated features.Reviewer #2 (Public review):

Summary and strengths:

O'Brien et al. present a compelling strategy to both understand rare disease that could have a neuronal focus and discover drugs for repurposing that can affect rare disease phenotypes. Using C. elegans, they optimize the Brown lab worm tracker and Tierpsy analysis platform to look at movement behaviors of 25 knockout strains. These gene knockouts were chosen based on a process to identify human orthologs that could underlie rare diseases. I found the manuscript interesting and a powerful approach to make genotype-phenotype connections using C. elegans. Given the rate that rare Mendelian diseases are found and candidate genes suggested, human geneticists need to consider orthologous approaches to understand the disease and seek treatments on a rapid time scale. This approach is one such way. Overall, I have a few minor suggestions and some specific edits.

Weaknesses:

(1) Throughout the text on figures, labels are nearly impossible to read. I had to zoom into the PDF to determine what the figure was showing. Please make text in all figures a minimum of 10 point font. Similarly, Figure 2D point type is impossible to read. Points should be larger in all figures. Gene names should be in italics in all figures, following C. elegans convention.

(2) I have a strong bias against the second point in Figure 1A. Sequencing of trios, cohorts, or individuals NEVER identifies causal genes in the disease. This technique proposes a candidate gene. Future experiments (oftentimes in model organisms) are required to make those connections to causality. Please edit this figure and parts of the text.

(3) How were the high-confidence orthologs filtered from 767 to 543 (lines 128-131)? Also, the choice of the final list of 25 genes is not well justified. Please expand more about how these choices were made.

(4) Figures 3 and 4, why show all 8289 features? It might be easier to understand and read if only the 256 Tierpsy features were plotted in the heat maps.

(5) The unc-80 mutant screen is clever. In the feature space, it is likely better to focus on the 256 less-redundant Tierpsy features instead of just a number of features. It is unclear to me how many of these features are correlated and not providing more information. In other words, the "worsening" of less-redundant features is far more of a concern than "worsening" of 1000 correlated features.

Reviewer #2 (Public review):

Summary:

The paper by Hydaiberdiev and Ovcharenko offers comprehensive analyses and insights about the 'high-occupancy target' (HOT) loci in the human genome. These are considered genomic regions that overlap with transcription factor binding sites. The authors provided very comprehensive analyses of the TF composition characteristics of these HOT loci. They showed that these HOT loci tend to overlap with annotated promoters and enhancers, GC-rich regions, open chromatin signals, and highly conserved regions and that these loci are also enriched with potentially causal variants with different traits.

Strengths:

Overall, the HOT loci' definition is clear and the data of HOT regions across the genome can be a useful dataset for studies that use HepG2 or K562 as a model. I appreciate the authors' efforts in presenting many analyses and plots backing up each statement.

Comments on revised version:

In the second round of review, I think the authors have sufficiently addressed all of my previous comments. The study itself is very comprehensive, tackling all aspects of the HOT loci, though I still find the paper to be unnecessarily long and long-winded. That said, being consistent with the long and detailed paper, the provided Github repository and Zenodo archive is well-documented. I appreciate that the authors include detailed readme about the different datafiles available for readers. The list of HOT loci is probably the most useful asset in this manuscript and the authors did a good job documenting data availability in both Github and Zenodo.

Reviewer #2 (Public Review):

Wang et al. investigated the role of dynein axonemal heavy chain 3 (DNAH3) in male infertility. They found that variants of DNAH3 were present in four infertile men, and the deficiency of DNAH3 in sperm affects sperm mobility. Additionally, they showed that Dnah3 knockout male mice are infertile. Furthermore, they demonstrated that DNAH3 influences inner dynein arms by regulating several DNAH proteins. Importantly, they showed that intracytoplasmic sperm injection (ICSI) can rescue the infertility in Dnah3 knockout mice and two patients with DNAH3 variants.

Strengths:

The conclusions of this paper are well-supported by data.

Weaknesses:

The sample/patient size is small; however, the findings are consistent with those of a recent study on DNAH3 in male infertility with 432 patients.

Reviewer #2 (Public Review):

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

Comments on latest version:

The term "invasion" has to be replaced with infection, as it doesn't have much meaning to this particular study. I already explained this point in the first review, but authors did not address it throughout the manuscript.

In fig. 1e there's no statistical analysis. How can one show measurements from multiple samples without statistical analysis? All the data points have to be shown in the graph and statistics performed. In the arg6-npr1 and snrk-npr1 pairs no nuclear marker is included. How can one know where the nucleus is, particularly in such poor quality low res. images? The nucleus marker has to be included in this analysis and shown. This is an important aspect of the study as nuclear localization of ATG6 is proposed to be essential for its new function. Co-localization provided in the fig. S2 cannot complement this analysis, particularly since no cytoplasmic fraction is present for NPR1-GFP in fig. S2.

In the alignment in fig 2c, it is not explained what are the species the atg6 is taken from. The predicted NLS has to be shown in the context of either the entire protein sequence alignment or at least individual domain alignment with the indication of conserved residues (consensus). They have to include more species in the analysis, instead of including 3 proteins from a single species. Also, the predicted NLS in atg6 doesn't really have the classical type architecture, which might be an indication that it is a weak NLS, consistent with the fact that the protein has significant cytoplasmic accumulation. They also need to provide the NLS prediction cut-off score, as this parameter is a measure of NLS strength.

Line 150: the NLS sequence "FLKEKKKKK" is a wrong sequence.

In fig. 3d no explanation for the error bars is included, and what type of statistical analysis is performed is not explained.

Reviewer #2 (Public review):

Summary:

The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).

Strengths:

The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.

Weaknesses:

The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be re-worked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.

Impact:

The authors have discovered that nocturnal bull ants, while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.

Reviewer #2 (Public review):

Summary:

The authors identified ORMDL3 as a negative regulator of the RLR pathway and anti-tumor immunity. Mechanistically, ORMDL3 interacts with MAVS and further promotes RIG-I for proteasome degradation. In addition, the deubiquitinating enzyme USP10 stabilizes RIG-I and ORMDL3 disturbs this process. Moreover, in subcutaneous syngeneic tumor models in C57BL/6 mice, they showed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8-positive T cells and IFN production in the tumor microenvironment (TME).

Strengths:

The paper has a clearly arranged structure and the English is easy to understand. It is well written. The results are clearly supporting the conclusion.

Reviewer #3 (Public review):

Summary:

An interesting feature of cilia in vertebrates and many, if not all, invertebrates is the striking heterogeneity of their lengths among different cell types. As mutations interfering with ciliary length usually impair ciliary functions, ciliary length appears to be tuned for proper ciliary functions in a given type of cells. Although ciliary length is known to be affected by multiple factors, including intraflagellar transport (IFT), a cilia-specific, train-like bidirectional transportation, and ciliary proteins regulating microtubule dynamics, how it is intrinsically controlled remains largely elusive.

