Reviewer #2 (Public review):

Summary:

Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in expression of HERVs in patients suffering from ME/CFS, FM or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables presents solid support for the findings. However, some statements made by the authors seem incomplete and would benefit by a more thorough literature review. Overall, this work will be of interest to the medical community seeking in better understanding the co-occurrence of these pathologies, hinting at a novel angle by integrating HERVs, which are often overlooked, into their assessment.

Strengths:

- The work is well-presented, allowing the reader to understand the overall workflow and how the specific aims contribute to filling the knowledge gap in the field.

- The analyses carried out to understand the potential impact on gene expression mediated by HERVs are in line with previous works, making it solid and robust in the context of this study.

Weaknesses:

- The authors claim to obtain genome-wide HERV expression profiles. However, the array used was developed using hg19, while the genomic analysis of this work are carried out using a liftover to hg38. It would improve the statement and findings to include a comparation of the differences in HERVs available in hg38, and how this could impact the "genome-wide" findings.

- The authors in some points are not thorough with the cited literature. Two examples are:<br /> (1) Lines 396-397 the authors say "the MLT1, usually found enriched near DE genes (Bogdan et al., 2020)". I checked the work by Bogdan, and they studied bacterial infection. A single work in a specific topic is not sufficient to support the statement that MLT1 is "usually" in close vicinity to differentially expressed genes. More works are needed to support this.<br /> (2) After the previous statement, the authors go on to mention "contributing to the coding of conserved lncRNAs (Ramsay et al., 2017)". First, lnc = long non-coding, so this doesn't make sense. Second, in the work by Ramsay they mention "that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved", which is different to what the authors in this study are trying to convey. Again, additional work and a rephrasing might help to support this idea.

- When presenting the clusters, the authors overlook the fact that cluster 4 is clearly control-specific, and fail to discuss what this means. Could this subset of HERV be used as bona fide markers of healthy individuals in the context of these diseases? Are they associated with DE genes? What could be the impact of such associations?

Appraisals on aims:

The authors set specific questions and presented the results to successfully answer them. The evidence is solid, with some weaknesses discussed above that will methodologically strengthen the work.

Likely impact of work on the field:<br /> This work will be of interest to the medical community looking for novel ways to improve clinical diagnosis. Although future works with a greater population size, and more robust techniques such as RNA-Seq, are needed, this is the first step in presenting a novel way to distinguish these pathologies.

It would be of great benefit to the community to provide a table/spreadsheet indicating the specific genomic locations of the HERVs specific to each condition. This will allow proper provenance for future researchers interesting in expanding on this knowledge, as these genomic coordinates will be independent of the technique used (as was the array used here).

Comments on revisions:

When addressing the comments made in the previous round, there are some answers that lack substance and don't seem to be incorporated in the manuscript. For example, the authors say:

Authors' response: This is an important point. However, the low number of probes (less than 100) that were excluded from our analysis by lack of correspondence with hg38 among the 1,290,800 probesets was interpreted as insignificant for "genome-wide" claims. An aspect that will be explained in the revised version of this manuscript.

I checked the revised manuscript with tracked changes, and there doesn't seem to be an updated explanation to this. In which lines is this explained?

For the other response:

Authors' response: Using control DE HERV as bona fide markers of healthy individuals seems like an interesting possibility worth exploring. Control DE HERV (cluster 4) associate with DE genes involved in apoptosis, T cell activation and cell-cell adhesion (modules 1 and 6). The impact of which deserves further study.

I couldn't find an updated mention of this in the discussion.

Another point that I raised was regarding the decision of using an FDR of 0.1 instead of 0.05. The authors only speculate about the impacts in their answer, while I believe that this could have been rigorously addressed. Since this was done in R, and DE analysis are relatively fast, I don't see a reason as to why this part was not repeated and discussed accordingly.

For other analyses, there doesn't seem to be a problem with using 0.05 as threshold. Examples of this are the "Overrepresentation functional analysis", or the "Statistical analysis" part of the methods they say "we used a Fisher exact test to calculate p-value, considering enriched in the provided list if an adjusted p-value (FDR) was less than 0.05".

Just to make this point clear: I'm not asking the authors to repeat all the work using the 0.05 FDR threshold, but rather that they are aware and conscious about the impact of this, and give an idea to the audience on how it would change the DE numbers. This would put in perspective the findings to any future reader.

I think that most of the other answers to both my previous concerns and the other reviewer's concerns are ok. My last outstanding concern is that the probe coordinates apparently can't be shared, which undermines a lot this study reproducibility, and its use by future researches which won't be able to compare their results to this study.

Reviewer #3 (Public review):

Summary

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation to the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network, constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience.

Strengths

Adaptive optics imaging of murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is a benefit for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. This model would potentially allow for controlling the size, depth and severity of the laser injury opening interesting avenues for future study.

The time-course, 2D and 3D spatial activation pattern of microglial activation are striking and provide an unprecedented view of the retinal response to mild injury.

Weaknesses

Generalization of the (lack of) neutrophil response to photoreceptor loss - there is ample evidence in literature that neutrophils are heavily recruited in response to severe retinal damage that includes photoreceptor loss. Why the same was not observed here in this article remains an open question. One could hypothesize that neutrophil recruitment might indeed occur under conditions that are more in line with the more extreme damage models, for example, with a stronger and global ablation (substantially more photoreceptor loss over a larger area). This parameter space is unwieldy and sufficiently large to address the question conclusively in the current article, i.e. how much photoreceptor loss leads to neutrophil recruitment? By the same token, the strong and general conclusion in the title - Photoreceptor loss does not recruit neutrophils - cannot be made until an exhaustive exploration be made of the same parameter space. A scaling back may help here, to reflect the specific, mild form of laser damage explored here, for instance - Mild photoreceptor loss does not recruit neutrophils despite...

EIU model - The EIU model was used as a positive control for neutrophil extravasation. Prior work with flow cytometry has shown a substantial increase in neutrophil counts in the EIU model. Yet, in all, the entire article shows exactly 2 examples in vivo and 3 ex vivo (Figure 7) of extravasated neutrophils from the EIU model (n = 2 mice). The general conclusion made about neutrophil recruitment (or lack thereof) is built partly upon this positive control experiment. But these limited examples, especially in the case where literature reports a preponderance of extravasated neutrophils, raise a question on the paradigm(s) used to evaluate this effect in the mild laser damage model.

Overall, the strengths outweigh the weaknesses, provided the conclusions/interpretations are reconsidered.

Reviewer #2 (Public review):

The authors have now addressed the most important points, and they include more comprehensive evaluation of their method and comparisons to other approaches for multiple datasets.

Some points would benefit from clarification:

- Figure 1B now compares "Otsu thresholding", "WNet 3D - No artifacts" and "WNet 3d". Why don't you also report the score for "Otsu thresholding - No Artifacts"? To my understanding this is a post-processing operation to remove small and very large objects, so it could easily be applied to the Otsu thresholding. Given the good results for Otsu thresholding alone (quite close F1-score to WNet 3d), it seems like DL might not really be necessary at all for this dataset and including "Otsu thresholding - No artifacts" would enable evaluating this point.

- CellPose and StarDist perform poorly in all the experiments performed by the authors. In almost all cases they underperform Otsu thresholding, which is in most cases on par with the WNet results (except for "Mouse Skull Nuclei CBG"). This is surprising and contradicts the collective expertise of the community: good CellPose and StarDist models can be trained for the 3D instance segmentation tasks studied here. Perhaps these methods were not trained in an optimal way. Seems unlikely that it is not possible to get much better CellPose or StarDist models for these tasks (current versions are on par or much worse than Otsu!), as I have applied both of these models successfully in similar settings. Specifically, it seems unlikely that the developers of CellPose or StarDist would obtain similarly poor scores on the same data (note I am not one of the developers).

The current experiments still highlight an interesting aspect: the problem of training / fine-tuning these methods correctly on new data and the technical challenges associated with this. But the reported results should by no means be taken as a fair assessment of the capabilities of StarDist or CellPose.

Please note that I did not have time to test the Napari plugin again, so I did not evaluate whether it improved in usability.

Reviewer #2 (Public review):

Summary:

In this manuscript, Zhang et al. examine neural activity in motor cortex as monkeys make reaches in a novel target interception task. Zhang et al. begin by examining the single neuron tuning properties across different moving target conditions, finding several classes of neurons: those that shift their preferred direction, those that change their modulation gain, and those that shift their baseline firing rates. The authors go on to find an interesting, tilted ring structure of the neural population activity, depending on the target speed, and find that 1) the reach direction has consistent positioning around the ring, and 2) the tilt of the ring is highly predictive of the target movement speed. The authors then model the neural activity with a single neuron representational model and a recurrent neural network model, concluding that this population structure requires a mixture of the three types of single neurons described at the beginning of the manuscript.

Strengths:

I find the task the authors present here to be novel and exciting. It slots nicely into an overall trend to break away from a simple reach-to-static-target tasks to better characterize the breadth of how motor cortex generates movements. I also appreciate the movement from single neuron characterization to population activity exploration, which generally serves to anchor the results and make them concrete. Further, the orbital ring structure of population activity is fascinating, and the modeling work at the end serves as a useful baseline control to see how it might arise.

Weaknesses:

While I find the behavioral task presented here to be excitingly novel, I find the presented analyses and results to be far less interesting than they could be. Key to this, I think, is that the authors are examining this task and related neural activity primarily with a single-neuron representational lens. This would be fine as an initial analysis, since the population activity is of course composed of individual neurons, but the field seems to have largely moved towards a more abstract "computation through dynamics" framework that has, in the last several years, provided much more understanding of motor control than the representational framework has. As the manuscript stands now, I'm not entirely sure what interpretation to take away from the representational conclusions the authors made (i.e. the fact that the orbital population geometry arises from a mixture of different tuning types). As such, by the end of the manuscript, I'm not sure I understand any better how motor cortex or its neural geometry might be contributing to the execution of this novel task.

Main Comments:

My main suggestions to the authors revolve around bringing in the computation through a dynamics framework to strengthen their population results. The authors cite the Vyas et al. review paper on the subject, so I believe they are aware of this framework. I have three suggestions for improving or adding to the population results:

(1) Examination of delay period activity: one of the most interesting aspects of the task was the fact that the monkey had a random-length delay period before he could move to intercept the target. Presumably, the monkey had to prepare to intercept at any time between 400 and 800 ms, which means that there may be some interesting preparatory activity dynamics during this period. For example, after 400ms, does the preparatory activity rotate with the target such that once the go cue happens, the correct interception can be executed? There is some analysis of the delay period population activity in the supplement, but it doesn't quite get at the question of how the interception movement is prepared. This is perhaps the most interesting question that can be asked with this experiment, and it's one that I think may be quite novel for the field--it is a shame that it isn't discussed.

(2) Supervised examination of population structure via potent and null spaces: simply examining the first three principal components revealed an orbital structure, with a seemingly conserved motor output space and a dimension orthogonal to it that relates to the visual input. However, the authors don't push this insight any further. One way to do that would be to find the "potent space" of motor cortical activity by regression to the arm movement and examine how the tilted rings look in that space. Presumably, then, the null space should contain information about the target movement. The ring tilt will likely be evident if the authors look at the highest variance neural dimension orthogonal to the potent space (the "null space")--this is akin to PC3 in the current figures, but it would be nice to see what comes out when you look in the data for it.

The authors attempt this sort of analysis in the supplement, alongside their dPCA results, but the results seem misinterpreted. The authors do identify one kind of output-potent space using the reach direction components of dPCA, and the reach directions are indeed aligned here. However, they then go on to interpret the target-velocity space as the output-null space, orthogonal to the potent space. There are two problems with this. 1) The target-velocity space is not necessarily orthogonal to the reach-direction space. This is a key aspect of dPCA--while the individual components within a particular marginalization space are orthogonal, the marginalization spaces themselves are not necessarily orthogonal unless they are forced to be (which the authors don't mention doing). 2) Even if the target-velocity space were orthogonal to the reach-direction space, it would not comprise the whole output-null space--such a null space would also include dimensions of neural population activity that have target-velocity/reach-direction interaction, which the authors show is a major component of neural population variance. Incidentally, the dPCA analysis the authors present shows what I would expect from their unsupervised results, but as it is written, the dPCA results are interpreted in a strange or potentially misleading way.

(3) RNN perturbations: as it's currently written, the RNN modeling has promise, but the perturbations performed don't provide me with much insight. I think this is because the authors are trying to use the RNN to interpret the single neuron tuning, but it's unclear to me what was learned from perturbing the connectivity between what seems to me almost arbitrary groups of neurons. It seems to me that a better perturbation might be to move the neural state before the movement onset to see how it changes the output. For example, the authors could move the neural state from one tilted ring to another to see if the virtual hand then reaches a completely different (yet predictable) target. Moreover, if the authors can more clearly characterize the preparatory movement, perhaps perturbations in the delay period would provide even more insight into how the interception might be prepared.

Reviewer #3 (Public review):

This work describes the tandem linkage of influenza hemagglutinin (HA) receptor binding domains of diverse subtypes to create 'beads on a string' (BOAS) immunogens. They show that these immunogens elicit ELISA binding titers against full-length HA trimers in mice, as well as varying degrees of vaccine mismatched responses and neutralization titers. They also compare these to BOAS conjugated on ferritin nanoparticles and find that this did not largely improve immune responses. This work offers a new type of vaccine platform for influenza vaccines, and this could be useful for further studies on the effects of conformation and immunodominance on the resulting immune response. 

Overall, the central claims of immunogenicity in a murine model of the BOAS immunogens described here are supported by the data. 

Strengths included the adaptability of the approach to include several, diverse subtypes of HAs. The determination of an optimal composition of strains in the 5-BOAS that overall yielded the best immune responses was an interesting finding and one that could also be adapted to other vaccine platforms. Lastly, as the authors discuss, the ease of translation to an mRNA vaccine is indeed a strength of this platform. 

One interesting and counter-intuitive result is the high levels of neutralization titers seen to vaccine-mismatched, group 2 H7 in the 5-BOAS group that differs from the 4-BOAS with the addition of a group 1 H5 RBD. At the same time, no H5 neutralization titers were observed for any of the BOAS immunogens, yet they were seen for the BOAS-NP. Uncovering where these immune responses are being directed and why these discrepancies are being observed would be informative future work. 

There are a few caveats in the data that should be noted: 

(1) 20 ug is a pretty high dose for a mouse and the majority of the serology presented is after 3 doses at 20 ug. By comparison, 0.5-5 ug is a more typical range (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380945/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9980174/). Also, the authors state that 20 ug per immunogen was used, including for the BOAS-NP group, which would mean that the BOAS-NP group was given a lower gram dose of HA RBD relative to the BOAS groups. 

(2) Serum was pooled from all animals per group for neutralization assays, instead of testing individual animals. This could mean that a single animal with higher immune responses than the rest in the group could dominate the signal and potentially skew the interpretation of this data. 

(3) In Figure S2, it looks like an apparent increase in MW by changing the order of strains here, which may be due to differences in glycosylation. Further analysis would be needed to determine if there are discrepancies in glycosylation amongst the BOAS immunogens and how those differ from native HAs. 

Comments on revisions:

The authors have addressed all concerns upon revision.

Reviewer #2 (Public review):

Recent findings in the field of motor learning have pointed to the combined action of multiple mechanisms that potentially contribute to changes in motor output during adaptation. A nearly ubiquitous motor learning process occurs via the trial-by-trial compensation of motor errors, often attributed to cerebellar-dependent updating. This error-based learning process is slow and largely unconscious. Additional learning processes that are rapid (e.g., explicit strategy-based compensation) have been described in discrete movements like goal-directed reaching adaptation. However, the role of rapid motor updating during continuous movements such as walking has been either under explored or inconsistent with those found during adaptation of discrete movements. Indeed, previous results have largely discounted the role of explicit strategy-based mechanisms for locomotor learning. In the current manuscript, Rossi et al. provide convincing evidence for a previously unknown rapid updating mechanism for locomotor adaptation. Unlike the now well-studied explicit strategies employed during reaching movements, the authors demonstrate that this stimulus-response mapping process is largely unconscious. The authors show that in approximately half of subjects, the mapping process appears to be memory based while the remainder of subjects appear to perform structural learning of the task design. The participants that learned using a structural approach had the capability to rapidly generalize to previously unexplored regions of the perturbation space.

One result that will likely be particularly important to the field of motor learning is the authors' quite convincing correlation between the magnitude of proprioceptive recalibration and the magnitude error-based updating. This result beautifully parallels results in other motor learning tasks and appears to provide a robust marker for the magnitude of the mapping process (by means of subtracting off the contribution of error-based motor learning). This is a fascinating result with implications for the motor learning field well beyond the current study.

A major strength of this manuscript is the large sample size across experiments and the extent of replication performed by the authors in multiple control experiments.

Finally, I commend the authors on extending their original observations via Experiment 2. While it seems that participants use a range of mapping mechanisms (or indeed a combination of multiple mapping mechanisms), future experiments may be able to tease apart why some subjects use memory versus structural mapping. A future ability to push subjects to learn structurally-based mapping rules has the potential to inform rehabilitation strategies.

Overall, the manuscript is well written, the results are clear, and the data and analyses are convincing.

Strengths:

(1) Convincing behavioral data supporting the existence of multiple learning processes during split-belt adaptation. Further convincing correlations typing the extent of forward-model based adaptation with proprioceptive recalibration.<br /> (2) The authors test a veritable "zoo" of prior motor learning models to show that these models do not account for their behavioral results.<br /> (3) The authors develop a convincing alternative model (PM-ReMap) that appears to account for their behavioral results by explicitly modeling forward-model based adaptation in parallel with goal remapping.

Reviewer #2 (Public Review):

Summary:

Naïve CD4 T cells in CD11c-Cre p28-floxed mice express highly elevated levels of proinflammatory IFNg and the transcription factor T-bet. This phenotype turned out to be imposed by thymic dendritic cells (DCs) during CD4SP T cell development in the thymus [PMID: 23175475]. The current study affirms these observations, first, by developmentally mapping the IFNg dysregulation to newly generated thymic CD4SP cells [PMID: 23175475], second, by demonstrating increased STAT1 activation being associated with increased T-bet expression in CD11c-Cre p28-floxed CD4 T cells [PMID: 36109504], and lastly, by confirming IL-27 as the key cytokine in this process [PMID: 27469302]. The authors further demonstrate that such dysregulated cytokine expression is specific to the Th1 cytokine IFNg, without affecting the expression of the Th2 cytokine IL-4, thus proposing a role for thymic DC-derived p28 in shaping the cytokine response of newly generated CD4 helper T cells. Mechanistically, CD4SP cells of CD11c-Cre p28-floxed mice were found to display epigenetic changes in the Ifng and Tbx21 gene loci that were consistent with increased transcriptional activities of IFNg and T-bet mRNA expression. Moreover, in autoimmune Aire-deficiency settings, CD11c-Cre p28-floxed CD4 T cells still expressed significantly increased amounts of IFNg, exacerbating the autoimmune response and disease severity. Based on these results, the investigators propose a model where thymic DC-derived IL-27 is necessary to suppress IFNg expression by CD4SP cells and thus would impose a Th2-skewed predisposition of newly generated CD4 T cells in the thymus, potentially relevant in autoimmunity.

Strengths:

Experiments are well-designed and executed. The conclusions are convincing and supported by the experimental results.

Weaknesses from the original round of review:

The premise of the current study is confusing as it tries to use the CD11c-p28 floxed mouse model to explain the Th2-prone immune profile of newly generated CD4SP thymocytes. Instead, it would be more helpful to (1) give full credit to the original study which already described the proinflammatory IFNg+ phenotype of CD4 T cells in CD11c-p28 floxed mice to be mediated by thymic dendritic cells [PMID: 23175475], and then, (2) build on that to explain that this study is aimed to understand the molecular basis of the original finding.

In its essence, this study mostly rediscovers and reaffirms previously reported findings, but with different tools. While the mapping of epigenetic changes in the IFNg and T-bet gene loci and the STAT1 gene signature in CD4SP cells are interesting, these are expected results, and they only reaffirm what would be assumed from the literature. Thus, there is only incremental gain in new insights and information on the role of DC-derived IL-27 in driving the Th1 phenotype of CD4SP cells in CD11c-p28 floxed mice.

Altogether, the major issues of this study remain unresolved:

(1) It is still unclear why the p28-deficiency in thymic dendritic cells would result in increased STAT1 activation in CD4SP cells. Based on their in vitro experiments with blocking anti-IFNg antibodies, the authors conclude that it is unlikely that the constitutive activation of STAT1 would be a secondary effect due to autocrine IFNg production by CD4SP cells. However, this possibility should be further tested with in vivo models, such as Ifng-deficient CD11c-p28 floxed mice. Alternatively, is this an indirect effect by other IFNg producers in the thymus, such as iNKT cells? It is necessary to explain what drives the STAT1 activation in CD11c-p28 floxed CD4SP cells in the first place.

(2) It is also unclear whether CD4SP cells are the direct targets of IL-27 p28. The cell-intrinsic effects of IL-27 p28 signaling in CD4SP cells should be assessed and demonstrated, ideally by CD4SP-specific deletion of IL-27Ra, or by establishing bone marrow chimeras of IL-27Ra germline KO mice.

[Editors' note: The resubmitted paper was minimally revised, and many of the initial concerns remain unresolved.]

Reviewer #2 (Public review):

Summary:

This is an interesting theoretical exploration of how a flexible protein domain, which has multiple DNA-binding sites along it, affects the stability of the protein-DNA complex. It proposes a mechanism ("octopusing") for protein doing a random walk while bound to DNA which simultaneously enables exploration of the DNA strand and stability of the bound state.

Strengths:

Stability of the protein-DNA bound state and the ability of the protein to perform 1d diffusion along the DNA are two properties of a transcription factor that are usually seen as being in opposition of each other. The octopusing mechanism is an elegant resolution of the puzzle of how both could be accommodated. This mechanism has interesting biological implications for the functional role of intrinsically disordered domains in transcription factor (TF) proteins. They show theoretically how these domains, if flexible and able to make multiple weak contacts with the DNA, can enhance the ability of the TF to efficiently find their binding site on the DNA from which they exert control over the transcription of their target gene. The paper concludes with a comparison of model predictions with experimental data which gives further support to the proposed model. Overall, this is an interesting and well executed theoretical paper that proposes an interesting idea about the functional role for IDR domains in TFs.

Weaknesses:

IDR domains are assumed flexible which I believe is not always the case. Also, I'm not sure how ubiquitous are the assumed binding sites on the DNA for multiple subdomains along the IDR. These assumptions though seem like interesting points of departure for further experiments.

Reviewer #2 (Public review):

In this manuscript, Hes et al. present a comprehensive multi-species atlas of the dorsal vagal complex (DVC) using single-nucleus RNA sequencing, identifying over 180,000 cells and 123 cell types across five levels of granularity in mice and rats. Intriguingly, the analysis uncovered previously uncharacterized cell populations, including Kcnj3-expressing astrocytes, neurons co-expressing Th and Cck, and a population of leptin receptor-expressing neurons in the rat area postrema, which also express the progenitor marker Pdgfra. These findings suggest species-specific differences in appetite regulation. This study provides a valuable resource for investigating the intricate cellular landscape of the DVC and its role in metabolic control, with potential implications for refining obesity treatments targeting this hindbrain region.

In line with previous work published by the PI, the topic is of clear scientific relevance, and the data presented in this manuscript are both novel and compelling. Additionally, the manuscript is well-structured, and the conclusions are robust and supported by the data. Overall, this study significantly enhances our understanding of the DVC and sheds light on key differences between rats and mice.

I applaud the authors for the depth of their analysis. However, I have a few major concerns, comments, and suggestions that should be addressed.

(1) If I understand the methodology correctly, mice were fasted overnight and then re-fed for 2 hours before being sacrificed (lines 91-92), which occurred 4 hours after the onset of the light phase (line 111). This means that the re-fed animals had access and consequently consumed food when they typically would not. While I completely recognize that every timepoint has its limitations, the strong influence of the circadian rhythm on the DVC gene expression (highlighted by the work published by Lukasz Chrobok), and the fact that timing of food/eating is a potent Zeitgeber, might have an impact on the analysis and should be mentioned as a potential limitation in the discussion (along with citing Dr Chrobok's work). Could this (i.e., eating during a time when the animals are not "primed by their own circadian clock to eat" potentially explain why the meal-related changes in gene expression were relatively small?

(2) In the Materials and Methods section, LiCl is mentioned as one of the treatment conditions; however, very little corresponding data are presented or discussed. Please include these results and elaborate on the rationale for selecting LiCl over other anorectic compounds.

(3) The number of animals used differs significantly between species, which the authors acknowledge as a limitation in the discussion. Since the authors took advantage of previously published mouse data sets (Ludwig and Dowsett data sets), I wonder if the authors could compare/integrate any rat data set currently available in rats as well to partially address the sample size disparity.

(4) Dividing cells in AP vs NTS vs DMX clusters and analyzing potential species differences would significantly enhance the quality of the manuscript, given the partially diverse functions of these regions. This could be done by leveraging existing published datasets that employed spatial transcriptomics or more classical methodologies (e.g., PMID: 39171288, PMID: 39629676, PMID: 38092916). I would be interested to hear the authors' perspective on the feasibility of such an analysis.

(5) Given the manuscript's focus on feeding and metabolism, I believe a more detailed description and comparison of the transcription profile of known receptors, neurotransmitters, and neuropeptides involved in food intake and energy homeostasis between mice and rats would add value. Adding a curated list of key genes related to feeding regulation would be particularly informative.

Reviewer #2 (Public review):

This work aims to study the evolution of nitrogenanses, understanding how their structure and function adapted to changes in the environment, including oxygen levels and changes in metal availability.

The study predicts > 5000 structures of nitrogenases, corresponding to extant, ancestral, and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive undertaking that is certain to be a resource for the community. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes.

The challenge with this study is that all (or nearly all) of the quantitative analyses presented are based on RMSD calculations, many of which are under 2 angstroms. For all intents and purposes, two structures with RMSD < 2 angstroms could be considered 'structurally identical'. A lot of insight generated is based on minuscule differences in RMSD, for which it is not clear that they are significantly different. The suggestion would be to find a way to evaluate the RMSD metric and determine whether these values, as obtained for structures being compared, are reliable. Some options are provided in earlier studies: PMID: 11514933, PMID: 17218333, PMID: 11420449, PMID: 8289285 (and others).

