Reviewer #1 (Public Review):

In this work, Roche et al. study a 13-year long time series of microbiome samples from wild baboons from Kenya. The data used in this work challenge a previous finding from the same authors that temporal dynamics in microbiome changes are largely individualized. Using a multinomial logistic-normal modeling approach, the authors detect that co-variance in temporal dynamics in microbial pair-wise associations among individuals occurs more frequently between relatives. Furthermore, the authors identify that microbial phylogenetic proximity is associated with consistent co-abundance changes over time and that their metric of universal microbial relationships is robust across hosts and is detected even in human longitudinal data. The authors conduct a thorough statistical revision of publicly available results, highlighting this time (e.g. compared to Björk et al, doi: 10.1038/s41559-022-01773-4) the consistently shared microbial properties between individuals, rather that the individual microbial signatures highlighted in their previous work.

Strengths:<br /> This work is foundational in its compelling effort to generate a rigorous method to evaluate co-abundance dynamics in longitudinal microbiome data. The approach taken will likely inspire developments that will sharpen the capacity to extract co-varying microbial features, taking into account seasonality, diet, age, relatedness, and more. To the best of my understanding, their hierarchical model integrated into the Gaussian process to analyze microbial dynamics is reasonably robust and they clearly explain the implementation. Furthermore, this work introduces and defines the concept of a universality score for microbial taxon pairs.<br /> Overall, the work presented is clear and convincing and provides tools for the community to benefit from both methods and results. Furthermore, conceptually, this work stresses the value of consistent and shared microbial dynamics in groups, which enriches our understanding of host-associated microbial ecology, otherwise understood to be largely dependent on external fluctuations.

Weakness:<br /> It is not entirely clear the extent to which the presented results revise, refute, or support the previously published analysis performed by the authors on the same dataset (doi: 10.1038/s41559-022-01773-4), which was more focused on individuality.

Reviewer #1 (Public Review):

The synaptonemal complex (SC) is a meiosis-specific tripartite chromosome structure for chromosome synapsis and regulates crossover formation essential for proper chromosome segregation during meiosis. In this interesting paper, the authors studied the dynamic behavior of two components of SC central regions, SYP-2 and SYP-3, in both oocytes and spermatocytes in C. elegans and also the effect of the dosage of the proteins on meiotic recombination and found sex-dimorphic and/or SYP-2/3 dosage sensitive dynamics of the SC components and proteins involved in crossover formation on the meiotic chromosomes, suggesting the intimate relationship between SC central regions and meiotic recombination.

Reviewer #1 (Public Review):

The authors set out to achieve two overarching objectives: 1) to demonstrate that BAFF is a bona fide senescence-associated factor and 2) to expand the field's understanding of senescence outcomes across cell types and models. By inducing senescence in a multitude of ways and across cell culture and animal models, the authors demonstrate that BAFF is robustly upregulated in response to senescence induction. Beyond mere association, by knocking down, overexpressing, or ectopically adding BAFF, the authors demonstrate that various senescence-associated phenotypes can be altered, suggesting that it is an effector of the senescent state. Moreover, by comparing transcriptomic and proteomic profiles in two very different types of cells-diploid WI-38 human fibroblasts and cancerous THP-1 monocytes-the authors identify two parallel trajectories, one involving p53 and one involving NF-kB.

Although trajectory differences may stem from cell type differences, it is possible that the cancerous vs non-cancerous status of the cell lines used may be a more important variable in this case. One question the reader may be left with is: would the two trajectories be different if non-cancerous monocytes with intact p53 were profiled?

Regardless, this study establishes a precedent for characterizing senescence responses in additional cell types of either healthy or diseased origin. Though a number of technical and statistical issues exist in the current version of the manuscript (i.e. use of only a single reference gene for RT-qPCR and inconsistent fold change thresholding in RNA-seq analyses), the results appear robust enough to remain statistically significant after modification. Moreover, many analyses are carried out at both the transcriptomic and proteomic levels with consistent results, highlighting the robustness of their observations.

Ultimately, the results strongly suggest that BAFF plays a senomorphic role in senescence, modulating downstream senescence-associated phenotypes, and may be an interesting candidate for senomorphic therapy.

Reviewer #1 (Public Review):

Cryo-EM structures of respiratory complex I have in recent years have a large impact on our understanding of its mechanism, regulation, assembly, and evolution. However, the coupling mechanism of complex I is still not clear, and controversies exist about whether certain conformations are part of the catalytic cycle or arise from the deactivation of the enzyme. Padavannil and colleagues now add to the story with the first structures of insect complex I, from the model organism Drosophila melanogaster. One of the rationales for choosing this organism is that it lacks the active-to-deactive (A-to-D) transition that prevents the enzyme from going in reverse, which should make the interpretation of any different conformations more straightforward.

The authors showed that the A-D transition seen in mammals and fungi was indeed not present in Drosophila complex I and they determined the cryo-EM structure. In contrast to especially mammalian complex I, which is often found in an "open" and a "closed" state, there was only a single conformation. Drosophila complex I has lost two accessory subunits compared to the mammalian complex, and several other subunits have lost or gained elements, with possible implications for the assembly, stability, or regulation of the complex. The interface of the two peripheral and membrane arms was poorly resolved. A focused classification on this region yielded distinct structures, differing in the angle of the two arms and in the presence or absence of an alpha helix at the N terminus of subunit NDUFS4 (the "lock helix"), a region that is not present in mammalian or yeast complex I. The authors observe a transition between two states named "closed" and "locked open" and speculate that the transition constitutes a deactivation mechanism in insect complex I.

The conclusions of the paper are for the most part solid and supported by the data. Only the interpretation of the significance of the "lock helix" is not convincing: without any evidence, it is assumed to be a regulatory element responsible for an off-pathway deactive state. The nomenclature "closed" and "locked open" is unfortunate, as most of the structural features that differ between the states are reversed compared to the mammalian closed and open states: the disorder of several loops in the quinone binding regions and the presence of absence of a π bulge in helix 4 of the ND6 subunit. Thus, the "locked open" state, which the authors assign as an off-pathway resting state, shares the features of the mammalian closed state, which in all catalysis models is considered an "active" state. An especially important feature in the closed state is the alpha-helical conformation of ND6-helix 4, which has been shown to support a water wire connection from the Q site to the membrane arm, suggesting a role in proton transfer. Conversely, all structures considered as possible D states in mammalian or yeast complex I are open and show disordered loops and a π bulge. These features as shared by the "closed" state of Drosophila, which is however assumed to be on-pathway.

Reviewer #1 (Public Review):

Most previous studies about burn injuries only considered systemic inflammation with analyses of blood specimens from patients. The current study is unique in the fact that it utilizes skin samples. The authors used single-cell analyses by flow cytometry and RNA-seq to characterize in detail the different T-cell populations. The differences are striking. Burned skins have higher degrees of CD69-negative T cells, which indicates that these are recruited from probably blood circulation. They are also substantially more responsive to stimulation by producing higher amounts of immunologic molecules, such as IFNG and TNFA. The results are compelling because they indicate that following burn injury, T cells infiltrate the lesions to potentially protect the damaged tissue from secondary infections.

However, there is an important aspect missing. What does induce T-cell infiltration into the burned skins? A potential explanation is that resident myeloid cells directly or indirectly promote chemokine-mediated recruitment of T cells.

Another important consideration is the impact on other leukocyte populations. While the study is well focused on T cells, the immune system consists of a complex network of cells and molecules that interact with each other. The study does not address myeloid cells and innate lymphoid cells, which could also play important roles and display altered functions in burned injuries.

Nevertheless, the study provides important information about the "activation" statuses of several T cell populations following burned injuries and could help guide the development of better treatments.

Reviewer #1 (Public Review):

The authors studied Eurasian perch in an experimental setup facilitated by a nuclear cooling plant to provide a natural laboratory. The heated area of the ecosystem raised in temperature by 8 degrees centigrade, while a reference area remained unheated. The authors provide a thorough and convincing description that the two areas are segregated such that individuals could not escape from one area to another prior to 2004, and such use data only until 2003 to test their hypotheses. The authors used both length-at-catch and age-increment data in a series of Bayesian mixed effects models to estimate the growth rate and length-at-age. They find that in the warmed area, both younger, smaller fish and older adults grew faster, contrary to the prediction of the temperature-size rule as well as many predictions and observations from other systems that fish reach smaller terminal body sizes in warmer environments due to increased metabolic demands. The authors furthermore combine the estimated body sizes with a mortality rate to determine the size-spectrum slope for both areas and determine the increased growth and increased mortality combine to essentially leave the size-spectrum slope observed in the ecosystem unchanged.

This is a thorough and interesting paper presented clearly and succinctly. These authors present a strong and thorough analysis of how temperature affects growth when all other ecosystem factors remain unchanged in a population. The dataset is a powerful one to support this type of analysis, and the statistical analysis methods the authors used appear to be robust and thorough. The diagnostics and visualizations are complete and inspire confidence in the convergence and accuracy of the modeling approach. The use of the size spectrum exponent to roll up individual-level changes across the population into a single metric was useful and interesting.

The estimates of the von Bertalanffy growth parameters in the results and discussion are less convincing than the growth increment and length-at-age estimates which seem much more robust. The presentation of estimates of the von Bertalanffy growth parameters in Figure S6 exhibit the high negative correlation between the k and L infinity parameters that are typical whenever multiple VBGF models are fit to subsets of data. It is difficult to determine which changes in parameters correspond to actual differences in early vs late life stage growth when, in any given year, if k is estimated low, L infinity will skew high simply due to the model structure. An example of this can be seen in 1995-1997 where L infinity is quite high but k is estimated quite low concurrently - in this case, it seems more reasonable to conclude the likelihood surface is quite flat between different parameter values than that fish suddenly reached a larger asymptotic size in these three years than all of the rest. The data in this case so strongly show larger growth in the heated area even without the VBGF results, and it would be more credible to base the discussion and results of this paper on the growth rate or observed length-at-age (e.g. Figure S4) estimates which are so clear.

Reviewer #1 (Public Review):

Caetano and colleagues describe the changes caused by periodontal inflammation in terms of tissue structure and provide additional evidence to understand the involvement of fibroblasts in altering the immune microenvironment.

While interesting and a concise study, the authors should improve their work on two major points:

1. To improve the resolution, the authors introduced a method that addresses improving the resolution by combining more information from the neighbour structure and the existing database. This raises the question of whether the lack of previous gingival tissue spatial transcriptome sequencing results weakens the reliability of this method. Does it miss the identification of some gingival tissue-specific cells? Is the failure to match two populations of fibroblasts between single-cell sequencing and spatial transcriptome sequencing of gingival tissue fibroblasts related to this?

2. Although the authors did the identification of the captured tissues, the results seem to require more analysis. Take Figure 5A as an example, there is a clear overlap between endothelial cells and basal cells. In addition, it is suggested that the authors indicate the specific location of the 10 clusters of cells in Figures 1D and 2C.