In the manuscript, the authors addressed this question from the angle of IFT by using zebrafish as a model organism. They demonstrated that ectopically expressed Ift88-GFP induced by heat shock treatment was able to sustain the normal development of and the cilia formation in ovl-/- zebrafish that would otherwise be dead by 7 dpf and lack of cilia due to the lack of Ift88, a critical component of IFT-B complex, suggesting a full function of the exogenous protein. They next live imaged Ift88-GFP in wild-type zebrafish larvae to visualize the IFT. Interestingly, they found that both anterograde and retrograde velocities of Ift88-GFP puncta differed in cilia of different cell types (crista, neuromast, pronephric duct, spinal chord, and epidermal cells) and displayed a positive correlation with the inherent length of the cilia. Similar results were obtained with ectopically expressed tdTomato-Ift43 driven by a beta-actin promoter. In the same cell type, however, the velocities of Ift88-GFP puncta did not alter in cilia of different lengths or at different developmental stages. Depletion of proteins such as Bbs4, Ttll3, Ttll6, and Ccp5 did not substantially alter the IFT velocities, excluding contributions of the BBSome or the enzymes involved in tubulin glycylation or glutamylation. They also used a cilia-localized ATP reporter to exclude the possibility of different ciliary ATP concentrations. When they compared the size of Ift88-GFP puncta in crista cilia, which are inherently long, and spinal chord cilia, which are relatively short, by imaging with a STED super-resolution microscope, they noticed a positive correlation between the puncta size, which presumably reflected the size of IFT trains, and the length of the cilia. Furthermore, in morphant larvae with slightly decreased Ift88 levels, judged by the grossly normal body axis, IFT particle sizes, their velocities, and ciliary lengths were all reduced as compared to control morphants. Therefore, they proposed that longer IFT trains facilitate faster IFT to result in longer cilia.

Strengths:

The authors demonstrated that: (1) both anterograde and retrograde IFT velocities can differ markedly in cilia of different cell types in zebrafish larvae; (2) specific IFT velocities are intrinsic to cell types; (3) IFT velocities in different types of cells are positively correlated with inherent ciliary lengths; and (4) IFT velocities are positively correlated with the size of IFT trains. These findings provide both new knowledge on IFT properties in zebrafish and insights that would facilitate understandings on mechanisms underlying the diversity of ciliary lengths in multicellular organisms. The experiments were carefully done and results are generally convincing. The imaging methods for tracing IFT in cilia of multiple cell types in zebrafish larvae are expected to be useful to other researchers in the field.

Weaknesses:

(1) Although the proposed model is reasonable, it is largely based on correlations.<br /> (2) The effects of anti-sense RNA-induced Ift88 downregulation on IFT and ciliary length are artificial. It is unclear whether the levels of one or more IFT components are indeed regulated to control IFT train sizes and ciliary lengths in physiological conditions. Similarly, whether IFT velocities are indeed dictated by the size of IFT trains remains to be clarified.<br /> (3) In the Discussion section, Kif17 is described as an important motor for IFT in mouse olfactory cilia. In the cited literature (Williams et al., 2014), however, Kif17 is reported to be dispensable for IFT in mouse olfactory cilia. This makes the discussions on Kif17 absurd.

Reviewer #2 (Public review):

Summary:

The authors provided benchmarking study results on tRNA-seq in terms of read alignment and quantification software with optimal parameterization. This result can be a useful guideline for choosing optimal parameters for tRNA-seq read alignment and quantification.

Strengths:

Benchmarking results for read alignment can be a useful guideline for choosing optimal parameters and mapping strategy (mapping to amino acid) for various tRNAseq.

Weaknesses:

Some explanation on sequencing data analysis pipeline is not clear for general readers.

Reviewer #3 (Public review):

Summary:

This work aims to demonstrate how recent advances in thermal stability assays can be utilised to screen chemical libraries and determine compound mechanism of action. Focusing on 96 compounds with known mechanisms of action, they use the PISA assay to measure changes in protein stability upon treatment with a high dose (10uM) in live K562 cells and whole cell lysates from K562 or HCT116. They intend this work to showcase a robust workflow which can serve as a roadmap for future studies.

Strengths:

The major strength of this study is the combination of live and whole cell lysates experiments. This allows the authors to compare the results from these two approaches to identify novel ligand-induced changes in thermal stability with greater confidence. More usefully, this also enables the authors to separate primary and secondary effects of the compounds within the live cell assay.

The study also benefits from the number of compounds tested within the same framework, which allows the authors to make direct comparisons between compounds.

These two strengths are combined when they compare between CHEK1 inhibitors and suggest that AZD-7762 likely induces secondary destabilisation of CRKL through off-target engagement with tyrosine kinases.

Weaknesses:

One of the stated benefits of PISA compared to the TPP in the original publication (Gaetani et al 2019) was that the reduced number of samples required allows more replicate experiments to be performed. Despite this, the authors of this study performed only duplicate experiments. They acknowledge this precludes use of frequentist statistical tests to identify significant changes in protein stability. Instead, they apply an 'empirically derived framework' in which they apply two thresholds to the fold change vs DMSO: absolute z-score (calculated from all compounds for a protein) > 3.5 and absolute log2 fold-change > 0.2. They state that the fold-change threshold was necessary to exclude non-specific interactors. While the thresholds appear relatively stringent, this approach will likely reduce the robustness of their findings in comparison to an experimental design incorporating more replicates. Firstly, the magnitude of the effect size should not be taken as a proxy for the importance of the effect. They acknowledge this and demonstrate it using their own data for PIK3CB and p38α inhibitors (Figure 2B-C). They have thus likely missed many small, but biological relevant changes in thermal stability due to the fold-change threshold. Secondly, this approach relies upon the fold-changes between DMSO and compound for each protein being comparable, despite them being drawn from samples spread across 16 TMT multiplexes. Each multiplex necessitates a separate MS run and the quantification of a distinct set of peptides, from which the protein-level abundances are estimated. Thus, it is unlikely the fold-changes for unaffected proteins are drawn from the same distribution, which is an unstated assumption of their thresholding approach. The authors could alleviate the second concern by demonstrating that there is very little or no batch effect across the TMT multiplexes. However, the first concern would remain. The limitations of their approach could have been avoided with more replicates and use of an appropriate statistical test. It would be helpful if the authors could clarify if any of the missed targets passed the z-score threshold but fell below the fold-change threshold.

The authors use a single, high, concentration of 10uM for all compounds. Given that many of the compounds may have low nM IC50s, this concentration could be orders of magnitude above the one at which they inhibit their target. This makes it difficult to assess the relevance of the off-target effects identified to clinical applications of the compounds or biological experiments. The authors acknowledge this and use ranges of concentrations for follow-up studies (e.g. Figure 2E-F). Nonetheless, this weakness is present for the vast bulk of the data presented.

Aims achieved, impact and utility:

The authors have achieved their main aim of presenting a workflow which serves to demonstrate the potential value of this approach. However, by using a single high dose of each compound and failing to adequately replicate their experiments and instead applying heuristic thresholds, they have limited the impact of their findings. Their results will be a useful resource for researchers wishing to explore potential off-target interactions and/or mechanisms of action for these 96 compounds but are expected to be superseded by more robust datasets in the near future. The most valuable aspect of the study is the demonstration that combining live cell and whole cell lysate PISA assays across multiple related compounds can help to elucidate the mechanisms of action.

Reviewer #2 (Public review):

Based on the controversy of whether the Desmodium intercrop emits bioactive volatiles that repel the fall armyworm, the authors conducted this study to assess the effects of the volatiles from Desmodium plants in the push-pull system on behavior of FAW oviposition. This topic is interesting and the results are valuable for understanding the push-pull system for the management of FAW, the serious pest. The methodology used in this study is valid, leading to reliable results and conclusions. I just have a few concerns and suggestions for improvement of this paper:

(1) The volatiles emitted from D. incanum were analyzed and their effects on the oviposition behavior of FAW moth were confirmed. However, it would be better and useful to identify the specific compounds that are crucial for the success of the push-pull system.

(2) That would be good to add "symbols" of significance in Figure 4 (D).

(3) Figure A is difficult for readers to understand.

