Reviewer #1 (Public Review):

Short Assessment

In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3. They demonstrate that circHIPK3 interacts with an RNA binding protein (IGF2BP2), sequestering it away from its target mRNAs. This interaction is shown to regulates the expression of hundreds of genes that share a specific sequence motif (11-mer motif) in their untranslated regions (3'-UTR), identical to one present in circHIPK3 where IGF2BP2 binds. The study further focuses on the specific case of STAT3 gene, whose mRNA product is found to be downregulated upon circHIPK3 depletion. This suggests that circHIPK3 sequesters IGF2BP2, preventing it from binding to and destabilizing STAT3 mRNA. The study presents evidence supporting this mechanism and discusses its potential role in tumor cell progression. These findings contribute to the growing complexity of understanding cancer regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.

Strengths:<br /> The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of studies. They provide both genome-wide analysis and a specific case (STAT3) which is relevant for cancer progression. Overall, the authors have significantly improved their manuscript in their revised version.

Weaknesses:<br /> While the authors have performed northern blots to measure circRNA levels, an estimation of the circRNA overexpression efficiency, namely the circular-to-linear expression ratio, would be desired. The seemingly contradictory effects of circHIPK3 and STAT3 depletion in cancer progression, are now addressed by the authors in their revised manuscript, incorporating potential reasons that might explain such complexity.

Major points about revised manuscript

(1) In Supplementary Figure S5H, the membrane may have been trimmed too closely to the circRNA band, potentially resulting in the absence of the linear RNA band. Could the authors provide a full image of the membrane that includes the loading points? Having access to the complete image would allow for a more comprehensive evaluation of the results, including the presence or absence of expected linear and circular RNA bands.

Reviewer #1 (Public Review):

Summary:

Authors were attempting to determine the extent that CIH altered swallowing motor function; specifically, the timing and probability of the activation of the larygneal and submental motor pools. The paper describes a variety of different motor patterns elicited by optogenetic activation of individual neuronal phenotypes within PiCo in a group of mice exposed to CIH. They show that there are a variety of motor patterns that emerge in CIH mice; this is apparently different than the more consistent motor patterns elicited by PiCo activation in normoxic mice (previously published)

Strengths:

The preparation is technically challenging and gives valuable information related to the role of PiCo in the pattern of motor activation involved in swallowing and its timing with phrenic activity. Genetic manipulations allow for the independent activation of the individual neuronal phenotypes of PiCo (glutamatergic, cholinergic) which is a strength.

Weaknesses:

(1) Comparisons made between experimental data acquired currently with those previously published are extremely problematic, with the potential confounding influence of changing environments, genetics and litter effects. For example, were the current mice tested at the same time as those exposed to normoxia? Are they littermates (or at least from the same colony) as those previously examined? If they were tested at the same time and age, then the authors should explicitly state this in the methods. The authors have provided no statistical analyses to determine whether there is an effect of CIH on the motor patterns. In short, how can they be sure that the phenomena they observe with respect to motor patterns is due to CIH?

(2) The data are descriptive in nature, reporting only differences (diversity) of motor patterns in this cohort of animals exposed to CIH. There is limited mechanistic insight into how PiCo manipulation alters the pattern and probability of motor activation. Can they utilize Fos or marker of activation within the nTS or other regions to provide initial insight? Or in another nucleus that contributes as part of the circuit.

(3) The differences between the genotypes (ChaTcre; Vglut2Cre; ChatCre:Vglut2FlpO) with regard to the probability of generating a swallow are not sufficiently discussed, in my view. If, as the authors state, it is "reasonable to suggest that CIH differentially affects" these populations, then what are some viable reasons? What are the known differences in these populations of neurons that could lead to variable responses? Do they project to different places?

(4) The Results section is difficult to follow and interpret. It would be beneficial to have a couple of sentences after each sub-section stating what the data actually mean. As of now it reads like a statistical report of the data with little "basic" interpretation of the data.

(5) I have a hard time understanding the functional significance of calculating and plotting the degree of correlation between shifting/delaying the following inspiratory burst and triggering a swallow.

Reviewer #1 (Public Review):

Summary:

In this work, the authors examine the activity and function of D1 and D2 MSNs in dorsomedial striatum (DMS) during an interval timing task. In this task, animals must first nose poke into a cued port on the left or right; if not rewarded after 6 seconds, they must switch to the other port. Critically, this task thus requires animals to estimate if at least 6 seconds have passed after the first nose poke - this is the key aspect of the task focused on here. After verifying that animals reliably estimate the passage of 6 seconds by leaving on average after 9 seconds, the authors examine striatal activity during this interval. They report that D1-MSNs tend to decrease activity, while D2-MSNs increase activity, throughout this interval. They suggest that this activity follows a drift-diffusion model, in which activity increases (or decreases) to a threshold after which a decision (to leave) is made. The authors next report that optogenetically inhibiting D1 or D2 MSNs, or pharmacologically blocking D1 and D2 receptors, increased the average wait time of the animals to 10 seconds on average. This suggests that both D1 and D2 neurons contribute to the estimate of time, with a decrease in their activity corresponding to a decrease in the rate of 'drift' in their drift-diffusion model. Lastly, the authors examine MSN activity while pharmacologically inhibiting D1 or D2 receptors. The authors observe most recorded MSNs neurons decrease their activity over the interval, with the rate decreasing with D1/D2 receptor inhibition.

Major strengths:

The study employs a wide range of techniques - including animal behavioral training, electrophysiology, optogenetic manipulation, pharmacological manipulations, and computational modeling. The behavioral task used by the authors is quite interesting and a nice way to probe interval timing in rodents. The question posed by the authors - how striatal activity contributes to interval timing - is of importance to the field and has been the focus of many studies and labs; thus, this paper can meaningfully contribute to that conversation. The data within the paper is presented very clearly, and the authors have done a nice job presenting the data in a transparent manner (e.g., showing individual cells and animals). Overall, the manuscript is relatively easy to read and clear, with sufficient detail given in most places regarding the experimental paradigm or analyses used.

Major weaknesses:

I perceive two major weaknesses. The first is the impact or contextualization of their results in terms of the results of the field more broadly. More specifically, it was not clear to me how the authors are interpreting the striatal activity in the context of what others have observed during interval timing tasks. In other words - what was the hypothesis going into this experiment? Does observing increasing/decreasing activity in D2 versus D1 support one model of interval timing over another, or does it further support a more specific idea of how DMS contributes to interval timing? Or was the main question that we didn't know if D2 or D1 neurons had differential activity during interval timing?

In the second, I felt that some of the conclusions suggested by the authors don't seem entirely supported by the data they present, or the data presented suggests a slightly more complicated story. Below I provide additional detail on some of these instances.

Regarding the results presented in Figures 2 and 3:

I am not sure the PC analysis adds much to the interpretation, and potentially unnecessarily complicates things. In particular, running PCA on a matrix of noisy data that is smoothed with a Gaussian will often return PCs similar to what is observed by the authors, with the first PC being a line up/down, the 2nd PC being a parabola that is up/down, etc. Thus, I'm not sure that there is much to be interpreted by the specific shape of the PCs here. I think an alternative analysis that might be both easier and more informative is to compute the slope of the activity of each neuron across the 6 seconds. This would allow the authors to quantify how many neurons increase or decrease their activity much like what is shown in Figure 2.

Relatedly, it seems that the data shown in Figure 2D *doesn't* support the authors' main claim regarding D2/D1 MSNs increasing/decreasing their activity, as the trial-by-trial slope is near 0 for both cell types.

Regarding the results in Figure 4:

The authors suggest that their data is consistent with a drift-diffusion model. However, it is unclear how well the output from the model fits the activity from neurons the authors recorded. Relatedly, it is unclear how the parameters were chosen for the D1/D2 versions of this model. I think that an alternate approach that would answer these questions is to fit the model to each cell, and then examine the best-fit parameters, as well as the ability of the model to predict activity on trials held out from the fitting process. This would provide a more rigorous method to identify the best parameters and would directly quantify how well the model captures the data.

Relatedly, looking at the raw data in Figure 2, it seems that many neurons either fire at the beginning or end of the interval, with more neurons firing at the end, and more firing at the beginning, for D2/D1 neurons respectively. Thus, it's not clear to me whether the drift-diffusion model is a good model of activity. Or, perhaps the model is supposed to be related to the aggregate activity of all D1/D2 neurons? (If so, this should be made more explicit. The comment about fitting the model directly to the data also still stands).

Further, it's unclear to me how, or why, the authors changed the specific parameters they used to model the optogenetic manipulation. Were these parameters chosen because they fit the manipulation data? This I don't think is in itself an issue, but perhaps should be clearly stated, because otherwise it sounds a bit odd given the parameter changes are so specific. It is also not clear to me why the noise in the diffusion process would be expected to change with increased inhibition.

Regarding the results in Figure 6:

My comments regarding the interpretation of PCs in Figure 2 apply here as well. In addition, I am not sure that examining PC2 adds much here, given that the authors didn't examine such nonlinear changes earlier in the paper.

A larger concern though that seems potentially at odds with the authors' interpretation is that there seems to be very little change in the firing pattern after D1 or D2 blockade. I see that in Figure 6F the authors suggest that many cells slope down (and thus, presumably, they are recoding more D1 cells), and that this change in slope is decreased, but this effect is not apparent in Figure 6C, and Figure 6B shows an example of a cell that seems to fire in the opposite direction (increase activity). I think it would help to show some (more) individual examples that demonstrate the summary effect shown by the authors, and perhaps the authors can comment on the robustness (or the variability) of this result.

Also, it seems that if the authors want to claim that this manipulation lowers the drift rate. I think to make this claim, they could fit the DDM model and examine whether D is significantly lower.

Regarding the results in Figure 7:

I am overall a bit confused about what the authors are trying to claim here. In Figure 7, they present data suggesting that D1 or D2 blockade disrupts their ability to decode time in the interval of interest (0-6 seconds). However, in the final paragraph of the results, the authors seem to say that by using another technique, they didn't see any significant change in decoding accuracy after D1 or D2 blockade. What do the authors make of this?

Impact:

The task and data presented by the authors are very intriguing, and there are many groups interested in how striatal activity contributes to the neural perception of time. The authors perform a wide variety of experiments and analysis to examine how DMS activity influences time perception during an interval-timing task, allowing for insight into this process. However, the significance of the key finding - that D2/D1 activity increases/ decreases with time - remains somewhat ambiguous to me. This arises from a lack of clarity regarding the initial hypothesis and the implications of this finding for advancing our understanding of striatal functions.

Reviewer #1 (Public Review):

Summary:

This is well-performed research with solid results and thorough control. The authors did a good job of finding the relationship between the 5-HT1A receptor and megakaryocytopoiesis, which demonstrated the potential of vilazodone in the management of thrombocytopenia. It emphasizes the regulatory mechanism of 5-HT1A receptor signaling on hematopoietic lineages, which could further advance the field of thrombocytopenia for therapeutic purposes.

Strengths:

This is a comprehensive and detailed research using multiple methods and model systems to determine the pharmacological effects and molecular mechanisms of vilazodone. The authors conducted in vitro experiments using HEL and Meg-01 cells and in vivo experiments using Zebrafish and Kunming-irradiated mice. The experiments and bioinformatics analysis have been performed with a high degree of technical proficiency. The authors demonstrated how vilazodone binds to 5-HTR1A and regulates the SRC/MAPK pathway, which is inhibited by particular 5-HTR1A inhibitors. The authors determined this to be the mechanistic underpinning for the effects of vilazodone in promoting megakaryocyte differentiation and thrombopoiesis.

Weaknesses:

(1) Which database are the drug test sets and training sets for the creation of drug screening models obtained from? What criteria are used to grade the results?<br /> (2) What is the base of each group in Figure 3b for the survival screening of zebrafish? The positivity rate of GFP-labeled platelets is too low, as indicated by the quantity of eGFP+ cells. What gating technique was used in Figure 3e?<br /> (3) In Figure 4C, the MPV values of each group of mice did not show significant downregulation or upregulation. Please explain the possible reasons.<br /> (4) The PPI diagram and the KEGG diagram in Figure 6 both provide a possible mechanism pathway for the anti-thrombocytopenia effect of vilazodone. How can the author analyze the differences in their results?<br /> (5) 5-HTR1A protein expression is measured only in the Meg-01 cells assay. Similar quantitation through western blot is not shown in other cell models.

Reviewer #1 (Public Review):

Summary:

In this manuscript the authors have applied an asymmetric split mNeonGreen2 (mNG2) system to human iPSCs. By integrating a constitutively expressed long fragment of mNG2 at the AAVS1 locus, this allows other proteins to be tagged through the use of available ssODN donors. This removes the need to generate long AAV donors for tagging, thus greatly facilitating high-throughput tagging efforts. The authors then demonstrate the feasibility of the method by successfully tagging 9 markers expressed in iPSC at various, and one expressed upon endoderm differentiation. Several additional differentiation markers were also successfully tagged but not subsequently tested for expression/visibility. As one might expect for high-throughput tagging, a few proteins, while successfully tagged at the genomic level, failed to be visible. Finally, to demonstrate the utility of the tagged cells, the authors isolated clones with genes relevant to cytokinesis tagged, and together with an AI to enhance signal to noise ratios, monitored their localization over cell division.

Strengths

Reviewer Comment: Characterization of the mNG2 tagged parental iPSC line was well and carefully done including validation of a single integration, the presence of markers for continued pluripotency, selected off-target analysis and G-banding-based structural rearrangement detection.<br /> The ability to tag proteins with simple ssODNs in iPSC capable of multi-lineage differentiation will undoubtedly be useful for localization tracking and reporter line generation.<br /> Validation of clone genotypes was carefully performed and highlights the continued need for caution with regards to editing outcomes.

Weaknesses

Reviewer Comment: IF and flow cytometry figures lack quantification and information on replication. How consistent is the brightness and localization of the markers? How representative are the specific images? Stability is mentioned in the text but data on the stability of expression/brightness is not shown.

Author Response: To address this comment, we have quantified the mean fluorescence intensity of the tagged cell populations in Fig. S3B-T. This data correlates well with the expected expression levels of each gene relative to the others (Fig. S3A), apart from CDH1 and RACGAP1, which are described in the discussion.

Reviewer Reply: Great, thanks.

Reviewer Comment: The localization of markers, while consistent with expectations, is not validated by a second technique such as antibody staining, and in many cases not even with Hoechst to show nuclear vs cytoplasmic.

Author Response: We find that the localization of each protein is distinct and consistent with previous studies. To address this comment, we have added an overlay of the green fluorescence images with brightfield images to better show the location of the tagged protein relative to the nuclei and cytoplasm. We have also added references to other studies that showed the same localization patterns for these proteins in iPSCs and other relevant cell lines.

Reviewer Reply: There was no question that the localization fit with expectations, however, this still doesn't show that in the same cell the tag is in the same spot. It would have been fairly simple to do for at least a handful of markers, image, fix and stain to demonstrate unequivocally the tag and protein are co-localized. Of course, this isn't damning by any means, it just would have been nice.

Reviewer Comment: For the multi-germ layer differentiation validation, NCAM is also expressed by ectoderm, so isn't a good solo marker for mesoderm as it was used. Indeed, the kit used for the differentiation suggests Brachyury combined with either NCAM or CXCR4, not NCAM alone.

Author Response: Since Brachyury is the most common mesodermal marker, we first tested differentiation using anti-Brachyury antibodies, but they did not work well for flow cytometry. We then switched to anti-NCAM antibodies. Since we used a kit for directed differentiation of iPSCs into the mesodermal lineage, NCAM staining should still report for successful differentiation. In the context of mixed differentiation experiments (embryoid body formation or teratoma assay), NCAM would not differentiate between ectoderm and mesoderm. The parental cells (201B7) have also been edited at the AAVS1 locus in multiple other studies, with no effect on their differentiation potential.

Reviewer Reply: This is placing a lot of trust in the kit that it only makes what it says it makes. It could have been measured by options other than flow such as qPCR, Western blot, or imaging, but fine.

Reviewer Comment: Only a single female parental line has been generated and characterized. It would have been useful to have several lines and both male and female to allow sex differences to be explored.

Author Response: We agree that it would be interesting (and important) to study differences in protein localization between female and male cell types, and from different individuals with different genetic backgrounds. We see our tool as opening a door for cell biology to move away from randomly collected, transformed, differentiated cell types to more directed comparative studies of distinct normal cell types. Since few studies of cell biological processes have been done in normal cells, a first step is to understand how processes compare in an isogenic background, then future studies can reveal how they compare with other individuals and sexes. We hope that either our group or others will continue to build similar lines so that these studies can be done.

Reviewer Reply: Fair enough.

Reviewer Comment: The AI-based signal to noise enhancement needs more details and testing. Such models can introduce strong assumptions and thus artefacts into the resolved data. Was the model trained on all markers or were multiple models trained on a single marker each? For example, if trained to enhance a single marker (or co-localized group of markers), it could introduce artefacts where it forces signal localization to those areas even for others. What happens if you feed in images with scrambled pixel locations, does it still say the structures are where the training data says they should be? What about markers with different localization from the training set. If you feed those in, does it force them to the location expected by the training data or does it retain their differential true localization and simply enhance the signal?

Author Response: The image restoration neural network was used as in Weigert et al., 2018. The model was trained independently for each marker. Each trained model was used only on the corresponding marker and with the same imaging conditions as the training images. From visual inspection, the fluorescent signal in the restored images was consistent with the signal in the raw images, both for interphase and mitotic cells. We found very few artefacts of the restoration (small bright or dark areas) that were discarded. We did not try to restore scrambled images or images of mismatched markers.

Reviewer Reply: I understand. What I'm saying is that for the restoration technique to be useful you need to know that it won't introduce artefacts if you have an unexpected localization. Think of it this way, if you already know the localization, then there's no point measuring it. If you don't, or there's a possibility that it is somewhere unexpected, then you need to know with confidence that your algorithm will be able to accurately detect that unexpected localization. As such, it would be extremely important to validate that your restoration algorithm will not bias the results to the expected localization if the true localization is unexpected/not seen in the training dataset. It would have been extremely trivial to run this analysis and I do not feel this comment has been in any way adequately addressed.

Reviewer #1 (Public Review):

Summary:

This study offers a comprehensive examination of the early postnatal development of the patch and matrix compartments within the striatum. These are segregated circuits within the striatum circuits with distinct embryonic origins and functional roles in mature brain physiology. Despite the recognized significance of these circuits, a comprehensive understanding of their postnatal maturation remains elusive.

Strengths:

The authors undertake a thorough investigation, characterizing the intrinsic properties of direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) across both matrix and striosome compartments throughout development. The authors identify the regulatory role of M1 receptors in modulating spontaneous activity in SPNs, and demonstrate the impact of chemogenetic inhibition of MOR-positive neurons during development on GABAergic synapses in substantia nigra pars compacta (SNc) dopamine (DA) neurons. These findings significantly advance our understanding of striatal development and function.

Weaknesses:

Certain methodological considerations warrant attention. Notably, the reliance on TdTomato expression for the identification of striosomes raises concerns, particularly regarding the substantial difference in slice thickness between the immunohistochemistry (IHC) images (50um) shown in Figure 2 and those utilized for whole-cell recordings (300um).

Enhanced clarification regarding the identification of cell patches is possible in the electrophysiology rig conditions. Using a widefield microscope rather than a confocal would strengthen the reliability of this methodology.

In the Ca2+ imaging experiments of Figure 2, striosomes were defined as the regions of brighter GCaMP fluorescence. This presents a potential limitation because it presupposes higher activity levels within patch cells, which is what the experiment is designed to test. Based on this criteria, neurons of this region will necessarily have more activity than in others.

There is also no information on how Ca2+ imaging traces were analyzed. In the examples provided, putative matrix neurons seem to exhibit different Ca2+ dynamics compared to striosome neurons. The plateau responses might reflect even higher activity than the transient signals observed in striosome neurons. It'll be important to know how the data was quantified. For example, calculations of F0 based on rolling functions tend to underestimate dF/F in traces like this. Calculations of the area under the curve can also provide valuable information in these cases.

There is no description of the 8mM KCl treatment in the methods. Was this only used for the Ca2+ imaging experiments? The percentage of active cells in Figures 2C-D is similar to or lower than that described in Figure 2B, which is confusing. Were recordings always performed in 8mM KCl?

Lastly, while the findings of Figure 6 suggest a deficit in striosomal inputs to SNc DA neurons, they do not conclusively demonstrate this point (DA neurons receive many sources of inhibition, and local interneurons in SNc are highly plastic). Given the availability of Opmr1-Cre mice and the utilization of multiple viruses in Figure 6 experiments, the inclusion of experiments employing ChR2 to directly assess striatal/striosome inputs would substantially strengthen this claim. This is the main claim stated in the manuscript title, so it is important to provide evidence of specific striatonigral deficits.

Reviewer #1 (Public Review):

Summary:

The authors seek to elucidate the structural role of N-glycosylation at the N343 position of the SARS-CoV-2 Spike protein's Receptor Binding Domain (RBD) and its evolution across different variants of concern (VoCs). Specifically, they aim to understand the impact of this glycosylation on the RBD's stability and function, which could have implications for the virus's infectivity and, eventually, the effectiveness of vaccines.

Strengths:

The major strength of the study stems from the molecular-level picture emerging from the use of over 45 μs of cumulative molecular dynamics (MD) simulations, including both conventional and enhanced sampling schemes, which provide detailed insights into the structural role of N343 glycosylation. The combination of these simulations with experimental assays, such as electron-spray ionization mass spectrometry (ESI-MS) for affinity measurements, bolsters the reliability of the findings. At the same time, one potential weakness is the inherent limitation of the current computational models to fully capture the complexities of in vivo systems. While the authors acknowledge the difficulty in completely gauging the N343 glycosylation's impact on RBD folding due to the dynamic nature of glycan structures, their computational/experimental approach lends support to their claims.

Weaknesses:

One potential weakness is the inherent limitation of computational models to fully capture the complexities of in vivo systems. While the authors acknowledge the difficulty in completely gauging the N343 glycosylation's impact on RBD folding due to the dynamic nature of glycan structures, their multi-faceted approach lends solid support to their claims.

Other Comments:

The study shows that N343 glycosylation plays a structural role in stabilizing the RBD across various SARS-CoV-2 strains. The removal of this glycan led to conformational changes that could affect the virus's infectivity. The results correlate with a reported reduction in viral infectivity upon deletion of glycosylation sites, supporting the authors' conclusion that N343 glycosylation is functionally essential for viral infection.

By providing molecular insights into the spike protein's architectural changes, the work could influence the design of more effective vaccines and therapeutic agents. The data and methods used could serve as a valuable resource for researchers looking into viral evolution, protein-glycan interactions, and the development of glycan-based interventions.

Reviewer #1 (Public Review):

Summary:

This is an interesting and potentially important paper, which however has some deficiencies.

Strengths:

A significant amount of potentially useful data.

Weaknesses:

One issue is a confusion of thermal stability with solubility. While thermal stability of a protein is a thermodynamic parameter that can be described by the Gibbs-Helmholtz equation, which relates the free energy difference between the folded and unfolded states as a function of temperature, as well as the entropy of unfolding. What is actually measured in PISA is a change in protein solubility, which is an empirical parameter affected by a great many variables, including the presence and concentration of other ambient proteins and other molecules. One might possibly argue that in TPP, where one measures the melting temperature change ∆Tm, thermal stability plays a decisive or at least an important role, but no such assertion can be made in PISA analysis that measures the solubility shift.

Another important issue is that the authors claim to have discovered for the first time a number of effects well described in prior literature, sometimes a decade ago. For instance, they marvel at the differences between the solubility changes observed in lysate versus intact cells, while this difference has been investigated in a number of prior studies. No reference to these studies is given during the relevant discussion.

The validity of statistical analysis raises concern. In fact, no calculation of statistical power is provided. As only two replicates were used in most cases, the statistical power must have been pretty limited. Also, there seems to be an absence of the multiple-hypothesis correction.

Also, the authors forgot that whatever results PISA produces, even at high statistical significance, represent just a prediction that needs to be validated by orthogonal means. In the absolute majority of cases such validation is missing.

Finally, to be a community-useful resource the paper needs to provide the dataset with a user interface so that the users can data-mine on their own.

Joint Public Review:

Summary:

Brauns et al. work to decipher the respective contribution of active versus passive contributions to cell shape changes during germ band elongation. Using a novel quantification tool of local tension, their results suggest that epithelial convergent extension results from internal forces.

Strengths:

The approach developed here, tension isogonal decomposition, is original and the authors made the demonstration that we can extract comprehensive data on tissue mechanics from this type of analysis.

They present an elegant diagram that quantifies how active and passive forces interact to drive cell intercalations.

The model qualitatively recapitulates the features of passive and active intercalation for a T1 event.

Regions of high isogonal strains are consistent with the proximity of known active regions.

They define a parameter (the LTC parameter) which encompasses the geometry of the tension triangles and allows the authors to define a criterium for T1s to occur.

The data are clearly presented, going from cellular scale to tissue scale, and integrating modeling approach to complement the thoughtful description of tension patterns.

Weaknesses:

The modeling is interesting, with the integration of tension through tension triangulation around vertices and thus integrating force inference directly in the vertex model. However, the authors are not using it to test their hypothesis and support their analysis at the tissue level. Thus, although interesting, the analysis at the tissue level stays mainly descriptive.

Major points:

(1) The authors mention that from their analysis, they can predict what is the tension threshold required for intercalations in different conditions and predict that in Snail and Twist mutants the T1 tension threshold would be around √2. Since movies of these mutants are most probably available, it would be nice to confirm these predictions.

(2) While the formalism is very elegant and convincing, and also convincingly allows making sense of the data presented in the paper, it is not all that clear whether the claims are compatible with previous experimental observations. In particular, it has been reported in different papers (including Collinet et al NCB 2015, Clement et al Curr Biol 2017) that affecting the initial Myosin polarity or the rate of T1s does not affect tissue-scale convergent extension. Analysis/discussion of the Tor phenotype (no extension with myosin anisotropy) and the Eve/Runt phenotype (extension without Myosin anisotropy), which seem in contradiction with an extension mostly driven by myosin anisotropy.

Reviewer #1 (Public Review):

The manuscript by Long et al. focused on SUL1, a gene encoding a sulfate transporter with signaling roles in yeast. The authors claim that the deletion of SUL1, rather than SUL2 (encoding a similar transporter), extended yeast replicative lifespan independent of sulfate transport. They also show that SUL1 loss-of-function mutants display decreased PKA activity, indicated by stress-protective carbohydrate accumulation, relevant transcription factor relocalization (measured during aging in single cells), and changes in gene expression. Finally, they show that loss of SUL1 increases autophagy, which is consistent with the longer lifespan of these cells. Overall, this is an interesting paper, but additional work should strengthen several conclusions, especially for the role of sulfate transport. Specific points include the following:

- What prompted the authors to measure the RLS of sul1 mutants? Prior systematic surveys of RLS in the same strain background (which included the same sul1 deletion strain they used) did not report lifespan extension in sul1 cells (PMID: 26456335).