Reviewer #1 (Public Review):

In this manuscript, the authors employed an adult-trained variational autoencoder deep learning model on a relatively large sample (over 700) of human fetal-neonatal resting fMRI data to enhance the individual non-linear compression of functional activity patterns of baby brains. This approach showed better performance in the reconstruction of functional fluctuation maps, age prediction accuracy, and age prediction generalizability in fetal and neonatal fMRI data compared with conventional linear models such as spatial independent component analysis. This method also revealed distinct baby brain functional networks spanning primary and high-level systems.

This is an inspired attempt to represent non-linear changes in fetal-neonatal brain fMRI data. Considering the high noises and inconsistent functional spatial distributions in baby fMRI images, stable and sensitive feature extraction approaches are urgently needed in the field of early brain studies. This work is well designed and well written in general.

Reviewer #1 (Public Review):

By performing immunopeptidomics of macrophages infected with virulent M. tuberculosis, the authors were able to appropriately address whether Mtb proteins are able to enter the MHC-I antigen processing pathway. Their interrogation provides convincing evidence that substrates of Mtb's type VII secretion systems (T7SS) are a significant contributor to the Mtb-derived peptides presented on MHC-I. Compelling data are provided to demonstrate that ESX-1 activity is required for the MHC-1 presentation of these newly identified peptides.

Strength:

Employing a virulent strain of Mtb for infection of human monocyte-derived macrophages to identify Mtb proteins that access the MHC-I antigen processing pathways and the associated mechanisms.

Weakness:

The immunogenicity of at least some of the identified peptides should have been evaluated.

Reviewer #1 (Public Review):

The accessory protein Orf3a from severe acute respiratory syndrome coronavirus (SARS-CoV-1 or SARS-CoV-2) was initially suggested to be a viroporin and function as a cation channel. In this study, Miller et.al performed a comprehensive structural and functional investigation of SARS-CoV-2 Orf3a utilizing a multidisciplinary approach, including extensive electrophysiological analysis using different systems and determination of multiple single-particle EM structures of the protein under different conditions. Their findings demonstrated that Orf3a has no channel function and is unlikely to be a viroporin. In addition, they tried, but failed to record any channel activity of Orf3a claimed in other studies. They demonstrated that large single-channel currents measured from vesicle-reconstituted Orf3a are due to transient membrane leakiness caused by high protein/lipid ratio and/or channel contamination. Furthermore, they found that SARS-CoV-2 Orf3a, but not SARS-CoV-1 Orf3a, interacts with VPS39, a host HOPS protein involved in autophagosome/late endosome fusion with the lysosome. They proposed that the interaction between SARS-CoV-2 Orf3a and VPS39 may function to assist with SARS-CoV-2 exit and host intracellular immune evasion. This is a meticulously executed research work. I appreciate the tremendous effort the authors spent in the study to clarify some misconceptions related to the role and function of Orfsa from coronavirus.

Reviewer #1 (Public Review):

The idea that because the hippocampal code generates responses that match the most needed variable for each task (time or distance) makes it a predictive code is not fully proved with the analyses provided in the manuscript. For example, in the elapsed time task, there are also place cells and in the fixed-distance travel there are also cells that encode other features. This, rather than a predictive code, can be a regular sample of the environment with an overrepresentation of the more salient variable that animals need to get in order to collect rewards. In addition, the analysis provided in the manuscript are rather simple, and better controls could be provided. Improving the analytical quantification of the results is necessary to support the main claim.

- What is the relationship of each type of cell with the speed of the animal?<br /> - What is the relationship with the n of trial that the animal has run (first 10 trials, last 10 trials..)?<br /> - What is the average firing rate of each neuron? Is there any relationship between intrinsic firing rate and the type of coding that the cell develops in each task?<br /> - What is the relation of the units of each type with LFP features (theta phase, ripple recruitment)?

Reviewer #1 (Public Review):

In this manuscript, the authors describe a one-step genome editing method to replace endogenous EB1 with their previously-developed light-sensitive variant, in order to examine the effect of acute and local optogenetic inactivation of EB1 in human neurons. They then attempt to assess the effects of EB1 inactivation on microtubule growth, F-actin dynamics, and growth cone advance and turning. They also perform these experiments in neurons that are lacking EB3, in order to determine whether EB1 can function in a direct and specific way without possible EB3 redundancy.

First, the experiments depicting the methodology are rigorous and compelling. Most previous studies of +TIP function use knockout or knockdown studies in which the proteins are inactivated over many hours or days in non-human systems. This is the first study to acutely and locally inactivate a +TIP in human neurons. While this group previously published the effects of replacing endogenous EB1 with the light-sensitive variant, the novelty in this current study is that they use a one-step gene editing replacement method (using CRISPR/Cas9) along with using human neurons derived from iPSCs. After proving their new experimental system works, the authors next seek to test the effect that acutely inactivating EB1 (alongside chronic EB3 knockdown) has on microtubule dynamics, and they observe a marked reduction in MT growth and MT length. They then seek to investigate whether F-actin dynamics are immediately affected by EB1 inactivation. While measured F-actin flow rates are not significantly affected, which leads the authors to conclude that EB1 inactivation does not have any immediate effect, the included figures and movies show a different phenotype, which is not discussed. Finally, they examine the effect of EB1 inactivation on growth cone advance and growth cone turning, and find that both are affected. However, the lack of certain controls in these final experiments (specifically for Figures 3, 4, and 5) reduces the strength of their findings.

Thus, the first part of this paper describing the new methodology is very compelling and should be of interest to a wide readership, while the second part describing the functional analysis is mostly solid, with very high-quality imaging data. However, additional analysis and controls would be needed to increase confidence in their conclusions.

1) Analysis of F-actin dynamics is not thorough and their claim is not completely supported by the data. Figure 3 only depicts F-actin dynamics data from growth cones of π-EB1 EB3-/- i3Neurons and does not control growth cones (to compare dark and light conditions). While their conclusion is that F-actin dynamics are not affected, there do appear to be immediate changes in the F-actin images, other than flow rates. For example, the F-actin bundles do not appear to emanate straight out with the light condition, compared to the dark condition. There also appears to be more F-actin intensity in the transition domain of the growth cone, compared to the dark condition. If the reason is due to the effects of four minutes of blue light exposure, this would be made clear by doing this experiment with control growth cones as well.

2) Analysis of the effect of EB1 inactivation on growth cone advance and growth cone turning. Figure 4C, showing the neurite unable to cross the blue light barrier, is potentially quite compelling data, but it would be even more convincing if there were also data showing that the blue light barrier has no effect on a control neurite. Given that a number of previous recent studies have shown a detrimental effect of blue light on neurons, it seems important to include these negative controls in this current study.

3) This concern also holds true for the final experiment, in which the authors examine whether localized blue light would lead to growth cone turning. The authors report difficulty with performing this technically challenging experiment of accurately targeting the light to only a localized region of the growth cone. Thus, the majority of the growth cones (72%) were completely retracted, and so only a small subset of growth cones showed turning. However, this data would be more compelling if there were also a control condition of blue light with neurons that are not expressing the light-inactivated EB1. Another useful control would be to examine whether precise region-of-interest blue light leads to localized loss of EGFP-Zdk1-EB1C on MT plus-ends within the growth cone, or if the loss extends throughout the growth cone. Either outcome would be helpful to potential readers.

Reviewer #1 (Public Review):

This study by Noonan et al. explores the role of TGFb signaling in melanoma. TGFb signaling in melanoma and in the tumor microenvironment is complex, acting as both a tumor suppressor and tumor promoter, as well as an immune suppressor. The authors identified a human TGFb-responsive genomic regulatory element that is activated in TGFb-treated melanoma cell lines. This human genomic regulatory element also functions in a zebrafish melanoma model (TIE:EGFP) in specific regions of advanced melanoma. The enhancer region is bound by SMAD2/3, JunB, and ATF3. The proposed model that TIE:EGFP+ melanoma cells are preferentially phagocytosed by macrophages suggests there is some signal specific to this subset of melanoma cells. How this subset of melanoma cells is phagocytosed by macrophages is still poorly understood and will require further investigation. In addition, the authors found that SATB2 overexpression drives the early onset of the TIE:EGFP reporter in melanoma. This novel zebrafish TGFb reporter line has provided unique insights into the dynamic in vivo interactions between melanoma cells and the microenvironment, as well as immune cells. This study will be of interest to researchers looking for novel signaling mechanisms of melanoma progression.

Reviewer #1 (Public Review):

The authors initiated the study motivated by the lack of knowledge about the molecular events downstream of the polarity effector Emx2 in the mammalian inner ear, hypothesizing that some of those molecular players will be found by sequencing cells that normally express Emx2 in ears from Emx2-mutant mice.

The hypothesis is sound, the technologies used are standard and well-established, and the presented data is of high quality. The results largely support the authors' conclusions. However, the authors have not formally demonstrated that Stk32A is a transcriptional target of Emx2. It is clear that it is positioned downstream of the events triggered by Emx2, and that it can reverse Emx2 activity, but the data do not support the claim that the kinase is under direct transcriptional control of Emx2.

The revelation that Stk32A has two separate functions in planar polarity is significant.

The results will have a significant impact in the field because it provides one of the more persuasive molecular links between Emx2 and the polarization machinery.

Reviewer #1 (Public Review):

This is a nicely written, very compelling manuscript, comprehensive in scope, that reaches new molecular and mechanistic conclusions on metal transport by Nramp on the basis of extensive crystallographic, molecular dynamics, and metal binding/transport assays. The higher resolution of the structures reported here provides new insights into metal (both Mn and Cd) coordination chemistry along the transport pathway which was generally missing (or incomplete) from previous structural analysis of this well-studied model bacterial system. The findings are strongly topical and likely applicable to other Nramps that are present in higher eukaryotes.

The new crystallography coupled with the molecular dynamics provides support for the overall transport pathway model. The conclusions are by and large strongly supported by the data. The figures are absolutely outstanding, and readily accessible even to the non-specialist. The authors identify a lower affinity "external" site which may function as an Mn transfer site that kinetically enhances Mn-binding to the cognate "orthosteric" site essential for transport across the membrane.

Minor weaknesses are the ITC experiments in general. The authors use these experiments to estimate binding affinities of the external and orthosteric sites in a variety of conformations. Although these data are extensive (there are many titrations here), the robustness of the fits to these data is not apparent from what is provided. Clearly the stoichiometry, and thus the binding model (one site vs. two independent sites) was assumed prior to the data fitting; the uncertainties in K are then quite large.

Reviewer #1 (Public Review):

Han and Eckstein asked human participants to follow the gaze of a person and to judge the presence/absence of a target person in videos. The videos contained a gazer and an additional person as gaze goal in present conditions. In absent conditions, this person was digitally removed from the video. The results show that participants use peripheral information about the most likely gaze goal to predictively execute a saccade towards the gaze goal before the gazer's head is oriented towards the goal. At the same time, foveal information about the head velocity of the gazer is processed, leading to more reverse saccades to the gazer when the head velocity of the gazer is low and/or when the head accelerates before the first saccade to the goal. Further control experiments show that the reverse saccades are effective in reducing the error of the following saccade because additional foveal information of the gazer's head direction is sampled. Predictive saccades are also observed when participants are not instructed to follow the gaze.