(4) It will be good to deeply discuss the functions of important volatile compounds identified here with comparison with results in previous studies in the discussion better.

Reviewer #2 (Public review):

Summary:

The authors set out to visualize the molecular architecture of the adult forebrain glutamatergic synapses in a near-native state. To this end, they use a rapid workflow to extract and plunge-freeze mouse synapses for cryo-electron tomography. In addition, the authors use knockin mice expression PSD95-GFP in order to perform correlated light and electron microscopy to clearly identify pre- and synaptic membranes. By thorough quantification of tomograms from plunge- and high-pressure frozen samples, the authors show that the previously reported 'post-synaptic density' does not occur at high frequency and therefore not a defining feature of a glutamatergic synapse.

Subsequently, the authors are able to reproduce the frequency of post-synaptic density when preparing conventional electron microscopy samples, thus indicating that density prevalence is an artifact of sample preparation. The authors go on to describe the arrangement of cytoskeletal components, membraneous compartments, and ionotropic receptor clusters across synapses.

Demonstrating that the frequency of the post-synaptic density in prior work is likely an artifact and not a defining feature of glutamatergic synapses is significant. The descriptions of distributions and morphologies of proteins and membranes in this work may serve as a basis for the future of investigation for readers interested in these features.

Strengths:

The authors perform a rigorous quantification of the molecular density profiles across synapses to determine the frequency of the post-synaptic density. They prepare samples using two cryogenic electron microscopy sample preparation methods, as well as one set of samples using conventional electron microscopy methods. The authors can reproduce previous reports of the frequency of the post-synaptic density by conventional sample preparation, but not by either of the cryogenic methods, thus strongly supporting their claim.

Reviewer #2 (Public review):

Summary:

In an effort to elucidate the role of the ECM protein Svep1 in lung development, Foxworth and colleagues have generated a Svep1 mutant (lacking exon 8). Based on their developmental analyses of branching morphogenesis and expression of epithelial, mesenchymal, and mesothelial markers in these mutants, the authors conclude that Svep1 is essential for normal lung growth, morphogenesis, and patterning. They propose that the Svep1 protein regulates, in part, FGF9 signalling. Overall, the paper demonstrates that the ECM is important for lung development and tries to implicate the ECM in the regulation of epithelial-mesenchymal interactions during lung development.

Strengths:

The strengths of this paper are the careful spatiotemporal characterization of 1) lung growth 2) branching morphogenesis 3) epithelial marker expression. The differential perturbation of growth and branching morphogenesis along the D-V axis and the progressive perturbation of branching morphogenesis are both very interesting and noteworthy phenotypes.

Weaknesses:

The weakness of this paper is that it does not present a convincing explanation for how Svep1 regulates any of the phenotypes described. In this regard, a demonstration of a genetic interaction between Svep1 and FGF9 mutants or a careful characterization of a tissue-specific knockdown of Svep1, could be insightful. In addition, a comparison of the phenotype of Svep1 mutants and the phenotypes of other mutants affecting ECM components would be worthwhile.

A minor weakness is that the title of the paper is not fully supported by the data presented. While the defects in the morphogenesis of the distal lung in Svep1 mutants presage a defect in alveolar differentiation, this cannot be formally demonstrated since the animals die soon after birth.

Reviewer #2 (Public review):

Summary:

Zhang et al. present very well-illustrated specimens of the artiopodan Cinderella eucalla from the Chengjiang Biota. Multiple specimens are shown with preserved appendages, which is rare for artiopodans and will greatly help our understanding of this taxon. The authors use CT scanning to reveal the ventral organization of this taxon. The description of the taxon needs some modification, specifically expansion of the gut and limb morphology. The conclusion that Cinderella was a fast-moving animal is very weak, comparisons with extant fast animals and possibly FEA analyses are necessary to support such a claim. Although the potential insights provided by such well-preserved fossils could be valuable, the claims made are tenuous and based on the available evidence presented herein.

Strengths:

The images produced through CT scanning specimens reveal the very fine detail of the appendages and are well illustrated. Specimens preserve guts and limbs, which are informative both for the phylogenetic position and ecology of this taxon. The limbs are very well preserved, with protopodite, exopodite, and endopodites visible. Addressing the weaknesses below will make the most of this compelling data that demonstrates the morphology of the limbs well.

Weaknesses:

Although this paper includes very well-illustrated fossils, including new information on the eyes, guts, and limbs of Cinderella, the data are not fully explained, and the conclusions are weakly supported.

The authors suggest the preservation of complex ramifying diverticular, but it should be better illustrated and the discussion of the gut diverticulae should be longer, especially as gut morphology can provide insights into the feeding strategy.

The conclusion that Cinderella eucalla was fast, sediment feeding in a muddy environment, is not well supported. These claims seem to be tenuously made without any evidence to support them. The authors should add a new section in the discussion focused on feeding ecology where they explicitly compare the morphology to suspension-feeding artiopodans to justify whether it fed that way or not. To further explore feeding, the protopodite morphology needs to be more carefully described and compared to other known taxa. The function of endites on the endopodite to stir up sediment for particle feeding in a muddy environment would also need to be more thoroughly discussed and compared with modern analogs. The impact of their findings is not highlighted in the discussion, which is currently more of a review of what has been previously said and should focus more on what insights are provided by the great fossils illustrated by the authors.

The authors argue that their data supports fast escaping capabilities, but it is not clear how they reached that conclusion based on the data available. Is there a way this can be further evaluated? The data is impressive, so including comparisons with extant taxa that display fast escaping strategies would help the authors make their case more compelling. The authors also claim that the limbs of Cinderella are strong, again this conclusion is unclear. Comparison with the limbs of other taxa to show their robustness would be useful. To actually test how these limbs deal with the force and strain applied to them by a sudden burst of movement, the authors could conduct Finite Element Analyses.

Reviewer #2 (Public review):

Summary:

More and more genes and genetic loci are being linked to bone fragility disorders like osteoporosis and osteogenesis imperfecta through GWAS and clinical sequencing. In this study, the authors seek to develop a pipeline for validating these new candidate genes using crispant screening in zebrafish. Candidates were selected based on GWAS bone density evidence (4 genes) or linkage to OI cases plus some aspect of bone biology (6 genes). NGS was performed on embryos injected with different gRNAs/Cas9 to confirm high mutagenic efficacy and off-target cutting was verified to be low. Bone growth, mineralization, density, and gene expression levels were carefully measured and compared across crispants using a battery of assays at three different stages.

Strengths:

(1) The pipeline would be straightforward to replicate in other labs, and the study could thus make a real contribution towards resolving the major bottleneck of candidate gene validation.

(2) The study is clearly written and extensively quantified.

(3) The discussion attempts to place the phenotypes of different crispant lines into the context of what is already known about each gene's function.

(4) There is added value in seeing the results for the different crispant lines side by side for each assay.

Weaknesses:

(1) The study uses only well-established methods and is strategy-driven rather than question/hypothesis-driven.

(2) Some of the measurements are inadequately normalized and not as specific to bone as suggested:

(a) The measurements of surface area covered by osteoblasts or mineralized bone (Figure 1) should be normalized to body size. The authors note that such measures provide "insight into the formation of new skeletal tissue during early development" and reflect "the quantity of osteoblasts within a given structure and [is] a measure of the formation of bone matrix." I agree in principle, but these measures are also secondarily impacted by the overall growth and health of the larva. The surface area data are normalized to the control but not to the size/length of each fish - the esr1 line in particular appears quite developmentally advanced in some of the images shown, which could easily explain the larger bone areas. The fact that the images in Figure S5 were not all taken at the same magnification further complicates this interpretation.

