Reviewer #3 (Public review):

Summary:

Chen and Phillips present intriguing work that extends our view on the C. elegans small RNA network significantly. While the precise findings are rather C. elegans specific there are also messages for the broader field, most notably the switching of small RNA populations bound to an argonaute, and RNA granules behavior depending on developmental stage. The work also starts to shed more light on the still poorly understood role of the CSR-1 argonaute protein and supports its role in the decay of maternal transcripts. Overall, the work is of excellent quality, and the messages have a significant impact.

Strengths:

Compelling evidence for major shift in activities of an argonaute protein during development, and implications for how small RNAs affect early development. Very balanced and thoughtful discussion.

Weaknesses:

Claims on col-localization of specific 'granules' are not well supported by quantitative data.

Reviewer #3 (Public review):

Summary:

Furman et al. investigated how exposure to prolonged pain impacts human alpha oscillations recorded by electroencephalography (EEG). Two experimental models of prolonged pain were employed in healthy participants, phasic heat pain (PHP) and capsaicin heat pain (CHP). 61 participants completed two identical study visits separated by at least 8 weeks. Peak alpha frequency was reliably slowed by exposure to prolonged pain, whereas overall alpha power was reliably reduced. Both effects appeared to reflect a specific decrease in higher frequency (10-12Hz) alpha activity. The authors suggest that slowing of alpha oscillations is a reliable neural correlate of pain exposure and that manipulation of alpha activity may hold promise for treating chronic pain.

Strengths:

The study uses a within-participants design to show that exposure to pain is associated with acute changes in both the power and frequency of alpha oscillations.

By employing two experimental models of pain exposure and two separate testing visits, the authors were able to show that the effects of pain exposure on alpha activity are replicable across models and time.

Rigorous analysis approaches are used throughout.

Weaknesses:

No a priori power analysis is presented and (due to exclusions) most of the analyses conducted included (sometimes considerably) fewer participants than the overall sample size.

It is not clear whether the power and frequency changes represent two sides of the same coin or whether they reflect distinct mechanisms. The authors suggest in the manuscript that both effects may be explained by decreased power in 'fast' (8-12 Hz) alpha activity, but at other times interpret the effects to potentially represent distinct mechanisms. It would be useful for the authors to further clarify their thoughts on this point.

The statistical significance of some of the effects was dependent on analysis choices such as the exact frequency range chosen to identify alpha peaks.

No control condition was used, and I was left wondering if the effects would be specific to painful stimuli, or would also see the same effects for pleasant or neutral somatosensory stimuli?

Reviewer #3 (Public review):

Asthma is a complex disease that includes endogenous epithelial, immune, and neural components that respond awkwardly to environmental stimuli. Small airborne particles with diameters in the range of 2.5 micrometers or less, so-called PM2.5, are generally thought to contribute to some forms of asthma. These forms of asthma may have increased numbers of neutrophils and/or eosinophils present in bronchoalveolar lavage fluid and are difficult to treat effectively as they tend to be poorly responsive to steroids. Here, Wang and colleagues build on a recent model that incorporated PM2.5 which was found to have a neutrophilic component. Wang altered the model to provide an extra kick via the incorporation of ovalbumin. Building on their prior expertise linking nociceptors and inflammation, they find that silencing TRPV1-expressing neurons either pharmacologically or genetically, abrogated inflammation and the accumulation of neutrophils. By examining bronchoalveolar lavage fluid, they found not only that levels of the number of cytokines were increased, but also that artemin, a protein that supports neuronal development and function, was elevated, which did not occur in nociceptor-ablated mice. They also found that alveolar macrophages exposed to PM2.5 particles had increased artemin transcription, suggesting a further link between pollutants, and immune and neural interactions.

There are substantial caveats that must be attached to the suggestions by the authors that targeting nociceptors might provide an approach to the treatment of neutrophilic airway inflammation in pollution-driven asthma in general and wildfire-associated respiratory problems in particular. These caveats include the uncertainty of the relevance of the conventional source of PM2.5, to pollution and asthma. According to the National Institute of Standards and Technology (NIST), the standard reference material (SRM) 2786 is a mix obtained from an air intake system in the Czech Republic. It is not clear exactly what is in the mix, and a recent bioRxiv preprint, https://www.biorxiv.org/content/10.1101/2023.08.18.553903v3.full.pdf reveals the presence of endotoxin. Care should thus be taken in interpreting data using particulate matter. Regarding wildfires, there is data that indicates that such exposure is toxic to macrophages. What impact might that then have on the production of cytokines, and artemin, in humans?

Reviewer #3 (Public review):

Summary:

Chemical communication is essential for the organization of eusocial insect societies. It is used in various important contexts, such as foraging and recruiting colony members to food sources. While such pheromones have been chemically identified and their function demonstrated in bioassays, little is known about their perception. Excellent candidates are the odorant receptors that have been shown to be involved in pheromone perception in other insects including ants and bees but not termites. The authors investigated the function of the odorant receptor PsimOR14, which was one of four target odorant receptors based on gene sequences and phylogenetic analyses. They used the Drosophila empty neuron system to demonstrate that the receptor was narrowly tuned to the trail pheromone neocembrene. Similar responses to the odor panel and neocembrene in antennal recordings suggested that one specific antennal sensillum expresses PsimOR14. Additional protein modeling approaches characterized the properties of the ligand binding pocket in the receptor. Finally, PsimOR14 transcripts were found to be significantly higher in worker antennae compared to soldier antennae, which corresponds to the worker's higher sensitivity to neocembrene.

Strengths:

The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size.

Weaknesses:

The manuscript would benefit from a more detailed explanation of the research advances this work provides. Stating that this is the first deorphanization of an odorant receptor in a clade is insufficient. The introduction primarily reviews termite chemical communication and deorphanization of olfactory receptors previously performed. Although this is essential background, it lacks a good integration into explaining what problem the current study solves.

Selecting target ORs for deorphanization is an essential step in the approach. Unfortunately, the process of choosing these ORs has not been described. Were the authors just lucky that they found the correct OR out of the 50, or was there a specific selection process that increased the probability of success?

The authors assigned antennal sensilla into five categories. Unfortunately, they did not support their categories well. It is not clear how they were able to differentiate SI and SII in their antennal recordings.

The authors used a large odorant panel to determine receptor tuning. The panel included volatile polar compounds and non-volatile non-polar hydrocarbons. Usually, some heat is applied to such non-volatile odorants to increase volatility for receptor testing. It is unclear how it is possible that these non-volatile compounds can reach the tested sensilla without heat application.

Reviewer #3 (Public review):

Summary:

This work presents the development, characterization, and use of new thin microendoscopes (500µm diameter) whose accessible field of view has been extended by the addition of a corrective optical element glued to the entrance face. Two micro endoscopes of different lengths (6.4mm and 8.8mm) have been developed, allowing imaging of neuronal activity in brain regions >4mm deep. An alternative solution to increase the field of view could be to add an adaptive optics loop to the microscope to correct the aberrations of the GRIN lens. The solution presented in this paper does not require any modification of the optical microscope and can therefore be easily accessible to any neuroscience laboratory performing optical imaging of neuronal activity.

Strengths:

(1) The paper is generally clear and well-written. The scientific approach is well structured and numerous experiments and simulations are presented to evaluate the performance of corrected microendoscopes. In particular, we can highlight several consistent and convincing pieces of evidence for the improved performance of corrected micro endoscopes:<br /> a) PSFs measured with corrected micro endoscopes 75µm from the centre of the FOV show a significant reduction in optical aberrations compared to PSFs measured with uncorrected micro endoscopes.<br /> b) Morphological imaging of fixed brain slices shows that optical resolution is maintained over a larger field of view with corrected micro endoscopes compared to uncorrected ones, allowing neuronal processes to be revealed even close to the edge of the FOV.<br /> c) Using synthetic calcium data, the authors showed that the signals obtained with the corrected microendoscopes have a significantly stronger correlation with the ground truth signals than those obtained with uncorrected microendoscopes.

(2) There is a strong need for high-quality micro endoscopes to image deep brain regions in vivo. The solution proposed by the authors is simple, efficient, and potentially easy to disseminate within the neuroscience community.

Weaknesses:

(1) Many points need to be clarified/discussed. Here are a few examples:

a) It is written in the methods: « The uncorrected microendoscopes were assembled either using different optical elements compared to the corrected ones or were obtained from the corrected probes after the mechanical removal of the corrective lens. »<br /> This is not very clear: the uncorrected microendoscopes are not simply the unmodified GRIN lenses?

b) In the results of the simulation of neuronal activity (Figure 5A, for example), the neurons in the center of the FOV have a very large diameter (of about 30µm). This should be discussed. Also, why is the optical resolution so low on these images?

c) It seems that we can't see the same neurons on the left and right panels of Figure 5D. This should be discussed.

d) It is not very clear to me why in Figure 6A, F the fraction of adjacent cell pairs that are more correlated than expected increases as a function of the threshold on peak SNR. The authors showed in Supplementary Figure 3B that the mean purity index increases as a function of the threshold on peak SNR for all micro endoscopes. Therefore, I would have expected the correlation between adjacent cells to decrease as a function of the threshold on peak SNR. Similarly, the mean purity index for the corrected short microendoscope is close to 1 for high thresholds on peak SNR: therefore, I would have expected the fraction of adjacent cell pairs that are more correlated than expected to be close to 0 under these conditions. It would be interesting to clarify these points.

e) Figures 6C, H: I think it would be fairer to compare the uncorrected and corrected endomicroscopes using the same effective FOV.

f) Figure 7E: Many calcium transients have a strange shape, with a very fast decay following a plateau or a slower decay. Is this the result of motion artefacts or analysis artefacts? Also, the duration of many calcium transients seems to be long (several seconds) for GCaMP8f. These points should be discussed.

g) The authors do not mention the influence of the neuropil on their data. Did they subtract the neuropil's contribution to the signals from the somata? It is known from the literature that the presence of the neuropil creates artificial correlations between neurons, which decrease with the distance between the neurons (Grødem, S., Nymoen, I., Vatne, G.H. et al. An updated suite of viral vectors for in vivo calcium imaging using intracerebral and retro-orbital injections in male mice. Nat Commun 14, 608 (2023). https://doi.org/10.1038/s41467-023-36324-3; Keemink SW, Lowe SC, Pakan JMP, Dylda E, van Rossum MCW, Rochefort NL. FISSA: A neuropil decontamination toolbox for calcium imaging signals. Sci Rep. 2018 Feb 22;8(1):3493. doi: 10.1038/s41598-018-21640-2. PMID: 29472547; PMCID: PMC5823956)<br /> This point should be addressed.

h) Also, what are the expected correlations between neurons in the pyriform cortex? Are there measurements in the literature with which the authors could compare their data?

(2) The way the data is presented doesn't always make it easy to compare the performance of corrected and uncorrected lenses. Here are two examples:

a) In Figures 4 to 6, it would be easier to compare the FOVs of corrected and uncorrected lenses if the scale bars (at the centre of the FOV) were identical. In this way, the neurons at the centre of the FOV would appear the same size in the two images, and the distances between the neurons at the centre of the FOV would appear similar. Here, the scale bar is significantly larger for the corrected lenses, which may give the illusion of a larger effective FOV.

b) In Figures 3A-D it would be more informative to plot the distances in microns rather than pixels. This would also allow a better comparison of the micro endoscopes (as the pixel sizes seem to be different for the corrected and uncorrected micro endoscopes).

(3) There seems to be a discrepancy between the performance of the long lenses (8.8mm) in the different experiments, which should be discussed in the article. For example, the results in Figure 4 show a considerable enlargement of the FOV, whereas the results in Figure 6 show a very moderate enlargement of the distance at which the person's correlation with the first ground truth emitter starts to drop.

a) There is also a significant discrepancy between measured and simulated optical performance, which is not discussed. Optical simulations (Figure 1) show that the useful FOV (defined as the radius for which the size of the PSF along the optical axis remains below 10µm) should be at least 90µm for the corrected microendoscopes of both lengths. However, for the long microendoscopes, Figure 3J shows that the axial resolution at 90µm is 17µm. It would be interesting to discuss the origin of this discrepancy: does it depend on the microendoscope used? Are there inaccuracies in the construction of the aspheric corrective lens or in the assembly with the GRIN lens? If there is variability between different lenses, how are the lenses selected for imaging experiments?

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors apply tissue expansion and tiling light sheet microscopy to study allometric growth and regeneration in planaria. They developed image analysis pipelines to help them quantify different neuronal subtypes and muscles in planaria of different sizes and during regeneration. Among the strengths of this work, the authors provide beautiful images that show the potential of the approaches they are taking and their ability to quantify specific cell types in relatively large numbers of whole animal samples. Many of their findings confirm previous results in the literature, which helps validate the techniques and pipelines they have applied here. Among their new observations, they find that the body wall muscles at the anterior and posterior poles of the worm are organized differently and show that the muscle pattern in the posterior head of beta-catenin RNAi worms resembles the anterior muscle pattern. They also show that glial cell processes appear to be altered in beta-catenin or insulin receptor-1 RNAi worms. Weaknesses include some over-interpretation of the data and lack of consideration or citation of relevant previous literature, as discussed below.

Strengths:

This method of tissue expansion will be useful for researchers interested in studying this experimental animal. The authors provide high-quality images that show the utility of this technique. Their analysis pipeline permits them to quantify cell types in relatively large numbers of whole animal samples.

The authors provide convincing data on changes in total neurons and neuronal sub-types in different-sized planaria. They report differences in body wall muscle pattern between the anterior and posterior poles of the planaria, and that these differences are lost when a posterior head forms in beta-catenin RNAi planaria. They also find that glial cell projections are reduced in insulin receptor-1 RNAi planaria.

Weaknesses:

The work would have been strengthened by a more careful consideration of previous literature. Many papers directly relevant to this work were not cited. Such omissions do the authors a disservice because in some cases, they fail to consider relevant information that impacts the choice of reagents they have used or the conclusions they are drawing.

For example, when describing the antibody they use to label muscles (monoclonal 6G10), they do not cite the paper that generated this reagent (Ross et al PMCID: PMC4307677), and instead, one of the papers they do cite (Cebria 2016) that does not mention this antibody. Ross et al reported that 6G10 does not label all body wall muscles equivalently, but rather "predominantly labels circular and diagonal fibers" (which is apparent in Figure S5A-D of the manuscript being reviewed here). For this reason, the authors of the paper showing different body wall muscle populations play different roles in body patterning (Scimone et al 2017, PMCID: PMC6263039, also not cited in this paper) used this monoclonal in combination with a polyclonal antibody to label all body wall muscle types. Because their "pan-muscle" reagent does not label all muscle types equivalently, it calls into question their quantification of the different body wall muscle populations throughout the manuscript. It does not help matters that their initial description of the body wall muscle types fails to mention the layer of thin (inner) longitudinal muscles between the circular and diagonal muscles (Cebria 2016 and citations therein).

Ipsilateral and contralateral projections of the visual axons were beautifully shown by dye-tracing experiments (Okamoto et al 2005, PMID: 15930826). This paper should be cited when the authors report that they are corroborating the existence of ipsilateral and contralateral projections.

The proportional decrease of neurons with growth in S. mediterranea was shown by counting different cell types in macerated planarians (Baguna and Romero, 1981; https://link.springer.com/article/10.1007/BF00026179) and earlier histological observations cited there. These results have also been validated by single-cell sequencing (Emili et al, bioRxiv 2023, https://www.biorxiv.org/content/10.1101/2023.11.01.565140v). Allometric growth of the planaria tail (the tail is proportionately longer in large vs small planaria) can explain this decrease in animal size. The authors never really discuss allometric growth in a way that would help readers unfamiliar with the system understand this.

In some cases, the authors draw stronger conclusions than their results warrant. The authors claim that they are showing glial-muscle interactions, however, they do not provide any images of triple-stained samples labeling muscle, neurons, and glia, so it is impossible for the reader to judge whether the glial cells are interacting directly with body wall muscles or instead with the well-described submuscular nerve plexus. Their conclusion that neurons are unaffected by beta-cat or inr-1 RNAi based on anti-phospho-Ser/Thr staining (Fig. 6E) is unconvincing. They claim that during regeneration "DV muscles initially regenerate into longitudinal fibers at the anterior tip" (line 373). They provide no evidence for such switching of muscle cell types, so it is unclear why they say this.

The authors show how their automated workflow compares to manual counts using PI-stained specimens (Figure S1T). I may have missed it, but I do not recall seeing a similar ground truth comparison for their muscle fiber counting workflow. I mention this because the segmented image of the posterior muscles in Figure 4I seems to be missing the vast majority of circular fibers visible to the naked eye in the original image.

It is unclear why the abstract says, "We found the rate of neuron cell proliferation tends to lag..." (line 25). The authors did not measure proliferation in this work and neurons do not proliferate in planaria.

It is unclear what readers are to make of the measurements of brain lobe angles. Why is this a useful measurement and what does it tell us?

The authors repeatedly say that this work lets them investigate planarians at the single-cell level, but they don't really make the case that they are seeing things that haven't already been described at the single-cell level using standard confocal microscopy.

Reviewer #3 (Public review):

Summary:

This paper demonstrates that membrane depolarization induces a small increase in cell entry into mitosis. Based on previous work from another lab, the authors propose that ERK activation might be involved. They show convincingly using a combination of assays that ERK is activated by membrane depolarization. They show this is Ca2+ independent and is a result of activation of the whole K-Ras/ERK cascade which results from changed dynamics of phosphatidylserine in the plasma membrane that activates K-Ras. Although the activation of the Ras/ERK pathway by membrane depolarization is not new, linking it to an increase in cell proliferation is novel.

Strengths

A major strength of the study is the use of different techniques - live imaging with ERK reporters, as well as Western blotting to demonstrate ERK activation as well as different methods for inducing membrane depolarization. They also use a number of different cell lines. Via Western blotting the authors are also able to show that the whole MAPK cascade is activated.

Weaknesses

A weakness of the study is the data in Figure 1 showing that membrane depolarization results in an increase of cells entering mitosis. There are very few cells entering mitosis in their sample in any condition. This should be done with many more cells to increase confidence in the results. The study also lacks a mechanistic link between ERK activation by membrane depolarization and increased cell proliferation.

The authors did achieve their aims with the caveat that the cell proliferation results could be strengthened. The results for the most part support the conclusions.

This work suggests that alterations in membrane potential may have more physiological functions than action potential in the neural system as it has an effect on intracellular signalling and potentially cell proliferation.

Reviewer #3 (Public Review):

Strengths:<br /> The study used optogenetics together with in vivo electrophysiology to monitor CGRP neuron activity in response to various aversive stimuli including robot chasing to determine whether they encode noxious stimuli differentially. The study used an interesting conditioning paradigm to investigate the role of CGRP neurons in the PBN in both freezing and flight behaviors.

Weakness:<br /> The major weakness of this study is that the chasing robot threat conditioning model elicits weak unconditioned and conditioned flight responses, making it difficult to interpret the robustness of the findings. Furthermore, the conclusion that the CGRP neurons are capable of inducing flight is not substantiated by the data. No manipulations are made to influence the flight behavior of the mouse. Instead, the manipulations are designed to alter the intensity of the unconditioned stimulus.

Reviewer #3 (Public Review):

The authors have performed well designed experiments that elucidate the protective role of Dapa in sepsis model of LPS. This model shows that Dapa works, in part, by increasing expression of the receptor LRP2 in the kidney, that maintains circulating ApoM levels. ApoM binds to S1P which then interacts with the S1P receptor stimulating cardiac function, epithelial and endothelial barrier function, thereby maintaining intravascular volume and cardiac output in the setting of severe inflammation. The authors used many experimental models, including transgenic mice, as well as several rigorous and reproducible techniques to measure the relevant parameters of cardiac, renal, vascular, and immune function. Furthermore, they employ a useful inhibitor of S1P function to show pharmacologically the essential role for this agonist in most but not all the benefits of Dapa. A strength of the paper is the identification of the pathway responsible for the cardioprotective effects of SGLT2is that may yield additional therapeutic targets. There are some weaknesses in the paper, such as, studying only male mice, as well as providing a power analysis to justify the number of animals used throughout their experimentation. Overall, the paper should have a significant impact on the scientific community because the SGLT2i drugs are likely to find many uses in inflammatory diseases and metabolic diseases. This paper provides support for an important mechanism by which they work in conditions of severe sepsis and hemodynamic compromise.

Reviewer #3 (Public Review):

The author proposed the minimum variance principle in the memory representation in addition to two alternative theories of the minimum energy and the maximum smoothness. The strength of this paper is the matching between the prediction data computed from the explicit equation and the behavioral data taken in different conditions. The idea of the weighting of multiple coordinate systems is novel and is also able to reconcile a debate in previous literature.

The weakness is that although each model is based on an optimization principle, but the derivation process is not written in the method section. The authors did not write about how they can derive these weighting factors from these computational principles. Thus, it is not clear whether these weighting factors are relevant to these theories or just hacking methods. Suppose the author argues that this is the result of the minimum variance principle. In that case, the authors should show a process of how to derive these weighting factors as a result of the optimization process to minimize these cost functions.

In addition, I am concerned that the proposed model can cancel the property of the coordinate system by the predicted variance, and it can work for any coordinate system, even one that is not used in the human brain. When the applied force is given in Cartesian coordinates, the directionality in the generalization ability of the memory of the force field is characterized by the kinematic relationship (Jacobian) between the Cartesian coordinate and the coordinate of interest (Cartesian, joint, and object) as shown in Equation 3. At the same time, when a displacement (epsilon) is considered in a space and a corresponding displacement is linked with kinematic equations (e.g., joint displacement and hand displacement in 2 joint arms in this paper), the generated variances in different coordinate systems are linked with the kinematic equation each other (Jacobian). Thus, how a small noise in a certain coordinate system generates the hand force noise (sigma_x, sigma_j, sigma_o) is also characterized by the kinematics (Jacobian). Thus, when the predicted forcefield (F_c, F_j, F_o) was divided by the variance (F_c/sigma_c^2, F_j/sigma_j^2, F_o/sigma_o^2, ), the directionality of the generalization force which is characterized by the Jacobian is canceled by the directionality of the sigmas which is characterized by the Jacobian. Thus, as it has been read out from Fig*D and E top, the weight in E-top of each coordinate system is always the inverse of the shift of force from the test force by which the directionality of the generalization is always canceled. Once this directionality is canceled, no matter how to compute the weighted sum, it can replicate the memorized force. Thus, this model always works to replicate the test force no matter which coordinate system is assumed. Thus, I am suspicious of the falsifiability of this computational model. This model is always true no matter which coordinate system is assumed. Even though they use, for instance, the robot coordinate system, which is directly linked to the participant's hand with the kinematic equation (Jacobian), they can replicate this result. But in this case, the model would be nonsense. The falsifiability of this model was not explicitly written.

Reviewer #3 (Public Review):

The ENANI-2019 study provides valuable insights into child nutrition, development, and metabolomics in Brazil, highlighting both challenges and opportunities for improving child health outcomes through targeted interventions and further research.

Strengths of the methods and results:<br /> (1) The study utilizes data from the ENANI-2019 cohort, which was already existing. This cohort choice allows for longitudinal assessments and exploration of associations between metabolites and developmental outcomes. In addition, there was conservation of resources which are scanty in all settings in the current scenario.<br /> (2) The study aims to investigate the relationship between circulating metabolites (exposure) and early childhood development (outcome), specifically developmental quotient (DQ). The objectives are clearly stated, which facilitates focused research questions and hypotheses. The population that is studied is clearly mentioned.<br /> (3) The study accessed a large number of children under five years, with blood collected from a final sample size of 5,004 children. The exclusion of infants under six months due to venipuncture challenges and lack of reference values highlights practical considerations in research design.<br /> The study sample reflects a diverse range of children in terms of age, sex distribution, weight status, maternal education, and monthly family income. This diversity enhances the generalizability of findings across different sociodemographic groups within Brazil.<br /> (4) The study uses standardized measures (e.g., DQ assessments) and chronological age. Confounding variables, such as child's age, diet quality, and nutritional status, are carefully considered and incorporated into analyses through a Directed Acyclic Graph (DAG). The mean DQ of 0.98 indicates overall developmental norms among the studied children, with variations noted across different demographic factors such as age, region, and maternal education. The prevalence of Minimum Dietary Diversity (MDD) being met by 59.3% of children underscores dietary patterns and their potential impact on health outcomes. The association between nutritional status (weight-for-height z-scores) and developmental outcomes (DQ) provides insights into the interplay between nutrition and child development.<br /> The study identified key metabolites associated with developmental quotient (DQ):<br /> Component 1: Branched-chain amino acids (Leucine, Isoleucine, Valine).<br /> Component 2: Uremic toxins (Cresol sulfate, Phenylacetylglutamine).<br /> Component 3: Betaine and amino acids (Glutamine, Asparagine).<br /> The study focused on several serum metabolites like PAG (phenylacetylglutamine), CS (p-cresyl sulfate), HA (hippuric acid), TMAO (trimethylamine-N-oxide), MeHis (methylhistidine), and Crtn (creatinine). These metabolites are implicated in various metabolic pathways linked to gut microbiota activity, amino acid metabolism, and dietary factors.<br /> These metabolites explained a significant portion of both metabolite variance (39.8%) and DQ variance (4.3%). The study suggests that these metabolites can be used as proxy measures of the gut microbiome in children.<br /> (5) The use of partial least square regression (PLSR) with cross-validation (80% training, 20% testing) which is a robust approach to identify metabolites predictive of DQ, which minimizes overfitting. This model allows for outliers to remain outliers for transparency.<br /> The Directed Acyclic Graph (DAG) identifies and adjusts for confounding variables (e.g., child's diet quality, nutritional status) and strengthens the validity of findings by controlling for potential biases. Developmental and gender differences were studied by testing interactions with the age of the child and the sex.<br /> Mediation analysis exploring metabolites as potential mediators provides insights into underlying pathways linking exposures (e.g., diet, microbiome) with DQ.<br /> The use of Benjamini-Hochberg correction for multiple comparisons and bootstrap tests (5,000 iterations) enhances the reliability of results by controlling false discovery rates and assessing significance robustly.