Strengths:

The study uses very clever experimental manipulations and analysis methods to understand when and where information is sampled for saccade programming. This is especially challenging because natural videos are used to investigate gaze control in an ecologically highly relevant scenario. Compared to previous studies on the sampling of information, in which mostly artificial and static targets were used, this is a large conceptual and methodological step forward and advances the state-of-the-art. The complex stimulus material is analysed using advanced AI techniques and traditional human annotations. Overall, the study contains a complex and rich data set that is created and analysed with innovative methods and it will certainly stimulate further research.

Weaknesses:

While the study uses clever and sophisticated manipulations to dissect the influence of different types of information on eye movement control, these manipulations inevitably lead to a few limitations of ecological validity, which might contribute to the findings:

1. Role of expectations: It seems that whenever there was a second person present in the video, it was always the gaze goal. This might influence the gaze dynamics of participants because participants can anticipate that the gazer will look towards the second person. This expectation might allow participants to infer the gaze goal with peripheral vision and reduce the necessity to rely on foveal information about the head direction of the gazer. Some or all of the differences between the present/absent conditions might actually reflect the effect of this expectation.

2. Absent videos: Absent videos were created by digitally removing the target/distractor person from the video. This is definitely useful to maximize the visual similarity of absent and present videos, but it also might lead to absent videos that do not contain a meaningful gaze goal in the scene. This can be seen in Figure 1e, where the gazer seems to look towards something that is outside of the video frame. This absence of a potential gaze goal might delay saccades and render them more variable, especially in terms of amplitude.

Reviewer #1 (Public Review):

This paper addresses the question of Prdm9-dependent hotspots and Prdm9 alleles evolution. Two properties underlie this question: the erosion of hotspots by biased gene conversion and the high mutation rate of the Prdm9 zinc finger domain. Here the authors include an additional recently observed property of Prdm9: its role in DSB repair, by enhancing DSB repair efficiency when binding on both homologs (symmetric sites). The status of symmetric binding depends on Prdm9 level and affinity, possibly other factors. The authors present a model for simulating Prdm9 and hotspots co-evolution based on several assumptions (Number of DSB independent of Prdm9, two types of hotspots, strong or weak; hotspots compete; at least one symmetric DSB is required on the smallest autosome). Although the in vivo context is obviously more complex, these assumptions are reasonable (except for the number of Prdm9 bound sites) as they qualitatively recapitulate or get close to what is known about the requirement for fertility. The model leads to several important conclusions and predictions that Prdm9 limits the number of sites used since such conditions are predicted to allow for a weaker contribution of asymmetric sites.

The presentation of the model is clear, but the results are difficult to follow and require many readings to follow the text and the associated figures.

A few specific points also require clarification:<br /> Competition: It seems that in the context defined Prdm9 is limiting (since most Prdm9 can be bound to all weak sites); in addition, it is not clear how the competition for DSB activity between Prdm9 sites is taken into account.

The number of Prdm9-bound sites in vivo is not known, thus several values must be tested.

It would be interesting to discuss the model prediction in the context of several observations published on hybrids with variable Prdm9 gene dosage.

Reviewer #1 (Public Review):

It is well established that valuation and value-based decision making is context-dependent. This manuscript presents the results of six behavioral experiments specifically designed to disentangle two prominent functional forms of value normalization during reward learning: divisive normalization and range normalization. The behavioral and modeling results are clear and convincing, showing that key features of choice behavior in the current setting are incompatible with divisive normalization but are well predicted by a non-linear transformation of range-normalized values.

Overall, this is an excellent study with important implications for reinforcement learning and decision-making research. The manuscript could be strengthened by examining individual variability in value normalization, as outlined below.

There is a lot of individual variation in the choice data that may potentially be explained by individual differences in normalization strategies. It would be important to examine whether there are any subgroups of subjects whose behavior is better explained by a divisive vs. range normalization process. Alternatively, it may be possible to compute an index that captures how much a given subject displays behavior compatible with divisive vs. range normalization. Seeing the distribution of such an index could provide insights into individual differences in normalization strategies.

One possibility currently not considered by the authors is that both forms of value normalization are at work at the same time. It would be interesting to see the results from a hybrid model.

Reviewer #1 (Public Review):

This paper establishes a strong case for the post-translational modification of C/EBPalpha to play a strong role in its effects, in this case, to promote macrophage differentiation in collaboration with PU.1. The cellular system being used for most of the experiments here takes advantage of the dual roles of PU.1 in B cells, which normally do not express C/EBP family factors, and in myeloid cells, which normally do express C/EBP family factors. The authors and others have previously shown that PU.1 and C/EBPalpha are very powerful collaborators, both needed to establish a macrophage identity. Thus, the title of the paper provocatively implies that the C/EBP modification that keeps it from being methylated on Arg35 works by increasing the re-distribution of PU.1 from B cells to myeloid gene sites in combination with C/EBP. Indeed, the authors show proximity ligation data to show that PU.1-C/EBPalpha juxtaposition is more frequent in the nucleus if C/EBPalpha cannot be Arg-methylated. The paper also shows careful and thorough characterization of the B to myeloid lineage conversion gene expression changes and the mapping of the Arg residues in C/EBPalpha that are most important to keep demethylation. Similarly, the paper provides strong evidence that it is Carm1, and not another protein arginine methyltransferase, that is responsible for the regulatory modification. This is a valuable and well-characterized demonstration of a mechanism that should be considered more generally as a regulator of transcription factor action.

Some weaknesses:

1. The mechanism proposed by the authors is that C/EBPalpha relocates PU.1 to macrophage sites and that C/EBPalpha R35A binds and relocates PU.1 more efficiently than wildtype, and this seems likely and appealing. However, it is not as strongly supported by data within the paper itself as the other points in the paper are. There is a puzzling gap in the data: no direct evidence is shown that C/EBPalpha is really relocating PU.1 from B cell to macrophage regulatory elements at all. Despite the figure titles (Fig. 4 and Fig. S4), there is no ChIP-seq data to show PU.1 binding sites before and after interaction with either wildtype or R35A mutant C/EBPalpha, just accessibility data. There is also a question of whether such a redistribution would occur fast enough to account for the impressive speed of the R35A mutant's other effects. These questions seem fairly straightforward to address. If relevant data could be added, it would greatly increase the impact and generality of the paper.

2. Also, there is evidence presented that the mutant C/EBPalpha still binds PU.1 at least as well as wildtype in co-immune precipitation and that the bands co-immune precipitated by the mutant may be about twofold stronger. However, this important interaction experiment is not done under quantitative titration conditions that would give confidence about the magnitude of the differences seen.

3. Finally, the effect of the mutation is assumed to be only on the interface for interaction between C/EBPalpha and PU.1 (or other co-factors). However, C/EBPalpha is such a short-lived protein that any modification that slightly increased its half-life could increase its potency. It seems important to present some quantitative protein staining evidence to clarify whether the steady-state level of C/EBPalpha in C/EBPalpha R35A-expressing cells is really unchanged from C/EBPalpha wild-type-expressing cells.

In summary, the authors have demonstrated an exciting and precise mechanism for modulating the effects of C/EBPalpha, but more direct evidence would be needed before concluding that this mechanism operates primarily by exposing a stronger interaction interface to speed up the relocation of PU.1 from B cell sites to macrophage sites.

Reviewer #1 (Public Review):

The manuscript seeks to address a major limitation in the study of SFRS1, a critical and well-studied alternative splicing factor. Specifically, this protein is insoluble at high concentrations due to liquid phase separation driven by the RS domain. This work tests the hypothesis that short RS repeat peptides might compete with intermolecular interactions that drive phase separation, thus solubilizing the protein sufficiently for biochemical and structural studies. The data convincingly show that short peptides with RS, ER, or DR repeats can render recombinant SRSF1 soluble. Moreover, the authors present well-resolved and assignable NMR spectra of SRSF1 dissolved with RS8 as a co-solute. Finally, the authors use paramagnetic relaxation enhancement experiments to map interactions between RS8 and SFRS1, which suggests that the interactions are driven by a combination of ionic interactions between arginine and acidic side chains, and pi-stacking interactions with surface-exposed hydrophobic residues.

The second aspect of this study seeks to identify features of proteins that make them more likely to undergo phase separations. Specifically, the authors use a bioinformatics approach to correlate the presence of RS repeats with the identity of proteins in three available databases of phase-separated material. In addition, the authors use molecular modeling and software tools to predict additional RRM domains that might prone to phase separation by looking specifically for those that are enriched in acidic amino acids with neighboring hydrophobics. These analyses are less convincing -the fold enrichment is small, the sample size decreases by a large amount with increasing repeat length, and it is not clear that the statistical tests performed correctly for multiple hypothesis testing. Moreover, the predictions of the model derived from the computational analyses have not been explicitly tested.

All told, the NMR and PRE data are convincing and the use of short RS, ER, and DR peptides as a co-solute for insoluble SR-domain proteins is novel and clever, but the computational analyses are incomplete and potentially over-interpreted.

Reviewer #1 (Public Review):

Lemerle et al utilize elegant imaging and molecular biology approaches to convincingly demonstrate the presence of Bin1 and caveolae containing rings capable of tubulation in developing muscle. The data is of fundamental potential significance as it advances our understanding of t-tubule biogenesis, which represents a major knowledge gap in muscle biology. The paper will be of broad interest to skeletal and cardiac muscle biologists and physiologists. The paper is well written, with a comprehensive yet concise introduction, clearly presented results, and an appropriate discussion. The imaging is spectacular, and the use of CLEM provides compelling validation of the protein constituents of ring structures identified via EM. When combined with time-lapse imaging, the combination of approaches provides powerful nanoscale structural information alongside temporal dynamics and live-cell confirmation of tubulating ability by Bin1-Cav3 containing rings. The data indicate that Bin1 is sufficient to generate circular structures that are subsequently decorated by caveolae which facilitate tubule formation at the membrane, and they support the requirement of both Bin1 and Cav3 for efficient tubule initiation and elongation. The authors also utilize myotubes from patients with cav3 mutations to explore whether altered ring formation may contribute to muscle pathology - however, this section requires additional controls and validation to confer pathological insight. Further, additional quantification of imaging data across the study is required to increase the rigor and strength of the conclusions of this work.

Reviewer #1 (Public Review):

The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second major strength is that in other to understand some of the physicochemical properties of the viral liquid inclusion, they used stimuli that have been very well studied, and thus one can really focus on a relatively easy interpretation of most of the data presented here.