It could also be valuable to focus more on site-specific RMSDs rather than Global RMSDs. The high conservation in the nitrogenases likely ensures that the global RMSDs will remain low across the family. Focusing on specific regions might reveal interesting differences between clades that are more informative regarding the evolution of structure in tandem with environment/time.

Reviewer #2 (Public review):

As remains obvious from my previous reviews, I still consider this to be an important paper and that is final and publishable in its current state.

In that previous review, I revealed my identity to help reassure the authors that I was doing my best to remain unbiased because I work in this area and some of the authors' results directly impact my prior research. I was genuinely excited to see the earlier preprint version of this paper when it first appeared. I get a lot of joy out of trying to - collectively, as a field - really understand the nature of our data, and I continue to commend the authors here for pushing at the sources of aperiodic activity!

In their manuscript, Schmidt and colleagues provide a very compelling, convincing, thorough, and measured set of analyses. Previously I recommended that the push even further, and they added the current Figure 5 analysis of event-related changes in the ECG during working memory. In my opinion this result practically warrants a separate paper its own!

The literature analysis is very clever, and expanded upon from any other prior version I've seen.

In my previous review, the broadest, most high-level comment I wanted to make was that authors are correct. We (in my lab) have tried to be measured in our approach to talking about aperiodic analyses - including adopting measuring ECG when possible now - because there are so many sources of aperiodic activity: neural, ECG, respiration, skin conductance, muscle activity, electrode impedances, room noise, electronics noise, etc. The authors discuss this all very clearly, and I commend them on that. We, as a field, should move more toward a model where we can account for all of those sources of noise together. (This was less of an action item, and more of an inclusion of a comment for the record.)

I also very much appreciate the authors' excellent commentary regarding the physiological effects that pharmacological challenges such as propofol and ketamine also have on non-neural (autonomic) functions such as ECG. Previously I also asked them to discuss the possibility that, while their manuscript focuses on aperiodic activity, it is possible that the wealth of literature regarding age-related changes in "oscillatory" activity might be driven partly by age-related changes in neural (or non-neural, ECG-related) changes in aperiodic activity. They have included a nice discussion on this, and I'm excited about the possibilities for cognitive neuroscience as we move more in this direction.

Finally, I previously asked for recommendations on how to proceed. The authors convinced me that we should care about how the ECG might impact our field potential measures, but how do I, as a relative novice, proceed. They now include three strong recommendations at the end of their manuscript that I find to be very helpful.

As was obvious from previous review, I consider this to be an important and impactful cautionary report, that is incredibly well supported by multiple thorough analyses. The authors have done an excellent job responding to all my previous comments and concerns and, in my estimation, those of the previous reviewers as well.

Reviewer #2 (Public review):

Summary:

In this work the authors show that dopaminergic neurons (DANs) from the DL1 cluster in Drosophila larvae are required for the formation of aversive memories. DL1 DANs complement pPAM cluster neurons which are required for the formation of attractive memories. This shows the compartmentalized network organization of how an insect learning center (the mushroom body) encodes memory by integrating olfactory stimuli with aversive or attractive teaching signals. Interestingly, the authors found that the 4 main dopaminergic DL1 neurons act partially redundant, and that single cell ablation did not result in aversive memory defects. However, ablation or silencing of a specific DL1 subset (DAN-f1,g1) resulted in reduced salt aversion learning, which was specific to salt but no other aversive teaching stimuli tested. Importantly, activation of these DANs using an optogenetic approach was also sufficient to induce aversive learning in the presence of high salt. Together with the functional imaging of salt and fructose responses of the individual DANs and the implemented connectome analysis of sensory (and other) inputs to DL1/pPAM DANs this represents a very comprehensive study linking the structural, functional and behavioral role of DL1 DANs. This provides fundamental insight into the function of a simple yet efficiently organized learning center which displays highly conserved features of integrating teaching signals with other sensory cues via dopaminergic signaling.

Strengths:

This is a very careful, precise and meticulous study identifying the main larval DANs involved in aversive learning using high salt as a teaching signal. This is highly interesting because it allows to define the cellular substrates and pathways of aversive learning down to the single cell level in a system without much redundancy. It therefore sets the basis to conduct even more sophisticated experiments and together with the neat connectome analysis opens the possibility to unravel different sensory processing pathways within the DL1 cluster and integration with the higher order circuit elements (Kenyon cells and MBONs). The authors' claims are well substantiated by the data and balanced, putting their data in the appropriate context. The authors also implemented neat pathway analyses using the larval connectome data to its full advantage, thus providing network pathways that contribute towards explaining the obtained results.

Weaknesses:

Previous comments were fully addressed by the authors.

Reviewer #2 (Public Review):

Summary:

In this work, Yuasa et al. aimed to study the spatial resolution of modulations in alpha frequency oscillations (~10Hz) within the human occipital lobe. Specifically, the authors examined the receptive field (RF) tuning properties of alpha oscillations, using retinotopic mapping and invasive electroencephalogram (iEEG) recordings. The authors employ established approaches for population RF mapping, together with a careful approach to isolating and dissociating overlapping, but distinct, activities in the frequency domain. Whereby, the authors dissociate genuine changes in alpha oscillation amplitude from other superimposed changes occurring over a broadband range of the power spectrum. Together, the authors used this approach to test how spatially tuned estimated RFs were when based on alpha range activity, vs. broadband activities (focused on 70-180Hz). Consistent with a large body of work, the authors report clear evidence of spatially precise RFs based on changes in alpha range activity. However, the size of these RFs were far larger than those reliably estimated using broadband range activity at the same recording site. Overall, the work reflects a rigorous approach to a previously examined question, for which improved characterization leads to improved consistency in findings and some advance of prior work.

Strengths:

Overall, the authors take a careful and well-motivated approach to data analyses. The authors successfully test a clear question with a rigorous approach and provide strong supportive findings. Firstly, well-established methods are used for modeling population RFs. Secondly, the authors employ contemporary methods for dissociating unique changes in alpha power from superimposed and concomitant broadband frequency range changes. This is an important confound in estimating changes in alpha power not employed in prior studies. The authors show this approach produces more consistent and robust findings than standard band-filtering approaches. As noted below, this approach may also account for more subtle differences when compared to prior work studying similar effects.

Original Weaknesses:

- Theoretical framing: The authors frame their study as testing between two alternative views on the organization, and putative functions, of occipital alpha oscillations: i) alpha oscillation amplitude reflects broad shifts in arousal state, with large spatial coherence and uniformity across cortex; ii) alpha oscillation amplitude reflects more specific perceptual processes and can be modulated at local spatial scales. However, in the introduction this framing seems mostly focused on comparing some of the first observations of alpha with more contemporary observations. Therefore, I read their introduction to more reflect the progress in studying alpha oscillations from Berger's initial observations to the present. I am not aware of a modern alternative in the literature that posits alpha to lack spatially specific modulations. I also note this framing isn't particularly returned to in the discussion. A second important variable here is the spatial scale of measurement. It follows that EEG based studies will capture changes in alpha activity up to the limits of spatial resolution of the method (i.e. limited in ability to map RFs). This methodological distinction isn't as clearly mentioned in the introduction, but is part of the author's motivation. Finally, as noted below, there are several studies in the literature specifically addressing the authors question, but they are not discussed in the introduction.

- Prior studies: There are important findings in the literature preceding the author's work that are not sufficiently highlighted or cited. In general terms, the spatio-temporal properties of the EEG/iEEG spectrum are well known (i.e. that changes in high frequency activity are more focal than changes in lower frequencies). Therefore, the observations of spatially larger RFs for alpha activities is highly predicted. Specifically, prior work has examined the impact of using different frequency ranges to estimate RF properties, for example ECoG studies in the macaque by Takura et al. NeuroImage (2016) [PubMed: 26363347], as well as prior ECoG work by the author's team of collaborators (Harvey et al., NeuroImage (2013) [PubMed: 23085107]), as well as more recent findings from other groups (Luo et al., (2022) BioRxiv: https://doi.org/10.1101/2022.08.28.505627). Also, a related literature exists for invasively examining RF mapping in the time-voltage domain, which provides some insight into the author's findings (as this signal will be dominated by low-frequency effects). The authors should provide a more modern framing of our current understanding of the spatial organization of the EEG/iEEG spectrum, including prior studies examining these properties within the context of visual cortex and RF mapping. Finally, I do note that the author's approach to these questions do reflect an important test of prior findings, via an improved approach to RF characterization and iEEG frequency isolation, which suggests some important differences with prior work.

- Statistical testing: The authors employ many important controls in their processing of data. However, for many results there is only a qualitative description or summary metric. It appears very little statistical testing was performed to establish reported differences. Related to this point, the iEEG data is highly nested, with multiple electrodes (observations) coming from each subject, how was this nesting addressed to avoid bias?

[Editors' note: the authors have addressed the original concerns.]

Reviewer #2 (Public review):

In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin (Nlgn) family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate astrocytic Neuroligin function in the current study. Using postnatal conditional deletion of Nlgn1-3 specifically from astrocytes in mice, Golf et al. show that virtually no changes in the expression of synaptic proteins or in the properties of synaptic transmission at either excitatory or inhibitory synapses are observed. Moreover, no alterations in the morphology of astrocytes themselves were found. To further extend this finding, the authors additionally analyzed human neurons co-cultured with mouse glia lacking expression of Nlgn1-4. No difference in excitatory synaptic transmission was observed between neurons cultured in the present of wildtype vs. Nlgn1-4 conditional knockout glia. The authors conclude that while Neuroligins are indeed expressed in astrocytes and are hence likely to play some role there, this role does not include any direct consequences on synaptic structure and function, in direct contrast to the model proposed by Stogsdill et al.

Overall, this is a strong study that addresses a fundamental and highly relevant question in the field of synaptic neuroscience. Neuroligins are not only key regulators of synaptic function, they have also been linked to numerous psychiatric and neurodevelopmental disorders, highlighting the need to precisely define their mechanisms of action. The authors take a wide range of approaches to convincingly demonstrate that under their experimental conditions, Nlgn1-3 are efficiently deleted from astrocytes in vivo, and that this deletion does not lead to major alterations in the levels of synaptic proteins or in synaptic transmission at excitatory or inhibitory synapses, or in the morphology of astrocytes. While the co-culture experiments are somewhat more difficult to interpret due to lack of a control for the effect of wildtype mouse astrocytes on human neurons, they are also consistent with the notion that deletion of Nlgn1-4 from astrocytes has no consequences for the function of excitatory synapses. Together, the data from this study provide compelling and important evidence that, whatever the role of astrocytic Neuroligins may be, they do not contribute substantially to synapse formation or function under the conditions investigated.

Reviewer #2 (Public review):

This study by Tardiff, Kang & Gold seeks to i) develop a normative account of how observers should adapt their decision-making across environments with different levels of correlation between successive pairs of observations, and ii) assess whether human decisions in such environments are consistent with this normative model. The authors first demonstrate that, in the range of environments under consideration here, an observer with full knowledge of the generative statistics should take both the magnitude and sign of the underlying correlation into account when assigning weight in their decisions to new observations: stronger negative correlations should translate into stronger weighting (due to the greater information furnished by an anticorrelated generative source), while stronger positive correlations should translate into weaker weighting (due to the greater redundancy of information provided by a positively correlated generative source). The authors then report an empirical study in which human participants performed a perceptual decision-making task requiring accumulation of information provided by pairs of perceptual samples, under different levels of pairwise correlation. They describe a nuanced pattern of results with effects of correlation being largely restricted to response times and not choice accuracy, which could be captured through fits of their normative model (in this implementation, an extension of the well-known drift diffusion model) to the participants' behaviour while allowing for mis-estimation of the underlying correlations. An intriguing result is that the observed pattern of behavioural effects is best explained by a model in which observers marginally underestimated the level of correlation between the generative sources, and that this bias affects behaviour through effects on stimulus encoding that then shape how the evidence furnished by each stimulus sample is weighted in decision formation.

As the authors point out in their very well-written paper, appropriate weighting of information gathered in correlated environments has important consequences for real-world decision-making. Yet, while this function has been well studied for 'high-level' (e.g. economic) decisions, how we account for correlations when making simple perceptual decisions on well-controlled behavioural tasks has not been investigated. As such, this study addresses an important and timely question that will be of broad interest to psychologists and neuroscientists. The computational approach to arrive at normative principles for evidence weighting across environments with different levels of correlation is elegant, makes strong connections with prior work in different decision-making contexts, and should serve as a valuable reference point for future studies in this domain. The empirical study is well designed and executed, and the modelling approach applied to these data showcases an impressively deep understanding of relationships between different parameters of the drift diffusion model and its novel application to this setting. Another strength of the study is that it is preregistered.

In my view, any major weaknesses of the study have been well addressed by the authors during review. An outstanding question that arises from the current work and remains unanswered here is around the (normative?) origin of the correlation underestimates, and the present work lays a strong foundation from which to pursue this question in the future.

Reviewer #2 (Public review):

Summary:

The authors aimed to elucidate the role of Ctla-4 in maintaining intestinal immune homeostasis by using a novel Ctla-4-deficient zebrafish model. This study addresses the challenge of linking CTLA-4 to inflammatory bowel disease (IBD) due to the early lethality of CTLA-4 knockout mice. Four lines of evidence were shown to show that Ctla-4-deficient zebrafish exhibited hallmarks of IBD in mammals: 1) impaired epithelial integrity and infiltration of inflammatory cells; 2) enrichment of inflammation-related pathways and the imbalance between pro- and anti-inflammatory cytokines; 3) abnormal composition of immune cell populations; and 4) reduced diversity and altered microbiota composition. By employing various molecular and cellular analyses, the authors established ctla-4-deficient zebrafish as a convincing model of human IBD.

Strengths:

The characterization of the mutant phenotype is very thorough, from anatomical to histological and molecular levels. The finding effectively established ctla-4 mutants as a novel zebrafish model for investigating human IBD. Evidence from the histopathological and transcriptome analysis was very strong and supports a severe interruption of immune system homeostasis in the zebrafish intestine. Additional characterization using sCtla-4-Ig further probed the molecular mechanism of the inflammatory response, and provided a potential treatment plan for targeting Ctla-4 in IBD models.

Weaknesses:

To probe the molecular mechanism of Ctla-4, the authors used a spectrum of antibodies that target Ctla-4 or its receptors. The phenotype assayed was lymphocyte proliferation, while it was the composition rather than number of immune cells that was observed to be different in the scRNASeq assay. Although sCtla-4 has an effect of alleviating the IBD-like phenotypes, I found this explanation a bit oversimplified.

Comments on revised version:

The authors have sufficiently addressed all my concerns and I don't have further suggestions.

Reviewer #2 (Public review):

Summary:

Chakravarty et al. propose a 'synchronized framework' for studying perceptual decision-making (DM) across species -namely humans, rats, and mice. Although all species shared hallmarks of evidence accumulation, the results highlighted species-specific differences. Humans were the slowest and most accurate, rats optimized the speed-accuracy tradeoff to maximize the reward rate and mice were the fastest but least accurate. In addition, while humans were better fit by a classic DDM with fixed bounds, rodents were better fit by a DDM with collapsing bounds. While comparing behavioral strategies in evidence accumulation tasks across species is an important and timely question, some of the presented differences across species lack a clear interpretation and could be simply caused by differences in the task design. There is important information and analyses missing about the DDM and the other models used, which lowers the confidence and enthusiasm about the results.

Strengths:

The comparison of behavior across species, including humans and commonly used laboratory species like rats and mice, is a fundamental step in neuroscience to establish more informed links between animal experiments and human cognition. In this work, Chakravarty et al. analyze and model the behavior of three species during the same evidence accumulation task. They draw conclusions about the different strategies used in each case.

Weaknesses:

Novelty:<br /> While quite relevant, some parts of the work presented are more novel than others. That EA drives choice behavior and these choices can be described with a DDM have been shown before (see e.g. (Kane et al. 2023; Brunton et al. in 2013; Pinto et al 2018)). The novelty here mostly lies in the comparison of three species in the same task and in fitting the same exact model (close quantitative comparison of behavioral strategies). However, some of the differences lack a clear interpretation. For instance, the values of some of the DDM fitted parameters between the three species are not ordered "as expected" (e.g. non-decision time or DDM BIC). Other comparison results completely lack an explanation (e.g. rats' RT are near optimal while humans and mice are not). The aspect that I found most novel and exciting is the application of HMMs to each of the species. However, this part comes at the end of the paper and has been done without sufficient depth. There is almost no explanation for the results. I would suggest the authors bring up this part and move back to other aspects which are, in my opinion, less novel or interpretable (e.g. results around the optimality of RT).

Task design:<br /> Since there is no fixation, the response time (RT) reflects both the evidence integration time plus the motor time (stimuli are played until a response is given). This design makes it hard to compare RTs between species. While humans just had to press a button, rodents had to move their whole bodies from a central port to a side port. When comparing rats and mice, their difference in size relative to port distance could explain different RTs. This could for example explain the large difference in non-decision time (ndt) in Figure 3F between mice and rats. Are the measurements of the rat and the mouse boxes comparable? The authors should explain this difference more openly and discuss its implications when interpreting the results. The Methods should also provide information about the distance between ports for each species. I also strongly recommend including a few videos of rats and mice performing the task to have a sense of the movements involved in the task in each species.

(1) DDM

Goodness of fit:<br /> The authors conclude that the three species use an accumulation of evidence strategy because they can fit a DDM. However, there is little information about the goodness of these fits. They only show the RT distributions for one example subject (too small to distinguish whether the fit of the histograms is good or not). We suggest they make a figure showing in more detail the match of the RT distributions across subjects (e.g. they can compare RT quartiles for data and model for the entire group of subjects). Then they provide BIC which is a measure that depends on the number of trials. Were the number of trials matched across subjects/species? Could the authors provide a measure independent of the number of trials (e.g. cross-validated log-likelihood per trial)? Moreover, is this BIC computed only on the RTs, mouse responses, or both?

Overparameterization:<br /> The authors chose to include as DDM parameters the variability of the initial offset, the variability in non-decision time, and the variability of the drift rate. Having so many parameters with just one stimulus condition (80:20 ratio of flashes) may lead to unidentifiability problems as recognized previously (e.g. see M. Jones (2021) here osf.io/preprints/psyarxiv/gja3u). Their parameter recovery Supplementary Figure 3 shows that at least two of these variability parameters can not be recovered. I also couldn't find the values of these parameters for the fitted DDM. So I was wondering the extent to which adding these parameters improves the fits and is overall necessary.

Tachometric curves:<br /> The authors show increasing tachometric curves (i.e. Accuracy vs RT) and use this finding as proof of accumulation. They fit these curves using a GAAM with little justification or detail (in fact the GAAM seems to over-fit the data a bit). The authors do not say, however, that the other model used, i.e. the DDM, may not reproduce these increasing tachometric curves because "in its basic form", the DDM gives flat tachometric curves. Does the DDM fitted to the individual RT and choice data capture the monotonic increase observed in the tachometric curves?

Correct vs Error trials:<br /> In a similar line, the authors do not test the fitted DDM separately in correct vs error trials, which is a classical distinction that most DDMs can't capture. It would be good to know if: (1) the RT in the data of correct vs error responses are similar (quantified in panel Figure 2B because in 2E it is not clear) and (2) the same trend between correct and error RTs are observed in the fitted DDMs.

Urgency model:<br /> It is not clear how the urgency model used works. The authors cite Ditterich (2006), but in that paper, the urgency signal was applied to a race model with two decision variables: the urgency signal "accelerated" both DVs equally and sped up the race without favoring one DV versus the other. In a one-dimensional DDM, it is not clear where the urgency is applied. We assume it is applied in the direction of the stimulus, but then it is unclear how the urgency knows about the stimulus, which is what the DDM is trying to estimate in the first place. The authors should explain this model in greater detail and try to resolve this question.

Despite finding differences between species, the analyses seem mostly exploratory instead of hypothesis-driven. There is little justification for why differences in some DDM parameters across species would be expected.

(2) GLM and HMM

The GLM fits show nicely that humans, rats, and mice weigh differently the total provided evidence (Figures 6C-D). This may be because the internal noise in the accumulation of evidence is higher but also it could simply be because animals do not weigh the evidence that is presented when they are already moving towards the side ports. A parsimonious alternative to the "more noisy" species is simply that they only consider the first part of the stimulus. Extending the GLM to capture the differential weighting of each sequential sample (what is called the Psychophysical kernel, PK) should be straightforward and would provide a more fair comparison between species (i.e. perhaps the slope of the psychometric curves is not that different, once evidence is weighted in each species with its corresponding PK.

Choice Bias:<br /> Panel 3G (DDM starting point) shows that both rats and mice are slightly but systematically biased to the Left (x0 < 0.5). Panel 6D "Bias" seems to be showing the absolute value of the GLM bias parameter. It would be nice to (i) show the signed GLM bias parameter. (ii) Compare that the biases computed in the DDM and GLM are comparable across species and subjects; it looks like from the GLM they are comparable in magnitude across species whereas the in DDM they weren't (mice had a much bigger |x0| in the DDM), (iii) explain (or at least comment) on why animals show a systematic bias to one side.

Reviewer #2 (Public review):

Summary:

This is an excellent paper that demonstrates Computational Modeling at its best. The authors propose a mechanism to provide flexibility to learn new information while preserving stability in neural networks by combining structural plasticity and synaptic plasticity.

Strengths:

An intriguing idea, that is well embedded in experimental data.

The problem posed is real, the model uses data to be designed and implemented yet adds to the data novel and useful insight. The project proposes a parsimonious explanation for why neurogenesis can be better than classical plasticity and how stability versus flexibility can be solved with this approach.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #2 (Public review):

Summary:

Shrews go through winter by shrinking their brain and most organs, then regrow them in the spring. The gene expression changes underlying this unusual brain size plasticity were unknown. Here, the authors looked for potential adaptations underlying this trait by looking at differential expression in the hypothalamus. They found enrichments for DE in genes related to the blood brain barrier and calcium signaling, as well as used comparative data to look at gene expression differences that are unique in shrews. This study leverages a fascinating organismal trait to understand plasticity and what might be driving it at the level of gene expression. This manuscript also lays the groundwork for further developing this interesting system.

Strengths:

One strength is that the authors used OU models to look for adaptation in gene expression. The authors also added cell culture work to bolster their findings.

Comments on revised version:

I think that the authors have made a strong revision. No other comments.

Reviewer #2 (Public review):

Summary:

This valuable work aims to infer, from microbiome data, microbial species interaction patterns associated with healthy and unhealthy human gut microbiomes. Using solid techniques from statistical physics, the authors propose that healthy and unhealthy microbiome interaction patterns substantially differ. Unhealthy microbiomes are closer to instability and single-strain dominance; whereas healthy microbiomes showcase near-neutral dynamics, mostly driven by demographic noise and immigration.

Strengths:

A well-written article, relatively easy to follow and transparent despite the high degree of technicality of the underlying theory. The authors provide a powerful inferring procedure, which bypasses the issue of having only compositional data.

Weaknesses:

(1) This sentence in the introduction seems key to me: "Focusing on single species properties as species abundance distribution (SAD), fail to characterise altered states of microbiome." Yet it is not explained what is meant by 'fail', and thus what the proposed approach 'solves'.

(2) Lack of validation, following arbitrary modelling choices made (symmetry of interactions, weak-interaction limit, uniform carrying capacity).<br /> Inconsistent interpretation of instability. Here, instability is associated with the transition to the marginal phase, which becomes chaotic when interaction symmetry is broken. But as the authors acknowledge, the weak interaction limit does not reproduce fat-tailed abundance distributions found in data. On the other hand, strong interaction regimes, where chaos prevails, tend to do so (Mallmin et al, PNAS 2024). Thus, the nature of the instability towards which unhealthy microbiomes approach is unclear.

(3) Three technical points about the methodology and interpretation.<br /> a) How can order parameters h and q0 can be inferred, if in the compositional data they are fixed by definition?<br /> b) How is it possible that weaker interaction variance is associated with approach to instability, when the opposite is usually true?<br /> c) Having an idea of what the empirical data compares to the theoretical fits would be valuable.

Implications:

As the authors say, this is a proof of concept. They point at limits and ways to go forward, in particular pointing at ways in which species abundance distributions could be better reproduced by the predicted dynamical models. One implication that is missing, in my opinion, is the interpretability of the results, and what this work achieves that was missing from other approaches (see weaknesses section above): what do we learn from the fact that changes in microbial interactions characterise healthy from unhealthy microbiota? For instance, what does this mean for medical research?

Reviewer #2 (Public review):

Summary:

This study shows that transcription factor Sp1 is required for correct ventral vs. dorsal targeting of limb-innervating LMC motor neurons using mouse and chick as model systems. In a wild-type embryo, lateral LMC axons specifically target dorsal muscles while medial LMC axons target ventral muscles. The authors convincingly show that this specificity is lost when Sp1 is knocked down or knocked out - axons of both lateral and medial LMC motor neurons project to both dorsal and ventral muscles in mutant conditions. The authors then conduct RNA-seq and ChIP experiments to show that Sp1 loss of function disrupts Ephrin-Epha receptor signaling pathway genes. These molecules are known to provide attractive or repulsive cues to guide LMC axons to their targets. The authors show that attraction/repulsion properties of medial and lateral LMC axons to specific Ephrin/Epha molecules are in fact disrupted in Sp1 mutants using ex vivo explant studies. Finally, the authors show that behaviors like coordinated movement and grip strength are also affected in Sp1 mutant mice. This study convincingly shows that Sp1 is important for correct circuit wiring of LMC neurons, and moves the field forward by elucidating a new level of transcriptional regulation required in this process. However, the claims made by the authors that the mode of Sp1-mediated regulation is through cis-attenuation of Epha activity is not well supported. These and additional strengths and weaknesses in approach and in data interpretation are discussed below.

Strengths:

(1) The study convincingly shows that wildtype levels of Sp1 are necessary for LMC axon targeting specificity. The combination of the following approaches is a strength:<br /> a) Both loss of function and gain of function experiments are performed for Sp1 and show complementary effects on the axon targeting phenotype.<br /> b) Retrograde labeling of LMC neurons from dorsal and ventral muscles shows that Sp1 mutants clearly lose the specificity of LMC axon targeting.<br /> c) The authors also use explant experiments to show that both loss of Sp1 and gain of Sp1 show clear changes in attraction and repulsion to specific ephrin and epha receptor molecules.<br /> d) The Sp1 loss and gain of function experiments are well controlled to show that the changes in axon wiring observed are not due to cell death, cell fate switches, or due to unequal numbers of medial and lateral LMC neurons being labeled in the experiments.