- Cells carrying a mutant Sul1 (E427Q), which was reported to be disrupted in sulfate transport, did not have a longer lifespan (Figure 1), leading them to conclude that "lifespan extension by SUL1 deletion is not caused by decreased sulfate uptake". They would need to measure sulfate uptake in the mutants they test to draw that conclusion firmly.

- Related to my previous point, another simple experiment would be to repeat the assays in Figure 1 with exogenous sulfur added to see if the lifespan extension is suppressed.

- There needs to be more information in the text or the methods about how they did the enrichment analysis in Figure 2B. P-values are typically insufficient, and adjusted FDR values are reported from standard gene ontology platforms (e.g., PANTHER).

- It is somewhat puzzling that relocalization of Msn2 was not seen in very old cells (past the 17th generation), but it was evident in younger cells. The authors could consider another possibility, that it was early and midlife experiences that made those cells live longer. Past that window, loss of Sul1 may have no impact on longevity. A conditional shutoff system to regulate SUL1 expression would be needed to test the above, albeit this is probably beyond the scope of this report.

- The connections between glucose restriction, autophagy, and sul1 (Figure 4) could be further tested by measuring the RLS of sul1 cells in glucose-restricted cells. If RLS is further extended by glucose restriction, then whatever effects they see should be independent of glucose restriction.

- They made and tested the double (sul1, msn2) mutants, but they should also test the sul1, msn4 combination since Msn4 functions similarly to Msn2.

Reviewer #1 (Public Review):

This is an interesting study investigating the mechanisms underlying membrane targeting of the NLRP3 inflammasome and reporting a key role for the palmitoylation-depalmitoylation cycle of cys130 in NRLP3. The authors identify ZDHHC3 and APT2 as the specific ZDHHC and APT/ABHD enzymes that are responsible for the s-acylation and de-acylation of NLRP3, respectively. They show that the levels of ZDHHC3 and APT2, both localized at the Golgi, control the level of palmitoylation of NLRP3. The S-acylation-mediated membrane targeting of NLRP3 cooperates with polybasic domain (PBD)-mediated PI4P-binding to target NLRP3 to the TGN under steady-state conditions and to the disassembled TGN induced by the NLRP3 activator nigericin.

However, the study has several weaknesses in its current form as outlined below.

(1) The novelty of the findings concerning cys130 palmitoylation in NLRP3 is unfortunately compromised by recent reports on the acylation of different cysteines in NLRP3 (PMID: 38092000), including palmitoylation of the very same cys130 in NLRP3 (Yu et al https://doi.org/10.1101/2023.11.07.566005), which was shown to be relevant for NLRP3 activation in cell and animal models. What remains novel and intriguing is the finding that NLRP3 activators induce an imbalance in the acylation-deacylation cycle by segregating NLRP3 in late Golgi/endosomes from de-acylating enzymes confined in the Golgi. The interesting hypothesis put forward by the authors is that the increased palmitoylation of cys130 would finally contribute to the activation of NLRP3. However, the authors should clarify the trafficking pathway of acylated-NLRP3. This pathway should, in principle, coincide with that of TGN46 which constitutively recycles from the TGN to the plasma membrane and is trapped in endosomes upon treatment with nigericin.

(2) To affect the S-acylation, the authors used 16 hrs treatment with 2-bromopalmitate (2-BP). In Figure 1f, it is quite clear that NLRP3 in 2-BP treated cells completely redistributed in spots dispersed throughout the cells upon nigericin treatment. What is the Golgi like in those cells? In other words, does 2-BP alter/affect Golgi morphology? What about PI4P levels after 2-BP treatment? These are important missing pieces of data since both the localization of many proteins and the activity of one key PI4K in the Golgi (i.e. PI4KIIalpha) are regulated by palmitoylation.

(3) The authors argue that the spots observed with NLRP-GFP result from non-specific effects mediated by the addition of the GFP tag to the NLRP3 protein. However, puncta are visible upon nigericin treatment, as a hallmark of endosomal activation. How do the authors reconcile these data? Along the same lines, the NLRP3-C130S mutant behaves similarly to wt NLRP3 upon 2-BP treatment (Figure 1h). Are those NLRP3-C130S puncta positive for endosomal markers? Are they still positive for TGN46? Are they positive for PI4P?

(4) The authors expressed the minimal NLRP3 region to identify the domain required for NLRP3 Golgi localization. These experiments were performed in control cells. It might be informative to perform the same experiments upon nigericin treatment to investigate the ability of NLRP3 to recognize activating signals. It has been reported that PI4P increases on Golgi and endosomes upon NG treatment. Hence, all the differences between the domains may be lost or preserved. In parallel, also the timing of such recruitment upon nigericin treatment (early or late event) may be informative for the dynamics of the process and of the contribution of the single protein domains.

(5) As noted above for the chemical inhibitors (1) the authors should check the impact of altering the balance between acyl transferase and de-acylases on the Golgi organization and PI4P levels. What is the effect of overexpressing PATs on Golgi functions?

Reviewer #1 (Public Review):

Summary<br /> In this work, Mouelhi et al investigated how the nucleus responds to long term confinement. They find that short-term confinement does not affect nuclear volume, whereas long-term confinement leads to a decrease in volume. The authors propose this decrease occurs after mitosis and relies on cPLA2 and myosin contractility.

Strengths

The ability to accurately control cell confinement allows authors to determine its effects on cellular function with high resolution. This provides a good addition to the existing collection of tools used for cellular micromanipulation. The results provided are relevant and timely and could help understand how cancer cells adapt to conditions of confinement.

Weaknesses

I have a few concerns which I believe should be addressed:

(1) It is unclear whether the authors took into consideration the contribution of nuclear blebs for nuclear volume measurements. This would be particularly relevant in situations of very strong confinement. Blebs were previously shown to affect volume (Mistriotis et al., JCB 2019). One could argue that the decreased nuclear volume was due to the increased blebbing observed in very strong confinements.

(2) From their experimental setup, it is unclear whether the reduced nuclear volume observed after confined cell division arises from a geometrical constraint or is due to an intrinsic nuclear feature. One could argue that cells exiting mitosis under confinement have clustered chromosomes and, therefore, will have decreased volume. This would imply that the nucleus is not "reset" but rather that a geometrical constraint is forcing nuclei to be smaller. One way to test this would be to follow individual cells under confinement, let them enter mitosis, and then release the confinement. If, under these conditions, the daughter nuclei are smaller, then it supports their model. If daughter nuclei recover to their initial value, then it´s simply due to a geometrical constraint that forces the clustering of chromosomes and the reassembly of the NE in a confined space.

(3) The authors claim that the nucleus adapts to confinement based on evidence that the nucleus no longer shrinks in the second division following the first division. I would argue no further decrease is possible because the DNA is already compacted in the smallest possible volume. If indeed nuclei are in a new homeostatic state as the authors claim, then one would expect nuclei to remain smaller even after confinement is removed. This analysis is missing.

(4) Also, if the authors want to claim that this is a mechanism used for cancer cells to adapt to confined situations as the title says, they need to show that normal, near-diploid cells do not behave in the same way. This analysis is missing.

(5) Authors state that "Loss of nuclear blebs is clearly linked to mitosis, suggesting that nuclear volume and nuclear envelope tension are tightly coupled, and supports the hypothesis that mitosis is a key regulator of nuclear envelope tension". I have a few issues with the way this sentence is written. Firstly, one could say that all nuclear structures (and not only blebs) are lost during mitosis because the nucleus disassembles. Hence, the new homeostatic state could be determined by envelope reassembly after mitosis and not mitosis itself. Secondly, I don´t understand why the loss of nuclear blebs suggests that volume and tension are tightly coupled. Thirdly, how can mitosis be a key regulator of nuclear envelope tension when the nucleus is disassembled during the process? These require clarification.

(6) The authors claim that, unlike previous studies (Lomakin et al), this work shows a "gradual nuclear adaptation". From their results, this is difficult to conclude simply because they do not analyse cPLA2 levels. This is solely based on indirect evidence obtained from cPLA2 inhibition. A gradual adaptation would mean that based on the level of confinement we would expect to have increasingly higher levels of cPLA2 (and therefore nuclear tension).

(7) The authors should refrain from saying that the mechanism behind DNA repair is coupled to the nuclear adaptation they show. There are several points regarding this statement. Firstly, increased DNA damage could be due to nuclear ruptures imposed by confinement at 2h. In fact, the authors show leakage of NLS from the nucleus after confinement (Figure S3A). Secondly, the decrease in DNA damage at 24h could be because these nuclei did not rupture. How can they ensure that cells with low DNA damage at 24h had increased DNA damage at 2h? Finally, one needs to confirm if the nuclei they are analysing at 24h did undergo a round of cell division previously. From the evidence provided, the authors cannot conclude that DNA damage regulation is occurring in confined cells. Moreover, cell cycle arrest is a known effect of DNA damage. Cells with high damage at 2h most likely are arrested or will present with increased mitotic errors (which the authors exclude from their analyses).

Reviewer #1 (Public Review):

Summary:

The authors aim to address a critical challenge in the field of bioinformatics: the accurate and efficient identification of protein binding sites from sequences. Their work seeks to overcome the limitations of current methods, which largely depend on multiple sequence alignments or experimental protein structures, by introducing GPSite, a multi-task network designed to predict binding residues of various molecules on proteins using ESMFold.

Strengths:

(1) Benchmarking. The authors provide a comprehensive benchmark against multiple methods, showcasing the performances of a large number of methods in various scenarios.

(2) Accessibility and Ease of Use. GPSite is highlighted as a freely accessible tool with user-friendly features on their website, enhancing its potential for widespread adoption in the research community.

Weaknesses:

(1) Lack of significant insights. The paper reproduces results and analyses already presented in previous literature, without providing significant novel analysis or interpretation. However, they show a novel method with an original approach.

The work is useful for the field, especially in disease mechanism elucidation and novel drug design. The availability of genome-scale binding residue annotations GPSite offers is a significant advancement.

Reviewer #1 (Public Review):

Summary:

This manuscript examined the impact of prenatal alcohol exposure on genome-wide DNA methylation in the brain and liver, comparing ethanol-exposed mice to unexposed controls. They also investigated whether a high-methyl diet (HMD) could prevent the DNA methylation alterations caused by alcohol. Using bisulfite sequencing (n=4 per group), they identified 78 alcohol-associated differentially methylated regions (DMRs) in the brain and 759 DMRs in the liver, of which 85% and 84% were mitigated by the HMD group, respectively. The authors further validated 7 DMRs in humans using previously published data from a Canadian cohort of children with FASD.

Overall, the findings from this study provide new insight into the impact of prenatal alcohol exposure, while also showing evidence for methyl-rich diets as an intervention to prevent the effects of alcohol on the epigenome. Some methodological concerns and confounders limit the robustness of these results, and should be addressed in future studies to further strengthen the conclusions of this study and its applicability to broader settings.

Strengths:

- The use of whole genome bisulfite sequencing allowed for the interrogation of the entire DNA methylome and DMR analysis, rather than a subset of CpGs.<br /> - The combination of data from animal models and humans allowed the authors to make stronger inferences regarding their findings<br /> - The authors investigated a potential mechanism (high methyl diet) to buffer against the effects of prenatal alcohol exposure, which increases the relevance and applicability of this research.

Weaknesses:

- The sample size was small for the epigenetic analyses, which limits the robustness of the findings.<br /> - The authors could not account for potential confounders in their analyses, including birthweight, alcohol levels, and sex. This is a particular problem for the high-methyl diet analyses, in which the alcohol-exposed mice consumed less alcohol than their non-diet counterparts.

Joint Public Review:

In this manuscript, Xue and colleagues investigate the fundamental aspects of cellular fate decisions and differentiation, focusing on the dynamic behaviour of gene regulatory networks. It explores the debate between static (noise-driven) and dynamic (signal-driven) perspectives within Waddington's epigenetic landscape, highlighting the essential role of gene regulatory networks in this process. The authors propose an integrated analysis of fate-decision modes and gene regulatory networks, using the Cross-Inhibition with Self-activation (CIS) network as a model. Through mathematical modelling, they differentiate two logic modes and their effect on cell fate decisions: requires both the presence of an activator and absence of a repressor (AA configuration) with one where transcription occurs as long the repressor is not the only species on the promoter (OO configuration).

The authors establish a relationship between noise profiles, logic-motifs, and fate-decision modes, showing that defining any two of these properties allows the inference of the third. They also identify, under the signal-driven mode, two fundamental patterns of cell fate decisions: either prioritising progression or accuracy in the differentiation process. The authors apply this analysis to available high-throughput datasets of cell fate decisions in hematopoiesis and embryogenesis, proposing the underlying driving force in each case and utilising the observed noise patterns to nominate key regulators.

The paper significantly advances our understanding of gene regulatory networks through a well-described computational study, where the authors rigorously evaluate assumptions in modelling. Particularly commendable is their introduction of the concept of combinatorial logic, exemplified by the double 'and' and double 'or' (AA/OO) logic motifs, which they successfully map to previously described cell fate decision processes. This theoretical and computational exploration sheds light on the dynamic landscape of epigenetic cell fate decisions, emphasising the role of combinatorial logic in coordinating noise and signal-driven processes. The thorough comparison of two model configurations underscores the importance of integration logic, contributing to a clearer understanding of gene regulatory network dynamics. Importantly, the results of the simulations are presented clearly, enhancing accessibility and intuitive understanding. The paper's strength also lies in its predictive power, as the authors use simulations to make insightful predictions about the regulatory organisation of stem cell differentiation systems. While the exploration is restricted to specific scenarios, these limitations serve to highlight areas for future research rather than detract from the paper's strengths.

While the paper presents an intriguing framework for understanding gene regulatory networks and cell fate decisions, there are some weaknesses that warrant attention. Firstly, the framework would benefit from validation with more experimental data and application to diverse systems beyond those explored in the study, such as de-differentiation in adult tissues and regeneration processes. Additionally, while the authors successfully make predictions about the regulatory organisation of stem cell differentiation systems, there is a lack of discussion regarding how perturbations in the regulatory network could affect cell fate decisions. Furthermore, the paper could be strengthened by addressing the effects of mutations and other perturbations that may significantly influence cell fate decision-making processes, thus enhancing the robustness of the findings. Finally, there are instances where the clarity of the writing could be improved to enhance understanding and accessibility for readers.

Reviewer #1 (Public Review):

Induction of beta cell regeneration is a promising approach for the treatment of diabetes. In this study, Massoz et.al., identified calcineurin (CaN) as a new potential modulator of beta cell regeneration by using zebrafish as model. They also showed that calcineurin (CaN) works together with Notch signaling to promote the beta cell regeneration. Overall, the paper is well organized, and technically sound. However, some evidences seem weak to get the conclusion.

Reviewer #1 (Public Review):

Summary:

The research study under review investigated the relationship between the gut and identified potential biomarkers derived from the nasopharyngeal and gut microbiota-based that could aid in predicting COVID-19 severity. The study reported significant changes in the richness and Shannon diversity index in nasopharyngeal microbiome associated with severe symptoms. The study showed a high abundance of Bacillota and Pesudomonadota in patients exhibiting severe symptomatology. Positive correlations were also found between Corynebacterium, Acinetobacter, Staphylococcus, and Veillonella, with the severity of SARS-CoV-2 infection.

Strengths:

The study successfully identified differences in the microbiome diversity that could indicate or predict disease severity. Furthermore, the authors demonstrated a link between individual nasopharyngeal organisms and the severity of SARS-CoV-2 infection. The density of the nasopharyngeal organism was shown to be a potential predictor of the severity of COVID-19.

Weaknesses:

The authors claimed an association between nasopharyngeal organisms and severity of SARS-CoV-2 infection but omitted essential data on the statistical significance of these associations between groups. The authors frequently referred to a p-value < 0.05 without presenting the actual p-values and percentages to show the significance of their results. The discussion is hard to understand (lacked clarity), as it contained an extensive literature review without discussing the study findings. A more focused discussion and results section on the main findings could have improved the overall readability of the paper. The role of potential confounders, such as HIV infection, and ethnicity which impacts the nasopharyngeal microbiome composition, was not included in the paper. Addressing the potential confounders would contribute to a more comprehensive understanding of the study's implications, specifically the role of the nasopharyngeal microbiome as a predictor of COVID-19 severity.

Joint Public Review:

This study investigates the role of Ikaros, a zinc finger family transcription factor related to Helios and Eos, in T-regulatory (Treg) cell functionality in mice. Through genome-wide association studies and chromatin accessibility studies, the authors find that Ikaros shares similar binding sites to Foxp3. Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibits Th1-like gene expression with abnormal expression of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signalling. Further, two models of inflammatory/ autoimmune diseases - Inflammatory Bowel Disease (IBD) and organ transplantation - are employed to examine the functional role of Ikaros in Treg-mediated immune suppression. The authors provide a detailed analysis of the epigenome and transcriptome of Ikaros-deficient Treg cells.

These studies establish Ikaros as a factor required in Treg for tolerance and the control of inflammatory immune responses. The data are of high quality. Overall, the study is well organized, and reports new data consolidating mechanistic aspects of Foxp3 mediated gene expression program in Treg cells.

Strengths:

The authors have performed biochemical studies focusing on mechanistic aspects of molecular functions of the Foxp3-mediated gene expression program and complemented these with functional experiments using two models of autoimmune diseases, thereby strengthening the study. The studies are comprehensive at both the cellular and molecular levels. The manuscript is well organized and presents a plethora of data regarding the transcriptomic landscape of these cells.

Weakness:

The findings of markedly increased percentages of activated conventional T cells (CD44hi), major increases in TFH cells, and elevated serum Ig levels indicate disrupted immune homeostasis even in the absence of overt autoimmune manifestations seen in histopathology. Thus, some of the observed genetic changes observed by the authors are likely Treg cell extrinsic. Further, clear conclusions from the genome-wide studies are lacking.

Reviewer #1 (Public Review):

The manuscript describes the development of a mouse model that co-expresses a fluorescent protein ZsGreen) marker in gene fusion with the FSHR gene.

The authors are correct in that there is a lack of reliable antibodies against many of the GPCR family members. The approach is novel and interesting, with the potential to help understand the expression pattern of gonadotropin receptors. There has been a very long debate about the expression of gonadotropin receptors in other tissues other than gonads. While their expression of the FSHR in some of those tissues has been detected by a variety of methods, their physiological, or pathophysiological, function(s) remain elusive.

The authors in this manuscript assume that the expression of ZsGren and the FSHR are equal. While this is correct genetically (transcription->translation) it does not go hand in hand with other posttranslational processes.

(1) One of the shocking observations in this manuscript is the expression of FSHR in Leydig cells. Other observations are in the osteoblasts and endothelial cells as well as epithelial cells in different organs. The expression of ZsGreen in these tissues seems high and one shall start questioning if there are other mechanisms at play here.

First, the turnover of fluorescent proteins is long, longer than 48h, which means that they accumulate at a different speed than the endogenous FSHR This means that ZsGreen will accumulate in time while the FSHR receptor might be degraded almost immediately. This correlated with mRNA expression (by the authors) but does not with the results of other studies in single-cell sequencing (see below).

The expression of ZsGreen in Leydig cells seems much higher than in Sertoli cells, this is "disturbing" to put it mildly. This is visible in both the ZsGreen expression and the FISH assay (Figure 2 B-D).

(2) The expression in WAT and BAT is also questionable as the expression of ZsGreen is high everywhere. That makes it difficult to believe that the images are truly informative. For example, the stainings of aorta show the ZsGreen expression where elastin and collagen fibres are - these are not "cells" and therefore are not expressing ZsGreen.

(3) FISH expression (for FSHR) in WT mice is missing.

Also, the tissue sections were stained with the IgG only (neg control) but in practice both the KI and the WT tissues should be stained with the primary and secondary antibodies. The only control that I could think of to truly get a sense of this would be a tagged receptor (N-terminal) that could then be analysed by immunohistochemistry.

(4) The authors also claim:<br /> To functionally prove the presence of FSHR in osteoblasts/osteocytes, we also deleted FSHR in osteocytes using an inducible model. The conditional knockout of FSHR triggered a much more profound increase in bone mass and decrease in fat mass than blockade by FSHR antibodies (unpublished data).

This would be a good control for all their images. I think it is necessary to make the large claim of extragonadal expression, as well as intragonadal such as Leydig cells.

(5) Claiming that the under-developed Leydig cells in FSHR KO animals are due to a direct effect of the FSHR, and not via a cross-talk between Sertoli and Leydig cells, is too much of a claim. It might be speculated to some degree but as written at the moment it suggests this is "proven".

(6) We also do not know if this FSHR expressed is a spliced form that would also result in the expression of ZsGreen but in a non-functional FSHR, or whether the FSHR is immediately degraded after expression. The insertion of the ZsGreen might have disturbed the epigenetics, transcription, or biosynthesis of the mRNA regulation.

(7) The authors should go through single-cell data of WT mice to show the existence of the FSHR transcript(s).<br /> For example here:<br /> https://www.nature.com/articles/sdata2018192

Reviewer #1 (Public Review):

As a pathogen, S. aureus has evolved strategies to evade the host's immune system. It effectively remains 'under the radar' in the host until it reaches high population densities, at which point it triggers virulence mechanisms, enabling it to spread within the host. The agr quorum sensing system is central to this process, as it coordinates the pathogen's virulence in response to its cell density.

In this study, Podkowik and colleagues suggest that cells activating agr signaling also benefit from protection against H2O2 stress, whereas inactivation of agr increases cell death. The underlying cause of this lack of protection is tied to an ATP deficit in the agr mutant, leading to increased glucose consumption and NADH production, ultimately resulting in a redox imbalance. In response to this imbalance, the agr mutant increases respiration, resulting in the endogenous production of ROS which synergizes with H2O2 to mediate killing of the agr mutant. Suppressing respiration in the agr mutant restored protection against H2O2 stress.

Additionally, the authors establish that agr-dependent protection against oxidative stress is also linked to RNAIII activation, and the subsequent block of Rot translation. However, the specific protective genes regulated by Rot remain unidentified. Thus, according to the evidence provided, agr triggers intrinsic mechanisms that not only decrease harmful ROS production within the cell but also alleviate its detrimental effects.

Interestingly, these protective mechanisms are long-lived, and guard the cells against external oxidative stressors such as H2O2, even after the agr system has been 'turned off' in the population.

Reviewer #1 (Public Review):

Summary:

Del Rosario et al characterized the extent and cell types of sibling chimerism in marmosets. To do so, they took advantage of the thousands of SNPs that are transcribed in single-nucleus RNA-seq (snRNA-seq) data to identify the sibling genotype of origin for all sequenced cells across 4 tissues (blood, liver, kidney, and brain) from many marmosets. They found that chimerism is prevalent and widespread across tissues in marmosets, which has previously been shown. However, their snRNA-seq approach allowed them to identify precisely which cells were of sibling origin, and which were not. In doing so they definitively show that sibling chimerism across tissues is limited to cells of myeloid and lymphoid lineages. The authors then focus on a large sample of microglia sequenced across many brain regions to quantify: (1) variation in chimerism across brain regions in the same individual, and (2) the relative importance of genetic vs. environmental context on microglia function/identity.

(1) Much like across different tissues in the same individual, they found that the proportion of chimeric microglia varies across brain regions collected from the same individuals (as well as differing from the proportion of sibling cells found in the blood of the same animals), suggesting that cells from different genetic backgrounds may differ in their recruitment and/or proliferation across regions and local tissue contexts, or that this may be linked to stochastic bottleneck effects during brain development.

(2) Their (admittedly smaller sample size) analyses of host-sibling gene expression showed that the local environment dominates genotype.

All told, this thoughtful and thorough manuscript accomplishes two important goals. First, it all but closes a previously open question on the extent and cell origins of sibling chimerism. Second, it sets the stage for using this unique model system to examine, in a natural context, how genetic variation in microglia may impact brain development, function, and disease.

The conclusions of this paper are well supported by the data, and the authors exert appropriate care when extrapolating their results that come from smaller samples. However, there are a few concerns that should be addressed.

The "modest correlation" mentioned in lines 170-172 does not take into account the uncertainty in estimates of each chimeric cell proportion (although the plot shows those estimates nicely). This is particularly important for the macrophages, which are far less abundant. Perhaps a more appropriate way to model this would be in a binomial framework (with a random effect for individuals of origin). Here, you could model the sibling identity of each macrophage as a function of the proportion of sibling-origin microglia and then directly estimate the percent variance explained.

A similar (albeit more complicated because of the number of regions being compared) approach could be applied to more rigorously quantify the variation in chimerism across brain regions (L198-215; Figure 4). This would also help to answer the question of whether specific brain regions are more "amenable" to microglia chimerism than others.

While the sample size is small, it would be exciting to see if any microglia eQTL are driven by sibling chimerism across the marmosets.

L290-292: The authors should propose ways in which they could test the two different explanations proposed in this paragraph. For instance, a simulation-based modeling approach could potentially differentiate more stochastic bottleneck effects from recruitment-like effects.

While intriguing, the gene expression comparison (Figure 5) is extremely underpowered. It would be helpful to clarify this and note the statistical thresholds used for identifying DEGs (the black points in the figure).

Reviewer #1 (Public Review):

Summary:<br /> The study presented by Atsumi et al. is about using smartphone-driven, community-sourced data to enhance biodiversity monitoring. The idea is to leverage the widespread use of smartphones to gather data from the community quickly, contributing to a more comprehensive understanding of biodiversity. The authors discuss the importance of ecosystem services linked to biodiversity and the threats posed by human activities. It emphasizes the need for comprehensive biodiversity data to implement the Kunming-Montreal Global Biodiversity Framework. The 'Biome' mobile app, launched in Japan, uses species identification algorithms and gamification to gather over 6 million observations since 2019. While community-sourced data may have biases, incorporating it into Species Distribution Models (SDMs) improves accuracy, especially for endangered species. The app covers urban-natural gradients uniformly, enhancing traditional survey data biased towards natural areas. Combining these sources provides valuable insights into species distributions for conservation, protected area designation, and ecosystem service assessment.

Strengths:

The use of a smartphone app ('Biome') for community-driven species occurrence data collection represents an innovative and inclusive approach to biodiversity monitoring, leveraging the widespread use of smartphones. The app has successfully accumulated a large volume of species occurrence data since its launch in 2019, showcasing its effectiveness in rapidly gathering information from diverse locations. Despite challenges with certain taxa, the study highlights high species identification accuracy, especially for birds, reptiles, mammals, and amphibians, making the 'Biome' app a reliable tool for species observation. The integration of community-sourced data into Species Distribution Models (SDMs) improves the accuracy of predicting species distributions. This has implications for conservation planning, including the designation of protected areas and assessment of ecosystem services. The rapid accumulation of data and advancements in machine learning methods open up opportunities for conducting time-series analyses, contributing to the understanding of ecosystem stability and interaction strength over time. The study emphasizes the collaborative nature of the platform, fostering collaboration among diverse stakeholders, including local communities, private companies, and government agencies. This inclusive approach is essential for effective biodiversity assessment and decision-making. The platform's engagement with various stakeholders, including local communities, supports biodiversity assessment, management planning, and informed decision-making. Additionally, the app's role in fostering nature-positive awareness in society is highlighted as a significant contribution to creating a sustainable society.