(b) Some of the genes evaluated by RT-PCR in Figure 2 are expressed in other tissues in addition to bone (as are the candidate genes themselves); because whole-body samples were used for these assays, there is a nonzero possibility that observed changes may be rooted in other, non-skeletal cell types.

(3) Though the assays evaluate bone development and quality at several levels, it is still difficult to synthesize all the results for a given gene into a coherent model of its requirement.

(4) Several additional caveats to crispant analyses are worth noting:

(a) False negatives, i.e. individual fish may not carry many (or any!) mutant alleles. The crispant individuals used for most assays here were not directly genotyped, and no control appears to have been used to confirm successful injection. The authors therefore cannot rule out that some individuals were not, in fact, mutagenized at the loci of interest, potentially due to human error. While this doesn't invalidate the results, it is worth acknowledging the limitation.

(b) Many/most loci identified through GWAS are non-coding and not easily associated with a nearby gene. The authors should discuss whether their coding gene-focused pipeline could be applied in such cases and how that might work.

Reviewer #2 (Public review):

Summary:

In this study, Solomon and colleagues demonstrate that trained immunity induced by BCG can reprogram myeloid cells within localised tissue, which can sustain prolonged protective effects. The authors further demonstrate an activation of STAT1-dependent pathways.

Strengths:

The main strength of this paper is the in-depth investigation of cell populations affected by BCG training, and how their transcriptome changes at different time points post-training. Through use of flow cytometry and sequencing methods, the authors identify a new cell population derived from classical monocytes. They also show that long-term trained immunity protection in the spleen is dependent on resident cells. Through sequencing, drug and recombinant inhibition of IFNg pathways, the authors reveal STAT1-dependent responses are required for changes in the myeloid population upon training, and recruitment of trained monocytes.

Weaknesses:

A significant amount of work has already been performed for this study. No significant weaknesses found.

Reviewer #2 (Public review):

Summary:

The authors investigate miRNA miR-195 in the context of B-cell development. They demonstrate that ectopic expression of miR-195 in hematopoietic progenitor cells can, to a considerable extent, override the consequences of deletion of Ebf1, a central B-lineage defining transcription factor, in vitro and upon short-term transplantation into immunodeficient mice in vivo. In addition, the authors demonstrate that the reverse experiment, genetic deletion of miR-195, has virtually no effect on B-cell development. Mechanistically, the authors identify Foxo1 phosphorylation as one pathway partially contributing to the rescue effect of miR-195. An additional analysis of epigenetics by ATACseq adds potential additional factors that might also contribute to the effect of ectopic expression of miR-195.

Strengths:

The authors employ a robust assay system, Ebf1-KO HPC, to test for B-lineage promoting factors. The manuscript overall takes on an interesting perspective rarely employed for the analysis of miRNA by overexpressing the miRNA of interest. Ideally, this approach may reveal, if not the physiological function of this miRNA, the role of distinct pathways in developmental processes.

Weaknesses:

At the same time, this approach constitutes a major weakness: It does not reveal information on the physiological role of miR-195. In fact, the authors themselves demonstrate in their KO approach, that miR-195 has virtually no role in B-cell development, as has been demonstrated already in 2020 by Hutter and colleagues. While the authors cite this paper, unfortunately, they do so in a different context, hence omitting that their findings are not original.

Conceptually, the authors stress that a predominant function of miRNA (in contrast to transcription factors, as the authors suggest) lies in fine-tuning. However, there appears to be a misconception. Misregulation of fine-tuning of gene expression may result in substantial biological effects, especially in developmental processes. The authors want to highlight that miR-195 is somewhat of an exception in that regard, but this is clearly not the case. In addition to miR-150, as referenced by the authors, also the miR-17-92 or miR-221/222 families play a significant role in B-cell development, their absence resulting in stage-specific developmental blocks, and other miRNAs, such as miR-155, miR-142, miR-181, and miR-223 are critical regulators of leukocyte development and function. Thus, while in many instances a single miRNA moderately affects gene expression at the level of an individual target, quite frequently targets converge in common pathways, hence controlling critical biological processes.

The paper has some methodological weaknesses as well: For the most part, it lacks thorough statistical analysis, and only representative FACS plots are provided. Many bar graphs are based on heavy normalization making the T-tests employed inapplicable. No details are provided regarding the statistical analysis of microarrays. Generation of the miR-195-KO mice is insufficiently described and no validation of deletion is provided. Important controls are missing as well, the most important one being a direct rescue of Ebf1-KO cells by re-expression of Ebf1. This control is critical to quantify the extent of override of Ebf1-deficiency elicited by miR-195 and should essentially be included in all experiments. A quantitative comparison is essential to support the authors' main conclusion highlighted in the title of the manuscript. As the manuscript currently stands, only negative controls are provided, which, given the profound role of Ebf1, are insufficient, because many experiments, such as assessment of V(D)J recombination, IgM surface expression, or class-switch recombination, are completely negative in controls. In addition, the authors should also perform long-term reconstitution experiments. While it is somewhat surprising that the authors obtained splenic IgM+ B cells after just 10 days, these experiments would be certainly much more informative after longer periods of time. Using "classical" mixed bone marrow chimeras using a combination of B-cell defective (such as mb1/mb1) bone marrow and reconstituted Ebf1-KO progenitors would permit much more refined analyses.

With regard to mechanism, the authors show that the Foxo1 phosphorylation pathway accounts for the rescue of CD19 expression, but not for other factors, as mentioned in the discussion. The authors then resort to epigenetics analysis, but their rationale remains somewhat vague. It remains unclear how miR-195 is linked to epigenetic changes.

Reviewer #2 (Public review):

Summary:

This manuscript describes a genetic tool utilizing mutant mitfa-Cas9 expressing zebrafish to knockout genes to analyze their function in melanocytes in a range of assays from developmental biology to tumorigenesis. Overall, the data are convincing and the authors cover potential caveats from their model that might impact its utility for future work.

Strengths:

The authors do an excellent job of characterizing several gene deletions that show the specificity and applicability of the genetic mitfa-Cas9 zebrafish to studying melanocytes.

Weaknesses:

Variability across animals not fully analyzed.

Reviewer #2 (Public review):

Summary:<br /> In this observational study, Barth et al. investigated the association between menopausal hormone therapy and brain health in middle- to older-aged women from the UK Biobank. The study evaluated detailed MHT data (never, current, or past user), duration of mHT use (age first/last used), history of hysterectomy with or without bilateral oophorectomy, APOEE4 genotype, and brain characteristics in a large, population-based sample. The researchers found that current mHT use (compared to never-users), but not past use, was associated with a modest increase in gray and white matter brain age gap (GM and WM BAG) and a decrease in hippocampal volumes. No significant association was found between the age of mHT initiation and brain measures among mHT users. Longer duration of use and older age at last MHT use post-menopause were associated with higher GM and WM BAG, larger WMH volumes, and smaller hippocampal volumes. In a sub-sample, after adjusting for multiple comparisons, no significant associations were found between detailed mHT variables (formulations, route of administration, dosage) and brain measures. The association between mHT variables and brain measures was not influenced by APOEE4 allele carrier status. Women with a history of hysterectomy with or without bilateral oophorectomy had lower GM BAG compared to those without such a history. Overall, these observational data suggest that the association between mHT use and brain health in women may vary depending on the duration of use and surgical history.