There are three major weaknesses in this article. The way it is written, especially at the beginning, is extremely confusing. First, I would suggest authors should check and review extensively for improvements to the use of English. In particular, the abstract and introduction are extremely hard to understand. Second, in the abstract and introduction, the authors use terms such as "hardening", "perturbing the type/strength of interactions", "stabilization", and "material properties", for just citing some terms. It is clear that the authors do know exactly what they are referring to, but the definitions come so late in the text that it all becomes confusing. The second major weakness is that there is a lack of deep discussion of the physical meaning of some of the measured parameters like "C dense vs inclusion", and "nuclear density and supersaturation". There is a need to explain further the physical consequences of all the graphs. Most of them are discussed in a very superficial manner. The third major weakness is a lack of analysis of phase separations. Some of their data suggest phase transition and/or phase separation, thus, a more in-deep analysis is required. For example, could they calculate the change of entropy and enthalpy of some of these processes? Could they find some boundaries for these transitions between the "hard" (whatever that means) and the liquid?

The authors have achieved almost all their goals, with the caveat of the third weakness I mentioned before. Their work presented in this article is of significant interest and can become extremely important if a more detailed analysis of the thermodynamics parameters is assessed and a better description of the physical phenomenon is provided.

Reviewer #1 (Public Review):

The manuscript by Shaikh and Sunagar addresses the question of the origin of spider venom proteins. It has been known for many years that an important component of spider venoms is a diverse group of small proteins known as disulfide-rich peptides (DRPs). However, it has not been clear whether this group of proteins has a common origin or evolved convergently in different lineages. The authors collected sequences of the genes encoding these proteins from publicly available genomes of spiders from a range of families. They aligned the sequences using the structural cysteines as guides and carried out a phylogenetic analysis of the different sequences, ultimately classifying the different proteins into over 50 super-families. One thing that is not clear from the text or from the references cited (I am not an expert on spider venom) is how many of these superfamilies were known before and how many are novel. There is also no clear indication of what criteria were used to define a subset of sequences as a superfamily. Nonetheless, the authors show that all these superfamilies have a single common ancestor, predating the divergence of araneomorphs and mygalomorphs and that the DRPs underwent independent diversification in each of these two lineages.

The authors also looked at selective forces acting on the sequences using dN/dS analyses. They reach the conclusion that there are different modes of selection acting on different sequences based on their role - defensive or predatory venoms - building on previous work by the lead author on venom sequence evolution in diverse animals.

All in all, this is an admirable piece of molecular evolution work, providing new data on the evolution of spider venom proteins. There are some confusions in terminology that need to be cleared up, and somewhat more context needs to be given for non-specialists as detailed in the points below:<br /> 1) Common names of the main spider infraorders should be given.<br /> 2) Opisthothelae is not the common ancestor of Mygalomorphae and Araneamorphae, but the clade that encompasses those two clades. This incorrect statement appears in several places. Further on, it is stated that Opisthothelae is the common ancestor of all extant spiders. This is wrong both from a terminological point of view (a clade cannot be ancestral to another clade) and from a factual point of view, since there are extant spiders not included in Opisthothelae.<br /> 3) Several proteins and proteins families are mentioned without being introduced, e.g. knottin. Please provide short descriptions.

Reviewer #1 (Public Review):

In this manuscript, the authors present a study on the humanized mouse model of HIV, in which the major focus is inflammasome activation during acute infection and the potential for blocking this activation. They describe the mouse model as sufficient to study inflammasome activity after HIV infection and proceed to demonstrate potentially beneficial effects of a caspase-1 inhibitor, VX-765, given during acute infection resulting in mildly reduced viral load and increased CD4 T cell preservation.

The authors clearly demonstrate established HIV infection in huNSG mice through detection of plasma viremia, viral RNA/DNA in tissue and depletion of CD4 T cells. Furthermore, they show moderate but inconsistent increases in expression of inflammasome-related genes using qPCR across multiple tissues and timepoints. As expected, the authors found increased levels of inflammatory cytokines, particularly IL-18, during acute viral infection.

Systemic IL-18 levels are significantly reduced by VX-765, demonstrating clear in vivo capacity to impact inflammasome-related cytokines. Furthermore, there appears to be a statistically significant preservation of CD4 T cell populations in VX-765-treated animals, although this preservation is inconsistent across different tissues and may not be to a biologically relevant degree. Finally, VX-765 appears to significantly decrease plasma viral load at day 22 post-infection and potentially results in lower HIV DNA levels in the spleen. Finally, the manuscript demonstrates reduced caspase-1 activity after VX-765 treatment, but this finding is limited to CD11c+ and CD14+ cells with no impact found within CD4 and CD8 T cells, and the authors acknowledge that they may be unable to detect caspase-1 activity in CD4 and CD8 T cells after HIV infection.

Although the study is interesting, there are several important comments and potential caveats/limitations that must be addressed, including for the correlations between AIM2 and IFI6 with viral loads and CD4 T cells that appear strongly driven by measurements at day 0 post-infection; multiple cytokine measurements that appear to be below the manufacturer's described limit of detection for the assay described; a lack of measurable caspase activity in T cells; some inconsistency between DNA or RNA content and plasma viremia.

Reviewer #1 (Public Review):

This is a very interesting paper showing that during amino acid starvation of Neurospora, the general amino acid control factors CPC-1 and CPC-3 are crucial to maintaining circadian rhythm at the levels of rhythmic growth and transcription of the FRQ gene. They show that deleting both genes leads to reduced and arrhythmic cell growth and FRQ transcription that can be accounted for by severely reduced occupancy of the FRQ promoter by the key transcription factor WCC. This defect in turn appears to result from diminished H3 acetylation of the FRQ promoter that was observed at least in the cpc-1 mutant, which is mediated by Gcn5. Thus, they show that Gcn5 occupancy at FRQ is rhythmic and impaired by cpc-1 knock-out, that CPC-1 occupies the FRQ promoter, and provide coIP evidence that Cpc-1 interacts with Gcn5 and Ada2 and, hence, could act directly to recruit these cofactors to the FRQ promoter. Importantly, they show that knock out of GCN5 eliminates rhythmic cell growth and FRQ expression (although surprisingly not FRQ mRNA abundance), as well as reducing H3ac levels and WCC binding at FRQ. They further show that TSA treatment can reverse the effects of histidine starvation on the circadian period in WT cells, and can partially restore rhythmic growth to histidine-starved cpc-3 cells, and that elimination of HDAC Hda1 increases H3ac at FRQ in WT cells. They provide some evidence that transcriptional activation of certain aa biosynthetic genes by CPC-1 is also rhythmic, although the evidence for this is not strong and it's unclear whether CPC-1 occupancy or its activation function would be periodic. They also did not address whether CPC-1 occupancy at FRQ is rhythmic.

This work is important in providing convincing evidence that CPC-1-mediated induction of transcription factor CPC-3 in starved Neurospora cells mediates CPC-1-mediated recruitment of Gcn5 and acetylation of the FRQ promoter, which counteracts the function of histone deacetylase HDA1 to maintain high occupancy of the transcription factor WCC and attendant circadian rhythm of FRQ transcription. Although the work does not identify new regulatory circuits, such as rhythmic transcription of FRQ, the role of Gcn5, Hda1, and promoter histone acetylation in supporting transcriptional activation, and the general amino acid control response to amino acid starvation are all well-established mechanisms, the work is significant in showing how these pathways and mechanisms are integrated to maintain circadian rhythm in the face of amino acid limitation.

There is an abundance of convincing experimental evidence provided to support the key claims just summarized above. However, there are a few instances in which additional experiments might be required to resolve a discrepancy in the data or provide stronger evidence to support a claim.

Reviewer #1 (Public Review):

Lin et al. characterise cellular pathologies in PLA2G6 mutant patient-derived neuronal cells (neuronal progenitor cells, NPCs, and IPSc-derived dopaminergic neurones) and a novel compound heterozygous PLA2G6 mutant mouse model. They build on their previous findings in an INAD fly model (lacking PLA2G6) to show that lysosomal and mitochondrial defects are evolutionary conserved in PLA2G6 deficiency. The authors proceed to use their INAD fly model and to screen a number of compounds that are predicted to modulate endo-lysosomal function using a bang sensitivity assay. They then show that the drugs that can rescue this fly behavioural phenotype also reduce LAMP2 expression in patient-derived NPCs on Western blot analysis. Lastly, the manuscript reports the creation of new genetic constructs that express human PLA2G6 and study expression levels in a human kidney cell line as well as in patent-derived NPCs. In the latter neuronal model, they show that expression of human PLA2G6 can rescue mitochondrial fragmentation associated with PLA2G6 loss-of-function. Lin et al then show that ICV (intracerebroventricular) and IV (intravenous) injection of a human PLA2G6-containing construct is able to partially rescue the rotarod phenotype in PLA2G6 transheterozygous PLA2G6 mutant mice between ~110 and 150 days. There is also an associated improvement in lifespan and body weight.

The strengths of this work are that the authors use a number of different model organism systems, including patient-derived neuronal cells, Drosophila models (INAD flies) and mouse models to study PLA2G6-associated neurodegeneration (PLAN) at the cellular level. They also screen drug compounds that are predicted to target endo-lysosomal trafficking and sphingolipid metabolic pathways to ameliorate PLAN, thus identifying potential new therapeutic strategies. The work in mice, showing that gene therapy with human PLA2G6 can rescue a behavioural phenotype and lifespan is the first proof-of-concept of such an advancement. This work will hopefully lead to further studies for optimisation toward clinical advancement.

The major weaknesses are that the pathogenic mechanisms shown in the patient-derived neuronal cells and mice do not extend as far as those previously shown in the fly model published by the authors. Of note, ceramide levels and retromer function are not studied, both key pathologies described in the previous fly models. In addition, the drug screening is limited by its testing in one fly behavioural assay and LAMP2 Western blot analysis on patient derived NPCs.

The results, in general, support the conclusions of the authors and represent well-performed work. However, the significance of elevated glucosylceramide levels is not clear in the present study. Although this was previously found to be elevated in INAD flies, it was ceramide levels that were thought to be the main toxic insult, with drugs aimed at reducing ceramide levels being shown to rescue INAD flies.

This work will no doubt be of significant interest to the field, confirming several previous findings in the Drosophila model of PLA2G6 (iPLA2-VIA) knockout. It also extends upon the fly work by identifying compounds that can be further studied for potential drug-re-purposing for the treatment of PLA2G6-associated disease. The gene therapy studies are also very interesting and a first proof-of-principle in PLAN using ICV and IV delivery in a mouse model.

Reviewer #1 (Public Review):

Targeting allosteric sites, including cryptic sites holds great potential for achieving drug design that distinguishes between isologous protein targets. Here, Meller et al seek to reveal the mechanisms by which blebbistatin, a selective allosteric inhibitor of myosins achieves selectivity between proteins with high structural and sequence similarity. Blebbistatin binds in a supposed cryptic pocket, and authors explore the hypothesis that this selectivity is modulated by dynamics of opening in the cryptic pocket. Studies use MD simulations to show that while cryptic pockets do not exist in experimental structures, they appear in simulations. Markov state models (MSM) generated from simulations are used to quantify probability of pocket opening. The same methods are used to show that ADP-bound myosins are more likely to open than ATP-bound state, consistent with higher blebbistatin binding affinity observed in the ADP-bound state. Myosin-II proteins are shown to have higher probability of opening than non-myosin-IIs, along with an observed correlation of probability with IC50. By docking blebbistatin into structures derived from MSMs, authors show a correlation between predicted binding affinity from docking and experimental binding. Binding was correctly predicted for a new isoform in a blind study, further establishing the utility of using conformational ensembles to predict sensitivity of blebbistatin binding to myosins.