Weaknesses:

While the studies make significant contributions to biodiversity monitoring, they also have some weaknesses. Firstly, relying on smartphone-driven, community-sourced data may introduce spatial and taxonomic biases. The 'Biome' app, for example, showed lower accuracy for certain taxa like seed plants, molluscs, and fishes, potentially impacting the reliability of the gathered data. Furthermore, the effectiveness of Species Distribution Models (SDMs) relies on the assumption that biases in community-sourced data can be adequately accounted for. The unique distribution patterns of the 'Biome' data, covering urban-natural gradients uniformly, might not fully represent the diversity of certain ecosystems, potentially leading to inaccuracies in the models. Moreover, the divergence in data distribution patterns along environmental gradients between 'Biome' data and traditional survey data raises concerns. The app data shows a more uniform distribution across natural-urban gradients, while traditional data is biased towards natural areas. This discrepancy may impact the representation of certain ecosystems and influence the accuracy of Species Distribution Models (SDMs). While the integration of 'Biome' data into SDMs improves accuracy, the study notes that controlling the sampling efforts is crucial. Spatially-biased sampling efforts in community-sourced data need careful consideration, and efforts to control biases are essential for reliable predictions.

Reviewer #1 (Public Review):

Summary:

The manuscript presents a short report investigating mismatch responses in the auditory cortex, following previous studies focused on the visual cortex. By correlating the mouse locomotion speed with acoustic feedback levels, the authors demonstrate excitatory responses in a subset of neurons to halts in expected acoustic feedback. They show a lack of responses to mismatch in the visual modality. A subset of neurons show enhanced mismatch responses when both auditory and visual modalities are coupled to the animal's locomotion.

While the study is well-designed and addresses a timely question, several concerns exist regarding the quantification of animal behavior, potential alternative explanations for recorded signals, correlation between excitatory responses and animal velocity, discrepancies in reported values, and clarity regarding the identity of certain neurons.

Strengths:

(1) Well-designed study addressing a timely question in the field.

(2) Successful transition from previous work focused on the visual cortex to the auditory cortex, demonstrating generic principles in mismatch responses.

(3) The correlation between mouse locomotion speed and acoustic feedback levels provides evidence for a prediction signal in the auditory cortex.

(4) Coupling of visual and auditory feedback shows putative multimodal integration in the auditory cortex.

Weaknesses:

(1) Lack of quantification of animal behavior upon mismatches, potentially leading to alternative interpretations of recorded signals.

(2) Unclear correlation between excitatory responses and animal velocity during halts, particularly in closed-loop versus playback conditions.

(3) Discrepancies in reported values in a few figure panels raise questions about data consistency and interpretation.

(4) Ambiguity regarding the identity of the [AM+VM] MM neurons.

Reviewer #1 (Public Review):

Summary:

The authors were trying to understand the relation between the development of large trunks and longirrostrine mandibles in bunodont proboscideans of Miocene, and how it reflects the variation in diet patterns.

Strengths:

The study is very well supported, written, and illustrated, with plenty Supplementary materials. The authors included all Asian bunodont proboscideans with long mandibles and I suggest that they should use the expression "bunodont proboscideans" instead of gomphotheres.

Weaknesses:

I believe that the only weakness is the lack of discussion comparing their results with the development of gigantism and long limbs in proboscideans from the same epoch.

The authors reviewed the manuscript according to my suggestions and responded well to all my comments.

Reviewer #1 (Public Review):

Summary:

This manuscript investigates the regulation of chlorophyll biosynthesis in rice embryos, focusing on the role of OsNF-YB7. The rigorous experimental approach, combining genetic, biochemical, and molecular analyses, provides a robust foundation for these findings. The research achieves its objectives, offering new insights into chlorophyll biosynthesis regulation, with the results convincingly supporting the authors' conclusions.

Strengths:

The major strengths include the detailed experimental design and the findings regarding OsNF-YB7's inhibitory role.

Weaknesses:

However, the manuscript's discussion on the practical implications for agriculture and the evolutionary analysis of regulatory mechanisms could be expanded.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e, and f) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that the global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control the translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

Strengths:

Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

Weaknesses:

The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging. To further support the authors' claims, several improvements are necessary, particularly in the methods of quantification and the points that should be demonstrated quantitatively. It is crucial to investigate the correlation between aging and the proteins eIF2β and eIF2α.

Reviewer #1 (Public Review):

Here the authors discuss mechanisms of ligand binding and conformational changes in GlnBP (a small E Coli periplasmic binding protein, which binds and carries L-glutamine to the inner membrane ATP-binding cassette (ABC) transporter). The authors have distinguished records in this area and have published seminal works. They include experimentalists and computational scientists. Accordingly, they provide comprehensive, high-quality, experimental and computational work.

They observe that apo- and holo- GlnBP does not generate detectable exchange between open and (semi-) closed conformations on timescales between 100 ns and 10 ms. Especially, the ligand binding and conformational changes in GlnBP that they observe are highly correlated. Their analysis of the results indicates a dominant induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. They then suggest that an approach resembling the one they undertook can be applied to other protein systems where the coupling mechanism of conformational changes and ligand binding.

They argue that the intuitive model where ligand binding triggers a functionally relevant conformational change was challenged by structural experiments and MD simulations revealing the existence of unliganded closed or semi-closed states and their dynamic exchange with open unbound conformations, discuss alternative mechanisms that were proposed, their merits and difficulties, concluding that the findings were controversial, which, they suggest is due to insufficient availability of experimental evidence to distinguish them. As to further specific conclusions they draw from their results, they determine that a conformational selection mechanism is incompatible with their results, but induced fit is. They thus propose induced fit as the dominant pathway for GlnBP, further supported by the notion that the open conformation is much more likely to bind substrate than the closed one based on steric arguments.

Considering the landscape of substrate-free states, in my view, the closed state is likely to be the most stable and, thus most highly populated. As the authors note and I agree that state can be sterically infeasible for a deep-pocketed substrate. As indeed they also underscore, there is likely to be a range of open states. If the populations of certain states are extremely low, they may not be detected by the experimental (or computational) methods. The free energy landscape of the protein can populate all possible states, with the populations determined by their relative energies. In principle, the protein can visit all states. Whether a particular state is observed depends on the time the protein spends in that state. The frequencies, or propensities, of the visits can determine the protein function. As to a specific order of events, in my view, there isn't any. It is a matter of probabilities which depend on the populations (energies) of the states. The open conformation that is likely to bind is the most favorable, permitting substrate access, followed by minor, induced fit conformational changes. However, a key factor is the ligand concentration. Ligand binding requires overcoming barriers to sustain the equilibrium of the unliganded ensemble, thus time. If the population of the state is low, and ligand concentration is high (often the case in in vitro experiments, and high drug dosage scenarios) binding is likely to take place across a range of available states.

This is however a personal interpretation of the data. The paper here, which clearly embodies massive careful, and high-quality work, is extensive, making use of a range of experimental approaches, including isothermal titration calorimetry, single-molecule Förster resonance energy transfer, and surface-plasmon resonance spectroscopy. The problem the authors undertake is of fundamental importance.

Reviewer #2 (Public Review):

Summary:

In this manuscript Nie et al investigate the effect of PARG KO and PARG inhibition (PARGi) on pADPR, DNA damage, cell viability and synthetic lethal interactions in HEK293A and Hela cells. Surprisingly, the authors report that PARG KO cells are sensitive to PARGi and show higher pADPR levels than PARG KO cells, which is abrogated upon deletion or inhibition of PARP1/PARP2. The authors explain the sensitivity of PARG KO to PARGi through incomplete PARG depletion and demonstrate complete loss of PARG activity when incomplete PARG KO cells are transfected with additional gRNAs in the presence of PARPi. Furthermore, the authors show that the sensitivity of PARG KO cells to PARGi is not caused by NAD depletion but by S-phase accumulation of pADPR on chromatin coming from unligated Okazaki fragments, which are recognized and bound by PARP1. Consistently, PARG KO or PARG inhibition show synthetic lethality with Pol beta, which is required for Okazaki fragment maturation. PARG expression levels in ovarian cancer cell lines correlate negatively with their sensitivity to PARGi.

Reviewer #1 (Public Review):

Summary:

The paper measures the prevalence and mortality of stroke and its comorbidities across geographic regions in order to find differences in risks that may lead to more effective guidance for these subpopulations. It also does a genetic analysis to look for variants that may drive these phenotypic variations.

Strengths:

The data provided here will provide a foundation for a lot of future research into the causes of the observed correlations as well as whether the observed differences in comorbidities across regions have clinically relevant effects on risk management.

Weaknesses:

As with any cross-national analysis of rates, the data is vulnerable to differences in classification and reporting across jurisdictions. Furthermore, given the increased death rate from COVID-19 associated with many of these comorbid conditions and the long-term effects of COVID-19 infection on vascular health, it is expected that many of the correlations observed in this dataset will shift along with the shifting health of the underlying populations.

Reviewer #1 (Public Review):

Summary:

The manuscript by Duilio M. Potenza et al. explores the role of Arginase II in cardiac aging, majorly using whole-body arg-ii knock-out mice. In this work, the authors have found that Arg-II exerts non-cell-autonomous effects on aging cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages. The authors have used arg II KO mice and an in vitro culture system to study the role of Arg II. The authors have also reported the cell-autonomous effect of Arg-II through mitochondrial ROS in fibroblasts that contribute to cardiac aging. These findings are sufficiently novel in cardiac aging and provide interesting insights. While the phenotypic data seems strong, the mechanistic details are unclear. How Arg II regulates the IL-1b and modulates cardiac aging is still being determined. The authors still need to determine whether Arg II in fibroblasts and endothelial contributes to cardiac fibrosis and cell death. This study also lacks a comprehensive understanding of the pathways modulated by Arg II to regulate cardiac aging.

Strengths:

This study provides interesting information on the role of Arg II in cardiac aging.

The phenotypic data in the arg II KO mice is convincing, and the authors have assessed most of the aging-related changes.

The data is supported by an in vitro cell culture system.

Weaknesses:

The manuscript needs more mechanistic details on how Arg II regulates IL-1b and modulates cardiac aging.

The authors used whole-body KO mice, and the role of macrophages in cardiac aging is not studied in this model. A macrophage-specific arg II Ko would be a better model.

Experiments need to validate the deficiency of Arg II in cardiomyocytes.

The authors have never investigated the possibility of NO involvement in this mice model.

A co-culture system would be appropriate to understand the non-cell-autonomous functions of macrophages.

The Myocardial infarction data shown in the mice model may not be directly linked to cardiac aging.

Reviewer #1 (Public Review):

Summary:

Semenova et al. have studied a large cross-sectional cohort of people living with HIV on suppressive ART, N=115, and performed high dimensional flow cytometry to then search for associations between immunological and clinical parameters and intact/total HIV DNA levels.

A number of interesting data science/ML approaches were explored on the data and the project seems a serious undertaking. However, like many other studies that have looked for these kinds of associations, there was not a very strong signal. Of course, the goal of unsupervised learning is to find new hypotheses that aren't obvious to human eyes, but I felt in that context, there were (1) results slightly oversold, (2) some questions about methodology in terms mostly of reservoir levels, and (3) results were not sufficiently translated back into meaning in terms of clinical outcomes.

Strengths:

The study is evidently a large and impressive undertaking and combines many cutting-edge statistical techniques with a comprehensive experimental cohort of people living with HIV, notably inclusive of populations underrepresented in HIV science. A number of intriguing hypotheses are put forward that could be explored further. Sharing the data could create a useful repository for more specific analyses.

Weaknesses:

Despite the detailed experiments and methods, there was not a very strong signal for the variable(s) predicting HIV reservoir size. The Spearman coefficients are ~0.3, (somewhat weak, and acknowledged as such) and predictive models reach 70-80% prediction levels, though sometimes categorical variables are challenging to interpret.

There are some questions about methodology, as well as some conclusions that are not completely supported by results, or at minimum not sufficiently contextualized in terms of clinical significance.

On associations: the false discovery rate correction was set at 5%, but data appear underdetermined with fewer observations than variables (144vars > 115ppts), and it isn't always clear if/when variables are related (e.g inverses of one another, for instance, %CD4 and %CD8).

The modeling of reservoir size was unusual, typically intact and defective HIV DNA are analyzed on a log10 scale (both for decays and predicting rebound). Also sometimes in this analysis levels are normalized (presumably to max/min?, e.g. S5), and given the large within-host variation of level we see in other works, it is not trivial to predict any downstream impact of normalization across population vs within-person.

Also, the qualitative characterization of low/high reservoir is not standard and naturally will split by early/later ART if done as above/below median. Given the continuous nature of these data, it seems throughout that predicting above/below median is a little hard to translate into clinical meaning.

Lastly, the work is comprehensive and appears solid, but the code was not shared to see how calculations were performed.

Reviewer #1 (Public Review):

Summary:

The current study aims to quantify associations between the regular use of proton-pump inhibitors (PPI) - defined as using PPI most days of the week during the last 4 weeks at one cross-section in time - with several respiratory outcomes up to several years later in time. There are 6 respiratory outcomes included: risk of influenza, pneumonia, COVID-19, other respiratory tract infections, as well as COVID-19 severity and mortality).

Strengths:

Several sensitivity analyses were performed, including i) estimation of the e-value to assess how strong unmeasured confounders should be to explain observed effects, ii) comparison with another drug with a similar indication to potentially reduce (but not eliminate) confounding by indication.

Weaknesses:

(1) The main exposure of interest seems to be only measured at one time-point in time (at study enrollment) while patients are considered many years at risk afterwards without knowing their exposure status at the time of experiencing the outcome. As indicated by the authors, PPI are sometimes used for only short amounts of time. It seems biologically implausible that an infection was caused by using PPI for a few weeks many years ago.

(2) Previous studies have shown that by focusing on prevalent users of drugs, one often induces several biases such as collider stratification bias, selection bias through depletion of susceptible, etc.

(3) It seems Kaplan Meier curves are not adjusted for confounding through e.g. inverse probability weighting. As such the KM curves are currently not informative (or the authors need to make clearer that curves are actually adjusted for measured confounding).

(4) Throughout the manuscript the authors seem to misuse the term multivariate (using one model with e.g. correlated error terms to assess multiple outcomes at once) when they seem to mean multivariable.

(5) Given multiple outcomes are assessed there is a clear argument for accounting for multiple testing, which following the logic of the authors used in terms of claiming there is no association when results are not significant may change their conclusions. More high-level, the authors should avoid the pitfall of stating there is evidence of absence if there is only an absence of evidence in a better way (no statistically significant association doesn't mean no relationship exists).

(6) While the authors claim that the quantitative bias analysis does show results are robust to unmeasured confounding, I would disagree with this. The e-values are around 2 and it is clearly not implausible that there are one or more unmeasured risk factors that together or alone would have such an effect size. Furthermore, if one would use the same (significance) criteria as used by the authors for determining whether an association exists, the required effect size for an unmeasured confounder to render effects 'statistically non-significant' would be even smaller.

(7) Some patients are excluded due to the absence of follow-up, but it is unclear how that is determined. Is there potentially some selection bias underlying this where those who are less healthy stop participating in the UK biobank?

(8) Given that the exposure is based on self-report how certain can we be that patients e.g. do know that their branded over-the-counter drugs are PPI (e.g. guardium tablets)? Some discussion around this potential issue is lacking.

(9) Details about the deprivation index are needed in the main text as this is a UK-specific variable that will be unfamiliar to most readers.

(10) It is unclear how variables were coded/incorporated from the main text. More details are required, e.g. was age included as a continuous variable and if so was non-linearity considered and how?

(11) The authors state that Schoenfeld residuals were tested, but don't report the test statistics. Could they please provide these, e.g. it would already be informative if they report that all p-values are above a certain value.

(12) The authors would ideally extend their discussion around unmeasured confounding, e.g. using the DAGs provided in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832226/, in particular (but not limited to) around severity and not just presence/absence of comorbidities.

(13) The UK biobank is known to be highly selected for a range of genetic, behavioural, cardiovascular, demographic, and anthropometric traits. The potential problems this might create in terms of collider stratification bias - as highlighted here for example: https://www.nature.com/articles/s41467-020-19478-2 - should be discussed in greater detail and also appreciated more when providing conclusions.

Reviewer #1 (Public Review):

The manuscript, "A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes," developed a unique culture platform with PEG hydrogel that facilitates the in-situ measurement of contractile dynamics of the engineered cardiac rings. The authors optimized the tissue seeding conditions, demonstrated tissue morphology with expressions of cardiac and fibroblast markers, mathematically modeled the equation to derive contractile forces and other parameters based on imaging analysis, and concluded by testing several compounds with known cardiac responses.

The authors answered my questions with appropriate experiments and explanation.

(1) This paper presents an intriguing platform that creates miniature cardiac rings with merely thousands of cardiomyocytes per tissue in a 96-well plate format. The shape of the ring and the squeezing motion can recapitulate the contraction of the cardiac chamber to a certain degree. However, Thavandiran et al. (PNAS 2013) created a larger version of the cardiac ring and found that electrical propagation revealed spontaneous infinite loop-like cycles of activation propagation traversing the ring. This model was used to mimic a reentrant wave during arrhythmia. Therefore, there are concerns about whether a large number of cardiac tissues experience arrhythmia due to geometry-induced re-entry current and cannot be used as a healthy tissue model.

In the new experiment, the authors demonstrated with voltage-sensitive dye that these miniaturized tissues do not experience any arrhythmia, potentially due to their small size.

(2) The platform can produce 21 cardiac rings per well in 96-well plates, with the throughput being the highest among competing platforms. The resulting tissues exhibit good sarcomere striation due to the strain from the pillars. However, emerging questions pertain to culture longevity and reproducibility among tissues. According to Figure 1E, uneven ring formation around the pillar leads to tissue thinning and breakage. Only 50% survival is observed after 20 days of culture in the optimized seeding group. Are there any strategies to improve this survival rate? Additionally, do the cardiac rings detach from the glass slides and roll up, given the two compartments with cardiac and fibroblast-rich regions where fibroblasts maintain attachment to the glass slides? Moreover, the standard deviation of force measurement is a quarter of the value, which is suboptimal given the high replicate number. As the platform utilizes imaging analysis to derive contractile dynamics, calibration based on the angle and distance of the camera lens to individual tissues should be conducted to reduce error. On the other hand, how reproducible are the pillars? It is highly recommended to mechanically evaluate the consistency of the hydrogel-based pillars across different wells and within wells to understand the variance.

The authors stated that the platform has been tested and improved with multiple cell lines to enhance tissue survival rates. The methodology of image capture and calculation of contractile dynamics were explained in detail to address concerns. Moreover, the reproducibility of the pillars was demonstrated by consistent results of Young's Modulus (AFM) from each pillar, showing low standard deviations.

(3) Does the platform allow the observation of non-synchronized beating when testing with compounds? This can be extremely important as the intended applications of this platform are drug testing and cardiac disease modeling. The author should elaborate on the method in the manuscript and explain the obtained results in detail.

Referring to Question #1, the platform does not present arrythmia potentially due to the small size of the tissue.

(4) The results of drug testing are interesting. Isoperenoral is typically causing positive chronotropic and positive inotropic responses, where inotropic responses are difficult to obtain due to low tissue maturity. It is inconsistent with other reported results that cardiac rings do not exhibit increased beating frequency, but slightly increased forces only.

The authors repeated the experiment with the same results and hypothesized that the results would be line-dependent, since the maturation of iPSC-CM is not consistent. The additional dose curves provided more information on the tissue behaviors against well-known compounds.

Overall, the manuscript is well-written, and the designed platform presents unique advantages for high-throughput cardiac tissue culture. The paper has adequate data to demonstrate the proof-of-concept study of the platform. The throughput, consistency of the tissue, and the potential integration of high-throughput automation would be the highlights of this platform.

Reviewer #1 (Public Review):

Thermogenic adipocyte activity associate with cardiometabolic health in humans, but decline with age. Identifying the underlying mechanisms of this decline is therefore highly important.

To address this task, Holman and co-authors present compelling data from their investigations of the effects of two major determinants of thermogenic activity: cold, which induce thermogenic de novo differentiation as well as conversion of dormant thermogenic inguinal adipocytes: and aging, which strongly reduce thermogenic activity. The authors study young and middle-aged mice at thermoneutrality and following cold exposure.

Using linage tracing, the authors conclude that the older group produce less thermogenic adipocytes from progenitor differentiation. However, they found no differences between thermogenic differentiation capacity between the age groups when progenitors are isolated and differentiated in vitro. This finding is consistent with previous findings in humans, demonstrating that progenitor cells derived from dormant perirenal brown fat of humans differentiate into thermogenic adipocytes in vitro. Taken together, this underscores that age-related changes in the microenvironment rather than autonomous alterations in the ASPCs explain the age related decline in thermogenic capacity, This is an important finding in terms of identifying new approaches to switch dormant adipocytes into an active thermogenic phenotype.

To gain insight into the age-related changes, the authors use single cell and single nuclei RNA sequencing mapping of their two age groups, comparing thermoneutral and cold conditions between the two groups. Interestingly, where the literature previously demonstrated that de novo lipogenesis (DNL) occurs in relation to thermogenic activation, the authors show that DNL in fact is activated in a white adipocyte cell type, whereas the beige thermogenic adipocytes form a separate cluster.

Considering recent findings, that adipose tissue contains several subtypes of ASPCs and adipocytes, mapping the changes at single cell resolution following cold intervention provides an important contribution to the field, in particular as an older group with limited thermogenic adaptation is analyzed in parallel with a younger, more responsive group. This model also allowed for detection of microenvironment as a determining factor of thermogenic response.

The use of only two time points (young and middle-aged) along the aging continuum limits the conclusions that can be made on aging as the only driver of the observed differences between the groups. Furthermore, as the authors also discuss, aging is a complex phenotype, and in this case the older mice were heavier and had larger fat depots, which should be taken into consideration when interpreting the data.

In conclusion, this study provides an important resource for further studies, which should investigate how the findings can be translated into humans for reactivation of dormant thermogenic fat and a potential improvement of metabolic health.

Reviewer #1 (Public Review):

Metzger et al develop a rigorous method filling an important unmet need in protein evolution - analysis of protein genetic architecture and evolution using data from combinatorially complete 20^N variant libraries. Addressing this need has become increasingly valuable, as experimental methods for generating these datasets expand in scope and scale. Their method integrates two key features - (1) it reports the effects of mutations relative to the average across all variants, rather than a particular genotype, making it useful for examining global genetic architecture, and (2) it does this for all possible 20 states at each site, in contrast to the binary analyses in prior work. These features are not individually novel but integrating them into a single analysis framework is novel and will be valuable to the protein evolution community. Using a previously published dataset generated by two of the authors, they conclude that (1) changes in function are largely attributable to pairwise but not higher-order interactions, and (2) epistasis potentiates, rather than constrains, evolutionary paths. These findings are well-supported by the data. Overall, this work has important implications for predicting the relationship between genotype and phenotype, which is of considerable interest to protein biochemistry, evolutionary biology, and numerous other fields.

Reviewer #1 (Public Review):

The authors have generated a set of yeast S. cerevisiae strains containing different numbers of chromosomes.<br /> Elimination of telomerase activates homologous recombination (HR) to maintain telomeres in cells containing the original 16 chromosomes. However, elimination of telomerase leads to circularization of cells containing a single or two chromosomes. The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They found that the subtelomeric sequences X and Y' are dispensable for cell proliferation and HR-mediated telomere maintenance in telomerase-minus SY12 cells. They conclude that subtelomeric X and Y' sequences do not play essential roles in both telomerase-proficient and telomerase-null cells and propose that these sequences represent remnants of genome evolution.

Interestingly, telomerase-minus SY12 generates survivors that are different from well-established Type I or Type II survivors. The authors uncover atypical telomere formation which does not depend on the Rad52 homologous recombination pathway.

Strengths:

The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They show that subtelomeres do not have essential roles in telomere maintenance and cell proliferation.

Weaknesses:

It is not fully addressed how atypical survivors are generated independently of Rad52-mediated homologous recombination.<br /> It remains possible that X and Y elements influence homologous recombination, type 1 and type 2 (type X), at telomeres. In particular, the presence of X and Y elements appears to be important for promoting type 1 recombination, although the authors conclude "Elimination of subtelomeric repeat sequences exerts little effect on telomere functions".

Reviewer #1 (Public Review):

Summary: In this article, Mirza et al developed a continuum active gel model of actomyosin cytoskeleton that account for nematic order and density variations in actomyosin. Using this model, they identify the requirements for the formation of dense nematic structures. In particular, they show that self-organization into nematic bundles requires both flow-induced alignment and active tension anisotropy in the system. By varying model parameters that control active tension and nematic alignment, the authors show that their model reproduces a rich variety of actomyosin structures, including tactoids, fibres, asters as well as crystalline networks. Additionally, discrete simulations are employed to calculate the activity parameters in the continuum model, providing a microscopic perspective on the conditions driving the formation of fibrillar patterns.

Strengths: The strength of the work lies in its delineation of the parameter ranges that generate distinct types of nematic organization within actomyosin networks. The authors pinpoint the physical mechanisms behind the formation of fibrillar patterns, which may offer valuable insights into stress fiber assembly. Another strength of the work is connecting activity parameters in the continuum theory with microscopic simulations.

Weaknesses: This paper is a very difficult read for nonspecialists, especially if you are not well-versed in continuum hydrodynamic theories. Efforts should be made to connect various elements of theory with biological mechanisms, which is mostly lacking in this paper. The comparison with experiments is predominantly qualitative. It is unclear if the theory is suited for in vitro or in vivo actomyosin systems. The justification for various model assumptions, especially concerning their applicability to actomyosin networks, requires a more thorough examination. The classification of different structures demands further justification. For example, the rationale behind categorizing structures as sarcomeric remains unclear when nematic order is perpendicular to the axis of the bands. Sarcomeres traditionally exhibit a specific ordering of actin filaments with alternating polarity patterns. Similarly, the criteria for distinguishing between contractile and extensile structures need clarification, as one would expect extensile structures to be under tension contrary to the authors' claim. Additionally, its unclear if the model's predictions for fiber dynamics align with observations in cells, as stress fibers exhibit a high degree of dynamism and tend to coalesce with neighboring fibers during their assembly phase. Finally, it seems that the microscopic model is unable to recapitulate the density patterns predicted by the continuum theory, raising questions about the suitability of the simulation model.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Liu et al. used scRNA-seq to characterize cell type-specific responses during allergic contact dermatitis (ACD) in a mouse model, specifically the hapten-induced DNFB model. Using the scRNA-seq data, they deconvolved the cell types responsible for the expression of major inflammatory cytokines such as IFNG (from CD4 and CD8 T cells), IL4/13 (from basophils), IL17A (from gd T cells), and IL1B from neutrophils and macrophages. They found the highest upregulation of a type 1 inflammatory response, centering around IFNG produced by CD4 and CD8 T cells. They further identified a subpopulation of dermal fibroblasts that upregulate CXCL9/10 during ACD and provided functional genetic evidence in their mouse model that disrupting IFNG signaling to fibroblasts decreases CD8 T cell infiltration and overall inflammation. They identify an increase in IFNG-expressing CD8 T cells in human patient samples of ACD vs. healthy control skin and co-localization of CD8 T cells with PDGFRA+ fibroblasts, which suggests this mechanism is relevant to human ACD. This mechanism is reminiscent of recent work (Xu et al., Nature 2022) showing that IFNG signaling in dermal fibroblasts upregulates CXCL9/10 to recruit CD8 T cells in a mouse model of vitiligo. Overall, this is a very well-presented, clear, and comprehensive manuscript. The conclusions of the study are mostly well supported by data, but some aspects of the work could be improved by additional clarification of the identity of the cell types shown to be involved, including the exact subpopulation discovered by scRNA-seq and the subtype of CD8 T cell involved. The study was limited by its use of one ACD model (DNFB), which prevents an assessment of how broadly relevant this axis is. The human sample validation is slightly circumstantial and limited by the multiplexing capacity of immunofluorescence markers.