(2) It is also convincing that Sp1 requires cofactors p300 and CBP for its function. In the absence of these cofactors, the gain of function phenotypes of Sp1 are subdued.

Weaknesses:

(1) The robustness of RNAseq and ChIP experiments is difficult to judge as methods are not described. For example, it is unclear if RNAseq is performed on purified motor neurons or on whole spinal cords. This is an important consideration as Sp1 is a broadly expressed protein.

(2) The authors state that expression of Ephrin A5 and Ephrin B2 is reduced based on RNAseq data, however, it is not shown that this reduction occurs specifically in LMC neurons.

(3) The authors show Sp1 ChIP peaks at Ephrin B2 promoter, but nothing is mentioned about peaks at Eprin A5 or other types of signaling molecules like Sema7a, which are also differentially expressed in Sp1 mutants. There is also no mention of the correlation between changes in gene expression seen in RNAseq data and the binding profile of Sp1 seen in ChIP data, which could help establish the robustness of these datasets.

(4) The authors conclude that Sp1 functions by activating Ephrin A5 in medial LMC and Ephrin B2 in lateral LMC. The argument, as I understand it, is that this activation leads to cis attenuation of their respective Epha receptors and therefore targeting the correct muscle. Though none of the data presented go against this hypothesis, this hypothesis is also not fully supported. Specifically:<br /> a) It would be important to know that modulation of Sp1 expression leads to changes in EphrinA5 and B2 in LMC lateral/medial neurons.<br /> b) It would also be important to show that none of the other changes caused by Sp1 are responsible for axon mistargeting by performing rescue experiments with Ephrin A5 and Ephrin B2.<br /> c) To make the most convincing case, experiments showing increased or decreased cis-binding of Ephrin molecules with Epha receptors would be necessary. This study would still be compelling without this last experiment, but the language in the abstract would need to be modulated.

(5) All behavior experiments are done in a pan-neuronal knockout of Sp1. As Sp1 is broadly expressed in neurons, a statement describing whether and why the authors think the phenotypes arise from Sp1's function in LMC motor neurons would be helpful. Experimentally, rescue experiments in which Sp1 is restored in LMC neurons or motor neurons would also make this claim more convincing.

Reviewer #2 (Public review):

Summary:

The manuscript presents findings that indicate a role in controlling Drosophila heart rate for a conserved miRNA (miR-238 in flies). Further, the manuscript localizes the relevant tissue for the function of this miRNA to a subset of neurons that are heavily involved in circadian regulation, thus presenting an interesting mechanistic link between the circadian system and heart rate. Either ubiquitous knockout or ubiquitous overexpression negatively impacts several aspects of heart performance, with a pronounced effect on heart rate. Interestingly, knockdowns in the heart itself are innocuous, but knockdown in LNvS neurons recapitulates the effect on heart rate. Authors use bioinformatics to identify the clockwork orange (cwo) gene as a potential target and validate that cwo expression is reduced when miR-238 is knocked down in LNvS neurons in vivo and also validate that cwo is regulated by miR-238 in cell culture luciferase assays. Exercise shows a modest ability to restore normal cwo expression and a trend toward an effect on survival, but shows a much stronger rescue of the heart rate phenotype.

Strengths:

Evidence is strong for the effect of miR-238 in pdf-positive neurons on the control of heart rate and for cwo as a downstream effector of miR-238.

Work to identify specific targets of miR-283 is well-done and successfully identified a key downstream regulator in cwo.

The potential mechanism using miR-238 to link circadian neurons to heart rate regulation is novel and exciting.

Weaknesses:

The evidence that this is related to normal aging is rather weak, and the effect of exercise on the observed parameters is small and not necessarily working through the miR-238/cwo mechanism.

The authors seem to be conflating two hypotheses in their interpretations. Is miR-283 working through circadian mechanisms or age-related mechanisms? While it is true that aging tends to reduce heart rate, I don't think that means that any intervention that reduces heart rate is causing "senescence". Similarly, reduced survival in miR-283 knockdown flies does not prove that miR-283 promotes healthy aging per se, just that miR-283 is required for health regardless of age.

Survival reduction is quite modest which does not necessarily support the idea that the bradycardia is causing major health issues or premature senescence for the flies. The interpretation of the longevity experiments throughout the manuscript seems overstated.

The study would benefit greatly from a direct test of the author's proposed pathway for exercise to improve bradycardia.

The statement in the discussion "inducing endurance exercise of anti gravity climbing in flies with miR-283 knockdown in LNvs can improve bradyarrhythmic features by decreasing brain miR-283 expression" is not fully supported by data in the paper. There is an association there, but it cannot be said to be the full cause (or even required) without doing more experiments

The summary figure includes both data-supported mechanistic relationships and mechanisms that are inferred or assumed.

Reviewer #2 (Public review):

Summary:

The goal of the study was to uncover the mechanisms mediating tactile-context-dependent locomotion modulation in C. elegans, which represents an interesting model of behavioral plasticity. Starting from a candidate genetic screen focusing on guanylate cyclase (GCY) mutants, the authors identified the AFD-specific gcy-18 gene as essential for tactile-context-dependent locomotion modulation. AFD is primarily characterized as a thermo-sensory neuron. However, key thermosensory transduction genes and the sensory ending structure of AFD were shown here to be dispensable for tactile-context locomotion modulation. AFD actuates tactile-context locomotion modulation via the cell-autonomous actions of GCY-18 and the CNG-3 cyclic nucleotide-gated channel, and via AFD's connection with AIB interneurons through electrical synapses. This represents a potentially relevant synaptic connection linking AFD to the mechanosensory-behavior circuit.

Strengths:

(1) The fact that AFD mediates tactile-context locomotion modulation is new, rather surprising, and interesting.

(2) The authors have combined a very clever microfluidic-based behavioral assay with a large set of genetic manipulations to dissect the molecular and cellular pathways involved. Rescue experiments with single-copy transgenes are very convincing.

(3) The study is very clearly written, and figures are nicely illustrated with diagrams that effectively convey the authors' interpretation.

Weaknesses:

(1) Whereas GCY-18 in AFD and the AFD-AIB synaptic connection clearly play a role in tactile-context locomotion modulation, whether and how they actually modulate the mechanosensory circuit and/or locomotion circuit remains unclear. The possibility of non-synaptic communication linking mechanosensory neurons and AFD (in either direction) was not explored. Thus, in the end, we have not learned much about what GCY-18 and the AFD-AIB module are doing to actuate tactile context-dependent locomotion modulation.

(2) The authors only focused on speed readout, and we don't know if the many behavioral parameters that are modulated by tactile context are also under the control of AFD-mediated modulation.

(3) The AFD-AIB gap junction reconstruction experiment was conducted in an innexin double mutant background, in which the whole nervous system's functioning might be severely impaired, and its results should be interpreted with this limitation in mind.

Reviewer #2 (Public review):

Summary:

This study presents a valuable characterization of the effects of intracranial theta-burst stimulation of the basolateral amygdala on single units spiking activity in several areas in the human brain, associated with memory processing. It is written clearly and concisely, allowing readers to fully understand the analysis used.

The authors used a visual recognition memory task previously employed by their group to characterize the effects of basolateral amygdala stimulation upon memory consolidation (Inman et al, 2018). This current report is an interesting analysis to complement the results reported in the 2018 paper.

Strengths:

Rare combination of human neurophysiology and behavior -<br /> The type of experiment performed in the manuscript, which contains both neurophysiological data, behavior, and a deep brain stimulation intervention (DBS), is incredibly rare, takes many years to accomplish with tight collaboration between clinical and research teams. Our understanding of spiking dynamics of human neurons is very limited, and this report is an important piece in the puzzle that allows DBS to be used in future interventions that will benefit patients' health.

Multiple brain areas included -<br /> It's important to note that the report analyzes brain areas with which the Amygdala has extensive connections (Fig. 1A) - Hippocampus, OFC, Amygdala, ACC. It seems that neurons in all these areas were modulated by the stimulation, except the ACC, in which firing rates were so low, that only a handful of neurons were included in the analysis. This is an important demonstration that low amplitude stimulation (even when reduced to 0.5mA) can travel far and wide across the human brain.

The experiment is cleverly designed to tease apart responses due to visual stimuli (image presentation) and electrical stimulation. Authors suggest that the units modulated by stimulation are largely distinct from those responsive to image offset during trials without stimulation. The subpopulation that responds strongly also tends to have a higher baseline of firing rate. It's important to add that the chosen modulation index is more likely to be significant in neurons with higher firing rates.

Weaknesses:

Readers can benefit from understanding with more details the locations chosen for stimulation - in light of previous studies that found differences between effects based on proximity to white matter (For example - PMID 32446925, Mohan et al, Brain Stimul. 2020 and PMID 33279717 Mankin et al Brain Stimul. 2021).

Reviewer #2 (Public review):

Summary:

This study from the CenGEN consortium addresses several limitations of single-cell RNA (scRNA) and bulk RNA sequencing in C. elegans with a focus on cells in the nervous system. scRNA datasets can give very specific expression profiles, but detecting rare and non-polyA transcripts is difficult. In contrast, bulk RNA sequencing on isolated cells can be sequenced to high depth to identify rare and non-polyA transcripts but frequently suffers from RNA contamination from other cell types. In this study, the authors generate a comprehensive set of bulk RNA datasets from 53 individual neurons isolated by fluorescence-activated cell sorting (FACS). The authors combine these datasets with a previously published scRNA dataset (Taylor et al., 2021) to develop a novel method, called LittleBites, to estimate and subtract contamination from the bulk RNA data. The authors validate the method by comparing detected transcripts against gold-standard datasets on neuron-specific and non-neuronal transcripts. The authors generate an "integrated" list of protein-coding expression profiles for the 53 neuron sub-types, with fewer but higher confidence genes compared to expression profiles based only on scRNA. Also, the authors identify putative novel pan-neuronal and cell-type specific non-coding RNAs based on the bulk RNA data. LittleBites should be generally useful for extracting higher confidence data from bulk RNA-seq data in organisms where extensive scRNA datasets are available. The additional confidence in neuron-specific expression and non-coding RNA expands the already great utility of the neuronal expression reference atlas generated by the CenGEN consortium.

Strengths:

The study generates and analyzes a very comprehensive set of bulk RNA datasets from individual fluorescently tagged transgenic strains. These datasets are technically challenging to generate and significantly expand our knowledge of gene expression, particularly in cells that were poorly represented in the initial scRNA-seq datasets. Additionally, all transgenic strains are made available as a resource from the Caenorhabditis Elegans Genetics Center (CGC).

The study uses the authors' extensive experience with neuronal expression to benchmark their method for reducing contamination utilizing a set of gold-standard validated neuronal and non-neuronal genes. These gold-standard genes will be helpful for benchmarking any C. elegans gene expression study.

Weaknesses:

The bulk RNA-seq data collected by the authors has high levels of contamination and, in some cases, is based on very few cells. The methodology to remove contamination partly makes up for this shortcoming, but the high background levels of contaminating RNA in the FACS-isolated neurons limit the confidence in cell-specific transcripts.

The study does not experimentally validate any of the refined gene expression predictions, which was one of the main strengths of the initial CenGEN publication (Taylor et al, 2021). No validation experiments (e.g., fluorescence reporters or single molecule FISH) were performed for protein-coding or non-coding genes, which makes it difficult for the reader to assess how much gene predictions are improved, other than for the gold standard set, which may have specific characteristics (e.g., bias toward high expression as they were primarily identified in fluorescence reporter experiments).

The study notes that bulk RNA-seq data, in contrast to scRNA-seq data, can be used to identify which isoforms are expressed in a given cell. However, no analysis or genome browser tracks were supplied in the study to take advantage of this important information. For the community, isoform-specific expression could guide the design of cell-specific expression constructs or for predictive modeling of gene expression based on machine learning.

Reviewer #2 (Public review):

Summary:

This study provides valuable context for ongoing research on the role of dopamine in memory and locomotion. DANs have been a fascinating area of study due to their complexity, and this work dissects specific DANs, exploring their roles in different memory-related behaviors while offering some explanations. The discussions provided by the authors effectively situates the study in the broader field of learning, memory, DAN circuitry and behavioral computation in insect brains. The study achieves what it sets out to and it does so unequivocally. The experiments were elegantly designed, leaving little room for doubt in the study's claims. However, the study lacks context regarding the molecular pathways underlying these results. While it strengthens current knowledge by providing robust evidence, it does little to explore the molecular mechanisms behind these effects.

Strengths:

(1) Experiment design is one of the strengths of this study. The experiments are thorough and cover the length and breadth of the core findings of the study. Although a lot of work has already been done in studying the role of dopamine in memory and locomotion, the dissection of the functions of distinct DANs in larvae has been done meticulously with well-structured experiments.<br /> (2) This study fits quite nicely into the puzzle of memory, especially in the context of Dopamine. Previous studies in *Drosophila* adults have shown the opposing roles of DANs in locomotion depending on the context of DAN activation. This study drives that point home for larvae, providing conclusive evidence in that regard.<br /> (3) The use of clear figures and simple language is one of the strengths of this paper. The figures are comprehensive, complete and manage to narrate the story by themselves. The flow of information is smooth. The simple and effective language used maintains scientific rigor while remaining accessible to those new to the field. A pleasant read.

Weaknesses:<br /> (1) The authors have done a great job at structuring the figures. But some main figures would benefit from including the controls instead of placing them in supplementary.<br /> (2) The paper would benefit from a deeper discussion regarding molecular mechanisms underlying their results. It would be interesting to see what the authors think about different Dopamine receptors and how they relate to the findings of this paper.<br /> (3) Throughout the paper, the authors have been clear and comprehensive, but in some cases, further explanation of their choices were missing. For example, the choice to compare bending and tail velocity over other parameters within the same clusters is unclear.

Reviewer #2 (Public review):

Summary:

The authors aimed to develop a novel and efficient method for SV detection, utilizing data from the 1000 Genomes Project (1KGP) for modeling and calibration. This method was subsequently validated using UK population data and applied to identify structural variants associated with specific disease phenotypes.

Strengths:

Third-generation single-molecule sequencing data offers several advantages over traditional high-throughput sequencing methods, particularly due to its long-read lengths, which provide valuable insights into significant forms of genomic variation. The authors have developed an efficient method for detecting structural variations and optimizing the utilization of genomic data. We hope that this method will continue to be refined, enabling researchers to more effectively leverage long-read data, high-throughput data, or even a synergistic combination of both.

Weaknesses:

Although this research contributes to our ability to more effectively utilize long-length and high-throughput data, there are some key issues that need to be addressed in terms of analyzing the specific results as well as writing the article.

Reviewer #3 (Public review):

Summary:

The goal of this study was to carry out an in-depth granular and unbiased phenotyping of peripheral blood circulating Tfh specific to two malaria vaccine candidates, PfSEA-1A and PfGARP, and correlate these with age (children vs adults) and protection from malaria (antibody titers against Plasmodium antigens.) Authors further attempted to identify any specific differences of the Tfh responses to these two distinct malaria antigens.

Strengths:

The authors had access to peripheral blood samples from children and adults living in a malaria-endemic region of Kenya. The authors studied these samples using in vitro restimulation in the presence of specific malaria antigens. Authors generated a very rich data set from these valuable samples using cutting-edge spectral flow cytometry and a 21-plex panel that included a variety of surface markers, cytokines and transcription factors.

Update following first revision (R1) of the manuscript:

The authors have made a great effort to comprehensively address comments raised by the reviewers. In particular, clearly showing expression of ICOS and Bcl6 on CXCR5+ cells greatly strengthens the case for defining these cells as Tfh-like circulatory lymphocytes (cTfh).

Weaknesses:

Update following first revision (R1) of the manuscript:

Unfortunately, my main concern remains. As it stands, the study is not really on antigen-specific T cells, but rather on the overall CD4 T cell compartment plus or minus antigenic stimulation. Although authors used an in vitro restimulation strategy with malaria antigens, they do not focus on cells de-novo expressing activation markers as a result of restimulation, neither they use tetramers to detect antigen-specific T cells. Moreover, their data shows that the number of CXCR5+ CD4 T cells de-novo expressing activation markers and/or cytokines as a result of their in vitro restimulation is negligible, even when using a prototypic superantigen (SEB).

Thus, no antigen-specific CXCR5+ CD4 T cells could be analysed with the data that the authors provide in this manuscript.

Reviewer #2 (Public review):

Summary:

Kapoor et. al. investigated the role of the mycobacterial protein Wag31 in lipid and peptidoglycan synthesis and sought to delineate the role of the N- and C- terminal domains of Wag31. They demonstrated that modulating Wag31 levels influences lipid homeostasis in M. smegmatis and cardiolipin (CL) localisation in cells. Wag31 was found to preferentially bind CL-containing liposomes, and deleting the N-terminus of the protein significantly decreased this interaction. Novel interactions between Wag31 and proteins involved in lipid metabolism and cell wall synthesis were identified, suggesting that Wag31 recruits proteins to the intracellular membrane domain by direct interaction.

Strengths:

(1) The importance of Wag31 in maintaining lipid homeostasis is supported by several lines of evidence.<br /> (2) The interaction between Wag31 and cardiolipin, and the role of the N-terminus in this interaction was convincingly demonstrated.

Weakness:

(1) Interactome analysis with truncated versions of the proteins could not be performed in M. smegmatis due to protein instability.

Reviewer #2 (Public review):

Summary:

In this work, Degutis and colleagues addressed an interesting issue related to the concurrent coding of sensory percepts and visual working memory contents in visual cortices. They used generalization analyses to test whether working memory representations change over time, diverge from sensory percepts, and vary across distraction conditions. Temporal generalization analysis demonstrated that off-diagonal decoding accuracies were lower than on-diagonal decoding accuracies, regardless of the presence of intervening distractions, implying that working memory representations can change over time. They further showed that the coding space for working memory contents showed subtle but statistically significant changes over time, potentially explaining the impaired off-diagonal decoding performance. The neural coding of sensory distractions instead remained largely stable. Generalization analyses between target and distractor codes showed overlaps but were not identical. Cross-condition decodings had lower accuracies compared to within-condition decodings. Finally, within-condition decoding revealed more reliable working memory representations in the condition with intervening random noises compared to cross-condition decoding using a trained classifier on data from the no-distraction condition, indicating a change in the VWM format between the noise distractor and no-distractor trials.

Strengths:

This paper demonstrates a clever use of generalization analysis to show changes in the neural codes of working memory contents across time and distraction conditions. It provides some insights into the differences between representations of working memory and sensory percepts, and how they can potentially coexist in overlapping brain regions.

Comments on revisions:

I appreciate the authors' efforts in addressing my previous concerns. The inclusion of additional analyses and data has strengthened the paper. I have no further concerns.

Reviewer #2 (Public review):

The study by Deganutti and co-workers is a methodological report on an adaptive sampling approach, multiple walker supervised molecular dynamics (mwSuMD), which represents an improved version of the previous SuMD.<br /> Case-studies concern complex conformational transitions in a number of G protein Coupled Receptors (GPCRs) involving long time-scale motions such as binding-unbinding and collective motions of domains or portions. GPCRs are specialized GEFs (guanine nucleotide exchange factors) of heterotrimeric Gα proteins of the Ras GTPase superfamily. They constitute the largest superfamily of membrane proteins and are of central biomedical relevance as privileged targets of currently marketed drugs.<br /> MwSuMD was exploited to address:

a) binding and unbinding of the arginine-vasopressin (AVP) cyclic peptide agonist to the V2 vasopressin receptor (V2R);<br /> b) molecular recognition of the β2-adrenergic receptor (β2-AR) and heterotrimeric GDP-bound Gs protein;<br /> c) molecular recognition of the A1-adenosine receptor (A1R) and palmotoylated and geranylgeranylated membrane-anchored heterotrimeric GDP-bound Gi protein;<br /> d) the whole process of GDP release from membrane-anchored heterotrimeric Gs following interaction with the glucagon-like peptide 1 receptor (GLP1R), converted to the active state following interaction with the orthosteric non-peptide agonist danuglipron.

The revised version has improved clarity and rigor compared to the original also thanks to the reduction in the number of complex case studies treated superficially.<br /> The mwSuMD method is solid and valuable, has wide applicability and is compatible with the most world-widely used MD engines. It may be of interest to the computational structural biology community.<br /> The huge amount of high-resolution data on GPCRs makes those systems suitable, although challenging, for method validation and development.<br /> While the approach is less energy-biased than other enhanced sampling methods, knowledge, at the atomic detail, of binding sites/interfaces and conformational states is needed to define the supervised metrics, the higher the resolution of such metrics is the more accurate the outcome is expected to be. Definition of the metrics is a user- and system-dependent process.

Reviewer #2 (Public review):

Zeeshan et al. investigate the function of the protein SUN1, a proposed nuclear envelope protein linking nuclear and cytoplasmic cytoskeleton, during the rapid male gametogenesis of the rodent malaria parasite Plasmodium berghei. They reveal that SUN1 localises to the nuclear envelope (NE) in male and female gametes and show that the male NE has unexpectedly high dynamics during the rapid process of gametogenesis. Using expansion microscopy, the authors find that SUN1 is enriched at the neck of the bipartite MTOC that links the intranuclear spindle to the basal bodies of the cytoplasmic axonemes. Upon deletion of SUN1, the basal bodies of the eight axonemes fail to segregate, no spindle is formed, and emerging gametes are anucleated, leading to a complete block in transmission. By interactomics the authors identify a divergent allantoicase-like protein, ALLAN, as a main interaction partner of SUN1 and further show that ALLAN deletion largely phenocopies the effect of SUN1.

Overall, the authors use an extensive array of fluorescence and electron microscopy techniques as well as interactomics to convincingly demonstrate that SUN1 and ALLAN play a role in maintaining the structural integrity of the bipartite MTOC during the rapid rounds of endomitosis in male gametogenesis.

Two suggestions for improvement of the work remain:

(1) Lipidomic analysis of WT and SUN1-knockout gametocytes before and after activation resulted in only minor changes in some lipid species. Without statistical analysis, it remains unclear if these changes are statistically significant and not rather due to expected biological variability. While the authors clearly toned down their conclusions in the revised manuscript, some phrasings in the results and the discussion still suggest that gametocyte activation and/or SUN1-knockout affects lipid composition. Similarly, some phrases suggest that SUN1 is responsible for the observed loops and folds in the NE and that SUN1 KO affects the NE dynamics. Currently, I do not think that the data supports these statements.

(2) It is interesting to note that ALLAN has a much more specific localisation to basal bodies than SUN1, which is located to the entire nuclear envelope. Knock out of ALLAN also exhibits a milder (but still striking) phenotype than knockout of SUN1. These observations suggest that SUN1 has additional roles in male gametogenesis besides its interaction with ALLAN, which could be discussed a bit more.

This study uses extensive microscopy and genetics to characterise an unusual SUN1-ALLAN complex, thus providing new insights into the molecular events during Plasmodium male gametogenesis, especially how the intranuclear events (spindle formation and mitosis) are linked to the cytoplasmic separation of the axonemes. The characterisation of the mutants reveals an interesting phenotype, showing that SUN1 and ALLAN are localised to and maintain the neck region of the bipartite MTOC. The authors here confirm and expand the previous knowledge about SUN1 in P. berghei, adding more detail to its localisation and dynamics, and further characterise the interaction partner ALLAN. Given the evolutionary divergence of Plasmodium, these results are interesting not only for parasitologists, but also for more general cell biologists.

Reviewer #2 (Public review):

Summary:

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

Strengths:

The authors provide compelling evidence that ornithine enhances the palatability of several chemical stimuli (i.e., IMP, MSG, MPG, Intralipos, sucrose, NaCl, quinine). Ornithine also increases CT nerve responses to MSG. Additionally, the authors provide evidence that the effects of ornithine are mediated by GPRC6A, a G-protein-coupled receptor family C group 6 subtype A, and that this receptor is expressed primarily in fungiform taste buds. Taken together, these results indicate that ornithine enhances the palatability of multiple taste stimuli in rats, and that the enhancement is mediated, at least in part, within fungiform taste buds. This finding could stand on its own. The question of whether ornithine produces these effects by eliciting kokumi-like perceptions (see below) should be presented as speculation in the Discussion section.

Weaknesses:

I am still unconvinced that the measurements in rats reflect the "kokumi" taste percept described in humans. The authors conducted long-term preference tests, 10-min avidity tests and whole chorda tympani (CT) nerve recordings. None of these procedures specifically model features of "kokumi" perception in humans, which (according to the authors) include increasing "intensity of whole complex tastes (rich flavor with complex tastes), mouthfulness (spread of taste and flavor throughout the oral cavity), and persistence of taste (lingering flavor)." While it may be possible to develop behavioral assays in rats (or mice) that effectively model kokumi taste perception in humans, the authors have not made any effort to do so. As a result, I do not think that the rat data provide support for the main conclusion of the study--that "ornithine is a kokumi substance and GPRC6A is a novel kokumi receptor."

Why are the authors hypothesizing that the primary impacts of ornithine are on the peripheral taste system? While the CT recordings provide support for peripheral taste enhancement, they do not rule out the possibility of additional central enhancement. Indeed, based on the definition of human kokumi described above, it is likely that the effects of kokumi stimuli in humans are mediated at least in part by the central flavor system.

The authors include (in the supplemental data section) a pilot study that examined the impact of ornithine on variety of subjective measures of flavor perception in humans. The presence of this pilot study within the larger rat study does not really make sense. If the human studies are so important, as the authors state, then why did the authors relegate them to the supplemental data section? Usually one places background and negative findings in this section of a paper. Accordingly, I recommend that the human data be published in a separate article.

Reviewer #2 (Public review):

Summary:

Consensus-independent component analysis and closely related methods have previously been used to reveal components of transcriptomic data which are not captured by principal component or gene-gene coexpression analyses.

Here, the authors asked whether applying consensus-independent component analysis (c-ICA) to published high-grade serous ovarian cancer (HGSOC) microarray-based transcriptomes would reveal subtle transcriptional patterns which are not captured by existing molecular omics classifications of HGSOC.