Significant correlations between serum metabolites and DQ, particularly negative associations with certain metabolites like PAG and CS, suggest potential biomarkers or pathways influencing developmental outcomes. Notably, these associations varied with age, suggesting different metabolic impacts during early childhood development.

Weaknesses:<br /> (1) The data collected was incomplete especially those related to breastfeeding history and birth weight. These have been mentioned in the limitations of the study but yet might have been potential confounders or even factors leading to the particular identified metabolite state of the population.<br /> (2) Other tests than mediation analysis might have been used to ensure reliability and robustness of the data. How data was processed, data cleaning methods, how outliers were handled and sensitivity analyses would ensure robustness of the findings.<br /> (3) The generalizability of the data is not sound especially considering the children mostly belonged to a higher socioeconomic group in Brazil with mother or caregiver education being above a certain level. Comparative studies with children from other socio-economic groups and other cohorts might have been useful. Consideration of sample size adequacy and power analysis might have helped in generalizing the findings.<br /> (4) Caution is needed in interpreting causality from this data because of the nature of the study design Discussing alternative explanations and potential confounding factors in more depth could strengthen the conclusions.

Appraisal<br /> (1) The aims of the study were to identify associations between children's serum metabolome and Early Childhood development. This aim was met. The results do confirm their conclusions.<br /> Impact of the work on the field

(1) Unless actual gut microbiome of children in this age group from gut bacteria examination or gastrointestinal examination of the gut of children, the causality of gut metabolome on early childhood development cannot be established with certainty. Because this may not be possible in every situation, proxy methods such as the one elucidated here might be useful, considering the risk-benefit ratio.<br /> (2) More research is needed on this theme through longitudinal studies to validate these findings and explore underlying pathways involving gut-brain interactions and metabolic dysregulation.<br /> Other readings: Readers are advised to read other research from other countries and other languages to understand the connection between gut microbiome, metabolite spectra, and child development. In addition to study the effect of these factors on child mental development too.

Readers might consider the following questions:<br /> (1) Should investigators study the families through direct observation of diet and other factors to look for a connection between food taken in and gut microbiome and child development?<br /> (2) Can an examination of the mother's gut microbiome influence the child's microbiome? Can the mother or caregiver's microbiome influence early childhood development?<br /> (3) Is developmental quotient enough to study early childhood development? Is it comprehensive enough?

Reviewer #3 (Public Review):

El Amri et al conducted an analysis on the function of marcks and marcksl in Xenopus spinal cord development and regeneration. Their study revealed these proteins are crucial for neurite outgrowth and cell proliferation, including Sox2+ progenitors. Furthermore, they suggested these genes may act through the PLD pathway. The study is well-executed with appropriate controls and validation experiments, distinguishing it from typical regeneration research by including behavioral assays. The manuscript is commendable for its quantifications, literature referencing, careful conclusions, and detailed methods. Conclusions are well-supported by the experiments performed in this study. Overall, this manuscript contributes to the field of spinal cord regeneration and sets a good example for future research in this area.

Reviewer #3 (Public review):

Summary:

Xiong et al. investigated the debated mechanism of PKA activation using hippocampal CA1 neurons under pharmacological and synaptic stimulations. Examining all major PKA-R isoforms in these neurons, they found that a portion of PKA-C dissociates from PKA-R and translocate into dendritic spines following norepinephrine bath application. Additionally, their use of a non-dissociable form of PKA demonstrates its essential role in structural long-term potentiation (LTP) induced by two-photon glutamate uncaging, as well as in maintaining normal synaptic transmission, as verified by electrophysiology. This study presents a valuable finding on the activation-dependent re-distribution of PKA catalytic subunits in CA1 neurons, a process vital for synaptic functionality. The robust evidence provided by the authors makes this work particularly relevant for biologists seeking to understand PKA activation mechanisms, its downstream effects, and synaptic plasticity.

Strengths:

The study is methodologically robust, particularly in the application of two-photon imaging and electrophysiology. The experiments are well-designed with effective controls and a comprehensive analysis. The credibility of the data is further enhanced by the research team's previous works in related experiments. The study provides sufficient evidence to support the classical model of PKA activation via dissociation in neurons.

Weaknesses:

No specific weaknesses are noted in the current study; future research could provide additional insights by exploring PKA dissociation under varied physiological conditions, particularly in vivo, to further validate and expand upon these findings.

Reviewer #3 (Public review):

Summary:

This study investigates the salt-dependent phase separation of A1-LCD, an intrinsically disordered region of hnRNPA1 implicated in neurodegenerative diseases. The authors employ all-atom molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which salt influences A1-LCD phase separation. Contrary to typical intrinsically disordered protein (IDP) behavior, A1-LCD phase separation is enhanced by NaCl concentrations above 100 mM. The authors identify two direct effects of salt: neutralization of the protein's net charge and bridging between protein chains, both promoting condensation. They also uncover an indirect effect, where high salt concentrations strengthen pi-type interactions by reducing water availability. These findings provide a detailed molecular picture of the complex interplay between electrostatic interactions, ion binding, and hydration in IDP phase separation.

Strengths:

• Novel Insight: The study challenges the prevailing view that salt generally suppresses IDP phase separation, highlighting A1-LCD's unique behavior.<br /> • Rigorous Methodology: The authors utilize all-atom MD simulations, a powerful computational tool, to investigate the molecular details of salt-protein interactions.<br /> • Comprehensive Analysis: The study systematically explores a wide range of salt concentrations, revealing a nuanced picture of salt effects on phase separation.<br /> • Clear Presentation: The manuscript is well-written and logically structured, making the findings accessible to a broad audience.

Weaknesses:

• Limited Scope: The study focuses solely on the truncated A1-LCD, omitting simulations of the full-length protein. This limitation reduces the study's comparative value, as the authors note that the full-length protein exhibits typical salt-dependent behavior. However, given the much larger size of the full-length protein, it is acceptable to omit it given the current computing resources available.

Overall, this manuscript represents a significant contribution to the field of IDP phase separation. The authors' findings provide valuable insights into the molecular mechanisms by which salt modulates this process, with potential implications for understanding and treating neurodegenerative diseases.

Reviewer #3 (Public review):

Summary:

The authors elucidated the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of abnormal enlarged vesicles (aberrant early endosomes) when USP8 function was lost. They showed that USP8 interacts with Rabx5 to dissociate it from early endosomes promoting the recruitment of the Rab7 GEF SAND-1/Mon1 and the maturation of the endosomes. The authors provided evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells.

Strengths:

The use of two models, C. elegans and a mammalian cell line to describe a similar mechanism.

Reviewer #3 (Public review):

Summary:<br /> In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the multisubunit eukaryotic translation initiation factor 3 is disrupted by knocking down individual subunits. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding modification enzymes in the ER and components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down, but retain elevated levels of activity.

Strengths:<br /> The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what transcripts undergo translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes. Changes observed by both ribosome profiling and western blotting supports their claims that eIF3 functions in mRNA-specific control of translation.

Weaknesses:<br /> (1) The paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is noted that the authors plan to address such outcomes of eIF3 dysregulation in future work, which will be of interest.

(2) The paper could also be strengthened if some of the experiments were performed in at least one other cell type to determine whether changes observed are general or cell-type specific. The authors discuss this issue and provide a literature citation to support a more general mechanism.

Reviewer #3 (Public review):

Summary:

This paper aims to address the problem of exploring potentially rewarding environments that contain the danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so.

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards.

Strengths:

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally, there is little to criticise about the study.

Weaknesses:

The extent of the testing in human participants is fairly limited but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later). The description of the models is also a little difficult to parse.

Reviewer #3 (Public review):

Summary:

The authors identify a novel relationship between exosome secretion and filopodia formation in cancer cells and neurons. They observe that multivesicular endosomes (MVE)-plasma membrane (PM) fusion is associated with filopodia formation in HT1080 cells and that MVEs are present in filopodia in primary neurons. Using overexpression and knockdown (KD) of Rab27/HRS in HT1080 cells, melanoma cells, and/or primary rat neurons, they found that decreasing exosome secretion reduces filopodia formation, while Rab27 overexpression leads to the opposite result. Furthermore, the decreased filopodia formation is rescued in the Rab27a/HRS KD melanoma cells by the addition of small extracellular vesicles (EVs) but not large EVs purified from control cells. The authors identify endoglin as a protein unique to small EVs secreted by cancer cells when compared to large EVs. KD of endoglin reduces filopodia formation and this is rescued by the addition of small EVs from control cells and not by small EVs from endoglin KD cells. Based on the role of filopodia in cancer metastasis, the authors then investigate the role of endoglin in cancer cell metastasis using a chick embryo model. They find that injection of endoglin KD HT1080 cells into chick embryos gives rise to less metastasis compared to control cells - a phenotype that is rescued by the co-injection of small EVs from control cells. Using quantitative mass spectrometry analysis, they find that thrombospondin type 1 domain containing 7a protein (THSD7A) is downregulated in small EVs from endoglin KD melanoma cells compared to those from control cells. They also report that THSD7A is more abundant in endoglin KD cell lysate compared to control HT1080 cells and less abundant in small EVs from endoglin KD cells compared to control cells, indicating a trafficking defect. Indeed, using immunofluorescence microscopy, the authors observe THSD7A-mScarlet accumulation in CD63-positive structures in endoglin KD HT1080 cells, compared to control cells. Finally, the authors determine that exosome-secreted THSD7A induces filopodia formation in a Cdc42-dependent mechanism.

Strengths:

(1) While exosomes are known to play a role in cell migration and autocrine signaling, the relationship between exosome secretion and the formation of filopodia is novel.

(2) The authors identify an exosomal cargo protein, THSD7A, which is essential for regulating this function.

(3) The data presented provide strong evidence of a role for endoglin in the trafficking of THSD7A in exosomes.

(4) The authors associate this process with functional significance in cancer cell metastasis and neurological synapse formation, both of which involve the formation of filopodia.

(5) The data are presented clearly, and their interpretation appropriately explains the context and significance of the findings.

Weaknesses:

(1) A better characterization of the nature of the small EV population is missing:

It is unclear why the authors chose to proceed to quantitative mass spectrometry with the bands in the Coomassie from size-separated EV samples, as there are other bands present in the small EV lane but not the large EV lane. This is important to clarify because it underlies how they were able to identify THSD7A as a unique regulator of exosome-mediated filopodia formation. Is there a reason why the total sample fractions were not compared? This would provide valuable information on the nature of the small and large EV populations.

(2) Data analysis and quantification should be performed with increased rigor:

a) Figure 1C - The optical and temporal resolution are insufficient to conclusively characterize the association between exosome secretion and filopodia. Specifically, the 10-second interval used in the image acquisitions is too close to the reported 20-second median time between exosome secretion and filopodia formation. Two-5 sec intervals should be used to validate this. It would also be important to correlate the percentage of filopodia events that co-occur with exosome secretion. Is this a phenomenon that occurs with most or only a small number of filopodia? Additionally, resolution with typical confocal microscopy is subpar for these analyses. TIRF microscopy would offer increased resolution to parse out secretion events. As the TIRF objective is listed in the Methods section, figure legends should mention which images were acquired using TIRF microscopy.

b) Figure 2 - It would be important to perform further analysis to concretely determine the relationship between exosome secretion and filopodia stability. Are secretion events correlated with the stability of filopodia? Is there a positive feedback loop that causes further filopodia stability and length with increased secretion? Furthermore, is there an association between the proximity of secretion with stability? Quantification of filopodia more objectively (# of filopodia/cell) would be helpful.

c) Figure 6 - Why use different gel conditions to detect THSD7A in small EVs from B16F1 cells vs HT1080 and neurons? Why are there two bands for THSD7A in panels C and E? It is difficult to appreciate the KD efficiency in E. The absence of a signal for THSD7A in the HT1080 shEng small EVs that show a signal for endoglin is surprising. The authors should provide rigorous quantification of the westerns from several independent experimental repeats.

(3) The study lacks data on the cellular distribution of endoglin and THSD7A:

a) Figure 6 - Is THSD7A expected to be present in the nucleus as shown in panel D (label D is missing in the Figure). It is not clear if this is observed in neurons. a Western of endogenous THSD7A on cell fractions would clarify this. The authors should further characterize the cellular distribution of THSD7A in both cell types. Similarly, the cellular distribution of endoglin in the cancer cells should be provided. This would help validate the proposed model in Figure 8.

b) Figure 7 - Although the western blot provides convincing evidence for the role of endoglin in THSD7A trafficking, the microscopy data lack resolution as well as key analyses. While differences between shSCR and shEng cells are clear visually, the insets appear to be zoomed digitally which decreases resolution and interferes with interpretation. It would be crucial to show the colocalization of endoglin and THSD7A within CD63-postive MVE structures. What are the structures in Figure 7E shSCR zoom1? It would be important to rule out that these are migrasomes using TSPAN4 staining. More information on how the analysis was conducted is needed (i.e. how extracellular areas were chosen and whether the images are representative of the larger population). A widefield image of shSCR and shEng cells and DAPI or HOECHST staining in the higher magnification images should be provided. Additionally, the authors should quantify the colocalization of external CD63 and mScarlet signals from many independently acquired images (as they did for the internal signals in panel F). Is there no external THSD7A signal in the shEng cells?

Reviewer #3 (Public review):

This manuscript studies the connection between neural activity collected through electrocorticography and hidden vector representations from autoregressive language models, with the specific aim of studying the influence of language model size on this connection. Neural activity was measured from subjects who listened to a segment from a podcast, and the representations from language models were calculated using the written transcription as the input text. The ability of vector representations to predict neural activity was evaluated using 10-fold cross-validation with ridge regression models.

The main results are that (as well summarized in section headings):

(1) Larger models predict neural activity better.

(2) The ability of language model representations to predict neural activity differs across electrodes and brain regions.

(3) The layer that best predicts neural activity differs according to model size, with the "SMALL" model showing a correspondence between layer number and the language processing hierarchy.

(4) There seems to be a similar relationship between the time lag and the ability of language model representations to predict neural activity across models.

Strengths:

(1) The experimental and modeling protocols generally seem solid, which yielded results that answer the authors' primary research question.

(2) Electrocorticography data is especially hard to collect, so these results make a nice addition to recent functional magnetic resonance imaging studies.

Weaknesses:

(1) The interpretation of some results seems unjustified, although this may just be a presentational issue.

a) Figure 2B: The authors interpret the results as "a plateau in the maximal encoding performance," when some readers might interpret this rather as a decline after 13 billion parameters. Can this be further supported by a significance test like that shown in Figure 4B?

b) Figure S1A: It looks like the drop in PCA max correlation is larger for larger models, which may suggest to some readers that the same trend observed for ridge max correlation may not hold, contra the authors' claim that all results replicate. Why not include a similar figure as Figure 2B as part of Figure S1?

(2) Discussion of what might be driving the main result about the influence of model size appears to be missing (cf. the authors aim to provide an explanation of what seems to drive the influence of the layer location in Paragraph 3 of the Discussion section). What explanations have been proposed in the previous functional magnetic resonance imaging studies? Do those explanations also hold in the context of this study?

(3) The GloVe-based selection of language-sensitive electrodes (at least to me) isn't explained/motivated clearly enough (I think a more detailed explanation should be included in the Materials and Methods section). If the electrodes are selected based on GloVe embeddings, then isn't the main experiment just showing that representations from larger language models track more closely with GloVe embeddings? What justifies this methodology?

(4) (Minor weakness) The main experiments are largely replications of previous functional magnetic resonance imaging studies, with the exception of the one lag-based analysis. Is there anything else that the electrocorticography data can reveal that functional magnetic resonance imaging data can't?

Reviewer #3 (Public review):

Summary:

The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:

The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1 and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs. In their revision, the authors have now improved the Rad9-AID methodology to place Rad9 in the context of DDC adaptation, as well as widening the association between adaptation and proximity to centromeres.

Weaknesses:

Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. Conclusions about the maintenance of G2/M in several mutant combinations could have been strengthened by adding simple microscopy experiments with DAPI staining. No clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block is given.

Reviewer #3 (Public review):

Diechsel et al. provide important and valuable insights into how Notch signaling is shut down in response to parasitic wasp infestation in order to suppress crystal cell fate and favor lamellocyte production. The study shows that CSL transcription factor Su(H) is phosphorylated at S269A in response to parasitic wasp infestation and this inhibitory phosphorylation is critical for shutting down Notch. The authors go on to perform a screen for kinases responsible for this phosphorylation and have identified Pkc53E as the specific kinase acting on Su(H) at S269A. Using analysis of mutants, RNAi and biochemistry-based approaches the authors convincingly show how Pkc53E-Su(H) interaction is critical for remodeling hematopoiesis upon wasp challenge. I find the study interesting, and the data presented supports the overall conclusions made by the authors. The authors have addressed all my comments satisfactorily in the revised submission.

Strengths:

The manuscript is well presented, and the conclusions made are backed by genetic, biochemical and molecular biology-based approaches. Overall, the authors convincingly demonstrate how Pkc53E mediated phosphorylated of Su(H) shuts down Notch signaling during wasp infestation in Drosophila.

Weaknesses:

The exact molecular trigger for activation of Pkc53E is still uncharacterized and it would be interesting to know how Pkc53E gets activated during wasp infestation and whether Pkc53E gets activated turning down Notch in other stress induced scenarios.

The authors have addressed comments satisfactorily. Overall, I think the findings are interesting and would be useful to the field of developmental biology and immunology and address an important gap in the field. The most significant conclusion from the work is how Notch acts as a molecular switch during parasitic wasp infestation.

Reviewer #3 (Public review):

This study attempted to investigate the relations between processing in the human brain during movie watching and corresponding thought processes. This is a highly interesting question, as movie watching presents a semi-constrained task, combining naturally occurring thoughts and common processing of sensory inputs across participants. This task is inherently difficult because in order to know what participants are thinking at any given moment, one has to interrupt the same thought process which is the object of study.

This study attempts to deal with this issue by aggregating staggered experience sampling data across participants in one behavioral study and using the population level thought patterns to model brain activity in different participants in an open access fMRI dataset.

The behavioral data consist of 120 participants who watched 3 11-minute movie clips. Participants responded to the mDES questionnaire: 16 visual scales characterizing ongoing thought 5 times, two minutes apart, in each clip. The 16 items are first reduced to 4 factors using PCA, and their levels are compared across the different movies. The factors are "episodic knowledge", "intrusive distraction", "verbal detail", and "sensory engagement". The factors differ between the clips, and distraction is negatively correlated with movie comprehension and sensory engagement is positively correlated with comprehension.

The components are aggregated across participants (transforming single subject mDES answers into PCA space and concatenating responses of different participants) and are used as regressors in a GLM analysis. This analysis identifies brain regions corresponding to the components. The resulting brain maps reveal activations that are consistent with the proposed mental processes (e.g. negative loading for intrusion in frontoparietal network, positive loadings for visual and auditory cortices for sensory engagement).

Then, the coordinates for brain regions which were significant for more than one component are entered into a paper search in neurosynth. It is not clear what this analysis demonstrates beyond the fact that sensory engagement contained both visual and auditory components.

The next analysis projected group-averaged brain activation onto gradients (based on previous work) and used gradient timecourses to predict the behavioral report timecourses. This revealed that high activations in gradient 1 (sensory→association) predicted high sensory engagement, and that "episodic knowledge" thought patterns were predicted by increased visual cortex activations. Then, permutation tests were performed to see whether these thought pattern related activations corresponded to well defined regions on a given cluster.

This paper is framed as presenting a new paradigm but it does little to discuss what this paradigm serves, what are its limitations and how it should have been tested. The novelty appears to be in using experience sampling from 1 sample to model the responses of a second sample.

What are the considerations for treating high-order thought patterns that occur during film viewing as stable enough to use across participants? What would be the limitations of this method? (Do all people reading this paper think comparable thoughts reading through the sections?) This is briefly discussed in the revised manuscript and generally treated as an opportunity rather than as a limitation.

In conclusion, this study tackles a highly interesting subject and does it creatively and expertly. It fails to discuss and establish the utility and appropriateness of its proposed method.

Reviewer #3 (Public review):

Summary:

The manuscript by Chen and colleagues explores the connections from cerebellar Purkinje cells to various brainstem nuclei. They combine two methods - presynaptic puncta labeling as putative presynaptic markers, and optogenetics, to test the anatomical projections and functional connectivity from Purkinje cells onto a variety of brainstem nuclei. Overall, their study provides an atlas of sorts of Purkinje cell connectivity to the brainstem, which includes a critical analysis of some of their own data from another publication. Overall, the value of this work is to both provide neural substrates by which Purkinje cells may influence the brainstem and subsequent brain regions independent of the deep cerebellar nuclei and also, to provide a critical analysis of viral-based methods to explore neuronal connectivity.

Strengths:

The strengths lie in the simplicity of the study, the number of cells patched, and the relationship between the presence of putative presynaptic puncta and electrophysiological results. This type of study is important and should provide a foundation for future work exploring cerebellar inputs and outputs. Overall, I think that the critique of viral-based methods to define connectivity, and a more holistic assessment of what connectivity is and how it should be defined is timely and warranted, as I think this is under-appreciated by many groups and overall, there is a good deal of research being published that do not properly consider the issues that this manuscript raises about what viral-based connectivity maps do and do not tell us.

Weaknesses:

While I overall liked the manuscript, I do have a few concerns that relate to interpretation of results, and discussion of technological limitations. The main concerns I have relate to the techniques that the authors use, and an insufficient discussion of their limitations. The authors use a Cre-dependent mouse line that expresses a synaptophysin-tomato marker, which the authors confidently state is a marker of synapses. This is misleading. Synaptophysin is a vesicle marker, and as such, labels axons, where vesicles are present in transit, and likely cell bodies where the protein is being produced. As such, the presence of tdtomato should not be interpreted definitively as the presence of a synapse. The use of vGAT as a marker, while this helps to constrain the selection of putative pre-synaptic sites, is also a vesicle marker and will likely suffer the same limitations (though in this case, the expression is endogenous and not driven by the ROSA locus). A more conservative interpretation of the data would be that the authors are assessing putative pre-synaptic sites with their analysis. This interpretation is wholly consistent with their findings showing the presence of tdtomato in some regions but only sparse connectivity - this would be expected in the event that axons are passing through. If the authors wish to strongly assert that they are specifically assessing synapses, a marker better restricted to synapses and not vesicles may be more appropriate.

Similarly, while optogenetics/slice electrophysiology remains the state of the art for assessing connectivity between cell populations, it is not without limitations. For example, connections that are not contained within the thickness of the slice (here, 200 um, which is not particularly thick for slice ephys preps) will not be detected. As such, the absence of connections is harder to interpret than the presence of connections. Slices were only made in the coronal plane, which means that if there is a particular topology to certain connections that is orthogonal to that plane, those connections may be under-represented. As such, all connectivity analyses likely are under-representations of the actual connectivity that exists in the intact brain. Therefore, perhaps the authors should consider revising their assessments of connections, or lack thereof, of Purkinje cells to e.g., LC cells. While their data do make a compelling case that the connections between Purkinje cells and LC cells are not particularly strong or numerous, especially compared to other nearby brainstem nuclei, their analyses do indicate that at least some such connections do exist. Thus, rather than saying that the viral methods such as rabies virus are not accurate reflections of connectivity - perhaps a more circumspect argument would be that the quantitative connectivity maps reported by other groups using rabies virus do not always reflect connectivity defined by other means e.g., functional connections with optogenetics. In some cases, the authors do suggest this (e.g."Together, these findings indicate that reliance on anatomical tracing experiments alone is insufficient to establish the presence and importance of a synaptic connection"), but in other cases, they are more dismissive of viral tracing results (e.g. "it further suggests that these neurons project to the cerebellum and were not retrogradely labeled"). Furthermore, some statements are a bit misleading e.g., mentioning that rabies methods are critically dependent on starter cell identity immediately following the citation of studies mapping inputs onto LC cells. While in general, this claim has merit, the studies cited (19-21) use Dbh-Cre to define LC-NE cells which does have good fidelity to the cells of interest in the LC. Therefore, rewording this section in order to raise these issues generally without proximity to the citations in the previous sentence may maintain the authors' intention without suggesting that perhaps the rabies studies from LC-NE cells that identified inputs from Purkinje cells were inaccurate due to poor fidelity of the Cre line. Overall, this manuscript would certainly not be the first report indicating that the rabies virus does not provide a quantitative map of input connections. In my opinion, this is still under-appreciated by the broad community and should be explicitly discussed. Thus, an acknowledgment of previous literature on this topic and how their work contributes to that argument is warranted.

Reviewer #3 (Public review):

Summary:

Yao et al use CHART to identify chromatin associated with Xist in female mouse ESCs, and, as control, male ESCs at various timepoints of differentiation. Besides binding of Xist to X chromosome regions they found significant binding to autosomes, concentrating mostly on promoter regions of around 100 autosomal genes, as elucidated by MACS. The authors went on to show that the RepB repeat is mostly responsible for these autosomal interactions using a female ESC line in which RepB is deleted. Evidence is provided that Xist interacts with active autosomal genes containing lower coverage of repressive marks H3K27me3 and H2AK119ub and that RepB dependent Xist binding leads to dampening of expression, but not silencing of autosomal genes. These results were confirmed by overexpression studies using transgenic ESCs with doxycycline-inducible Xist as well as via a small molecule inhibitor of Xist (X1), inducing/inhibiting the dampening of autosomal genes, respectively. Finally, using MEFs and Xist mutants RepB or RepE the authors provide evidence that Xist is bound to autosomal genes in cells after the XCI process but appears not to affect gene expression. The data presented appear generally clear and consistent and indicate some differences between human and mouse autosomal regulation by Xist.