Reviewer #1 (Public Review):

This study provides further detailed analysis of recently published Fly Atlas datasets supplemented with newly generated single cell RNA-seq data obtained from 6,000 testis cells. Using these data, the authors define 43 germline cell clusters and 22 somatic cell clusters. This work confirms and extends previous observations regarding changing gene expression programs through the course of germ cell and somatic cell differentiation.

This study makes several interesting observations that will be of interest to the field. For example, the authors find that spermatocytes exhibit sex chromosome specific changes in gene expression. In addition, comparisons between the single nucleus and single cell data reveal differences in active transcription versus global mRNA levels. For example, previous results showed that (1) several mRNAs remain high in spermatids long after they are actively transcribed in spermatocytes and (2) defined a set of post-meiotic transcripts. The analysis presented here shows that these patterns of mRNA expression are shared by hundreds of genes in the developing germline. Moreover, variable patterns between the sn- and sc-RNAseq datasets reveals considerable complexity in the post-transcriptional regulation of gene expression.

Overall, this paper represents a significant contribution to the field. These findings will be of broad interest to developmental biologists and will establish an important foundation for future studies. However, several points should be addressed.

In figure 1, I am struck by the widespread expression of vasa outside of the germ cell lineage. Do the authors have a technical or biological explanation for this observation? This point should be addressed in the paper with new experiments or further explanation in the text.

The proposed bifurcation of the cyst cells into head and tail populations is interesting and worth further exploration/validation. While the presented in situ hybridization for Nep4, geko, and shg hint at differences between these populations, double fluorescent in situs or the use of additional markers would help make this point clearer. Higher magnification images would also help in this regard.

Reviewer #1 (Public Review):

Wolfram syndrome 1 (WS1) is a rare genetic disorder characterized by diabetes mellitus, various neurological dysfunction, and blindness caused by optic atrophy. The primary impact of this paper is the characterization of vision loss, retinal dysfunction, and retinal ganglion cell (RGC) degeneration in the Wfs1exon8del murine model of WS1 combined with the generation of -omics datasets at RNA and protein levels. Based largely on a qualitative assessment of select targets, the authors propose mechanisms that could increase RGC susceptibility in WS1 pathology.

Strengths:

1. This study determines that Wfs1exon8del mice exhibit progressive disruption of RGC function similar to that reported in the Wfs1exon5del rat model.<br /> 2. This study performs an in-depth assessment of retinal anatomy, including in vivo OCT and FA, which provides analysis of both vascular and neural elements of pathology.<br /> 3. TEM and immunohistochemical assessment of optic nerve anatomy and RGC soma elucidate a timeline of degenerative events that begins with myelin thinning and axon pathology followed by axon and soma loss.<br /> 4. RNA sequencing and proteomic profiling provide a global assessment of potentially relevant pathways associated with retinal and optic nerve pathology induced by Wfs1 deletion.

Weaknesses:

1. Mechanisms are generally inferred from previous literature rather than demonstrated directly in this model.<br /> 2. Diverse phenotypes were noted in oligodendrocytes, astrocytes, Muller cells, and microglia. It is difficult to piece together the significance of these observations and their relationship to the RGC degeneration noted in earlier figures. There is a sense that the surface is skimmed for each of these.<br /> 3. ERG and f-VEP data do not rule out the possibility that the electrophysiological function of the retina is abnormal from birth.<br /> 4. Only positive correlations with existing literature are discussed.

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!- Comment : 30 to 1 ratio of unequal exchange to North to South Aid - structural unfairness baked into the system - the North knowingly benefits - Knowing this, it can be argued that North states are structurally corrupt and morally bankrupt knowingly imposing this mass suffering upon the south

Reviewer #1 (Public Review):

The current study proposed a drug discovery pipeline to accelerate the process of identifying drug candidates for LCA10 patients using cells from mouse retinal organoid for initial screening, human patient iPSC-derived retinal organoid for further testing, and then mouse mutants for in vivo validation. Reserpine was identified as the top candidate, possibly through modulating proteostasis and autophagy to promote cilium assembly. The study was with high translational value. However, the rationale using dissociated cells from mouse retinal organoid for initial drug screening needs to be justified. In addition, the consistency of phenotypic characteristics in human patient iPSC-derived retinal organoid needs to be reported. It was unclear if the rescued phenotypic changes were from the drug effects or a result of phenotypic variations in organoids.

Reviewer #1 (Public Review):

The manuscript by Webb et al., describes a proband with biallelic variants in the MCAT gene. The proband presents with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Subsequent studies in isolated lymphoblasts and fibroblast samples from the proband unveil combined OXPHOS deficiency. Although the manuscript is of interest and adds translational impact to the previous work of the authors, a few issues remain unresolved. The study would benefit from additional functional tests of the variant MCAT allele.

Reviewer #1 (Public Review):

Roncaioli et al. build upon their previous findings showing that the NAIP/NLRC4 inflammasome confers host resistance to oral Shigella flexneri infection. They investigate the role of additional programmed cell death pathways in the host response to Shigella infection in mice. They find that in the absence of the NAIP inflammasome, caspase-11 contributes but in a limited capacity due to OspC3 antagonism of caspase-11 activity. Furthermore, in the absence of both NAIP and caspase-11, TNF-mediated caspase-8 activation contributes. Thus, the authors conclude that there is a hierarchy of cell death pathways involving caspase-1, 11, and 8 that all contribute to restrict Shigella infection in mice. Overall, the manuscript is well-written, the studies are logically presented and well-designed, and the data largely support the authors' conclusions. The findings will be of great interest to the field. I have a few suggestions for improving the manuscript.

Reviewer #1 (Public Review):

Latshaw and colleagues show that interfering with the function of the tyramine receptor, AmTYR1, causes a precipitous decline in responses to olfactory stimuli, a decline consistent with AmTYR1's proposed involvement in the regulation of inhibitory networks within the antennal lobes (primary olfactory centres) of the honey bee brain. Interestingly, impacts on odour learning of disrupting AmTYR1 function are enhanced by repeatedly exposing bees to an odour without reinforcement ('familiarization'). The authors argue that disruption of AmTYR1 signalling increases the expression of latent inhibition without affecting appetitive conditioning. The results do not, in my view, support this claim. Nevertheless, the disruption of tyramine signalling in the bee brain clearly packs a powerful punch.

Strengths:<br /> Repeated presentation of an odour without reinforcement slows subsequent learning of an association between the odour in question and food reward (latent inhibition). This study's aim was to investigate the mechanisms that underlie individual differences in this trait.

The authors select honey bee lines showing high and low levels of latent inhibition and use QTL mapping to identify genes potentially responsible for this trait. Amtyr1 is identified, a gene that encodes a tyramine receptor the authors have shown elsewhere is expressed in the brain, including on presynaptic terminals of olfactory sensory neurons in the antennal lobes (primary olfactory centres of the brain). Using the tyramine receptor blocker, yohimbine, and Amtyr1 knockdown with dsiRNA, Latshaw et al. show that disruption of tyramine signalling via AmTYR1 receptors inhibits dramatically the magnitude of responses to odour signals at the level of the antennal lobes. Odour learning appears to remain largely intact unless, prior to conditioning, bees are exposed repeatedly to puffs of odour without reinforcement (a situation expected to induce latent inhibition). As a result of familiarization, learning not only of the familiarized odour, but also of novel odours declines dramatically. These findings are fascinating and consistent with the hypothesis that AmTYR1 is involved in regulating inhibitory networks within the AL.

Weaknesses:<br /> The authors argue that disruption of AmTYR1 signalling increases the expression of latent inhibition without affecting appetitive conditioning. The results, in my view, do not support this conclusion. Electrophysiological recordings from the AL show that blocking AmTYR1 function causes significant non-odour-specific suppression of responses at the level of the antennal lobes, a result that would be predicted if inhibiting AmTYR1 function increased lateral inhibition (as opposed to latent inhibition) globally in the antennal lobe.

Under these conditions, the consequences of odour familiarization would, I believe, be predicted by current models of inhibitory networks in antennal lobes. A schematic of olfactory circuits within the antennal lobes, and the location of AmTYR1 receptors within this network, would assist in enabling readers to navigate these complex networks and interpret the interesting findings presented in this study. While the stated aim was to investigate the mechanisms that underlie individual differences in latent inhibition, this goal seems to be lost along the way.

Reviewer #1 (Public Review):

In their paper "Spatiotemporal Ecological Chaos Enables Gradual Evolutionary Diversification Without Niches or Tradeoffs", Mahadevan, Pearce, and Fisher build on previous works to explore a compelling potential answer (what they term a "scenario") to an important open and fascinating question: what gives rise to micro strain-level diversity?

Naively, the ecological principle of competitive exclusion would suggest that closely related strains should not be able to co-exist. However, Fisher and collaborators have previously proposed an interesting and potentially novel and powerful solution to this paradox. If the species have (extremely) anti-correlated species-species interactions (i.e more A helps B but more B hurts A) then there is a reasonably large set of parameters under which you can have infinite diversity due to spatial-temporal chaos (STC). This is really an interesting and compelling picture.

The purpose of this paper is to explore a natural follow-up question: does the STC phase still support infinite diversity even when communities are assembled using evolution, or more accurately undergo evolution starting with a sufficiently large randomly assembled community? In other words, is STC still a reasonable explanation for strain-level diversity once evolution is considered? This is an extremely interesting question since evolution and ecology are so deeply intertwined at the time scales on which strains evolve. The work is especially impressive due to the extreme dearth of analytic and computational tools to really understand eco-evolutionary dynamics.

Reviewer #1 (Public Review):

This is one of the most careful analyses of sexual dimorphism in dinosaurs, based on a remarkable assemblage of 61 ornithomimosaur fossils from the Early Cretaceous of western France. The dimorphism is expressed in variations in the shaft curvature and the distal epiphysis width, analysed appropriately here and plausible because these are the kinds of morphological features that vary between males and females among birds and crocodilians, among others.

In the Introduction, it is right to highlight the shortage of convincing cases of demonstrated sexual dimorphism (SD) in dinosaurs. But note the points made by Hone, Saitta and others that SD can exist in many species today without major morphological differences, making it hard to demonstrate in fossils with such types of dimorphism. Also, some proposed statistical tests to ensure that SD has been convincingly demonstrated in fossils are so stringent they would be hard ever to pass (requiring enormous and constant morphological distinctiveness). In other words, we are conditioned not to find SD in dinosaurs, and yet may be massively under-reporting it because of preservation difficulties (of course) but also because of some overly rigorous demands for proof. These issues help argue that the current study is especially valuable because the data set is large (itself a rarity), and 3D bone shape analysis and proper statistical testing have been applied.