Strengths:

Through deep characterization of the in vivo ACD model, the authors were able to determine which cell types were expressing the major cytokines involved in ACD inflammation, such as IFNG, IL4/13, IL17A, and IL1B. These analyses are well-presented and thoughtful, showing first that the response is IFNG-dominant, then focusing on deeper characterization of lymphocytes, myeloid cells, and fibroblasts, which are also validated and complemented by FACS experiments using canonical markers of these cell types as well as IF staining. Crosstalk analyses from the scRNA-seq data led the authors to focus on IFNG signaling fibroblasts, and in vitro experiments demonstrate that CXCL9 and CXCL10 are expressed by fibroblasts stimulated by IFNG. In vivo functional genetic evidence demonstrates an important role for IFNG signaling in fibroblasts, as KO of Ifngr1 using Pdgfra-Cre Ifngr1 fl/fl mice, showed a reduction in inflammation and CD8 T cell recruitment.

Weaknesses:

The use of one model limits an understanding of how broad this fibroblast-T cell axis is during ACD. However, the authors chose the most commonly employed model and cited additional work in a vitiligo model (another type 1 immune response). The identity of the involved fibroblasts and T cells in the mouse model is difficult to assess as scRNA-seq identified subpopulations of these cell types, but most work in the Pdgfra-Cre Ifngr1 fl/fl mice used broad markers for these cell types as opposed to matched subpopulation markers from their scRNA-seq data. Human patient samples of ACD were co-stained with two markers at a time, demonstrating the presence of CD8+IFNG+ T cells, PDGFRA+CXCL10+ fibroblasts, and co-localization of PDGFRA+ fibroblasts and CD8+ T cells. However, no IF staining demonstrates co-expression of all 4 markers at once; thus, the human validation of co-localization of CD8+IFNG+ T cells and PDGFRA+CXCL10+ fibroblasts is ultimately indirect, although not a huge leap of faith. Although n=3 samples of healthy control and ACD samples are used, there is no quantification of any results to demonstrate the robustness of differences.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

The identification of similar mutations in non-receptor tyrosine kinases (NRTKs) in two different families with familial SLE is a significant finding in human disease. Furthermore, the paper provides a detailed analysis of the molecular mechanisms behind the impairment of efferocytosis caused by mutations in ACK1 and BRK.

Weaknesses:

A critical point in this paper is whether the loss of function of ACK1 or BRK contributes to the onset of familial SLE. The authors emphasize that inhibitors of ACK1/BRK worsened IgG deposition in the kidneys in a pristane-induced SLE model, which contributes not to the onset but to the exacerbation of SLE, thus only partially supporting their claim.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors employed direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. They conclude that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Contrary to the literature, they found that, unlike typical RNA decay models in normal conditions, stress-induced RNA decay is dependent on XRN1 but does not depend on the removal of the poly(A) tail. The findings presented are interesting but a substantial amount of work is needed to fully establish these paradigm-shifting findings.

Strengths:

These are paradigm-shifting observations using cutting-edge technologies.

Weaknesses:

The conclusions do not appear to be fully supported by the data presented.

Reviewer #1 (Public Review):

In this study Cacho-Navas et al. describes the role of ICAM-1 expressed on the apical membrane of bile canaliculi and its function to control the homeostasis of the bile canaliculi (BCs). This is a previously unrecognized function of this protein in hepatocytes. The same authors have previously shown that basolateral ICAM-1 plays a role in controlling lymphocyte adhesion to hepatocytes during inflammation and that this interaction is responsible on the loss of polarity of hepatocytes during the disease.<br /> In this new study they show that ICAM-1, is mainly localized in the apical domain of the BC and in association with EBP-50, comunicates with the subapical acto-myosin ring to regulate the size and morphology of the BC.<br /> In this study they used the well-known immortal cell line of liver cells (HepG2) in which they knocked-out ICAM-1 using CRISPR-Cas9 editing and hepatic organoid derived from WT and ICAM-1-KO mice. alternating knocking-out as well as rescue experiments they show that in the absence of apical ICAM-1, the BC dimension and shape are altered.<br /> The conclusions of the study are sufficiently supported by the data.

Comments on revision:

The authors have addressed most of the reviewer's comments in the re-submission, however the use of the organoids as a model to study bile canaliculi is still not convincing.<br /> The HA-4 staining and the space wehere CFDA is secreted does not overlap considering the nuclei position in the middle z-stack section. Also, the interdigitations between cells identified by EM do not form an enclosed space as we should expect for a bile canaliculi.<br /> I understand that other studies have used these organoids to show some hepatocytic functions but at the same time none has characterized before the formation of bile canaliculi as suggested in this study. Therefore a characterization showing the expression of specific markers (i.e mrp2, bsep) should be provided to support this claim.<br /> I would suggest the authors to carefully read the helpful review by Marsee et al., Cell Stem Cell 2021 that clearly and carefully address the classification and validation of liver organoids from experts in the field.

DOI: 10.1016/j.celrep.2024.113955

Resource: ZFIN_ZDB-ALT-110124-1

Curator: @abever99

SciCrunch record: RRID:ZFIN_ZDB-ALT-110124-1

What is this?

Reviewer #1 (Public Review):

Tu et al investigated how LFPs recorded simultaneously with rsfMRI explain the spatiotemporal patterns of functional connectivity in sedated and awake rats. They find that connectivity maps generated from gamma band LFPs (from either area) explain very well the spatial correlations observed in rsfMRI signals, but that the temporal variance in rsfMRI data is more poorly explained by the same LFP signals. The authors excluded the effects of sedation in this effect by investigating rats in the awake state (a remarkable feat in the MRI scanner), where the findings generally replicate. The authors also performed a series of tests to assess multiple factors (including noise, outliers, and nonlinearity of the data) in their analysis.

This apparent paradox is then explained by a hypothetical model in which LFPs and neurovascular coupling are generated in some sense "in parallel" by different neuron types, some of which drive LFPs and are measured by ePhys, while others (nNOS, etc.) have an important role in neurovascular coupling but are less visible in Ephys data. Hence the discrepancy is explained by the spatial similarity of neural activity but the more "selective" LFPs picked up by Ephys account for the different temporal aspects observed.

This is a deep, outstanding study that harnesses multidisciplinary approaches (fMRI and ephys) for observing brain activity. The results are strongly supported by the comprehensive analyses done by the authors, which ruled out many potential sources for the observed findings. The study's impact is expected to be very large.

There are very few weaknesses in the work, but I'd point out that the 1-second temporal resolution may have masked significant temporal correlations between LFPs and spontaneous activity, for instance, as shown by Cabral et al Nature Communications 2023, and even in earlier QPP work from the Keilholz Lab. The synchronization of the LFPs may correlate more with one of these modes than the total signal. Perhaps a kind of "dynamic connectivity" analysis on the authors' data could test whether LFPs correlate better with the activity at specific intervals. However, this could purely be discussed and left for future work, in my opinion.

Reviewer #1 (Public Review):

Summary:

In this paper, Song, Shi, and Lin use an existing deep learning-based sequence model to derive a score for each haplotype within a genomic region, and then perform association tests between these scores and phenotypes of interest. The authors then perform some downstream analyses (fine-mapping, various enrichment analyses, building polygenic scores) to ensure that these associations are meaningful. The authors find that their approach allows them to find additional associations, the associations have biologically interpretable enrichments in terms of tissues and pathways, and can slightly improve polygenic scores when combined with standard SNP-based PRS.

Strengths:

- I found the central idea of the paper to be conceptually straightforward and an appealing way to use the power of sequence models in an association testing framework.

- The findings are largely biologically interpretable, and it seems like this could be a promising approach to boost power for some downstream applications.

Weaknesses:

- While not a weakness of the manuscript, the proposed method is computationally intensive.

Reviewer #1 (Public Review):

In this study, Hoops et al. showed that Netrin-1 and UNC5c can guide dopaminergic innervation from nucleus accumbens to cortex during adolescence in rodent models. They found that these dopamine axons project to the prefrontal cortex in a Netrin-1 dependent manner and knocking down Netrin-1 disrupted motor and learning behaviors in mice. Furthermore, the authors used hamsters, a seasonal model that is affected by the length of daylight, to demonstrate that the guidance of dopamine axons is mediated by the environmental factor such as daytime length and in sex dependent manner.

Regarding the cell type specificity of Netrin-1 expression, the authors began by stating "this question is not the focus of the study and we consider it irrelevant to the main issue we are addressing, which is where in the forebrain regions we examined Netrin-1+ cells are present." This statement contradicts the exact issue regarding the specificity issue I raised. They then went on to show the RNAscope data for Netriin-1 in Figure 2, which showed Netrin-1 mRNA was actually expressed quite ubiquitously in anterior cingulate cortex, dorsopeduncular cortex, infralimbic cortex, prelimbic cortex, etc. In addition, contrary to the authors' statement that Netrin-1 is a "secreted protein", the confocal images in Figure 1 in the rebuttal letter actually show Netrin-1 present in "granule-like" organelles inside the cytoplasm of neurons. Finally, the authors presented Figure 7 to indicate the location where virus expressing Netrin-1 shRNA might be located. Again, the brain region targeted was quite focal and most likely did not cover all the Netrin-1+ brain regions in Figure 2. Collectively, these results raised more questions regarding the specificity of Netrin-1 expression in brain regions that are behaviorally relevant to this study.

With respect to the effectiveness of Netrin-1 knockdown in the animals in this study, the authors cited data in HEK293 cells (Figure 5), which did not include any statistics, and previously published in vivo data in a separate, independent study (Figure 6). They do not provide any data regarding the effectiveness of Netrin-1 knockdown in THIS study.

Similar concerns regarding UNC5C knockdown (points #6, #7, and #8) were not adequately addressed.

In brief, while this study provides a potential role of Netrin-1-UNC5C in target innervation of dopaminergic neurons and its behavioral output in risk-taking, the data lack sufficient evidence to firmly establish the cause-effect relationship.

Reviewer #1 (Public Review):

In this paper, the authors developed an image analysis pipeline to automatically identify individual ‎‎neurons within a population of fluorescently tagged neurons. This application is optimized to deal with ‎‎multi-cell analysis and builds on a previous software version, developed by the same team, to resolve ‎‎individual neurons from whole-brain imaging stacks. Using advanced statistical approaches and ‎‎several heuristics tailored for C. elegans anatomy, the method successfully identifies individual ‎‎neurons with a fairly high accuracy. Thus, while specific to C. elegans, this method can ‎become ‎instrumental for a variety of research directions such as in-vivo single-cell gene expression ‎analysis ‎and calcium-based neural activity studies.‎

Reviewer #1 (Public Review):

In this manuscript, Yao et al. explored the transcriptomic characteristics of neural stem cells (NSCs) in the human hippocampus and their changes under different conditions using single-nucleus RNA sequencing (snRNA-seq). They generated single-nucleus transcriptomic profiles of human hippocampal cells from neonatal, adult, and aging individuals, as well as from stroke patients. They focused on the cell groups related to neurogenesis, such as neural stem cells and their progeny. They revealed genes enriched in different NSC states and performed trajectory analysis to trace the transitions among NSC states and towards astroglial and neuronal lineages in silico. They also examined how NSCs are affected by aging and injury using their datasets and found differences in NSC numbers and gene expression patterns across age groups and injury conditions. One major issue of the manuscript is questionable cell type identification. For example, more than 50% of the cells in the astroglial lineage clusters are NSCs, which is extremely high and inconsistent with classic histology studies.

While the authors have made efforts to address previous critics, major concerns have not been adequately addressed, including a very limited sample size and patient information. In addition, some analytical approaches are still questionable and the authors acknowledge some issues they cannot address. Therefore, while the topic is interesting, some results are preliminary and some conclusions are not fully supported by the data presented.

Reviewer #1 (Public Review):

The authors show that concurrently presenting foreign words and their translations during sleep leads to the ability to semantically categorize the foreign words above chance. Specifically, this procedure was successful when stimuli were delivered during slow oscillation troughs as opposed to peaks, which has been the focus of many recent investigations into the learning & memory functions of sleep. Finally, further analyses showed that larger and more prototypical slow oscillation troughs led to better categorization performance, which offers hints to others on how to improve or predict the efficacy of this intervention.

Comments on the revised version:

I applaud the authors on a nice rebuttal. Many responses use solid arguments based on the existing literature, such as their response regarding the possibility that low-level acoustic characteristics explaining EEG differences between conditions. Their new analyses also clarify the paper. Additionally, I appreciate their labeling their more speculative claims as such. Below are my remaining thoughts:

Major point:

The largest remaining issue for me regards the term 'episodic'. Before I begin, I should say that I imagine the authors have thought considerably about this definition and may disagree with what I will say. That would be fine - it's their choice at this journal. My main point in writing this is to help them clarify their case further. R3 had a similar concern on the first round of review, and I imagine others holding the "traditional" view of episodic memory would be similarly skeptical. If the authors have a great rebuttal to these points, I imagine it will address others' concerns too.<br /> I believe I understand the authors' argument: I read the Henke (2010, Nature Reviews Neuroscience) piece years back with great interest and again now, and I've gone back to read their other papers cited in this manuscript. Again, I applaud the authors on producing a large collection of fascinating findings expanding knowledge of what can be accomplished via unconscious learning. That includes this paper! But I still disagree with the term 'episodic' for what is measured here. The authors state in the Methods section that they prompted participants to 'guess whether the presented pseudoword designates an animal, a tool, or a place'. IMHO, the main issue of using 'episodic' is the nature of the memory representation - 'guessing' does not ask participants anything about the source (the who-what-when-why-where) of the information (anything about an episode).<br /> Notably, it does seem to fit their own definition from Henke (2010). Rapid? I believe so - 4 trial-learning is fairly quick. Certainly, there are studies of supposed episodic memory that use a few rounds of learning the same stimuli (rather than single trial learning) and one can still get away with calling the nature of the memories 'episodic'. Flexible? I believe the authors mean that their task is flexible because participants learn a category exemplar during sleep (e.g., 'aryl'-'bird') but then only respond based on its category membership ('animal'?). If this is the case, I agree that the representations are flexible. Reliant on the 'episodic memory system' (lines 495-9)? Reasonably likely, given their prior findings (e.g., Züst et al., 2019). However, there is considerable data suggesting the hippocampus contributes to functions beyond episodic memory, including statistical learning (e.g., Schapiro et al., 2013, Current Biology), motor learning (e.g., Schendan et al., 2003, Neuron; Dohring et al., 2017, Cortex; Jacobacci et al., 2020, PNAS), attention (e.g., Aly & Turk-Browne, 2016, Cerebral Cortex), perception (e.g., Lee et al., 2012), and semantic memory (e.g., Cutler et al., 2019, Frontiers in Human Neuroscience). Therefore, given that the hippocampus contributes to other tasks too, saying the task is episodic in part because it likely relies on the hippocampus (the 'episodic memory system') is an incorrect reverse inference. But regardless of this concern, it seems true to me that the term fits 'episodic' according to Henke (2010).<br /> So, it seems I'm raising an issue with this entire way of defining memory. IMHO, the biggest issue is that there is no reason to assume the participant relies upon any source-related information in making their guess. There is room in the field for a new type of rapid, unconscious, flexible, hippocampal-dependent learning that does not need to align with the term, 'episodic', for it to be important and fascinating! The term, 'episodic', is convenient for a reason - namely, for labeling the behavioral output of what it measures, not the process that underlies it. The authors have continually made an excellent case for rapid, unconscious, flexible, hippocampal-dependent learning, and it would seem even more beneficial for the field for the authors to just call this its own thing.

A related point:<br /> - I see that the authors do not use 'episodic' in prior papers with similar tasks (e.g., Züst et al., 2019), and I am curious if anything changed in their thinking or why they use the term now. They can ignore this if they'd like, but it would perhaps give useful context.

Other points:<br /> IMHO, the issue of repeated tests is more legitimate than the authors suggest. They state in their response letter, "However, recent literature suggests that retrieval practice is only beneficial when corrective feedback is provided (Belardi et al., 2021; Metcalfe, 2017)." This is incorrect. While retrieval practice is often less effective without feedback, it can be effective without feedback if retrieval accuracy is high and if the experimenters later employ a long enough retention interval to witness long-term effects. This is clear in various papers (e.g., Roediger & Karpicke, 2006, Psychological Science; Karpicke & Roediger, 2008, Science) and there is a nice theoretical model explaining how these complex effects could arise (Halamish & Bjork, 2011, JEP:LMC; Kornell et al., 2011, JML). The authors do not heavily rely on this in their paper, but they could consider tempering their claims that it is 'unlikely' (line 509) that delayed retrieval was affected by the first retrieval.<br /> The authors claim that fast spindles are part of a speculative model underlying their learning effects (lines 605-6). However, they did not find any differential spindle effects in determining later performance, so they could consider keeping just points #1&2 or mentioning that spindles differ by condition but may not directly influence the learning effects here.

Reviewer #1 (Public Review):

This manuscript from Clayton and co-authors, entitled "Mechanism of dimer selectivity and binding cooperativity of BRAF inhibitors", aims to clarify the molecular mechanism of BRAF dimer selectivity. Indeed, first-generation BRAF inhibitors, targeting monomeric BRAFV600E, are ineffective in treating resistant dimeric BRAF isoforms. Here, the authors employed molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric BRAF, in the presence and absence of inhibitors. Multi-microsecond MD simulations showed an inward shift of the αC helix in the BRAFV600E mutant dimer. This helped in identifying a hydrogen bond between the inhibitors and the BRAF residue Glu501 as critical for dimer compatibility. The stability of the aforementioned interaction seems to be important to distinguish between dimer-selective and equipotent inhibitors.

The study is overall valuable and robust. The authors used the recently developed particle mesh Ewald constant pH molecular dynamics, a state-of-the-art method, to investigate the correct histidine protonation considering the dynamics of the protein. Then, multi-microsecond simulations showed differences in the flexibility of the αC helix and DFG motif. The dimerization restricts the αC position in the inward conformation, in agreement with the result that dimer-compatible inhibitors can stabilize the αC-in state. Noteworthy, the MD simulations were used to study the interactions between the inhibitors and the protein, suggesting a critical role for a hydrogen bond with Glu501. Finally, simulations of a mixed state of BRAF (one protomer bound to the inhibitor and the other apo) indicate that the ability to stabilize the inward αC state of the apo protomer could be at the basis of the positive cooperativity of PHI1.

One potential weakness in the manuscript is the lack of reported uncertainties related to the analyzed quantities. Providing this information would significantly enhance the clarity regarding the reliability of the analyses and the confidence in the claims presented.

Reviewer #1 (Public Review):

Zhang et al. tackle the important topic of primate-specific structural features of the brain and the link with functional specialization. The authors explore and compare gyral peaks of the human and macaque cortex through non-invasive neuroimagery, using convincing techniques that have been previously validated elsewhere. They show that nearly 60% of the macaque peaks are shared with humans, and use a multi-modal parcellation scheme to describe the spatial distribution of shared and unique gyral peaks in both species.

The claim is made that shared peaks are mainly located in lower-order cortical areas whereas unique peaks are located in higher-order regions, however, no systematic comparison is made. The authors then show that shared peaks are more consistently found across individuals than unique peaks, and show a positive but small and non-significant correlation between cross-individual counts of the shared peaks of the human and the macaque i.e. the authors show a non-significant trend for shared peaks that are more consistently found across humans to be those that are also more found across macaques.

In order to identify if unique and shared peaks could be identified based on the structural features of the cortical regions containing them, the authors compared them with t-tests. A correction for multiple comparisons should be applied and t-values reported. Graph-theoretical measures were applied to functional connectivity datasets (resting-state fMRI) and compared between unique and shared peak regions for each species separately. Again the absence of multiple comparison correction and t-values make the results hard to interpret. The same comment applies to the analysis reporting that shared peaks are surrounded by a larger number of brain regions than unique peaks. Finally, the potentially extremely interesting results about differential human gene expression of shared and unique peaks regions are not systematically reported e.g. the 28 genes identified are not listed and the selection procedure of 7 genes is not fully reported.

The paper is well written and the methods used for data processing are very compelling i.e. the peak cluster extraction pipeline and cross-species registration.

Comments on revision:

The authors have convincingly addressed all my previous concerns such that, as the revised paper stands now, the presented results provide solid support for the conclusions of the authors. The revised paper is now of interest for a large part of the neuroscience community and specifically for those interested in primate-specific structural features of the brain and the link with functional specialization.

Reviewer #1 (Public Review):

Summary:

The authors re-analysed the data of a previous study in order to investigate the relation between asymmetries of subcortical brain structures and the hemispheric lateralization of alpha oscillations during visual spatial attention. The visual spatial attention task crossed the factors of target load and distractor salience, which made it possible to also test the specificity of the relation of subcortical asymmetries to lateralized alpha oscillations for specific attentional load conditions. Asymmetry of globus pallidus, caudate nucleus, and thalamus explained inter-individual differences in attentional alpha modulation in the left versus right hemisphere. Multivariate regression analysis revealed that the explanatory potential of these regions' asymmetries varies as a function of target load and distractor salience.

In the revision of the article, the authors addressed my concerns.

However, my concern with regard to the statistical analysis of the specificity of certain subcortical regions predicting HLM seems to be not fully addressed. The authors added an additional statistical analysis for "testing the null hypothesis that a given regressor does not impact all dependent variables". To my understanding, this is a somewhat unusual definition of a null hypothesis. Typically, the null hypothesis is the hypothesis of no effect, meaning here it should state that the effect is the same across predictors.

In the new statistical analysis, the authors seem to take non-significant results (p>.05) as evidence for the specificity of subcortical regions in predicting HLM. The rationale of this statistical approach is difficult to follow and was somewhat unclear to me.

A much simpler and more straight-forward approach would be to contrast beta-estimates per subcortical region between experimental conditions. For instance, if the beta estimates in the thalamus for the "low-load target, non-salient distractor" condition would be significantly larger than the beta estimates for the other conditions, this would speak to specificity.

Reviewer #1 (Public Review):

SUMMARY:

Parkinson's disease (PD) and other synucleinopathies, including Parkinson's Disease Dementia (PDD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA), pose significant challenges for early diagnosis, as their clinical manifestations often emerge after substantial neurodegeneration has occurred. In this context, the Alpha-Synuclein Seeding Amplification Assay (SAA) has garnered considerable attention for its potential as a diagnostic tool, capable of detecting pathological forms of alpha-synuclein (αSyn) even before the onset of classical clinical symptoms and signs. The assay exploits αSyn's intrinsic property to convert healthy forms into pathological ones, subsequently amplifying these pathological forms for visualization. This study aims to investigate the efficacy of SAA in accurately identifying subtypes of synucleinopathies, including PD, PDD, DLB, and MSA. To achieve this, the results from the patient brain-derived αSyn SAA are compared with those obtained through conformational stability assays, immunolabeling, and electron microscopy. Study shows that brain-derived αSyn fibrils exhibit significant differences across various synucleinopathies in their conformation, biochemical profile and phosphorylation patterns. Importantly, the SAA method appears to fall short in capturing these distinctions.

The study's findings are highly relevant given the rapidly advancing landscape of utilizing the SAA for the diagnosis and differentiation of various forms of PD and synucleinopathies using patient biofluids. It is somewhat surprising that the authors primarily characterize SAA as a research tool without delving into its potential as a biomarker detection assay, especially in the context of the field's excitement about its diagnostic applications. Additionally, a missed opportunity lies in not referencing a recent study that employed SAA successfully to diagnose PD and subtype the condition using a vast sample size. To further strengthen the results, the inclusion of healthy control brains in the biochemical and immunostaining/immunoblot experiments would provide more robust comparisons. Overall, the authors have conducted their experiments diligently, and their study offers valuable insights that align with the ongoing efforts to enhance early diagnosis and subtype differentiation in the domain of synucleinopathies.

STRENGTH:

The strengths of this research article are indeed notable and contribute to the credibility and significance of the study:

Important Research Question: The study addresses a crucial question in the field of neurodegenerative diseases by evaluating the effectiveness of the αSyn SAA in diagnosing and differentiating synucleinopathies. This question is of significant clinical and scientific interest.<br /> Comprehensive Introduction: The article provides a thorough and well-structured introduction to the topic with an illustration, setting the stage for the research. It ensures that readers, including those unfamiliar with the subject matter, can grasp the context and significance of the study.<br /> Use of Patient Brain Tissue: The use of patient-derived brain tissue samples from various synucleinopathies, including PD, PDD, DLB, and MSA, enhances the clinical relevance and applicability of the findings.<br /> Replication and Statistical Significance: Conducting the experiments six times for each sample demonstrates the rigor of the study and the robustness of the results, and increases the confidence in the conclusions drawn.<br /> Clarity in Experimental Results and Discussion: The authors have presented the experimental results in a clear and understandable manner. I was personally impressed by images showing twisted and straight conformations of αSyn, as well as immunogold labeling for phosphorylation of αSyn, which aids in conveying the findings effectively to the readers. The results clearly show distinct differences in the characteristics of αSyn fibrils across different synucleinopathies. It also highlights the more aggressive seeding capacities and higher biochemical stability of αSyn in PDD and DLB patients, offering valuable insights into the pathophysiology of these conditions. The authors also clearly show that SAA fails in differentiating the disease types within the synucleinopathies.<br /> Clinical relevance: The study underscores the importance of considering complementary diagnostic methods alongside SAA for a more comprehensive understanding of synucleinopathy subtypes. The study might also play an important role in potential FDA approval of SAA as a diagnostic tool for synucleinopathies, especially for PD.<br /> These strengths collectively make the study a valuable contribution to the field of neurodegenerative diseases, shedding light on the limitations and potential applications of SAA in the diagnosis and differentiation of various synucleinopathies.

WEAKNESS:

While this study is overall robust, there are several aspects that could further enhance the quality and interpretation of the findings.