Strengths:

Regulation of autosomal gene expression by Xist is a "big deal" as misregulation of this lncRNA causes developmental defects and human disease. Moreover, this finding may explain sex-specific developmental differences between the sexes. The results in this manuscript identify specific mouse autosomal genes bound by Xist and decipher critical Xist regions that mediate this binding and gene dampening. The methods used in this study are appropriate, and the overall data presented appear convincing and are consistent, indicating some differences between human and mouse autosomal regulation by Xist.

Weaknesses:

(1) The figure legends and/or descriptions of data are often very short lacking detail, and this unnecessarily impedes the reading of the manuscript, in particular the figures would benefit not only from more detailed descriptions/explanations of what has been done but also what is shown. This will facilitate the reading and overall comprehension by the reader. One out of many examples: In Fig S1B in the CHART data at d4 and d7 there is not only signal in female WT Xist antisense but also in female sense control. For a reader that is not an expert in XCI it would be helpful to point out in the legend that this signal corresponds to the lncRNA Tsix (I suppose), that is transcribed on the other strand.

(2) Different scales are used in the lower panels of Figures 1A and 2A, which makes it difficult to directly compare signals between the different differentiation stages.

(3) In this study some of the findings on mouse cells contrast previously published results in human ESCs: 1) Xist binding occurs preferentially to promoters in mice, not in human. 2) Binding of Xist is mostly detected in polycomb-depleted regions in mice but there is a positive correlation between Xist RNA and PRC2 marks in human ESCs. These differences are surprising but may be very interesting and relevant. While I am aware that this might be a difficult task, it would be helpful to experimentally address this issue in order to distinguish whether species specific and/or methodological differences between the studies are responsible for these differences.

Reviewer #3 (Public review):

This study was focused on the conserved mechanisms across the Transmembrane Channel/Scramblase superfamily, which includes members of the TMEM16, TMEM63/OSCA, and TMC families. In previous work, the authors have studied the role of the inner activation gate of these proteins. Here, the authors show that the introduction of mutations at the TM4-TM6 interface, which are close to the inactivation gate, can disrupt gating and confer scramblase activity to non-scramblases proteins.

Overall, the confocal imaging experiments, patch clamping experiments, and data analysis are performed well and in line with standard methods. The molecular dynamics simulation work is focused but adds supportive evidence to their findings. Although there could have been more extensive molecular analysis to bolster the authors' arguments on the role of the TM4-TM6 interface (e.g. evaluate effects of size/hydrophobicity, double mutants, cross-linking, more in-depth simulation data), there is adequate evidence to conclude that certain residues at this interface is critical to ion conduction and phospholipid scramblase activity. The data presented only adds incremental depth of knowledge for each individual channel, but together, they show this to be true for conserved TM4 residues across TMEM16F, TMEM16A, OSCA1.2, and TMEM63A proteins. This breadth of data is a major strength of this paper, and provides strong evidence for a coupled pathway for ion conduction and phospholipid transport, though the underlying biophysical mechanism is still speculative and remains to be elucidated.

Reviewer #3 (Public review):

Summary:

In this manuscript, Outla Z et al described the analysis of plectin in HCC pathogenesis. Specifically, it was found that elevated plectin levels in liver tumors, correlated with poor prognosis for HCC patients. Mechanistically, it showed that plectin-dependent disruption of cytoskeletal networks leads to the attenuation of oncogenic FAK, MAPK/Erk, and PI3K/AKT signals. Finally, the authors showed that plectin inhibitor plecstatin-1 (PST) is well-tolerated and capable of overcoming therapy resistance in HCC.

Strengths:

The studies of plectin are not entirely novel (Pubmed: 36613521). Nevertheless, the current manuscript provides a much more detailed mechanistic study and the results have translational implications. Additional strengths include convincing cell biology data, such as plectin regulates cytoskeletal networks, and HCC migration/invasion.

Weaknesses:

Multiple major issues are noted, and the conclusion is not well supported by the data presented.

(1) The rationale for using Huh7 cells in the manuscript is not well explained as it has the lowest plectin expression levels.

(2) The KO cell experiments should be supplemented with overexpression experiments.

(3) There is significant concern that while ablation of Ple led to reduced tumor number, these mice had larger tumors. The data indicate that plectin may have distinct roles in HCC initiation versus progression. The data are not well explained and do not fully support that plectin promotes hepatocarcinogenesis.

(4) Figure 3 showed that plectin does not regulate p-FAK/FAK expression. Therefore, the statement that plectin regulates the FAK pathway is not valid. Furthermore, there are too many variables in turns of p-AKT and p-ERK expression, making the conclusion not well supported.

(5) The studies of plecstatin-1 in HCC should be expanded to a panel of human HCC cells with various plectin expression levels in turns of cell growth and cell migration. The IC50 values should be determined and correlate with plectin expression.

(6) One of the major issues is the mechanistic studies focusing on plectin regulating HCC migration/metastasis, whereas the in vivo mouse studies focus on HCC formation (Figures 3 and 7). These are distinct processes and should not be mixed.

(7) Figure 7B showed that Ple KO mice were treated with PST, but the data are not presented in the manuscript. Tumor cell proliferation and apoptosis rates should be analyzed as well.

(8) The status of FAK, AKT, and ERK pathway activation was not analyzed in mouse liver samples. In Figure 7D, most of the adjusted p-values are not significant.

(9) There is no evidence to support that PST is capable of overcoming therapy resistance in HCC. For example, no comparison with the current standard care was provided in the preclinical studies.

Reviewer #3 (Public review):

Summary:

In this work, the authors aims and efforts point towards evaluating the interaction mechanisms between viral protein integrase (IN) and viral DNA. They develop a multifaceted approach to probe the effect that IN has on the formation and structure of IN-DNA complexes under different environmental conditions to determine the role of IN in early stages of infection. HIV infection is considered a global pandemic with huge challenges in both treatment and prevention. This work presents a step towards understanding the mechanisms in early infection and thus prevention.

The experimental work is carried out using single molecule imaging and force spectroscopy, alongside computational verification using Monte-Carlo simulations. The authors use a range of well-established methods to quantitatively evaluate this, pushing forward the current state of the art.

The paper shows that in the presence of IN, DNA is compacted into a condensate in a biphasic manner, first forming a 'semi-compact' rosette condensate followed by a fully compacted condensate. As HIV DNA must be fully compacted to enter the cell nucleus for infection, this work describes the importance of the role of IN and the conditions required for it to reach a full condensate, and hence provides a new understanding on the early role of IN in infection. Furthermore, the authors show that the semi-compact rosette condensate (i.e. the first phase) is susceptible to IN inhibitors whereas the second compaction phase is insusceptible. This work provides us with information that using inhibitors in the early stages of IN-DNA interaction, infection may be prevented.

Strengths:

The authors present a strong piece of work, using current experimental and computational methods to investigate IN-DNA interactions and to convincingly describe their experimental observations. Firstly the data and analysis shown from AFM and MT experiments convincingly show a two-phase compaction of DNA upon interaction with IN. The authors use Monte-Carlo simulations to model DNA-IN interactions, specifically showing that their experimental results of a two-phase compaction can only be observed via simulations if IN-IN attraction is included.

The authors aim of showing the effect of IN on the compaction of DNA was achieved successfully using AFM and MT. Furthermore, the works show clearly the susceptibility of the partially compacted DNA-IN core to inhibitors. Overall the conclusions in this paper are supported well by their experimental data and it is likely that this paper will not only be used as a model for future experimental work to explore other retroviral nucleoprotein condensation but also to develop a deeper understanding of the role of IN-inhibitors infection prevention.

Finally, the article is written very coherently and is well supported by critical analysis of their findings and appropriate referencing to supplementary figures.

Overall, this article is very worthy and through extensive and detailed work the authors probe difficult questions regarding HIV infection, which currently poses a huge global risk. The work completed by the authors substantially advances our understanding of HIV infection and can be used by those in the future to probe this question further.

Weaknesses:

Important aspects of the methodologies in this paper are not described in detail. For example, force volume curves have been used to evaluate the mechanical properties of the DNA-IN complex. Force-volume measurements are prone to a number of errors, particularly relating to data acquisition and analysis. The methodology presented is not clear on how the data is acquired, whether statically or in amplitude modulation, which affects analysis and interpretation. Although the authors do recognise some of the difficulties with force curve analysis, a more rigorous study could have been provided with citations to additional relevant literature (particularly taking note of the methods).

A minor point is that it is not clear that the AFM imaging is performed in air, in contrast to AFM force spectroscopy in liquid, which could affect the interpretation of the data and therefore comparisons which are drawn between the two. This is made more challenging as the methodology for the compaction measurements is not described in the methods, and the code is not provided. The source code should be made open-access and available to enable the work to be better understood and reproduced.

Reviewer #3 (Public review):

Summary:

The authors develop a method to visually analyze micronuclei using automated methods. The authors then use these methods to isolate MN post-photoactivation and analyze transcriptional changes in cells with and without micronuclei of RPE-1 cells. The authors observe in RPE-1 cells that MN-containing cells show similar transcriptomic changes as aneuploidy, and that MN rupture does not lead to vast changes in the transcriptome.

Strengths:

The authors develop a method that allows for automating measurements and analysis of micronuclei. This has been something that the field has been missing for a long time. Using such a method has the potential to advance micronuclei biology. The authors also develop a method to identify cells with micronuclei in real time and mark them using photoconversion and then isolate them via FACS. The authors use this method to study the transcriptome. This method is very powerful as it allows for the sorting of a heterogenous population and subsequent analysis with a much higher sample number than could be previously done.

Weaknesses:

The major weakness of this paper is that the results from the RNA-seq analysis are difficult to interpret as very few changes are found to begin with between cells with MN and cells without. The authors have to use a 1.5-fold cut-off to detect any changes in general. This is most likely due to the sequencing read depth used by the authors. Moreover, there are large variances between replicates in experiments looking at cells with ruptured versus intact micronuclei. This limits our ability to assess if the lack of changes is due to truly not having changes between these populations or experimental limitations. Moreover, the authors use RPE-1 cells which lack cGAS, which may contribute to the lack of changes observed. Thus, it is possible that these results are not consistent with what would occur in primary tissues or just in general in cells with a proficient cGAS/STING pathway.

Reviewer #3 (Public review):

Summary:

In this contribution, the authors report atomistic, coarse-grained, and lattice simulations to analyze the mechanism of supercomplex (SC) formation in mitochondria. The results highlight the importance of membrane deformation as one of the major driving forces for SC formation, which is not entirely surprising given prior work on membrane protein assembly, but certainly of major mechanistic significance for the specific systems of interest.

Strengths:

The combination of complementary approaches, including an interesting (re)analysis of cryo-EM data, is particularly powerful and might be applicable to the analysis of related systems. The calculations also revealed that SC formation has interesting impacts on the structural and dynamical (motional correlation) properties of the individual protein components, suggesting further functional relevance of SC formation. Overall, the study is rather thorough and highly creative, and the impact on the field is expected to be significant.

Weaknesses:

In general, I don't think the work contains any obvious weaknesses, although I was left with some questions.

Reviewer #3 (Public review):

Summary:

Type VI secretion systems (T6SS) are employed by bacteria to inject competitor cells with numerous effector proteins. These effectors can kill injected cells via an array of enzymatic activities. A common class of T6SS effector are peptidoglycan (PG) lysing enzymes. In this manuscript, the authors characterize a PG-lysing effector-TseP-from the pathogen Aeromonas dhakensis. While the C-terminal domain of TseP was known to have lysozyme activity, the N-terminal domain was uncharacterized. Here, the authors functionally characterize TsePN as a zinc-dependent amidase. This discovery is somewhat novel because it is rare for PG-lysing effectors to have amidase and lysozyme activity.

In the second half of the manuscript, the authors utilize a crystal structure of the lysozyme TsePC domain to inform the engineering of this domain to lyse gram-positive peptidoglycan.

Strengths:

The two halves of the manuscript considered together provide a nice characterization of a unique T6SS effector and reveal potentially general principles for lysozyme engineering.

Weaknesses:

The advantage of fusing amidase and lysozyme domains in a single effector is not discussed but would appear to be a pertinent question. Labeling of the figures could be improved to help readers understand the data.

Reviewer #3 (Public review):

Summary:

The authors provide an interesting and novel approach, RCSP, to determining what they call the "root causal genes" for a disease, i.e. the most upstream, initial causes of disease. RCSP leverages perturbation (e.g. Perturb-seq) and observational RNA-seq data, the latter from patients. They show using both theory and simulations that if their assumptions hold then the method performs remarkably well, compared to both simple and available state-of-the-art baselines. Whether the required assumptions hold for real diseases is questionable. They show superficially reasonable results on AMD and MS.

Strengths:

The idea of integrating perturbation and observational RNA-seq dataset to better understand the causal basis of disease is powerful and timely. We are just beginning to see genome-wide perturbation assay, albeit in limited cell-types currently. For many diseases, research cohorts have at least bulk observational RNA-seq from a/the disease-relevant tissue(s). Given this, RCSP's strategy of learning the required causal structure from perturbations and applying this knowledge in the observational context is pragmatic and will likely become widely applicable as Perturb-seq data in more cell-types/contexts becomes available.

The causal inference reasoning is another strength. A more obvious approach would be to attempt to learn the causal network structure from the perturbation data and leverage this in the observational data. However, structure learning in high-dimensions is notoriously difficult, despite recent innovations such as differentiable approaches. The authors notice that to estimate the root causal effect for a gene X, one only needs access to a (superset of) the causal ancestors of X: much easier relationships to detect than the full network.

The applications are also reasonably well chosen, being some of the few cases where genome-scale perturb-seq is available in a roughly appropriate (see below) cell-type, and observational RNA-seq is available at a reasonable sample size.

Weaknesses:

Several assumptions of the method are problematic. The most concerning is that the observational expression changes are all causally upstream of disease. There is work using Mendelian randomization (MR) showing that the _opposite_ is more likely to be true: most differential expression in disease cohorts is a consequence rather than a cause of disease (https://www.nature.com/articles/s41467-021-25805-y). Indeed, the oxidative stress of AMD has known cellular responses including the upregulation of p53. The authors need to think carefully about how this impacts their framework. Can the theory say anything in this light? Simulations could also be designed to address robustness.

A closely related issue is the DAG assumption of no cycles. This assumption is brought to bear because it required for much classical causal machinery, but is unrealistic in biology where feedback is pervasive. How robust is RCSP to (mild) violations of this assumption? Simulations would be a straightforward way to address this.

The authors spend considerable effort arguing that technical sampling noise in X can effectively be ignored (at least in bulk). While the mathematical arguments here are reasonable, they miss the bigger picture point that the measured gene expression X can only ever be a noisy/biased proxy for the expression changes that caused disease: 1) Those events happened before the disease manifested, possibly early in development for some conditions like neurodevelopmental disorders. 2) bulk RNA-seq gives only an average across cell-types, whereas specific cell-types are likely "causal". 3) only a small sample, at a single time point, is typically available. Expression in other parts of the tissue and at different times will be variable.

My remaining concerns are more minor.

While there are connections to the omnigenic model, the latter is somewhat misrepresented. 1) The authors refer to the "core genes" of the omnigenic model as being at the end (longitudinally) of pathogenesis. The omnigenic model makes no statements about temporally ordering: in causal inference terminology the core genes are simply the direct cause of disease. 2) "Complex diseases often have an overwhelming number of causes, but the root causal genes may only represent a small subset implicating a more omnigenic than polygenic model" A key observation underlying the omnigenic model is that genetic heritability is spread throughout the genome (and somewhat concentrated near genes expressed in disease relevant cell types). This implies that (almost) all expressed genes, or their associated (e)SNPs, are "root causes".

The claim that root causal genes would be good therapeutic targets feels unfounded. If these are highly variable across individuals then the choice of treatment becomes challenging. By contrast the causal effects may converge on core genes before impacting disease, so that intervening on the core genes might be preferable. The jury is still out on these questions, so the claim should at least be made hypothetical.

The closest thing to a gold standard I believe we have for "root causal genes" is integration of molecular QTLs and GWAS, specifically coloc/MR. Here the "E" of RCSP are explicitly represented as SNPs. I don't know if there is good data for AMD but there certainly is for MS. The authors should assess the overlap with their results. Another orthogonal avenue would be to check whether the root causal genes change early in disease progression.

The available perturb-seq datasets have limitations beyond on the control of the authors. 1) The set of genes that are perturbed. The authors address this by simply sub-setting their analysis to the intersection of genes represented in the perturbation and observational data. However, this may mean that a true ancestor of X is not modeled/perturbed, limiting the formal claims that can be made. Additionally, some proportion of genes that are nominally perturbed show little to no actual perturbation effect (for example, due to poor guide RNA choice) which will also lead to missing ancestors.

The authors provide no mechanism for statistical inference/significance for their results at either the individual or aggregated level. While I am a proponent of using effect sizes more than p-values, there is still value in understanding how much signal is present relative to a reasonable null.

I agree with the authors that age coming out of a "root cause" is potentially encouraging. However, it is also quite different in nature to expression, including being "measured" exactly. Will RCSP be biased towards variables that have lower measurement error?

Finally, it's a stretch to call K562 cells "lymphoblasts". They are more myeloid than lymphoid.

Reviewer #3 (Public review):

In the current manuscript, Matsuo-Takasaki et al. demonstrate that the addition of PKCβ and WNT signaling pathway inhibitors to suspension cultures of iPSCs effectively suppresses spontaneous differentiation. These conditions are well-suited for the large-scale expansion of iPSCs. The authors have shown that, under these conditions, they can successfully perform single-cell cloning, direct cryopreservation, and iPSC derivation from PBMCs. Furthermore, they provide a comprehensive characterization of iPSCs grown in these conditions, including assessments of undifferentiated stem cell markers and genetic stability.

They have elegantly demonstrated that iPSCs cultured in these conditions can differentiate into derivatives of all three germ layers. By differentiating iPSCs into dopaminergic neural progenitors, cardiomyocytes, and hepatocytes, the authors show that differentiation is comparable to that of adherent cultures. This new method of expanding iPSCs has significant potential for clinical applications. The authors also tested these conditions in multiple cell lines and observed consistent results.

Although the authors have elaborated on the mechanism to some extent-suggesting that PKCβ and WNT signaling pathway inhibition suppresses differentiation and shifts cells toward a naïve pluripotency state in suspension cultures-further research is needed to fully understand this process. Nevertheless, their findings are promising and will be beneficial for producing scalable amounts of iPSCs in controlled conditions.

Reviewer #3 (Public review):

Summary:

The authors report the performance of a series of machine learning models inferred from a large-scale dataset and externally validated with an independent cohort of patients, to predict the risk of post-stroke epilepsy. Some of the reported models have very good explicative performance, and seem to have very good predictive ability.

Strengths:

The models have been derived from real-world large-scale data.

Performances of the best-performing models seem to be very good according to the external validation results.

Early prediction of risk of post-stroke epilepsy would be of high interest to implement early therapeutic interventions that could improve prognosis.

Code is publicly available. The authors also stated that the datasets used are available on request.

Weaknesses:

The writing of the article may be significantly improved.

Although the external validation is appreciated, cross-validation to check robustness of the models would also be welcome.

External validation results may be biased/overoptimistic, since the authors informed that "The external validation cohort focused more on collecting positive cases 80 to examine the model's ability to identify positive samples", which may result in overoptimistic PPV and Sensitivity estimations. The specificity for the external validation set has not been disclosed.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The study has several weaknesses: it provides limited novel insights into pathways mediating transcriptional regulation of cold-inducible genes, as IRE-1 and XBP-1 are already well-known responders to endoplasmic reticulum stress, including that induced by cold. Additionally, the weak cold sensitivity phenotype observed in ire-1 mutants casts doubt on the pathway's key role in cold adaptation. The study also overlooks previous research (e.g. PMID: 27540856) that links IRE-1 to SKN-1, another major stress-responsive pathway, potentially missing important interactions and mechanisms involved in cold adaptation.

Reviewer #3 (Public review):

Summary:

Day et al. introduced high-throughput expansion microscopy (HiExM), a method facilitating the simultaneous adaptation of expansion microscopy for cells cultured in a 96-well plate format. The distinctive features of this method include: 1) the use of a specialized device for delivering a minimal amount (~230 nL) of gel solution to each well of a conventional 96-well plate, and 2) the application of the photochemical initiator, Irgacure 2959, to successfully form and expand toroidal gel within each well.

Addition upon revision:

Overall, the authors have adequately addressed most of the concerns raised. There are a few minor issues that require attention.

Minor comments:

Figure S10: There appears to be a discrepancy in the panel labeling. The current labels are E-H, but it is unclear whether panels A-D exist. Also, this reviewer thought that panels G and H would benefit from statistical testing to strengthen the conclusions. As a general rule for scientific graph presentation, the y-axis of all graphs should start at zero unless there is a compelling reason not to do so.

Editor note: this comment has been addressed in the latest version.

Reviewer #3 (Public review):

Summary:

Tsingos et al. seek to advance beyond the current paradigm that proliferation of malignant cells in T-cell acute lymphoblastic leukemia occurs in a cell-autonomous fashion. Using a computational agent-based model and experimental validation, they show instead that cell proliferation also depends on interaction with thymic epithelial cells (TEC) in the thymic niche. One key finding is that a dense TEC network inhibits the proliferation of malignant cells and favors the proliferation of normal cells, whereas a sparse TEC network leads to rapid expansion of malignant thymocytes.

Strengths:

A key strength of this study is that it combines computational modeling using an agent-based model with experimental work. The original modeling and novel experimental work strengthen each other well. In the agent-based model, the authors also tested the effects of varying a few key parameters of cell proliferation.

Weaknesses:

A minor weakness is that the authors did not conduct a global sensitivity analysis of all parameters in their agent-based model to show that the model is robust to variation, which would demonstrate that their results would still hold under a reasonable level of variation in the model and model parameters. This is a minor point, and such a supporting study would end in an appendix or supplement.

Reviewer #3 (Public review):

Summary:

In this work, the authors proposed that the mechano-gated ion channel Piezo1 enhances GLP-1 production and secretion possibly through stimulating Ca2+-CaMKKbeta-CaMKIV-mTORC1 signaling pathway. By using intestinal L cell-specific piezo1 knock-out mice, intestinal bead implantation mice model, and the chemical agonist Yoda1, the authors claimed that piezo1 promotes pro-glucagon expression, GLP-1 production and secretion. In sorted primary intestinal L cells and STC-1 cells, the authors validated that CaMKKbeta-CaMKIV-mTORC1 signaling pathway positively regulated GLP-1 production and secretion. This study provides new evidence about the specific role of piezo1 in intestinal L cells, broadening the understanding of metabolic functions of piezo1.

Strengths:

The new concept and innovative in vivo and in vitro models.

Weaknesses:

Although the authors have addressed most of the issues in the revised manuscript, there are still some questions that need to be clarified.

(1) This study claimed that piezo1 enhances proglucagon expression, GLP-1 production and secretion through Ca2+-CaMKKbeta-CaMKIV-mTORC1 signaling pathway, which is a highly time-consuming process. However, as a mechano-gated ion channel, it should exert functions promptly. Is it possibly that piezo1 directly stimulates GLP-1 release by influx of Ca2+? if so, have authors measured intracellular Ca2+ concentration?<br /> (2) The authors proposed that the CaMKKbeta-CaMKIV-mTORC1 signaling pathway mediated the effects of piezo1. However, the data is not convincing. At least, chemical inhibitors of CaMKKbeta/CaMKIV/mTORC1 should be used in intL-piezo1 KO mice or STC-1 cells to see if piezo1-induced GLP-1 secretion was abrogated by these chemical inhibitors.<br /> (3) According to previous studies of the team, piezo1 could enhance insulin, ghrelin and GLP-1 secretion while inhibit glucagon production in pancreatic α-cells. In a recent work, the authors found that piezo1 in enterocytes suppresses nutrient absorption. Why an ion channel has these various effects in different cells? What is the fundamental and common mechanism underlying its metabolic functions? Its value as a drug target? These questions need to be discussed in more details.

Reviewer #3 (Public review):

Summary:

In this paper, the authors measured neural activity (using MEG) and eye gaze while individuals listened to speech from either one or two speakers, which sometimes contained semantic incongruencies.

The stated aim is to replicate two previous findings by this group: (1) that there is "ocular speech tracking" (that eye-movements track the audio of the speech), (2) that individual differences in neural response to tones that are predictable vs. not-predictable in their pitch is linked to neural response to speech. In addition, here they try to link the above two effects to each other, and to link "attention, prediction, and active sensing".

Strengths:

This is an ambitious project, that tackles an important issue and combines different sources of data (neural data, eye-movements, individual differences in another task) in order to obtain a comprehensive "model" of the involvement of eye-movements in sensory processing.

The authors use many adequate methods and sophisticated data-analysis tools (including MEG source analysis and multivariate statistical models) in order to achieve this.

Weaknesses:

Although I sympathize with the goal of the paper and agree that this is an interesting and important theoretical avenue to pursue, I am unfortunately not convinced by the results and find that many of the claims are very weakly substantiated in the actual data.

Since most of the analyses presented here are derivations of statistical models and very little actual data is presented, I found it very difficult to assess the reliability and validity of the results, as they currently stand. I would be happy to see a thoroughly revised version, where much more of the data is presented, as well as control analyses and rigorous and well-documented statistical testing (including addressing multiple comparisons).

These are the main points of concern that I have regarding the paper, in its current format.

(1) Prediction tendencies - assessed by listening to sequences of rhythmic tones, where the pitch was either "predictable" (i.e., followed a fixed pattern, with 25% repetition) or "unpredictable" (no particular order to the sounds). This is a very specific type of prediction, which is a general term that can operate along many different dimensions. Why was this specific design selected? Is there theoretical reason to believe that this type of prediction is also relevant to "semantic" predictions or other predictive aspects of speech processing?