It's interesting the dinosaur example shows the same two dimorphic traits (femoral obliquity = bicondylar angle; width of distal epiphysis = bicondylar breadth) seen in mammals (MS, lines 117-123), where the femur angle may vary because of the need for broader hips in the female to accommodate the birth canal, and yet dinosaurs laid eggs. These are small dinosaurs, so perhaps their eggs were relatively large in proportion to body size. Perhaps the authors could comment on this. There is some discussion with regard to modern birds at MS lines 187-199.

Reviewer #1 (Public Review):

Here the authors sought to understand how BPGM/2,3-BPG levels are involved in adaptive responses to hypoxia and whether they are involved in fetal growth restriction. In the current state, I find the data to be confusing and lacking in mechanistic data to justify that increased BPGM is an adaptive response to hypoxia. While the authors find increased staining for the enzyme BPGM in SpA-TGCs after hypoxia, they did not assess 2,3-BPG in cord blood. This would show that increased enzymatic levels have a downstream impact. MRI experiments assessing placental and fetal haemoglobin-oxygenation, showed no differences. Human FGR samples, however, showed reduced 2,3-BPG in cord blood. Further evidence is required to show hypoxia increases BPGM as a compensatory mechanism to permit adequate 2,3-BPG and placental-fetal oxygenation levels as the authors claim. Additional experiments that demonstrate that BPGM is advantageous in the context of hypoxia would strengthen the authors arguments, and would provide a novel mechanism for adaptive responses to hypoxia in the placenta which is highly interesting.

Reviewer #1 (Public Review):

This manuscript describes a new method to perform online movement correction and extraction of calcium signals from a miniscope. The efficiency of the algorithm is tested by quantifying the accuracy of animal location decoding from hippocampal place cells. The online decoding happens with virtually no delay which is promising for closed-loop methods. It seems to be superior to online decoding without motion correction, which was the state of the art.

The strength of this technique is therefore that it achieves real-time processing.<br /> The weakness of the study is the lack of comparison of the decoding accuracy with what can be obtained with electrophysiological state of the art, which prevents really estimating how precise the technique is.

Although less critical, there is no demonstration of a closed-loop application. Real-time position decoding is technically nice, but the position can be obtained from tracking the animal so it is practically useless. It is also clear that decoding position on a linear track is easier than on a 2D arena, therefore it is difficult to estimate how much the efficiency of the method can be challenged in harder settings.

Thus despite its technical excellence, the impact of this method seems weak.

Reviewer #1 (Public Review):

In this study, the authors collected mandibular alveolar bone samples from control mice and the mice with apical periodontitis (AP) and performed single-cell RNA sequencing (scRNA-Seq) experiment. Using the data from cell subsets of the mandibular alveolar bone, the authors compared the expression profiles of the mice with AP with those from control mice. They also determined the relationship between MSCs and immune cells and confirmed the role of a subset of MSCs in inflammation. In addition, the authors demonstrated MSC differentiation potential to mature osteoblasts during AP inflammation. Using transgenic mouse models and samples derived from patients with chronic AP, they further confirmed the findings of scRNA-Seq data. Taken together, these results reveal the heterogeneity and interactions of alveolar bone cells during periodontitis inflammation. One of their key findings is to identify a subset of MSC cells and their differentiation in the inflamed tissues.

Reviewer #1 (Public Review):

This study intended to identify the metabolic at-risk profile within PLWH on ART, by integrating and analyzing the multiomics data from multi-omics including untargeted plasma metabolomic, lipidomic, and fecal 16s microbiome. The overall strength of the study is the long-term treatment (~15 years) of the study subjects with well-recovered CD4 cell count and viral suppression. The integration and analysis of multi-omics data using similarity network fusion and factor analysis, etc. to group or differentiate HIV patients are informative and useful. The weakness of the study is the lack of presentation of comparability between patients and healthy controls and the use of multiple regression analysis for controlling potential confounders.

Reviewer #1 (Public Review):

Complex I deficiency is associated with multiple diseases ranging from devastating inborn errors of metabolism to more common ailments associated with aging. It has long been known that Complex I transports electrons through a series of iron-sulfur clusters; however, the consequences of the likely dysfunction of these cofactors in models of Complex I deficiency have been surprisingly unexplored. The authors of this manuscript explore the contributions of iron to pathophysiology seen in a mouse model of Complex I deficiency, the Ndufs4 knockout model. Specifically, the authors hypothesize "that Complex I deficiencies may alter normal cellular or regional iron distribution which contributes to mitochondrial disease progression."

The authors begin by convincingly demonstrating that modulating iron availability affects clasping - a marker of neurodegeneration - as well as lifespan. Using either an iron-chelating agent or a low iron diet, the authors delay the onset of clasping (i.e., neurodegeneration) and extend lifespan in Ndufs4 knockout mice. As expected, a limited iron diet causes anemia, but does not affect overall weight in either wild type or Ndufs4 knockout mice, suggesting the diet is tolerated despite the hematological defects. To begin to understand the molecular mechanisms underlying these phenotypes, the authors quantify total iron levels across tissues, and surprisingly find tissue-specific effects of chelation and/or diet-induced modulation of total iron levels. These data suggest that a low iron diet rescues iron levels in the liver, kidney, and duodenum, but not in the brain, which is surprising given the rescue of the clasping phenotype. To follow up on this, the authors look at multiple metals whose uptake can be affected by altered iron homeostasis, and find select defects in other tissues, such as elevated zinc in the brains of knockout mice relative to wild-type controls and elevated manganese in Ndufs4 KO tissues. However, the effects of such metal imbalances were not further explored. The authors then show that the imbalance in liver free iron could cause oxidative stress and reprogram the cellular program of iron transport. Interestingly, these phenotypes are found in the liver but not the brain, consistent with the tissue-specific changes in metals found in previous experiments. Collectively, the data presented by the authors convincingly demonstrate that: 1. Complex I deficiency causes defects in metal homeostasis, albeit in a tissue-specific manner; 2. In tissues harboring elevated iron levels in Ndufs4 knockout mice, this leads to altered iron regulation pathways as well as elevated oxidative stress; and 3. That, despite the tissue specificity of the molecular changes in iron homeostasis, limiting iron uptake in Ndufs4 knockout mice rescues neurodegeneration and extends lifespan in mice. While these data support the authors' hypothesis that "Complex I deficiencies may alter... regional iron distribution which contributes to mitochondrial disease progression," the authors do not test the role of altered cellular distribution of iron within this model.

Overall, the data presented strongly suggest that modulating iron intake may ameliorate the pathophysiology associated with Complex I dysfunction. However, the specific tissues in which these benefits may occur, and the molecular mechanisms underlying this therapeutic benefit are not fully established in this study. Furthermore, the benefits of modulating iron levels as a potential therapeutic strategy for Complex I dysfunction would need to be balanced with potential complications, such as anemia.

Reviewer #1 (Public Review):

The manuscript by Seroussi et al. presents a comprehensive analysis of the expression and function of the entire, extensive family of Argonaute (AGO) proteins in C. elegans. Using genome editing methods, the authors fused tags to 19 endogenous argonaute genes to allow for visualization of their expression patterns in live worms and immunoprecipitation assays to detect RNA partners. Furthermore, they analyzed how the loss of specific AGOs impacts smRNA populations as well as fertility, developmental, and pathogen susceptibility phenotypes. The methods are rigorous and care was taken to maintain the functionality of tagged proteins. This study offers an extremely valuable resource in its comprehensive evaluation of all AGOs in an organism and the resulting datasets and reagents. Furthermore, the authors provide thoughtful analyses of their own data pointing out surprises and follow-up experiments to support their interpretations. A good example is the isolation of miRNAs in the ERGO-1 IP, which provide compelling evidence to indicate that this result is likely due to co-IP of ALG-1/2 on transcripts also bound by ERGO-1 rather than a new role for ERGO-1 in directly binding miRNAs. The authors are also to be commended on the clear and engaging figures, which are often difficult to produce from largely genomics data. Overall, this is a highly significant body of work that provides extensive datasets and reagents that will propel the smRNA field forward faster.

Reviewer #1 (Public Review):

This work identified a novel gene Belly roll (Bero) as a key protein that controls nociceptive escape behavior in Drosophila larvae using genome-wide association analysis. The authors show that constitutive deletion of Bero by CRISPR or RNAi knockdown of Bero expression shows enhanced escape rolling behavior induced by heat probe stimulation. They then showed that Bero is expressed specifically in dimm' pepti, Eh+, Ilp, and AbLK neurons. Next, they demonstrated that Bero RNAi knockdown specifically in ABLk neurons showed the mutant phenotype. Furthermore, they found that ABLK neurons exhibit spontaneous activity and, that Bero RNAi knockdown inhibited this spontaneous activity and increased the response latency to nociceptive stimulation in ABLK neurons.

Strong points of this study: Authors identify a novel gene as a key protein that negatively controls nociceptive escape behavior in Drosophila. The data were presented clearly and the approach to identifying the gene was unique.

Weakness of this study:<br /> I think if authors are able to link the variable bero expression levels in each GNP line and ABLK neural activity, the physiological function of Bero will be strong. I also appreciate the proposed model in the supplemental figure although it has not shown the evidence to link stress and Bero in this study.

Reviewer #1 (Public Review):

This study combines the biologging method with captive experiments and DNA metabarcoding to detail the hunting behavior of a bat species in the wild. Specifically, it shows that bats use two foraging strategies (echolocating small prey in the air and capturing large ground prey with passive listening) with different success rates and energetic gains. This result highlights that a species believed to be a specialist forager can, in fact, have mixed strategies depending on the condition and environment.

The detailed foraging behavior they show for such a small animal is impressive. A combination of several different methods, including captive experiments, is a major strength of the paper. I especially like the mastication sound analysis, although I don't know how new it is. However, I have a major concern about the presentation of this study. The manuscript is apparently written for a bat community, and it's hard to understand the significance of the results in the field of animal ecology.

Reviewer #1 (Public Review):

Recent studies indicate that osteoblasts formed during endochondral bone formation have arisen from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). In this study, through a single-cell transcriptomics approach, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts at postnatal and adult stages. HC-specific Mmp14 knockout mice had increased bone mass phenotype. The authors found that MMP14 cleaves the extracellular domain of PTH1R and inhibits PTH signaling. They also found that HC-derived osteoblasts contribute about 50% of osteogenesis promoted by the treatment with PTH 1-34 and this response was amplified in Mmp14 knockout mice. MMP14 controls PTH signaling through both HC- and non-HC-derived osteoblasts. The authors concluded that they have identified a novel mechanism of MMP14-mediated PTH signaling in the osteoblast lineage cells.

Reviewer #1 (Public Review):

King et al. provide an interesting reanalysis of existing fMRI data with a novel functional connectivity modeling approach. Three connectivity models accounting for the relationship between cortical and cerebellar regions are compared, each representing a hypothesis. Evidence is presented that - contrary to a prominent theoretical account in the literature - cortical connectivity converges on cerebellar regions, such that the cerebellum likely integrates information from the cortex (rather than forming parallel loops with the cortex). If true, this would have large implications for understanding the likely computational role of the cerebellum in influencing cortical functions. Further, this paper provides a unique and potentially groundbreaking set of methods for testing alternate connectivity hypotheses in the human brain. However, it appears that insufficient details were provided to properly evaluate these methods and their implications, as described below.