Statistical associations of these (hitherto masked) transcriptional components with prognostic outcomes in HGSOC would lead to additional insights into underlying mechanisms and, coupled with corroborating evidence from spatial transcriptomics, are proposed for further investigation.

This approach is complementary to existing transcriptomics classifications of HGSOC.

The authors have previously applied the same approach in colorectal carcinoma (for example, Knapen et al. (2024) Commun. Med).

Strengths:

Overall, this study describes a solid data-driven description of c-ICA-derived transcriptional components that the authors identified in HGSOC microarray transcriptomics data, supported by detailed methods and supplementary documentation.

The biological interpretation of transcriptional components is convincing based on (data-driven) permutation analysis and a suite of analyses of association with copy-number, gene sets, and prognostic outcomes.<br /> The resulting annotated transcriptional components have been made available in a searchable online format.

For the highlighted transcriptional component which has been annotated as related to synaptic signalling, the detection of the transcriptional component among 11 published spatial transcriptomics samples from ovarian cancers is compelling and supports the need for further mechanistic follow-up.

Further comments:

This revised version includes a suite of comparisons between the c-ICA-derived components and existing published transcriptomic/genomic-based classifications of ovarian cancers. Newly described components will require experimental validation, as acknowledged by the authors.

Here, the authors primarily interpret the c-ICA transcriptional components as a deconvolution of bulk transcriptomics due to the presence of cells from tumour cells and the tumour microenvironment.<br /> In this revised version, the authors additionally investigate their TC scores in single cells from a published HGSOC single-cell RNAseq dataset, highlighting examples of TC scores within and between cell types.

c-ICA is not explicitly a deconvolution method with respect to cell types: the transcriptional components do not necessarily correspond to distinct cell types, and may reflect differential dysregulation within a cell type. This application of c-ICA for the purpose of data-driven deconvolution of cell populations is distinct from other deconvolution methods which explicitly use a prior cell signature matrix.

Reviewer #2 (Public review):

Summary:

While the phylogenetic position of Acoels (and Xenacoelomorpha) remains still debated, investigations of various representative species are critical to understanding their overall biology.

Hofstenia is an Acoels species that can be maintained in laboratory conditions and for which several critical techniques are available. The current manuscript provides a comprehensive and widely descriptive investigation of the productive system of Hofstenia miamia.

Strengths:

(1) Xenacoelomorpha is a wide group of animals comprising three major clades and several hundred species, yet they are widely understudied. A comprehensive state-of-the-art analysis on the reprodutive system of Hofstenia as representative is thus highly relevant.

(2) The investigations are overall very thorough, well documented, and nicely visualised in an array of figures. In some way, I particularly enjoyed seeing data displayed in a visually appealing quantitative or semi-quantitative fashion.

(3) The data provided is diverse and rich. For instance, the behavioral investigations open up new avenues for further in-depth projects.

Weaknesses:

While the analyses are extensive, they appear in some way a little uni-dimensional. For instance the two markers used were characterized in a recent scRNAseq data-set of the Srivastava lab. One might have expected slightly deeper molecular analyses. Along the same line, particularly the modes of spermatogenesis or oogenesis have not been further analysed, nor the proposed mode of sperm-storage.

Reviewer #2 (Public review):

Summary:

The authors recently uncovered a novel nested gene, Aff3ir, and this work sets out to study its function in endothelial cells further. Based on differences in expression correlating with areas of altered shear stress, they investigate a role for the isoform Aff3ir-ORF2 in endothelial activation and development of atherosclerosis downstream of disturbed shear stress. Using a knockout mouse model and in vivo overexpression experiments, they demonstrate a strong potential for Aff3ir-ORF2 to alleviate atherosclerosis. They find that Aff3ir-ORF2 interacts with the pro-inflammatory transcription factor IRF5 and retains it in the cytoplasm, hence preventing upregulation of inflammation-associated genes. The data expands our knowledge of IRF5 regulation which could be relevant to researchers studying various inflammatory diseases as well as adding to our understand of atherosclerosis development.

Strengths:

The in vivo data is convincing using immunofluorescence staining to assess AFF3ir-ORF2 expression, a knockout mouse model, overexpression and knockdown studies and rescue experiments in combination with two atherosclerotic models to demonstrate that Aff3ir-ORF2 can lessen atherosclerotic plaque formation in ApoE-/- mice.

Weaknesses:

The effect on atherosclerosis is clear and there is sufficient evidence to conclude that this is the result of reduced endothelial cell activation. However, other cell types such as smooth muscle cells or macrophages could be contributing to the effects observed. The mouse model is a global knockout and the shRNA knockdowns (Fig. 5) and overexpression data in Figure 2 are not cell type-specific. Only the overexpression construct in Figure 6 uses an ICAM-2 promoter construct, which drives expression in endothelial cells, though leaky expression of this promoter has been reported in the literature.

The in vitro experiments are solidly executed, but most experiments are performed in mouse embryonic fibroblasts (MEFs) and results extrapolated to endothelial cell responses. However, several key experiments are repeated in HUVEC, thereby making a solid case that Aff3ir-ORF2 can regulate IRF5 in both MEFs and HUVEC. It is important to note that the sequence of AFF3ir-ORF2 is not conserved in humans and lacks an initiation codon, hence the regulatory pathway is not conserved. However, the overexpression studies in HUVEC suggest that mouse AFF3ir-ORF2 can also regulate human IRF5 and hence the mechanism retains relevance for possible human health interventions.

Overall, the paper succeeds in demonstrating a link between Aff3ir-ORF2 and atherosclerosis. The study shows a functional interaction between Aff3ir-ORF2 and IRF5 in embryonic fibroblasts, but makes a solid case that this mechanism is relevant for atherosclerosis development via endothelial cell activation.

Reviewer #2 (Public review):

This is an interesting and important work from Shihabeddin et al, to identify master regulators for rod photoreceptor regenerations in a zebrafish model of Retinitis Pigmentosa. Building on their scRNA-seq data, Shihabeddin et al dissected the progenitor cell types and performed trajectory analyses to predict transcription factors that apparently drive the progenitor proliferation and differentiation into rod photoreceptors. Their analyses predicted e2f1, e2f2, and e2f3 as critical drivers of progenitor proliferation, Prdm1a as a driver of rod photoreceptor differentiation, and SP1 as a driver of rod photoreceptor maturation. Genetic experiments provide clear support for the roles of e2fs in progenitor proliferation. It's also apparent from Figure 8 that prdm1 knockdown appears to cause a decrease in rhodopsin expression. By colocalizing BrdU and Retp1, the authors inferred that the apparent "new rods" (which exhibit mixed BrdU and Retp1 signal) are decreased with prdm1, providing further support. Overall I found the work to be interesting, rigorous, and informative for the community.

I have a few suggestions for the authors to consider:

(1) Perhaps the authors can consider explaining why the Prdm1a knock-down cells would have a higher Retp1 signal per cell in Fig 9B. Is this a representative picture? This appears to contradict Figure 8's conclusion, although I could tell that the number of Retp1+ cells in the ONL appears to be lower.

(2) The authors noted "Surprisingly, the knockdown of prdm1a resulted in a significantly higher number of rhodopsin-positive cells in the INL (p=0.0293)", while it appears in Figure 9B, 9C that the difference is 2 cells vs 0 in a rightly broader field. It seems to be too strong of a statement for this effect.

(3) It appears to this reviewer that the proteomic data didn't reveal much in line with the overall hypothesis or the mechanism, and it's unclear why the authors went for proteomics rather than bulk RNA-seq or ChIP-seq for a transcription factor knock-down experiment. Overall this is a minor point.

Reviewer #2 (Public review):

Summary:

In the manuscript, the authors seek to discover putative gene regulatory interactions underlying the lineage bifurcation process of neural progenitor cells in the embryonic mouse anterior brainstem into GABAergic and glutamatergic neuronal subtypes. The authors analyze single-cell RNA-seq and single-cell ATAC-seq datasets derived from the ventral rhombomere 1 of embryonic mouse brainstems to annotate cell types and make predictions or where TFs bind upstream and downstream of the effector TFs using computational methods. They add data on the genomic distributions of some of the key transcription factors and layer these onto the single-cell data to get a sense of the transcriptional dynamics.

Strengths:

The authors use a well-defined fate decision point from brainstem progenitors that can make two very different kinds of neurons. They already know the key TFs for selecting the neuronal type from genetic studies, so they focus their gene regulatory analysis squarely on the mechanisms that are immediately upstream and downstream of these key factors. The authors use a combination of single-cell and bulk sequencing data, prediction and validation, and computation.

Weaknesses:

The study generates a lot of data about transcription factor binding sites, both predicted and validated, but the data are substantially descriptive. It remains challenging to understand how the integration of all these different TFs works together to switch terminal programs on and off.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors were providing a proof of concept that they can identify and mutate a cholesterol-binding site of a high-interest class B receptor, the GLP-1R, and functionally characterize the impact of this mutation on receptor behavior in the membrane and downstream signaling with the intent that similar methods can be useful to optimize small molecules that as ligands or allosteric modulators of GLP-1R can improve the therapeutic tools targeting this signaling system.

Strengths:

The majority of results on receptor behavior are elucidated in INS-1 cells expressing the wt or mutant GLP-1R, with one experiment translating the findings to primary mouse beta-cells. I think this paper lays a very strong foundation to characterize this mutation and does a good job discussing how complex cholesterol-receptor interactions can be (ie lower cholesterol binding to V229A GLP-1R, yet increased segregation to lipid rafts). Table 1 and Figure 9 are very beneficial to summarize the findings. The lower interaction with cholesterol and lower membrane diffusion in V229A GLP-1R resembles the reduced diffusion of wt GLP-1R with simv-induced cholesterol reductions, by presumably decreasing the cholesterol available to interact with wt GLP-1R. The effects of this mutation are not due to differences in Ex-4:recepotor affinity. I think this paper will be of interest to many physiologists who may not be familiar with many of the techniques used in this paper and the authors largely do a good job explaining the goals of using each method in the results section. While not necessary for this paper, a comparison of islet cholesterol content after this cholesterol diet vs the more typical 60% HFD used in obesity research would be beneficial for GLP-1 physiology research broadly to take these findings into consideration with model choice.

Weaknesses:

There are no obvious weaknesses in this manuscript and overall, I believe the authors achieved their aims and have demonstrated the importance of cholesterol interactions on GLP-1R functioning in beta-cells.

Certainly many follow-up experiments are possible from these initial findings and of primary interest is how this mutation affects insulin homeostasis in vivo under different physiological conditions. One of the biggest pathologies in insulin homeostasis in obesity/t2d is an elevation of baseline insulin release (as modeled in Fig 1E) that renders the fold-change in glucose stimulated insulin levels lower and physiologically less effective. Future work by the authors may determine the effects of the GLP-1R V229A mutation on insulin secretion responses under diet-induced metabolic stress conditions. Furthermore, the authors may additionally investigate if V229A would have the same impact in a different cell type, especially in neurons, with implications in the regulation of satiation, gut motility, and especially nausea, which are of high translational interest.

The comparison is drawn in the discussion between this mutation and ex4-phe1 to have biased agonism towards Gs over beta-arrestin signaling. Ex4-phe1 lowered pica behavior (a proxy for nausea) in the authors previously co-authored paper on ex4-phe1 (PMID 29686402) and drawing a parallel for this mutation or modification of cholesterol binding to potentially mitigate nausea is a novel direction.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

After revision, this paper has no outstanding weaknesses. Points where further investigation is needed have been clearly identified.

Reviewer #2 (Public review):

Summary

The manuscript presents valuable findings, particularly in the crystal structure of the Sld3CBD-Cdc45 interaction and the identification of additional sequences involved in their binding. The modeling of the Sld7-Sld3CBD-CDC45 subcomplex is novel, and the results provide insights into potential conformational changes that occur upon interaction. Although the single-stranded DNA binding data from Sld3 of different species is a minor weakness, the experiments support a model in which the release of Sld3 from the complex may be promoted by its binding to origin single-stranded DNA exposed by the helicase.

Strengths

• The Sld3CBD-Cdc45 structure is a novel contribution, revealing critical residues involved in the interaction.<br /> • The model structures generated from the crystal data are well presented and provide valuable insights into the interaction sequences between Sld3 and Cdc45.<br /> • The experiments testing the requirements for interaction sequences are thorough and conducted well, with clear figures supporting the conclusions.<br /> • The conformational changes observed in Sld3 and Cdc45 upon binding are interesting and enhance our understanding of the interaction.<br /> • The modeling of the Sld7-Sld3CBD-CDC45 subcomplex is a new and valuable addition to the field.<br /> • The proposed model of Sld3 release from the complex through binding to single stranded DNA at the origin is intriguing.

Weaknesses

• The section on the binding of Sld3 complexes to origin single-stranded DNA is somewhat weakened by the use of Sld3 proteins from different species. The comparisons between Sld3-CBD, Sld3CBD-Cdc45, and Sld7-Sld3CBD-Cdc45 involve complexes from different species, limiting the comparisons' value.<br /> • Although the study reveals that Sld3 binds to different residues of Cdc45 than those previously shown to bind Mcm or GINS, the data in the paper do not shed any additional light on how GINS and Sld3 binding to Cdc45 or Mcms. would affect each other. Other previous research has suggested that the binding of GINS and Sld3 to Mcm or Cdc45 may be mutually exclusive. The authors acknowledge that a structural investigation of Sld3, Sld7, Cdc45, and MCM during the stage of GINS recruitment will be a significant goal for future research.

Reviewer #2 (Public review):

Summary:

This work investigates the roles of TANGO2 orthologs in different model systems and suggests bioenergetic dysfunction and oxidative stress (and not heme metabolism) as crucial pathways in TANGO2 deficiency disorders (TDD). Specifically, studies in C. elegans showed that the lack of TANGO2 ortholog activity (i) does not provide a survival benefit upon toxic heme exposure; (ii) results in a series of defects related to energy levels (reduced pharyngeal pumping, lawn avoidance, poor motility, and low brood size); (iii) reduces the fluorescence of the heme analog ZnMP in the intestine. Furthermore, upon oxidative stress, one TANGO2 ortholog, hrg-9, is upregulated compared to control conditions. Additional studies on yeast and zebrafish models failed to replicate prior findings on heme distribution and muscle integrity.

These findings have a clear therapeutic impact, as TDD currently has no cure but only symptom-managing treatments. Identifying the correct pathway to correct the disease is pivotal to finding a cure.

Although compelling, the authors' primary claim is based on indirect evidence that only hints toward it. Unfortunately, I do not see any direct and convincing evidence linking TANGO2 orthologs to bioenergetic and oxidative stress pathways.

Strengths:

(1) The study refutes and extends previous findings, highlighting new aspects of TANGO2's roles in cell physiology.

(2) The use of different model systems to address the main research questions is useful.

(3) The results suggest a broader impact than previously described, somewhat supporting the novelty of the study.

Weaknesses:

(1) The manuscript is written mainly as a criticism of a previously published paper. Although reproducibility in science is an issue that needs to be acknowledged, a manuscript should focus on the new data and the experiments that can better prove and strengthen the new claims.

(2) The current presentation of the logic of the study and its results does not help the authors deliver their message, although they possess great potential.

(3) The study is missing experiments to link hrg-9 and hrg-10 more directly to bioenergetic and oxidative stress pathways.

Reviewer #2 (Public review):

Summary:

The Activity-by-Contact (ABC) model is a relatively widespread model of enhancer-gene regulation. This model leverages CRISPRi data to predict whether a gene is regulated by a given enhancer. To make this possible, this model accounts for the activity of an enhancer and its contact frequency with a target promoter in order to produce an "ABC score". However, while quantitative in its ability to predict enhancer-promoter regulation, this model is mostly phenomenological and does not commit to specific molecular mechanisms.

In this manuscript, the authors formalize the molecular and mathematical assumptions made by the ABC model. Specifically, they demonstrate a basic set of assumptions that can be made to arrive at the ABC model's mathematical structure. The resulting default model (basically, a null model) places particular emphasis on the requirement that gene activation and enhancer-gene communication must be independent and at a steady state. The authors leverage and extend a graph-based formalism they have previously spearheaded to show the generality of their conclusions with respect to different molecular realizations of the process by which enhancers interact with their promoters.

Previously published works have found that specific models of how multiple enhancers communicate with the same gene can result in additive mRNA production rates. Here, the authors demonstrate that steady-state mRNA levels are additive regardless of the specific Markovian model for how any individual enhancer communicates with the gene, as long as the model follows the basic assumptions of their default model.

By coarse-graining, both gene activation and enhancer-gene communication to simple two-state models, the authors then clearly demonstrate that the mathematical structure of the ABC model emerges. This mathematical structure implies that the ABC score summed over all the enhancers regulating a given gene must equal 1. However, experimental measurements show values ranging from 0 to 3. The authors show that, in order to explain these experimental deviations with respect to the theory, at least one of the assumptions of the default model must be broken. They demonstrate that either invoking enhancer cooperativity in mRNA production rates or breaking the assumption that individual enhancers communicate with the gene independently can explain existing experimental data.

Strengths:

By demonstrating that the mathematical structure of the ABC model emerges from a set of basic assumptions including the independence of gene activation and enhancer-gene communication, the authors succeeded in their aim to put the ABC model on a formal and molecular footing. Since some experimental results do not agree with the ABC model, the authors importantly demonstrated which assumptions of the model can be broken to explain such data. The theoretical work in this manuscript is written in a reasonably accessible manner that features how a graph theory-based approach to modeling biochemical networks can result in general statements about biological phenomena.

Weaknesses:

While the authors discuss a number of experimental techniques that can be used to test the validity of their model, a more specific discussion of proposed experiments could have strengthened the impact of the paper by providing explicit opportunities for dialogue with experimentalists.

Reviewer #2 (Public review):

Summary:

This study profiles the genome-wide distribution of DNA methylation using methylation capture sequencing in four stages of male germ cells: Thy1+ (undifferentiated spermatogonia), Kit+ (differentiated spermatogonia), pachytene spermatocytes, and round spermatids. These analyses revealed site-specific loss of DNA methylation in pachytene cells compared with differentiating spermatogonia. Integrated analysis using published datasets indicates that hypomethylated sites correlate with nucleosome retention sites and bivalent histone methylation sites in sperm.

Strengths:

The methyl-seq approach provides a comprehensive profile of DNA methylation in male germ cells. The concept that DNA hypomethylation in meiotic cells precedes histone modification and histone retention in sperm is interesting.

Weaknesses:

(1) In the title, the word "presets" should be changed to "precedes" or "correlates with". Preset means a causal relationship, which is not the case. This needs to be changed throughout the manuscript. For example, in the abstract, "predetermine" needs to be changed to "precede".

(2) The statement that "Based on these results, we propose that meiosis is a process of epigenetic reprogramming that sets up embryonic gene regulation" (lines 94-95) is a speculation that in the opinion of this reviewer should be removed from the text. It is too broad and not supported by the data presented.

(3) Figure 1B: details are missing. How many cells were analyzed/used? How many times was this experiment done [(The number of experiments (n)]? Were the changes statistically significant (Lines 109-111)?

(4) Figure 1A and Figure 1D: These seem to be contradictory. According to Figure 1D, leptotene/zygotene spermatocytes show bright 5mC staining. However, the diagram in 1A shows delayed recovery of DNA methylation. The authors should clarify this. It appears that 5mC was high in Kit+ spermatogonia and leptotene/zygotene spermatocytes, and then decreased in pachytene spermatocytes.

(5) L121-122: Statement: These results suggest that 5mC levels change dynamically during spermatogenesis before and after the transient reduction of DNA methylation in the premeiotic S phase. In order to make this claim about the premeiotic S phase, I suggest performing 5mC staining in premeiotic S phase cells, which can be pulse-labelled with BrdU or cite a reference if available.

Reviewer #2 (Public review):

Summary:

This important study by Turner et al. examines the functional role of a sparse but unique population of neurons in the cortex that express Nitric oxide synthase (Nos1). To do this, they pharmacologically ablate these neurons in the focal region of whisker-related primary somatosensory (S1) cortex using a saponin-substance P conjugate. Using widefield and 2-photon microscopy, as well as field recordings, they examine the impact of this cell-specific lesion on blood flow dynamics and neuronal population activity. Locally within the S1 cortex, they find changes in neural activity patterns, decreased delta band power, and reduced sensory-evoked changes in blood flow (specifically eliminating the sustained blood flow change after stimulation). Surprisingly, given the tiny fraction of cortical neurons removed by the lesion, they also find far-reaching effects on neural activity patterns and blood volume oscillations between the cerebral hemispheres.

Strengths:

This was a technically challenging study and the experiments were executed in an expert manner. The manuscript was well written and I appreciated the cartoon summary diagrams included in each figure. The analysis was rigorous and appropriate. Their discovery that Nos1 neurons can have far-reaching effects on blood flow dynamics and neural activity is quite novel and surprising (to me at least) and should seed many follow-up, mechanistic experiments to explain this phenomenon. The conclusions were justified by the convincing data presented.

Weaknesses:

I did not find any major flaws in the study. I have noted some potential issues with the authors' characterization of the lesion and its extent. The authors may want to re-analyse some of their data to further strengthen their conclusions. Lastly, some methodological information was missing, which should be addressed.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

The authors addressed the remarks on the previous version very well. Addressing the two points below would further increase the quality of the manuscript.

(1) In the previous version the authors mentioned that their results are also consistent with the disposable soma theory: "These results are also consistent with the disposable soma theory that suggests aging as an outcome tradeoff between an organism's investment in reproduction and somatic maintenance and repair."

Although the antagonistic pleiotropy and disposable soma theories describe different mechanisms, both provide frameworks for understanding how genes linked to fertility influence health. The antagonistic pleiotropy theory posits that genes enhancing fertility early in life may have detrimental effects later. In contrast, the disposable soma theory suggests that energy allocation involves a trade-off, where investment in fertility comes at the expense of somatic maintenance, potentially leading to poorer health in later life.

To strengthen the manuscript, a discussion section should be added to clarify the overlap and distinctions between these two evolutionary theories and suggest directions for future research in disentangling their specific mechanisms.

(2) In response to the question why the authors did not include age at menopause in addition to the already included age at first child and age at menarche the following explanation was provided: "Our manuscript focuses on the antagonistic pleiotropy theory, which posits that inherent trade-off in natural selection, where genes beneficial for early survival and reproduction (like menarche and childbirth) may have costly consequences later. So, we only included age at menarche and age at first childbirth as exposures in our research."

It remains, however, unclear why genes beneficial for early survival and reproduction would be reflected only in age at menarche and age at first childbirth, but not in age at menopause. While age at menarche marks the onset of fertility, age at menopause signifies its end. Since evolutionary selection acts directly until reproduction is no longer possible (though indirect evolutionary pressures persist beyond this point), the inclusion of additional fertility-related measures could have strengthened the analysis. A more detailed justification for focusing exclusively on age at menarche and first childbirth would enhance the clarity and rigor of the manuscript.

Reviewer #2 (Public review):

Summary:

The manuscript by Hathaway et al. describes a set of elegant behavioral experiments designed to understand which aspects of cue-reward contingencies drive risky choice behavior. The authors developed several clever variants of the well-established rodent gambling task (also developed by this group) to understand how audiovisual cues alter learning, choice behavior, and risk. Computational and sophisticated statistical approaches were used to provide evidence that: (1) audiovisual cues drive risky choice if they are paired with rewards and decrease risk if only paired with loss, (2) pairing cues with rewards reduces learning from punishment, and (3) differences in risk-taking seem to be present early on in training.

Strengths:

The paper is well-written, the experiments are well-designed, and the results are highly interesting, particularly for understanding how cues can motivate and invigorate normal and abnormal behavior.

Weaknesses:

Additional support and evidence are needed for the claims made by the authors. Some of the statements are inconsistent with the data and/or analyses or are only weakly supportive of the claims.

Reviewer #2 (Public review):

Summary:

This work addresses the question of whether artificial deep neural network models of the brain could be improved by incorporating top-down feedback, inspired by the architecture of the neocortex.

In line with known biological features of cortical top-down feedback, the authors model such feedback connections with both, a typical driving effect and a purely modulatory effect on the activation of units in the network.

To assess the functional impact of these top-down connections, they compare different architectures of feedforward and feedback connections in a model that mimics the ventral visual and auditory pathways in the cortex on an audiovisual integration task.

Notably, one architecture is inspired by human anatomical data, where higher visual and auditory layers possess modulatory top-down connections to all lower-level layers of the same modality, and visual areas provide feedforward input to auditory layers, whereas auditory areas provide modulatory feedback to visual areas.

First, the authors find that this brain-like architecture imparts the models with a light visual bias similar to what is seen in human data, which is the opposite in a reversed architecture, where auditory areas provide a feedforward drive to the visual areas.

Second, they find that, in their model, modulatory feedback should be complemented by a driving component to enable effective audiovisual integration, similar to what is observed in neural data.

Last, they find that the brain-like architecture with modulatory feedback learns a bit faster in some audiovisual switching tasks compared to a feedforward-only model.

Overall, the study shows some possible functional implications when adding feedback connections in a deep artificial neural network that mimics some functional aspects of visual perception in humans.

Strengths:

The study contains innovative ideas, such as incorporating an anatomically inspired architecture into a deep ANN, and comparing its impact on a relevant task to alternative architectures.

Moreover, the simplicity of the model allows it to draw conclusions on how features of the architecture and functional aspects of the top-down feedback affect the performance of the network.

This could be a helpful resource for future studies of the impact of top-down connections in deep artificial neural network models of the neocortex.

Weaknesses:

Overall, the study appears to be a bit premature, as several parts need to be worked out more to support the claims of the paper and to increase its impact.

First, the functional implication of modulatory feedback is not really clear. The "only feedforward" model (is a drive-only model meant?) attains the same performance as the composite model (with modulatory feedback) on virtually all tasks tested, it just takes a bit longer to learn for some tasks, but then is also faster at others. It even reproduces the visual bias on the audiovisual switching task. Therefore, the claims "Altogether, our results demonstrate that the distinction between feedforward and feedback inputs has clear computational implications, and that ANN models of the brain should therefore consider top-down feedback as an important biological feature." and "More broadly, our work supports the conclusion that both the cellular neurophysiology and structure of feed-back inputs have critical functional implications that need to be considered by computational models of brain function" are not sufficiently supported by the results of the study. Moreover, the latter points would require showing that this model describes neural data better, e.g., by comparing representations in the model with and without top-down feedback to recorded neural activity.