(2) On the same point - I was disappointed that the results of "prediction tendencies" were not reported in full, but only used later on to assess correlations with other metrics. Even though this is a "replication" of previous work, one would like to fully understand the results from this independent study. On that note, I would also appreciate a more detailed explanation of the method used to derive the "prediction tendency" metric (e.g, what portion of the MEG signal is used? Why use a pre-stimulus and not a post-stimulus time window? How is the response affected by the 3Hz steady-state response that it is riding on? How are signals integrated across channels? Can we get a sense of what this "tendency" looks like in the actual neural signal, rather than just a single number derived per participant (an illustration is provided in Figure 1, but it would be nice to see the actual data)? How is this measure verified statistically? What is its distribution across the sample? Ideally, we would want enough information for others to be able to replicate this finding).

(3) Semantic violations - half the nouns ending sentences were replaced to create incongruent endings. Can you provide more detail about this - e.g., how were the words selected? How were the recordings matched (e.g., could they be detected due to audio editing?)? What are the "lexically identical controls that are mentioned"? Also, is there any behavioral data to know how this affected listeners? Having so many incongruent sentences might be annoying/change the nature of listening. Were they told in advance about these?

(4) TRF in multi-speaker condition: was a univariate or multivariate model used? Since the single-speaker condition only contains one speech stimulus - can we know if univariate and multivariate models are directly comparable (in terms of variance explained)? Was any comparison to permutations done for this analysis to assess noise/chance levels?

(5) TRF analysis at the word level: from my experience, 2-second segments are insufficient for deriving meaningful TRFs (see for example the recent work by Mesik & Wojtczak). Can you please give further details about how the analysis of the response to semantic violations was conducted? What was the model trained on (the full speech or just the 2-second long segments?) Is there a particular advantage to TRFs here, relative - say - to ERPs (one would expect a relatively nice N400 response, not)? In general, it would be nice to see the TRF results on their own (and not just the modulation effects).

(6) Another related point that I did not quite understand - is the dependent measure used for the regression model "neural speech envelope tracking" the r-value derived just from the 2sec-long epochs? Or from the entire speech stimulus? The text mentions the "effect of neural speech tracking" - but it's not clear if this refers to the single-speaker vs. two-speaker conditions or to the prediction manipulation. Or is it different in the different analyses? Please spell out exactly what metric was used in each analysis.

Reviewer #3 (Public review):

Summary:

Cholecystokinin (CCK) is highly expressed in auditory thalamocortical (MGB) neurons and CCK has been found to shape cortical plasticity dynamics. In order to understand how CCK shapes synaptic plasticity in the auditory thalamocortical pathway, they assessed the role of CCK signaling across multiple mechanisms of LTP induction with the auditory thalamocortical (MGB - layer IV Auditory Cortex) circuit in mice. In these physiology experiments that leverage multiple mechanisms of LTP induction and a rigorous manipulation of CCK and CCK-dependent signaling, they establish an essential role of auditory thalamocortical LTP on the co-release of CCK from auditory thalamic neurons. By carefully assessing the development of this plasticity over time and CCK expression, they go on to identify a window of time that CCK is produced throughout early and middle adulthood in auditory thalamocortical neurons to establish a window for plasticity from 3 weeks to 1.5 years in mice, with limited LTP occurring outside of this window. The authors go on to show that CCK signaling and its effect on LTP in the auditory cortex is also capable of modifying frequency discrimination accuracy in an auditory PPI task. In evaluating the impact of CCK on modulating PPI task performance, it also seems that in mice <1.5 years old CCK-dependent effects on cortical plasticity are almost saturated. While exogenous CCK can modestly improve discrimination of only very similar tones, exogenous focal delivery of CCK in older mice can significantly improve learning in a PPI task to bring their discrimination ability in line with those from young adult mice.

Strengths:

(1) The clarity of the results along with the rigor multi-angled approach provide significant support for the claim that CCK is essential for auditory thalamocortical synaptic LTP. This approach uses a combination of electrical, acoustic, and optogenetic pathway stimulation alongside conditional expression approaches, germline knockout, viral RNA downregulation, and pharmacological blockade. Through the combination of these experimental configures the authors demonstrate that high-frequency stimulation-induced LTP is reliant on co-release of CCK from glutamatergic MGB terminals projecting to the auditory cortex.

(2) The careful analysis of the CCK, CCKB receptor, and LTP expression is also a strength that puts the finding into the context of mechanistic causes and potential therapies for age-dependent sensory/auditory processing changes. Similarly, not only do these data identify a fundamental biological mechanism, but they also provide support for the idea that exogenous asynchronous stimulation of the CCKBR is capable of restoring an age-dependent loss in plasticity.

(3) Although experiments to simultaneously relate LTP and behavioral change or identify a causal relationship between LTP and frequency discrimination are not made, there is still convincing evidence that CCK signaling in the auditory cortex (known to determine synaptic LTP) is important for auditory processing/frequency discrimination. These experiments are key for establishing the relevance of this mechanism.

Weaknesses:

(1) Given the magnitude of the evoked responses, one expects that pyramidal neurons in layer IV are primarily those that undergo CCK-dependent plasticity, but the degree to which PV-interneurons and pyramidal neurons participate in this process differently is unclear.

(2) While these data support an important role for CCK in synaptic LTP in the auditory thalamocortical pathway, perhaps temporal processing of acoustic stimuli is as or more important than frequency discrimination. Given the enhanced responsivity of the system, it is unclear whether this mechanism would improve or reduce the fidelity of temporal processing in this circuit. Understanding this dynamic may also require consideration of cell type as raised in weakness #1.

(3) In Figure 1, an example of increased spontaneous and evoked firing activity of single neurons after HFS is provided. Yet it is surprising that the group data are analyzed only for the fEPSP. It seems that single-neuron data would also be useful at this point to provide insight into how CCK and HFS affect temporal processing and spontaneous activity/excitability, especially given the example in 1F.

(4) The authors mention that CCK mRNA was absent in CCK-KO mice, but the data are not provided.

(5) The circuitry that determines PPI requires multiple brain areas, including the auditory cortex. Given the complicated dynamics of this process, it may be helpful to consider what, if anything, is known specifically about how layer IV synaptic plasticity in the auditory cortex may shape this behavior.

Reviewer #3 (Public review):

Summary:

In this paper, Tanaka and colleagues address the role played by the C-C chemokine receptor 4 (CCR4) in developing early atherosclerotic plaques using ApoE-deficient mice fed with a standard chow diet as a model. Since CCR4 is expressed in several T CD4+ lymphocyte subsets, the authors examined the consequences of CCR4 deficiency on the differentiation profile and traffic of T CD4+ lymphocytes. By histological analysis of aortic lesions, they demonstrated that the absence of CCR4 promoted the development of early atherosclerosis, characterized by an inflammatory reaction with increased levels of macrophages and T CD4+ inflammatory lymphocytes while decreased collagen content. Using flow cytometry together with mRNA expression analysis for identifying T CD4+ cell subsets, the authors found that the accelerated aortic inflammation induced by CCR4 deficiency correlated with higher proliferation of T CD4+ cells in lymphoid tissues, favouring the expansion of the pro-inflammatory effector Th1 cell subset, typically found in atherosclerotic lesions. Interestingly, the increased T CD4+ cell response occurred despite the expansion of T CD4+ Foxp3+ regulatory cells (Treg), which were in higher numbers in the lymphoid tissues of CCR4-deficient mice, suggesting the absence of CCR4 interfered with the regulatory actions of Treg cells. Using in vitro and or in vivo approaches, the authors found evidence of CCR4 requirement for Treg suppressive activity and migratory capacity to inflamed aortic areas, contributing to why CCR4 deficiency induced an augmented Th1/Treg ratio in the aortic lesions. These findings might not be surprising considering the demonstrated involvement of CCR4 in driving Treg migration to inflamed tissues in immune-related pathological models and Treg-dendritic cell contact for imprinting suppressive signals. However, in previous studies using a murine model of advanced atherosclerosis, neither hematopoietic nor systemic CCR4 deficiency altered the development of the aortic lesions. The authors included a thoughtful discussion about hypothetical mechanisms explaining these contrasting results, highlighting putative differences in the role played by the CCL17/CCL22-CCR4 axis along the stages of atherosclerosis development in this murine model.

Major strengths and weaknesses:

The main effects of CCR4 deficiency on early atherosclerosis development and Treg functional loss are valuable and supported by collected data. In vivo studies for comparing Treg-tissue accumulation or atherosclerotic lesions in Apoe-/- mice that received Treg derived from Apoe-/- or Apoe-/-Ccr4-/- mice, strengthening results. However, an incomplete description of methods (particularly flow cytometry) and data analysis weakens some conclusions of this study. Readers should note some inconsistencies in the T CD4+ response analysis in different tissues. In aortic lesions, but not in lymphoid tissues (peripheral, para-aortic, and spleen), the ratio Th1/Treg was used for evaluating the effect of CCR4 deficiency on the profile of Th cell subsets. In lymphoid tissues, increments in the frequency of both effector Th1 and Treg were observed in CCR4-deficient Apoe-/- mice compared to CCR4-sufficient Apoe-/- mice. Therefore, it is not convincing that CCR4-deficiency shifts Th1 cell/Treg balance toward Th1 cell responses in all lymphoid tissues; this claim needs to be revised by the authors. The Treg dysfunction, caused by CCR4 deficiency, enhanced T CD4+ activation and might have amplified rather than shifted, the typical biased Th1-mediated inflammatory response observed in the lymphoid tissues of hypercholesterolemic mice. A different scenario emerged in aortic lesions, where recruitment of effector Th1 cells, but not of additional effector T CD4+ cell subsets expanded in lymphoid tissues, leading to a higher Th1/Treg balance. Also, effector Th17 cells seem to predominate among effector TCD45+CD3+CD4+ cells in the aorta of Apoe-/- mice, and the Th1/Th17 balance appears to have increased as a consequence of CCR4 deficiency as well. Modulation of Th1/Th17 balance might be responsible for changes in the type and functional properties of recruited inflammatory cells in the aorta.

Study limitations:

This investigation has some limitations. Current tools for single-cell characterization have revealed the phenotypic heterogeneity and dynamics of aortic leukocytes, including T cells, which are among the principal aortic leukocytes found in mouse and human atherosclerotic lesions (doi:10.1161/CIRCRESAHA.117.312513). The flow cytometry analysis applied in this study cannot distinguish the generation of particular phenotypes within T CD4+ subsets, including putative phenotypes of no-suppressive T cells expressing low levels of Foxp3, as seems could occur in other chronic inflammatory disorders (doi: 10.1038/nm.3432; doi: 10.1172/JCI79014). Limitations due to the use of a complete CCR4 knockout mouse and putative differences in CCR4-mediated mechanisms along atherosclerosis stages and in human atherosclerosis were commented on by the authors in the discussion.

Global Impact

This work opens the way for a deeper analysis of the contribution of CCR4 and its ligands to the activation and differentiation of T CD4+ lymphocytes during atherosclerosis development, with these lymphocytes being fundamental players in the generation of pro-atherogenic and anti-atherogenic immune responses. Differences in the mechanisms mediated by the CCL17/CCL22-CCR4 axis among early and advanced atherosclerosis highlight the complex landscape to examine and validate in human samples and the need to achieve a deep knowledge for identifying genuine and safe targets capable of promoting protective anti-atherogenic immune responses.

Reviewer #3 (Public review):

Summary:

The authors suggest a new biomarker of chronic back pain with an option to predict a result of treatment.

Strengths:

The results were reproduced in three studies.

Weaknesses:

The number of participants is still low, an explanation of microstructure changes was not given, and some technical drawbacks are presented.

Reviewer #3 (Public review):

Summary:

The work shows how learned assembly structure and its influence on replay during spontaneous activity can reflect the statistics of stimulus input. In particular, stimuli that are more frequent during training elicit stronger wiring and more frequent activation during replay. Past works (Litwin-Kumar and Doiron, 2014; Zenke et al., 2015) have not addressed this specific question, as classic homeostatic mechanisms forced activity to be similar across all assemblies. Here, the authors use a dynamic gain and threshold mechanism to circumnavigate this issue and link this mechanism to a cellular monitoring of membrane potential history.

Strengths:

(1) This is an interesting advance, and the authors link this to experimental work in sensory learning in environments with non-uniform stimulus probabilities.

(2) The authors consider their mechanism in a variety of models of increasing complexity (simple stimuli, complex stimuli; ignoring Dale's law, incorporating Dale's law).

(3) Links a cellular mechanism of internal gain control (their variable h) to assembly formation and the non-uniformity of spontaneous replay activity. Offers a promise of relating cellular and synaptic plasticity mechanisms under a common goal of assembly formation.

Weaknesses:

(1) However, while the manuscript does show that assembly wiring does follow stimulus likelihood, it is not clear how the assembly specific statistics of h reflect these likelihoods. I find this to be a key issue.

(2) The authors model does take advantage of the sigmoidal transfer function, and after learning an assembly is either fully active or near fully silent (Fig. 2a). This somewhat artificial saturation may be the reason that classic homeostasis is not required, since runaway activity is not as damaging to network activity.

(3) Classic mechanisms of homeostatic regulation (synaptic scaling, inhibitory plasticity) try to ensure that firing rates match a target rate (on average). If the target rate is the same for all neurons then having elevated firing rates for one assembly compared to others during spontaneous activity would be difficult. If these homeostatic mechanisms were incorporated, how would they permit the elevated firing rates for assemblies that represent more likely stimuli?

Reviewer #3 (Public review):

In this manuscript, Nishi et al. propose a new model to explain the previously reported myeloid-biased hematopoiesis associated with aging. Traditionally, this phenotype has been explained by the expansion of myeloid-biased hematopoietic stem cell (HSC) clones during aging. Here, the authors question this idea and show how their Hoxb5 reporter model can discriminate long-term (LT) and short-term (ST) HSC and characterized their lineage output after transplant. From these analyses, the authors conclude that changes during aging in the LT/ST HSC proportion explain the myeloid bias observed.

Although the topic is appropriate and the new model provides a new way to think about lineage-biased output observed in multiple hematopoietic contexts, some of the experimental design choices, as well as some of the conclusions drawn from the results could be substantially improved. Also, they do not propose any potential mechanism to explain this process, which reduces the potential impact and novelty of the study.

The authors have satisfactorily replied to some of my comments. However, there are multiple key aspects that still remain unresolved.

Reviewer #3 (Public review):

Summary:

The manuscript is evaluating changes in dopamine signaling in the nucleus accumbens following pair bonding and exposure to various stimuli in mandarin voles. In addition, the authors present chemogenetic data that demonstrate excitation and inhibition of D1 and D2 MSN affect pair bond formation.

Strengths:

The experimental designs are strong. The approaches are innovative and use cutting-edge methods. The manuscript is well written.

Weaknesses:

The statistical results are not presented, and not all statistical analyses are appropriate. Additionally, some details of methods are absent.

Reviewer #3 (Public review):

Summary:

The ability of cardiac cells to regenerate has been the object of intense (and sometimes controversial) research in biology. While lower organisms can robustly undergo cardiac regeneration by reactivation of embryonic cardiogenic pathway, this ability is strongly reduced in mice, both temporally and qualitatively. Finding a way to derive precursor cells with regenerative ability from differentiated cells in mammals has been challenging.

Zhou, He and colleagues hypothesized that ISL-1-positive cells would show regenerative capacity and developed a small molecules screen to dedifferentiate cardiomyocytes (CM) to ISL1-positive precursor cells. Using hESC-derived CM, authors found that the combination of both, WNT activation (CHIR99021) and p300 acetyltransferase inhibition (A-485) (named 2C protocol) induces CM dedifferentiation to regenerative cardiac cells (RCCs). RCCs are proliferative and re-express embryonic cardiogenic genes while decreasing expression of more mature cardiac genes, bringing them towards a more precursor-like state. RCCs were able to differentiate to CM, smooth muscle cells and endothelial cells, highlighting their multipotent property. In vivo administration of 2C in rats and mice had protective effects upon myocardial infarction.

Mechanistically, authors report that 2C protocol drives CM-specific transcriptional and epigenetic changes.

Strengths:

The authors made a great effort to validate their data using orthogonal ways, and several hESC lines. The use of lineage tracing convincingly showed a dedifferentiation from CM. They translate their findings into an in vivo model of myocardial injury, and show functional cardiac regeneration post injury. They also showed that 2C could surprisingly be used as preventive treatment. Together their data may suggest a regenerative effect of 2C both in vitro and in vivo settings. If confirmed, this study might unlock therapeutic strategy for cardiac regeneration.

Weaknesses:

Updated General comments:

Experimental design & Interpretation

(1) The titration provided by the author following the first round of revision is puzzling to me. Based on the authors explanation, the initial screen was performed using 10uM of A-485, allowing the authors to choose CHIR + A-485 as a combination of drugs increasing Isl1-positive cells. However, in the titration provided, the combination of CHIR + 10uM of A-485 (used during the screen) shows *no* increase of the percentage of Isl-1-positive cells compared to DMSO control. How is that possible? Can the authors provide a transparent explanation of the experimental design for their screen. How was A-485 isolated from the 4000+ compounds tested if it does not show any effect on the titration? This titration raises significant concerns about the rational of following up with the combination of compounds.

(2) The authors have not really addressed the concern raised earlier. If only ~1% of the cells de-differentiate and become Isl-positive, how can anybody quantify a nuclear/cytosolic ratio at the global population and show statistical significant when only 1% of the cells should be different?

(3) Authors now provide a quantification of the effect of I-BET-762 (Supp 1H). While the authors state " [the combination of CHIR + I-BET-762] was less effective than A-485 in combination with CHIR99021", the figure provided does not test that. A side-by-side comparaison of the effect of A485 and I-BET should have been performed on the same graph. I-BET increases by 4 fold, while A-485 increases by 5-fold, which, based on the variation of their data, will unlikely be statistically different. The rational for disregarding the effect of I-BET-762 is therefore weakened.

(4) Why NR2F2 is statistically significant in one set of experiments (Fig 2 - Fig. supplement 1) and then non-significant in another set (Fig. 1G) using the exact same experiment design (NC vs 2C for 60h) and similar statistical test applied?

Statistics & Data Acquisition

(1) Authors should refrain from deriving statistics from 2 biological repeats (Figure 3G).<br /> (2) Authors still do not state whether the normality of their data was tested.<br /> (3) What is the rational for using a two-way ANOVA for Fig 3G? Authors are only comparing the effect of their treatment for each marker. Same question for most panels from Figure 1, Fig 2C, 2F, and throughout the manuscript. This needs clarification/justification especially because in other experiments, they used multiple unpaired t-test (Fig 2 - Fig. supplement 1).

Others

(1) Authors should try to make their manuscript colorblind-friendly: No modification added following this comment.

Reviewer #4 (Public review):

Summary:

The manuscript by Graça et al. explores the role of MftG in the ethanol metabolism of mycobacteria. The authors hypothesise that MftG functions as a mycofactocin dehydrogenase, regenerating mycofactocin by shuttling electrons to the respiratory chain of mycobacteria. Although the study primarily uses M. smegmatis as a model microorganism, the findings have more general implications for understanding mycobacterial metabolism. Identifying the specific partner to which MftG transfers its electrons within the respiratory chain of mycobacteria would be an important next step, as pointed out by the authors.

Strengths:

The authors have used a wide range of tools to support their hypothesis, including co-occurrence analyses, gene knockout and complementation experiments, as well as biochemical assays and transcriptomics studies.<br /> An interesting observation that the mftG deletion mutant grown on ethanol as the sole carbon source exhibited a growth defect resembling a starvation phenotype.<br /> MftG was shown to catalyse the electron transfer from mycofactocinol to components of the respiratory chain, highlighting the flexibility and complexity of mycobacterial redox metabolism.

Weaknesses:

Could the authors elaborate more on the differences between the WT strains in Fig. 3C and 3E? in Fig. 3C, the ethanol concentration for the WT strain is similar to that of WT-mftG and ∆mftG-mftG, whereas the acetate concentration in thw WT strain differs significantly from the other two strains. How this observation relates to ethanol oxidation, as indicated on page 12.<br /> The authors conclude from their functional assays that MftG catalyses single-turnover reactions, likely using FAD present in the active site as an electron acceptor. While this is plausible, the current experimental set up doesn't fully support this conclusions, and the language around this claim should be softened.<br /> The authors suggest in the manuscript that the quinone pool (page 24) may act as the electron acceptor from mycofactocinol, but later in in the discussion section (page 30) they propose cytochromes as the potential recipients. If the authors consider both possibilities valid, I suggest discussing both options in the manuscript.

Reviewer #3 (Public review):

Here, Wang et al. aim to clarify the role of the centrosome and conserved polarity regulators in apical membrane formation during the polarization of MDCK cells cultured in 3D. Through well-presented and rigorous studies, the authors focused on the emergence of polarity as a single MDCK cell divided in 3D culture to form a two-cell cyst with a nascent lumen. Focusing on these very initial stages, rather than in later large cyst formation as in most studies, is a real strength of this study. The authors found that conserved polarity regulators Gp135/podocalyxin, Crb3, Cdc42, and the recycling endosome component Rab11a all localize to the centrosome before localizing to the apical membrane initiation site (AMIS) following cytokinesis. This protein relocalization was concomitant with a repositioning of centrosomes towards the AMIS. In contrast, Par3, aPKC, and the junctional components E-cadherin and ZO1 localize directly to the AMIS without first localizing to the centrosome. Based on the timing of the localization of these proteins, these observational studies suggested that Par3 is upstream of centrosome repositioning towards the AMIS and that the centrosome might be required for delivery of apical/luminal proteins to the AMIS.

To test this hypothesis, the authors generated numerous new cell lines and/or employed pharmacological inhibitors to determine the hierarchy of localization among these components. They found that removal of the centrosome via centrinone treatment severely delayed and weakened the delivery of Gp135 to the AMIS and single lumen formation, although normal lumenogenesis was apparently rescued with time. This effect was not due to the presence of CEP164, ODF2, CEP120, or Pericentrin. Par3 depletion perturbed the repositioning of the centrosome towards the AMIS and the relocalization of the Gp135 and Rab11 to the AMIS, causing these proteins to get stuck at the centrosome. Finally, the authors culture the MDCK cells in several ways (forced aggregation and ECM depleted) to try and further uncouple localization of the pertinent components, finding that Par3 can localize to the cell-cell interface in the absence of cell division. Par3 localized to the edge of the cell-cell contacts in the absence of ECM and this localization was not sufficient to orient the centrosomes to this site, indicating the importance of other factors in centrosome recruitment.

Together, these data suggest a model where Par3 positions the centrosome at the AMIS and is required for the efficient transfer of more downstream polarity determinants (Gp135 and Rab11) to the apical membrane from the centrosome. The authors present solid and compelling data and are well-positioned to directly test this model with their existing system and tools. In particular, one obvious mechanism here is that centrosome-based microtubules help to efficiently direct the transport of molecules required to reinforce polarity and/or promote lumenogenesis. This model is not really explored by the authors except by Pericentrin and subdistal appendage depletion and the authors do not test whether these perturbations affect centrosomal microtubules. Exploring the role of microtubules in this process could considerably add to the mechanisms presented here. In its current state, this paper is a careful observation of the events of MCDK polarization and will fill a knowledge gap in this field. However, the mechanism could be significantly bolstered with existing tools, thereby elevating our understanding of how polarity emerges in this system.

Reviewer #3 (Public review):

In the manuscript titled "Heat Shock Factor Regulation of Antimicrobial Peptides Expression Suggests a Conserved Defense Mechanism Induced by Febrile Temperature in Arthropods", the authors investigate the role of heat shock factor 1 (HSF1) in regulating antimicrobial peptides (AMPs) in response to viral infections, particularly focusing on febrile temperatures. Using shrimp (Litopenaeus vannamei) and Drosophila S2 cells as models, this study shows that HSF1 induces the expression of AMPs, which in turn inhibit viral replication, offering insights into how febrile temperatures enhance immune responses. The study demonstrates that HSF1 binds to heat shock elements (HSE) in AMPs, suggesting a conserved antiviral defense mechanism in arthropods. The findings are informative for understanding innate immunity against viral infections, particularly in aquaculture. However the logical flow of the paper can be improved.

Reviewer #3 (Public review):

This manuscript presents a number of interesting findings that have the potential to increase our understanding of the mechanism underlying homeostatic synaptic plasticity (HSP). The data broadly support that Rab3A plays a role in HSP, although the site and mechanism of action remain uncertain.

The authors clearly demonstrate the Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength seems already elevated. In this context, it is unclear if the plasticity is absent or just occluded by a ceiling effect due the synapses already being strengthened. Occlusion may also occur in the mixed cultures, with Rab3A missing from neurons but not astrocytes. The authors do appropriately discuss both options. There are also differences in genetic background between the Rab3A KO and Earlybird mutants that could also impact the results, which are also noted. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between synaptic strength during HSP and AMPA receptor trafficking and conclude that trafficking may not be solely responsible for the changes in synaptic strength.

Strengths:

This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is likely only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms.