Clinical Data on Patient Brain Samples: The inclusion of specific details such as post-mortem intervals and the age at disease onset for patient brain samples would be valuable. These factors could significantly affect the quality of the tissues and their relevance to the study. Moreover, given the large variation in disease duration between PD and PDD, it's important to consider disease duration as a potential confounding factor, especially when concluding that PDD patients have a more severe form of synucleinopathy compared to PD.<br /> Inclusion of Healthy Controls in Multiple Tests: Given the importance of healthy controls in scientific studies, especially those involving human brain samples, the authors could consider using healthy controls in more tests to strengthen the robustness of the findings. Expanding the use of healthy controls in biochemical profiling and phosphorylation profiles would provide a better basis for comparison and clarify the significance of results in a disease context.<br /> This will help the authors to elaborate on the interpretation of results, for example, in Figure 3, where the authors claim that PD brains show mostly monomeric αSyn forms (line 119 and 120, and also in 222 and 223). Whether it implies the absence of alpha-syn pathology in PD brains? If there are differences from healthy controls? What are these low molecular weight bands (<15kD) (line 125-126) and whether they are also present in healthy controls? Also, we do not have a perfect pS129-specific (anti-p𝛼Syn) antibody. They are known for non-specific labeling. Investigating the phosphorylation levels in healthy controls and comparing them to PD brains, especially considering the predominance of monomeric (healthy αSyn?) in PD brains, would help clarify the observed changes.<br /> Age of Healthy Controls: Providing information about the age at death for healthy controls is crucial, as age can impact the accumulation of αSyn. Also include if the brain samples were age-matched, or analyses were age-adjusted.<br /> Braak Staging Discrepancy: The study reports the same Braak staging for both PD and PDD, despite the significant difference in disease duration. Maybe other reviewers with clinical experience might have a better take on this. This observation merits discussion in the paper, allowing readers to better understand the implications of this finding.<br /> Citation of Relevant Studies: The paper should consider citing and discussing a recent celebrated study on PD biomarkers that used thousands of cerebrospinal fluid (CSF) samples from different PD patient cohorts to demonstrate the effectiveness of SAA as a biochemical assay for diagnosing PD and its subtypes (https://doi.org/10.1016/S1474-4422(23)00109-6).<br /> In summary, these suggestions aim to enhance the study's quality and the clarity of its findings, ultimately contributing to a more comprehensive understanding of synucleinopathies and the diagnostic potential of SAA.

Reviewer #1 (Public Review):

Summary:

Songbirds provide a tractable system to examine neural mechanisms of sequence generation and variability. In past work, the projection from LMAN to RA (output of the anterior forebrain pathway) was shown to be critical for driving vocal variability during babbling, learning, and adulthood. LMAN is immediately adjacent to MMAN, which projects to HVC. MMAN is less well understood but, anatomically, appears to resemble LMAN in that it is the cortical output of a BG-thalamocortical loop. Because it projects to HVC, a major sequence generator for both syllable phonology and sequence, a strong prediction would be that MMAN drives sequence variability in the same way that LMAN drives phonological variability. This hypothesis predicts that MMAN lesions in a Bengalese finch would reduce sequence variability. Here, the authors test this hypothesis. They provide a surprising and important result that is well motivated and well analyzed: MMAN lesions increase sequence variability - this is exactly the opposite result from what would be predicted based on the functions of LMAN.

Strengths:

(1) A very important and surprising result shows that lesions of a frontal projection from MMAN to HVC, a sequence generator for birdsong, increase syntactical variability.

(2) The choice of Bengalese finches, which have complex transition structures, to examine the mechanisms of sequence generation, enabled this important discovery.

(3) The idea that frontal outputs of BG-cortical loops can generate vocal variability comes from lesions/inactivations of a parallel pathway from LMAN to RA. The difference between MMAN and LMAN functions is striking and important.

Reviewer #1 (Public Review):

Summary:

This manuscript reports a series of experiments examining category learning and subsequent generalization of stimulus representations across spatial and nonspatial domains. In Experiment 1, participants were first trained to make category judgments about sequences of stimuli presented either in nonspatial auditory or visual modalities (with feature values drawn from a two-dimensional feature manifold, e.g., pitch vs timbre), or in a spatial modality (with feature values defined by positions in physical space, e.g., Cartesian x and y coordinates). A subsequent test phase assessed category judgments for 'rotated' exemplars of these stimuli: i.e., versions in which the transition vectors are rotated in the same feature space used during training (near transfer) or in a different feature space belonging to the same domain (far transfer). Findings demonstrate clearly that representations developed for the spatial domain allow for representational generalization, whereas this pattern is not observed for the nonspatial domains that are tested. Subsequent experiments demonstrate that if participants are first pre-trained to map nonspatial auditory/visual features to spatial locations, then rotational generalization is facilitated even for these nonspatial domains. It is argued that these findings are consistent with the idea that spatial representations form a generalized substrate for cognition: that space can act as a scaffold for learning abstract nonspatial concepts.

Strengths:

I enjoyed reading this manuscript, which is extremely well written and well presented. The writing is clear and concise throughout, and the figures do a great job of highlighting the key concepts. The issue of generalization is a core topic in neuroscience and psychology, relevant across a wide range of areas, and the findings will be of interest to researchers across areas in perception and cognitive science. It's also excellent to see that the hypotheses, methods and analyses were pre-registered.

The experiments that have been run are ingenious and thoughtful; I particularly liked the use of stimulus structures that allow for disentangling of one-dimensional and two-dimensional response patterns. The studies are also well powered for detecting effects of interest. The model-based statistical analyses are thorough and appropriate throughout (and it's good to see model recovery analysis too). The findings themselves are clear-cut: I have little doubt about the robustness and replicability of these data.

Weaknesses:

In my original review I raised a concern related to a potential alternative interpretation of the findings: the idea that participants have substantial experience of representing space in terms of multiple, independent, and separable dimensions, whereas this may not be the case for the visual and auditory stimuli used here. As I noted in that prior review, on this view "the impact of spatial pre-training and (particularly) mapping is simply to highlight to participants that the auditory / visual stimuli comprise two separable (and independent) dimensions."

In addressing this point, the authors note that performance in the visual/auditory "mapping" task in Experiments 2c and 3c suggests that most participants were paying attention to both dimensions of auditory and visual stimuli. I agree that seems to have been the case. But there is a difference between making use of information from both dimensions, and realizing that ***the two dimensions are separable and independent*** (which is what is required for rotational generalization in this task).

As an analogy, suppose I have a task where participants have to map a pillow and a shuttlecock to category A, and a surfboard and a bicycle to category B. A participant could learn to do this just by memorizing the correct response for each item considered as a "whole thing". Or they could realize that the items contain component information, learning that "things with feathers" belong in category A, and "things that can carry people" go in category B. Performance may be the same in both cases, but the underlying process is quite different.

The "attention to dimensions" account that I advanced in my previous review was referring to something more like the latter (feathers/vehicle) case: that spatial pre-training helps people to understand that items can be decomposed into separable pieces of information. Above-chance performance in the visual-auditory mapping task does not (necessarily) demonstrate this ability because it could reflect memorization of "whole" stimuli rather than reflecting decomposition into separable component parts. I agree that it does at least show that participants were paying attention to and making use of information from both dimensions when making their mapping decisions; it's just that they may not have *realized* that they were using information from two separable dimensions.

Reviewer #1 (Public Review):

Summary:

This is an interesting report examining activity patterns in mouse ACC and in the OFC neurons projecting to ACC. In addition, the effects of inactivation are examined. In aggregate, the results provide new and interesting information about these two brain areas and they translate motivation into action - a function that it seems intuitively plausible that ACC might perform but, despite this intuition, there have been comparatively few direct tests of the idea and little is known of the specific mechanisms. The study is performed carefully and is written up clearly. There were just a few points where I wondered if a little more clarification might be helpful.

Strengths:

The combination of recording and inactivation/inhibition experiments and the combination of investigation of ACC neurons and of OFC regions projecting to ACC are very impressive.

Weaknesses:

These are all minor points of clarification.

(1) An important conclusion (Figure 4) is that when mice are trained to run through no reward (N) cues in order to reach reward (R) cues, the OFC neurons projecting to ACC each respond to different specific events in a manner that ensures that collectively they tile the extended behavioural sequence. What I was less sure of was whether the ACC neurons do the same or not. Figure 3 suggests that on average ACC neurons maintain activity across N cues in order to get to R cues but I was not sure whether this was because all individual neurons did this or whether some had activity patterns like the OFC neurons projecting to ACC.

(2) Figure 1 versus Figure 2: There does not seem to be a particular motivation for whether chemogenetic inactivation or optogenetic inhibition were used in different experiments. I think that this is not problematic but, if I am wrong and there were specific reasons for performing each experiment in a certain way, then further clarification as to why these decisions were made would be useful. If there is no particular reason, then simply explaining that this is the case might stop readers from seeking explanations.

(3) P5, paragraph 2. The authors argue that OFC and anteriomedial (AM) thalamic inputs into ACC are especially important for mediating motivation through N cues in order to reach R cues. Is this based on a statistical comparison between the activity in OFC or AM inputs as opposed to the other inputs?

(4) P3, paragraph 2. Some papers by Khalighinejad and colleagues (eg Neuron 2020, Current Biology, 2022) might be helpful here in as much as they assess ACC roles in determining action frequency, initiation, and speed and mediating the relationship between reward availability and action frequency and speed.

(5) Paragraph 1 "This learning is of a more deliberate, informed nature than habitual learning, as they are sensitive to the current value of outcomes and can lead to a novel sequence of actions for a desired outcome1-3." Should "they" be "it"?

Reviewer #1 (Public Review):

Summary:

Wang and colleagues present a study aimed at demonstrating the feasibility of repeated ultrasound localization microscopy (ULM) recording sessions on mice chronically implanted with a cranial window transparent to ultrasound. They provided quantitative information on their protocol, such as the required number of contrast-enhancing microbubbles (MBs) to get a clear image of the vasculature of a brain coronal section. Also, they quantified the co-registration quality over time-distant sessions and the vasodilator effect of isoflurane.

Strengths:

The study showed a remarkable performance in recording precisely the same brain coronal section over repeated imaging sessions. In addition, it sheds light on the vasodilator effect of isoflurane (an anesthetic whose effects are not fully understood) on the different brain vasculature compartments, although, as the authors stated, some insights in this aspect have already been published with other imaging techniques. The experimental setting and protocol are very well described.

Weaknesses:

While the title is fair with respect to the data shown, in the summary and the rest of the paper, the comparison between anesthetized and awake conditions is systematically stated, while more caution should be used.

First, isoflurane is one of the (many) anesthetics commonly used in pre-clinical research, and its effect on the brain vasculature cannot be generalized to all the anesthetics. Indeed, other anesthesia approaches do not produce evident vasodilation; see ketamine + medetomidine mixtures. Second, the imaged awake state is head-fixed and body-constrained in mice. A condition that can generate substantial stress in the animals. In this study, there is no evaluation of the stress level of the mice. In addition, the awake imaging sessions were performed a few minutes after the mouse woke up from isoflurane induction, which is necessary to inject the MB bolus. It is known that the vasodilator effects of isoflurane last a long time after its withdrawal. This aspect would have influenced the results, eventually underestimating the difference with respect to the awake state.

These limitations should be clearly described in the Discussion.

Looking at Figure 2e, it takes more than 5' to reach the 5 Millions MB count useful for good imaging. However, the MB count per pixel drops to a few % at that time. This information tells me that (i) repeated measurements are feasible but with limited brain coverage since a single 'wake up' is needed to acquire a single brain section and (ii) this approach cannot fit the requirements of functional ULM that requires to merge the responses to multiple stimuli to get a complete functional image. Of course, a chronic i.v. catheter would fix the issue, but this configuration is not trivial to test in the experimental setup proposed by the authors, hindering the extension of the approach to fULM.

Statistics are often poor or not properly described. The legend and the text referring to Figure 2 do not report any indication of the number of animals analyzed. I assume it is only one, which makes the findings strongly dependent on the imaging quality of THAT mouse in THAT experiment. Three mice have been displayed in Figure 3, as reported in the text, but it is not clear whether it is a mouse for each shown brain section. Figure 5 reports quantitative data on blood vessels in awake VS isoflurane states but: no indication about the number of tested mice is provided, nor the number of measured blood vessels per type and if statistics have been done on mice or with a multivariate method. Also, a T-test is inappropriate when the goal is to compare different brain regions and blood vessel types. Similar issues partially apply to Figure 6, too.

Reviewer #2 (Public Review):

The authors combine genetic tools, dye fills and connectome analysis techniques to generate a "first-of-its-kind", near complete, synaptic resolution map of the head bristle neurons of Drosophila. While some of the BMN anatomy was already known based on previous work by the authors and other researchers, this is the first time a near complete map has been created for the head BMNs at electron microscopy resolution.

Strengths:

(1) The authors cleverly use techniques that allow moving back and forth between periphery (head bristle location) and brain, as well as moving between light microscopy and electron microscopy data. This allows them to first characterize the pathways taken by different head BMNs to project to the brain and also characterize anatomical differences among individual neurons at the level of morphology and connectivity.<br /> (2) The work is very comprehensive and results in a near complete map of all head BMNs.<br /> (3) Authors also complement this anatomical characterization with a first-level functional analysis using optogenetic activation of BMNs that results in expected directed grooming behavior.

Weaknesses:<br /> (1) While not strictly needed here, it could help provide context if authors revealed some of the important downstream pathways that could explain optogenetics behavioral phenotypes: This point was addressed by authors in the revisions and I agree a detailed description of downstream circuits is not needed at this point.<br /> (2) In contrast to the rigorous quantitative analysis of the anatomical data, the behavioral data is analyzed using much more subjective methods. While I do not think it is necessary to perform a rigorous analysis of behaviors in this anatomy focused manuscript, the conclusions based on behavioral analysis should be treated as speculative in the current form e.g. calling "nodding + backward motions" as an avoidance response is not justified as it currently stands. Strong optogenetic activation could lead to sudden postural changes that due to purely biomechanical constraints could lead to a couple of backward steps as seen in the example videos. Moreover since the quantification is manual, it is not clear what the analyst interprets as backward walking or nodding. Interpretation is also concerning because controls show backward walking (although in fewer instances based on subjective quantification): This point was addressed by the authors during revisions and I'm mostly satisfied with their response, where authors agree that the behavioral results are currently used to speculate about the role of BMNs in aversive behaviors. Still, the fact that controls show some "backward motions" is a bit concerning when talking about "significant differences" between control and test groups based on manual annotations and I would recommend future studies focusing on these behaviors to use more unbiased quantitative analysis wherever possible.

Summary:

The authors end up generating a near-complete map of head BMNs that will serve as a long-standing resource to the Drosophila research community. This will directly shape future experiments aimed at modeling or functionally analyzing the head grooming circuit to understand how somatotopy guides behaviors. I appreciate the authors taking the time to revise the manuscript and address reviewer concerns.

Reviewer #1 (Public Review):

Summary:

The motivating questions are an accurate reflection of the current state of knowledge surrounding striatal pathway function. The comparisons of pathway function across striatal subregion, activation & inhibition, and task context are laudable and extremely important for advancing the subfield. Had these manipulations, to the largest extent possible been performed in single animals (e.g. activate dSPNs of DMS or DLS in the same mouse across the 3 tasks), this would have significantly strengthened the impact and conclusions that could be drawn by making this set of studies even more so internally consistent and directly comparable. While this is no longer possible, a conceptually related and fantastic contribution to the subfield (and likely beyond in terms of Opto manipulations of brain areas) would be to directly demonstrate that within their studies their DMS pathway manipulations do not impact nearby DLS activity (and vice versa). This is a significant and non-essential request. More feasibly and reasonably, it would be fantastic and strengthen the conclusions here to more fully detail their opsin expression patterns in DMS vs DLS groups and perhaps attempt to relate individual opsin profiles and fiberoptic targeting with behavioral outcomes across tests.

Strengths:

A comprehensive and paired comparison of inhibition and activation of striatal pathways across subregions and tasks is a very important and meaningful step towards reconciling contradictory results on striatal pathway function that are observed across labs (who typically focus on one subregion, one task setting, and often do not directly report comparisons of activation and inhibition).

Weaknesses:

Figure 1A - the example DMS vs DLS opsin expression and fiber targeting are not terribly convincing that the manipulations will be specific to each subregion (the example in Figure 2A is a little better but I have a similar concern still). The specificity of these manipulations is key to interpretation and conclusions and I strongly feel they should be strengthened here. The best evidence would be direct neural recordings (light in DMS, no effect in DLS, and vice versa), but this is a tall ask and not expected. The next best option, which is readily feasible, is to show not only fiberoptic targeting summaries (as in Figure 1A, Figure 2A) but also a summary of opsin spread for all animals (especially given the two examples appear to have significant spread across DMS and DLS). It would be of great benefit to the field to have these in the Allen Common Coordinate Framework. It would also be fine and useful to utilize the authors' current classical histological atlas alignment methods (e.g. Paxinos pdf). These histological summary figures would also benefit from being larger and more visible (perhaps as separate supplemental figures associated with the main figures).

Related to the above, it is a concern that the classic view is supported or not because of individual variations in virus/fiber targeting to striatal subregions which likely have greater granularity than the traditional dorsal medial vs lateral (e.g. Hunnicutt et al 2016, Foster et al 2021, Hintiryan et al 2016). Although there may not be enough animals or variation in targeting in the present study to find meaningful relationships, it would strengthen the paper and be a great benefit to the field to know whether for key findings if the strength of behavioral effects correlated with anterior/posterior or medial/lateral or dorsal/ventral fiberoptic coordinates (or the volume of opsin expression profiles).

Conceptually, a clear new idea or integrative interpretation of prior work (nor even the large body of results within this work) comes to the fore, save for the already appreciated fact that the classic view of opposing pathways is sometimes supported and sometimes not. Two tangible suggestions that I believe would facilitate the influence of this study - (1) can the authors more thoughtfully bridge the logical steps in their results sections and the prior context around them (some topic sentences jump right into results, e.g. line 195: "The inhibition experiment showed), and (2) in discussion, rather than emphasizing when/where the classic view is supported and not, more content on precisely why would be helpful. Some questions more specifically, if DMS/DLS pathway activation/inhibition is *mostly* oppositely appetitive/aversive, what does that mean in the context of spontaneous or reward-guided locomotion? Self-initiated pathway activation/inhibition is in part learned (with very intriguing differences across pathways in the expression across learning) - how should we think about striatal pathway function with regards to learning, spontaneous/innate behaviors, vs over-trained behaviors? When the classic view fails in the dorsal striatum - why? And is a complimentary "model" an actual alternative concept, a distinct mode of circuit function, or just a negative result on the classic view?

Reviewer #1 (Public Review):

Gazula and co-workers presented in this paper a software tool for 3D structural analysis of human brains, using slabs of fixed or fresh brains. This tool will be included in Freesurfer, a well-known neuroimaging processing software. It is possible to reconstruct a 3D surface from photographs of coronal sliced brains, optionally using a surface scan as model. A high-resolution segmentation of 11 brain regions is produced, independent of the thickness of the slices, interpolating information when needed. Using this method, the researcher can use the sliced brain to segment all regions, without the need of ex vivo MRI scanning.

The software suite is freely available and includes 3 modules. The first accomplishes preprocessing steps, for correction of pixel sizes and perspective. The second module is a registration algorithm that registers a 3D surface scan obtained prior to sectioning (reference) to the multiple 2D slices. It is not mandatory to scan the surface, -a probabilistic atlas can also be used as reference- however the accuracy is lower. The third module uses machine learning to perform the segmentation of 11 brain structures in the 3D reconstructed volume. This module is robust, dealing with different illumination conditions, cameras, lens and camera settings. This algorithm ("Photo-SynthSeg") produces isotropic smooth reconstructions, even in high anisotropic datasets (when the in-plane resolution of the photograph is much higher than the thickness), interpolating the information between slices.

To verify the accuracy and reliability of the toolbox, the authors reconstructed 3 datasets, using real and synthetic data. Real data of 21 postmortem confirmed Alzheimer's disease cases from the Massachusetts Alzheimer's Disease Research Center (MADRC)and 24 cases from the AD Research at the University of Washington(who were MRI scanned prior to processing)were employed for testing. These cases represent a challenging real-world scenario. Additionally, 500 subjects of the Human Connectome project were used for testing error as a continuous function of slice thickness. The segmentations were performed with the proposed deep-learning new algorithm ("Photo-SynthSeg") and compared against MRI segmentations performed to "SAMSEG" (an MRI segmentation algorithm, computing Dice scores for the segmentations. The methods are sound and statistically showed correlations above 0.8, which is good enough to allow volumetric analysis. The main strengths of the methods are the datasets used (real-world challenging and synthetic) and the statistical treatment, which showed that the pipeline is robust and can facilitate volumetric analysis derived from brain sections and conclude which factors can influence in the accuracy of the method (such as using or not 3D scan and using constant thickness).

Although very robust and capable of handling several situations, the researcher has to keep in mind that processing has to follow some basic rules in order for this pipeline to work properly. For instance, fiducials and scales need to be included in the photograph, and the slabs should be photographed against a contrasting background. Also, only coronal slices can be used, which can be limiting for certain situations.

The authors achieved their aims, and the statistical analysis confirms that the machine learning algorithm performs segmentations comparable to the state-of-the-art of automated MRI segmentations.<br /> Those methods will be particularly interesting to researchers who deal with post-mortem tissue analysis and do not have access to ex vivo MRI. Quantitative measurements of specific brain areas can be performed in different pathologies and even in the normal aging process. The method is highly reproducible, and cost-effective since allows the pipeline to be applied by any researcher with small pre-processing steps.

Reviewer #1 (Public Review):

The authors have developed and optimized a footprinting assay to monitor the recruitment of mRNAs to a reconstituted translation initiation system. This assay is named Recruitment-Sequencing (Rec-Seq) and enables the analysis of many purified mRNAs in the reconstituted system.

This system possesses the ability to determine how competition occurs between mRNAs for the initiation machinery. This is the first approach using a reconstituted system that enables this important feature, and this is an important advance for the field.

Using purified mRNAs in a fully reconstituted system together with the ability to monitor start site selection is an important advance. The method enables one to observe for the first time how competition between mRNAs is altered in response to the absence or presence of different initiation components or accessory proteins.

Start site fidelity in purified reconstituted systems can be altered in different buffer conditions and by the concentration of various initiation factors involved in start site fidelity. Future experiments will reveal how these variables can regulate start site selection in this powerful system.

Comments on revised version:

The authors have addressed all of my original comments. This is an impressive manuscript.

Reviewer #1 (Public Review):

Summary:

The CPC plays multiple essential roles in mitosis such as kinetochore-microtubule attachment regulation, kinetochore assembly, spindle assembly checkpoint activation, anaphase spindle stabilization, cytokinesis, and nuclear envelope formation, as it dynamically changes its mitotic localization: it is enriched at inner centromeres from prophase to metaphase but it is relocalized at the spindle midzone in anaphase. The business end of the CPC is Aurora B and its allosteric activation module IN-box, which is located at the C-terminus of INCENP. In most well-studied eukaryotic species, Aurora B activity is locally controlled by the localization module of the CPC, Survivin, Borealin and the N-terminal portion of INCENP. Survivin and Borealin, which bind the N-terminus of INCENP, recognize histone residues that are specifically phosphorylated in mitosis, while anaphase spindle midzone localization is supported by the direct microtubule-binding capacity of the SAH (single alpha helix) domain of INCENP and other microtubule-binding proteins that specifically interact with INCENP during anaphase, which are under the regulation of CDK activity. One of these examples includes the kinesin-like protein MKLP2 in vertebrates. Trypanosoma is an evolutionarily interesting species to study mitosis since its kinetochore and centromere proteins do not show any similarity to other major branches of eukaryotes, while orthologs of Aurora B and INCENP have been identified. Combining molecular genetics, imaging, biochemistry, cross-linking IP-MS (IP-CLMS), and structural modeling, this manuscript reveals that two orphan kinesin-like proteins KIN-A and KIN-B act as localization modules of the CPC in Trypanosoma brucei. The IP-CLMS, AlphaFold2 structural predictions, and domain deletion analysis support the idea that (1) KIN-A and KIN-B form a heterodimer via their coiled-coil domains, (2) Two alpha helices of INCENP interact with the coiled-coil of the KIN-A-KIN-B heterodimer, (3) conserved KIN-A C-terminal CD1 and CD2 interact with the heterodimeric KKT9-KKT11 complex, which is a submodule of the KKT7-KKT8 kinetochore complex composed of KKT7, KKT8, KKT9, KKT11, and KKT12 unique to Trypanosoma, (4) KIN-A and KIN-B coiled-coil domains and the KKT7-KKT8 complex are required for CPC localization at the centromere, (5) CD1 and CD2 domains of KIN-A support its centromere localization. The authors further introduced a KIN-A rigor mutant and knocked-down wild-type KIN-A to show that the ATPase activity of KIN-A seems dispensable for centromere targeting but critical for spindle midzone enrichment of the CPC. The imaging data of the KIN-A rigor mutant suggest that dynamic KIN-A-microtubule interaction is required for metaphase alignment of the kinetochores and proliferation. Overall, the study reveals novel pathways of CPC localization regulation via KIN-A and KIN-B by multiple complementary approaches.

Strengths:

The major conclusion is collectively supported by multiple approaches, combining CRISPR-mediated gene deletion and complementation/site specific genome engineering, epistasis analysis of cellular localization, AlphaFold2 structure prediction of protein complexes, IP-CLMS and biochemical reconstitution (the complex of KKT8, KKT9, KKT11 and KKT12)

Weaknesses:

Minor weakness. The authors imply that KIN-A, but not KIN-B, interacts with microtubules based on microtubule pelleting assay (Fig. S6), but the substantial insoluble fractions of 6HIS-KINA and 6HIS-KIN-B make it difficult to conclusively interpret the data. It is possible that these two proteins are not stable unless they form a heterodimer.

Reviewer #1 (Public Review):

Summary:

This is a follow-up study to the authors' previous eLife report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

Strengths:

Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Sang et al. proposed a pair of IR60b-expressing pharyngeal neurons in Drosophila use IR25a, IR76b, and IR60b channels to detect high Na+ and limit its consumption. Some of the key findings that support this thesis are: 1) animals that lacked any one of these channels - or with their IR60b-expressing neurons selectively silenced - showed much reduced rejection of high Na+, but restored rejection when these channels were reintroduced back in the IR60b neurons; 2) animals with TRPV artificially expressed in their IR60b neurons rejected capsaicin-laced food whereas WT did not; 3) IR60b-expressing neurons exhibited increased Ca2+ influx in response to high Na+ and such response went away when animals lacked any of the three channels.

The experiments were thorough and well designed and further improved after revision. The results are compelling and support the main claim. The development and the use of the DrosoX two-choice assay put forward for a more quantitative and automatic/unbiased assessment for ingestion volume and preference.

Joint Public Review:

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

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

Reviewer #4 (Public Review):

Summary:

Walker et al. investigated the function of TMEM127 on RET regulation and function that could contribute to the development of pheochromocytoma (PCC). The authors showed that deletion of TMEM127 causes RET protein accumulation on the cell surface and, thereby, increased its constitutive ligand-independent activity and downstream signaling. They also unveiled the mechanism of how TMEM127 regulates cell membrane dynamics, particularly focusing on clathrin distribution and its effects on cargo internalization.