Second, the analyses are not supported by supplementary material, hence it is difficult to evaluate parts of the claims. For example, it would be helpful to investigate the impact of the process time after which the output is taken for evaluation of the model. This is especially important because in recurrent and feedback models the convergence should be checked, and if the network does not converge, then it should be discussed why at which point in time the network is evaluated.

Third, the descriptions of the models in the methods are hard to understand, i.e., parameters are not described and equations are explained by referring to multiple other studies. Since the implications of the results heavily rely on the model, a more detailed description of the model seems necessary.

Lastly, the discussion and testable predictions are not very well worked out and need more details. For example, the point "This represents another testable prediction flowing from our study, which could be studied in humans by examining the optical flow (Pines et al., 2023) between auditory and visual regions during an audiovisual task" needs to be made more precise to be useful as a prediction. What did the model predict in terms of "optic flow", how can modulatory from simple driving effect be distinguished, etc.

Reviewer #2 (Public review):

In this manuscript, the authors present an approach to correct GRIN lens aberrations, which primarily cause a decrease in signal-to-noise ratio (SNR), particularly in the lateral regions of the field-of-view (FOV), thereby limiting the usable FOV. The authors propose to mitigate these aberrations by designing and fabricating aspherical corrective lenses using ray trace simulations and two-photon lithography, respectively; the corrective lenses are then mounted on the back aperture of the GRIN lens.

This approach was previously demonstrated by the same lab for GRIN lenses shorter than 4.1 mm (Antonini et al., eLife, 2020). In the current work, the authors extend their method to a new class of GRIN lenses with lengths exceeding 6 mm, enabling access to deeper brain regions as most ventral region of the mouse brain. Specifically, they designed and characterized corrective lenses for GRIN lenses measuring 6.4 mm and 8.8 mm in length. Finally, they applied these corrected long micro-endoscopes to perform high-precision calcium signal recordings in the olfactory cortex.

Compared with alternative approaches using adaptive optics, the main strength of this method is that it does not require hardware or software modifications, nor does it limit the system's temporal resolution. The manuscript is well-written, the data are clearly presented, and the experiments convincingly demonstrate the advantages of the corrective lenses.

The implementation of these long corrected micro-endoscopes, demonstrated here for deep imaging in the mouse olfactory bulb, will also enable deep imaging in larger mammals such as rats or marmosets.

Comments on revisions:

The authors have clearly addressed all my comments.

Reviewer #2 (Public Review):

Summary:

The authors aimed to explore the role of climbing fibers (CFs) in cerebellar learning, with a focus on optokinetic reflex (OKR) adaptation. Their goal was to understand how CF activity influences memory acquisition, memory consolidation, and memory retrieval by optogenetically suppressing CF inputs at various stages of the learning process.

Strengths:

The study addresses a significant question in the cerebellar field by focusing on the specific role of CFs in adaptive learning. The authors use optogenetic tools to manipulate CF activity. This provides a direct method to test the causal relationship between CF activity and learning outcomes.

Weaknesses:

Despite shedding light on the potential role of CFs in cerebellar learning, the study is hampered by significant methodological issues that question the validity of its conclusions. The absence of detailed evidence on the effectiveness of CF suppression and concerns over tissue damage from optogenetic stimulation weakens the argument that CFs are not essential for memory consolidation. These challenges make it difficult to confirm whether the study's objectives were fully met or if the findings conclusively support the authors' claims. The research commendably attempts to unravel the temporal involvement of CFs in learning but also underscores the difficulties in pinpointing specific neural mechanisms that underlie the phases of learning. Addressing these methodological issues, investigating other signals that might instruct consolidation, and understanding CFs' broader impact on various learning behaviors are crucial steps for future studies.

[Editors' note: we have included the original concerns, which the Reviewing Editor agrees with. Methodological concerns remain after revisions.]

Reviewer #2 (Public review):

Summary:

Nagarajan et al. investigate the role of the anterior cingulate cortex (ACC) in vocal development of infant marmoset monkeys using lesions in this brain area. Many previous studies show that ACC plays an important role in volitional and emotion-driven vocal behavior in mammals. The experiments Nagarajan et al. performed strengthen the long-standing hypothesis that ACC influences the development of social-vocal behavior in non-human primates. Furthermore, their anatomical studies support the idea of cortical structures exerting cognitive control over subcortical networks for innate vocalization, and thus, enabling mammals to perform flexible social-vocal communication.

Strengths:

Many invasive behavioral studies in monkeys often use 2-3 animals. The authors used a sufficiently high number of animals for their experiments. This increases the power of their conclusions.

The study also investigates the impact of ACC lesions on downstream areas important for innate vocal production. This adds further evidence to the role of ACC on influencing these subcortical regions during vocal development and vocal behavior in general.

Weaknesses:

The study only provides data up to the 6th week after birth. Given the plasticity of the cortex, it would be interesting to see if these impairments in vocal behavior persist throughout adulthood or if the lesioned marmosets will recover their social-vocal behavior compared to the control animals. The authors give a reasonable explanation for why they did not provide this data.

Even though this study focuses entirely on the development of social vocalizations, providing data about altered social non-vocal behaviors that accompany ACC lesions is missing. This data can provide further insights and generate new hypothesis about the exact role of ACC in social-vocal development. For example, do these marmosets behave differently towards their conspecifics or family members and vice versa, and is this an alternate cause for the observed changes in social-vocal development? Unfortunately, the authors are unable to provide that data. Hopefully, this will be the goal of future studies.

Reviewer #2 (Public review):

Summary:

It is generally believed that higher-order areas in the prefrontal cortex guide selection during working memory and attention through signals that selectively recruiting neuronal populations in sensory areas that encode the relevant feature. In this work, Parto-Dezfouli and colleagues tested how these prefrontal signals influence activity in visual area V4 using a spatial working memory task. They recorded neuronal activity from visual area V4 and found that information about visual features at the behaviorally relevant part of space during the memory period is carried in a spatially selective manner in the timing of spikes relative to a beta oscillation (phase coding) rather than in the average firing rate (rate code). The authors further tested whether there is a causal link between prefrontal input and the phase encoding of visual information during the memory period. They found that indeed inactivation of the frontal eye fields, a prefrontal area known to send spatial signal to V4, decreased beta oscillatory activity in V4 and information about the visual features. The authors went one step further to develop a neural model that replicated the experimental findings and suggested that changes in the average firing rate of individual neurons might be a result of small changes in the exact beta oscillation frequency within V4. These data provide important new insights on the possible mechanisms through which top-down signals can influence activity in hierarchically lower sensory areas and can therefore have a significant impact on the Systems, Cognitive and Computational Neuroscience fields.

Strengths:

This is a well-written paper with a well-thought-out experimental design. The authors used a smart variation of the memory-guided saccade task to assess how information about the visual features of stimuli is encoded during the memory period. By using a grating of various contrasts and orientations as the background the authors ensured that bottom-up visual input would drive responses in visual area V4 in the delay period, something that is not commonly done in experimental settings in the same task. Moreover, one of the major strengths of the study is the use of different approaches including analysis of electrophysiological data using advanced computational methods of analysis, manipulation of activity through inactivation of prefrontal cortex to establish causality of top-down signals on local activity signatures (beta oscillations, spike locking and information carried) as well as computational neuronal modeling. This has helped extend an observation into a possible mechanism well supported by the results.

Weaknesses:

Although the authors provide support for their conclusions from different approaches, a few conceptual gaps make it harder for the reader to appreciate the mechanisms that lead to the observed results and evaluate whether and how these may apply to other cases of top-down control. The fact that the visual features under study were behaviorally irrelevant make it difficult to appreciate the relevance of the finding and its relation to top-down spatial attention mechanisms that involve similar/overlapping circuits. In the same vein, the use of the memory-guided saccade task has certain disadvantages in the context of this study. Although delay activity is interpreted as memory activity by the authors, it is in principle possible that it reflects preparation for the upcoming saccade, spatial attention (particularly since there is a stimulus in the RF) etc. This could potentially change the conclusion and perspective.

Moreover, encoding of the two visual features that are manipulated in the context of the study (contrast and orientation) seems to be affected differently in certain cases, which leaves a reader wondering about the source of this variability.

Finally, although the study provides evidence in favor of a role of FEF in influencing phase coding of visual features in V4 in beta frequencies, important analysis that could have revealed the long-range mechanisms of such an effect including the analysis of intra-FEF and interareal (FEF-V4) neuronal interactions is missing from this paper

Reviewer #2 (Public review):

Summary:

While selective attention is a crucial ability of human beings, previous studies on selective attention are primarily conducted in a strictly controlled context, leaving a notable gap in underlying the complexity and dynamic nature of selective attention in a naturalistic context. This issue is particularly important for classroom learning in individuals with ADHD, as selecting the target and ignoring the distractions are pretty difficult for them but are the pre-requirement of effective learning. The authors of this study have addressed this challenge using a well-motivated study. I believe the findings of this study will be a nice addition to the fields both cognitive neuroscience and educational neuroscience.

Strengths:

To achieve the purpose of setting up a naturalistic context, the authors have based their study on a novel Virtual Reality platform. This is clever as it is usually difficult to perform such a study in the real classroom. Moreover, various techniques such as brain imaging, eye-tracking and physiological measurement are combined to collect multi-level data. They found that, different from the controls, individuals with ADHD had higher neural responses to the irrelevant rather than the target sounds, reduced speech tracking of the teacher. Additionally, the power of alpha-oscillations and frequency of gaze-shifts away from the teacher are found to be associated with the ADHD symptoms. These results provide new insights into the mechanism of selective attention among ADHD populations.

Weaknesses:

It is worth noting that nowadays there has been some studies trying to do so in the real classroom, and thus the authors should acknowledge the difference between the virtual and real classroom context and foresee the potential future changes.<br /> The approach of combining multi-level data owns advantage to obtain reliable results, but also raises significant difficult for the readers to understand the main results.

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

As expected, individuals with ADHD showed anomalous pattern of neural responses, and eye-tracking pattern, compared to the controls. But there are also some similarities between groups such as amount of time paying attention to teachers, etc. In general, their conclusions are supported.

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

The findings are an extension of previous efforts in understanding selective attention in the naturalistic context. The findings of this study are particularly helpful in inspiring teacher's practice and advancing the research of educational neuroscience. This study demonstrates, again, that it is important to understand the complexity of cognitive process in the naturalistic context.

Comments on revisions:

The authors have appropriately responded to my concerns. I do not have other comments. I do hope to see more data and results from the authors in future.

Reviewer #2 (Public review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population level activity in CA1.

Comments on revisions:

I have no further major requests and thank the authors for the additional data and analyses.

Reviewer #2 (Public review):

Summary:

The basal ganglia is often understood within a reinforcement learning (RL) framework, where dopamine neurons convey a reward prediction error which modulates cortico-striatal connections onto spiny projection neurons (SPNS) in the striatum. However, current models of plasticity rules are inconsistent with learning in a reinforcement learning framework.

This paper proposes a new model that describes how distinct learning rules in direct and indirect pathway striatal neurons allows them to implement reinforcement learning models. It proposes that two distinct component of striatal activity affect action selection and learning. They show that the proposed implementation allows learning in simple tasks and is consistent with experimental data from calcium imaging data in direct and indirect SPNs in freely moving mouse.

Strengths:

Despite the success of reward prediction errors at characterizing the responses of dopamine neurons as the temporal difference error within an RL framework, the implementation of RL algorithms in the rest of the basal ganglia has been unclear. A key missing aspect has been the lack of a RL implementation that is consistent with the distinction of direct- and indirect SPNs. This paper proposes a new model that is able to learn successfully in simple RL tasks and explains recent experimental results.

The author shows that their proposed model, unlike previous implementations, this model can perform well in RL tasks. The new model allows them to make experimental predictions. They test some of these predictions and show that the dynamics of dSPNs and iSPNs correspond to model predictions.

More generally, this new model can be used to understand striatal dynamics across direct and indirect SPNs in future experiments.

Weaknesses:

The authors could characterize better the reliability of their experimental predictions and the description of the parameters of some of the simulations

The authors propose some ideas about how the specificity of the striatal efferent inputs but should highlight better that this is a key feature of the model whose anatomical implementation has yet to be resolved.

Comments on revisions:

I thank the authors for their response to public and private reviews and for the clarifications and changes to the manuscript which have strengthened it. I understand the inability to implement some of the proposed additional simulation due to authors having left academia and the request for a version of record.

Reviewer #2 (Public review):

Summary:

In this study, Goyal et al demonstrate that the assembly of proteins with polyphosphate into either condensates or aggregates can reveal information on the initial protein ensemble. They show that, unlike DNA, polyphosphate is able to effectively discriminate against initial protein ensembles with different conformational heterogeneity, structure, and compactness. The authors further show that the protein native ensemble is vital on whether polyphosphate induces phase separation or aggregation, whereas DNA induces a similar outcome regardless of the initial protein ensemble. This work provides a way to improve our mechanistic understanding of how conformational transitions of proteins may regulate or drive LLPS condensate and aggregate assemblies within biological systems.

Strengths:

This is a thoroughly conducted study that provides an alternative route for inducing phase separation that is more informative on the initial protein ensemble involved. This is particularly useful and a complementary means to investigate the role played by protein dynamics and plasticity in phase transitions. The authors use an appropriate set of techniques to investigate unique phase transitions within proteins induced by polyphosphates. An alternative protein system is used to corroborate their findings that the unique assemblies induced by polyphosphates when compared to DNA are not restricted to a single system. The work here is well-documented, easy to interpret, and of relevance for the condensate community.

Weaknesses:

The major weakness of this manuscript is that it is unclear if the information on the initial protein conformational ensemble can be determined solely from the assembly and maturation behavior and the discrimination abilities of polyphosphates. In both systems studied (CytR and FruR), polyphosphate discriminates and results in unique assemblies and maturation behaviors based on the initial protein ensemble. However, it seems the assembly and maturation behavior are not a direct result of the degree of conformational dynamics and plasticity in the initial protein. In the case of CytR, the fully-folded system forms condensates that resolubilize, while the highly disordered state immediately aggregates. Whereas, in the case of FruR, the folded state induces spontaneous aggregation, and the more dynamic, molten globular, system results in short-lived condensates. These results seem to suggest the polyphosphates' ability to discriminate between the initial protein ensemble may not be able to reveal what that initial protein ensemble is unless it is already known.

Reviewer #2 (Public review):

Summary:

The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection, thus suggesting a remodeling of the A13 connectome. Whether this remodelling contributes to pro-locomotor effects of the photostimulation of the A13 region remains unknown as causality was not addressed.

Strengths:

Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients. The study also provides a description of the A13 region connectome pertaining to motor behaviors and how it changes after a dopaminergic lesion. Although there is no causal link between anatomical and behavioral data, it raises interesting questions for further studies.

Weaknesses:

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, some uncertainty remains regarding the phenotype of neurons underlying recovery of akinesia and improvement of bradykinesia.

Figure 4 is improved, but the results from the correlation analyses remain difficult to interpret, as they may reflect changes in various impaired brain regions independently of the A13 region. While the analysis offers a snapshot of correlated changes within the connectome, it does not identify which specific cell or axonal populations are actually increasing or decreasing. Although functional MRI connectome analyses are well-established, anatomical data seem less suitable for this purpose. How can one interpret correlated changes in anatomical inputs or outputs between two distinct regions?

Figure 5 is also improved, but there is room for further enhancement. As currently presented, it is difficult to distinguish the differences between the sham and 6-OHDA groups. The first column could compare afferents, while the second column could compare efferents. Given the small sample size, it would be more appropriate to present individual data rather than the mean and standard deviation.

Appraisal and impact

Although the behavioral experiments are convincing, the low number of animals in the anatomical studies is insufficient to make any relevant statistical conclusions due to extremely low statistical power.

Reviewer #2 (Public review):

I appreciate the author's responses to my original review. This is a comprehensive analysis of CAPE on C. difficile activity. It seems like this compound affects all aspects of C. difficile, which could make it effective during infection but also make it difficult to understand the mechanism. Even considering the authors responses, I think it is critical for the authors to work on the conclusions regarding the infection model. There is some protection from disease by CAPE but some parameters are not substantially changed. For instance, weight loss is not significantly different in the C. difficile only group versus the C. difficile + CAPE group. Histology analysis still shows a substantial amount of pathology in the C. difficile + CAPE group. This should be discussed more thoroughly using precise language.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase the likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

The sample size was relatively small and may have been underpowered to find differences in outcomes (i.e., food choice behaviors). Participants were all women with BN, which limits the generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. Moreover, it is unclear how long the negative affect persisted during the actual task. It is possible that any increases in negative affect would have dissipated by the time participants were engaged in the decision-making task.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of vasopressin and vasopressin receptor 1A gene expression in the mouse brain using a high-resolution in situ hybridization method called RNAscope. Overall, the findings are useful in identifying brain regions expressing Avpr1a transcript. The impact of findings is decreased by incomplete or inadequate data analysis due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference. A comprehensive overview of Avpr1a expression in the mouse brain has the potential to be highly informative and impactful. The current manuscript used RNAscope (a proprietary method of in situ hybridization) to assess the transcript abundance of Avp (arginine vasopressin, a neuropeptide) and its receptor (Avpr1a). The style of graphs, limited use of photomicrographs, and low number of subjects all combine to limit the impact of the dataset. The finding of Avp-expressing cells outside of the hypothalamus and extended amygdala is poorly documented but would be novel. The Avpr1a data suggest expression in numerous brain regions. However, the data presented are difficult to interpret, with every value being an extremely small density value for a large swath of the brain. How many cells are impacted? Are puncta spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield? For a descriptive study, there is minimal statistical inference and relatively little description. The authors make a case for the novel nature of the work but do not seem, at times, to recognize a robust literature developed over the last 50 years. In conclusion, the experimental data are important and informative; however, the low number of subjects, lack of statistical power, limited description of individual brain regions, and poor quality and design of data figures reduce the overall impact.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring the function of vasopressin in the mammalian brain and developing hypotheses about cell - and circuit-level function.

Weaknesses:

(1) The style and type of data presentation, focusing on the density of individual mRNA transcript across a whole brain region, seemed incomplete in so far as the data presentation did not provide a clear visualization of the distribution of Avpr1a-expressing cells or transcript itself. However, knowing which brain regions do express transcript is itself informative.

(2) The manuscript strongly emphases on the possibility of sex differences in Avp and Avpr1a expression. However, the low number of animals used does not provide adequate statistical power to make strong inferences regarding sex differences in the data.

(3) The manuscript's methods are minimal but adequate to understand data acquisition. The description of how quantitative analyses were conducted is inadequate and would be impossible to replicate beyond identifying the program used.

Reviewer #2 (Public review):

Summary:

The authors perform a series of studies to follow up on their previous work, which established a role for dorsal raphe dopamine neurons (DRN) in the regulation of social-isolation-induced rebound in mice. In the present study, Lee et. al, use a combination of modern circuit tools to investigate putatively distinct roles of DRN dopamine transporting containing (DAT) projections to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP). Notably, they reveal that optogenetic stimulation of distinct pathways confers specific behavioral states, with DRNDAT-BLP driving aversion, DRNDAT-BNST regulating non-social exploratory behavior, and DRNDAT-CeA promoting social ability. A combination of electrophysiological studies and in situ hybridization studies reveal heterogenous dopamine and neuropeptide expression and different firing properties, providing further evidence of pathway-specific neural properties. Lastly, the authors combine optogenetics and calcium imaging to resolve social encoding properties in the DRNDAT-CeA pathway, which correlates observed social behavior to socially engaged neural ensembles.

Collectively, these studies provide an interesting way of dissecting out separable features of a complex multifaceted social-emotional state that accompanies social isolation and the perception of 'loneliness.' The main conclusions of the paper provide an important and interesting set of findings that increase our understanding of these distinct DRN projections and their role in a range of social (e.g., prosocial, dominance), non-social, and emotional behaviors. However, as noted below, the examination of these circuits within a homeostatic framework is limited given that a number of the datasets did not include an isolated condition. The DRNDAT-CeA pathway was investigated with respect to social homeostatic states in the present study for some of the datasets.

Strengths:

(1) The authors perform a comprehensive and elegant dissection of the anatomical, behavioral, molecular, and physiological properties of distinct DRN projections relevant to social, non-social, and emotional behavior, to address multifaceted and complex features of social state.

(2) This work builds on prior findings of isolation-induced changes in DRN neurons and provides a working framework for broader circuit elements that can be addressed across the social homeostatic state.

(3) This work characterizes a broader circuit implicated in social isolation and provides a number of downstream targets to explore, setting a nice foundation for future investigation.

(4) The studies account for social rank and anxiety-like behavior in several of the datasets, which are an important consideration to the interpretation of social motivation states, especially in male mice with respect to dominance behavior.

Weaknesses:

(1) The conceptual framework of the study is based on the premise of social isolation and perceived 'loneliness' under the framework of social homeostasis, analogous to hunger. In this framework, social isolation should provoke an aversive state and compensatory social contact behavior. In the authors' prior work, they demonstrate synaptic changes in DRN neurons and social rebound following acute social isolation. Thus, the prediction would be that downstream projections also would show state-dependent changes as a function of social housing conditions (e.g., grouped vs. isolated). In the current paper, a social isolation condition was not included for the majority of the studies conducted (e.g., Figures 1-6 do not include an isolated condition, Figures 7-8 do include an isolated condition). Thus, while Figure 1-6 adds a very interesting and compelling set of data that is of high value to the social behavior field with respect to social and emotional processing and general circuit characterization, these studies do not directly investigate the impacts of dynamic social homeostatic state. The main claim of the paper, including the title (e.g., separable DRN projections mediate facets of loneliness-like state), abstract, intro, and discussion presents the claim of this work under the framework of dynamic social homeostatic states, which should be interpreted with caution, as the majority of the work in the paper did not include a social isolation comparison.

(2) In Figure 1, the authors confirm co-laterals in the BNST and CeA via anatomical tracing studies. The goal of the optogenetic studies is to dissociate the functional/behavioral roles of distinct projections. However, one limitation of optogenetic projection targeting is the possibility of back-propagating action potentials (stimulation of terminals in one region may back-propagate to activate cell bodies, and then afferent projections to other regions), and/or stimulation of fibers of passage. Therefore, one limitation in the dataset for the optogenetic stimulation studies is the possibility of non-specific unintended activation of projections other than those intended (e.g., DRNDAT-CeA). This can be dealt with by administering lidocaine to prevent back-propagating action potentials.

(3) It is unclear from the test, but in the subjects' section of the methods, it appears that only male animals were included in the study, with no mention of female subjects. It should be clear to the reader that this was conducted in males only if that is the case, with consideration or discussion, about female subjects and sex as a biological variable.

(4) Averaged data are generally reported throughout the study in the form of bar graphs, across most figures. Individual data points would increase the transparency of the data.

Reviewer #2 (Public review):

The authors tested tactile acuity on the breast of females using several tasks and reported overall low acuity compared to the back, which is typically considered to have the worst acuity of all body parts. Moreover, there was evidence that acuity is worse the larger the breast; this finding mirrors similar findings for the hand and therefore suggests that the number of tactile sensors is fixed and must be distributed across a larger extent of skin when a body part is larger, thus resulting in comparably lower tactile acuity.

Strengths:

I find this an interesting paper with results that are relevant to the tactile community. The authors apply several tasks allowing them to link the paper with previous results. The methodology and psychophysical analysis are sound.

Weaknesses:

The analysis of localization error direction, with the result that the nipple area may be a landmark for tactile localization, is interesting and aligns the paper with some other recent papers that have suggested that such landmarks should exist. However, there are major issues with methodology and statistics, so that currently the conclusions are not supported.

In the following, line numbers refer to the re-formatted manuscript provided by the authors upon request and are mentioned for them to find the relevant passages faster.

(1) Comments on analysis of tactile acuity:

- I had a hard time understanding some parts of the report. What is meant by "broadly no relationship" in line 137?

- It is suggested that spatial expansion (which is correlated with body part size) is related between medial breast and hand - is this to say that women with large hands have large medial breast size? Nipple size was measured, but hand size was not measured, is this correct?

- It is furthermore unclear how the authors differentiate medial breast and NAC. The sentence in lines 140-141 seems to imply the two terms are considered the same, as a conclusion about NAC is drawn from a result about the medial breast. This requires clarification.

- Finally, given that the authors suspect that overall localization ability (or attention) may be overshadowed by a size effect, would not an analysis be adequate that integrates both, e.g. a regression with multiple predictors?

(2) Comments on analysis of "The nipple is a unit":

- Statistics in this section are not adequately described and may be partly false.

- In the paragraph about testing quadrants of the nipple, it is stated that only 3 of 10 participants barely outperformed chance with a p < 0.01. It is unclear how a significant t-test is an indication of "barely above chance".

- The final part of the paragraph on nipple quadrants (starting line 176) explains that there was a trend (4 of 10 participants) for lower tactile acuity being related to the inability to differentiate quadrants. It seems to me that such a result would not be expected: The stated hypothesis is that all participants have the same number of tactile sensors in their nipple and areola, independent of NAC size. In this section, participants determine the quadrant of a single touch. Theoretically, all participants should be equally able to perform this task, because they all have the same number of receptors in each quadrant of nipple and areola. Thus, the result in Figure 2C is curious.

(3) Comments on analysis of "Absolute localization on the breast is anchored to the nipple"

- Again, there are things that are unclear with the statistics and description of the analysis.

- This section reports an Anova (line 193/194) with a factor "participant". This doesn't appear sensible. Please clarify. The factor distance is also unclear; is this a categorical or a continuous variable? Line 400 implies a 6-level factor, but Anovas and their factors, respectively, are not described in methods (nor are any of the other statistical approaches).

- The analysis on imprecision using mean pairwise error (line 199) is unclear: does pairwise refer to x/y or to touch vs. center of the nipple?

- p8, upper text, what is meant by "relative over-representation of the depth axis"? Does this refer to the breast having depth but the equivalent area on the back not having depth? What are the horizontal planes (probably meant to be singular?) - do you simply mean that depth was ignored for the calculation of errors? This seems to be implied in Figure 3AB.