However, the conclusions on the partial dissociation of AMPAR trafficking and synaptic response are made from somewhat weaker data. On average, across 3 culture sets, they saw similar magnitude of change in mEPSC amplitude and GluA2 cluster area and integral, but the GluA2 data was not significant. This is likely due to the nature of the datasets. Their imaging method involves only assessing puncta pairs (GluA2/VGlut1) clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, with usually less than 20 synapses per neuron analyzed (as stated by the authors). The mEPSC recordings will be averaging across several hundred events, which likely represent a hundred or more synapses given reasonable expectations on release probability. It has been reported, in work from this lab as well as by direct monitoring of tagged AMPARs during HSP (Wang, et al., 2019), that individual synapses are quite variable in their response. So there will almost necessarily be higher variability in the imaging data due to the smaller number of synapses sampled. The overall trends, though, are in alignment with previous data implicating receptor trafficking as the mechanism for HSP. However, the authors go on to evaluate each of the individual cultures, where 2 show similar changes between the mEPSC data and GluA2 clusters, and 1 culture showing little/no change in GluA2 clusters. The n's are very low here, and none of the datasets are significant. They want to conclude for this culture, there was a change in mEPSC amplitude that was not accompanied by a change in GluA2 at synaptic sites. But these data are collected from different coverslips, and due to the low n's, the potential under-sampling of the GluA2 clusters, and neuron-to-neuron variability, it is very hard to distinguish if this apparent difference is a methodological issue rather than a biological one. Much stronger data would be necessary to conclude that additional factors beyond receptor trafficking are required for HSP.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a frequency effect that is unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. However the change in frequency seems to argue (as the authors do) that some synapses only have CP-AMPARs, while the rest of the synapses have few or none. Another possibility is that there are pre-synaptic NASPM-sensitive receptors that influence release probability. Further, the amplitude data show a strong trend towards smaller amplitude following NASPM treatment (Fig 3B). The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. The decrease on average is larger in the TTX neurons, and some cells show a strong effect. It is possible there is some heterogeneity between neurons on whether GluA1/A2 heteromers or GluA1 homomers are added during HSP. This would impact the weakly supported conclusions about the GluA2 imaging vs mEPSC amplitude data.

Unaddressed issues that would greatly increase the impact of the paper:

(1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role. They could use sparse knock-down of Rab3A, or simply mix cultures from KO and WT mice (with appropriate tags/labels). The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. The more support for their suggestion of a pre-synaptic site of control, the better.

(2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at these synapses.

Reviewer #3 (Public review):

The authors apply multivoxel decoding analyses from fMRI during reward feedback about the cues previously chosen that led to that feedback. They compare two versions of the task - one in which the feedback is provided about the current trial, and one in which the feedback is provided about the previous trial. Reward probability changes slowly over time, so subjects need to identify which cues are leading to reward at a given time. They find that evidence for recall of the cue in the lateral orbitofrontal cortex (lOFC) and hippocampus (HC). They also find that in the second condition, where feedback is for the one-back trial, this representation is mediated by the lateral frontal pole (FPl).

Overall, the analyses are clean and elegant and seem to be complete. I have only a few comments.

(1) They do find (not surprisingly) that the one-back task is harder. It would be good to ensure that the reason that they had more trouble detecting direct HC & lOFC effects on the harder task was not because the task is harder and thus that there are more learning failures on the harder one-back task. (I suspect their explanation that it is mediated by FPl is likely to be correct. But it would be nice to do some subsampling of the zero-back task [matched to the success rate of the one-back task] to ensure that they still see the direct HC and lOFC there).

(2) The evidence that they present in the main text (Figure 3) that the HC and lOFC are mediated by FPl is a correlation. I found the evidence presented in Supplemental Figure 7 to be much more convincing. As I understand it, what they are showing in SF7 is that when FPl decodes the cue, then (and only then) HC and lOFC decode the cue. If my understanding is correct, then this is a much cleaner explanation for what is going on than the secondary correlation analysis. If my understanding here is incorrect, then they should provide a better explanation of what is going on so as to not confuse the reader.

(3) I like the idea of "credit spreading" across trials (Figure 1E). I think that credit spreading in each direction (into the past [lower left] and into the future [upper right]) is not equivalent. This can be seen in Figure 1D, where the two tasks show credit spreading differently. I think a lot more could be studied here. Does credit spreading in each of these directions decode in interesting ways in different places in the brain?

Reviewer #3 (Public review):

This study provides a useful insight into the proteomic analysis of several human induced pluripotent (hiPSC) and human embryonic stem cell (hESC) lines. Although the study is largely descriptive with limited validation of the differences found in the proteomic screen, the findings provide a solid platform for further mechanistic discovery.

Reviewer #3 (Public review):

Summary:

The manuscript by Witten et al., aims to investigate the link between acuity thresholds (and hyperacuity) and retinal sampling. Specifically, using in vivo foveal cone-resolved imaging and simultaneous microscopic photo stimulation, the researchers examined visual acuity thresholds in 16 volunteers and correlated them with each individual's retinal sampling capacity and the characteristics of ocular drift.

First, the authors found that although visual acuity was highly correlated with the individual spatial arrangement of cones, for all participants, visual resolution exceeded the Nyquist sampling.

Thus, the researchers hypothesized that this increase in acuity, which could not be explained in terms of spatial encoding mechanisms, might result from exploiting the spatiotemporal characteristics of the visual input associated with the dynamics of the fixational eye movements (and ocular drift in particular).

The authors reported a correlation between acuity threshold and drift amplitude, suggesting that the visual system benefits from transforming spatial input into a spatiotemporal flow. Finally, they showed that drift, contrary to the traditional view of it as random involuntary movement, appears to exhibit directionality: drift tends to move stimuli to higher cone density areas, therefore enhancing visual resolution.

I find the work of broad interest, its methods are clear, and the results solid.

Reviewer #3 (Public review):

Summary:

This paper examined the role of nucleus reuniens (RE) projections to dorsal CA1 neurons in context fear extinction learning. First, they show that RE neurons send excitatory projections to the stratum oriens (SO) and the stratum lacunosum moleculare (SLM), but not the stratum radiatum (SR). After context fear conditioning, the synaptic connections between RE and dCA1 neurons in the SLM (but not the SO) are weakened (reduced bouton and spine density) after mice undergo context fear conditioning. This weakening is reversed by extinction learning, which leads to enhanced synaptic connectivity between RE inputs and dendrites in the SLM. Control experiments demonstrate that the observed changes are due to extinction and not caused by simple exposure to the context. Extinction learning also induced increases in the size (volume and surface area) of the post-synaptic density (PSD) in SLM. To establish the functional role of RE inputs to dCA1, the researchers used an inhibitory DREADD to silence this pathway during extinction learning. They observe that extinction memory (measured 2-hours or 24-hours later) is impaired by this inhibition. Control experiments show that the extinction memory deficit is not simply due to increased freezing caused by inactivation of the pathway or injections of CNO. Inhibiting the RO projection during extinction learning also reduced the levels of PSD-95 protein levels in the spines of dCA1 neurons.

Strengths:

Based on their results, the authors conclude that, "the RE→SLM pathway participates in the updating of fearful context value by actively regulating CFE-induced molecular and structural synaptic plasticity in the SLM.". I believe the data are generally consistent with this hypothesis, although there is an important control condition missing from the behavioral experiments.

Weaknesses:

(1) A defining feature of extinction learning is that it is context specific (Bouton, 2004). It is expressed where it was learned, but not in other environments. Similarly, it has been shown that internal contexts (or states) also modulate the expression of extinction (Bouton, 1990). For example, if a drug is administered during extinction learning, it can induce a specific internal state. If this state is not present during subsequent testing, the expression of extinction is impaired just as it is when the physical context is altered (Bouton, 2004). It is possible that something similar is happening in Figure 6. In these experiments, CNO is administered to inactivate the RE-dCA1 projection during extinction learning. The authors observe that this manipulation impairs the expression of extinction the next day (or 2-hours later). However, the drug is not given again during the test. Therefore, it is possible that CNO (and/or inactivation of the RE-dCA1 pathway) induces a state change during extinction that is not present during subsequent testing. Based on the literature cited above, this would be expected to disrupt fear extinction as the authors observed. To determine if this alternative explanation is correct, the researchers need to add groups that receive CNO during extinction training and subsequent extinction testing. If the deficits in extinction expression reported in Figure 6 result from a state change, then these groups should not exhibit an impairment. In contrast, if the authors' account is correct, then the expression of extinction should still be disrupted in mice that receive CNO during training and testing.

(2) In their analysis of dCA1 synapses after contextual fear extinction (CFE) (Figure 4), the authors should have compared Ctx and Ctx-Ctx animals against naïve animals (as they did in Figure 3) when comparing 5US and Ext with naïve animals. Otherwise, the authors cannot make the following conclusion; "since changes of SLM synapses were not observed in the animals exposed to the familiar context that was not associated with the USs, our data support the role of the described structural plasticity at the RE→SLM synapses in CFE, rather than in processing contextual information in general.".

(3) In the materials and methods section, the description of cannula placements is confusing and needs to be rewritten.

Reviewer #3 (Public review):

Summary:

This manuscript by McDougal et al, demonstrates species-specific activities of diverse IFIT1 orthologs and seeks to utilize evolutionary analysis to identify key amino acids under positive selection that contribute to the antiviral activity of this host factor. While the authors identify amino acid residues as important for the antiviral activity of some orthologs and propose a possible mechanism by which these residues may function, the significance or applicability of these findings to other orthologs is unclear. However, the subject matter is of interest to the field, and these findings could be significantly strengthened with additional data.

Strengths:

Assessment of multiple IFIT1 orthologs shows the wide variety of antiviral activity of IFIT1, and identification of residues outside of the known RNA binding pocket in the protein suggests additional novel mechanisms that may regulate IFIT1 activity.

Weaknesses:

Consideration of alternative hypotheses that might explain the variable and seemingly inconsistent antiviral activity of IFIT1 orthologs was not really considered. For example, studies show that IFIT1 activity may be regulated by interaction with other IFIT proteins but was not assessed in this study.

Given that there appears to be very little overlap observed in orthologs that inhibited the viruses tested, it's possible that other amino acids may be key drivers of antiviral activity in these other orthologs. Thus, it's difficult to conclude whether the findings that residues 362/4/6 are important for IFIT1 activity can be broadly applied to other orthologs, or whether these are unique to human and chimpanzee IFIT1. Similarly, while the hypothesis that these residues impact IFIT1 activity in an allosteric manner is an attractive one, there is no data to support this.

Reviewer #3 (Public review):

Summary:

In this manuscript, Yip and colleagues incorporated the pipette cleaning technique into their existing dual-patch robotic system, "the PatcherBot", to allow sequential patching of more cells for synaptic connection detection in living brain slices. During dual-patching, instead of retracting all two electrodes after each recording attempt, the system cleaned just one of the electrodes and reused it to obtain another recording while maintaining the other. With one new patch clamp recording attempt, new connections can be probed. By placing one pipette in front of the other in this way, one can "walk" across the tissue, termed "patch-walking." This application could allow for probing additional neurons to test the connectivity using the same pipette in the same preparation.

Strengths:

Compared to regular dual-patch recordings, this new approach could allow for probing more possible connections in brain slices with dual-patch recordings, thus having the potential to improve the efficiency of identifying synaptic connections

Weaknesses:

While this new approach offers the potential to increase efficiency, it has several limitations that could curtail its widespread use.

Loss of Morphological Information: Unlike traditional multi-patch recording, this approach likely loses all detailed morphology of each recorded neuron. This loss is significant because morphology can be crucial for cell type verification and understanding connectivity patterns by morphological cell type.

Spatial Restrictions: The robotic system appears primarily suited to probing connections between neurons with greater spatial separation (~100µm ISD). This means it may not reliably detect connections between neurons in close proximity, a potential drawback given that the connectivity is much higher between spatially close neurons. This limitation could help explain the low connectivity rate (5%) reported in the study.

Limited Applicability: While the approach might be valuable in specific research contexts, its overall applicability seems limited. It's important to consider scenarios where the trade-off between efficiency and specific questions that are asked.<br /> Scalability Challenges: Scaling this method beyond a two-pipette setup may be difficult. Additional pipettes would introduce significant technical and logistical complexities.

Reviewer #3 (Public review):

In this study, O'Brien et al. address the need for scalable and cost-effective approaches to finding lead compounds for the treatment of the growing number of Mendelian diseases. They used state-of-the-art phenotypic screening based on an established high-dimensional phenotypic analysis pipeline in the nematode C. elegans.

First, a panel of 25 C. elegans models was created by generating CRISPR/Cas9 knock-out lines for conserved human disease genes. These mutant strains underwent behavioral analysis using the group's published methodology. Clustering analysis revealed common features for genes likely operating in similar genetic pathways or biological functions. The study also presents results from a more focused examination of ciliopathy disease models.

Subsequently, the study focuses on the NALCN channel gene family, comparing the phenotypes of mutants of nca-1, unc-77, and unc-80. This initial characterization identifies three behavioral parameters that exhibit significant differences from the wild type and could serve as indicators for pharmacological modulation.

As a proof-of-concept, O'Brien et al. present a drug repurposing screen using an FDA-approved compound library, identifying two compounds capable of rescuing the behavioral phenotype in a model with UNC80 deficiency. The relatively short time and low cost associated with creating and phenotyping these strains suggest that high-throughput worm tracking could serve as a scalable approach for drug repurposing, addressing the multitude of Mendelian diseases. Interestingly, by measuring a wide range of behavioural parameters, this strategy also simultaneously reveals deleterious side effects of tested drugs that may confound the analysis.

Considering the wealth of data generated in this study regarding important human disease genes, it is regrettable that the data is not made accessible to researchers less versed in data analysis methods. This diminishes the study's utility. It would have a far greater impact if an accessible and user-friendly online interface were established to facilitate data querying and feature extraction for specific mutants. This would empower researchers to compare their findings with the extensive dataset created here.

Another technical limitation of the study is the use of single alleles. Large deletion alleles were generated by CRISPR/Cas9 gene editing. At first glance, this seems like a good idea because it limits the risk that background mutations, present in chemically-generated alleles, will affect behavioral parameters. However, these large deletions can also remove non-coding RNAs or other regulatory genetic elements, as found, for example, in introns. Therefore, it would be prudent to validate the behavioral effects by testing additional loss-of-function alleles produced through early stop codons or targeted deletion of key functional domains.

Reviewer #3 (Public review):

Summary:

Hudaiberdiev and Ovcharenko investigate regions within the genome where a high abundance of DNA associated proteins are located and identify DNA sequence feature enriched in these regions, their conservation in evolution, and variation in disease. Using ChIP-seq binding profiles of over 1,000 proteins in three human cell lines (HepG2, K562, and H1) as a data source they're able to identify nearly 44,000 high-occupancy target loci (HOT) that form at promoter and enhancer regions, thus suggesting these HOT loci regulate housekeeping and cell identity genes. Their primary investigative tool is HepG2 cells, but they employ K562 and H1 cells as tools to validate these assertions in other human cell types. Their analyses use RNA pol II signal, super enhancer, regular enhancer and epigentic marks to support the identification of these regions. The work is notable, in that it identifies a set of proteins that are invariantly associated with high-occupancy enhancers and promoters and argues for the integration of these molecules at different genomic loci. These observations are leveraged by the authors to argue HOT loci as potential sites of transcriptional condensates, a claim that they provide information in support of. Transcriptional condensates are an important "family" of condensates, regulating different types of genes and this work supports the hypothesis that they possess similar protein partner molecules as those thought to define such bodies.

Reviewer #3 (Public Review):

Summary:

(1) To further explore the genetic basis of asthenoteratozoospermia, the authors performed whole-exome sequencing analyses among infertile males affected by asthenoteratozoospermia. Four unrelated Han Chinese patients were found to carry biallelic variations of DNAH3, a gene encoding IDA-associated protein.<br /> (2) To verify the function of IDA associated protein DNAH3, the authors generated a Dnah3-KO mouse model and revealed that the loss of DNAH3 leads to severe male infertility as a result of the severe reduction in sperm movement with the abnormal IDA and mitochondrion structures.<br /> (3) Mechanically, they confirmed decreased expression of IDA-associated proteins (including DNAH1, DNAH6 and DNALI1) in the spermatozoa from patients with DNAH3 mutations and Dnah3-KO male mice.<br /> (4) Then, they also found that male infertility caused by DNAH3 deficiency could be rescued by intracytoplasmic sperm injection (ICSI) treatment in humans and mice.

Strengths:

(1) In addition to existing research, the authors provided novel variants of DNAH3 as important factors leading to asthenoteratozoospermia. This further expands the spectrum of pathogenic variants in asthenoteratozoospermia.<br /> (2) By mechanistic studies, they found that DNAH3 deficiency led to decreased expression of IDA-associated proteins, which may be used to explain the disruption of sperm motility and reduced fertility caused by DNAH3 deficiency.<br /> (3) Then, successful ICSI outcomes were observed in patients with DNAH3 mutations and Dnah3 KO mice, which will provide an important reference for genetic counselling and clinical treatment of male infertility.

Reviewer #3 (Public review):

Summary:

This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.

Strengths:

The study was conducted carefully and is clearly explained here.

Weaknesses:

The revised manuscript is much improved.

Reviewer #3 (Public review):

Summary:

The authors use cryo-electron tomography to thoroughly investigate the complexity of purified, excitatory synapses. They make several major interesting discoveries: polyhedral vesicles that have not been observed before in neurons; analysis of the intermembrane distance, and a link to potentiation, essentially updating distances reported from plastic-embedded specimen; and find that the postsynaptic density does not appear as a dense accumulation of proteins in all vitrified samples (less than half), a feature which served as a hallmark feature to identify excitatory plastic-embedded synapses.

Strengths:

(1) The presented work is thorough: the authors compare purified, endogenously labeled synapses to wild-type synapses to exclude artifacts that could arise through the homogenation step, and, in addition, analyse plastic embedded, stained synapses prepared using the same quick workflow, to ensure their findings have not been caused by way of purification of the synapses. Interestingly, the 'thick lines of PSD' are evident in most of their stained synapses.

(2) I commend the authors on the exceptional technical achievement of preparing frozen specimens from a mouse within two minutes.

(3) The approaches highlighted here can be used in other fields studying cell-cell junctions.

(4) The tomograms will be deposited upon publication which will enable neurobiologists and researchers from other fields to carry on data evaluation in their field of expertise since tomography is still a specialized skill and they collected and reconstructed over 100 excellent tomograms of synapses, which generates a wealth of information to be also used in future studies.

(5) The authors have identified ionotropic receptor positions and that they are linked to actin filaments, and appear to be associated with membrane and other cytosolic scaffolds, which is highly exciting.

(6) The authors achieved their aims to study neuronal excitatory synapses in great detail, were thorough in their experiments, and made multiple fascinating discoveries. They challenge dogmas that have been in place for decades and highlight the benefit of implementing and developing new methods to carefully understand the underlying molecular machines of synapses.

Weaknesses:

The authors show informative segmentations in their figures but none have been overlayed with any of the tomograms in the submitted videos. It would be helpful for data evaluation to a broad audience to be able to view these together as videos to study these tomograms and extract more information. Deposition of segmentations associated with the tomgrams would be tremendously helpful to Neurobiologists, cryo-ET method developers, and others to push the boundaries.

Impact on community:

The findings presented by Peukes et al. pertaining to synapse biology change dogmas about the fundamental understanding of synaptic ultrastructure. The work presented by the authors, particularly the associated change of intermembrane distance with potentiation and the distinct appearance of the PSD as an irregular amorphous 'cloud' will provide food for thought and an incentive for more analysis and additional studies, as will the discovery of large membranous and cytosolic protein complexes linked to ionotropic receptors within and outside of the synaptic cleft, which are ripe for investigation. The findings and tomograms available will carry far in the synapse fields and the approach and methods will move other fields outside of neurobiology forward. The method and impactful results of preparing cryogenic, unlabeled, unstained, near-native synapses may enable the study of how synapses function at high resolution in the future.

Reviewer #3 (Public review):

This paper provides an interesting description of the ventral parts of the Cambrian xandarellid Cindarella eucalla, derived from exceptionally preserved specimens of the Chengjiang Biota. These morphological data are useful for our broad understanding and future research on Xandarellida, and are generally well-represented in the description and accompanying figures. The strengths of this work rest in this morphological description of exceptional fossil material, and this is generally well supported. In addition, the authors put this description in the context of the morphology of other xandarellids and Cambrian arthropod groups, with most of these parallels being useful and reasonably supported, though in several places homology is assumed and this currently lacks evidence. The manuscript goes on to use these morphological data and comparisons to other groups (particularly trilobites) to make suggestions for the ecology of Cindarella eucalla and other xandarellids. The majority of my comments on this work relate to this latter aim - the ecological conclusions drawn are generally derived through morphological comparisons, where a specific morphology has been suggested as an adaption to a particular ecological function in another extinct arthropod group. However, the original suggestions for ecological function are untested, and so remain hypotheses. Despite this, they are frequently presented as truisms to enable ecological conclusions to be drawn for Cindarella eucalla. I have listed my comments and queries on the study below for the authors to address or respond to, and I hope they are useful to the authors.

Comments:

There are a number of ecological and functional morphology conclusions stated that seem put too strongly to be considered sufficiently supported by the evidence given. These relate to both the description of C. eucalla, and comparisons to other extinct arthropod taxa (notably trilobites). Many of these latter statements are assumptions of functional morphology, and should not be repeated as truisms, rather than they represent suggested functions and ecologies based on the known morphological descriptions. This aspect occurs throughout the article, and, for me, is the primary concern.

Reviewer #3 (Public review):

Summary:

The manuscript "Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility" describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). The authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observed varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes.

A major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested.

A major weakness is a lack of new insights into underlying mechanisms that may contribute to disease phenotypes, nor any clear commonalities across gene sets. This was most evident in the qRT-PCR analysis of select skeletal developmental genes, which all showed varied changes in fold and direction, but with little insight into the implications of the results.

Ultimately, the authors were able to show their approach is capable of connecting candidate genes with perturbation of skeletal phenotypes. It was surprising that all four GWAS candidate genes (which presumably were lower confidence) also produced a result. These authors have previously demonstrated that crispants recapitulate skeletal phenotypes of stable mutant lines for a single gene, somewhat reducing the novelty of the study.

Reviewer #3 (Public review):

Summary:

In this study, Miyatake et al. present the interesting finding that ectopic expression of miR-195 in EBF1-deficient hematopoietic progenitor cells can partially rescue their developmental block and allow B cells to progress to a B220+ CD19+ cells stage. Notably, this is accompanied by an upregulation of B-cell-specific genes and, correspondingly, a downregulation of T, myeloid, and NK lineage-related genes, suggesting that miR-195 expression is at least in part equivalent to EBF1 activity in orchestrating the complex gene regulatory network underlying B cell development. Strengthening this point, ATAC sequencing of miR-195-expressing EBF1-deficient B220+CD19+ cells and a comparison of these data to public datasets of EBF1-deficient and -proficient cells suggest that miR-195 indirectly regulates gene expression and chromatin accessibility of some, but not all regions regulated by EBF1.

Mechanistically, the authors identify a subset of potential target genes of miR-195 involved in MAPK and PI3K signalling. Dampening of these pathways has previously been demonstrated to activate FOXO1, a key transcription factor for early B cells downstream of EBF1. Accordingly, the authors hypothesize that miR-195 exerts its function through FOXO1. Supporting this claim, also exogenous FOXO1 expression is able to promote the development of EBF1-deficient cells to the B220+CD19+ stage and thus recapitulates the miR-195 phenotype.

Strengths:

The strength of the presented study is the detailed assessment of the altered chromatin accessibility in response to ectopic miR-195 expression. This provides insight into how miR-195 impacts the gene regulatory network that governs B-cell development and allows the formation of mechanistic hypotheses.

Weaknesses:

The key weakness of this study is that its findings are based on the artificial and ectopic expression of a miRNA out of its normal context, which in my opinion strongly limits the biological relevance of the presented work.

While the authors performed qPCRs for miR-195 on different B cell populations and show that its relative expression peaks in early B cells, it remains unclear whether the absolute miR-195 expression is sufficiently high to have any meaningful biological activity. In fact, other miRNA expression data from immune cells (e.g. DOI 10.1182/blood-2010-10-316034 and DOI 10.1016/j.immuni.2010.05.009) suggest that miR-195 is only weakly, if at all, expressed in the hematopoietic system.

The authors support their finding by a CRISPR-derived miR-195 knockout mouse model which displays mild, but significant differences in the hematopoietic stem cell compartment and in B cell development. However, they fail to acknowledge and discuss a lymphocyte-specific miR-195 knockout mouse that does not show any B cell defects in the bone marrow or spleen and thus contradicts the authors' findings (DOI 10.1111/febs.15493). Of note, B-1 B cells in particular have been shown to be elevated upon loss of miR-15-16-1 and/or miR-15b-16-2, which contradicts the data presented here for loss of the family member miR-195.

A second weakness is that some claims by the authors appear overstated or at least not fully backed up by the presented data. In particular, the findings that miR-195-expressing cells can undergo VDJ recombination, express the pre-BCR/BCR and class switch needs to be strengthened. It would be beneficial to include additional controls to these experiments, e.g. a RAG-deficient mouse as a reference/negative control for the ddPCR and the surface IgM staining, and cells deficient in class switching for the IgG1 flow cytometric staining.

Moreover, the manuscript would be strengthened by a more thorough investigation of the hypothesis that miR-195 promotes the stabilization and activity of FOXO1, e.g. by comparing the authors' ATACseq data to the FOXO1 signature.

Reviewer #3 (Public review):

Summary:

Perlee et al. present a method for generating cell-type restricted knockouts in zebrafish, focusing on melanocytes. For this method, the authors knock-in a Cas9 encoding sequence into the mitfa locus. This mitfaCas9 line has restricted Cas9 expression, allowing the authors to generate melanocyte-specific knockouts rapidly by follow-up injection of sgRNA expressing transposon vectors.

The paper presents some interesting vignettes to illustrate the utility of their approach. These include 1) a derivation of albino mutant fish as a demonstration of the method's efficiency, 2) an interrogation and novel description of tuba1a as a potential non-autonomous contributor to melanocyte dispersion, and 3) the generation of sox10 deficient melanoma tumors that show "escape" of sox10 loss through upregulation of sox9. The latter two examples highlight the usefulness of cell-type targeted knockouts (Body-wide sox10 and tuba1a loss elicit developmental defects). Additionally, the tumor models involve highly multiplexed sgRNAs for tumor initiation which is nicely facilitated by the stable Cas9.