Strengths:

They showed that the deletion of TMEM127 affected multiple classes of transmembrane proteins, including RTKs (RET, EGFR), cell adhesion molecules (N-Cadherin, Integrin Beta-3), and carrier proteins (Transferrin Receptor-1), suggesting a global effect on cell surface proteins. This, at least in part, may explain how TMEM127 mutations act as drivers in PCC as well as in other cancers, such as renal cell carcinoma, where RET is not highly expressed. Overall, these findings provide new insights into the understanding of pheochromocytoma pathogenesis and potentially other cancers.

Weaknesses:

The major weakness of this study is the lack of human PCC cell lines for evaluating the function of TMEM127. Currently, the cell line models for pheochromocytoma are unavailable, and manipulation of patient-derived organoids is challenging. To complement this weakness, they provided immunohistochemical data showing that RET protein is highly expressed in TMEM127-mutant PCC.

Furthermore, some of the authors in this manuscript recently published a paper titled 'TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation' (Guo et al. Cell Reports 42, 113070, 2023, which is also cited in the current manuscript). In this manuscript, they showed that TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127. In general, the ubiquitination of proteins is highly specific to each molecule. In the current version of the manuscript, there is no description of the relevance between these two potentially different mechanisms (clathrin-mediated or ubiquitin-mediated) of accumulating RET and/or other proteins mentioned in two separate papers. I believe the authors should at least discuss this.

Reviewer #2 (Public Review):

The manuscript from deHaro-Arbona et al, entitled "Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind", uses single molecule microscopy imaging in live tissues to understand the dynamics and molecular determinants of transcription factor recruitment to the E(spl)-C locus in Drosophila salivary gland cells under Notch-ON and -OFF conditions. Previous studies have identified the major players that are involved in transcription regulation in the Notch pathway, as well as the importance of general transcriptional coregulators, such as CBP/P300 and the Mediator CDK module, but the detailed steps and dynamics involved in these processes are poorly defined. The authors present a wealth of single molecule data that provides significant insights into Notch pathway activation, including:

(1) Activation complexes, containing CSL and Mam, have slower dynamics than the repressor complexes, containing CSL and Hairless.<br /> (2) Contribution of CSL, NICD, and Mam IDRs to recruitment.<br /> (3) CSL-Mam slow-diffusing complexes are recruited and form a hub of high protein concentrations around the target locus in Notch-ON conditions.<br /> (4) Mam recruitment is not dependent on transcription initiation or RNA production.<br /> (5) CBP/P300 or its associated HAT activity is not required for Mam recruitment<br /> (6) Mediator CDK module and CDK8 activity is required for Mam recruitment, and vice-versa, but not CSL recruitment.<br /> (7) Mam is not required for chromatin accessibility but is dependent on CSL and NICD.<br /> (8) CSL recruitment and increased chromatin accessibility persist after NICD removal and loss of Mam, which confers a memory state that enables rapid re-activation in response to subsequent Notch activation<br /> (9) Differences in the proportions of nuclei with both Pol II and with Mam enrichment, which results in transcription being probabilistic/stochastic. These data demonstrate that presence of Mam-complexes is not sufficient to drive all the steps required for transcription in every Notch-ON nucleus.<br /> (10) The switch from more stochastic to robust transcription initiation was elicited when ecdysone was added.

Overall, the manuscript is well written, concise, and clear, and makes significant contributions to the Notch field, which are also important for a general understanding of transcription factor regulation and behavior in the nucleus. The authors have satisfactorily addressed all my criticisms of their initial submission and therefore congratulate the authors on an excellent paper.

Reviewer #1 (Public Review):

In this paper, N'Guessan et al report a study of expression QTL (eQTL) mapping in yeast using single cells. The authors make use of advances in single-cell RNAseq (scRNAseq) in yeast to increase the efficiency with which this type of analysis can be undertaken. Building on prior research led by the senior author that entailed genotyping and fitness profiling of almost 100,000 cells derived from a cross between two yeast strains (BY and RM) they performed scRNAseq on a subset of 4,489 individual cells. To address the sparsity of genotype data in the expression profiling they used a Hidden Markov Model (HMM) to infer genotypes and then identify the most likely known lineage genotype from the original dataset. To address the relationship between variance in fitness and gene expression the authors partition the variance to investigate the sources of variation. They then perform eQTL mapping and study the relationship between eQTL and fitness QTL identified in the earlier study.

This paper seeks to address the challenging question of how quantitative trait variation and expression variation are related. scRNAseq represents an appealing approach to eQTL mapping as it is possible to simultaneously genotype individual cells and measure expression in the same cell. As eQTL mapping requires large sample sizes to identify statistical relationships, this approach is likely to dramatically increase the statistical power of such studies. However, there are several technical challenges associated with scRNAseq and the authors' study is focused on addressing those challenges. Although the authors present results suggesting the feasibility of the approach there are limitations in the conclusions that can be drawn in the current study owing to the lack of clarity in the presentation of the results. Ultimately, this study presents a proof of concept with limited novel biological insights that would nonetheless make a useful contribution to the literature if the following major points were addressed:

(1) There is insufficient information provided about the nature of data. At a minimum, the following information should be provided to enable assessment of the study: What is the total library size, how many genes are identified per cell, how many UMIs are found per cell, what is the doublet rate, and how are doublets identified (e.g. on the basis of heterozygous calls at polymorphic loci?), how many times is each genotype observed, and how many polymorphic sites are identified per cell that are the basis of genotype inferences?

(2) The prior study analyzed 18 different conditions, whereas this study only assays expression in a single condition. However, the power of the authors' approach is that its efficiency enables testing eQTLs in multiple conditions. The study would be greatly strengthened through analysis of at least one more condition, and ideally several more conditions. The previous fitness study would be a useful guide for choosing additional conditions as identifying those conditions that result in the greatest contrasts in fitness QTL would be best suited to testing the generalizations that can be drawn from the study.

(3) Alternatively, the authors could demonstrate the power of their approach by applying it to a cross between two other yeast strains. As the cross between BY and RM has been exhaustively studied, applying this approach to a different cross would increase the likelihood of making novel biological discoveries.

(4) Figure 1 is misleading as A presents the original study from 2022 without important details such as how genotypes were identified. It is unclear what the barcode is in this study and how it is used in the analysis. Is the barcode for each lineage transcribed so that it is identified in the scRNAseq data? Or, does the barcode in B refer to the cell index barcode? A clearer presentation and explanation of terms are needed to understand the method.

(5) The rationale for the analysis reported in Figure 2B is unclear. The fitness data are from the previous study and the goal is to estimate the heritability using the genotyping data from the scRNAseq data. What is the explanation for why the data don't agree for only one condition, i.e. 37C? And, what are we to understand from the overall result?

(6) Figure 3 presents an analysis of variance partitioning as a Venn diagram. This summarized result is very hard to understand in the absence of any examples of what the underlying raw data look like. For example, what does trait variation look like if only genotype explains the variance or if only gene expression explains the variance? The presented highly summarized data is not intuitive and its presentation is poor - the result that is currently provided would be easier to read in a table format, but the reader needs more information to be able to interpret and understand the result.

(7) I am concerned about the conclusions that can be drawn about expression heritability. The authors claim that expression heritability is correlated with expression levels. It seems likely that this reflects differing statistical power. How can this possibility be excluded?

(8) Conversely, the authors claim that the genes with the lowest heritability are genes involved in the cell cycle. However, uniquely in scRNAseq, cell cycle regulated genes appear to have the highest variance in the data as they are only expressed in a subset of cells. Without incorporating this fact one would erroneously conclude that the variation is not heritable. To test the heritability of cell cycle regulation genes the authors should partition the cells into each cell cycle stage based on expression.

(9) I do not understand Figure S5 and how eQTL sites are assigned to these specific classes given that the authors say that causative variation cannot be resolved because of linkage disequilibrium.

(10) The paragraph starting at line 305 is very confusing. In particular, the authors state that they identify a hotspot of regulation at the mating type locus. It is not obvious why this would be the case. Moreover, they claim that they find evidence for both MATa and MATalpha gene expression. Information is not provided about how segregants were isolated, but assuming that the authors did not dissect 25,000 tetrads to obtain 100,000 segregants I would infer that random spore using SGA was used. In that case, all cells should be MATa. The authors should clarify and explain this observation.

(11) Ultimately, it is not clear what new biological findings the authors have made. There are no novel findings with respect to causative variation underlying eQTLs and I would encourage the authors to make clearer statements in their abstract, introduction, and conclusion about the key discoveries. E.g. What are the "new associations between phenotypic and transcriptomic variations" mentioned in the abstract?

The following minor points should be addressed:

(1) The segregants should be referred to as F2 segregants as they are derived from an F1 cross.

(2) The connections to eQTLs in other organisms should be addressed in the introduction and conclusion. For example, in humans, there has been little evidence for trans eQTLs in contrast to what has been found in yeast.

(3) The authors state that an advantage of scRNAseq over bulk is that it captures rare cell populations (line 79), but this advantage is not exploited in this study.

(4) The authors use ~5% of the lineages from the original study. There is no rationale for why this is an appropriate sample size. Is there an argument for using more cells in eQTL mapping or conversely could the authors ask if fewer cells would provide similar conclusions by downsampling?

(5) I do not agree that the use of UMIs overcomes the challenges of low sequencing depth. UMIs mitigate the possible technical artifacts due to massive PCR amplification.

(6) There is an inadequate reference to prior work on scRNAseq in yeast that established the methods used by the authors and eQTL mapping in human cells using scRNAseq.

(7) The use of empty quotes in Figure 4A is confusing and an alternative presentation method should be used.

(8) The authors speculate about the use of predicted fitness instead of observed fitness, but this is something they could explicitly address in their current study.

Reviewer #1 (Public Review):

Summary:

This is an experimentally soundly designed work and a very well-written manuscript. There is a very clear logic that drives the reader from one experiment to the next, the experimental design is clearly explained throughout and the relevance of the acquired data is well analyzed and supports the claims made by the authors. The authors made an evident effort to combine imaging, genetic, and molecular data to describe previously unknown early embryonic movement patterns and to identify regulatory mechanisms that control several aspects of it.

Strengths:

The authors develop a new method to analyze, quantitatively, the onset of movement during the latter embryonic stages of Drosophila development. This setup allows for a high throughput analysis of general movement dynamics based on the capture of variations of light intensity reflected by the embryo. This setup is capable of imaging several embryos simultaneously and provides a detailed measure of movement over time, which proves to be very useful for further discoveries in the manuscript. This setup already provides a thorough and quantifiable description of a process that is little known and identifies two different phases during late embryonic movements: a myogenic phase and a neurogenic phase, which they elegantly prove is dependent on neuronal activity by knocking down action potentials across the nervous system.

However, in this system, movement is detected as a whole, and no further description of the type of movement is provided beyond frequency and amplitude; it would be interesting to know from the authors if a more precise description of the movements that take place at this stage can be achieved with this method (e.g. motion patterns across the A-P body axis).

Importantly, this highly quantitative experimental setup is an excellent system for performing screenings of motion regulators during late embryonic development, and its use could be extended to search for different modulators of the process, beyond miRNAs (genetic mutants, drugs, etc.).

Using their newly established motion detection pipeline, the authors identify miR-2b-1 as required for proper larval and embryonic motion, and identify an overall reduction in the quantity of both myogenic and neurogenic movements, as well as an increased frequency in neurogenic movement "pulses".

Focusing on the neurogenic movement phenotype the authors use in situ probes and perform RT-PCR on FACS-sorted CNS cells to unambiguously detect miR-2b-1 expression in the embryonic nervous system. The neurogenic motion defects observed in miR-2b-1 mutant embryos and early larvae can be completely rescued by the expression of ectopic miR-2b-1 specifically in the nervous system, providing solid evidence of the requirement and sufficiency of miR-2b-1 expressed in the nervous system to regulate these phases of movement.

To explore the mechanism through which miR-2b-1 impacts embryonic movement, the authors use a state-of-the-art bioinformatic approach to identify potential targets of miR-2b-1, and find that the expression levels of an uncharacterized gene, CG3638, are indeed regulated by miR-2b-1. Furthermore, they prove that by knocking down the expression of CG3638 in a miR-2b-1 mutant background, the neurogenic embryonic movement defects are rescued, pointing that the repression of CG3638 by miR-2b-1 is necessary for correct motion patterns in wild-type embryos. Therefore, this paper provides the first functional characterization of CG3638, and names this gene Janus.

Finally, the authors aim to discriminate which elements of the embryonic motor system miR-2b-1/Janus are required. Using directed overexpression of miR-2b-1 and Janus knockdown in the motor neurons and the chordotonal (sensory) organs, they prove that the miR-2b-1/Janus regulatory axis is specifically required in the sensory organs to promote normal embryonic and larval movement.

Weaknesses:

The authors do not describe properly how the miRNA screening was performed and just claim that only miR-2b-1 mutants presented a defective motion phenotype in early L1. How many miRNAs were tested, and how candidates were selected is never explicitly mentioned in the text or the Methods section.

The initial screening to identify miRNAs involved in motion behaviors is performed in early larval movement. The logic presented by the authors is clear - it is assumed that early larval movement cannot proceed normally in the absence of previous embryonic motion - and ultimately helped them identify a miRNA required for modulation of embryonic movement. However, it is possible that certain miRNAs play a role in the modulation of embryonic movement while being dispensable for early L1 behaviors. Such regulators might have been missed with the current screening setup.

Although similar changes to those described for the neurogenic phase of embryonic movement are described for the myogenic phase in miR-2b-1 mutants (reduction in motion amplitude), this phenotype goes unexplored. This is not a big issue, as the authors convincingly demonstrate later that miR-2b-1 is specifically required in the nervous system for proper embryonic and larval movement, and the effects of miR-2b-1 on myogenic movement might as well be the focus of future work. However, it will be interesting to discuss here the implications of a reduced myogenic movement phase, especially as miR-2b-1 is specifically involved in regulating the activity of the chordotonal system - which precisely detects early myogenic movements.

FACS-sorting of neuronal cells followed by RT-PCR convincingly detects the presence of miR-2b-1 in the embryonic CNS. However, control of non-neuronal cells would be required to explore whether miR-2b-1 is not only present but enriched in the nervous system compared to other tissues. This is also the case in the miR-2b-1 and Janus expression analysis in the chordotonal organs: a control sample from the motor neurons would help discriminate whether miR-2b-1/Janus regulatory axis is specifically enriched in chordotonal organs or whether both genes are expressed throughout the CNS but operate under a different regulation or requirements for the movement phenotypes.

Reviewer #1 (Public Review):

Summary:

This manuscript employs yolk sac visceral endoderm cells as a novel model for studying endosomal fusion, observing two distinct fusion behaviors: quick homotypic fusion between late endosomes, and slower heterotypic fusion between late endosomes and lysosomes. The mathematical modeling suggests that vesicle size critically influences the mode of fusion. Further investigations reveal that actin filaments are dynamically associated with late endosomal membranes, and are oriented in the x-y plane and along the apical-basal axis. Actin and Arf2/3 were shown to appear at the rear end of the endosomes along the moving direction suggesting polymerization of actin may provide force for the movement of endosomes. Additionally, the authors found that actin dynamics regulate homotypic and heterotypic fusion events in a different manner. The authors also provide evidence to suggest that Cofilin-dependent actin dynamics are involved in late endosome fusion.

Strengths:

The unique feature of this study is that the authors use yolk sac visceral endoderm cells to study endosomal fusion. Yolk sac visceral endoderm cells have huge endocytic vesicles, endosomes, and lysosomes, offering an excellent system to explore endosomal fusion dynamics and the assembly of cellular factors on membranes. The manuscript provides a valuable and convincing observation of the modes of endosomal fusion and the roles of actin dynamics in this process, and the conclusions of the study are justified by the data.

Weaknesses:

While the study offers compelling observations, it falls short of delivering clear mechanistic insights. Key questions remain unaddressed, such as the functional significance of actin filaments that extend apically in positioning late endosomes, the ways in which actin dynamics influence fusion events, and the functional implications of the slower bridge fusion process.

Reviewer #1 (Public Review):

This work provides new mechanistic insights into the competitive inhibition in the mammalian P2X7 receptors using structural and functional approaches. The authors solved the structure of panda (pd) P2X7 in the presence of the classical competitive antagonists PPNDS and PPADS. They find that both the drugs bind to the orthosteric site employed by the physiological agonist ATP. However, owing to the presence of a single phosphate group, they prevent movements in the flipper domain required for channel opening. The authors performed structure based mutational analysis together with electrophysiological characterization to understand the subtype specific binding of these drugs. It is known from previous studies that P2X1 and P2X3 are more sensitive to these drugs as compared to P2X7, hence, the residues adjacent to the ATP binding site in pdP2X7 were mutated to those present in P2X1. They observed that mutations of Q143, I214 and Q248 into lysine (hP2X1) increased the P2X7 sensitivity to PPNDS, whereas in P2X1, mutations of these lysines to alanine reduced sensitivity to PPNDS, suggesting that these key residues contribute to the subunit specific sensitivity to these drugs. Similar experiments were done in hP2X3 to demonstrate its higher sensitivity to PPNDS. This preprint provides a useful framework for developing subtype specific drugs for the family of P2X receptor channels, an area that is currently relatively unexplored.<br /> The conclusions of the paper are well supported.

The revised manuscript is well written and presents its data with more clarity.

DOI: 10.1016/j.isci.2024.109293

Resource: (ZFIN Cat# ZDB-ALT-130122-1,RRID:ZFIN_ZDB-ALT-130122-1)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-130122-1

What is this?

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Reviewer #1 (Public Review):

Using a pharmacological and knock-down approach, the authors could demonstrate that ROCK activity is required for the normal development of the larval skeleton. The presence of ROCK in the pluteus stage depends on the activity of VEGF that is responsible for the formation of the tubular syncytial sheath of the calcifying primary mesenchyme cells in which the skeleton forms. The importance of ROCK in skeleton formation was confirmed in cell culture experiments, demonstrating that ROCK inhibition leads to decreased elongation and abnormal branching of spicules. µCT analyses underline this finding demonstrating that the inhibition of ROCK mainly affects elongation of spicules while growth in girth is little affected. F-actin labeling experiments could demonstrate that ROCK inhibition interferes with the organization of the actomyosin network in the early phase of skeleton formation, while f-actin organization in the tips of the elongating spicule is unaffected by the pharmacological inhibition of ROCK. Finally, ROCK inhibition strongly affects the expression of major regulatory and calcification-related genes in the calcifying cells. Based on these findings the authors propose a model for the regulatory interaction between the skeletogenic GRN, ROCK and the f-actin system relevant for skeletogenesis.

Comments on revised version:

In their manuscript Hijaze et al. adequately addressed the majority of my previous concerns in a satisfactory manner. In particular, they validated their morpholino knock-down experiments by explaining how they determined the optimal concentrations and provided an immunohistological evidence for the reduction in ROCK protein abundance. The authors also added new antibody stainings providing evidence that ROCK and F-actin do not interact directly but likely through other kinases that modulate f-actin, and that the localization of f-actin at the spicule tips remains unaffected by the knock-down. In addition, the authors revised their discussion to not overstate their observations, and by focusing on the potential mechanisms by which ROCK may affect biomineralization (i.e. mechano sensing and exocytosis of vesicles). Here I would like to add, that f-actin mediated exocytosis does not necessarily target mineral baring vesicles but may also promote the exocytosis of matrix proteins that are essential for the normal formation of the spicules and that are an integral component of other biominerals, as well. I strongly encourage the authors to continue on this exciting research, including the development of methods to analyze the molecular mechanisms that control vesicular trafficking in mineralizing systems.

Reviewer #1 (Public Review):

Summary:

The authors want to understand fundamental steps in ligand binding to muscle nicotinic receptors using computational methods. Overall, although the work provides new information and support for existing models of ligand activation of this receptor type, some limitations in the methods and approach mean that the findings are not as conclusive as hoped.

Strengths:

The strengths include the number of ligands tried, and the comparison to the existing mature analysis of receptor function from the senior author's lab.

Weaknesses:

The weakness are the brevity of the simulations, the concomitant lack of scope of the simulations, the lack of depth in the analysis and the incomplete relation to other relevant work. The free energy methods use seem to lack accuracy - they are only correct for 2 out of 4 ligands.

Reviewer #1 (Public Review):

Summary:

The work by Joseph et al "Impact of the clinically approved BTK inhibitors on the conformation of full-length BTK and analysis of the development of BTK resistance mutations in chronic lymphocytic leukemia" seeks to comparatively analyze the effect of a range of covalent and noncovalent clinical BTK inhibitors upon BTK conformation. The novel aspect of this manuscript is that it seeks to evaluate the differential resistance mutations that arise distinctly from each of the inhibitors.

Strengths:

This is an exciting study that builds upon the fundamental notion of ensemble behavior in solutions for enzymes such as BTK. The HDX-MS and NMR experiments are adequately and comprehensively presented.

Weaknesses:

While I commend the novelty of the study, the absence of important controls greatly tempers my enthusiasm for this work. As stated in the abstract, there are no broad takeaways for how resistance mutation bias operated from this study, although the mechanism of action of 2 common resistance mutations is useful. How these 2 resistance mutations connect to ensemble behavior, is not obvious. This is partly because BTK does not populate just binary "open"/"closed" conformations, but there are likely multiple intermediate conformations. Each inhibitor appears to preferentially "select" conformations by the authors' own assessment (line 236) and this carries implications for the emergence of resistance mutations. The most important control that would help is to use ADP or nonhydrolyzable and ATP as a baseline to establish the "inactive" and "active" conformations. All of the HDX-MS and NMR studies use protein that has no nucleotide present. A major question that remains is whether each of the inhibitors preferentially favors/blocks ADP or ATP binding. This then means it is not equivalent to correlate functional kinase assay conditions with either HDX-MS or NMR experiments.

Reviewer #1 (Public Review):

The Calcium Homeostasis Modulators (CALHM) are a family of large pore channels, of which the physiological role of CALHM1 and 3 is well understood, in particular their key role in taste sensation via the release of the neurotransmitter ATP. The activation mechanism of CALHM1 involves membrane depolarization and a decrease in extracellular Ca concentration, allowing the passage of large cellular metabolites. However, the activation mechanism and physiological roles of other family members are much less well understood. Many structures of homomeric CALHM proteins have been determined, revealing distinct oligomeric assemblies despite a common transmembrane domain topology. CALHM1 and 3 have been shown functionally to form heteromeric assemblies with properties distinct from those of homomeric CALHM1. However, the structural basis of heteromeric CALHM1 and 3 remains unexplored.

In this paper, Drozdzyk et al. present an important study on the structures of heteromeric channels composed of CALHM2 and CALHM4, extending the structural understanding of the CALHM family beyond homomeric channels. The study relies primarily on cryo-EM. Despite the inherent challenges of structural determination due to the similar structural features of CALHM2 and CALHM4, the authors innovatively use synthetic nanobodies to distinguish between the subunits. Their results show a broad distribution of different heteromeric assemblies, with CALHM4 conformation similar to its homomeric form and CALHM2 conformation influenced by its proximity to CALHM4, and provide detailed insights into the interaction between CALHM2 and CALHM4.

The manuscript is well-structured and presents clear results that support the conclusions drawn. The discovery of heteromeric CALHM channels, although currently limited to an overexpressed system, represents a significant advance in the field of large-pore channels and will certainly encourage further investigation into the physiological relevance and roles of heteromeric CALHM channels. The manuscript would benefit from further insight into the functional properties of these heteromeric channels. However, this is not a weakness as the identification of precise activation stimuli for CALHM2 and 4 is beyond the scope of this work.

A challenge noted is the wide distribution of heteromeric assemblies in the 3D classification, resulting in insufficient particles for high-resolution structure determination of each assembly. The authors choose to combine particles from assemblies with 2-4 copies of CALHM4, which reveals the interface between CALHM2 and 4 but may compromise the quality of structural details. I recommend an alternative data processing strategy. First, refine particles with 2-4 CALHM4 subunits with symmetry imposed. This is followed by symmetry expansion, signal subtraction of two adjacent subunits, and subsequent classification and refinement of the subtracted particles. This approach, while not guaranteed, can potentially provide a clearer definition of CALHM2 and CALHM4 interfaces and show whether CALHM2 subunits adopt different conformations based on their proximity to CALHM4 subunits.

Reviewer #1 (Public Review):

Summary:

This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with amplitude comparable to aspartate, and cells can quickly adapt (and generally over-adapt). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.

Strengths:

The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH. The updated manuscript controls for the impact of pH on the fluorescent protein brightness that can bias the measured FRET signal. After correction the response amplitude and sharpness (hill coefficient) are comparable to conventional chemoattractants (e.g. aspartate), indicating the general mechanisms underlying the response may be similar. The authors suggest that the biphasic response of Tar mutants may be due to pH influencing the activity of other enzymes (CheA, CheR or CheB), which will be an interesting direction for future study.

Weaknesses:

The measured response may be biased by adaptation, especially for weak potassium signals. For other attractant stimuli, the response typically shows a low plateau before it recovers (adapts). In the case of potassium, the FRET signal does not have an obvious plateau following the stimuli of small potassium concentrations, perhaps due to the faster adaptation compared to other chemoattractants. It is possible cells have already partially adapted when the response reaches its minimum, so the measured response may be a slight underestimate of the true response. Mutants without adaptation enzymes appear to be sensitive to potassium only at much larger concentrations, where the pH significantly disrupts the FRET signal; more accurate measurements would require development of new mutants and/or measurement techniques.

Reviewer #1 (Public Review):

Summary:

This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

Strengths:

The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of<br /> PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP level. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

Weaknesses:

It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

Reviewer #1 (Public Review):

Summary:

This work describes the mechanism of protein disaggregation by the ClpL AAA+ protein of Listeria monocytogenes. Using several model subtrate proteins the authors first show that ClpL possesses a robust disaggregase activity that does not further require the endogenous DnaK chaperone in vitro. In addition, they found that ClpL is more thermostable than the endogenous L. monocytogenes DnaK and has the capacity to unfold tightly folded protein domains. The mechanistic basis for the robust disaggregase activity of ClpL was also dissected in vitro and in some cases, supported by in vivo data performed in chaperone-deficient E. coli strains. The data presented show that the two AAA domains, the pore-2 site and the N-terminal domain (NTD) of ClpL are critical for its disaggregase activity. Remarkably, grafting the NTD of ClpL to ClpB converted ClpB into an autonomous disaggregase, highlighting the importance of such a domain in the DnaK-independent disaggregation of proteins. The role of the ClpL NTD domain was further dissected, identifying key residues and positions necessary for aggregates recognition and disaggregation. Finally, using sets of SEC and negative staining EM experiments combined with conditional covalent linkages and disaggregation assays the authors found that ClpL shows significant structural plasticity, forming dynamic hexameric and heptameric active single rings that can further form higher assembly states via their middle domains.

Strengths:

The manuscript is well written and the experimental work well executed. It contains a robust and complete set of in vitro data that push further our knowledge of such important disaggregases. It shows the importance of the atypical ClpL N-terminal domain in the disaggregation process as well as the structural malleability of such AAA+ proteins. More generally, this work expands our knowledge of heat resistance in bacterial pathogens.