- Lines 232-241, I cannot follow the conclusions drawn here. First, it is not clear to a reader what the aim of the presented analyses is: what are you looking for when you analyze the vectors? Second, "vector strength" should be briefly explained in the main text. Third, it is not clear how the final conclusion is drawn. If there is a bias of all locations towards the nipple, then a point closer to the nipple cannot exhibit a large bias, because the nipple is close-by. Therefore, one would expect that points close to the nipple exhibit smaller errors, but this would not imply higher acuity - just less space for localizing anything. The higher acuity conclusion is at odds with the remaining results, isn't it: acuity is low on the outer breast, but even lower at the NAC, so why would it be high in between the two?

(4) Comments on the Discussion:

The discussion makes some concrete suggestions for sensors in implants (line 283). It is not clear how the stated numbers were computed. Also, why should 4 sensors nipple quadrants receive individual sensors if the result here was that participants cannot distinguish these quadrants?

Additional comments:

I would find it interesting to know whether participants with small breast measurement delta had breast acuity comparable to the back. Alternatively, it would be interesting to know whether breast and back acuity are comparable in men. Such a result would imply that the torso has uniform acuity overall, but any spatial extension of the breast is unaccounted for. The lowest single participant data points in Figure 1B appear similar, which might support this idea.

Reviewer #2 (Public review):

Summary:

The author presents a transport-based morphometry (TBM) approach for the discovery of non-contrast computed tomography (NCCT) markers of hematoma expansion risk in spontaneous intracerebral hemorrhage (ICH) patients. The findings demonstrate that TBM can quantify hematoma morphological features and outperforms existing clinical scoring systems in predicting 24-hour hematoma expansion. In addition, the inversion model can visualize features, which makes it interpretable. In conclusion, this research has clinical potential for ICH risk stratification, improving the precision of early interventions.

Strengths:

TBM quantifies hematoma morphological changes using the Wasserstein distance, which has a well-defined physical meaning. It identifies features that are difficult to detect through conventional visual inspection (such as peripheral density distribution and density heterogeneity), which provides evidence supporting the "avalanche effect" hypothesis in hematoma expansion pathophysiology.

Weaknesses:

(1) As a methodology-focused study, the description of the methods section somewhat lacks depth and focus, which may make it challenging for readers to fully grasp the overall structure and workflow of the approach. For instance, the manuscript lacks a systematic overview of the entire process, from NCCT image input to the final prediction output. A potential improvement would be to include a workflow figure at the beginning of the manuscript, summarizing the proposed method and subsequent analytical procedures. This would help readers better understand the mechanism of the model.

(2) The description of the comparison algorithms could be more detailed. Since TBM directly utilizes NCCT images as input for prediction, while SVM and K-means are not inherently designed to process raw imaging data, it would be beneficial to clarify which specific features or input data were used for these comparison models. This would better highlight the effectiveness and advantages of the TBM method.

(3) The relatively small training and testing dataset may limit the model's performance and generalizability. Notably, while the study mentions that 1,066 patients from the ERICH dataset met the inclusion criteria, only 170 were randomly selected for the test set. Leveraging the full 1,066 ERICH cases for model training and internal validation might potentially enhance the model's robustness and performance.

(4) Some minor textual issues need to be checked and corrected, such as line 16 in the abstract "Incorporating these traits into a v achieved an AUROC of 0.71 ...".

(5) Some figures need to be reformatted (e.g., the x-axis in Figure 2 a is blocked).

Reviewer #2 (Public review):

Summary:

The paper describes an effort to identify the factors responsible for intron retention and alternate exon splicing in a complex system known to be regulated by the O-GlcNAc cycling system. The CRISPR/Cas9 system was used to identify potential factors. The bioinformatic analysis is sophisticated and compelling. The conclusions are of general interest and advance the field significantly.

Strengths:

- Exhaustive analysis of potential splicing factors in an unbiased screen.<br /> - Extensive genome wide bioinformatic analysis.<br /> - Thoughtful discussion and literature survey

Weaknesses:

- No firm evidence linking SFSWA to an O-GlcNAc specific mechanism.<br /> - Resulting model leaves many unanswered questions.

Comments on revisions:

I think the authors have adequately dealt with the overall reviewer's comments.

Reviewer #2 (Public review):

Summary:

ACVR2A is one of a handful of genes for which significant correlations between associated SNPs and the incidences of preeclampsia have been found in multiple populations. It is one of the TGFB family receptors, and multiple ligands of ACVR2A, as well as its coreceptors and related inhibitors, have been implicated in placental development, trophoblast invasion, and embryo implantation. This useful study builds on this knowledge by showing that ACVR2A knockout in trophoblast-related cell lines reduces trophoblast invasion, which could tie together many of these observations. The implication of cross-talk between the WNT and ACRV2A/SMAD2 pathways is an important contribution to the understanding of the regulation of trophoblast function.

Strengths:

(1) ACVR2A is one of very few genes implicated in preeclampsia in multiple human populations, yet its role in pathogenesis is not very well studied and this study begins to address that hole in our knowledge.

(2) ACVR2A is also indirectly implicated in trophoblast invasion and trophoblast development via its connections to many ligands, inhibitors, and coreceptors, suggesting its potential importance.

(3) The authors have used multiple cell lines to verify their most important observations.

Editors' note: Following the first round of peer review, the original reviewers were not available to review the revised manuscript. As several specific weakness detailed by the reviewers were largely addressed in the revised manuscript, they are not included here.

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.

The authors have effectively addressed some key questions I raised. However, I still have some lingering concerns regarding the mechanisms underlying AOAH's effects.

(1) AOAH is expressed in the intestine, where it may inactivate LPS before it enters systemic circulation. In Fig. 3F, fecal LPS is significantly higher in Aoah⁻/⁻ mice compared to Aoah⁺/⁺ mice, indicating that AOAH in the intestine reduces bioactive LPS levels at the source. This implies that differences in hepatic LPS levels are already influenced by the gut environment, raising doubts about how much Kupffer cells contribute to inactivating LPS in the liver.

(2) The reliance on Kupffer cell depletion with clodronate-liposomes may overestimate the role of Kupffer cells because clodronate does not exclusively target hepatic Kupffer cells. Clodronate liposomes are taken up by macrophages systemically, potentially depleting macrophages in other organs, including the intestine and circulation. This means observed effects could also be due to loss of AOAH activity in non-hepatic macrophages.

Reviewer #2 (Public review):

Summary:

This paper provides a valuable addendum to the findings described in Hamilton et al. 2020 (https://doi.org/10.7554/eLife.56582). In the earlier paper, the authors reconstituted the budding yeast centromeric nucleosome together with parts of the budding yeast kinetochore and tested which elements are required and sufficient for force transmission from microtubules to the nucleosome. Although budding yeast centromeres are defined by specific DNA sequences, this earlier paper did not use centromeric DNA but instead the generic Widom 601 DNA. The reason is that it has so far been impossible to stably reconstitute a budding yeast centromeric nucleosome using centromeric DNA.

In this new study, the authors now report that they were able to replace part of the Widom 601 DNA with centromeric DNA from chromosome 3. This makes the assay more closely resemble the in vivo situation. Interestingly, the presence of the centromeric DNA fragment makes one type of minimal kinetochore assembly, but not the other, withstand stronger forces.

Which kinetochore assembly turned out to be affected was somewhat unexpected, and can currently not be reconciled with structural knowledge of the budding yeast centromere/kinetochore. This highlights that, despite recent advances (e.g. Guan et al., 2021; Dendooven et al., 2023), aspects of budding yeast kinetochore architecture and function remain to be understood and that it will be important to dissect the contributions of the centromeric DNA sequence.

In the future, it will be interesting to pinpoint which interactions contribute to the enhanced force resistance in the presence of centromeric DNA.

Strength:

- The paper demonstrates that centromeric DNA can increase the attachment strength between budding yeast microtubules and centromeric nucleosomes.

Weakness:

- How centromeric DNA exerts this effect remains unclear.

Comments on revisions:

I appreciate the authors' detailed response and their decision to list all the tested in chimeras in Table 3.

All my prior comments have been addressed.

Reviewer #2 (Public review):

The Gram-positive cell wall contains for a large part of TAs, and is essential for most bacteria. However, TA biosynthesis and regulation is highly understudied because of the difficulties in working with these molecules. This study closes some of our important knowledge gaps related to this and provides new and improved methods to study TAs. It also shows an interesting role for TAs in maintaining a 'periplasmic space' in Gram positives. Overall, this is an important piece of work. Future work will need to address the possible causal link between TAs and periplasmic space, for instance using complemented mutants and CEMOVIS. It will be interesting to see what happens with the periplasmic space in other mutants besides TA or also in strains with capsules/without capsules and in PG mutants, or in lafB (essential for production of another glycolipid) mutants. Overall, I support the publication of this revised work as it pioneers some new methods that will definitively move the field forward.

Reviewer #2 (Public review):

Summary

In this paper, the function of trpγ in lipid metabolism was investigated. The authors found that lipid accumulation levels were increased in trpγ mutants and remained high during starvation; the increased TAG levels in trpγ mutants were restored by the expression of active AMPK in DH44 neurons and oral administration of the anti-diabetic drug metformin. Furthermore, oral administration of lipase, TAG and free fatty acids effectively restored survival of trpγ mutants under starvation conditions. These results indicate that TRPv plays an important role in the maintenance of systemic lipid levels through the proper expression of lipase. Furthermore, authors have shown that this function is mediated by DH44R2. This study provides an interesting finding in that the neuropeptide DH44 released from the brain regulates lipid metabolism through a brain-gut axis, acting on the receptor DH44R2 expressed in gut cells.

Strengths

Using Drosophila genetics, careful analysis of which cells express trpγ regulates lipid metabolism is performed in this study. The study supports its conclusions from various angles, including not only TAG levels, but also fat droplet staining and survival rate under starved conditions, and oral administration of substances involved in lipid metabolism.

Weaknesses

The function of lipases, as well as identification of cell types, in the DH44R2-expressing cells in the gut can be investigated.

Reviewer #2 (Public review):

The authors developed an algorithm that allows to deconvolute plasmid sequences from a mixture of plasmids that have been sequenced by nanopore long read technology. As library preparations and barcoding of individual samples increases sequencing costs, the algorithm bypasses this need and thus decreases time on sample prep and sequencing costs. In a first step, the tool assesses which of the plasmid constructions can be mixed in a single library preparation by calculating a distance matrix between the reference plasmid and the constructions producing sequence clusters. The user is given groups of plasmids, from different clusters, to be pooled together for sequencing. After sequencing, the algorithm deconvolutes the reads by classifying them based on alignments to the reference sequence. A Bayesian analysis approach is used to obtain a consensus sequence and quality scores.

Strengths

The authors exploit one of the main advantages of long read sequencing that is to accurately resolve regions of high complexity, as regularly found in plasmids, and developed a tool that can validate plasmid constructions by reducing sequencing costs. Multiple plasmids (up to six) can be analyzed simultaneously in a single library without the need of sample barcoding, also reducing sample preparation time. Although inserts must be different, just 2 bases difference would be enough for correct assignation. Maximizes cost-efficiency for projects that require large amounts of plasmid constructions and high-throughput validation. The algorithm also allows for linear DNA analysis offering extra flexibility.

Reviewer #3 (Public review):

Summary:

The authors sought to understand the molecular mechanisms that cells use to survive cold temperatures by studying gene expression regulation in response to cold in C. elegans. They determined whether gene expression changes during cold adaptation occur primarily at the transcriptional level and identified specific pathways, such as the unfolded protein response pathway, that are activated to possibly promote survival under cold conditions.

Strengths:

Effective use of bulk RNA sequencing (RNA-seq) to measure transcript abundance and ribosome profiling (ribo-seq) to assess translation rates, providing a comprehensive view of gene expression regulation during cold adaptation. This combined approach allows for correlation between mRNA levels and their translation, thereby offering evidence for the authors' conclusion that transcriptional regulation is the primary mechanism of cold-specific gene expression changes.

Weaknesses:

Many aspects of the weakness have been addressed by the revision. Still, the weak cold sensitivity phenotype observed in ire-1 mutants suggests the ER-UPR pathway's role is likely minor, modulatory or there is an unknown compensatory mechanism responsible for surviving cold.

Reviewer #2 (Public Review):

In this manuscript, Hsu et al. used scRNA-seq to profile germ cells isolated from zebrafish ovaries. They identified the transcriptional profile of germ cells representing the early stages of oogenesis, from germline stem cells to newly formed follicle stage oocytes. They identified foxl2l as a gene expressed in probable oocyte progenitor cells, one of the least understood germ cell stages in the ovary. To understand to role of Foxl2l in oogenesis, they produced loss-of-function mutations in foxl2l using CRISPR/Cas9. They found that all foxl2l mutants are males as adults, suggesting that Foxl2l is required for oogenesis. To gain more insights, they performed scRNA-seq on cells isolated from 28 dpf foxl2l mutant ovaries and found that in the absence of foxl2l, germ cells appear to arrest as early progenitors. These results argue that Foxl2l, like its medaka homolog Foxl3, is necessary for promoting oocyte vs. spermatocyte differentiation during the oocyte progenitor stage.

Reviewer #2 (Public review):

Summary:

In Cholesterol Taste Avoidance in Drosophila melanogaster, Pradhan et al. used behavioral and electrophysiological assays to demonstrate that flies can: (1) detect cholesterol through a subset of bitter-sensing gustatory receptor neurons (GRNs) and (2) avoid consuming food with high cholesterol levels. Mechanistically, they identified five members of the IR family as necessary for cholesterol detection in GRNs and for the corresponding avoidance behavior. Ectopic expression experiments further suggested that Ir7g + Ir56d or Ir51b + Ir56d may function as tuning receptors for cholesterol detection, together with the Ir25a and Ir76b co-receptors.

Strengths:

The experimental design of this study was logical and straightforward. Leveraging their expertise in the Drosophila taste system, the research team identified the molecular and cellular basis of a previously unrecognized taste category, expanding our understanding of gustation. A key strength of the study was its combination of electrophysiological recordings with behavioral genetic experiments.

Weaknesses:

My primary concern with this study is the lack of a systematic survey of the IRs of interest in the labellum GRNs. Consequently, there is no direct evidence linking the expression of putative cholesterol IRs to the B GRNs in the S6 and S7 sensilla.

Specifically, the authors need to demonstrate that the IR expression pattern explains cholesterol sensitivity in the B GRNs of S6 and S7 sensilla, but not in other sensilla. Instead of providing direct IR expression data for all candidate IRs (as shown for Ir56d in Figure 2-figure supplement 1F), the authors rely on citations from several studies (Lee, Poudel et al. 2018; Dhakal, Sang et al. 2021; Pradhan, Shrestha et al. 2024) to support their claim that Ir7g, Ir25a, Ir51b, and Ir76b are expressed in B GRNs (Lines 192-194). However, none of these studies provide GAL4 expression or in situ hybridization data to substantiate this claim.

Without a comprehensive IR expression profile for GRNs across all taste sensilla, it is difficult to interpret the ectopic expression results observed in the B GRN of the I9 sensillum or the A GRN of the L-sensillum (Figure 4). It remains equally plausible that other tuning IRs-beyond the co-receptor Ir25a and Ir76b-could interact with the ectopically expressed IRs to confer cholesterol sensitivity, rather than the proposed Ir7g + Ir56d or Ir51b + Ir56d combinations.

Reviewer #2 (Public review):

This study examined the role of CRF neurons in the BNST in both phasic and sustained fear in males and females. The authors first established a differential fear paradigm whereby shocks were consistently paired with tones (Full) or only paired with tones 50% of the time (Part), or controls who were exposed to only tones with no shocks. Recall tests established that both Full and Part conditioned male and female mice froze to the tones, with no difference between the paradigms. Additional studies using the NSF and startle test, established that neither fear paradigm produced behavioral changes in the NSF test, suggesting that these fear paradigms do not result in an increase in anxiety-like behavior. Part fear conditioning, but not Full, did enhance startle responses in males but not females, suggesting that this fear paradigm did produce sustained increases in hypervigilance in males exclusively. Photometry studies found that while undifferentiated BNST neurons all responded to shock itself, only Full conditioning in males lead to a progressive enhancement of the magnitude of this response. BNST neurons in males, but not females, were also responsive to tone onset in both fear paradigms, but only in Full fear did the magnitude of this response increase across training. Knockdown of CRF from the BNST had no effect on fear learning in males or females, nor any effect in males on fear recall in either paradigm, but in females enhanced both baseline and tone-induced freezing only in Part fear group. When looking at anxiety following fear training, it was found in males that CRF knockdown modulated anxiety in Part fear trained animals and amplified startle in Full trained males but had no effect in either test in females. Using 1P imaging, it was found that CRF neurons in the BNST generally decline in activity across both conditioning and recall trials, with some subtle sex differences emerging in the Part fear trained animals in that in females BNST CRF neurons were inhibited after both shock and omission trials but in males this only occurred after shock and not omission trials. In recall trials, CRF BNST neuron activity remained higher in Part conditioned mice relative to Full conditioned mice.

Overall, this is a very detailed and complex study that incorporates both differing fear training paradigms and males and females, as well as a suite of both state-of-the-art imaging techniques and gene knockdown approaches to isolate the role and contributions of CRF neurons in the BNST to these behavioral phenomena. The strengths of this study come from the thorough approach that the authors have taken, which in turn helped to elucidate nuanced and sex specific roles of these neurons in the BNST to differing aspects of phasic and sustained fear. More so, the methods employed provide a strong degree of cellular resolution for CRF neurons in the BNST. In general, the conclusions appropriately follow the data, although the authors do tend to minimize some of the inconsistencies across studies, although this has now been addressed to some degree. The discussion has also been improved to now address some of the inconsistencies in the data head on. Discussion of a few other points is below:

- Given the focus on CRF neurons in the BNST, it was unclear why the photometry studies were performed in undifferentiated BNST neurons as opposed to CRF neurons specifically, although the authors have now explained this in better depth making this clearer to the reader.

- The CRF KD studies are interesting, but it remains speculative as to whether these effects are mediated locally in the BNST or due to CRF signaling at downstream targets. As the literature on local pharmacological manipulation of CRF signaling within the BNST seems to be largely performed in males, the addition of pharmacological studies here would benefit this to help to resolve if these changes are indeed mediated by local impairments in CRF release within the BNST or not. While it is not essential to add these experiments, the authors have addressed this point in the discussion and highlighted studies like this as necessary in future work.

- The authors have addressed the difference between arousal and anxiety by expanding the discussion to include more focus on the behavioral measures. The CRF KD data are still somewhat confusing but better contextualized now. Overall, the manuscript has been improved by the revisions and edits the authors have made.

Reviewer #2 (Public review):

Strengths:

The authors have done a nice job providing additional data in response to reviewer feedback. I appreciate that accuracy plots are now included, as well as a separate analysis where differences in parameter estimates are performed for participants whose accuracy data were above chance levels. I also appreciate the new figure with the sphere ROIs for each participant, as they help us appreciate anatomical variability in the peak response separately for each task.

I have four concerns related to the weaknesses of the study:

(1) Although the results still hold when removing participants whose accuracy was 50% or less, a major limitation of this study is that participants made a button press response only to the last trial in a block. This is problematic because a participant could get all trials in a block correct except for the last one, or a participant could get all trials in a block wrong, and performance would be considered equivalent-as a consequence, it is not possible for one to know if participants who are at chance are performing differently from participants who are not at chance, and it is not possible to control for variance in reaction time (a concern also raised by reviewer 3).

(2) My second concern relates to the way in which the data are interpreted based on thresholding. There is above-threshold activation in the left SMG for all tasks except the fluid cognition task. The z-scores associated with significant voxels in Figure 3 are very strong (minimum z is 6). If one were to relax the threshold of the group level maps to, e.g., p < .001, uncorrected, FDR q < .05, or FWER of .10, there will be overlapping voxels outside the SMG. The discussion of the left SMG in the manuscript is prominent and narrowly construed-the left SMG is discussed as if it were 'the' region: "This confirms that the technical-reasoning network depends upon the recruitment of the left area PF, even if additional cognitive processes involving other peripheral brain areas can be engaged depending on the task" (pp. 9). My intuition is there will be numerous other areas of overlap when using a threshold that is still highly significant (e.g., z = 3 or 4). So, for proponents of the technical reasoning hypothesis, is there a counterfactual or alternative brain area/network/system not in the left SMG?

(3) I like the new Figure 6 because it shows variability in the location of the peak coordinate at the level of single participants. And, indeed, there's considerable variability that is typical when localizing ROIs in single participants. My concern is the level at which hypothesis testing is performed. An independent SMG ROI is used to extract parameter estimates and correlate responses between tasks to show a pattern of correlation that comports with a technical reasoning model of left SMG function. This is a fine approach but it does not rule out the so-called 'same region different function' interpretation because it relies on correlation-one cannot reverse infer that the left SMG is carrying out the same function across different tasks because the response in that area is more strongly correlated between certain tasks. This finding points to that possibility and makes interesting predictions for future studies to pursue, but it cannot tell us whether common functions in the left SMG are involved in each task. E.g., one interesting prediction for future studies is to test if patients with lesions to this site are disproportionately more inaccurate in the experimental condition of the mechanical problem solving task, the psychotechnical task, the mentalizing task, but not the fluid cognition task.

(4) I appreciated the approach to testing the adjacency interpretation by showing the sphere and peak Y coordinate across the tasks. It is interesting that across the groups, there is no difference in the peak Y coordinate of the psychotechnical task and both conditions of the mentalizing task, whereas the peak Y coordinate in the fluid intelligence task is more anterior in the post-central gyrus across participants (why is that?). But why restrict the analysis to just the Y coordinate? A rigorous way to test the adjacency hypothesis is to compute Euclidean distance among X, Y, and Z coordinates between any two tasks collected in the same participant. One can then test if the Euclidean distance between, e.g., the psychotechnical task and one condition of the mentalizing task is smaller than the Euclidean distance between the psychotechnical task and the fluid cognition task. Similarly, one can test whether Euclidean distance between the INT and PHY conditions of the mentalizing task is smaller than the Euclidean distance between the INT and psychotechnical task or PHY and psychotechnical task. There is no justification to restrict this analysis to the anterior-posterior dimension only.

Reviewer #2 (Public review):

Summary:

This manuscript provides experimental evidence on circadian behavioural cycles in Antarctic krill. The krill were obtained directly from krill fishing vessels and the experiments were carried out on board using an advanced incubation device capable of recording activity levels over a number of days. A number of different experiments were carried out where krill were first exposed to simulated light:dark (L:D) regimes for some days followed by continuous darkness (DD). These were carried out on krill collected during late autumn and late summer. A further set of experiments was performed on krill across three different seasons (summer, autumn, winter), where incubations were all DD conditions. Activity was measured as the frequency by which an infrared beam close to the top of the incubation tube was broken over unit time. Results showed that patterns of increased and decreased activity that appeared synchronised to the LD cycle persisted during the DD period. This was interpreted as evidence of the operation of an internal (endogenous) clock. The amplitude of the behavioural cycles decreased with time in DD, which further suggests that this clock is relatively weak. The authors argued that the existence of a weak endogenous clock is an adaptation to life at high latitudes since allowing the clock to be modulated by external (exogenous) factors is an advantage when there is a high degree of seasonality. This hypothesis is further supported by seasonal DD experiments which showed that the periodicity of high and low activity levels differed between seasons.

Strengths:

Although there has been a lot of field observations of various circadian type behaviour in Antarctic krill, relatively few experimental studies have been published considering this behaviour in terms of circadian patterns of activity. Krill are not a model organism and obtaining them and incubating them in suitable conditions are both difficult undertakings. Furthermore, there is a need to consider what their natural circadian rhythms are without the overinfluence of laboratory-induced artefacts. For this reason alone, the setup of the present study is ideal to consider this aspect of krill biology. Furthermore, the equipment developed for measuring levels of activity is well-designed and likely to minimise artefacts.

Reviewer #2 (Public review):

Summary:

Kaya et al uncover an intriguing relationship between hippocampal sharp wave-ripple production and peripheral hormone exposure, food intake, and lateral hypothalamic function. These findings significantly expand our understanding of hippocampal function beyond mnemonic processes and point a direction for promising future research.

Strengths:

Some of the relationships observed in this paper are highly significant. In particular, the inverse relationship between GLP1/Leptin and Insulin/Ghrelin are particularly compelling as this aligns well with opposing hormone functions on satiety.

Reviewer #2 (Public review):

Summary:

While bacteria have the ability to induce genes in response to specific stresses, they also use the General Stress Response (GSR) to deal with growth conditions that presumably include a larger range of stresses (for instance, stationary phase growth). The activation of GSR-specific sigma factors is frequently at the heart of the induction of a GSR. Given the range of stresses that can lead to GSR induction, the regulatory inputs are frequently complex. In B. subtilis, the stressosome, a multi-protein complex, contains a set of proteins that, upon appropriate stresses, initiate partner switching cascades that free the sigma B sigma factor from an anti-sigma. The focus here is on the mode of activation of RsbU, a serine/threonine phosphatase of the PPM family, leading to sigB activation. RbsT, a component of the degradosome interacts with RsbU upon stress, activating the phosphatase activity. Once active, RsbU dephosphorylates its target (RsbV, an anti-antisigma), which in turn binds the anti-sigma. The conclusion is that flexible linker domains upstream of the phosphatase domain are the target for activation, resulting in a crossed-linker dimeric structure. The authors then use the information on RsbU to suggest that parallel approaches may be used to activate PPM phosphatases for the GSR response in other bacteria.

Strengths and Weaknesses:

(1) A strength of the work is the combination of modeling, genetics and biochemical approaches to support the idea that the flexibility of the linker of the RsbU phosphatase is critical to signalling and that this changes as a result of interactions of the signaling protein RsbT.

(2) The impact of the work, beyond better understanding of this particular signalling system, lies in the suggested parallels with other GSR system regulators in a range of bacteria. The work here provides fairly clear indications of what mutational changes would be most likely to test the model.

(3) Assuming that these predictions are shown to be correct in future work, that will leave as an intriguing question why this particular geometry has been conserved in GSR - whether they emerge from a common ancestor (found where?) and/or there is some characteristic (flexibility of modulating the response?) that is particularly important for GSR signal input. Coupled with this will be further understanding of how the linker and/or interacting proteins change in different systems.