Strengths:

The study has several strengths, including a large, population-based sample of women in the UK, and comprehensive details of demographic variables such as menopausal status, history of oophorectomy/hysterectomy, genetic risk factors for Alzheimer's disease (APOE ε4 status), age at mHT initiation, age at last use, duration of mHT, and brain imaging data (hippocampus and WMH volume).

In a sub-sample, the study accessed detailed mHT prescription data (formulations, route of administration, dosage, duration), allowing the researchers to study how these variables were associated with brain health outcomes. This level of detail is generally missing in observational studies investigating the association of mHT use with brain health.

Weaknesses:

While the study has many strengths, it also has some weaknesses. As highlighted in an editorial by Kantarci & Manson (2023), women with symptoms such as subjective cognitive problems, sleep disturbances, and elevated vasomotor symptoms combined with sleep disturbances tend to seek mHT more frequently than those without these symptoms. The authors of this study have also indicated that the need of mHT use which might be associated with these symptoms may be indicators of preexisting neurological changes, potentially reflecting worse brain health scores, including higher BAG and lower hippocampal volume and/or higher WMH. However, among current users, how many of these women have these symptoms could not be reported in the study. Women with these vasomotor symptoms who are using mHT are more likely to stay longer in the healthcare system compared with those without these symptoms and no MHT use history. The authors noted that the UK Biobank lacks detailed information on menopausal symptoms and perimenopausal staging, limiting the study's ability to understand how these variables influence outcomes.

Earlier observational studies have reported conflicting results regarding the association between mHT use and the risk of dementia and brain health. Contrary to some observational studies, three randomized trials (WHI, KEEPS, ELITE) (Espeland et al 2013, Gleason et al 2015; Henderson et al 2016) demonstrated neither beneficial nor harmful effects of mHT (with varying doses and formulations) when initiated closer to menopause (<5 years). While strong efforts were made to run proper statistical analyses to investigate the association between mHT use and brain health, these results reflect mainly associations, but not causal relationships as also stated by the authors.

Furthermore, observational studies have intrinsic limitations, such as a lack of control over switching mHT doses and formulations, a lack of laboratory measures to confirm mHT use, and reliance on self-reported data, which may not always be reliable. The authors caution that these findings should not guide individual-level decisions regarding the benefits versus risks of mHT use. However, the study raises new questions that should be addressed by randomized clinical trials to investigate the varying effects of MHT on brain health and dementia risk.

Reviewer #2 (Public review):

Summary:

The manuscript by Hanna et al., addresses the question of energy metabolism in the retina, a neuronal tissue with an inordinately high energy demand. Paradoxically, the retina appears to employ to a large extent glycolysis to satisfy its energetic needs, even though glycolysis is far less efficient than oxidative phosphorylation (OXPHOS). The focus of the present study is on the early development of the retina and the retinal progenitor cells (RPCs) that proliferate and differentiate to form the seven main classes of retinal neurons. The authors use different genetic and pharmacological manipulations to drive the metabolism of RPCs or the retina towards higher or lower glycolytic activity. The results obtained suggest that increased glycolytic activity in early retinal development produces a more rapid differentiation of RPCs, resulting in a more rapid maturation of photoreceptors and photoreceptor segment growth. The study is significant in that it shows how metabolic activity can determine cell fate decisions in retinal neurons.

Strengths:

This study provides important findings that are highly relevant to the understanding of how early metabolism governs the development of the retina. The outcomes of this study could be relevant also for human diseases that affect early retinal development, including retinopathy of maturity where an increased oxygenation likely causes a disturbance of energy metabolism.

Weaknesses:

The restriction to only relatively early developmental time points makes it difficult to assess the consequences of the different manipulations on the (more) mature retina. Notably, it is conceivable that early developmental manipulations, while producing relevant effects in the young post-natal retina, may "even out" and may no longer be visible in the mature, adult retina.

Reviewer #2 (Public review):

Summary:

Siddiqui et al. show that C. elegans prefers certain bacterial strains that have been supplemented with the essential amino acid (EEA) leucine. They convincingly show that some leucine enriched bacteria stimulate the production of isoamyl alcohol (IAA). IAA is an attractive odorant that is sensed by the AWC. The authors an identify a receptor, SRD-12, that is expressed in the AWC chemosensory neurons and is required for chemotaxis to IAA. The authors propose that IAA is a predominant olfactory cue that determines diet preference in C. elegans. Since leucine is an EAA, the authors propose that worm IAA sensing allows the animal provides a proxy mechanism to identify EAA rich diets.

Strengths:

The authors propose IAA as a predominant olfactory cue that determines diet preference in C. elegans providing molecular mechanism underlying diet selection. They show that wild isolates of C. elegans have a strong chemotactic response to IAA indicating that IAA is an ecologically relevant odor for the worm. The paper is well written, and the presented data are convincing and well organized. This is an interesting paper that connects chemotactic response with bacterially produced odors and thus provides an understanding of how animals adapt their foraging behavior through the perception of molecules that may indicate the nutritional value.

Weaknesses:

Major:

While I do like the way the authors frame C. elegans IAA sensing as mechanisms to identify leucine (EAA) rich diets it is not fully clear whether bacterial IAA production is a proxy for bacterial leucine levels.

(1) Can the authors measure leucine (or other EAA) content of the different CeMbio strains? This would substantiate the premise in the way they frame this in the introduction. While the authors convincingly show that leucine supplementation induces IAA production in some strains, it is not clear if there are lower leucine levels in the different in non-preferred strains.

(2) It is not clear whether the non-preferred bacteria in Figure 1A and 1B have the ability to produce IAA. To substantiate the claim that C. elegans prefers CEent1, JUb66, and BIGb0170 due to their ability to generate IAA from leucine, it would measure IAA levels in non-preferred bacteria (+ and - leucine supplementation). If the authors have these data it would be good to include this.

(3) The authors would strengthen their claim if they could show that deletion or silencing ilvE enzyme reduces IAA levels and eliminates the increased preference upon leucine supplementation.

(4) While the three preferred bacteria possess the ilvE gene, it is not clear whether this enzyme is present in the other non-preferred bacterial strains. As far as I know, the CeMbio strains have been sequenced so it should be easy to determine if the non-preferred bacteria possess the capacity to make IAA. Does the expression of ilvE in e.g. E. coli increase its preference index or are the other genes in the biosynthesis pathway missing?

(5) It is strongly implied that leucine-rich diets are beneficial to the worm. Do the authors have data to show the effect on leucine supplementation on C. elegans healthspan, life-span or broodsize?

Other comments:

Page 6. Figure 2c. While the authors' conclusions are correct based on AWC expts. it would be good at this stage to include the possibility that odors that enriched in the absence of leucine may be aversive.

Page 6. IAA increases 1.2-4 folds upon leucine supplementation. If the authors perform a chemotaxis assay with just IAA with 1-2-4 fold differences do you get the shift in preference index as seen with the bacteria? i.e. is the difference in IAA concentration sufficient to explain the shift in bacterial PI upon leucine supplementation? Other attractants such as Acetoin and isobutanol go up in -Leu conditions.

Page 14-15. The authors identify a putative IAA receptor based on expression studies. I compliment the authors for isolating two CRISPR deletion alleles. They show that the srd-12 mutants have obvious defects in IAA chemotaxis. Very few ligand-odorant receptors combinations have been identified so this is an important discovery. CenGen data indicate that srd-12 is expressed in a limited set of neurons. Did the authors generate a reporter to show the expression of srd-12? This is a simple experiment that would add to the characterization of the SRD-12 receptor. Rescue experiments would be nice even though the authors have independent alleles. To truly claim that SRD-12 is the ligand for IAA and activates the AWC neurons would require GCamp experiments in the AWC neuron or heterologous expression system. I understand that GCamp imaging might not be part of the regular arsenal of the lab but it would be a great addition (even in collaboration with one of the many labs that do this regularly). Comparing AWC activity using GCaMP in response IAA-producing bacteria with high leucine levels in both wild-type and SRD-12-deficient backgrounds, would further support their narrative. I leave that to the authors.