Strengths:

The approach is clever and could prove very useful for studying melanocytes and other cell types. As the authors hint at in their discussion, this approach would become even more powerful with the generation of other Cas9-restricted lineages so a single sgRNA construct can be screened across many lineages rapidly (or many sgRNA and fish lines screened combinatorially).

The biological findings used to demonstrate the power of the approach are interesting in their own right. If it proves true, tuba1a's non-autonomous effects on melanosome dispersion are striking, and this example demonstrates very nicely how one could use Perlee et al.'s approach to search for other non-autonomous mechanisms systematically. Similarly, the observation of the sox9 escape mechanism with sox10 loss is a beautiful demonstration of the relevance of SOX10/SOX9's reciprocal regulation in vivo. This system would be a very nice model for further interrogating mechanisms/interventions surrounding Sox10 in melanoma.

Finally, the figure presentation is very nice. This work involves complex genetic approaches including multiple fish generations and multiplexed construct injections. The vector diagrams and breeding schemes in the paper make everything very clear/"grok-able," and the paper was enjoyable to read.

Weaknesses:

The mitfa-driven GFP on their sgRNA-expressing cassette is elegant, but it makes one wonder why the endogenous knock-in is necessary. It would strengthen the motivation of the work if the authors could detail the potential advantages and disadvantages of their system compared to expressing Cas9 with a lineage-specific promoter from a transposon in their introduction or discussion.

Related to the above - is mitfa haplosufficient? If the mitfaCas9/+ fish have any notable phenotypes, it would be worth noting for others interested in using this approach to study melanoma and pigmentation.

A core weakness (and also potential strength) of the system is that introduced edits will always be non-clonal (Fig 2H/I). The activity of individual sgRNAs should always be validated in the absence of any noticeable phenotype to interpret a negative result. Additionally, caution should be taken when interpreting results from rare events involving positive outgrowth (like tumorogenesis) to account for the fact many cells in the population might not have biallelic null alleles (i.e., 100% of the gene product removed).

Along those lines: in my opinion, the tuba1a results are the most provocative finding in the paper, but they lack key validation. With respect to cutting activity, the Alt-R and transgenic sgRNA expression approaches are not directly comparable. Since there is no phenotype in the melanocyte specific tuba1a knockouts, the authors must confirm high knockout efficiency with this set of reagents before making the claim there is a non-autonomous phenotype. This can be achieved with GFP+ sorting and NGS like they performed with their albino melanocytes.

The whole-body tuba1a knockout phenotype is expected to be pleiotropic, and this expectation might mask off-target effects. Controls for knockout specificity should be included. For instance, confidence in the claims would greatly increase if the dispersed melanosome phenotype could be recovered with guide-resistant tuba1a re-expression and if melanocyte-restricted tuba1a re-expression failed to rescue. As a less definitive but adequate alternative, the authors could also test if another guide or a morpholino against tuba1a phenocopies the described Alt-R edited fish.

I have similar questions about the sox10 escapers, but these suggestions are less critical for supporting the authors claims (especially given the nice staining). Are the sox10 tumors relatively clonal with respect to sox10 mutations? And are the sox10 tumor mutations mostly biallelic frameshifts or potential missense mutations/single mutations that might not completely remove activity? I am particularly curious as SOX10 doesn't seem to be completely absent (and is still very high in some nuclei) in the immunohistochemistry.

Reviewer #3 (Public review):

In this study Barth et al. present results of detailed analyses of the relationships between menopausal hormone therapy (MHT), APOE ε4 genotype, and measures of anatomical brain age in women in the UK Biobank. While past studies have investigated the links between some of these variables (including works by the authors themselves), this new study adds more detailed MHT variables, surgical status, and additional brain aging measures. The UK biobank sample is large, but it is a population cohort and many of the MHT measures are self-reported (as the authors point out). However, the authors present a solid analysis of the available information which shows associations between MHT user status, length of MHT use, as well as surgical status with brain age. However, as the authors themselves state, the results do not unequivocally support the neuroprotective or adverse effect of MHT on the brain. I think this work strengthens the case for the need of better-designed longitudinal studies investigating the effect of MHT on the brain in the peri/post-menopausal stage.

Strengths:

The authors addressed the statistical analyses rigorously. For example, multiple testing corrections, outlier removal, and sensitivity analysis were performed carefully. Ample background information is provided in the introduction allowing even individuals not familiar with the field to understand the motivation behind the work. The discussion section also does a great job of addressing open questions and limitations. Very detailed results of all statistical tests are provided either in the main text or in the supplementary information.

Weaknesses:

For me, the biggest weakness was the presentation of the results. As many variables are involved and past studies have investigated several of these questions, it would have helped to better clarify the analysis and questions that are addressed by this study in particular and what sets this work apart from past studies. The information is present in the manuscript but better organization might have helped. For example, a figure depicting the key questions near the beginning of the manuscript would have been very helpful for me. The Tables also contain a lot of information but I wonder if there might be a way to capture the most relevant information more succinctly (either in Table format or in a figure) for the main text.<br /> Another concern I had was the linear models investigating the effects of these MHT variables on the brain age gap. The authors have included "age" as one of the parameters in this analysis. I wonder if adding a quadratic age factor age2 in the model might have improved the fit since many brain phenotypes tend to show quadratic brain age effects in the 40 to 80-year age range.

Reviewer #3 (Public review):

Summary:

This study examines the metabolic regulation of progenitor proliferation and differentiation in the developing retina. The authors observe dynamic changes in glycolytic gene expression in retinal progenitors and use various strategies to test the role of glycolysis. They find that elevated glycolysis in Pten-cKO retinas results in alteration of RPC fate, while inhibition of glycolysis has converse effects. They specifically test the role of elevated glycolysis using dominant active cytoPFKB3, which demonstrates the selective effects of elevated glycolysis on progenitor proliferation and rod differentiation. They then show that elevated glycolysis modulates both pHi and Wnt signaling, and provide evidence that these pathways impact proliferation and differentiation of progenitors, particularly affecting rod photoreceptor differentiation.

Strengths:

This is a compelling and rigorous study that provides an important advance in our understanding of metabolic regulation of retina development, addressing a major gap in knowledge. A key strength is that the study utilizes multiple genetic and pharmacological approaches to address how both increased or decreased glycolytic flux affect retinal progenitor proliferation and differentiation. They discover elevated Wnt signaling pathway genes in Pten cKO retina, revealing a potential link between glycolysis and Wnt pathway activation. Altogether the study is comprehensive and adds to the growing body of evidence that regulation of glycolysis plays a key role in tissue development.

Weaknesses:

(1) Following the expression of cytoPFKB3, which results in increased glycolytic flux, BrDU labeling was performed at e12.5 and increased labeled cells were detected in the outer nuclear layer. However whether these are cones or rods is not established. The rest of the analysis is focused on the precocious maturation of rhodopsin-labeled outer segments, and the major conclusions emphasize rod photoreceptor differentiation. Therefore it is unclear whether there is an effect on cone differentiation for either Pten cKO or cytoPFKB3 transgenic retina. It is also not established whether rods are born precociously. Presumably, this would be best detected by BrDU labeling at later embryonic stages.

(2) The authors find that there is upregulation of multiple Wnt pathway components in Pten cKO retina. They further show that inhibiting Wnt signaling phenocopies the effects of reducing glycolysis. However, they do not test whether pharmacological inhibition of Wnt signaling reverses the effects of high glycolytic activity in Pten cKO retinas. Thus the argument that Wnt is a key downstream effector pathway regulating rod photoreceptor differentiation is weak.

(3) The use of sodium acetate to force protein acetylation is quite non-specific and will have effects beyond beta-catenin acetylation (which the authors acknowledge). Thus it is a stretch to state that "forced activation of beta-catenin acetylation" mimics the impact of Pten loss/high glycolytic activity in RPCs since the effects could be due to acetylation of other proteins.

Reviewer #3 (Public review):

In this manuscript by Goldblatt et al. the authors study the development of a well-known sensorimotor system, the vestibulo-ocular reflex circuit, using Danio rerio as a model. The authors address whether motor neurons within this circuit are required to determine the identity, upstream connectivity and function of their presynaptic partners, central projection neurons. They approach this by generating a CRISPR-mediated knockout line for the transcription factor phox2a, which specifies the fate of extraocular muscle motor neurons. After showing that phox2a knockout ablates these motor neurons, the authors show that functionally, morphologically, and transcriptionally, projection neurons develop relatively normally.

Overall, the authors present a convincing argument for the dispensability of motor neurons in the wiring of this circuit, although their claims about the generalizability of their findings to other sensorimotor circuits should be tempered. The study is comprehensive and employs multiple methods to examine the function, connectivity and identity of projection neurons.

Comments on the revised version:

The authors have addressed all my previous concerns.

Reviewer #3 (Public review):

Summary:

The manuscript by Jaime Tobon and Moser uses patch-clamp electrophysiology in cochlear preparations to probe the pre- and post-synaptic specializations that give rise to diverse activity of spiral ganglion afferent neurons (SGN). The experiments are quite an achievement! They use paired recordings from pre-synaptic cochlear inner hair cells (IHC) that allow precise control of voltage and therefore calcium influx, with post-synaptic recordings from type I SGN boutons directly opposed to the IHC for both presynaptic control of membrane voltage and post-synaptic measurement of synaptic function with great temporal resolution.

Any of these techniques by themselves are challenging, but the authors do them in pairs, at physiological temperatures, and in hearing animals, all of which combined make these experiments a real tour de force. The data is carefully analyzed and presented, and the results are convincing. In particular, the authors demonstrate that post-synaptic features that contribute to the spontaneous rate (SR) of predominantly monophasic post-synaptic currents (PSCs), shorter EPSC latency, and higher PSC rates are directly paired with pre-synaptic features such as a lower IHC voltage activation and tighter calcium channel coupling for release to give a higher probability of release and subsequent increase in synaptic depression. Importantly, IHCs paired with Low and High SR afferent fibers had the same total calcium currents, indicating that the same IHC can connect to both low and high SR fibers. These fibers also followed expected organizational patterns, with high SR fibers primarily contacting the pillar IHC face and low SR fibers primarily contacting the modiolar face. The authors also use in vivo-like stimulation paradigms to show different RRP and release dynamics that are similar to results from SGN in vivo recordings. Overall, this work systematically examines many features giving rise to specializations and diversity of SGN neurons.

Reviewer #3 (Public review):

Summary:

This study used transcranial direct current stimulation administered using small 'high-definition' electrodes to modulate neural activity within the non-human primate prefrontal cortex during both wakefulness and anaesthesia. Functional magnetic resonance imaging (fMRI) was used to assess the neuromodulatory effects of stimulation. The authors report on the modification of brain dynamics during and following anodal and cathodal stimulation during wakefulness and following anodal stimulation at two intensities (1 mA, 2 mA) during anaesthesia. This study provides some possible support that prefrontal direct current stimulation can alter neural activity patterns across wakefulness and sedation in monkeys. However, the reported findings need to be considered carefully against several important methodological limitations.

Strengths:

A key strength of this work is the use of fMRI-based methods to track changes in brain activity with good spatial precision. Another strength is the exploration of stimulation effects across wakefulness and sedation, which has the potential to provide novel information on the impact of electrical stimulation across states of consciousness.

Weaknesses:

The lack of a sham stimulation condition is a significant limitation, for instance, how can the authors be sure that results were not affected by drowsiness or fatigue as a result of the experimental procedure?

In the anaesthesia condition, the authors investigated the effects of two intensities of stimulation (1 mA and 2 mA). However, a potential confound here relates to the possibility that the initial 1 mA stimulation block might have caused plasticity-related changes in neural activity that could have interfered with the following 2 mA block due to the lack of a sufficient wash-out period. Hence, I am not sure any findings from the 2 mA block can really be interpreted as completely separate from the initial 1 mA stimulation period, given that they were administered consecutively. Several previous studies have shown that same-day repeated tDCS stimulation blocks can influence the effects of neuromodulation (e.g., Bastani and Jaberzadeh, 2014, Clin Neurophysiol; Monte-Silva et al., J. Neurophysiology).

The different electrode placement for the two anaesthetised monkeys (i.e., Monkey R: F3/O2 montage, Monkey N: F4/O1 montage) is problematic, as it is likely to have resulted in stimulation over different brain regions. The authors state that "Because of the small size of the monkey's head, we expected that tDCS stimulation with these two symmetrical montages would result in nearly equivalent electric fields across the monkey's head and produce roughly similar effects on brain activity"; however, I am not totally convinced of this, and it really would need E-field models to confirm. It is also more likely that there would in fact be notable differences in the brain regions stimulated as the authors used HD-tDCS electrodes, which are generally more focal.

Given the very small sample size, I think it is also important to consider the possibility that some results might also be impacted by individual differences in response to stimulation. For instance, in the discussion (page 9, paragraph 2) the authors contrast findings observed in awake animals versus anaesthetised animals. However, different monkeys were examined for these two conditions, and there were only two monkeys in each group (monkeys J and Y for awake experiments [both male], and monkeys R and N [male and female] for the anaesthesia condition). From the human literature, it is well known that there is a considerable amount of inter-individual variability in response to stimulation (e.g., Lopez-Alonso et al., 2014, Brain Stimulation; Chew et al., 2015, Brain Stimulation), therefore I wonder if some of these differences could also possibly result from differences in responsiveness to stimulation between the different monkeys? At the end of the paragraph, the authors also state "Our findings also support the use of tDCS to promote rapid recovery from general anesthesia in humans...and suggest that a single anodal prefrontal stimulation at the end of the anesthesia protocol may be effective." However, I'm not sure if this statement is really backed-up by the results, which failed to report "any behavioural signs of awakening in the animals" (page 7)?

Reviewer #3 (Public review):

The authors make use of a large dataset of reaches from several studies run in their lab to try to identify the source of direction-dependent radial reaching errors. While this has been investigated by numerous labs in the past, this is the first study where the sample is large enough to reliably characterize isometries associated with these radial reaches to identify possible sources of errors.

The sample size is impressive, but the authors should include confidence intervals and ideally, the distribution of responses across individuals along with average performance across targets. It is unclear whether the observed "averaged function" is consistently found across individuals, or if it is mainly driven by a subset of participants exhibiting large deviations for diagonal movements. Providing individual-level data or response distributions would be valuable for assessing the ubiquity of the observed bias patterns and ruling out the possibility that different subgroups are driving the peaks and troughs. It is possible that the Transformation or some other model (see below) could explain the bias function for a substantial portion of participants, while other participants may have different patterns of biases that can be attributable to alternative sources of error.

The different datasets across different experimental settings/target sets consistently show that people show fewer deviations when making cardinal-directed movements compared to movements made along the diagonal when the start position is visible. This reminds me of a phenomenon referred to as the oblique effect: people show greater accuracy for vertical and horizontal stimuli compared to diagonal ones. While the oblique effect has been shown in visual and haptic perceptual tasks (both in the horizontal and vertical planes), there is some evidence that it applies to movement direction. These systematic reach deviations in the current study thus may reflect this epiphenomenon that applies across modalities. That is, estimating the direction of a visual target from a visual start position may be less accurate, and may be more biased toward the horizontal axis, than for targets that are strictly above, below, left, or right of the visual start position. Other movement biases may stem from poorer estimation of diagonal directions and thus reflect more of a perceptual error than a motor one. This would explain why the bias function appears in both the in-lab and on-line studies although the visual targets are very different locations (different planes, different distances) since the oblique effects arise independent of plane, distance, or size of the stimuli.

When the start position is not visible like in the Vindras study, it is possible that this oblique effect is less pronounced; masked by other sources of error that dominate when looking at 2D reach endpoint made from two separate start positions, rather than only directional errors from a single start position. Or perhaps the participants in the Vindras study are too variable and too few (only 10) to detect this rather small direction-dependent bias.

A bias in estimating visual direction or visual movement vector is a more realistic and relevant source of error than the proposed visual bias model. The Visual Bias model is based on data from a study by Huttenlocher et al where participants "point" to indicate the remembered location of a small target presented on a large circle. The resulting patterns of errors could therefore be due to localizing a remembered visual target, or due to relative or allocentric cues from the clear contour of the display within which the target was presented, or even movements used to indicate the target. This may explain the observed 4-peak bias function or zig-zag pattern of "averaged" errors, although this pattern may not even exist at the individual level, especially given the small sample size. The visual bias source argument does not seem well-supported, as the data used to derive this pattern likely reflects a combination of other sources of errors or factors that may not be applicable to the current study, where the target is continuously visible and relatively large. Also, any visual bias should be explained by a coordinates centre on the eye and should vary as a function of the location of visual targets relative to the eyes. Where the visual targets are located relative to the eyes (or at least the head) is not reported.

The Proprioceptive Bias Model is supposed to reflect errors in the perceived start position. However, in the current study, there is only a single, visible start position, which is not the best design for trying to study the contribution. In fact, my paradigms also use a single, visual start position to minimize the contribution of proprioceptive biases, or at least remove one source of systematic biases. The Vindras study aimed to quantify the effect of start position by using two sets of radial targets from two different, unseen start positions on either side of the body midline. When fitting the 2D reach errors at both the group and individual levels (which showed substantial variability across individuals), the start position predicted most of the 2D errors at the individual level - and substantially more than the target direction. While the authors re-plotted the data to only illustrate angular deviations, they only showed averaged data without confidence intervals across participants. Given the huge variability across their 10 individuals and between the two target sets, it would be more appropriate to plot the performance separately for two target sets and show confidential intervals (or individual data). Likewise, even the VT model predictions should differ across the two targets set since the visual-proprioceptive matching errors from the Wang et al study that the model is based on, are larger for targets on the left side of the body.

I am also having trouble fully understanding the V-T model and its associated equations, and whether visual-proprioception matching data is a suitable proxy for estimating the visuomotor transformation. I would be interested to first see the individual distributions of errors and a response to my concerns about the Proprioceptive Bias and Visual Bias models.

Reviewer #3 (Public review):

Summary:

This study reveals that sound exposure enhances drug delivery to the cochlea through the non-selective action of outer hair cells. The efficiency of sound-facilitated drug delivery is reduced when outer hair cell motility is inhibited. Additionally, low-frequency tones were found to be more effective than broadband noise for targeting substances to the cochlear apex. Computational model simulations support these findings.

Strengths:

The study provides compelling evidence that the broad action of outer hair cells is crucial for cochlear fluid circulation, offering a novel perspective on their function beyond frequency-selective amplification. Furthermore, these results could offer potential strategies for targeting and optimizing drug delivery throughout the cochlear spiral.

Weaknesses:

The primary weakness of this paper lies in the surgical procedure used for drug administration through the round window. Opening the cochlea can alter intracochlear pressure and disrupt the traveling wave from sound, a key factor influencing outer hair cell activity. However, the authors do not provide sufficient details on how they managed this issue during surgery. Additionally, the introduction section needs further development to better explain the background and emphasize the significance of the work.

Reviewer #3 (Public review):

Summary:

Authors try to challenge the mainstream scientific as well as popularly held view that Inattentional Blindness (IB) signifies subjects having no conscious awareness of what they report not seeing (after being exposed to unexpected stimuli). They show that even when subjects indicate NOT having seen the unexpected stimulus, they are at above chance level for reporting features such as location, color or movement of these stimuli. Also, they show that 'not seen' responses are in part due to a conservative bias of subjects, i.e. they tend to say no more than yes, regardless of actual visibility. Their conclusion is that IB may not (always) be blindness, but possibly amnesia, uncertainty etc.

Strengths:

A huge pool of (25.000) subjects is used. They perform several versions of the IB experiments, both with briefly presented stimuli (as the classic Mack and Rock paradigm), as well as with prolonged stimuli moving over the screen for 5 seconds (a bit like the famous gorilla version), and all these versions show similar results, pointing in the same direction: above chance detection of unseen features, as well as conservative bias towards saying not seen.

Weaknesses:

Results are all significant but effects are not very strong, typically a bit above chance. Also, it is unclear what to compare these effects to, as there are no control experiments showing what performance would have been in a dual task version where subjects have to also report features etc for stimuli that they know will appear in some trials

There are quite some studies showing that during IB, neural processing of visual stimuli continues up to high visual levels, for example, Vandenbroucke et al 2014 doi:10.1162/jocn_a_00530 showed preserved processing of perceptual inference (i.e. seeing a kanizsa illusion) during IB. Scholte et al 2006 doi: 10.1016/j.brainres.2005.10.051 showed preserved scene segmentation signals during IB. Compared to the strength of these neural signatures, the reported effects may be considered not all that surprising, or even weak.

Reviewer #3 (Public review):

Summary:

The authors first tested whether EAA supplementation increases olfactory preference for bacterial food for a variety of bacterial strains. Of the EAAs, they found only leucine supplementation increased olfactory preference (within a bacterial strain), and only for 3 of the bacterial strains tested. Leucine itself was not found to be intrinsically attractive.

They determined that leucine supplementation increases isoamyl alcohol (IAA) production in the 3 preferred bacterial strains. They identify the biochemical pathway that catabolizes leucine to IAA, showing that a required enzyme for this pathway is upregulated upon supplementation.

Consistent with earlier studies, they find that AWC olfactory neuron is primarily responsible for increased preference for IAA-producing bacteria.

Testing volatile compounds produced by bacteria and identified by GC/MS, and identified several as attractive, most of them require AWC for the full effect. Adaptation assays were used to show that odorant levels produced by bacterial lawns were sufficient to induce olfactory adaptation, and adaptation to IAA reduced chemotaxis to leucine-supplemented lawns. They then showed that IAA attractiveness is conserved across wild strains, while other compounds are more variable, suggesting IAA is a principal foraging cue.

Finally, using the CeNGEN database, they developed a list of candidate IAA receptors. Using behavioral tests, they show that mutation of srd-12 greatly impairs IAA chemotaxis without affecting locomotion or attraction to another AWC-sensed odor, PEA.

Comments

This study will be of great interest in the field of C. elegans behavior, chemical senses and chemical ecology, and understanding of the sensory biology of foraging.

Strengths:

The identification of a receptor for IAA is an excellent finding. The combination of microbial metabolic chemistry and the use of natural bacteria and nematode strains makes an extremely compelling case for the ecological and adaptive relevance of the findings.

Weaknesses:

AWC receives synaptic input from other chemosensory neurons, and thus could potentially mediate navigation behaviors to compounds detected in whole or in part by those neurons. Language concluding detection by AWC should be moderated (e.g. p9 "worms sense an extensive repertoire...predominantly using AWC") unless it has been demonstrated.

srd-12 is not exclusively expressed in AWC. Normally, cell-specific rescue or knockdown would be used to demonstrate function in a specific cell. The authors should provide such a demonstration or explain why they are confident srd-12 acts in AWC.

A comparison of AWC's physiological responses between WT and srd-12, preferably in an unc-13 background, would be nice. Even further, the expression of srd-12 in a different neuron type and showing that it confers responsiveness to IAA (in this case, inhibition) would be very convincing.

Reviewer #3 (Public review):

Summary:

In this work, Rossi et al. use a novel split-belt treadmill learning task to reveal distinct sub-components of gait adaptation. The task involved following a standard adaptation phase with a "ramp-down" phase that helped them dissociate implicit recalibration and more deliberate SR map learning. Combined with modeling and re-analysis of previous studies, the authors show multiple lines of evidence that both processes run simultaneously, with implicit learning saturating based on intrinsic learning constraints and SR learning showing sensitivity to a "perceptual" error. These results offer a parallel with work in reaching adaptation showing both explicit and implicit processes contributing to behavior; however, in the case of gait adaptation the deliberate learning component does not appear to be strategic but is instead a more implicit SR learning processes.

Strengths:

(1) The task design is very clever and the "ramp down" phase offers a novel way to attempt to dissociate competing models of multiple processes in gait adaptation.

(2) The analyses are thorough, as is the re-analysis of multiple previous data sets.

(3) The querying of perception of the different relative belt speeds is a very nice addition, allowing the authors to connect different learning components with error perception.

(4) The conceptual framework is compelling, highlighting parallels with work in reaching but also emphasizing differences, especially w/r/t SR learning versus strategic behaviors. Thus the discovery of an SR learning process in gait adaptation would be both novel and also help conjoin different siloed subfields of motor learning research.

Weaknesses:

(1) The behavior in the ramp-down phase does indeed appear to support multiple learning processes. However, I may have missed something, but I have a fundamental worry about the specific modeling and framing of the "SR" learning process. If I correctly understand, the SR process learns by adjusting to perceived L/R belt speed differences (Figure 7). What is bugging me is why that process would not cause the SR system to still learn something in the later parts of the ramp-down phase when the perceived speed differences flip (Figure 4). I do believe this "blunted learning" is what the SR component is actually modeled with, given this quote in the caption to Figure 7: "When the perturbation is perceived to be opposite than adaptation, even if it is not, mapping is zero and the Δ motor output is constant, reflecting recalibration adjustments only." It seems a priori odd and perhaps a little arbitrary to me that a SR learning system would just stop working (go to zero) just because the perception flipped sign. Or for that matter "generalize" to a ramp-up (i.e., just learn a new SR mapping just like the system did at the beginning of the first perturbation). What am I missing that justifies this key assumption? Or is the model doing something else? (if so that should be more clearly described).

(2) A more minor point, but given the sample size it is hard to be convinced about the individual difference analysis for structure learning (Figure 5). How clear is it that these two groups of subjects are fully separable and not on a continuum? The lack of clusters in another data set seems like a somewhat less than convincing control here.