Reviewer #2 (Public review):

Summary:

Lim et al. have developed a self-amplifying RNA (saRNA) design that incorporates immunomodulatory viral proteins, and show that the novel design results in enhanced protein expression in vitro in mouse primary fibroblast-like synoviocytes. They test constructs including saRNA with the vaccinia virus E3 protein and another with E3, Toscana virus NS protein and Theiler's virus L protein (E3 + NS + L), and another with srIκBα-Smad7-SOCS1. They have also tested whether ML336, an antiviral, enables control of transgene expression.

Strengths:

The experiments are generally well-designed and offer mechanistic insight into the RNA-sensing pathways that confer enhanced saRNA expression. The experiments are carried out over a long timescale, which shows the enhance effect of the saRNA E3 design compared to the control. Furthermore, the inhibitors are shown to maintain the cell number, and reduce basal activation factor-⍺ levels.

Weaknesses:

One limitation of this manuscript is that the RNA is not well characterized; some of the constructs are quite long and the RNA integrity has not been analyzed. Furthermore, for constructs with multiple proteins, it's imperative to confirm the expression of each protein to confirm that any therapeutic effect is from the effector protein (e.g. E3, NS, L). The ML336 was only tested at one concentration; it is standard in the field to do a dose-response curve. These experiments were all done in vitro in mouse cells, thus limiting the conclusion we can make about mechanisms in a human system.

Reviewer #2 (Public review):

This manuscript presents a compelling application of NAD(P)H fluorescence lifetime imaging (FLIM) to study metabolic activity in the Drosophila brain. The authors reveal regional differences in oxidative and glycolytic metabolism, with a particular focus on the mushroom body, a key structure involved in associative learning and memory. In particular, they identify metabolic shifts in α/β Kenyon cells following classical conditioning, consistent with their established role in energy-demanding middle- and long-term memories.

These results highlight the potential of label-free FLIM for in-vivo neural circuit studies, providing a powerful complement to genetically encoded sensors. This study is well-conducted and employs rigorous analysis, including careful curve fitting and well-designed controls, to ensure the robustness of its findings. It should serve as a valuable technical reference for researchers interested in using FLIM to study neural metabolism in vivo. Overall, this work represents an important step in the application of FLIM to study the interactions between metabolic processes, neural activity, and cognitive function.

Reviewer #2 (Public review):

Summary:

The revised paper by Kim et al. reports two disease mutations in proBMP4, S91C and E93G, disrupt the FAM20C phosphorylation site at Ser91, blocking the activation of proBMP4 homodimers, while still allowing BMP4/7 heterodimers to function. Analysis of DMZ explants from Xenopus embryos expressing the proBMP4 S91C or E93G mutants showed reduced expression of pSmad1 and tbxt1. The expert amphibian tissue transplant studies were expanded to in vivo studies in Bmp4S91C/+ and Bmp4E93G/+ mice, highlighting the impact of these mutations on embryonic development, particularly in female mice, consistent with patient studies. Additionally, studies in mouse embryonic fibroblasts (MEFs) demonstrated that the mutations did not affect proBMP4 glycosylation or ER-to-Golgi transport but appeared to inhibit the furin-dependent cleavage of proBMP4 to BMP4. Based on these findings and AI modeling using AlphaFold of proBMP4, the authors speculate that pSer91 influences access of furin to its cleavage site at Arg289AlaLysArg292 in a new "Ideas and Speculation" section. Overall, the authors addressed the reviewers' comments, improving the presentation.

Strengths:

The strengths of this work continue to lie in the elegant Xenopus and mouse studies that elucidate the impact of the S91C and E93G disease mutations on BMP signaling and embryonic development. Including an "Ideas and Speculation" subsection for mechanistic ideas reduces some shortcomings regarding the analysis of the underlying mechanisms.

Weaknesses:

(Minor) In Figure S1 and lines 165-174 and 179-180, the authors should consider that, unlike the wild-type protein (Ser), which can be reversibly phosphorylated or dephosphorylated, phosphomimic mutations are locked into mimicking either the phosphorylated state (Asp) or the non-phosphorylated state (Ala). Consequently, if the S91D mutant exhibits lower activity than WT, it could imply that S91D interferes with other regulatory constraints, as the authors suggest. However, it may also be inhibiting activation. Therefore, caution is warranted when comparing S91D with S91C to conclude that Ser91 phosphorylation increases BMP4 activity. While additional experiments are not necessary, further consideration is essential.

In Figure 4, panels A, E, and I, the proBMP bands in the mouse embryonic lysates and MEFs expressing the mutations show a clear size shift. Are these shifts a cause or a consequence of the lack of cleavage? Regardless, the size shifts should be explicitly noted.

(Minor) In line 314, the authors should consider modifying the wording to: "is required for modulating proprotein convertase..."

(Minor) In lines 394-399, the authors cleverly speculate that pS91 interacts with Arg289-the essential P4 arginine for furin processing. If so, this interaction could hinder the cleavage of proBMP4, as indicated by the results in Figure S1. The discussion would benefit from considering that, contrary to their favored model, dephosphorylation at Ser91 might actually facilitate cleavage.

Reviewer #2 (Public review):

In this revision the authors have made a solid effort to address each of the points raised by all three reviewers. Due to the fact that animals in this study were freely moving, and there has not been any high-speed video recordings to measure whisker movements or other possible stimulus-induced motor effects it is still not possible to rule out motor effects completely. However, the fact that the multisensory enhancements are stimulus specific, much stronger in the multisensory case than the visual only condition, and short in latency it does seem the most parsimonious explanation is likely that these responses are visual in nature.

The delayed auditory stimulus offers some explanation for the very small latency difference between audio and visual stimulus elements. Studies using LED flashes in rat V2 report latencies around ~50 ms (e.g. 2017 paper from Brian Allman's group). The response latencies for visual stimuli in this manuscript are of this order of magnitude, albeit still shorter than that (which presumably means they don't originate from V2).

There are still parts of the manuscript that are inappropriately causal - e.g. line 283 "this suggests that strong multisensory integration is critical for behavior" - it could just as well be the case that high attention / motivation / arousal leads to both strong integration and good behavior.

for - program event selection - 2025 - April 2 - 10:30am-12pm GMT - Skoll World Forum - From Inner Work to Global Impact - Stop Reset Go Deep Humanity / cosmolocal - LCE - relevant to - event time conflict - with Building Citizen-Led Movements - solution - watch one live and the other recorded

meeting notes - see below

ANNIKA: - inner work helps us stay sane dealing with the chaos in our work - healing is not fixing - hope is a muscle, go to the "hope gym" - not just personal but collective

EDWIN: - inner WORK - constant, continuous work - how do you scale these things? Is it wrong term to use? Mechanistic? - how do we move to global impact? We don't know yet

LOUISE - inner work saved my - orientate inside away from trauma architecture - colonized and colonizer energies - they longed to be in union - be with all parts of myself - allow alchemy on the outside to the inside - liberate myself from my trauma structures and unfold myself - we cannot be a restorer unless we do that inner work - systeming - verbalizing / articulating it - we are all actors in creating the system - question - where am i systeming from? - answer - I am an interbeing - Am i systeming from the interbeing space or the trauma architecture space? - Where am I seeding from? What energy do I put into my work? - system is not concrete and fixed but fluid - fielding - bringing different human fields together - I can work with hatred and rage on the inside and transmute it so that I don't add to it on the outside

JOHN: - stuck systems and lens of trauma can help us get unstock - 70% of people have experienced trauma - trauma is part of the human experience - people make up systems - so traumatized people makes traumatized systems - fight, flight and freeze happens at both levels - at system level, its fractally similiar - disembodied from wisdom - in state of survival and fear - fixing things - until we deal with the trauma in the people, we will continue to have traumatized systems - More work won't help if it's coming from traumatized people

EDWIN - incremental change - something holding us back - built upon these traumas - Economic metrics are out of touch with how the trauma affects systems - Journey - awareness first, then understanding and inner transformation and finally change - Discussion with funders - most are still stuck in old paradigm of metrics, audits, etc - this comes with trauma because we have no trust on who is on the other side - a big part of the system is built on mistrust, creates more gaps between us - need to become anti-fragile

ANNIKA - Funders have lack of trust because inner work hasn't been done on both sides - As a funder, we really try to create a space of trust - Think of the language we use to be inclusive - How do we make inner work a part of the operating system of how we work? - We looked at 500 mental health organizations over the years - It's so urgent now that we align our work

EDWIN - We have a lot of half-formed thoughts - It's very complex and nobody has cracked it - We have a phrase at Axum that we move at the speed of trust - To do something different, they need to trust you - When I think of the discussions I've had with heads of states and CEOs, these meaningful inner ideas are not often brought up

LAURA - When there's no trust, even if there is no danger, the trauma is still brought up - We need to shift our lens on trauma and become aware of when trauma emerges - quote - inner condition of the intervener determines the success of intervention - Bill O'Brien

LOUISE - I work a lot with nervous system and body system - We need small changes in our nervous system - If I try to do something big, I can re-traumatize myself - We also have a collective nervous system - Restore love to all parts of your system first - Make friends with trees to seed actions from union

JOHN - Become aware of my own trauma triggers - When we see an outsized reaction, we can guess that person is undergoing personal trauma - A settled body settles bodies - If we are calm, it helps calm others

LAURA - Feel where we don't feel grounded, where we shame ourselves, feel compassion there

QUESTIONS - See below

• mushrooms and ayuahuasca - is it helpful?

• A lot of women forget the feminine energy to climb the ladder and get sick?

• backlash - feels like white men were being pushed to do work they weren't ready to do so now reclaiming their comfortable traumatized space

• how early do we start to teach this knowledge?

• How do organizations hold space for the enormous trauma that the US govt is manufacturing. We need to build this practice into organizations to help deal with the onslaight

• Youth are so hungry for being in the presence of others who are wise, compassionate. We can't move faster than the speed of trust but it needs to become accessible.

ANSWERS - See below

LOUISE - Organizations have a huge role to play at this time - We want to reconfigure and transform the trauma - Deep forming teams in organizations to help transform - Trauma fields want to come through human nervous systems to transform - We are both feminine and masculine and the masculine wounding is very important and needs to find the feminine - We cannot go away by ourselves to heal from patriarchy, colonialism energies

ANNIKA - In terms of how we fund, can we fund differently? We need to fund these spaces

EDWIN - I sit on board of Wellbeing project - changemakers go through burnout - how do we prevent this and create a container that can sustain them? - Weve brought 20,000 people in summits who have affected 3 million people. Please come to the Hurts summit in Czec and Wellbeing project - When pendulum swings back from individual space, we should be like a spiral

JOHN - In systems change spaces, trauma is seldom spoken of. - Systems work will not work if we ignore trauma - This is critical

LOUISE - Arundhati Roy - Another world is not only possible but is on its way. On a quiet day, I can hear it breathing.

Reviewer #2 (Public review):

This work set out to identify all extracellular matrix proteins and associated factors present within the starlet sea anemone Nematostella vectensis at different life stages. Combining existing genomic and transcriptomic datasets, alongside new mass spectometry data, the authors provide a comprehensive description of the Nematostella matrisome. In addition, immunohistochemistry and electron microscopy were used to image whole mount and de-cellularized mesoglea from all life stages. This served to validate the de-cellularization methods used for proteomic analyses, but also resulted in a very nice description of mesoglea structure at different life stages. A previously published developmental cell type atlas was used to identify the cell type specificity of the matrisome, indicating that the core matrisome is predominantly expressed in the gastrodermis, as well as cnidocytes. The analyses performed were rigorous and the results were clear, supporting the conclusions made by the authors.

Reviewer #2 (Public review):

Summary:

This manuscript presents a valuable single-cell RNA-seq study on Trypanosoma cruzi, an important human parasite. It investigates the expression heterogeneity of surface proteins, particularly those from the trans-sialidase-like (TcS) superfamily, within amastigote and trypomastigote populations. The findings suggest a previously underappreciated level of diversity in TcS expression, which could have implications for understanding parasite-host interactions and immune evasion strategies. The use of single-cell approaches to delve into population heterogeneity is strong. However, the study does have some limitations that need to be addressed.

The focus on single-cell transcriptional heterogeneity in surface proteins, especially the TcS family, in T. cruzi is novel. Given the important role of these proteins in parasite biology and host interaction, the findings have potential significance.

Strengths:

The key finding of heterogeneous TcS expression in trypomastigotes is well-supported. The analysis comparing multigene families, single-copy genes, and ribosomal proteins highlights the unusual nature of the variation in surface protein-coding genes.

Weaknesses:

While the manuscript identifies TcS heterogeneity, the functional implications of the different expression profiles remain speculative. The authors state it may reflect differences in infectivity, but no direct experimental evidence supports this.

The manuscript lacks any functional validation of the single-cell findings. For instance, do the trypomastigote subpopulations identified based on TcS expression exhibit differences in infectivity, host cell tropism, or immune evasion? Such experiments would greatly strengthen the study.

The authors identify a subpopulation of TcS genes that are highly expressed in many cells. However, it is unclear if these correspond to previously characterized TcS members with specific functions.<br /> The authors hypothesize that observed heterogeneity may relate to chromatin regulation. However, the study does not directly address these mechanisms. There are interesting connections to be made with what they identify as the colocalization of genes within chromatin folding domains, but the authors do not fully explore this. It would be insightful to address these mechanisms in future work.

The merging of technical replicates needs further justification and explanation as they were not processed through separate experimental conditions. While barcodes were retained, it would be informative to know how well each technical replicate corresponds with the other. If both datasets were sequenced on the same lane, the inclusion of technical replicates adds noise to the analysis.<br /> While the number of cells sequenced (3192) seems reasonable, it's not clear how much the conclusions are affected by the depth of sequencing. A more detailed description of the sequencing depth and its impact on gene detection would be valuable.

While most of the methods are clear, the way in which the subsampled gene lists were generated could be more thoroughly described, as some details are not clear for the subsampling of single-copy genes.

Some of the figures are difficult to interpret. For example, the color scaling in the heatmap of Supplementary Figure 3B is not self-explanatory and it is hard to extract meaningful conclusions from the graph.

Reviewer #2 (Public review):

Summary:

The author proposes a novel method for mapping single-cell data to specific locations with higher resolution than several existing tools.

Strengths:

The spatial mapping tests were conducted on various tissues, including the mouse cortex, human PDAC, and intestinal villus.

Weaknesses:

(1) Although the researchers claim that glmSMA seamlessly accommodates both sequencing-based and image-based spatial transcriptomics (ST) data, their testing primarily focused on sequencing-based ST data, such as Visium and Slide-seq. To demonstrate its versatility for spatial analysis, the authors should extend their evaluation to imaging-based spatial data.

(2) The definition of "ground truth" for spatial distribution is unclear. A more detailed explanation is needed on how the "ground truth" was established for each spatial dataset and how it was utilized for comparison with the predicted distribution generated by various spatial mapping tools.

(3) In the analysis of spatial mapping results using intestinal villus tissue, only Figure 3d supports their findings. The researchers should consider adding supplemental figures illustrating the spatial distribution of single cells in comparison to the ground truth distribution to enhance the clarity and robustness of their investigation.

(4) The spatial mapping tests were conducted on various tissues, including the mouse cortex, human PDAC, and intestinal villus. However, the original anatomical regions are not displayed, making it difficult to directly compare them with the predicted mapping results. Providing ground truth distributions for each tested tissue would enhance clarity and facilitate interpretation. For instance, in Figure 2a and Supplementary Figures 1 and 2, only the predicted mapping results are shown without the corresponding original spatial distribution of regions in the mouse cortex. Additionally, in Figure 3c, four anatomical regions are displayed, but it is unclear whether the figure represents the original spatial regions or those predicted by glmSMA. The authors are encouraged to clarify this by incorporating ground truth distributions for each tissue.

(5) The cell assignment results from the mouse hippocampus (Supplementary Figure 6) lack a corresponding ground truth distribution for comparison. DG and CA cells were evaluated solely based on the gene expression of specific marker genes. Additional analyses are needed to further validate the robustness of glmSMA's mapping performance on Slide-seq data from the mouse hippocampus.

(6) The tested spatial datasets primarily consist of highly structured tissues with well-defined anatomical regions, such as the brain and intestinal villus. It remains unclear whether glmSMA can be effectively applied to tissue types where anatomical regions are not distinctly separated, such as liver tissue. Further evaluation of such tissues would help determine the method's broader applicability.

Reviewer #2 (Public review):

Summary:

This study introduces a new metric for assessing the efficacy of rotavirus vaccines through the genetic distance clustering of strains. The authors analyzed variations in vaccine protection using whole genome sequencing.

Strengths:

Evaluating vaccine efficacy using whole genome sequencing can enhance our understanding of how pathogen evolution influences disease transmission and control.

Weaknesses:

While the study proposed a new method for evaluating vaccine efficacy using genetic information, its weaknesses arise from the insufficient evidence that analyses based on whole genome sequencing are more reliable than those that rely solely on VP7 and VP4 genotypes.

Though most cases received the RV5 vaccine (n=119 compared to n=30 for RV1), Figure 2 and the primary focus of the paper concentrate on RV1, as the authors identified a stronger association with genetic distance.

Additionally, it is unclear whether the difference between the two groups (j=0 versus j=1) is statistically significant for the analysis based on genetic distance to the RV1 strain, as well as for that based on minimum genetic distance to any of the RV5 vaccine strains. In both cases, the confidence intervals show substantial overlap.

The authors do not seem to have used a criterion for model selection based on the number of clusters; therefore, k=2 may not represent the optimal number of clusters, particularly in relation to the genetic distance associated with the RV5 vaccine (Figure 1B), which does not appear to show a bimodal distribution.

Finally, outcomes for RV1 are highly associated with both homotypic and heterotypic antibody responses (Supplemental Figure 10), which have already been shown to impact vaccine effectiveness (The Pediatric Infectious Disease Journal 40(12):p 1135-1143, 2021, doi:10.1097/INF.0000000000003286). Given this strong association, the benefit of using genetic distance is unclear, as the GxPx genotype serves as a good proxy for genetic similarity.

Reviewer #2 (Public review):

Summary:

The manuscript, "scRNA+TCR-seq Reveals the Proportion and Characteristics of Dual TCR Treg Cells in Mouse Lymphoid and Non-lymphoid Tissues" by Xu and Peng, et al. investigates whether co-expression of 2 T cell receptor (TCR) clonotypes can be detected in FoxP3+ regulatory CD4+ T cells (Tregs) and if it is associated with identifiable phenotypic effects. This paper presents data reanalyzing publicly available single-cell TCR sequencing and transcriptional analysis, convincingly demonstrating that dual TCR co-expression can be detected in Tregs, both in peripheral circulation as well as among Tregs in tissues. They then compare metrics of TCR diversity between single-TCR and dual TCR Tregs, as well as between Tregs in different anatomic compartments, finding the TCR repertoires to be generally similar though with dual TCR Tregs exhibiting a less diverse repertoire and some moderate differences in clonal expansion in different anatomic compartments. Finally, they examine the transcriptional profile of dual TCR Tregs in these datasets, finding some potential differences in the expression of key Treg genes such as Foxp3, CTLA4, Foxo3, Foxo1, CD27, IL2RA, and Ikzf2 associated with dual TCR-expressing Tregs, which the authors postulate implies a potential functional benefit for dual TCR expression in Tregs.

Strengths:

This report examines an interesting and potentially biologically significant question, given recent demonstrations that dual TCR co-expression is a much more common phenomenon than previously appreciated (approximately 15-20% of T cells) and that dual TCR co-expression has been associated with significant effects on the thymic development and antigenic reactivity of T cells. This investigation leverages large existing datasets of single-cell TCRseq/RNAseq to address dual TCR expression in Tregs. The identification and characterization of dual TCR Tregs is rigorously demonstrated and presented, providing convincing new evidence of their existence.

Weaknesses:

The existence of dual TCR expression by Tregs has previously been demonstrated in mice and humans (Reference #18 and Tuovinen. 2006. Blood. 108:4063; Schuldt. 2017. J Immunol. 199:33, both omitted from references). The presented results should be considered in the context of these prior important findings.

This demonstration of dual TCR Tregs is notable, though the authors do not compare the frequency of dual TCR co-expression by Tregs with non-Tregs. This limits interpreting the findings in the context of what is known about dual TCR co-expression in T cells.

Comparison of gene expression by single- and dual TCR Tregs is of interest, but as presented is difficult to interpret. Statistical analyses need to be performed to provide statistical confidence that the observed differences are true.

The interpretations of the gene expression analyses are somewhat simplistic, focusing on the single-gene expression of some genes known to have a function in Tregs. However, the investigators miss an opportunity to examine larger patterns of coordinated gene expression associated with developmental pathways and differential function in Tregs (Yang. 2015. Science. 348:589; Li. 2016. Nat Rev Immunol. Wyss. 2016. 16:220; Nat Immunol. 17:1093; Zenmour. 2018. Nat Immunol. 19:291).

Reviewer #2 (Public review):

Summary:

The manuscript presents a valuable contribution to the field of ACE structural biology and dynamics by providing the first complete full-length dimeric ACE structure in four distinct states. The study integrates cryo-EM and molecular dynamics simulations to offer important insights into ACE dynamics. The depth of analysis is commendable, and the combination of structural and computational approaches enhances our understanding of the protein's conformational landscape. However, the strength of evidence supporting the conclusions needs refinement, particularly in defining key terms, improving structural validation, and ensuring consistency in data analysis. Addressing these points through major revisions will significantly improve the clarity, rigor, and accessibility of the study to a broader audience, allowing it to make a stronger impact in the field.

Strengths:

The integration of cryo-EM and MD simulations provides valuable insights into ACE dynamics, showcasing the authors' commitment to exploring complex aspects of protein structure and function. This is a commendable effort, and the depth of analysis is appreciated.

Weaknesses:

Several aspects of the manuscript require further refinement to improve clarity and scientific rigor as detailed in my recommendations for the authors.

Reviewer #2 (Public review):

Summary:

In this paper, Andriani and colleagues are examining the potential role of Zn flux in sperm and its effect on Slo3 channels. This is an interesting question that is likely critical to how sperm function properly and Slo3 channels are a possible candidate for a downstream molecule that is impacted by Zn. In this paper, the authors use Zn imaging, sperm motility assays, and electrophysiology to show that Zn flux impacts sperm function. They then go on to look at the impact Zn has on Slo3 current and propose a binding site based on MD simulations. While the ideas are interesting, the experiments are not well described in many places making understanding the results very difficult. In addition, critical controls are missing throughout the paper.

Strengths:

The question of how Zn flux impacts membrane potential and sperm motility is an important one. Moreover, Slo3 presents an interesting candidate or the target of Zn regulation. The combination of methods used here also has the potential to uncover mechanisms of Zn regulation of Slo3.

Weaknesses:

Much of the paper lacks experimental description which makes interpretation quite difficult, or a detailed discussion is missing. Examples include:

(1) Figure 1, particularly the Zn imaging, is not sufficiently described. How is the fluorescence intensity measured? A representative ROI? The whole tail and head? Are the sperm immobile? If not, there is evidence that motion artifacts can significantly distort these sorts of measures from Calcium measurements in Cilia. Were there controls done? Is the small amount of Zn seen in the tail above the background?

(2) The second half of Figure 1 is also not well described. What is the extracellular solution in the recordings? When you apply the Zn ionophore, do you expect influx or efflux? I assume efflux is based on the conclusions but this should be discussed explicitly.

(3) Figure 2H labels the Y axis, "normalized current". Normalized to what? Why do neither of the curves end at 1? A better description of what this figure represents is needed.

(4) The alpha fold simulations are not well described. How many Zn binding sites were found? Are all of the histidine mutations in Figure 4 Supplement 1 the ones that were found?

(5) There is no discussion of physiological intracellular Zn concentration. How much Zn is inside the sperm? How much if likely Free vs buffered? Is 100uM a reasonable physiological concentration?

There are a number of areas where the interpretation is not well supported by the data including:

(6) You say in the Figure 4 supplement, that "we did not observe any significant decrease in the percentage of current inhibition." But that is a pretty misleading statement. There are large changes (increases) in the amount of zinc inhibition. These might be allosteric changes but I don't think you can safely eliminate these as relevant Zn binding sites. Also, some of these mutations appear to allow at least some unbinding of Zn.

(7) Following up on the above point, it seems unfair to conclude that the D162S, E169A, and E205 mutants are part of the inhibitory binding site for Zn when the mutation has no effect on inhibition and only an effect on the washout. The mutations on the intracellular side also had an impact on the washout so it seems equally likely that they are the critical residues based on your data.

(8) Nowhere in the paper do you make the specific link between Zn flux and membrane hyperpolariation via Slo3. You show that Zn flux changes the ability of the sperm to hyperpolarize and you show that Slo3 is inhibited by Zn but the connection between the two is not demonstrated. There appears to be a specific Slo3 blocker. If you use this in sperm, do you no longer see the Zn effect?

(9) In the second half of Figure 1, the authors suggest that there is "no hyperpolization in 100uM Zn. That is not really true. It is reduced but not absent.

(10) The claim that Lrcc52 with Slo3 shows a higher current inhibition at pH 7.5 than pH 8 is not well supported because there are only 3 replicates in the 7.5 case. In addition, the claim is made in the test that 100uM ZnCl2 "already inhibited mSlo3+Lrcc52 at pH7.5", contrasted with mSlo3 alone, is not tested statistically.

In a number of places, better controls are needed.

(11) How specific is this effect for Zn? Mg2+, for instance, is also a divalent cation that is in the hundreds of uM range inside the cell. Does it exert the same effect? Each ion certainly has unique preferred coordination geometries, does your predicted binding with MD show what you might expect for tetrahedral coordination with Zn? Did you test other divalent cations functionally or in silicon?

(12) For the VCF experiments, a significantly higher concentration of Zn was used (10mM). What is the reason for this? There is no discussion of how much a "puff" is. Assuming you are using the RNA injector it is probably on the order of 50nL or less. Assuming the volume of an oocyte is 1uL that would argue that the final concentration is 500uM or higher. But this is also complicated by potential local effects of high Zn at the injection site, artifacts of injecting that much metal, and the fact that a great deal of the Zn will likely be bound to other things inside the cell. Better controls are needed for this experiment.

Reviewer #2 (Public review):

Summary:

In this paper, the authors report on the genomic correlates of the transition to the pathogenic lifestyle in Sordariomycetes. The pathogenic lifestyle was found to be better explained by the number of genes, and in particular effectors and tRNAs, but this was modulated by the type of interacting host (insect or not insect) and the ability to be vectored by insects.

Strengths:

The main strength of this study lies in the size of the dataset, and the potentially high number of lifestyle transitions in Sordariomycetes.