Minor:

Page 4 "These results suggested that worms can forage for diets enriched in specific EAA, leucine...." More precise at this stage would be to state " These results indicated that worms can forage for diets supplemented with specific EAA...".

Page 5."these findings suggested that worms not only rely on odors to choose between two bacteria but also to find leucine enriched bacteria" This statement is not clear to me and doesn't follow the data in Fig. S2. Preferred diets in odorant assays are the IAA producing strains.

Page 5. Figure S2A provides nice and useful data that can be part of the main Figure 1.

Reviewer #2 (Public review):

Summary:

This study was designed to test the hypothesis that motor neurons play a causal role in circuit assembly of the vestibulo-ocular reflex circuit, which is based on the retrograde model proposed by Hans Straka. This circuit consists of peripheral sensory neurons, central projection neurons, and motor neurons. The authors hypothesize that loss of extraocular motor neurons, through CRISPR/Cas9 mutagenesis of the phox2a gene, will disrupt sensory selectivity in presynaptic projection neurons if the retrograde model is correct.

Account of the major strengths and weaknesses of the methods and results:

The work presented is impressive in both breadth and depth, including the experimental paradigms. Overall, the main results were that the loss of function paradigm to eliminate extraocular motor neurons did not 1) alter the normal functional connections between peripheral sensory neurons and central projection neurons, 2) affect the position of central projection neurons in the sensorimotor circuit, or 3) significantly alter the transcriptional profiles of central projection neurons. Together, these results strongly indicate that retrograde signals from motor neurons are not required for the development of the sensorimotor architecture of the vestibulo-ocular circuit.

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

The results of this study showed that extraocular motor neurons were not required for central projection neuron specification in the vestibulo-ocular circuit, which countered the prevailing retrograde hypothesis proposed for circuit assembly. A concern is that the results presented may be limited to this specific circuit and may not be generalizable to other circuit assemblies, even to other sensorimotor circuits.

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

As mentioned above, this study sheds valuable new insights into the developmental organization of the vestibulo-ocular circuit. However, different circuits likely utilize various mechanisms, extrinsic or intrinsic (or both), to establish proper functional connectivity. So, the results shown here, although they begin to explain the developmental organization of the vestibulo-ocular circuit, whether generalizable to other circuits is debatable. At a minimum, this study provides a starting point for the examination of the patterning of connections in this and other sensorimotor circuits.

Reviewer #2 (Public review):

Summary:

Using in vitro and in vivo approaches, the authors first demonstrate that BEST4 inhibits intestinal tumor cell growth and reduces their metastatic potential, possibly via downstream regulation of TWIST1.

They further show that HES4 positively upregulates BEST4 expression, with direct interaction with BEST4 promoter region and protein. The authors further expand on this with results showing that negative regulation of TWIST1 by HES4 requires BEST4 protein, with BEST4 required for TWIST1 association with HES4. Reduction of BEST1 expression was shown in CRC tumor samples, with correlation of BEST4 mRNA levels with different clinicopathological factors such as sex, tumor stage and lymph node metastasis, suggesting a tumor-suppressive role of BEST4 for intestinal cancer.

Strengths:

• Good quality western blot data<br /> • Multiple approaches were used to validate the findings<br /> • Logical experimental progression for readability<br /> • Human patient data / In vivo murine model / Multiple cell lines were used, which supports translatability/reproducibility of findings

Weaknesses:

• Figure quality should still be improved<br /> • The discussion should still be improved

Reviewer #2 (Public review):

Summary:

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

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

Reviewer #3 (Public review):

Summary:

This paper presents convincing data from technically demanding dual whole cell patch recordings of stellate cells in medial entorhinal cortex slice preparations during optogenetic stimulation of PV+ interneurons. The authors show that the patterns of postsynaptic activation are consistent with dual recorded cell close to each other receiving shared inhibitory input and sending excitatory connections back to the same PV neurons, supporting a circuitry in which clusters of stellate cells and PV+IN interact with each other with much weaker interactions between clusters. These data are important to our understanding of the dynamics of functional cell responses in the entorhinal cortex. The experiments and analysis are quite complex and would benefit from some revisions to enhance clarity.

Strengths:

These are technically demanding experiments, but the authors show quite convincing differences in the correlated response of cell pairs that are close to each other in contrast to an absence of correlation in other cell pairs at a range of relative distances. This supports their main point of demonstrating anatomical clusters of cells receiving shared inhibitory input.

Weaknesses:

The overall technique is complex, but the authors have made every effort to present this in a clear manner. In addition, due to this being a slice preparation they cannot directly relate the inhibitory interactions to the functional properties of grid cells which was possible in a complementary approach using 2-photon in vivo imaging by Heys, Rangarajan and Dombeck, 2014.

Reviewer #2 (Public review):

Summary:

In this paper by de Guglielmo and colleagues, the authors were interested in analyzing addiction-like behaviors using a very large number of heterogeneous outbred rats in order to determine the relationships among these behaviors. The paper used both males and females on the order of hundreds of rats, allowing for detailed and complex statistical analyses of the behaviors. The rats underwent cocaine self-administration, first via 2-hour access and then via 6-hour access. The rats also underwent a test of punishment resistance in which footshocks were administered a portion of the times a lever was pressed. The authors also conducted a progressive ratio test to determine the break point for "giving up" pressing the lever and a bottle-brush test to determine the rats "irritability". Ultimately, principal component analysis revealed that escalation of intake during 6-hour access, punishment resistance, and breakpoint all loaded onto the same principal component. Moreover, the authors also identified a subgroup of "resilient" rats that qualitatively differed from the "vulnerable" rats and also identified sex differences in their work.

Strengths:

The use of heterogeneous rats and the use of so many rats are major strengths for this paper. Moreover, the statistical analyses are particular strengths as they enabled the identification of the three measures as likely reflecting a single underlying construct. The behavioral methods themselves are also strong, as the authors used behavioral measures commonly used in the field that will enable comparison with the field at large. In general, the results support the conclusions and provide a wealth of data to the field. The addition of effect sizes is also a strength, as this provides critical information to other researchers.

Additionally, the changes made to the manuscript are another strength, as the authors clearly took the reviewers' points seriously and made strong efforts incorporate the reviewers' ideas.

The manuscript also uses both males and females and provides a good analysis of how findings differed by sex as well as how large the effect sizes were for those differences.

Reviewer #2 (Public review):

General comments:

The authors investigated the effects of tDCS on brain dynamics in awake and anesthetized monkeys using functional MRI. They claim that cathodal tDCS disrupts the functional connectivity pattern in awake monkeys while anodal tDCS alters brain patterns in anesthetized monkeys. This study offers valuable insight into how brain states can influence the outcomes of noninvasive brain stimulation. However, there are several aspects of the methods and results sections that should be improved to clarify the findings.

Major comments

(1) For the anesthetized monkeys, the anode location differs between subjects, with the electrode positioned to stimulate the left DLFPC in monkey R and the right DLPFC in monkey N. The authors mention that this discrepancy does not result in significant differences in the electric field due to the monkeys' small head size. However, this is not correct, as placing the anode on the left hemisphere would result in much lower EF in the right DLPFC compared to placing the anode on the right side. Running an electric field simulation would confirm this. Additionally, the small electrode size suggested by the Easy cap configuration for NHP appears sufficient to focally stimulate the targeted regions. If this interpretation is correct, the authors should provide additional evidence to support their claim, such as a computational simulation of the EF distribution.