Strengths:<br /> • Use of a large task battery performed by every participant, increasing confidence in the generality of the results across a variety of cognitive functions.<br /> • Multiple regression was used to reduce the chance of confounding (false connections driven by a third region) in the functional connectivity estimates.<br /> • A focus on the function and connectivity of the cerebellum is important, given that it is clearly essential for a wide variety of cognitive processes but is studied much less often than the cortex.<br /> • The focus on clear connectivity-based hypotheses and clear descriptions of what would be expected in the results if different hypotheses were true.<br /> • Generalization of models to a completely held-out dataset further increases confidence in the generalizability of the models.

Concerns:<br /> • The main conclusion of the paper (including in the title) involves a directional inference, and yet it is notoriously difficult to make directional inferences with fMRI. The term "input" into the cerebellum is repeatedly used to describe the prediction of cerebellar activity based on cortical activity, and yet the cerebellum is known to form loops with the cortex. With the slow temporal resolution of fMRI it is typically unclear what is the "input" versus the "output" in the kinds of predictions used in the present study. Critically, this may mean that a cerebellar region could receive input from a single cortical region (i.e., the alternate hypothesis supposedly ruled out by the present study), then output to multiple cortical regions, likely resulting (using the fMRI-based approach used here) in a faulty inference that convergent signals from cortex drove the results. On pg. 4 it is stated: "We chose this direction of prediction, as the cerebellar BOLD signal overwhelmingly reflects mossy-fiber input, with minimal contribution from cerebellar output neurons, the Purkinje cells (Mathiesen et al., 2000; Thomsen et al., 2004)." First, it would be good to know how certain this is in 2022, given the older references and ongoing progress in understanding the relationship between neuronal activity and the BOLD signal (e.g., Drew 2019). Second, given that it's likely that activity in the mossy-fiber inputs has an impact on Purkinje cell outputs, and that some cortical activity supposedly reflects cerebellar output, it is possible that FC could also reflect the opposite direction (cerebellumcortex). It would seem important to consider these possibilities in the interpretation of the results.<br /> • It would be helpful to have more details included in the "Connectivity Models" sub-section of the Methods section. The GLM-based connectivity approach is highly non-standard, such that more details on the logic behind it and any validation of the approach would be helpful. More specifically, it would be helpful to have clarity on how this form of functional connectivity relates to more standard forms, such as Pearson correlation and perhaps less standard multiple regression (or partial correlation) approaches. If I understand this approach correctly, each cortical parcel's time series is modulated (up or down) using that parcel's task-evoked beta weights, then "normalized" by the standard deviation of that parcel's time series, with the resulting time series then used in a multiple regression model to explain variance in a given cerebellar voxel's time series. It would be helpful if each of these steps were better explained and justified. For example, it is unclear what modulation of the cortical parcel time series by task-related beta weights does to the functional connectivity estimates, and thus how they should be interpreted.<br /> • It appears that task-related functional connectivity is used in the present study, and yet the potential for task-evoked activations to distort such connectivity estimates does not appear to be accounted for (Norman-Haignere et al. 2012; Cole et al. 2019). For example, voxel A may respond to just the left hemifield of visual space while voxel B may respond to just the right hemifield of visual space, yet their correlation will be inflated due to task-evoked activity for any centrally presented visual stimuli. There are multiple methods for accounting for the confounding effect of task-evoked activations, none of which appear to be applied here. For example, the following publications include some options for reducing this confounding bias: (Cole et al. 2019; Norman-Haignere et al. 2012; Ito et al. 2020; Rissman, Gazzaley, and D'Esposito 2004; Al-Aidroos, Said, and Turk-Browne 2012). If this concern does not apply in the current context it would be important to explain/show why.<br /> • It is stated (pg. 21): "To reduce the influence of these noise correlations, we used a "crossed" approach to train the models: The cerebellar time series for the first session was predicted by the cortical time series from the second session, and vice-versa (see Figure 1). This procedure effectively negates the influence of noise processes, given that noise processes are uncorrelated across sessions." However, this does not appear to be strictly true, given that the task design (parts of which repeat across sessions) could interact with sources of noise. For example, task instruction cues (regardless of the specific task) likely increase arousal, which likely increases breathing and heart rates known to impact global fMRI BOLD signals. The current approach likely reduces the impact of noise relative to other approaches, but such strong certainty that noise processes are uncorrelated across sessions appears to be unwarranted.<br /> • It appears possible that the sparse cerebellar model does worse simply because there are fewer predictors than the alternate models. It would be helpful to verify that the methods used, such as cross-validation, rule out (or at least reduce the chance) that this result is a trivial consequence of just having a different number of predictors across the tested models. It appears that the "model recovery" simulations may rule this out, but it is unclear how these simulations were conducted. Additional details in the Methods section would be important for evaluating this portion of the study.

References:

Al-Aidroos, Naseem, Christopher P. Said, and Nicholas B. Turk-Browne. 2012. "Top-down Attention Switches Coupling between Low-Level and High-Level Areas of Human Visual Cortex." Proceedings of the National Academy of Sciences of the United States of America 109 (36): 14675-80.<br /> Cole, Michael W., Takuya Ito, Douglas Schultz, Ravi Mill, Richard Chen, and Carrisa Cocuzza. 2019. "Task Activations Produce Spurious but Systematic Inflation of Task Functional Connectivity Estimates." NeuroImage 189 (April): 1-18.<br /> Drew, Patrick J. 2019. "Vascular and Neural Basis of the BOLD Signal." Current Opinion in Neurobiology 58 (October): 61-69.<br /> Ito, Takuya, Scott L. Brincat, Markus Siegel, Ravi D. Mill, Biyu J. He, Earl K. Miller, Horacio G. Rotstein, and Michael W. Cole. 2020. "Task-Evoked Activity Quenches Neural Correlations and Variability in Large-Scale Brain Systems." PLoS Computational Biology. https://doi.org/10.1101/560730.<br /> Norman-Haignere, S. V., G. McCarthy, M. M. Chun, and N. B. Turk-Browne. 2012. "Category-Selective Background Connectivity in Ventral Visual Cortex." Cerebral Cortex 22 (2): 391-402.<br /> Rissman, Jesse, Adam Gazzaley, and Mark D'Esposito. 2004. "Measuring Functional Connectivity during Distinct Stages of a Cognitive Task." NeuroImage 23 (2): 752-63.

!- David Hume : book 1 and 2 - book 1 explains what persons are - book 2 explains that persons don't have selves

Reviewer #1 (Public Review):

The authors sought to define the molecular mechanism of activation of the thrombopoietin receptor (TpoR), a very important cytokine receptor that regulates megakaryocyte differentiation and platelet production. They conducted a thorough series of experiments combining mutagenesis experiments with sophistical biological assays and that also includes solid-state NMR structural measurements. This work builds on a body of previous studies of TpoR from this group and from others. They focused both on (1) the role and impact of W515 located in the juxtamembrane cytosolic domain and (2) the impact of introducing either Asn at sites in the transmembrane domain to induce various dimerization modes, or insertion of pairs of Ala residues to induce helical rotation to the TM domain. There is a lot of nice data in this paper, which is fairly intricate - a tough read, but that's because it's a complicated system. The writing is excellent.

This paper presents a model for receptor activation in which the inactive receptor is the monomeric form of the receptor in which the juxtamembrane domain, including W515, maintains a helical structure. Activation of the receptor triggers dimerization of the transmembrane domain and loss of helicity of the juxtamembrane segment, which facilitates optimal interactions of the kinase domains with their JACK2 domain phosphorylation substrates.

There is a lot to like in this careful work and the resulting manuscript. There is one major shortcoming in this manuscript, which concerns W515. It is known that mutation of W515 to any of 17 of the canonical amino acids, including Phe, is sufficient to trigger homodimerization and receptor activation. The authors present some evidence that the phenomenon behind this is that mutation of W515 to almost any other residues disrupts the helical secondary structure of the critical juxtamembrane segment, which promotes dimerization and receptor activation. What I find puzzling is why a Trp at site 515 promotes helix formation, but nearly all other amino acids at this site disrupt helix formation. This strongly suggests the side chain of W515 must be interacting with another domain of the protein in the inactive state, in a manner that is responsible for how Trp stabilizes the juxtamembrane helix which is a central feature that helps define that state. I think that for this paper, this dangling missing piece of their mechanistic model should be resolved.

Reviewer #1 (Public Review):

The manuscript by Xu et. al. does a very thorough characterization and molecular dissection of the role of SSH2 in spermatogenesis. Loss of SSh2 in germ cells results in germ cell arrest In step2-3 spermatids and eventually leads to germ cell loss by apoptosis. Molecular characterization of the mutant mice shows that the loss of SSH2 prevents the fusion of proacrosomal vesicles leading to the formation of a fragmented acrosome. The fragmentation of the acrosome is due to the impaired actin bundling and dephosphorylation of COFILIN. In short, this is a comprehensive body of work.

Reviewer #1 (Public Review):

This study demonstrates that MALPs (Marrow Adipogenic Lineage Progenitors), which were previously described by these authors and constitute approximately 0.5% of bone marrow mesenchymal cells, are major producers of Csf1 (M-CSF) in murine bone marrow. The initial discovery of Csf1 in MALPs occurred during review of scRNA-seq datasets. Here the authors show that deletion of Csf1 in MALPs with AdipoQ-Cre increased trabecular bone mass in long bones, but not vertebrae, and reduced the number of osteoclasts on trabecular bone surfaces. Cortical bone was not altered. Deletion of Csf1 with Adipo-Cre also prevented LPS-induces osteolysis and reduced numbers of hematopoietic progenitors and F4/80+ macrophages. Strengths of this study include use of two CKO lines (Adipo-Cre and Prx-Cre) to understand the relative contribution of MALPs to Csf1 levels in the bone marrow, examination of bone mass in both long bones and vertebrae at several ages, challenging bone responses in Csf1 CKOAdipoQ mice with LPS-induced osteolysis, and studying the effect of Csf1 deletion in MALPs on hematopoiesis and vasculature. Mechanical studies of bone strength were not included but would be necessary to determine if deletion of Csf1 in MALPs is sufficient to cause osteopetrosis. Additional information on other molecular changes Csf1 CKOAdipoQ mice would provide insights into how deletion of Csf1 in MALPs affects bone remodeling. Overall, this is a very important study that will have a high impact on the field because it is challenging the paradigm that osteoblasts and osteocytes are the major sources of M-CSF in the bone marrow.

Reviewer #1 (Public Review):

In the current study, the authors reanalyze a prior dataset testing effects of D2 antagonism on choices in a delay discounting task. While the prior report using standard analysis, showed no effects, the current study used a DDM to examine more carefully possible effects on different subcomponents of the decision process. This approach revealed contrasting effects of D2 blockade on the effect of reward size differences and bias. Effects were uncorrelated, suggesting separate mechanisms perhaps. The authors speculate that these opposing effects explain the variability in effects across studies, since they mean that effects would depend on which of these factors is more important in a particular design. Overall the study is novel and well-executed, and the explanation offers interesting insight into neural processes.