Reviewer #3 (Public Review):

Hüttemeister et. al. describe a study where researchers utilized a genetic modification technique to knockin a red fluorescence protein variant mCherry into titin, a giant muscle protein, at the Z-disk in order to investigate skeletal muscle development and remodeling. The study revealed that titin's integration into the sarcomere is tightly regulated during muscle development, and its mobility allows for a homogeneous distribution of titin after cell fusion, which is crucial for syncytium formation and skeletal muscle maturation. Furthermore, in adult mice with mCherry-tagged titin, the researchers observed the process of muscle injury treatment by implanting myoblasts containing titin tagged with another fluorescent protein, eGFP. This experiment provided insights into how myocytes integrate, fuse, and contribute to the continuous myofilament system across cell boundaries during muscle regeneration. Interestingly, the behavior of titin proteins differed between immature primary cells and adult muscle tissue. The manuscripts point our interesting observation that develop treatment protocols that target the early postnatal patient or consider in utero cell therapy approaches based on controlling the ratio of therapeutic to diseased cells. though the approach is very interesting, the paper is very qualitative in its approaches. Community will benefit from better quantification of data as most of them are microscopic data that requires quantification.

Reviewer #3 (Public Review):

Fu et al. present a multi-model study using goose and mouse that investigates the protective effects of Lactobacillus plantarum against hyperuricaemia. They highlight this strain's significance and clarify its role in responding to intestinal nucleoside levels and affecting uric acid metabolism through modulation of host signaling pathways.

Strengths:<br /> (1) Fu et al. created two animal models for validation, yielding more reliable and extensive data. In addition, the in vitro tests were repeatedly tested by a multitude of methods, proving to be convincing.<br /> (2) This study integrates microbiomics, whole genomics, in vitro bacterial culture, and metabolomics, providing a wealth of data and valuable insights for future research.

Weakness:<br /> Fu et al. clearly described the role of Lactobacillus plantarum, but it is also important to explore its other mechanisms influencing uric acid metabolism in the host. While changes in hepatic and renal uric acid metabolism were confirmed, the gut's role in this process deserves investigation, particularly regarding whether Lactobacillus plantarum or its metabolites act within the gut. The authors have effectively conveyed the story outlined in the article's title, and the remainder can be explored later. In addition, further discussion is needed to highlight how this strain of Lactobacillus plantarum differs from other Lactobacillus strains or how it innovatively functions differ from some literature reported.

Reviewer #3 (Public review):

Summary:

In this manuscript, Devakinandan and colleagues have undertaken a thorough characterization of the cell types of the mouse vomeronasal organ, focusing on the vomeronasal sensory neurons (VSNs). VSNs are known to arise from a common pool of progenitors that differentiate into two distinct populations characterized by the expression of either the G protein subunit Gnao1 or Gnai2. Using single-cell RNA sequencing followed by unsupervised clustering of the transcriptome data, the authors identified three Gnai2+ VSN subtypes and a single Gnao1+ VSN type. To study VSN developmental trajectories, Devakinandan and colleagues took advantage of the constant renewal of the neuronal VSN pool, which allowed them to harvest all maturation states. All neurons were re-clustered and a pseudotime analysis was performed. The analysis revealed the emergence of two pools of Gap43+ clusters from a common lineage, which differentiate into many subclusters of mature Gnao1+ and Gnai2+ VSNs. By comparing the transcriptomes of these two pools of immature VSNs, the authors identified a number of differentially expressed transcription factors in addition to known markers. Next, by comparing the transcriptomes of mature Gnao1+ and Gnai2+ VSNs, the authors report an enrichment of ER-related genes in Gnao1+ VSNs. Using electron microscopy, they found that this enrichment was associated with specific ER morphology in Gnao1+ neurons. Finally, the authors characterized chemosensory receptor expression and co-expression (as well as H2-Mv proteins) in mature VSNs, which recapitulated known patterns.

Strengths:

The data presented here provide new and interesting perspectives on the distinguishing features between Gnao1+ and Gnai2+ VSNs. These features include newly identified markers, such as transcription factors, as well as an unsuspected ER-related peculiarity in Gnao1+ neurons, consisting in a hypertrophic ER and an enrichment in ER-related genes. In addition, the authors provide a comprehensive picture of specific co-expression patterns of V2R chemoreceptors and H2-Mv genes.

Importantly, the authors provide a browser (scVNOexplorer) for anyone to explore the data, including gene expression and co-expression, number and proportion of cells, with a variety of graphical tools (violin plots, feature plots, dot plots, ...).

Reviewer #3 (Public review):

Summary:

Understanding the mechanisms whereby animals restrict the timing of their reproduction according to day length is a critical challenge given that many of the most relevant species for agriculture are strongly photoperiodic. However, the principal animal models capable of detailed genetic analysis do not respond to photoperiod so this has inevitably limited progress in this field. The fish model medaka occupies a uniquely powerful position since its reproduction is strictly restricted to long days and it also offers a wide range of genetic tools for exploring, in depth, various molecular and cellular control mechanisms.

For these reasons, this manuscript by Tagui and colleagues is particularly valuable. It uses the medaka to explore links bridging photoperiod, feeding behaviour, and reproduction. The authors demonstrate that in female, but not male medaka, photoperiod-induced reproduction is associated with an increase in feeding, presumably explained by the high metabolic cost of producing eggs on a daily basis during the reproductive period. Using RNAseq analysis of the brain, they reveal that the expression of the neuropeptides agrp and npy that have been previously implicated in the regulation of feeding behaviour in mice are upregulated in the medaka brain during exposure to long photoperiod conditions. Unlike the situation in mice, these two neuropeptides are not co-expressed in medaka neurons, and food deprivation in medaka led to increases in agrp but also a decrease in npy expression. Furthermore, the situation in fish may be more complicated than in mice due to the presence of multiple gene paralogs for each neuropeptide. Exposure to long-day conditions increases agrp1 expression in medaka as the result of increases in the number of neurons expressing this neuropeptide, while the increase in npyb levels results from increased levels of expression in the same population of cells. Using ovariectomized medaka and in situ hybridization assays, the authors reveal that the regulation of agrp1 involves estrogen acting via the estrogen receptor esr2a. Finally, a loss of agrp1 function mutant is generated where the female mutants fail to show the characteristic increase in feeding associated with long-day enhanced reproduction as well as yielding reduced numbers of eggs during spawning.

Strengths:

This manuscript provides important foundational work for future investigations aiming to elucidate the coordination of photoperiod sensing, feeding activity, and reproduction function. The authors have used a combination of approaches with a genetic model that is particularly well suited to studying photoperiodic-dependent physiology and behaviour. The data are clear and the results are convincing and support the main conclusions drawn. The findings are relevant not only for understanding photopriodic responses but also provide more general insight into links between reproduction and feeding behaviour control.

Weaknesses:

Some experimental models used in this study, namely ovariectomized female fish and juvenile fish have not been analysed in terms of their feeding behaviour and so do not give a complete view of the position of this feeding regulatory mechanism in the context of reproduction status. Furthermore, the scope of the discussion section should be expanded to speculate on the functional significance of linking feeding behaviour control with reproductive function.

Reviewer #3 (Public review):

Summary:

In the manuscript entitled "Human Brain Barcodes", the author sought to use single-cell CpG methylation information to trace cell lineages in the human brain.

Strengths:

Tracing cell lineages in the human brain is important but technically challenging. Lineage tracing with single-cell CpG methylation would be interesting if convincing evidence exists.

Weaknesses:

As the author noted, "DNA methylation patterns are usually copied between cell division, but the replication errors are much higher compared to base replication". This unstable nature of CpG methylation would introduce significant problems in inferring the true cell lineage. The unreliable CpG methylation status also raises the question of what the "Barcodes" refer to in the title and across this study. Barcodes should be stable in principle and not dynamic across cell generations, as defined in Reference#1. It is not convincing that the "dynamic" CpG methylation fits the "barcodes" terminology. This problem is even more concerning in the last section of results, where CpG would fluctuate in post-mitotic cells.

Reviewer #3 (Public review):

Summary:

The goal of the paper is to examine the objective function of total reward rate in an environment to understand the behavior of humans and animals in two types of decision-making tasks: (1) stay/forgo decisions and (2) simultaneous choice decisions. The main aims are to reframe the equation of optimizing this normative objective into forms that are used by other models in the literature like subjective value and temporally discounted reward. One important contribution of the paper is the use of this theoretical analysis to explain apparent behavioral inconsistencies between forgo and choice decisions observed in the literature.

Strengths:

The paper provides a nice way to mathematically derive different theories of human and animal behavior from a normative objective of global reward rate optimization. As such, this work has value in trying to provide a unifying framework for seemingly contradictory empirical observations in literature, such as differentially optimal behaviors in stay-forgo v/s choice decision tasks. The section about temporal discounting is particularly well motivated as it serves as another plank in the bridge between ecological and economic theories of decision-making.

Weaknesses:

One broad issue with the paper is readability. Admittedly, this is a complicated analysis involving many equations that are important to grasp to follow the analyses that subsequently build on top of previous analyses.

But, what's missing is intuitive interpretations behind some of the terms introduced, especially the apportionment cost without referencing the equations in the definition so the reader gets a sense of how the decision-maker thinks of this time cost in contrast with the opportunity cost of time.

Re-analysis of some existing empirical data through the lens of their presented objective functions, especially later when they describe sources of error in behavior.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors used structural biology approaches to determine the molecular mechanism underlying the inactivation of the PIEZO1 ion channel. To this end, the authors presented structures of human PIEZO1 and its slow-inactivating mutants. The authors also determined the structures of these PIEZO1 constructs in complexes with the auxiliary subunit MDFIC, which substantially slows down PIEZO1 inactivation. From these structures, the authors suggested an anti-correlation between the inactivation kinetics and the resting curvature of PIEZO1 in detergent. The authors also observed a unique feature of human PIEZO1 in which the lipid molecules plugged the channel pore. The authors proposed that these lipid molecules could stabilize human PIEZO1 in a prolonged inactivated state.

Strengths:

Notedly, this manuscript reported the first structures of a human PIEZO1 channel, its channelopathy mutants, and their complexes with MDFIC. The evidence that lipid molecules could occupy the channel pore of human PIEZO1 is solid. The authors' proposals to correlate PIEZO1 resting curvature and pore-resident lipid molecules with the inactivation kinetics are novel and interesting.

Weaknesses:

However, in my opinion, additional evidence is needed to support the authors' proposals.

(1) The authors determined the apo structure of human PIEZO1, which showed a more flattened architecture than that of the mouse PIEZO1. Functionally, the inactivation kinetics of human PIEZO1 is faster than its mouse counterpart. From this observation (and some subsequent observations such as the complex with MDFIC), the authors proposed the anti-correlation between curvature and inactivation kinetics. However, the comparison between human and mouse PIEZO1 structure might not be justified. For example, the human and mouse structures were determined in different detergent environments, and the choice of detergent could influence the resting curvature of the PIEZO structures.

(2) Related to point 1), the 3.7 Å structure of the A1988V mutant presented by the authors showed a similar curvature as the WT but has a slower inactivating kinetics.

(3) Related to point 1), the authors stated that human PIEZO1 might not share the same mechanism as mouse PIEZO1 due to its unique properties. For example, MDFIC only modifies the curvature of human PIEZO1, and lipid molecules were only observed in the pore of the human PIEZO1. Therefore, it may not be justified to draw any conclusions by comparing the structures of PIEZO1 from humans and mice.

(4) Related to point 1), it is well established that PIEZO1 opening is associated with a flattened structure. If the authors' proposal were true, in which a more flattened structure led to faster inactivation, we would have the following prediction: more opening is associated with faster inactivation. In this case, we would expect a pressure-dependent increase in the inactivation kinetics. Could the authors provide such evidence, or provide other evidence along this direction?

(5) In Figure S2, the authors showed representative experiments of the inactivation kinetics of PIEZO1 using whole-cell poking. However, poking experiments have high cell-to-cell variability. The authors should also show statics of experiments obtained from multiple cells.

(6) In Figure 2 and Figure 5, when the authors show the pore diameter, it could be helpful to also show the side chain densities of the pore lining residues.

(7) The authors observed pore-plugging lipids in slow inactivating conditions such as channelopathy mutations or in complex with MDFIC. The authors propose that these lipid molecules stabilize a "deep resting state" of PIEZO1, making it harder to open and harder to inactivate once opened. This will lead to the prediction that the slow-inactivating conditions will lead to a higher activation threshold, such as the mid-point pressure in the activation curve. Is this true?

Reviewing Editor (Public Review):

Summary:

In this well-written paper, a pharmacological experiment is described in which a large group of volunteers is tested on a novel probabilistic reversal learning task with different levels of noise, once after intake of methamphetamine and once after intake of placebo. The design includes a separate baseline session, during which performance is measured. The key result is that drug effects on learning rate variability depend on performance in this separate baseline session.

The approach and research question are important, the results will have an impact, and the study is executed according to current standards in the field. Strengths include the interventional pharmacological design, the large sample size, the computational modeling, and the use of a reversal-learning task with different levels of noise.

(i) One novel and valuable feature of the task is the variation of noise (having 70-30 and 80-20 conditions). This nice feature is currently not fully exploited in the modeling of the task and the data. For example, recently reported new modeling approaches for disentangling two types of uncertainty (stochasticity vs volatility) could be usefully leveraged here (by Piray and Daw, 2021, Nat Comm). The current 'signal to noise ratio' analysis that is targeting this issue relies on separately assessing learning rates on true reversals and learning rates after misleading feedback, in a way that is experimenter-driven. As a result, this analysis cannot capture a latent characteristic of the subject's computational capacity.

(ii) An important caveat is that all the drug x baseline performance interactions, including for the key computational eta parameter did not reach the statistical threshold, and only tended towards significance.

(iii) Both the overlap and the differences between the current study and previous relevant work (that is, how this goes beyond prior studies in particular Rostami Kandroodi et al, which also assessed effects of catecholaminergic drug administration as a function of baseline task performance using a probabilistic reversal learning task) are not made explicit, particularly in the introduction.

(iv) In the discussion, it is stated that the existing literature has, to date, overlooked baseline performance effects, but this is not true in the general sense, given that an accumulating number of studies have shown that the effects of drugs like MA depend on baseline performance on working memory tasks, which often but certainly not always correlates positively with performance on the task under study.

Reviewer #3 (Public review):

Summary of the Study:

The authors investigate the organization of the human pulvinar by analyzing DWI, fMRI, and PET data. The authors explore the hypothesis of the "replication principle" in the pulvinar.

Strengths and Weaknesses of the Methods and Results:

The study effectively integrates diverse imaging modalities to provide a view of the pulvinar's organization. The use of analysis techniques, such as diffusion embedding-driven gradients combined with detailed interpretations of the pulvinar, is a strength.

Even though the study uses the best publicly available resolution possible with current MR-technology, the pulvinar is densely packed with many cell bodies, requiring even higher spatial resolution. In addition, the model order selection of gradients may vary with the acquired data quality. Therefore, the pulvinar's intricate organization needs further exploration with even higher spatial resolution to capture gradients closer to the biological organization of the pulvinar.

Appraisal of the Study's Aims and Conclusions:

The authors delineate the gradient organization of the pulvinar. The study provides a basis for understanding the pulvinar's role in mediating brain network communication.

Impact and Utility of the Work:

This work contributes to the field by offering insights into pulvinar organization.

Reviewer #3 (Public review):

The current study examined 13 monosomic yeast strains that lost different individual chromosomes. By comparing the fitness of monosomic strains and several heterozygous deletion strains, the authors observed strong positive epistasis for fitness. The transcriptomes of monosomic strains indicated that general gene-dose compensation is not the reason for fitness gains. On the other hand, gene expression of ribosomal proteins was up-regulated and proteasome subunit expression was down-regulated in all tested monosomic strains. The authors speculated that overexpression in combination with decreased degradation of the insufficient proteins might explain the positive epistasis observed in monosomic strains. This study investigates an important biological question and has some interesting results. However, I have some reservations about the data interpretations listed below.

(1) In Figure 3b (and line 179), the authors stated that those haploinsufficient genes were not transcribed at elevated rates, but almost half of them are in reddish colors (indicating that the expression is higher than 1-fold). Obviously, many haploinsufficient genes are up-regulated in monosomic strains. What the data really show is that the level of overexpression is not correlated with the fitness effect of the deletion (since all the p values are not significant). The authors need to correct their conclusions.<br /> (2) Why are some monosomic strains removed from the transcriptomics analysis, especially when the chromosome IV and XV strains show very strong positive epistasis? The authors need to provide an explanation here.<br /> (3) The authors stated that diploidy observed in chromosome VII and XIII strains were due to endoreplication after losing the marked chromosomes (lines 97 and 117). Isn't chromosome missegregation an equally possible explanation? Since monosomic cells are generated by chromosome missegregation during mitosis, another chromosome missegregation event may occur to rescue the fitness (or viability) of monosomic cells in these strains.

Comments for the revised version:

The authors have addressed all my previous concerns and I have no further questions.

Reviewer #3 (Public review):

Summary:

Yang et al reported in this paper that TGF-beta induces SIRT4 activation, TGF-beta activated SIRT4 then modulates U2AF2 alternative splicing, U2AF2 in turn causes CCN2 for expression. The mechanism is described as this: mitochondrial SIRT4 transport into the cytoplasm in response to TGF-β stimulation, phosphorylated by ERK in the cytoplasm, and pathway and then undergo nuclear translocation by forming the complex with importin α1. In the nucleus, SIRT4 can then deacetylate U2AF2 at K413 to facilitate the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Moreover, they used exosomes to deliver Sirt4 antibodies to mitigate renal fibrosis in a mouse model. TGF-beta has been widely reported for its role in fibrosis induction.

Strengths:

TGF-beta induction of SIRT4 translocation from mitochondria to nuclei for epigenetics or gene regulation remains largely unknown. The findings presented here that SIRT4 is involved in U2AF2 deacetylation and CCN2 expression are interesting.

Comments on revised version:

I went through the revised manuscript and the letter from the authors. I have no further concerns.

Reviewer #3 (Public review):

Summary:

Regulated metabolism has only recently been recognized as a key component of cancer biology, and even more recently recognized as a significant modulator of the tumor microenvironment (TME). TAMs in the TME play a major role in supporting cancer cell survival and growth/spread, as well as generating an immunosuppressive ME to suppress anti-tumor immunity. Specific regulation of lipid metabolism in this context, in particular how lipids are stored and subsequently mobilized for metabolism, is largely unexplored - especially in the immunological components of the TME.

In this manuscript, the authors build on their previous observations that the fatty acid-binding protein FABP4 plays an important role in macrophage function and that FABP4 expression in tumor associated macrophages (TAM) promotes breast cancer progression. They demonstrate:

(1) Unlike saturated fatty acids (FA), unsaturated FA promotes lipid droplet (LD) accumulation in murine macrophages. LD is the primary intracellular storage depot for FA.

(2) Unsaturated FA activates the FABP4-C/EBPalpha axis to upregulate transcription of the enzymes involved in the synthesis of neutral triacylglycerol (TAG) is an essential step in the formation of the neutral lipid core of LD. It should be noted that the authors speculate that UFA-activated FABP4 translocates to the nucleus to activate PPARgamma, which is known to induce C/EBPalpha expression, but do not directly test the involvement of PPARgamma in this axis.

(3) FABP4 deficiency compromises unsaturated FA-mediated lipid accumulation and utilization in murine macrophages.

(4) FABP4-mediated lipid metabolism in macrophages (TAMS) contributes to breast cancer metastasis, in in-vitro of tumor migration induced by murine macrophages and in correlative studies from human patient breast cancer biopsies.

From these studies, the manuscript concludes that FABP4 plays a pivotal role in mediating lipid droplet formation and lipolysis in TAM, which provides lipids to breast cancer cells that contribute to their growth and metastasis.

These are significant findings, as they provide new insight into the mechanistic regulation of TAM biology via regulation of lipid metabolism, as well as define new biomarkers and potential novel therapeutic targets.

The findings are strong in the studies that mechanistically define the role of FAB4 in lipid accumulation and utilization in murine macrophages. However, evidence is less compelling regarding TAM biology and human breast cancer in 3 main areas:

First, while there is clear in vitro evidence that co-cultured murine macrophages genetically deficient in FABP4 (or their conditioned media) do not enhance breast cancer cell motility and invasion, these macrophages are not bonafide TAM - which may have different biology. The use of actual TAM in these experiments would be more compelling. Perhaps more importantly, there is no in vivo data in tumor-bearing mice that macrophage deficiency of FABP4 affects tumor growth or metastasis - which are doable experiments given the availability of the FABP4 KO mice.

Second, no data is presented that the mechanisms/biology that are elegantly demonstrated in the murine macrophages also occur in human macrophages - which would be foundational to translating these findings into human breast cancer. It seems like straightforward in vitro studies in human monocytes/macrophages could be done to recapitulate the main characteristics seen in the murine macrophages.

Third, while the data from the human breast cancer specimens is very intriguing, it is difficult to ascertain how accurate IHC is in determining that the CD163+ cells (TAM) are in fact the same cells expressing FABP4 - which is the central premise of these studies. Demonstrating that IHC has the resolution to do this would be important. Additionally, the in vitro characterization of FABP4 expression in human macrophages would also add strength to these findings.

In summary, the strengths of this manuscript are the significance of metabolic regulation of the immune tumor microenvironment (TME), and the careful mechanistic delineation of FABP4 involvement in mediating lipid droplet formation and lipolysis in murine macrophages. The weaknesses of the work are the lack of direct experimental evidence that human macrophages behave in the same way as murine macrophages, the incomplete characterization of the role of FABP4 expression in TAM in modulating tumor growth in vivo (in murine models), and whether it can be definitively determined that FABP4 is being primarily expressed in the CD163+ macrophages in human breast cancer samples.

Strengths:

(1) Regulated metabolism has only recently been recognized as a key component of cancer biology, and even more recently recognized as a significant modulator of the tumor microenvironment (TME). TAMs in the TME play a major role in supporting cancer cell survival and growth/spread, as well as generating an immunosuppressive ME to suppress anti-tumor immunity.

(2) Regulation of lipid metabolism in this context is largely unexplored, especially in the immunological components of the TME.

(3) The work builds on the authors' previous work on the role of FABP4 plays an important role in macrophage function including FABP4 expression in TAM promotes breast cancer progression (Hao et al, Cancer Res 2018). This paper identified FABP4-expressing macrophages as being pro-tumorigenic via upregulation of IL-6STAT3 signaling.

(4) The careful and thorough mechanistic delineation of FABP4 involvement in mediating lipid droplet formation and lipolysis in murine macrophages.

(5) The intriguing observations that FABP4-mediated lipid metabolism in macrophages contributes to breast cancer metastasis, in in vitro of tumor migration induced by murine macrophages and in correlative studies from human patient breast cancer biopsies that CD163+ cell numbers (putatively TAM) and FABP4 expression was associated with increased metastatic disease and poor overall survival.

(6) Identification of FABP4 both a prognostic biomarker and a potential therapeutic target to modulate the pro-tumor immune TME.

Weaknesses:

(1) While the authors speculate that UFA-activated FABP4 translocates to the nucleus to activate PPARgamma, which is known to induce C/EBPalpha expression, they do not directly test involvement of PPARgamma in this axis.

(2) While there is clear in vitro evidence that co-cultured murine macrophages genetically deficient in FABP4 (or their conditioned media) do not enhance breast cancer cell motility and invasion, these macrophages are not bonafide TAM - which may have different biology. Use of actual TAM in these experiments would be more compelling. Perhaps more importantly, there is no in vivo data in tumor bearing mice that macrophage-deficiency of FABP4 affects tumor growth or metastasis.

(3) Related to this, the authors find FABP4 in the media and propose that macrophage secreted FABP4 is mediating the tumor migration - but don't do antibody neutralizing experiments to directly demonstrate this.

(4) No data is presented that the mechanisms/biology that are elegantly demonstrated in the murine macrophages also occurs in human macrophages - which would be foundational to translating these findings into human breast cancer.

(5) While the data from the human breast cancer specimens is very intriguing, it is difficult to ascertain how accurate IHC is in determining that the CD163+ cells (TAM) are in fact the same cells expressing FABP4 - which is central premise of these studies. Demonstration that IHC has the resolution to do this would be important. Additionally, the in vitro characterization of FABP4 expression in human macrophages would also add strength to these findings.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Weaknesses:

(1) At the beginning of the results section, the authors say "We have expected lipids as ligands". It is not quite clear why these could not have been other substances. It is because they were expected to bind in the lipophilic membrane anchors? Various lipophilic and hydrophilic ligands are known for GPCR which also have transmembrane domains. Maybe 1-2 additional sentences could be helpful here.

(2) In stably transfected HEK cells expressing mAC3 or mAC5, they have used only one dose of isoproterenol (2.5 uM) for submaximal AC activation. The reference 28 provided here (PMID: 33208818) did not specifically look at Iso and endogenous beta2 adrenergic receptors expressed in HEK cells. As far as I remember from the old pharmacological literature, this concentration is indeed submaximal in receptor binding assays but regarding AC activity and cAMP generation (which happen after signal amplification with a so-called receptor reserve), lower Iso amounts would be submaximal. When we measure cAMP, these are rather 10 to 100 nM but no more than 1 uM at which concentration response dependencies usually saturate. Have the authors tried lower Iso concentrations to prestimulate intracellular cAMP formation? I am asking this because, with lower Iso prestimulation, the subsequent stimulatory effects of AC ligands could be even greater.

(3) The authors refer to HEK cell models as "in vivo". I agree that these are intact cells and an important model to start with. It would be very nice to see the effects of the new ligands in other physiologically relevant types of cells, and how they modulate cAMP production under even more physiological conditions. Probably, this is a topic for follow-up studies.

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

The authors have achieved their aims to a very high degree, their results do nicely support their conclusions. There is only one point (various classical GPCR concentrations, please see above) that would be beneficial to address.

Without any doubt, this is a groundbreaking study that will have profound implications in the field for the next years/decades. Since it is now clear that mammalian adenylyl cyclases are receptors for aliphatic fatty acids and anandamide, this will change our view on the whole signaling pathway and initiate many new studies looking at the biological function and pathophysiological implications of this mechanism. The manuscript is outstanding.