(2) For the anesthetized monkeys, the authors applied 1 mA tDCS first, followed by 2 mA tDCS. A 20-minute stimulation duration of 1 mA tDCS is strong enough to produce after-effects that could influence the brain state during the 2 mA tDCS. This raises some concerns. Previous studies have shown that 1 mA tDCS can generate EF of over 1 V/m in the brain, and the effects of stimulation are sensitive to brain state (e.g., eye closed vs. eye open). How do the authors ensure that there are no after-effects from the 1 mA tDCS? This issue makes it challenging to directly compare the effects of 1 mA and 2 mA stimulation.

(3) The occurrence rate of a specific structural-functional coupling pattern among random brain regions shows significant effects of tDCS. However, these results seem counterintuitive. It is generally understood that noninvasive brain stimulation tends to modulate functional connectivity rather than structural or structural-functional connectivity. How does the occurrence rate of structural-functional coupling patterns provide a more suitable measure of the effectiveness of tDCS than functional connectivity alone? I would recommend that the authors present the results based on functional connectivity itself. If there is no change in functional connectivity, the relevance of changes in structural-functional coupling might not translate into a meaningful alteration in brain function, making it unclear how significant this finding is without corresponding functional evidence.

(4) The authors recorded data from only two monkeys, which may limit the investigation of the group effects of tDCS. As the number of scans for the second monkey in each consciousness condition is lower than that in the first monkey, there is a concern that the main effects might primarily reflect the data from a single monkey. I suggest that the authors should analyze the data for each monkey individually to determine if similar trends are observed in both subjects.

(5) Anodal tDCS was only applied to anesthetized monkeys, which limits the conclusion that the authors are aiming for. It raises questions about the conclusion regarding brain state dependency. To address this, it would be better to include the cathodal tDCS session for anesthetized monkeys. If cathodal tDCS changes the connectivity during anesthesia, it becomes difficult to argue that the effects of cathodal tDCS varies depending on the state of consciousness as discussed in this paper. On the other hand, if cathodal tDCS would not produce any changes, the conclusion would then focus on the relationship between the polarity of tDCS and consciousness. In that case, the authors could maintain their conclusion but might need to refine it to reflect this specific relationship more accurately.

Reviewer #2 (Public review):

Summary:

This work examines an important question in the planning and control of reaching movements - where do biases in our reaching movements arise and what might this tell us about the planning process? They compare several different computational models to explain the results from a range of experiments including those within the literature. Overall, they highlight that motor biases are primarily caused by errors in the transformation between eye and hand reference frames. One strength of the paper is the large number of participants studied across many experiments. However, one weakness is that most of the experiments follow a very similar planar reaching design - with slicing movements through targets rather than stopping within a target. Moreover, there are concerns with the models and the model fitting. This work provides valuable insight into the biases that govern reaching movements, but the current support is incomplete.

Strengths:

The work uses a large number of participants both with studies in the laboratory which can be controlled well and a huge number of participants via online studies. In addition, they use a large number of reaching directions allowing careful comparison across models. Together these allow a clear comparison between models which is much stronger than would usually be performed.

Weaknesses:

Although the topic of the paper is very interesting and potentially important, there are several key issues that currently limit the support for the conclusions. In particular I highlight:

Almost all studies within the paper use the same basic design: slicing movements through a target with the hand moving on a flat planar surface. First, this means that the authors cannot compare the second component of a bias - the error in the direction of a reach which is often much larger than the error in reaching direction. Second, there are several studies that have examined biases in three-dimensional reaching movements showing important differences to two-dimensional reaching movements (e.g. Soechting and Flanders 1989). It is unclear how well the authors' computational models could explain the biases that are present in these much more common-reaching movements.

The model fitting section is under-explained and under-detailed currently. This makes it difficult to accurately assess the current model fitting and its strength to support the conclusions. If my understanding of the methods is correct, then I have several concerns. For example, the manuscript states that the transformation bias model is based on studies mapping out the errors that might arise across the whole workspace in 2D. In contrast, the visual bias model appears to be based on a study that presented targets within a circle (but not tested across the whole workspace). If the visual bias had been measured across the workspace (similar to the transformation bias model), would the model and therefore the conclusions be different? There should be other visual bias models theoretically possible that might fit the experimental data better than this one possible model. Such possibilities also exist for the other models.

Although the authors do mention that the evidence against biomechanical contributions to the bias is fairly weak in the current manuscript, this needs to be further supported. Importantly both proprioceptive models of the bias are purely kinematic and appear to ignore the dynamics completely. One imagines that there is a perceived vector error in Cartesian space whereas the other imagines an error in joint coordinates. These simply result in identical movements which are offset either with a vector or an angle. However, we know that the motor plan is converted into muscle activation patterns which are sent to the muscles, that is, the motor plan is converted into an approximation of joint torques. Joint torques sent to the muscles from a different starting location would not produce an offset in the trajectory as detailed in Figure S1, instead, the movements would curve in complex patterns away from the original plan due to the non-linearity of the musculoskeletal system. In theory, this could also bias some of the other predictions as well. The authors should consider how the biomechanical plant would influence the measured biases.

Reviewer #2 (Public review):

Summary:

Recent cochlear micromechanical measurements in living animals demonstrated outer hair cell-driven broadband vibration of the reticular lamina that contradicts frequency-selective cochlear amplification. The authors hypothesized that motile outer hair cells can drive cochlear fluid circulation. This hypothesis was tested by observing the effects of acoustic stimuli and salicylate, an outer hair cell motility blocker, on kainic acid-induced changes in the cochlear nucleus activities. It was found that acoustic stimuli can reduce the latency of the kainic acid effect, and a low-frequency tone is more effective than broadband noise. Salicylate reduced the effect of acoustic stimuli on kainic acid-induced changes. The authors also developed a computational model to provide the physical basis for interpreting experimental results. It was concluded that experimental data and simulations coherently indicate that broadband outer hair cell action is for cochlear fluid circulation.

Strengths:

The major strengths of this study include its high significance and the combination of electrophysiological recording of the cochlear nucleus responses with computational modeling. Cochlear outer hair cells have been believed to be responsible for the exceptional sensitivity, sharp tuning, and huge dynamic range of mammalian hearing. Recent observation of the broadband reticular lamina vibration contradicts frequency-specific cochlear amplification. Moreover, there is no effective noninvasive approach to deliver the drugs or genes to the cochlea for treating sensorineural hearing loss, one of the most common auditory disorders. These important questions were addressed in this study by observing outer hair cells' roles in the cochlear transport of kainic acid. The well-established electrophysiological method for recording cochlear nucleus responses produced valuable new data, and the purposely developed computational model significantly enhanced the interpretation of the data.

The authors successfully tested their hypothesis, and both the experimental and modeling results support the conclusion that active outer hair cells can drive cochlear fluid circulation in the living cochlea.<br /> Findings from this study will help auditory scientists understand how the outer hair cells contribute to cochlear amplification and normal hearing.

Weaknesses:

While the statement "The present study provides new insights into the nonselective outer hair cell action (in the second paragraph of Discussion)" is well supported by the results, the authors should consider providing a prediction or speculation of how this hair cell action enhances cochlear sensitivity. Such discussion would help the readers better understand the significance of the current work.