Weaknesses:

There is no specific weakness in this work, although it would have helped to have a drawing model showing how ClpL performs protein disaggregation based on their new findings. The function of the higher assembly states of ClpL remains unresolved and will need further extensive research. Similarly, it will be interesting in the future to see whether the sole function of the plasmid encoded ClpL is to cope with general protein aggregates under heat stress.

Joint Public Review:

Summary:

Previously, this group showed that Tgfbr1 regulates the reorganization of the epiblast and primitive streak into the chordo-neural hinge and tailbud during the trunk-to-tail transition. Gdf11 signaling plays a crucial role in orchestrating the transition from trunk to tail tissues in vertebrate embryos, including the reallocation of axial progenitors into the tailbud and Tgfbr1 plays a key role in mediating its signaling activity. Progenitors that contribute to the extension of the neural tube and paraxial mesoderm into the tail are located in this region. In this work, the authors show that Tgfbr1 also regulates the reorganization of the posterior primitive streak/base of allantois and the endoderm as well.

By analyzing the morphological phenotypes and marker gene expression in Tgfbr1 mutant mouse embryos, they show that it regulates the merger of somatic and splanchnic layers of the lateral plate mesoderm, the posterior streak derivative. They also present evidence suggesting that Tgfbr1 acts upstream of Isl1 (key effector of Gdf11 signaling for controlling differentiation of lateral mesoderm progenitors) and regulates the remodelling of the major blood vessels, the lateral plate mesoderm and endoderm associated with the trunk-to-tail transition. Through a detailed phenotypic analysis, the authors observed that, similarly to Isl1 mutants, the lack of Tgfbr1 in mouse embryos hinders the activation of hindlimb and external genitalia maker genes and results in a failure of lateral plate mesoderm layers to converge during tail development. As a result, they interpret that ventral lateral mesoderm, which generates the peri cloacal mesenchyme and genital tuberculum, fails to specify.

They also show defects in the morphogenesis of the dorsal aorta at the trunk/tail juncture, resulting in an aberrant embryonic/extraembryonic vascular connection. Endoderm reorganization defects following abnormal morphogenesis of the gut tube in the Tgfbr1 mutants cause failure of tailgut formation and cloacal enlargement. Thus, Tgfbr1 activity regulates the morphogenesis of the trunk/tail junction and the morphogenetic switch in all germ layers required for continuing post-anal tail development. Taken together with the previous studies, this work places Gdf11/8 - Tgfbr1 signaling at the pivot of trunk-to-tail transition and the authors speculate that critical signaling through Tgfbr1 occurs in the posterior-most part of the caudal epiblast, close to the allantois.

Strengths:

The data shown is solid with excellent embryology/developmental biology. This work demonstrates meticulous execution and is presented in a comprehensive and coherent manner. Although not completely novel, the results/conclusions add to the known function of Gdf11 signaling during the trunk-to-tail transition.

Weaknesses:

The authors rely on the expression of a small number of key regulatory genes to interpret the developmental defects. The alternative possibilities remain to be ruled out thoroughly. The manuscript is also quite descriptive and would benefit from more focused highlighting of the novelty regarding the absence of Tgfbr1 in the mouse embryo. They should also strengthen some of their conclusions with more details in the results.

Reviewer #1 (Public Review):

Summary:<br /> In the present study, Rincon-Torroella et al. developed ME3BP-7, a microencapsulated formulation of 3BP, as an agent to target MCT1 overexpressing PDACs. They provided evidence showing the specific killing of PDAC cells with MCT1 overexpressing in vitro, along with demonstrating the safety and anti-tumor efficacy of ME3BP-7 in PDAC orthotopic mouse models.

Strengths:<br /> * Developed a novel agent.<br /> * Well-designed experiments and an organized presentation of data that support the conclusions drawn.

Weaknesses:<br /> There are some minor issues that could enhance the clarity and completeness of the study:

(1) Statistical results should be visually presented in Figure 4 and Figure S1.

(2) Given the tumor heterogeneity and the identification of focal high expression of MCT1 in Figure 7 and Figure S5B, it is suggested that the authors include the results of immunohistochemical (IHC) analysis of MCT1 expression in both control and ME3BP-7 treated tumor tissues. This addition may offer insight into whether the remaining tumors are composed of PDAC cells with negative MCT1 expression, while the cells with relatively high levels of MCT1 expression were eliminated by ME3BP-7 treatment.

(3.)The authors are encouraged to discuss the future directions for improving the efficacy of this study. For example, exploring the combination of ME3BP-7 with a glutaminase-1 inhibitor (PMID 37891897) could be a valuable avenue for further research.

Reviewer #1 (Public Review):

Summary:

This work presents an in-depth characterization of the factors that influence the structural dynamics of the Clostridium botulinum guanidine-IV riboswitch (riboG). Using a single-molecule FRET, the authors demonstrate that riboG undergoes ligand and Mg2+ dependent conformational changes consistent with the dynamic formation of a kissing loop (KL) in the aptamer domain. Formation of the KL is attenuated by Mg2+ and Gua+ ligand at physiological concentrations as well as the length of the RNA. Interestingly, the KL is most stable in the context of just the aptamer domain compared to longer RNAs capable of forming the terminator stem. To attenuate transcription, binding of Gua+ and formation of the KL must occur rapidly after transcription of the aptamer domain but before transcription of the rest of the terminator stem.

Strengths:

(1) Single-molecule FRET microscopy is well suited to unveil the conformational dynamics of KL formation and the authors provide a wealth of data to examine the effect of the ligand and ions on riboswitch dynamics. The addition of complementary transcriptional readthrough assays provides further support for the author's proposed model of how the riboswitch dynamics contribute to function.

(2) The single-molecule data strongly support that the effect of Gua+ ligand and Mg2+ influence the RNA structure differently for varying lengths of the RNA. The authors also demonstrate that this is specific for Mg2+ as Na+ and K+ ions have little effect.

(3) The PLOR method utilized is clever and well adapted for both dual labeling of RNAs and examining RNA at various lengths to mimic co-transcriptional folding. Using PLOR, they demonstrate that a change in the structural dynamics and ligand binding can occur after the extension of the RNA transcript by a single nucleotide. Such a tight window of regulation has intriguing implications for kinetically controlled riboswitches.

Weaknesses:

(1) The authors use only one mutant to confirm that their FRET signal indicates the formation of the KL. Importantly, this mutation does not involve the nucleotides that are part of the KL interaction. It would be more convincing if the authors used mutations in both strands of the KL and performed compensatory mutations that restore base pairing. Experiments like this would solidify the structural interpretation of the work, particularly in the context of the full-length riboG RNA or in the co-transcriptional mimic experiments, which appear to have more conformational heterogeneity.

(2) The existence of the pre-folded state (intermediate FRET ~0.5) is not well supported in their data and could be explained by an acquisition artifact. The dwell times are very short often only a single frame indicating that there could be a very fast transition (< 0.1s) from low to high FRET that averages to a FRET efficiency of 0.5. To firmly demonstrate that this intermediate FRET state is metastable and not an artifact, the authors need to perform measurements with a faster frame rate and demonstrate that the state is still present.

(3) The PLOR method employs a non-biologically relevant polymerase (T7 RNAP) to mimic transcription elongation and folding near the elongation complex. T7 RNAP has a shorter exit channel than bacterial RNAPs and therefore, folding in the exit channel may be different between different RNAPs. Additionally, the nascent RNA may interact with bacterial RNAP differently. For these reasons, it is not clear how well the dynamics observed in the T7 ECs recapitulate riboswitch folding dynamics in bacterial ECs where they would occur in nature.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Zeng and Staley provide a valuable analysis of the molecular requirements for the export of a reporter mRNA that contains a lariat structure at its 5' end in the budding yeast S. cerevisiae. The authors provide evidence that this is regulated by the main mRNA export machinery (Yra1, Mex67, Nab2, Npl3, Tom1, and Mlp1). Of note, Mlp1 has been mainly implicated in the nuclear retention of unspliced pre-mRNA (i.e. quality control), and relatively little has been done to investigate its role in mRNA export in budding yeast.

Strengths:

There is relatively little information in the current literature about the nuclear export of splicing intermediates. This paper provides one of the first analyses of this process and dissects the molecular components that promote this form of RNA export. Overall, the strength of the data presented in the manuscript is solid. The paper is well written and the message is clear and of general interest to the mRNA community.

Weaknesses:

There are three problems with the paper, although these are not major and likely would not affect the final model as most aspects of the molecular details are confirmed by multiple complementary assays.

(1) The brG reporter produces both unspliced pre-mRNA and a lariat-containing intermediate RNA. Based on the primer extension assay the authors claim that only 33% of the final product is in pre-mRNA form and that this "is insufficient to account for the magnitude of the cytoplasmic signal from the brG reporter (83%)". Nevertheless, it is possible that primer extension is incomplete or that the lariat-containing RNA is inaccessible for smFISH. The authors could easily perform a dual smFISH experiment (similar to Adivarahan et l., Molecular Cell 2018) where exon 1 is labelled with probes of one color, and the region that overlaps the lariat-containing intermediate is labelled with probes of a second color. If the authors are correct, then one-third of the smFISH foci should have both labels and the rest would have only the second label. This would also confirm that the latter (i.e. the lariat-containing RNAs) are exported to the cytoplasm. Using this approach, the authors could then show that MLP1-depletion (or depletion of any of the other factors) affect(s) one pool of RNAs (i.e. those that are lariat-containing) but not the other (i.e. pre-mRNA). Including these experiments would make the evidence for their model more convincing.

(2) In some cases, the number of smFISH foci appears to change drastically depending on the genetic background. This could either be due to the stochastic nature of mRNA expression between cells or reflect real differences between the genetic backgrounds that could alter the interpretation of the other observations.

(3) The authors state in the discussion that "the general mRNA export pathway transports discarded lariat intermediates into the cytoplasm". Although this appears to be the case for the reporters that are investigated in this paper, I don't think that the authors should make such a broad sweeping claim. It may be that some discarded lariat intermediates are exported to the cytoplasm while others are targeted for nuclear retention and/or decay.

Reviewer #1 (Public Review):

Summary:

The major result in the manuscript is the observation of the higher order structures in a cryoET reconstruction that could be used for understanding the assembly of toroid structures. The cross-linking ability of ZapD dimers result in bending of FtsZ filaments to a constant curvature. Many such short filaments are stitched together to form a toroid like structure. The geometry of assembly of filaments - whether they form straight bundles or toroid like structures - depends on the relative concentrations of FtsZ and ZapD.

Strengths:

In addition to a clear picture of the FtsZ assembly into ring-like structures, the authors have carried out basic biochemistry and biophysical techniques to assay the GTPase activity, the kinetics of assembly, and the ZapD to FtsZ ratio.

Weaknesses:

The discussion does not provide an overall perspective that correlates the cryoET structural organisation of filaments with the biophysical data.

The crosslinking nature of ZapD is already established in the field. The work carried out is important to understand the ring assembly of FtsZ. However, the availability of the cryoET observations can be further analysed in detail to derive many measurements that will help validate the model, and obtain new insights.

Reviewer #1 (Public Review):

Summary:

Hippo pathway activity is required for pancreas morphogenesis, but its role in endocrine pancreas function remains elusive. The author aims to study the function of the TEAD1 gene in b-cells.

Strengths:

The authors generated TEAD1 conditional knockout animals by crossing the TEAD1f/f mice with three Cre strains (RIP-Cre, Ins1-Cre, and MIP-CreERT). In all of them, the KO animals showed progressive loss of insulin secretion with normal beta cell mass. Further characterization of the animals indicated glucose-induced insulin secretion defect and increased beta cell proliferation rate. RNA-Seq and ChIP-Seq experiments identified Pdx1, MafA, and Glut2, etc. as direct targets of TEAD1, which might be responsible for the insulin secretion defect in the animals. Of interest, the authors also uncovered the cell cycle-related gene p16 as a direct target of TEAD1. Reduction of p16 is likely to drive the beta cell proliferation in the TEAD1 knockout model. Thus, they proposed that TEAD1 is a regulator of the proliferative quiescence process in beta cells. Overall, the evidence provided by the authors is highly relevant and supports their conclusion.

Weaknesses:

(1) The authors don't explicitly mention that some results appeared in a previous publication (https://doi.org/10.1093/nar/gkac1063) from them.

(2) The authors begin their story by introducing TEAD1 as part of the Hippo pathway. They showed Taz expression data in Figure 1. Did they do any experiments to detect Taz in their TEAD1 model? Did the authors detect any expression changes in CTGF following TEAD1 knockout? I could not see this changed. The phenotype characterization data presented here contrasts with what has been shown in TAZ b-cell knockout mice (https://doi.org/10.1101/2022.05.31.494216). Based on the data presented here, Hippo is not involved, which should at least be discussed in length.

(3) Figure 1B - TAZ staining looks different in the three-month age group.

(4) TEAD ChIP-seq data doesn't look very convincing to me. It's hard to tell whether those highlighted regions in Figures 3A and 5J were signals or background noise. Although the authors also performed ChIP-qPCR in MIN6, it's unclear whether these binding events occur in vivo. The analysis of ChIP-seq dataset is limited as well. How many peaks called? What proportion of differentially expressed genes are bound by TEAD1? Was TEAD1 also detectable at NGN3 and NEUROD1 gene regions? If acquiring enough cells is not possible, the authors could try CUT&RUN or CUT&Tag to improve the data quality.

(5) The authors should perform RNA-seq or gene expression studies in MIP-CreERT to confirm, which could help narrow down the actual targets of TEAD1 as well.

(6) Figure 6 - the experiment lacks a control: Ezh2 beta cell KO. In addition to p16, Ezh2, and PRC2 have other targets in beta-cells, the authors could not rule out the contribution of those to the phenotype, so the implication of this experiment is vague.

Reviewer #1 (Public Review):

Summary:

Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI oncogene. Ewing sarcoma cells are addicted to this chimeric transcription factor, which represents a strong therapeutic vulnerability. Unfortunately, targeting EWS-FLI has proven to be very difficult, and a better understanding of how this chimeric transcription factor works is critical to achieving this goal. Towards this perspective, the group had previously identified a DBD-𝛼4 helix (DBD) in FLI that appears to be necessary to mediate EWS-FLI transcriptomic activity. Here, the authors used multi-omic approaches, including CUT&tag, RNAseq, and MicroC to investigate the impact of this DBD domain. Importantly, these experiments were performed in the A673 Ewing sarcoma model where endogenous EWS-FLI was silenced, and EWS-FLI-DBD proficient or deficient isoforms were re-expressed (isogenic context). They found that the DBD domain is key to mediating EWS-FLI cis activity (at msat) and to generating the formation of specific TADs. Furthermore, cells expressing DBD-deficient EWS-FLI display very poor colony-forming capacity, highlighting that targeting this domain may lead to therapeutic perspectives.

Strengths:

The group has strong expertise in Ewing sarcoma genetics and epigenetics and also in using and analyzing this model (Theisen et al., 2019; Boone et al., 2021; Showpnil et al., 2022).

They aim at better understanding how EWS-FLI mediated its oncogenic activity, which is critical to eventually identifying novel therapies against this aggressive cancer.

They use the most recent state-of-the-art omics methods to investigate transcriptome, epigenetics, and genome conformation methods. In particular, Micro-C enables achieving up to 1kb resolved 3D chromatin structures, making it possible to investigate a large number of TADs and sub-TADs structures where EWS-FLI1 mediates its oncogenic activity.

They performed all their experiments in an Ewing sarcoma genetic background (A673 cells) which circumvents bias from previously reported approaches when working in non-orthologous cell models using similar approaches.

Weaknesses:

The main weakness comes from the poor reproducibility of Micro-C data. Indeed, it appears that the distances/clustering observed between replicates are typically similar or even larger than between biological conditions. For instance, in Figure 1B, I do not see any clustering when considering DBD1, DBD2, DBD+1, DBD+2.

Lanes 80-83: "KD replicates clustered together with DBD replicate 1 on both axes and with DBD replicate 2 on the y-axis. DBD+ replicates, on the other hand, clustered away from both KD and DBD replicates. These observations suggest that the global chromatin structure of DBD replicates is more similar to KD than DBD+ replicates."

When replacing DBD replicate 1 with DBD replicate 2, their statement would not be true anymore.

Additional replicates to clarify this aspect seem absolutely necessary since those data are paving the way for the entire manuscript.

Similarly:<br /> - In Figure 1C, how would the result look when comparing DBD2/KD2/DBD+2? Same when comparing DBD 1 with KD1 and DBD+1. Would the difference go in the same direction?<br /> - Figure 1D-E. How would these plots look like when comparing each replicate to each other's? How much difference would be observed when comparing, for instance, DBD1/DBD2 ? or DBD1/DBD+1?<br /> - Figure 2: again, how would these analyses look like when performing the analysis with only DBD1/DBD+1/KD1 or DBD2/DBD+2/KD?

Another major question is the stability of EWS-FLI DBD vs EWS-FLI DBD+ proteins. Indeed, it seems that they have more FLAG (i.e., EWS-FLI) peaks in the DBD+ condition compared to the DBD condition (Figure 2B). In the WB, FLAG intensities seem also higher (2/3 replicates) in DBD+ condition compared to the DBD condition (Figure S1B).

Would it be possible that DBD+ is just more expressed or more stable than DBD? The higher stability of the re-expressed DBD+ could also partially explain their results independently of the 3D conformational change. In other words, can they exclude that DBD+ and DBD binding are not related to their respective protein stability or their global re-expression levels?

Surprisingly, WB FLI bands in DBD+ conditions are systematically (3/3 replicates) fainter than in DBD conditions (Figure S1B). How do the authors explain these opposite results between FLI and FALG in the WB?

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors provide strong evidence that the cell surface E3 ubiquitin ligases RNF43 and ZNRF3, which are well known for their role in regulating cell surface levels of WNT receptors encoded by FZD genes, also target EGFR for degradation. This is a newly identified function for these ubiquitin ligases beyond their role in regulating WNT signaling. Loss of RNF43/ZNRF3 expression leads to elevated EGFR levels and signaling, suggesting a potential new axis to drive tumorigenesis, whereas overexpression of RNF43 or ZNRF3 decreases EGFR levels and signaling. Furthermore, RNF43 and ZNRF3 directly interact with EGFR through their extracellular domains.

Strengths:

The data showing that RNF43 and ZNRF3 interact with EGFR and regulate its levels and activity are thorough and convincing, and the conclusions are largely supported.

Weaknesses:

While the data support that EGFR is a target for RNF43/ZNRF3, some of the authors' interpretations of the data on EGFR's role relative to WNT's roles downstream of RNF43/ZNRF3 are overstated. The authors, perhaps not intentionally, promote the effect of RNF43/ZNRF3 on EGFR while minimizing their role in WNT signaling. This is the case in most of the biological assays (cell and organoid growth and mouse tumor models). For example, the conclusion of "no substantial activation of Wnt signaling" (page 14) in the prostate cancer model is currently not supported by the data and requires further examination. In fact, examination of the data presented here indicates effects on WNT/b-catenin signaling, consistent with previous studies.<br /> Cancers in which RNF43 or ZNRF3 are deleted are often considered to be "WNT addicted", and inhibition of WNT signaling generally potently inhibits tumor growth. In particular, treatment of WNT-addicted tumors with Porcupine inhibitors leads to tumor regression. The authors should test to what extent PORCN inhibition affects tumor (and APC-min intestinal organoid) growth. If the biological effects of RNF43/ZNRF3 loss are mediated primarily or predominantly through EGFR, then PORCN inhibition should not affect tumor or organoid growth.

Reviewer #1 (Public Review):

Wang and colleagues conducted a study to determine the neurotransmitter identity of all neurons in C. elegans hermaphrodites and males. They used CRISPR technology to introduce fluorescent gene expression reporters into the genomic loci of NT pathway genes. This approach is expected to better reflect in vivo gene expression compared to other methods like promoter- or fosmid-based transgenes, or available scRNA datasets. The study presents several noteworthy findings, including sexual dimorphisms, patterns of NT co-transmission, neuronal classes that likely use NTs without direct synthesis, and potential identification of unconventional NTs (e.g. betaine releasing neurons). The data is well-described and critically discussed, including a comparison with alternative methods. Although many of the observations and proposals have been previously discussed by the Hobert lab, the current study is particularly valuable due to its comprehensiveness. This NT atlas is the most complete and comprehensive of any nervous system that I am aware of, making it an extremely useful tool for the community.

Reviewer #1 (Public Review):

Summary:

Dong et al here have studied the impact of the small Ras-like GTPase Rab10 on the exocytosis of dense core vesicles (DVC), which are important mediators of neuropeptide signaling in the brain. They use optical imaging to show that lentiviral depletion of Rab10 in mouse hippocampal neurons in culture independent of the established defects in neurite outgrowth hamper DCV exocytosis. They further demonstrate that such defects are paralleled by changes in ER morphology and defective ER-based calcium buffering as well as reduced ribosomal protein expression in Rab10-depleted neurons. Re-expression of Rab10 or supplementation of exogenous L-leucine to restore defective neuronal protein synthesis rescues impaired DCV secretion. Based on these results they propose that Rab10 regulates DCV release by maintaining ER calcium homeostasis and neuronal protein synthesis.

Strengths:

This work provides interesting and potentially important new insights into the connection between ER function and the regulated secretion of neuropeptides via DCVs. The authors combine advanced optical imaging with light and electron microscopy, biochemistry, and proteomics approaches to thoroughly assess the effects of Rab10 knockdown at the cellular level in primary neurons. The proteomic dataset provided may be valuable in facilitating future studies regarding Rab10 function. This work will thus be of interest to neuroscientists and cell biologists.

Weaknesses:

While the main conclusions of this study are comparably well supported by the data, I see three major weaknesses:

(1) For some of the data the statistical basis for analysis remains unclear. I.e. is the statistical assessment based on N= number of experiments or n = number of synapses, images, fields of view etc.? As the latter cannot be considered independent biological replicates, they should not form the basis of statistical testing.

(2) As it stands the paper reports on three partially independent phenotypic observations, the causal interrelationship of which remains unclear. Based on prior studies (e.g. Mercan et al 2013 Mol Cell Biol; Graves et al JBC 1997) it is conceivable that defective ER-based calcium signaling and the observed reduction in protein synthesis are causally related. For example, ER calcium release is known to promote pS6K1 phosphorylation, a major upstream regulator of protein synthesis and ribosome biogenesis. Conversely, L-leucine supplementation is known to trigger calcium release from ER stores via IP3Rs. Given the reported impact of Rab10 on axonal transport of autophagosomes and, possibly, lysosomes via JIP3/4 or other mediators (see e.g. Cason and Holzbaur JCB 2023) and the fact that mTORC1, the alleged target of leucine supplementation, is located on lysosomes, which in turn form membrane contacts with the ER, it seems worth analyzing whether the various phenotypes observed are linked at the level of mTORC1 signaling.

(3) The claimed lack of effect of Rab10 depletion on SV exocytosis is solely based on very strong train stimulation with 200 Aps, a condition not very well suited to analyze defects in SV fusion. The conclusion that Rab10 loss does not impact SV fusion thus seems premature.

Reviewer #1 (Public Review):

Summary:

Zhang et al., investigated the relationship between monocular and binocular responses of V1 superficial-layer neurons using two-photon calcium imaging. They found a strong relationship in their data: neurons that exhibited a greater preference for one eye or the other (high ocular dominance) were more likely to be suppressed under binocular stimulation, whereas neurons that are more equivalently driven by each other (low ocular dominance) were more likely to be enhanced by binocular stimulation. This result chiefly demonstrates the relationship between ocular dominance and binocular responses in V1, corroborating what has been shown previously using electrophysiological techniques with now much finer spatial resolution. The binocular responses were well-fitted by a model that institutes divisive normalization between the eyes that accounts for both the suppression and enhancement phenomena observed in the subpopulation of binocular neurons. In so doing, the authors reify the importance of incorporating ocular dominance in computational models of binocular combination.

The conclusions of this paper are well supported by the data. The authors deftly contextualize these important findings in the literature while also acknowledging the limitations of the methodology employed. Future work would do well to combine the spatial power of 2P imaging with the temporal power of electrophysiology to assess ocular dominance-dependent binocular combination across the V1 laminar microcircuit.

Strengths:

The two-photon imaging technique used to resolve the activity of individual neurons within intact brain tissue grants a host of advantages. Foremost, two-photon imaging confers considerably high spatial resolution. As a result, the authors were able to sample and analyze the activity from thousands of verified superficial-layer V1 neurons. The animal model used, awake macaques, is also highly relevant for the study of binocular combination. Macaques, like humans, are binocular animals, meaning they have forward-facing eyes that confer overlapping visual fields. Importantly, macaque V1 is organized into cortical columns that process specific visual features from the separate eyes just like in humans. In combination with a powerful imaging technique, this allowed the authors to evaluate the monocular and binocular response profiles of V1 neurons that are situated within neighboring ocular dominance columns, a novel feat. To this aim, the approach was well-executed and should instill confidence in the notion that V1 neurons combine monocular information in a manner that is dependent on the strength of their ocular dominance.

Weaknesses:

This study suffers no major weaknesses. The authors address the limitations of the methodology and have calibrated the interpretations accordingly.

Reviewer #1 (Public Review):

Summary:

This study examines lipid profiles in cancer patients treated with the neurotoxic chemotherapy paclitaxel. Multiple methods, including machine learning as well as more conventional statistical modelling, were used to classify lipid patterns before and after paclitaxel treatment and in conjunction with neuropathy status. Lipid profiles before and after paclitaxel therapy were analysed from 31 patients. The study aimed to characterize from the lipid profile if plasma samples were collected pre-paclitaxel or post-paclitaxel and their relevance to neuropathy status. Sphingolipids including sphinganine-1-phosphate (SA1P) differed between patients with and without neuropathy. To examine the potential role of SA1P, it was applied to murine primary sensory neuron cultures, and produced calcium transients in a proportion of neurons. This response was abolished by the application of a TRPV1 antagonist. The number of neurons responding to SA1P was partially reduced by the sphingosine 1-phosphate receptor (S1PR1) modulator fingolimod.

Strengths:

The strengths of this study include the use of multiple methods to classify lipid patterns and the attempt to validate findings from the clinical cohort in a preclinical model using primary sensory neurons.

Weaknesses:

There are a number of weaknesses in the study. The small sample size is a significant limitation of the study. Out of 31 patients, only 17 patients were reported to develop neuropathy, with significant neuropathy (grade 2/3) in only 5 patients. The authors acknowledge this limitation in the results and discussion sections of the manuscript, but it limits the interpretation of the results. Also acknowledged is the limited method used to assess neuropathy.

Potentially due to this small number of patients with neuropathy, the machine learning algorithms could not distinguish between samples with and without neuropathy. Only selected univariate analyses identified differences in lipid profiles potentially related to neuropathy.