Reviewer #3 (Public review):

Summary:

In this study the authors use the zebrafish model and in vitro co-cultures with human cell lines, to study how keratinocytes modulate the early stages of melanoma development/migration. The authors demonstrate that keratinocytes undergo an EMT-like transformation in the presence of melanoma cells which leads to a reduction in melanoma cell migration. This EMT transformation occurs via Twist; and resulted in an improvement in OS in zebrafish melanoma models. Authors suggest that the limitation of melanoma cell migration by Twist-overexpressing keratinocytes was through altered cell-cell interactions (Jam3b) that caused a physical blockage of melanoma cell migration.

Strengths:

The authors describe a new cross-talk between melanoma and its major initial microenvironment: the keratinocytes and how instructed by melanoma cells keratinocytes undergo an EMT transformation, which then controls melanoma migration.

Overall, the paper is very well written, and the results are clearly organized and presented.

Weaknesses:

(1) To really show their last point it would be important to CRISPR KO Jam3b in melanoma with twist OE keratinocytes, in vivo or in vitro.

(2) The use of patient biopsies from early-stage melanomas vs healthy tissue to assess if there is a similar alteration of morphology of adjacent keratinocytes and an increase in vimentin in human samples would strengthen the author's findings.

(3) The cell-cell junctions and borders between cells (melanoma/ keratinocytes) should be characterized better, with cellular and sub-cellular resolution. Since melanocytes can "touch" with their dendrites ~40 keratinocytes - can authors expand and explain better their model? Can this explain that in some images we cannot observe a direct interface between the cells?

Reviewer #3 (Public review):

In this study, the authors employed the protein complex structure prediction tool AlphaFold-Multimer to obtain a predicted structure of the protein complex composed of ULK1-ATG13-FIP200 and validated the structure using mutational analysis. This complex plays a central role in the initiation of autophagy in mammals. Previous attempts at resolving its structure have failed to obtain high-resolution structures that can reveal atomic details of the interactions within the complex. The results obtained in this study reveal extensive binary interactions between ULK1 and ATG13, between ULK1 and FIP200, and between ATG13 and FIP200, and pinpoint the critical residues at each interaction interface. Mutating these critical residues led to the loss of binary interactions. Interestingly, the authors showed that the ATG13-ULK1 interaction and the ATG13-FIP200 interaction are partially redundant for maintaining the complex.

The experimental data presented by the authors are of high quality and convincing. However, given the core importance of the AlphaFold-Multimer prediction for this study, I recommend the authors improve the presentation and documentation related to the prediction, including the following:

(1) I suggest the authors consider depositing the predicted structure to a database (e.g. ModelArchive) so that it can be accessed by the readers.

(2) I suggest the authors provide more details on the prediction, including explaining why they chose to use the 1:1:2 stoichiometry for ULK1-ATG13-FIP200 and whether they have tried other stoichiometries, and explaining why they chose to use the specific fragments of the three proteins and whether they have used other fragments.

(3) I suggest the authors present the PAE plot generated by AlphaFold-Multimer in Figure S1. The PAE plot provides valuable information on the prediction.

Reviewer #3 (Public review):

In this study, Bison et al. analyzed the role of the GATA6 transcription factor in patterning the early mesoderm and generating cardiomyocytes, using human embryonic stem cell differentiation assays and patient-derived hiPSCs with heart defects associated with mutations in the GATA6 gene. They identified a novel role for GATA6 in regulating genes involved in the WNT and BMP pathways -findings not previously noted in earlier analyses of GATA6 mutant hiPSCs during early cardiac mesoderm specification (Sharma et al., 2020). Modulation of the WNT and BMP pathways may partially rescue early cardiac mesoderm defects in GATA6 mutant hESCs. These results provide significant insights into how GATA6 loss-of-function and heterozygous mutations contribute to heart defects.

I have the following comments:

(1) Throughout the manuscript, Bison et al. alternate between different protocols to generate cardiomyocytes, which creates some confusion (e.g., Figure 1 vs. Supplemental Figure 2A). The authors should provide a clear justification for using alternative protocols.

(2) The authors should characterise the mesodermal identity and cardiomyocyte subtypes generated with the activin/BMP-induction protocol thoroughly and clarify whether defects in the expression of BMP and WNT-related gene affect the formation of specific cardiomyocyte subtypes in a chamber-specific manner. This analysis is important, as Sharma et al. suggested a role for GATA6 in orchestrating outflow tract formation, and Bison et al. similarly identified decreased expression of NRP1, a gene involved in outflow tract septation, in their GATA6 mutant cells.

(3) The authors developed an iPSC line derived from a congenital heart disease (CHD) patient with an atrial septal defect and observed that these cells generate cTnnT+ cells less efficiently. However, it remains unclear whether atrial cardiomyocytes (or those localised specifically at the septum) are being generated using the activin/BMP-induction protocol and the patient-derived iPSC line.

(4) The authors should also justify the necessity of using the patient-derived line to further analyse GATA6 function.

(5) Figure 3 suggests an enrichment of paraxial mesoderm genes in the context of GATA6 loss-of-function, which is intriguing given the well-established role of GATA6 in specifying cardiac versus pharyngeal mesoderm lineages in model organisms. Could the authors expand their analysis beyond GO term enrichment to explore which alternative fates GATA6 mutant cells may acquire? Additionally, how does the potential enrichment of paraxial mesoderm, rather than pharyngeal mesoderm, relate to the initial mesodermal induction from their differentiation protocol? Could the authors also rule out the possibility of increased neuronal cell fates?

Reviewer #3 (Public review):

Summary:

Day et al. introduced high-throughput expansion microscopy (HiExM), a method facilitating the simultaneous adaptation of expansion microscopy for cells cultured in a 96-well plate format. The distinctive features of this method include: 1) the use of a specialized device for delivering a minimal amount (~230 nL) of gel solution to each well of a conventional 96-well plate, and 2) the application of the photochemical initiator, Irgacure 2959, to successfully form and expand toroidal gel within each well.

Addition upon revision:

Overall, the authors have adequately addressed most of the concerns raised. There are a few minor issues that require attention.

Minor comments:

Figure S10: There appears to be a discrepancy in the panel labeling. The current labels are E-H, but it is unclear whether panels A-D exist. Also, this reviewer thought that panels G and H would benefit from statistical testing to strengthen the conclusions. As a general rule for scientific graph presentation, the y-axis of all graphs should start at zero unless there is a compelling reason not to do so.

Reviewer #3 (Public Review):

Summary:

The authors set out to devise a system for the neural and behavioral study of socially cooperative behaviors in nonhuman primates (common marmosets). They describe instrumentation to allow for a "cooperative pulling" paradigm, the training process, and how both behavioral and neural data can be collected and analyzed. This is a valuable approach to an important topic, as the marmoset stands as a great platform to study primate social cognition. Given that the goals of such a methods paper are to (a) describe the approach and instrumentation, (b) show the feasibility of use, and (c) quantitatively compare to related approaches, the work is easily able to meet those criteria. My specific feedback on both strengths and weaknesses is therefore relatively limited in scope and depth.

Strengths:

The device is well-described, and the authors should be commended for their efforts in both designing this system but also in "writing it up" so that others can benefit from their R&D.

The device appears to generate more repetitions of key behavior than other approaches used in prior work (with other species).

The device allows for quantitative control and adjustment to control behaviour.

The approach also supports the integration of markerless behavioral analysis as well as neurophysiological data.

Reviewer #3 (Public Review):

Summary:

The work submitted by Dr. Jeong-Oh Shin and co-workers aims to investigate the therapeutic efficacy of rhPTH(1-34) and R25CPTH(1-34) on bone regeneration and osseointegration of titanium implants using a postmenopausal osteoporosis animal model.

In my opinion the findings presented are not strongly supported by the provided data since the methods utilized do not allow to significantly support the primary claims.

Strengths:

Strengths include certain good technologies utilized to perform histological sections (i.e. the EXAKT system).

Weaknesses:

Certain weaknesses continue to significantly lower the enthusiasm for this work. Most important: the limited number of samples/group. In fact, as presented, the work has an n=4 for each treatment group. This limited number of samples/group significantly impairs the statistical power of the study. In addition, the implants were surgically inserted following a "conventional implant surgery", implying that no precise/guided insertion was utilized. This weakness is, in my opinion, particularly significant since the amount of bone osteointegration may greatly depend on the bucco-lingual positioning of each implant at the time of the surgical insertion (which should, therefore, be precisely standardized across all animals and for all surgical procedures).

Reviewer #3 (Public review):

Summary:

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

Strengths

(1) The data clearly show that the repositioned Mcm helicase fires earlier than the Mcm in the wild type position.<br /> (2) The study identifies a specific role for Fun30 in replication timing and an effect on nucleosome occupancy around the newly positioned Mcm helicase in sir2 cells.

Weaknesses

(1) It is unclear which strains were used in each experiment.<br /> (2) The relevance of the fun30 phospho-site mutant (S20AS28A) is unclear.<br /> (3) For some experiments (Figs. 3, 4, 6) it is unclear whether the data are reproducible and the differences significant. Information about the number of independent experiments and quantitation is lacking. This affects the interpretation, as fun30 seems to affect the +3 nucleosome much more than let on in the description.

Reviewer #3 (Public review):

Summary:

In the present manuscript, the authors propose that soluble Uric acid (sUA) is an enzymatic inhibitor of the NADase CD38 and that it controls levels of NAD modulating inflammatory response. Although interesting the studies are at this stage preliminary and validation is needed.

Strengths:

The study characterizes the potential relevance of sUA in NAD metabolism.

Comment on revised version:

The authors have responded the majority of my criticism.

Reviewer #3 (Public review):

The manuscript "Regeneration following tissue necrosis is mediated by non-apoptotic caspase activity" by Klemm et al. is an exploration of what happens to a group of cells that experience caspase activation after necrosis occurs some distance away from the cells of interest. These experiments have been conducted in the Drosophila wing imaginal disc, which has been used extensively to study the response of a developing epithelium to damage and stress. The authors revise and refine their earlier discovery of apoptosis initiated by necrosis, here showing that many of those presumed apoptotic cells do not complete apoptosis. Thus, the most interesting aspect of the paper is the characterization of a group of cells that experience mild caspase activation in response to an unknown signal, followed by some effector caspase activation and DNA damage, but that then recover from the DNA damage, avoid apoptosis, and proliferate instead. Many questions remain unanswered, including the signal that stimulates the mild caspase activation, and the mechanism through which this activation stimulates enhanced proliferation.

The authors should consider answering additional questions, clarifying some points, and making some minor corrections:

Major concerns affecting the interpretation of experimental results:

Expression of STAT92E RNAi had no apparent effect on the ability of hinge cells to undergo NiA, leading the authors to conclude that other protective signals must exist. However, the authors have not shown that this STAT92E RNAi is capable of eliminating JAK/STAT signaling in the hinge under these experimental conditions. Using a reporter for JAK/STAT signaling, such as the STAT-GFP, as a readout would confirm the reduction or elimination of signaling. This confirmation would be necessary to support the negative result as presented.

Similarly, the authors should confirm that the Zfh2 RNAi is reducing or eliminating Zfh2 levels in the hinge under these experimental conditions, before concluding that Zfh2 does not play a role in stopping hinge cells from undergoing NiA.

EdU incorporation was quantified by measuring the fluorescence intensity of the pouch and normalizing it to the fluorescence intensity of the whole disc. However, the images show that EdU fluorescence intensity of other regions of the disc, especially the notum, varied substantially when comparing the different genetic backgrounds (for example, note the substantially reduced EdU in the notum of Figure 3 B' and B'). Indeed, it has been shown that tissue damage can lead to suppression of proliferation in the notum and elsewhere in the disc, unless the signaling that induces the suppression is altered. Therefore, the normalization may be skewing the results because the notum EdU is not consistent across samples, possibly because the damage-induced suppression of proliferation in the notum is different across the different genetic backgrounds.

The authors expressed p35 to attempt to generate "undead cells". They take an absence of mitogen secretion or increased proliferation as evidence that undead cells were not generated. However, there could be undead cells that do not stimulate proliferation non-autonomously, which could be detected by the persistence of caspase activity in cells that do not complete apoptosis. Indeed, expressing p35 and observing sustained effector caspase activation could help answer the later question of what percentage of this cell population would otherwise complete apoptosis (NiA, rescued by p35) vs reverse course and proliferate (NiCP, unaffected by p35).

It is unclear if the authors' model is that the NiCP cells lead to autonomous or non-autonomous cell proliferation, or both. Could the lineage-tracing experiments and/or the experiments marking mitosis relative to caspase activity answer this question?

Many of the conclusions rely on single images. Quantification of many samples should be included wherever possible.

Why does the reduction of Dronc appear to affect regenerative growth in females but not males?

Reviewer #3 (Public review):

The authors developed a useful methodology for generating all combinations of multiple reagents using standard lab equipment. This methodology has clear uses for studying microbial ecology as they demonstrated. The methodology will likely be useful for other types of experiments that require exhaustive testing of all possible combinations of a given set of reagents (e.g., drug-drug antagonism and synergy).

The authors provided a useful R script that generates a detailed experimental protocol for building the desired combination from any number of reagents. The produced document is useful and has clear instructions. The output of the computer script will be strengthened if graphical output is also provided (similar to the one provided in Figure 1C).

The authors show that the error rate of the method doesn't go up with the number of combinations using dyes (Figure 2).

The authors demonstrate the value of their methodology for studying interactions within microbial consortia by assembling all possible combinations of eight strains of Pseudomonas aeruginosa. The value of their methodology for this application is well-founded. However, it is also unclear why specific experimental choices were made for this application. It is unclear why authors continue to show the absorbance measurements of strain assemblies over the entire wavelength spectrum and not just for ABS 600 nm (Figures 3 and 4). It is also unclear why the authors provided information on the "sum of the three spectra" as this reference line is meaningless and not a reasonable null model for estimating how well specific strain combinations will grow together.

Figure 5 illustrates the various analysis types that can be performed on the data collected from growing combinations of eight Pseudomonas aeruginosa strains. It is a very informative figure since it provides a "roadmap" on the various ways in which the dataset produced can be explored. The information in Figures 5 and S6 will likely be very useful for a wide audience.

Reviewer #3 (Public review):

Summary:

Using flies, Kazama et al. combined behavioral analysis, electrophysiological recordings, and calcium imaging experiments to elucidate how odors activate gustatory receptor neurons (GRNs) and elicit a proboscis extension response, which is interpreted as a feeding response.

The authors used DeepLabCut v2.0 to estimate the extension of the proboscis, which represents an unbiased and more precise method for describing this behavior compared to manual scoring.

They demonstrated that the probability of eliciting a proboscis extension increases with higher odor concentrations. The most robust response occurs at a 0.5 v/v concentration, which, despite being diluted in the air stream, remains a relatively high concentration. Although the probability of response is not particularly high it is higher than control stimuli. Notably, flies respond with a proboscis extension to both odors that are considered positive and those regarded as negative.

The authors used various transgenic lines to show that the response is mediated by GRNs. Specifically, inhibiting Gr5a reduces the response, while inhibiting Gr66a increases it in fed flies. Additionally, they find that odors induce a strong positive response in both types of GRNs, which is abolished when the labella of the proboscis are covered. This response was also confirmed through electrophysiological tip recordings.

Finally, the authors demonstrated that the response increases when two stimuli of different modalities, such as sucrose and odors, are presented together, suggesting clear multimodal integration.

Strengths:

The integration of various techniques, that collectively support the robustness of the results.

The assessment of electrophysiological recordings in intact animals, preserving natural physiological conditions.

Weaknesses:

The behavioral response is observed in only a small proportion of animals.

Reviewer #3 (Public review):

Summary:

The study investigates reinforcement learning across the lifespan with a large sample of participants recruited for an online game. It finds that children gradually develop their abilities to learn reward probability, possibly hindered by their immature spatial processing and probabilistic reasoning abilities. Motor noise, reinforcement learning rate, and exploration after a failure all contribute to children's subpar performance.

Strengths:

(1) The paradigm is novel because it requires continuous movement to indicate people's choices, as opposed to discrete actions in previous studies.

(2) A large sample of participants were recruited.

(3) The model-based analysis provides further insights into the development of reinforcement learning ability.

Weaknesses:

(1) The adequacy of model-based analysis is questionable, given the current presentation and some inconsistency in the results.

(2) The task should not be labeled as reinforcement motor learning, as it is not about learning a motor skill or adapting to sensorimotor perturbations. It is a classical reinforcement learning paradigm.

Reviewer #3 (Public review):

Summary:

In this very thorough manuscript, the authors provide further evidence that the lectin-24A gene in Drosophila melanogaster is directly involved in the anti-parasitoid wasp humoral immune reaction.

Strengths:

In this study in particular they use a fluorescent reporter and promoter-bashing to determine how this gene is regulated. They find that JAK/STAT, Pannier, and NF-κB signaling are integral to the regulation of lectin-24A and to the humoral anti-parasitoid immune response. These claims are well supported by the experimental design, results, and analysis.

Weaknesses:

A bit of clarity is needed regarding Figure 4a as well as on the rationale for the lengths of the promoter intervals used.

Reviewer #3 (Public review):

Summary:<br /> The overarching goal of the authors was to understand whether emotional information conveyed through point-light biological motion can trigger automatic physiological responses, as reflected in pupil size.

Strengths:<br /> This manuscript has several noticeable strengths: it addresses an intriguing research question that fills that gap in existing literature, presents a clear and accurate presentation of the current literature, and conducts a series of experiments and control experiments with adequete sample size. Yet, it also entails several noticeable limitations - especially in the study design and statistical analyses.

Assessment of the revision:

The authors have done a thorough job revising the manuscript, effectively addressing all of my previous concerns.

Reviewer #3 (Public review):

Summary:

Das et al. discovered a maternal role for Caspar (Casp), the Drosophila orthologue of human Fas-associated factor-1 (FAF1), in embryonic development and germ cell formation. They find that Casp interacts with Transitional endoplasmic reticulum 94 (TER94). Loss of Casp or TER94 leads to partial embryonic lethality, correlated with aberrant centrosome behavior and cytoskeletal abnormalities. This suggests that Casp, along with TER94, promotes embryonic development through a still unidentified mechanism. They also find that Casp regulates germ cell number by controlling a key determinant of germ cell formation, Oskar, through its negative regulator, Smaug.

Strengths:

Overall, the experiments are well-conducted, and the conclusions of this paper are mostly well-supported by data.

Weaknesses:

Some additional controls could be included, and the language could be clarified for accuracy.

Reviewer #3 (Public review):

Summary:

Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also suggest that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. The work also shows that different VMH populations are activated by fresh and old saliva, demonstrating that these stimuli create qualitatively different neural activity profiles. However, the exact components that differ between fresh and old saliva remain unknown and may be identified in future studies.

Strengths:

(1) Predator saliva is a stimulus of high ethological relevance<br /> (2) The authors performed a careful quantification of fos induction across the anterior-posterior axis<br /> (3) Authors show that different VMH populations are activated by fresh and old saliva

Weaknesses:

(1) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing neural activity of relevant cell bodies and axonal projections

(2) Some of the findings are disconnected from the story. For example, the authors show V2R-A4-expressing cells are activated by predator odors, but the causal role of these cells in generating defensive actions is not shown

Reviewer #3 (Public review):

Summary:

Using a mouse model of Staphylococcus aureus gut colonization, Lejeune et al. demonstrate that the microbiome, immune system, and sex are important contributing factors for whether this important human pathogen persists in the gut. The work begins by describing differential gut clearance of S. aureus in female B6 mice bred at NYU compared to those from Jackson Laboratories (JAX). NYU female mice cleared S. aureus from the gut but NYU male mice and mice of both sexes from JAX exhibited persistent gut colonization. Further experimentation demonstrated that differences between staphylococcal gut clearance in NYU and JAX female mice were attributed to the microbiome. However, NYU male and female mice harbor similar microbiomes, supporting the conclusion that the microbiome cannot account for the observed sex-dependent clearance of S. aureus gut colonization. To identify factors responsible for female clearance of S. aureus, the authors performed RNAseq on intestinal epithelial cells and cells enriched within the lamina propria. This analysis revealed sex-dependent transcriptional responses in both tissues. Genes associated with immune cell function and migration were distinctly expressed between the sexes. To determine which immune cell types contribute to S. aureus clearance Lejeune et al employed genetic and antibody-mediated immune cell depletion. This experiment demonstrated that CD4+ IL17+ cells and neutrophils promote the elimination of S. aureus from the gut. Subsequent experiments, including the use of the 'four core genotype model' were conducted to discern between the roles of sex chromosomes and sex hormones. This work demonstrated that sex-chromosome-linked genes are not responsible for clearance, increasing the likelihood that hormones play a dominant role in controlling S. aureus gut colonization.

Strengths:

A strength of the work is the rigorous experimental design. Appropriate controls were executed and, in most cases, multiple approaches were conducted to strengthen the authors' conclusions. The conclusions are supported by the data.

The following suggestions are offered to improve an already strong piece of scholarship.

Weaknesses:

The correlation between female sex hormones and the elimination of S. aureus from the gut could be further validated by quantifying sex hormones produced in the four core genotype mice in response to colonization. Additionally, and this may not be feasible, but according to the proposed model administering female sex hormones to male mice should decrease colonization. Finally, knowing whether the quantity of IL-17a CD4+ cells change in the OVX mice has the potential to discern whether abundance/migration of the cells or their activation is promoted by female sex hormones.

In the Discussion, the authors highlight previous work establishing a link between immune cells and sex hormone receptors, but whether the estrogen (and progesterone) receptor is differentially expressed in response to S. aureus colonization could be assessed in the RNAseq dataset. Differential expression of known X and Y chromosome-linked genes were discussed but specific sex hormones or sex hormone receptors, like the estrogen receptor, were not. This potential result could be highlighted.

Reviewer #3 (Public review):

Summary:

Avila and colleagues investigate the role of glutamate signaling in the dorsomedial striatum in a treadmill-based task where rats learn to turn or stop their walking based on learning cue-associations that allow them to acquire rewards. Phenotypic variation in Pavlovian conditioned sign and goal-tracking behavior was examined, where behavioral differences in stopping and turning were observed. Glutamate signals in the DMS were recorded during the treadmill task and were related to features of cue-controlled movement, with a stronger relationship seen for goal trackers. Finally, chemogenic inhibition of prelimbic neurons projecting to the DMS (the predicted source of those glutamate signals), preferentially affected cued movement in goal trackers. The authors couch these experiments in the context of cognitive control-attentional mechanisms, movement disorders, and individual differences in cue reactivity.

Strengths:

Overall these studies are interesting and are of general relevance to a number of research questions in neurology and psychiatry. The assessment of the intersection of individual differences in cue-related learning strategies with movement-related questions - in this case, cued turning behavior - is an interesting and understudied question. The link between this work and growing notions of corticostriatal control of action selection makes it timely.

Weaknesses:

The clarity of the manuscript could be improved in several places, including in the graphical visualization of data. It is sometimes difficult to interpret the glutamate results, as presented, in the context of specific behavior, for example.

Reviewer #3 (Public review):

Summary:

Loss of cell attachment to extracellular matrix (ECM) triggers aniokis (a type of programmed cell death), and resistance to aniokis plays a role in cancer development. However, mechanisms underlying anoikis resistance, and the precise role of F-actin, are not fully known.

Here authors describe the formation of a new organelle, giant unilocular vacuole (GUVac), in cells whose F-actin is disrupted during loss of matrix attachment. GUVac formation (diameter >500 nm) resulted from a previously unrecognised macropinocytosis-like process, characterized by inwardly curved micron-sized plasma membrane invaginations, dependent on F-actin depolymerization, septin recruitment and PI(3)P. Finally, the authors show GUVac formation after loss of matrix attachment promotes resistance to anoikis.

From these results, authors conclude that GUVac formation promotes cell survival in environments where F-actin is disrupted and conditions of cell stress.

Strengths:

The manuscript is clear and well-written, figures are all presented at a very high level.

A variety of cutting edge cell biology techniques (eg time-lapse imaging, EM, super-resolution microscopy) are used to study the role of cytoskeleton in GUVac formation, discovering (i) a macropinocytosis-like process dependent on F-actin depolymerisation, SEPT6 recruitment and PI(3)P contributes to GUVac formation, and (ii) GUVac formation is associated with resistance to cell death.

Experimental work was advanced in response to reviewers' comments, improving the manuscript message and mechanistic advance.

Weaknesses:

The manuscript is highly reliant on the use of drugs, or combinations of drugs, for long periods of time (6hr, 18hr). However, in the revised manuscript, authors test conclusions drawn from experiments involving drugs using other canonical cell biology approaches.

The molecular characterisation of GUVacs has been advanced, although not fully resolved.

The authors show (mostly using pharmacological inhibition) that F-actin is key for GUVac formation. The precise role of F-actin / GUVac formation in anoikis resistance will be the focus of future work.

Reviewer #3 (Public review):

Abidi et al. investigated the role of Notch signalling for sebaceous gland differentiation and sebocyte progenitor proliferation in adult mouse skin. By injecting antagonising antibodies against different Notch receptors and ligands into mice, the authors identified that the Notch1 receptor and, to a lesser extent, Notch2 receptor, as well as the Notch ligand Jagged2, contribute to the regulation of sebaceous gland differentiation. In situ hybridisation confirmed that treatment with anti-Jagged2 dramatically reduced the number of basal sebocytes staining for the transcriptionally active intracellular domain of Notch1. Loss of Notch activity in sebocyte progenitors robustly inhibited sebaceous gland differentiation. Under these conditions, the number of sebocyte progenitors marked by Lrig1 was not affected, while the number of proliferating basal sebocytes was increased. Upon recovery of Notch activity, sebaceous gland differentiation could likewise be recovered. By suggesting that Notch activity in sebocyte progenitors is required to balance proliferation and differentiation, these data bring valuable new and relevant findings for the skin field on the sebaceous gland homeostasis.