Reviewer #2 (Public review):

In this study, Nartker et al. examine how much observers are conscious of using variations of classic inattentional blindness studies. The key idea is that rather than simply asking observers if they noticed a critical object with one yes/no question, the authors also ask follow-up questions to determine if observers are aware of more than the yes/no questions suggest. Specifically, by having observers make forced choice guesses about the critical object, the authors find that many observers who initially said "no" they did not see the object can still "guess" above chance about the critical object's location, color, etc. Thus, the authors claim, that prior claims of inattentional blindness are mistaken and that using such simple methods has led numerous researchers to overestimate how little observers see in the world. To quote the authors themselves, these results imply that "inattentionally blind subjects consciously perceive these stimuli after all... they show sensitivity to IB stimuli because they can see them."

Before getting to a few issues I have with the paper, I do want to make sure to explicitly compliment the researchers for many aspects of their work. Getting massive amounts of data, using signal detection measures, and the novel use of a "super subject" are all important contributions to the literature that I hope are employed more in the future.

Main point 1: My primary issue with this work is that I believe the authors are misrepresenting the way people often perform inattentional blindness studies. In effect, the authors are saying, "People do the studies 'incorrectly' and report that people see very little. We perform the studies 'correctly' and report that people see much more than previously thought." But the way previous studies are conducted is not accurately described in this paper. The authors describe previous studies as follows on page 3:

"Crucially, however, this interpretation of IB and the many implications that follow from it rest on a measure that psychophysics has long recognized to be problematic: simply asking participants whether they noticed anything unusual. In IB studies, awareness of the unexpected stimulus (the novel shape, the parading gorilla, etc.) is retroactively probed with a yes/no question, standardly, "Did you notice anything unusual on the last trial which wasn't there on previous trials?". Any subject who answers "no" is assumed not to have any awareness of the unexpected stimulus.

If this quote were true, the authors would have a point. Unfortunately, I do not believe it is true. This is simply not how many inattentional blindness studies are run. Some of the most famous studies in the inattentional blindness literature do not simply as observes a yes/no question (e.g., the invisible gorilla (Simons et al. 1999), the classic door study where the person changes (Simons and Levin, 1998), the study where observers do not notice a fight happening a few feet from them (Chabris et al., 2011). Instead, these papers consistently ask a series of follow-up questions and even tell the observers what just occurred to confirm that observers did not notice that critical event (e.g., "If I were to tell you we just did XYZ, did you notice that?"). In fact, after a brief search on Google Scholar, I was able to relatively quickly find over a dozen papers that do not just use a yes/no procedure, and instead as a series of multiple questions to determine if someone is inattentionally blind. In no particular order some papers (full disclosure: including my own):

(1) Most et al. (2005) Psych Review<br /> (2) Drew et al. (2013) Psych Science<br /> (3) Drew et al. (2016) Journal of Vision<br /> (4) Simons et al. (1999) Perception<br /> (5) Simons and Levin (1998) Perception<br /> (6) Chabris et al. (2011) iPerception<br /> (7) Ward & Scholl (2015) Psych Bulletin and Review<br /> (8) Most et al. (2001) Psych Science<br /> (9) Todd & Marois (2005) Psych Science<br /> (10) Fougnie & Marois (2007) Psych Bulletin and Review<br /> (11) New and German (2015) Evolution and Human Behaviour<br /> (12) Jackson-Nielsen (2017) Consciousness and cognition<br /> (13) Mack et al. (2016) Consciousness and cognition<br /> (14) Devue et al. (2009) Perception<br /> (15) Memmert (2014) Cognitive Development<br /> (16) Moore & Egeth (1997) JEP:HPP<br /> (17) Cohen et al. (2020) Proc Natl Acad Sci<br /> (18). Cohen et al. (2011) Psych Science

This is a critical point. The authors' key idea is that when you ask more than just a simple yes/no question, you find that other studies have overestimated the effects of inattentional blindness. But none of the studies listed above only asked simple yes/no questions. Thus, I believe the authors are mis-representing the field. Moreover, many of the studies that do much more than ask a simple yes/no question are cited by the authors themselves! Furthermore, as far as I can tell, the authors believe that if researchers do these extra steps and ask more follow-ups, then the results are valid. But since so many of these prior studies do those extra steps, I am not exactly sure what is being criticized.

To make sure this point is clear, I'd like to use a paper of mine as an example. In this study (Cohen et al., 2020, Proc Natl Acad Sci USA) we used gaze-contingent virtual reality to examine how much color people see in the world. On the critical trial, the part of the scene they fixated on was in color, but the periphery was entirely in black and white. As soon as the trial ended, we asked participants a series of questions to determine what they noticed. The list of questions included:

(1) "Did you notice anything strange or different about that last trial?"<br /> (2) "If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?"<br /> (3) "If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?"<br /> (4) "Did you notice anything different about the colors in the last scene?"<br /> (5) We then showed observers the previous trial again and drew their attention to the effect and confirmed that they did not notice that previously.<br /> In a situation like this, when the observers are asked so many questions, do the authors believe that "the inattentionally blind can see after all?" I believe they would not say that and the reason they would not say that is because of the follow-up questions after the initial yes/no question. But since so many previous studies use similar follow-up questions, I do not think you can state that the field is broadly overestimating inattentional blindness. This is why it seems to me to be a bit of a straw-man: most people do not just use the yes/no method.

Main point 2: Let's imagine for a second that every study did just ask a yes/no question and then would stop. So, the criticism the authors are bringing up is valid (even though I believe it is not). I am not entirely sure that above chance performance on a forced choice task proves that the inattentionally blind can see after all. Could it just be a form of subliminal priming? Could there be a significant number of participants who basically would say something like, "No I did not see anything, and I feel like I am just guessing, but if you want me to say whether the thing was to the left or right, I will just 100% guess"? I know the literature on priming from things like change and inattentional blindness is a bit unclear, but this seems like maybe what is going on. In fact, maybe the authors are getting some of the best priming from inattentional blindness because of their large sample size, which previous studies do not use.<br /> I'm curious how the authors would relate their studies to masked priming. In masked priming studies, observers say the did not see the target (like in this study) but still are above chance when forced to guess (like in this study). Do the researchers here think that that is evidence of "masked stimuli are truly seen" even if a participant openly says they are guessing?

Main point 3: My last question is about how the authors interpret a variety of inattentional blindness findings. Previous work has found that observers fail to notice a gorilla in a CT scan (Drew et al., 2013), a fight occurring right in front of them (Chabris et al., 2011), a plane on a runway that pilots crash into (Haines, 1991), and so forth. In a situation like this, do the authors believe that many participants are truly aware of these items but simply failed to answer a yes/no question correctly? For example, imagine the researchers made participants choose if the gorilla was in the left or right lung and some participants who initially said they did not notice the gorilla were still able to correctly say if it was in the left or right lung. Would the authors claim "that participant actually did see the gorilla in the lung"? I ask because it is difficult to understand what it means to be aware of something as salient as a gorilla in a CT scan, but say "no" you didn't notice it when asked a yes/no question. What does it mean to be aware of such important, ecologically relevant stimuli, but not act in response to them and openly say "no" you did not notice them?

Overall: I believe there are many aspects of this set of studies that are innovative and I hope the methods will be used more broadly in the literature. However, I believe the authors misrepresent the field and overstate what can be interpreted from their results. While I am sure there are cases where more nuanced questions might reveal inattentional blindness is somewhat overestimated, claims like "the inattentionally blind can see after all" or "Inattentionally blind subjects consciously perceive thest stimuli after all" seem to be incorrect (or at least not at all proven by this data).