Three sphingolipid mediators including SA1P differed between patients with and without neuropathy at the end of treatment. These sphingolipids were elevated at the end of treatment in the cohort with neuropathy, relative to those without neuropathy. However, across all samples from pre to post-paclitaxel treatment, there was a significant reduction in SA1P levels. It is unclear from the data presented what the underlying mechanism for this result would be. If elevated SA1P is associated with neuropathy development, it would be expected to increase in those who develop neuropathy from pre to post-treatment time points.

Primary sensory neuron cultures were used to examine the effects of SA1P application. SA1P application produced calcium transients in a small proportion of sensory neurons. It is not clear how this experimental model assists in validating the role of SA1P in neuropathy development as there is no assessment of sensory neuron damage or other hallmarks of peripheral neuropathy. These results demonstrate that some sensory neurons respond to SA1P and that this activity is linked to TRPV1 receptors. However, further studies will be required to determine if this is mechanistically related to neuropathy.

Impact:

Taken in total, the data presented do not provide sufficient evidence to support the contention that SA1P has an important role in paclitaxel-induced peripheral neuropathy. Further, the results do not provide evidence to support the use of S1PR1 receptor antagonists as a therapeutic strategy. It is important to be careful with language use in the discussion, as the significance of the present results is overstated.

However, based on the results of previous studies, it is likely that sphingolipid metabolism plays a role in chemotherapy-induced peripheral neuropathy. Based on this existing evidence, the S1PR1 receptor antagonist fingolimod has already been examined in experimental models and clinical trials. Further work is needed to examine the links between lipid mediators and neuropathy development and identify additional strategies for intervention.

Reviewer #2 (Public Review):

Summary:

Previous work has shown subjects can use a form of short-term sensory memory, known as 'iconic memory', to accurately remember stimuli over short periods of time (several hundred milliseconds). Working memory maintains representations for longer periods of time but is strictly limited in its capacity. While it has long been assumed that sensory information acts as the input to working memory, a process model of this transfer has been missing. To address this, Tomic and Bays present the Dynamic Neural Resource (DyNR) model. The DyNR model captures the dynamics of processing sensory stimuli, transferring that representation into working memory, the diffusion of representations within working memory, and the selection of a memory for report.

The DyNR model captures many of the effects observed in behavior. Most importantly, psychophysical experiments found the greater the delay between stimulus presentation and the selection of an item from working memory, the greater the error. This effect also depended on working memory load. In the model, these effects are captured by the exponential decay of sensory representations (i.e., iconic memory) following the offset of the stimulus. Once the selection cue is presented, residual information in iconic memory can be integrated into working memory, improving the strength of the representation and reducing error. This selection process takes time, and is slower for larger memory loads, explaining the observation that memory seems to decay instantly. The authors compare the DyNR model to several variants, demonstrating the importance of persistence of sensory information in iconic memory, normalization of representations with increasing memory load, and diffusion of memories over time.

Strengths:

The manuscript provides a convincing argument for the interaction of iconic memory and working memory, as captured by the DyNR model. The analyses are cutting-edge and the results are well captured by the DyNR model. In particular, a strength of the manuscript is the comparison of the DyNR model to several alternative variants.

The results provide a process model for interactions between iconic memory and working memory. This will be of interest to neuroscientists and psychologists studying working memory. I see this work as providing a foundation for understanding behavior in continuous working memory tasks, from either a mechanistic, neuroscience, perspective or as a high-water mark for comparison to other psychological process models.

Finally, the manuscript is well written. The DyNR model is nicely described and an intuition for the dynamics of the model are clearly shown in Figures 2 and 4.

Weaknesses:

The manuscript appropriately acknowledges and addresses several minor weaknesses that are due to the limited ability of the approach to disambiguate underlying neural mechanisms. Nevertheless, the manuscript adds significant value to the literature on working memory.

Reviewer #1 (Public Review):

Summary:

This study assumes but also demonstrates that auditory rhythm processing is produced by internal oscillating systems and evaluates the properties of internal oscillators across individuals. The authors designed an experiment and performed analyses that address individuals' preferred rate and flexibility, with a special focus on how much past rhythms influence subsequent trials. They find evidence for such historical dependence and show that we adapt less well to new rhythms as we age. While I have some doubts about the entrainment-based interpretation of the results, this work offers a useful contribution to our understanding of individual differences in rhythm processing regardless.

Strengths:

The inclusion of two tasks -- a tapping and a listening task -- complement each other methodologically. By analysing both the production and tracking of rhythms, the authors emphasize the importance of the characteristics of the receiver, the external world, and their interplay. The relationship between the two tasks and components within tasks are explored using a range of analyses. The visual presentation of the results is very clear. The age-related changes in flexibility are useful and compelling.

The paper includes a discussion of the study assumptions, and it contextualizes itself more explicitly as taking entrainment frameworks as a starting point. As such, even if the entrainment of oscillators cannot be decisively shown, it is now clear that this is nevertheless adopted as a useful theoretical lens.

Weaknesses:

The newly included analyses that justify an entrainment or oscillator-based interpretation of the result could be presented in a clearer manner so that readers can parse their validity better. For example, in line with an entrainment interpretation, the regression lines in Figure 2B show accuracy increases as the IOI moves towards the preferred rate -- but then beyond the preferred rate, accuracy appears to increase further still. Furthermore, the additional analyses on harmonic relationships could be enriched with justification and explanation of each of its steps.

Reviewer #1 (Public Review):

Summary:

The paper presents a nice study investigating the impairments of biological motion perception in individuals with ADHD in comparison with neurotypical controls. Motivated by the idea that there is a relationship between biological motion perception and social capabilities, the authors investigated the impairments of local and global (holistic) biological motion perception, the diagnosis status, and several additional behavioral variables that are affected in ADHS (IQ, social responsiveness, and attention / impulsivity). As well local as global biological motion perception is impaired in ADHD individuals. In addition, the study demonstrates a significant correlation between local biological motion perception skills and the social responsiveness score in the ADHD group, but not in controls. A path analysis in the ADHD group suggests that general performance in<br /> biological motion perception is influenced mainly by global biological motion perception performance and attentional and perceptual reasoning skills.

Strengths:

It is true that there exists not much work on biological motion perception and ADHD. Therefore, the presented study contributes an interesting new result to the biological motion literature, and adds potentially also new behavioral markers for this clinical group. The design of the study is straightforward and technically sound, and the drawn conclusions are supported by the presented results.

Weaknesses:

Some of the claims about the relationship between genetic factors and ADHD and the components of biological motion processing have to remain speculative at this point because genetic influences were not explicitly tested in this paper. Specifically, the hypothesis that the perception of human social interaction is critically based on a local mechanism for the detection of asymmetry in foot trajectories of walkers (this is what 'BL-local' really measures), or on the detection of live agents in cluttered scenes seems not very plausible.

Based on my last comments, now the discussion has been changed in a way that tries to justify the speculative claims by citing a lot of other speculative papers, which does not really address the problem. For example, the fact that chicks walk towards biological motion stimuli is interesting. To derive that this verifies a fundamental mechanism in human biological motion processing is extremely questionable, given that birds do not even have a cortex. Taking the argumentation of the authors serious, one would have to assume that the 'Local BM' mechanism is probably located in the mesencephalon in humans, and then would have to interact in some way with social perception differences of ADHD children. To me all this seems to make very strong (over-)claims. I suggest providing a much more modest interpretation of the interesting experimental result, based on what has been really experimentally shown by the authors and closely related other data, rather than providing lots of far-reaching speculations.

In the same direction, in my view, go claims like 'local BM is an intrinsic trait' (L. 448) , which is not only imprecise (maybe better 'mechanisms of processing of local BM cues') but also rather questionable. Likely, this' local processing of BM' is a lower level mechanisms, located probably in early and mid-levels of the visual cortex, with a possible influence of lower structures. It seems not really plausible that this is related to a classical trait variables in the sense of psychology, like personality, as seems to be suggested here. Also here I suggest a much more moderate and less speculative interpretation of the results.

Reviewer #1 (Public Review):

This paper presents a cognitive model of out-of-distribution generalisation, where the representational basis is grid-cell codes. In particular, the authors consider the tasks of analogies, addition, and multiplication, and the out-of-distribution tests are shifting or scaling the input domain. The authors utilise grid cell codes, which are multi-scale as well as translationally invariant due to their periodicity. To allow for domain adaptation, the authors use DPP-A which is, in this context, a mechanism of adapting to input scale changes. The authors present simulations results demonstrating this model can perform out-of-distribution generalisation to input translations and re-scaling, whereas other models fail.

This paper makes the point it sets out to - that there are some underlying representational bases, like grid cells, that when combined with a domain adaptation mechanism, like DPP-A, can facilitate out-of-generalisation. I don't have any issues with the technical details.

The paper nicely demonstrates how neural codes can be transformed into a common representational space so that analogies, and presumably other useful tasks/computations, can be performed.

Reviewer #1 (Public Review):

It is known that aberrant habit formation is a characteristic of obsessive-compulsive disorder (OCD). Habits can be defined according to the following features (Balleine and Dezfouli, 2019): rapid execution, invariant response topography, action 'chunking' and resistance to devaluation.

The extent to which OCD behavior is derived from enhanced habit formation relative to deficits in goal-directed behavior is a topic of debate in the current literature. This study examined habit-learning specifically (cf. deficits in goal-directed behavior) by regularly presenting, via smartphone, sequential learning tasks to patients with OCD and healthy controls. Participants engaged in the tasks every day over the course of a month. Automaticity, including the extent to which individual actions in the sequence become part of a unified 'chunk', was an important outcome variable. Following the 30 days of training, in-laboratory tasks were then administered to examine 1) if performing the learned sequences themselves had become rewarding 2) differences in goal-directed vs. habitual behavior.

Several hypotheses were tested, including:<br /> Patients would have impaired procedural learning vs. healthy volunteers (this was not supported, possibly because there were fewer demands on memory in the task used here)<br /> Once the task had been learned, patients would display automaticity faster (unexpectedly, patients were slower to display automaticity)<br /> Habits would form faster under a continuous (vs. variable) reinforcement schedule

Exploratory analyses were also conducted: an interesting finding was that OCD patients with higher self-reported symptoms voluntarily completed more sessions with the habit-training app and reported a reduction in symptoms.

Strengths

This paper is well situated theoretically within the habit learning/OCD literature.<br /> Daily training in a motor-learning task, delivered via smartphone, was innovative, ecologically valid and more likely to assay habitual behaviors specifically. Daily training is also more similar to studies with non-humans, making a better link with that literature. The use of a sequential-learning task (cf. tasks that require a single response) is also more ecologically valid.<br /> The in-laboratory tests (after the 1 month of training) allowed the researchers to test if the OCD group preferred familiar, but more difficult, sequences over newer, simpler sequences.

Weaknesses

The authors were not able to test one criterion of habits, namely resistance to devaluation, due to the nature of the task.<br /> The sample size was relatively small. Some potentially interesting individual differences within the OCD group could have been examined more thoroughly with a bigger sample (e.g., preference for familiar sequences). A larger sample may have allowed the statistical testing of any effects due to medication status.

The authors achieved their aims in that two groups of participants (patients with OCD and controls) engaged with the task over the course of 30 days. The repeated nature of the task meant that 'overtraining' was almost certainly established, and automaticity was demonstrated. This allowed the authors to test their hypotheses about habit learning. The results are supportive of the author's conclusions.

This article is likely to be impactful -- the delivery of a task across 30 days to a patient group is innovative and represents a new approach for the study of habit learning that is superior to an in-laboratory approach.

An interesting aspect of this manuscript is that it prompts a comparison with previous studies of goal-directed/habitual responding in OCD that used devaluation protocols, and which may have had their effects due to deficits in goal-directed behavior and not enhanced habit learning per se.

"Il résulte donc de ce qui précède, qu’en l’absence d’obstacle juridique, l’organe délibératif de l’EPLE est parfaitement libre d’adopter le principe d’une répartition de l’année scolaire en deux semestres, au lieu de trois trimestres. Une fois cette résolution arrêtée, il conviendra également de modifier en conséquence le règlement intérieur de l’établissement."

Reviewer #1 (Public Review):

Summary:

In this study, participants completed two different tasks. A perceptual choice task in which they compared the sizes of pairs of items and a value-different task in which they identified the higher value option among pairs of items with the two tasks involving the same stimuli. Based on previous fMRI research, the authors sought to determine whether the superior frontal sulcus (SFS) is involved in both perceptual and value-based decisions or just one or the other. Initial fMRI analyses were devised to isolate brain regions that were activated for both types of choices and also regions that were unique to each. Transcranial magnetic stimulation was applied to the SFS in between fMRI sessions and it was found to lead to a significant decrease in accuracy and RT on the perceptual choice task but only a decrease in RT on the value-different task. Hierarchical drift-diffusion modelling of the data indicated that the TMS had led to a lowering of decision boundaries in the perceptual task and a lower of non-decision times on the value-based task. Additional analyses show that SFS covaries with model-derived estimates of cumulative evidence and that this relationship is weakened by TMS.

Strengths:

The paper has many strengths including the rigorous multi-pronged approach of causal manipulation, fMRI and computational modelling which offers a fresh perspective on the neural drivers of decision making. Some additional strengths include the careful paradigm design which ensured that the two types of tasks were matched for their perceptual content while orthogonalizing trial-to-trial variations in choice difficulty. The paper also lays out a number of specific hypotheses at the outset regarding the behavioural outcomes that are tied to decision model parameters and are well justified.

Weaknesses:

Unless I have missed it, the SFS does not actually appear in the list of brain areas significantly activated by the perceptual and value tasks in Supplementary Tables 1 and 2. Its presence or absence from the list of significant activations is not mentioned by the authors when outlining these results in the main text. What are we to make of the fact that it is not showing significant activation in these initial analyses?

The value difference task also requires identification of the stimuli, and therefore perceptual decision-making. In light of this, the initial fMRI analyses do not seem terribly informative for the present purposes as areas that are activated for both types of tasks could conceivably be specifically supporting perceptual decision-making only. I would have thought brain areas that are playing a particular role in evidence accumulation would be best identified based on whether their BOLD response scaled with evidence strength in each condition which would make it more likely that areas particular to each type of choice can be identified. The rationale for the authors' approach could be better justified.

TMS led to reductions in RT in the value-difference as well as the perceptual choice task. DDM modelling indicated that in the case of the value task, the effect was attributable to reduced non-decision time which the authors attribute to task learning. The reasoning here is a little unclear. If task learning is the cause, then why are similar non-decision time effects not observed in the perceptual choice task? Given that the value-task actually requires perceptual decision-making, is it not possible that SFS disruption impacted the speed with which the items could be identified, hence delaying the onset of the value-comparison choice?

The sample size is relatively small. The authors state that 20 subjects is 'in the acceptable range' but it is not clear what is meant by this.

Reviewer #1 (Public Review):

Summary and strength:

The authors undertook to assemble and annotate the genome sequence of the Malabar grouper fish, with the aim of providing molecular resources for fundamental and applied research. Even though this is more mainstream, the task is still daunting and labor-intensive. Currently, high-quality and fully annotated genome sequences are of strategic importance in modern biology. The authors make use of the resource to address the endocrine control of an ecologically and developmentally relevant life cycle transition, metamorphosis. As opposed to amphibian and flat fish where body plan changes, fish metamorphosis is anatomically more subtle and much less known, although it is clear that thyroid hormone (TH) signaling is a key player. The authors thus provide a repertoire of TH-relevant gene expression changes during development and across metamorphosis and correlate developmental stages with changes in gene expression. Overall, this work has a strong potential to meet its target.

Weaknesses:

The manuscript needs proper editing and is not complete. Some wordings lack precision and make it difficult to follow (e.g. line 98 "we assembled a chromosome-scale genome of ..." should read instead "we assembled a chromsome-scla genome sequence of ...". Also, panel Figure 2E is missing.

The shortcomings of the manuscripts are not limited to the writing style, and important technical and technological information is missing or not clear enough, thereby preventing a proper evaluation of the resolution of the genomic resources provided:

- Several RNASeq libraries from different tissues have been built to help annotate the genome and identify transcribed regions. This is fine. But all along the manuscript, gene expression changes are summarized into a single panel where it is not clear at all which tissue this comes from (whole embryo or a specific tissue ?), or whether it is a cumulative expression level computed across several tissues (and how it was computed) etc. This is essential information needed for data interpretation.

- The bioinformatic processing, especially of the assemble and annotation, is very poorly described. This is also a sensitive topic, as illustrated by the numerous "assemblathon" and "annotathon" initiatives to evaluate tools and workflows. Importantly, providing configuration files and in-depth description of workflows and parameter settings is highly recommended. This can be made available through data store services and documents even benefit from DOIs. This provides others with more information to evaluate the resolution of this work. No doubt that it is well done,<br /> but especially in the field of genome assembly and annotation, high resolution is VERY cost and time-intensive. Not surprisingly, most projects are conditioned by trade-offs between cost, time, and labor. The authors should provide others with the information needed to evaluate this.

- Quantifications of T3 and T4 levels look fairly low and not so convincing. The work would clearly benefit from a discussion about why the signal is so low and what are the current technological limitations of these quantifications. This would really help (general) readers.

- Differential analysis highlights up to ~ 15,000 differentially expressed genes (DEG), out of a predicted 26k genes. This corresponds to more than half of all genes. ANOVA-based differential analysis relies on the simple fact that only a minority of genes are DEG. Having >50% DEG is well beyond the validity of the method. This should be addressed, or at least discussed.

Reviewer #1 (Public Review):

Summary:

In their manuscript, "Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway", authors Isotani et al claimed that this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggest the use of DBZ as a potential therapeutic strategy for treating intestinal tumors. However, the presented data do not support the primary claims.

Weaknesses:

My main reservation is that the quality of the results presented in the manuscript may not fully substantiate their conclusions. For instance, in Figure 2 A and B, it is challenging to discern a healthy organoid. This is significant, as the entirety of Figure 2 and several panels in Figures 3 - 5 are based on these organoid assays. Additionally, there seems to be a discrepancy in the quality of results from the western blot, as the lanes of actin do not align with other proteins (Figure 6B).

Reviewer #1 (Public Review):

Summary:

Cognitive and brain development during the first two years of life is vast and determinant for later development. However, longitudinal infant studies are complicated and restricted to occidental high-income countries. This study uses fNIRS to investigate the developmental trajectories of functional connectivity networks in infants from a rural community in Gambia. In addition to resting-state data collected from 5 to 24 months, the authors collected growing measures from birth until 24 months and administrated an executive functioning task at 3 or 5 years old.

The results show left and right frontal-middle and right frontal-posterior negative connections at 5 months that increase with age (i.e., become less negative). Interestingly, contrary to previous findings in high-income countries, there was a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months. Additionally, the study describes that some connectivity patterns related to better cognitive flexibility at pre-school age.

Strengths:

- The authors analyze data from 204 infants from a rural area of Gambia, already a big sample for most infant studies. The study might encourage more research on different underrepresented infant populations (i.e., infants not living in occidental high-income countries).

- The study shows that fNIRS is a feasible instrument to investigate cognitive development when access to fMRI is not possible or outside a lab setting.

- The fNIRS data preprocessing and analysis are well-planned, implemented, and carefully described. For example, the authors report how the choices in the parameters for the motion artifacts detection algorithm affect data rejection and show how connectivity stability varies with the length of the data segment to justify the threshold of at least 250 seconds free of artifacts for inclusion.

- The authors use proper statistical methods for analysis, considering the complexity of the dataset.

Weaknesses:

- No co-registration of the optodes is implemented. The authors checked for correct placement by looking at pictures taken during the testing session. However, head shape and size differences might affect the results, especially considering that the study involves infants from 5 months to 24 months and that the same fNIRS array was used at all ages.

- The authors regress the global signal to remove systemic physiological noise. While the authors also report the changes in connectivity without global signal regression, there are some critical differences. In particular, the apparent decrease in frontal inter-hemispheric connections is not present when global signal regression is omitted, even though it is present for deoxy-Hb. The authors use connectivity results obtained after applying global signal regression for further analysis. The choice of regressing the global signal is questionable since it has been shown to introduce anti-correlations in fMRI data (Murphy et al., 2009), and fNIRS in young infants does not seem to be highly affected by physiological noise (Emberson et al., 2016). Systemic physiological noise might change at different ages, which makes its remotion critical to investigate functional network development. However, global signal regression might also affect the data differently. The study would have benefited from having short separation channels to measure the systemic psychological component in the data.

- I believe the authors bypass a fundamental point in their framing. When discussing the results, the authors compare the developmental trajectories of the infants tested in a rural area of Gambia with the trajectories reported in previous studies on infants growing in occidental high-income countries (likely in urban contexts) and attribute the differences to adverse effects (i.e., nutritional deficits). Differences in developmental trajectories might also derive from other environmental and cultural differences that do not necessarily lead to poor cognitive development.

- While the study provides a solid description of the functional connectivity changes in the first two years of life at the group level, the evidence regarding the links between adverse situations, developmental trajectories, and later cognitive capacities is weaker. The authors find that early restricted growth predicts specific connectivity patterns at 24 months and that certain connectivity patterns at specific ages predict cognitive flexibility. However, the link between development trajectories (individual changes in connectivity) with growth and later cognitive capacities is missing. To address this question adequately, the study should have compared infants with different growing profiles or those who suffered or did not from undernutrition. However, as the authors discussed, they lacked statistical power.

Reviewer #1 (Public Review):

The authors sought to test whether anterior insular cortex neurons increase or decrease firing during fear behavior and freezing, bi-directionally control fear via separate, anatomically defined outputs. Using a fairly simple behavior where mice were exposed to tone-shock pairings, they found roughly equal populations that do indeed either increase or decrease firing during freezing. Next, they sought to test whether these distinct populations may also have distinct outputs. Using retrograde tracers they found that the anterior insular cortex contains non-overlapping neurons which project to the mediodorsal thalamus or amygdala. Mediodorsal thalamus-projecting neurons tended to cluster in deep cortical layers while amygdala-projecting neurons were primarily in more superficial layers. Stimulation of insula-thalamus projection decreased freezing behavior, and stimulation of insula-amygdala projections increased fear behavior. Given that the neurons that increased firing were located in deep layers, that thalamus projections occurred in deep layers, and that stimulation of insula-thalamus neurons decreased freezing, the authors concluded that the increased firing neurons may be thalamus projections. Similarly, given that decreased-firing neurons tended to occur in more superficial layers, that insula-amygdala projections were primarily superficial, and that insula-amygdala stimulation increased freezing behavior, authors concluded that the decreased firing cells may be amygdala projections. The study has several strengths though also some caveats.

Strengths:

The potential link between physiological activity, anatomy, and behavior is well laid out and is an interesting question. The activity contrast between the units that increase/decrease firing during freezing is clear.

It is nice to see the recording of extracellular spiking activity, which provides a clear measure of neural output, whereas similar studies often use bulk calcium imaging, a signal that rarely matches real neural activity even when anatomy suggests it might (see London et al 2018 J Neuro - there are increased/decreased spiking striatal populations, but both D1 and D2 striatal neurons increase bulk calcium).

Weaknesses:

The link between spiking, anatomy, and behavior requires assumptions/inferences: the anatomically/genetically defined neurons which had distinct outputs and opposite behavioral effects can only be assumed the increased/decreased spiking neurons, based on the rough area of the cortical layer they were recorded.

The behavior would require more control to fully support claims about the associative nature of the fear response (see Trott et al 2022 eLife) - freezing, in this case, could just as well be nonassociative. In a similar vein, fixed intertrial intervals, though common practice in the fear literature, pose a problem for neurophysiological studies. The first is that animals learn the timing of events, and the second is that neural activity is dynamic and changes over time. Thus it is very difficult to determine whether changes in neural activity are due to learning about the tone-shock contingency, timing of the task, simply occur because of time and independently of external events, or some combination of the above.

Reviewer #1 (Public Review):

Summary:

Cheong et al. use a synapse-resolution wiring map of the fruit fly nerve cord to comprehensively investigate circuitry between descending neurons (DNs) from the brain and motor neurons (MNs) that enact different behaviours. These neurons were painstakingly identified, categorised, and linked to existing genetic driver lines; this allows the investigation of circuitry to be informed by the extensive literature on how flights walk, fly, and escape from looming stimuli. New motifs and hypotheses of circuit function were presented. This work will be a lasting resource for those studying nerve cord function.

Strengths:

The authors present an impressive amount of work in reconstructing and categorising the neurons in the DN to MN pathways. There is always a strong link between the circuitry identified and what is known in the literature, making this an excellent resource for those interested in connectomics analysis or experimental circuits neuroscience. Because of this, there are many testable hypotheses presented with clear predictions, which I expect will result in many follow-up publications. Most MNs were mapped to the individual muscles that they innervate by linking this connectome to pre-existing light microscopy datasets. When combined with past fly brain connectome datasets (Hemibrain, FAFB) or future ones, there is now a tantalising possibility of following neural pathways from sensory inputs to motor neurons and muscle.

Weaknesses:

As with all connectome datasets, the sample size is low, limiting statistical analyses. Readers should keep this in mind, but note that this is the current state-of-the-art. Some figures are weakened by relying too much on depictions of wiring diagrams as evidence of circuit function, similarity between neuropils, etc. without additional quantitative justification.

Reviewer #1 (Public Review):

Summary:

This paper reports the first results on the effects of a novel waveform for weak transcranial magnetic stimulation, which they refer to as "perturbation" (kTMP). The waveform is sinusoidal at kHz frequency with subthreshold intensities of 2V/m, instead of the suprathreshold pulses used in conventional TMS (~100V/m). The effect reported here concerns motor-evoked potentials (MEPs) elicited on the hand with single-pulse TMS. These MEPs are considered a marker of "corotico-spinal excitability. The manuscripts report that kTMP at 3.5kHz enhances MEPs with a medium effect size, and reports independent replications of this fining on 3 separate cohorts of subjects (N=16, 15, 16). This result is important for the field of non-invasive brain stimulation. The evidence in support of this claim is compelling.

Strengths:

• This is a novel modality for non-invasive brain stimulation.

• Knowing the history in this field, is likely to lead to a large number of follow-up studies in basic and clinical research.

• The modality cases practically no sensation which makes it perfectly suitable for control conditions. Indeed, the study itself used a persuasive double-blinding procedure.

• The replication of the main result in two subsequent experiments is very compelling.

• The effect size of Cohen's d=0.5 is very promising.

• It is nice the E-fields were actually measured on a phantom, not just modeled.

Weakness:

• The within-subject design may have carry-over effects, although a 2-day gap is probably enough for washout.

• It would have been nice to assess washout by comparing the per-conditions between days. Particularly problematic are the paired-pulse effects that are done within sessions in experiments 2 and 3 which could have carried over to the main metric of interest, which was the single pulse MEP.

• Statistical analysis combining Experiments 1, 2, and 3 is a little muddled.

• Related, the biorxiv version history of this work as experiments 1-3 came together to point to diverging results, and changing analysis methods. Specifically, an earlier version of the work claims that modulated kHz sinusoids are more effective than un-modulated sinusoids, yet the current version says that no differences were detected - which seems consistent with the data presented in this version. However, it does raise concerns about analytic methods, which seem to have shifted over time.

• While sensation has been documented nicely, it does not seem like blinding has been directly assessed, by asking participants at the end which group they thought to be in.