Weaknesses:

The main strength of the study is not the clarity of the conclusions.

(1) This is due firstly to the presentation of the hypotheses. The introduction is poorly structured and contradictory in some places. It is also incomplete since, for example, fungus-insect associations are not mentioned in the introduction even though they are explicitly considered in the analyses.

(2) The lack of clarity also stems from certain biases that are challenging to control in microbial comparative genomics. Indeed, defining lifestyles is complicated because many fungi exhibit different lifestyles throughout their life cycles (for instance, symbiotic phases interspersed with saprotrophic phases). In numerous fungi, the lifestyle referenced in the literature is merely the sampling substrate (such as wood or dung), which doesn't mean that this substrate is a crucial aspect of the life cycle. This issue is discussed by the authors, but they do not eliminate the underlying uncertainties.

Reviewer #2 (Public review):

Summary:

The paper introduces the IBEX Knowledge-Base (KB), a shared online resource designed to help scientists working with immunofluorescence imaging. It acts as a central hub where researchers can find and share information about reagents, protocols, and imaging methods. The KB is not static like traditional publications; instead, it evolves as researchers contribute new findings and refinements. A key highlight is that it includes results of both successful and unsuccessful experiments, helping scientists avoid repeating failed experiments and saving time and resources. The platform is built on open-access tools ensuring that the information remains available to everyone. Overall, the KB aims to collaboratively accelerate research, improve reproducibility, and reduce wasted effort in imaging experiments.

Strengths:

(1) The IBEX KB is built entirely on open-source tools, ensuring accessibility and long-term sustainability. This approach aligns with FAIR data principles and ensures that the KB remains adaptable to future advancements.

(2) The KB also follows strict data organization standards, ensuring that all information about reagents and protocols is clearly documented and easy to find with little ambiguity.

(3) The KB allows scientists to report both positive and negative results, reducing duplication of effort and speeding up the research process.

(4) The KB is helpful for all researchers, but even more so for scientists in resource-limited settings. It provides guidance on finding affordable alternatives to expensive or discontinued reagents, making it easier for researchers with fewer resources to perform high-quality experiments.

(5) The KB includes a community discussion forum where scientists can ask for advice, share troubleshooting tips, and collaborate with others facing similar challenges.

Weaknesses:

(1) The potential impact of IBEX KB is very clear. However, the paper would benefit by also discussing more on KB maintenance and outreach, and how higher participation could be incentivized.

(2) Use of resources like GitHub may limit engagement from non-coding members of the scientific community. Will there be alternative options like a user-friendly web interface to contribute more easily?

Reviewer #2 (Public review):

Summary:

The authors harness single-cell RNAseq data from zebrafish and mice to identify Oct4 as a candidate driver of neurogenesis. They then use adeno-associated virus vectors to show that while Oct4 overexpression alone converts rare adult Müller glia (MG) to bipolar cells, it synergizes with Notch pathway inhibition to cause this neurogenesis (achieved by Cre-mediated knockout of Rbpj floxed allele). Importantly, they genetically lineage-mark adult MG using a GLAST-CreER transgene and a Sun-GFP reporter, so that any non-MG cells that convert can be identified unambiguously. This is crucial because several high-profile papers made erroneous claims using short promoters in the viral delivery vector itself to mark MG, but those promoters are leaky and mark other non-MG cell types, making it impossible to definitively state whether manipulations studied were actually causing neurogenesis, or were merely the result of expression in pre-existing neurons. Once the authors establish Oct4 + RbpjKO synergy they use snRNAseq/ATACseq to identify known and novel transcription factors that could play a role in driving neurogenesis.

Strengths:

The system to mark MG is stringent, so the authors are studying transdifferentiation, not artifactual effects due to leaky viral promoters. The synergy between Oct4 and Notch pathway blockade is notable. The single-cell results add the potential involvement of new players such as Rfx4 in adult-MG-neurogenesis.

Weaknesses:

The existing version is difficult to read due to an unusually high number of text errors (e.g. references to the wrong figure panels etc.). A fuller explanation for the fraction of non-MG cells seen in control scRNAseq assays is required, particularly because the neurogenic trajectory which is enhanced in the Oct4/Rbpj-KO context is also evident in the control retina. Claims regarding the involvement of transcription factors in adult neurogenesis (such as Rfx4) need to be toned down unless they are backed up with functional data. It is possible that such factors are important, but equally, they may have no role or a redundant role, and without functional tests, it's impossible to say one way or the other.

Overall, the authors achieved what they set out to do, and have made new insights into how neurogenesis can be stimulated in MG. Ultimately, a major long-term goal in the field is to replace lost photoreceptors as this is most relevant to many human visual disorders, and while this paper (like all others before it) does not generate rods or cones, it opens new strategies to coax MG to form a related neuronal cell type. Their approach underscores the benefits of using a gold-standard approach for lineage tracing.

Reviewer #2 (Public review):

Summary:

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 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 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. These weaknesses were properly discussed throughout the article. The manuscript has 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. 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. The authors also highlighted that these results don't reflect causal relationships. 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):

The authors have conducted a valuable comparative analysis of perturbation responses in three nonlinear kinetic models of E. coli central carbon metabolism found in the literature. They aimed to uncover commonalities and emergent properties in the perturbation responses of bacterial metabolism. They discovered that perturbations in the initial concentrations of specific metabolites, such as adenylate cofactors and pyruvate, significantly affect the maximal deviation of the responses from steady-state values. Furthermore, they explored whether the network connectivity (sparse versus dense connections) influences these perturbation responses. The manuscript is reasonably well written.

Comments on latest version:

The authors have adequately addressed my concerns.

Reviewer #2 (Public review):

Park et al. set out to test two competing hypotheses about the role of the medial prefrontal cortex (PFC) in cognitive control, the ability to use task-relevant cues and ignore task-irrelevant cues to guide behavior. The "central computation" hypothesis assumes that cognitive control relies on computations performed by the PFC, which then interacts with other brain regions to accomplish the task. Alternatively, the "local computation" hypothesis suggests that computations necessary for cognitive control are carried out by other brain regions that have been shown to be essential for cognitive control tasks, such as the dorsal hippocampus and the thalamus. If the central computation hypothesis is correct, PFC lesions should disrupt cognitive control. Alternatively, if the local computation hypothesis is correct, cognitive control would be spared after PFC lesions. The task used to assess cognitive control is the active place avoidance task in which rats must avoid a sector of a rotating arena using the stationary room cues and ignoring the local olfactory cues on the rotating platform. Performance on this task has previously been shown to be disrupted by hippocampal lesions and hippocampal ensembles dynamically represent the room and arena depending on the animal's proximity to the shock zone. They found no group (lesion vs. sham) differences in the three behavioral parameters tested: distance traveled, latency to enter the shock zone, and number of shock zone entries for both the standard task and the "conflict" task in which the shock zone was rotated by 180 degrees. The only significant difference was the savings index; the lesion group entered the new shock zone more often than the sham group during the first 5 minutes of the second conflict session. This deficit was interpreted as a cognitive flexibility deficit rather than a cognitive control failure. Next, the authors compared cytochrome oxidase activity between sham and lesion groups in 14 brain regions and found that only the amygdala shows significant elevation in the lesion vs. sham group. Pairwise correlation analysis revealed a striking difference between groups, with many correlations between regions lost in the lesion group (between reuniens and hippocampus, reuniens and amygdala and a correlation between dorsal CA1 and central amygdala that appeared in the lesion group and were absent in the sham group. Finally, the authors assessed dorsal hippocampal representations of the spatial frame (arena vs. room) and found no differences between lesion and sham groups. The only difference in hippocampal activity was reduced overdispersion in the lesion group compared to the sham group on the pretraining session only and this difference disappeared after the task began. Collectively, the authors interpret their findings as supporting the local computation hypothesis; computations necessary for cognitive control occur in brain regions other than the PFC.

Strengths:

The data were collected in a rigorous way with experimental blinding and appropriate statistical analyses.<br /> Multiple approaches were used to assess differences between lesion and sham groups, including behavior, metabolic activity in multiple brain regions, and hippocampal single unit recording.

Weaknesses:

Only male rats were used with no justification provided for excluding females from the sample.

The conceptual framework used to interpret the findings was to present two competing hypotheses with mutually exclusive predictions about the impact of PFC lesions on cognitive control. The authors then use mainly null findings as evidence in support of the local computation hypothesis. They acknowledge that some people may question the notion that the active place avoidance task indeed requires cognitive control, but then call the argument "circular" because PFC has to be involved in cognitive control. This assertion does not address the possibility that the active place avoidance task simply does not require cognitive control.

The authors did not link the CO activity with the behavioral parameters even though the CO imaging was done on a subset of the animals that ran the behavioral task nor do they make any attempt to interpret these findings in light of the two competing hypotheses posed in the introduction. Moreover, the discussion is lacking any mechanistic interpretations of the findings. For example, there are no attempts to explain why amygdala activity and its correlation with dCA1 activity might be higher in the PFC lesioned group.

Publishing null results is important to avoid wasting animals, time, and money. This study's results will have a significant impact on how the field views the role of the PFC in cognitive control. Whether or not some people reject the notion that the active place avoidance task measures cognitive control, the findings are solid and can serve as a starting point for generating hypotheses about how brain networks change when deprived of PFC input.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

Comments on revisions:

The authors addressed the reviews from the first submission which has substantially strengthened the conclusions of the study, including acknowledgement of unanswered questions for future studies to address.

Reviewer #2 (Public review):

Summary:

The authors provide an important summary of ten years of Brain Initiative funding including a description of the historical development of the initiative, the specific funding mechanisms utilized, and examples of grants funded and work produced. The authors also conduct analyses of the impact on overall funding in Systems and Computational Neuroscience, the raw and field normalized bibliographic impact of the work, the social media impact of the funded work, and the popularity of some tools developed.

Strengths:

This is a useful perspective on an important funding initiative over a ten-year period. It is clearly written and the illustrations and analyses are mostly useful for understanding the impact of the initiative.

Weaknesses:

The major limitation is that the bibliographic analysis does not provide a comparison group of funded grants. Because work that successfully competes for funding is likely to be more impactful than all work in a given area, the normalization of citations to field medians may reflect this "grant review" effect, rather than anything special about the Brain Initiative. Hopefully, this speculation is incorrect (I would guess that it is), but it would be helpful to try to demonstrate this more directly by including a funded comparison group.

There are also minor inconsistencies in the numbering of the figures that need to be cleared up.

Reviewer #2 (Public review):

Summary:

The authors review the history of the team projects within the Brain initiative and analyze their success in progression to additional rounds of funding and their bibliographic impact.

Strengths:

The history of the team projects and the fact that many had renewed funding and produced impactful papers is well documented.

Weaknesses:

The core bibliographic and funding impact results have largely been reported in the companion manuscript and so represent "double dipping" I presume the slight disagreement in the number of grants (by one) represents a single grant that was not deemed to address systems/computational neuroscience. The single figure is relatively uninformative. The domains of study are sufficiently large and overlapping that there seems to be little information gained from the graphic and the Sankey plot could be simply summarized by rates of competing success.

Reviewer #2 (Public review):

Summary:

In this study, Mondal and co-authors present the development of a computational model of homeostatic plasticity incorporating activity-dependent regulation of gating properties (activation, inactivation) of ion channels. The authors show that, similar to what has been observed for activity-dependent regulation of ion channel conductances, implementing activity-dependent regulation of voltage sensitivity participates in the achievement of a target phenotype (bursting or spiking). The results however suggest that activity-dependent regulation of voltage sensitivity is not sufficient to allow this and needs to be associated with the regulation of ion channel conductances in order to reliably reach the target phenotype. Although the implementation of this biologically relevant phenomenon is undeniably relevant, the main conclusions of the paper and the insights brought by this computational work are difficult to grasp.

Strengths:

(1) Implementing activity-dependent regulation of gating properties of ion channels is biologically relevant.

(2) The modeling work appears to be well performed and provides results that are consistent with previous work performed by the same group.

Weaknesses:

(1) The writing is rather confusing, and the state of the art explaining the need for the study is unclear.

(2) The main outcomes and conclusions of the study are difficult to grasp. What is predicted or explained by this new version of homeostatic regulation of neuronal activity?

Reviewer #2 (Public review):

Summary

This paper reports histological, PET imaging, functional, and behavioural data evaluating the longevity of AAV2 infection in multiple brain areas of macaques in the context of DREADD experiments. The central aim is to provide unprecedented information about how long the expression of HM4di or HM3dq receptors is expressed and efficient in modulating brain functions after vector injections. The data show peak expression after 40 to 60 days of vector injection, and stable expressions for up to 1.5 years for hM4di, and that hM3dq remained mostly at 75% of peak after a year, declining to 50% after 2 years. DREADDs effectively modulated neuronal activity and behaviour for approximately two years, evaluated with behavioral testings, neural recordings, or FDG-PET. A statistical evaluation revealed that vector titers, DREADD type, and tags contribute to the measured peak level of DREADD expression.

The article presents a thorough discussion of the limitations and specificities of chemogenetic approaches in monkeys.

Strength

These are unique data, in non-human primates (NHP), an animal model that not only features physiological and immunological characteristics similar to humans but also contribute to neurobiological functional studies on a long timescale with experiments spanning months or years. This evaluation of the long-term efficacy of DREADDs will be very important for all laboratories using this approach in NHP but also for future use of such approach in experimental therapies. The longevity estimates are based on multiple approaches including behavioural and neurophysiological ones, thus providing information on the functional efficacy of DREADD expression.

Performing such evaluation requires specific tools like PET imaging that very few monkey labs have access to in the world. This study was done by the laboratory that has developed the radiotracer c11-DCZ used here, a radiotracer binding selectively to DREADDs and providing, using PET, quantitative in vivo measures of DREADD expression. This study and its data should thus be a reference in the field, providing estimates to plan future chemogenetic experiments.

Publishing databases of experimental outcomes in NHP DREADD experiments is crucial for the community because such experiments are rare, expensive, and long. It contributes to refining experiments and reducing the number of animals overall used in the domain.

Weaknesses

This study is a meta-analysis of several experiments performed in one lab. The good side is that it combined a large amount of data that might not have been published individually; the downside is that all things were not planned and equated, creating a lot of unexplained variances in the data. This was yet judiciously used by the authors, but one might think that planned and organized multicentric experiments would provide more information and help test more parameters, including some related to inter-individual variability, and particular genetic constructs.

Reviewer #2 (Public review):

In addition to the study by Kaletsky et al. (2025), I read the bioRxiv and eLife versions, as well as the eLife reviewer comments, for Gainey et al. (2024), to which Kaletsky et al. respond.

Kaletsky et al. provide detailed, rigorous, and reproducible protocols and results. The authors point out the critical methods that the Hunter group failed to follow/confirm (e.g. azide to paralyze animals during pathogenic learning/memory assays; the expression of the P11 small RNA that is both necessary and sufficient for TEI of avoidance behavior; a single condition for training - PA14 grown on plates at 25°C and training at 20°C for 24 hr - that the Hunter lab did not follow and could not reproduce). The Kaletsky et al. response is evidence-based, fair, level-headed and unbiased, which is in contrast to the Gainey et al. paper.

Reading the eLife review of Gainey et al., I note that the reviewers repeatedly pointed out that authors did not follow published protocols by the Murphy lab.

Public response by Gainey et al. to Reviewer 2: "It remains possible that we misunderstood the published Murphy lab protocols, but we were highly motivated to replicate the results so we could use these assays to investigate the reported RNAi-pathway dependent steps, thus we read every published version with extreme care."

Public response by Gainey et al. to Reviewer 3: "We agree that our study was not exhaustive in our exploration of variables that might be interfering with our ability to detect F2 avoidance."

Gainey et al. provide reasons/excuses for why they did not follow published methods - notably their subjective decision to exclude the paralyzing agent sodium azide from their choice assays, but their abstract reads "We conclude that this example of transgenerational inheritance lacks robustness." I strongly disagree with this conclusion.

Reviewer #2 (Public review):

Parkes et al. combined real-time keypoint tracking with transdermal activation of sensory neurons to examine the effects of recruitment of sensory neurons in freely moving mice. This builds on the authors' previous investigations involving transdermal stimulation of sensory neurons in stationary mice. They illustrate multiple scenarios in which their engineering improvements enable more sophisticated behavioral assessments, including (1) stimulation of animals in multiple states in large arenas, (2) multi-animal nociceptive behavior screening through thermal and optogenetic activation, and (3) stimulation of animals running through maze corridors. Overall, the experiments and the methodology, in particular, are written clearly. However, there are multiple concerns and opportunities to fully describe their newfound capabilities that, if addressed, would make it more likely for the community to adopt this methodology:

The characterization of laser spot size and power density is reported as a coefficient of variation, in which a value of ~3 is interpreted as uniform. My interpretation would differ - data spread so that the standard deviation is three times larger than the mean indicates there is substantial variability in the data. The 2D polynomial fit is shown in Figure 2 - Figure Supplement 1A and, if the fit is good, this does support the uniformity claim (range of spot size is 1.97 to 2.08 mm2 and range of power densities is 66.60 to 73.80 mW). The inclusion of the raw data for these measurements and an estimate of the goodness of fit to the polynomials would better help the reader evaluate whether these parameters are uniform across space and how stable the power density is across repeated stimulations of the same location. Even more helpful would be an estimate of whether the variation in the power density is expected to meaningfully affect the responses of ChR2-expressing sensory neurons.

While the error between the keypoint and laser spot error was reported as ~0.7 to 0.8 mm MAE in Figure 2L, in the methods, the authors report that there is an additional error between predicted keypoints and ground-truth labeling of 1.36 mm MAE during real-time tracking. This suggests that the overall error is not submillimeter, as claimed by the authors, but rather on the order of 1.5 - 2.5 mm, which is considerable given the width of a hind paw is ~5-6 mm and fore paws are even smaller. In my opinion, the claim for submillimeter precision should be softened and the authors should consider that the area of the paw stimulated may differ from trial to trial if, for example, the error is substantial enough that the spot overlaps with the edge of the paw.

As the major advance of this paper is the ability to stimulate animals during ongoing movement, it seems that the Figure 3 experiment misses an opportunity to evaluate state-dependent whole-body reactions to nociceptor activation. How does the behavioral response relate to the animal's activity just prior to stimulation?

Given the characterization of full-body responses to activation of TrpV1 sensory neurons in Figure 4 and in the authors' previous work, stimulation of TrpV1 sensory neurons has surprisingly subtle effects as the mice run through the alternating T maze. The authors indicate that the mice are moving quickly and thus that precise targeting is required, but no evidence is shared about the precision of targeting in this context beyond images of four trials. From the characterization in Figure 2, at max speed (reported at 241 +/- 53 mm/s, which is faster than the high speeds in Figure 2), successful targeting occurs less than 50% of the time. Is the initial characterization consistent with the accuracy in this context? To what extent does inaccuracy in targeting contribute to the subtlety of affecting trajectory coherence and speed? Is there a relationship between animal speed and disruption of the trajectory?

Reviewer #2 (Public review):

- Approach

In this study, Yogesh et al. aimed at characterizing hemodynamic occlusion in two photon imaging, where its effects on signal fluctuations are underappreciated compared to that in wide field imaging and fiber photometry. The authors used activity-independent GFP fluorescence, GCaMP and GRAB sensors for various neuromodulators in two-photon and widefield imaging during a visuomotor context to evaluate the extent of hemodynamic occlusion in V1 and ACC. They found that the GFP responses were comparable in amplitude to smaller GCaMP responses, though exhibiting context-, cortical region-, and depth-specific effects. After quantifying blood vessel diameter change and surrounding GFP responses, they argued that GFP responses were highly correlated with changes in local blood vessel size. Furthermore, when imaging with GRAB sensors for different neuromodulators, they found that sensors with lower dynamic ranges such as GRAB-DA1m, GRAB-5HT1.0, and GRAB-NE1m exhibited responses most likely masked by the hemodynamic occlusion, while a sensor with larger SNR, GRAB-ACh3.0, showed much more distinguishable responses from blood vessel change. They thoroughly investigate other factors that could contribute to these signals and demonstrate hemodynamic occlusion is the primary cause.

- Impact of revision

This is an important update to the initial submission, adding much supplemental imaging and population data that provide greater detail to the analyses and increase the confidence in the authors conclusions.

Specifically, inclusion of the supplemental figures 1 and 2 showing GFP expression across multiple regions and the fluorescence changes of thousands of individual neurons provides a clearer picture of how these effects are distributed across the population. Characterization of brain motion across stimulation conditions in supplemental figure 5 provides strong evidence that the fluorescence changes observed in many of the conditions are unlikely to be primarily due to brain motion associated imaging artifacts. The role of vascular area on fluorescence is further supported by addition of new analyses on vasoconstriction leading to increased fluorescence in Figures 4C1-4, complementing the prior analyses of vasodilation.

The expansion of the discussion on other factors that could lead to these changes is thorough and welcome. The arguments against pH playing a factor in fluorescence changes of GFP, due to insensitivity to changes in the expected pH range are reasonable, as are the other discussed potential factors.

With respect to the author's responses to prior critique, we agree that activity dependent hemodynamic occlusion is best investigated under awake conditions. Measurement of these dynamics under anesthesia could lead to an underestimation of their effects. Isoflurane anesthesia causes significant vasodilation and a large reduction in fluorescence intensity in non-functional mutant GRABs. This could saturate or occlude activity dependent effects.

- Strengths

This work is of broad interest to two photon imaging users and GRAB developers and users. It thoroughly quantifies the hemodynamic driven GFP response and compares it to previously published GCaMP data in a similar context, and illustrates the contribution of hemodynamic occlusion to GFP and GRAB responses by characterizing the local blood vessel diameter and fluorescence change. These findings provide important considerations for the imaging community and a sobering look at the utility of these sensors for cortical imaging.

Importantly, they draw clear distinctions between the temporal dynamics and amplitude of hemodynamic artifacts across cortical regions and layers. Moreover, they show context dependent (Dark versus during visual stimuli) effects on locomotion and optogenetic light-triggered hemodynamic signals.

The authors suggest that signal to noise ratio of an indicator likely affects the ability to separate hemodynamic response from the underlying fluorescence signal. With a new analysis (Supplemental Figure 4) They show that the relative degree of background fluorescence does not affect the size of the artifact.

Most of the first generation neuromodulator GRAB sensors showed relatively small responses, comparable to blood vessel changes in two photon imaging, which emphasizes a need for improved the dynamic range and response magnitude for future sensors and encourages the sensor users to consider removing hemodynamic artifacts when analyzing GRAB imaging data.

- Weaknesses

The largest weakness of the paper remains that, while they convincingly quantify hemodynamic artifacts across a range of conditions, they provide limited means of correcting for them. However they now discuss the relative utility of some hemodynamic correction methods (e.g. from Ocana-Santero et al., 2024).

The paper attributes the source of 'hemodynamic occlusion' primarily to blood vessel dilation, but leaves unanswered how much may be due to shifts in blood oxygenation. Figure 4 directly addresses the question of how much of the signal can be attributed to occlusion by measuring the blood vessel dilation, and has been improved by now showing positive fluorescence effects with vasoconstriction. They now also discuss the potential impact of oxygenation.

Along these lines, the authors carefully quantified the correlation between local blood vessel diameter and GFP response (or neuropil fluorescence vs blood vessel fluorescence with GRAB sensors). We are left to wonder to what extent does this effect depend on proximity to the vessels? Do GFP/ GRAB responses decorrelate from blood vessel activity in neurons further from vessels (refer to Figure 5A and B in Neyhart et al., Cell Reports 2024)? The authors argue that the primary impact of occlusion is from blood vessels above the plane of imaging, but without a vascular reconstruction, their evidence for this is anecdotal.

The choice of ACC as the frontal region provides a substantial contrast in location, brain movement, and vascular architecture as compared to V1. As the authors note, ACC is close to the superior sagittal sinus and thus is the region where the largest vascular effects are likely to occur. A less medial portion of M2 may have been a more appropriate comparison. The authors now include example imaging fields for ACC and interesting out-of-plane vascular examples in the supplementary figures that help assess these impacts.

-Overall Assessment

This paper is an important contribution to our understanding of how hemodynamic artifacts may corrupt GRAB and calcium imaging, even in two-photon imaging modes. While it would be wonderful if the authors were able to demonstrate a reliable way to correct for hemodynamic occlusion which did not rely on doing the experiments over with a non-functional sensor or fluorescent protein, the careful measurement and reporting of the effects here is, by itself, a substantial contribution to the field of neural activity imaging. It's results are of importance to anyone conducting two-photon or widefield imaging with calcium and GRAB sensors and deserves the attention of the broader neuroscience and in-vivo imaging community.

Reviewer #3 (Public review):

This paper addresses a long-standing problem in microbiology: the evolution of bacterial cell shape. Bacterial cells can take a range of forms, among the most common being rods and spheres. The consensus view is that rods are the ancestral form and spheres the derived form. The molecular machinery governing these different shapes is fairly well understood but the evolutionary drivers responsible for the transition between rods and spheres is not. Enter Yulo et al.'s work. The authors start by noting that deletion of a highly conserved gene called MreB in the Gram-negative bacterium Pseudomonas fluorescens reduces fitness but does not kill the cell (as happens in other species like E. coli and B. subtilis) and causes cells to become spherical rather than their normal rod shape. They then ask whether evolution for 1000 generations restores the rod shape of these cells when propagated in a rich, benign medium.

The answer is no. The evolved lineages recovered fitness by the end of the experiment, growing just as well as the unevolved rod-shaped ancestor, but remained spherical. The authors provide an impressively detailed investigation of the genetic and molecular changes that evolved. Their leading results are:

(1) The loss of fitness associated with MreB deletion causes high variation in cell volume among sibling cells after cell division;<br /> (2) Fitness recovery is largely driven by a single, loss-of-function point mutation that evolves within the first ~250 generations that reduces the variability in cell volume among siblings;<br /> (3) The main route to restoring fitness and reducing variability involves loss of function mutations causing a reduction of TPase and peptidoglycan cross-linking, leading to a disorganized cell wall architecture characteristic of spherical cells.

The inferences made in this paper are on the whole well supported by the data. The authors provide a uniquely comprehensive account of how a key genetic change leads to gains in fitness and the spectrum of phenotypes that are impacted and provide insight into the molecular mechanisms underlying models of cell shape.