Reviewer #1 (Public Review):

In this manuscript, Richardson et al. describe the repertoire characteristics of Ky mice that carry human immunoglobulin heavy (IgH) and light chain (Igk and l) genes. Immunophenotyping revealed no abnormalities in B cell subsets in the bone marrow, spleen, or lymph nodes of Ky mice (Fig. 1) and the light chain k/l ratio was similar to that observed in humans. Bulk RNA-seq showed some differences in VH, DH, and JH utilization in Ky mice compared to humans (Fig. 2). Ky sequences also had slightly shorter CDRH3 regions (Fig. 3) with a diversity index lying between that of mice and humans (Fig. 4). Use of a novel algorithm further substantiated similarities between Ky mice and human-derived sequences. The authors conclude that Ky mice are suitable to study human immune responses.

Richardson et al. have compiled a comprehensive dataset of Ig sequences from Ky mice to compare with human repertoires. The differences in gene segment utilization are potentially interesting but there is little discussion of the ramifications of these differences during immune responses. They briefly stated that previously published studies of immunizations in Ky mice support their conclusion that these mice respond like humans. However, no details were provided, and it is difficult to assess how similarly Ky mice respond to specific antigens compared to humans. Given the substantial amount of single-cell RNA-Seq data that is now available for responses against the SARS-CoV2 spike, this may be a good antigen to test in Ky mice. Finally, I defer to computational experts to speak to the novelty and validity of conclusions regarding diversity and structural variability in Ky mice compared to humans.

Reviewer #1 (Public Review):

This study will be of interest to those who want to understand how non-pharmaceutical interventions in response to epidemics in human populations (here using the example of SARS-CoV-2 in the Netherlands) might be designed to reduce negative societal impacts through subnational implementation. In most countries, including the Netherlands, non-pharmaceutical interventions such as movement restrictions and school closures were implemented simultaneously throughout entire jurisdictions. The authors of this paper investigated whether subnational heterogeneities in the prevalence of infection could be exploited to develop local level control measures that varied according to local changes in prevalence of infection, an innovation that could potentially reduce negative societal impacts of interventions while maintaining similar levels of epidemiological control. Using simulations from a carefully parameterised agent based model of SARS-CoV-2 transmission in the first wave in Netherlands , the authors generated convincing evidence to suggest that this would be, at least in theory, possible, though practical difficulties of implementing such a control policy were not explored.

Reviewer #1 (Public Review):

The data that is presented is quite clear, and expected given the prior in vitro work, as well as prior work in vivo with helminth infection and BCG vaccination. Overall, it is important to demonstrate that observations from in vitro experiments are relevant in vivo, however, there are concerns with the design of this study which limits its impact. In addition, the study confirms what is expected from prior work, but falls short of adding any new mechanistic insight.

In terms of the in vivo experimental design, it is unclear why the authors chose to administer BCG IP, when the vaccine is given SC (and then based on more recent data, IV could be arguably interesting and relevant). The focus on the peritoneum limits the potential application of these findings to address the important question of the effects of helminth infection on BCG vaccine responses. The ultimate in vivo experiment to be able to demonstrate a physiological relevance of the mechanisms explored here would be to see what the effect was on Mtb infection in the lung.

The authors do report different responses in the spleen and lymphnode, which is interesting, but lines 336-337 accurately point out that compartmentalized overexpression of IL-10 in the spleens but not the lymph nodes has been described in mice with chronic schistosomiasis. Mechanistic insight into this phenomenon was lacking, and the relevance to Mtb infection is still unknown.

Reviewer #1 (Public Review):

COVID-19 severity has been previously linked to a genetic region on chromosome 3 introgressed from Neandertals. The authors use several computational methods to, within this region, identify specific regions that putatively regulate gene expression, and to identify genes within these regions associated with COVID-19 severity. The use of several complementary computational approaches is a major strength of the paper as it bolsters confidence in the findings and narrows the search for significant genomic regions down to most likely candidates. They find 14 genes that exhibit expression regulated by the identified introgressed genomic regions. Among these are several chemokine receptors including two - CCR1 and CCR5 - whose upregulation is associated with severe COVID-19. The authors then use functional genomics to determine whether the identified regions do regulate gene expression.

In contrast to the robustness of the computational findings, the authors' MPRA results are less robust with respect to the significance of the paper to clinical severity of COVID-19. The MPRA shows that the computational methods were reasonably effective at identifying regulatory elements within the introgressed region (53%). The authors then focus on emVars where the H.n. allele differentially regulates expression and identify 4 putative emVars that may regulate expression of CCR1 and CCR5. However, the authors found in their MPRA that these emVars downregulate reporter gene expression, whereas the genes of interest CCR1 and CCR5 are upregulated during severe COVID.

This result highlights the principal weakness of using the MPRA in this context, as it assumes that reporter gene expression using a minimal promoter has identical regulatory determinants as expression of the gene of interest. Its strength is the high-throughput nature of the assay, but its weakness is the lack of specificity with respect to the question at hand. This lack of specificity mitigates the impact of the functional aspect of the work. The authors' computational findings certainly bolster previous work that H.n. introgressed alleles are associated with COVID-19 severity and that this association may be at least partly dependent on gene expression differences between the archaic and modern alleles. However, the specific question at hand, whether chemokine receptor expression is linked to the clinical phenotype, remains unaddressed.

Ultimately the authors results support the conclusions that the 4 emVars identified do regulate gene expression. However, the hypothesis that these specific regions are linked to COVID-19 severity is not supported. The authors' speculation as to why their results may differ from the observed upregulation during disease is intriguing, but lacks support.

Reviewer #1 (Public Review):

Implementation of host-directed therapies (HDTs) could alter the global trajectory of tuberculosis and several HDTs are under investigation. In this manuscript, sertraline (SRT) is proposed as an adjunctive therapy with the mechanism proposed to be due to possible effects on host immune cells. However, the mechanism by which SRT exerts its potentiating effects on Mtb growing in macrophages or in mice is unclear. Schump et al (2017) had previously demonstrated that SRT has a weak anti-tubercular effect in vitro but that this is further enhanced in macrophages simply because SRT acts as a weak base that accumulates in phagosomes. SRT, however, has immune effects as well, as demonstrated by its inhibition of IRF3 dependent gene expression by virtue of its inhibition of PI3K signaling (part of the TLR3 pathway)(Zhu et al., 2010). In this work, a variety of phenotypes are demonstrated for SRT but it is never conclusively demonstrated that the potentiating action of SRT can be ascribed to effects on type I interferon production. It should also be noted that while type I interferon production is generally accepted to promote Mtb growth and dissemination, type I interferons also have a positive role to play in immune regulation (see PMID 29666166). Comparing SRT to inhibitors such as BX795 and Isoliquiritigenin does not establish that the observed effects act through a common mechanism, especially in light of the fact that BX795 has a greater effect on inhibiting IRF3-dependent transcription than SRT but has weaker potentiating effects against Mtb in macrophages. BX795 also inhibits IRF3 transcription but through a different pathway. The role of cGAS/STING is also explored. The cGAS/STING pathway also results in IRF3-dependent signaling but this is through another upstream pathway where the cGAS/STING is activated by dsDNA and bacterial cyclic dinucleotides. Previous studies have shown that cGAS is protective in mice (Collins et al., 2015) which would suggest that inhibition of this pathway and possibly all pathways that mediate IRF3 signaling, would be detrimental to the host. The authors suggest that SRT enhances inflammasome activation but the data supporting this is not convincing and the control Isoliquiritigenin, a NLRP3 inflammasome inhibitor, potentially has other effects. In addition, NLRP3 inflammasomes appear to enhance Mtb growth and spread in host cells (see Beckwith et al. 2020) which would counter the argument that NLRP3 inflammasomes are central to SRT effects. Overall, studies to demonstrate that the activity of SRT can be directly to inhibition of SRT signaling would corroborate the hypothesis that SRT acts as an HDT.

Nevertheless, the fact that SRT has an effect and somehow potentiates the activity of drugs in macrophages and in mice is an important demonstration and a highlight of this work. The demonstration that SRT also acts on Mtb in different physiological states as well as a drug-tolerant clinical strain, is also an important advance.

Reviewer #1 (Public Review):

The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency.

The major strengths of this paper include comprehensive pharmacological and genetic methods, and the combination of histology and functional electrophysiological measures of neuronal function. While it is not clear the mechanism by which heme oxygenase-2 deficiency might occur in motor neuron pools, or the relevance to human disease, the authors identify several targetable molecules in the hypoglossal motor neurons that are candidates for future study.

Furthermore, the work completed here may be relevant to other diseases in which motor neuron signal transmission is a key contributor.

Reviewer #1 (Public Review):

The manuscript discussed the combination use of pyrotinib, tamoxifen, and dalpiciclib against HER2+/HR+ breast cancer cells. Through a series of in vitro drug sensitivity studies and in vivo drug susceptibility studies, the authors revealed that pyrotinib combined with dalpiciclib exhibits better therapeutic efficacy than the combination use of pyrotinib with tamoxifen. Moreover, the authors found that CALML5 may serve as a biomarker in the treatment of HER2+/HR+ breast cancer.

The authors provide solid evidence for the following:<br /> 1. The combination use of pyrotinib with dalpiciclib exhibits better therapeutic efficacy than the combination use of pyrotinib with tamoxifen.<br /> 2. Nuclear ER distribution is increased upon anti-HER2 therapy and could be partially abrogated by the treatment of dalpiciclib.<br /> 3. CALML5 may serve as a putative risk biomarker in the treatment of HER2+/HR+ breast cancer.

The manuscript has significant strengths and several weaknesses. The strengths include the identification of the novel role of dalpiciclib in the treatment of HER2+/HR+ breast cancer. Moreover, the authors provide solid evidence that the combined use of dalpiciclib with pyrotinib significantly decreased the total and nuclear expression of ER. The main weakness of the manuscript is that the manuscript is difficult to read due to language inconsistency. In addition, some figure captions and figure legends should be carefully amended.

Reviewer #1 (Public Review):

Prostate cancer is the most common cancer and the second most common cause of cancer death in men. Hence, there continues to be a pressing need for new diagnostic and therapeutic approaches for this disease, as well as better prognostic biomarkers to guide treatment. In this manuscript, Lauer et al. show increased expression of PCA3 and decreased expression of PRUNE2 in prostate cancer compared with the adjacent normal prostate across all tumor grades and stages. And there was no association between the relative gene expression levels of PCA3 or PRUNE2 and time to disease recurrence. These findings suggest a role for PCA3 and PRUNE2 in prostate cancer. And most conclusions of this paper are supported by data.

Reviewer #1 (Public Review):

Sonobe et al provide compelling data on the translation initiation codons required for PR and PG production from the antisense C4G2 repeat expansion associated with C9orf72-ALS/FTD. The strengths of this study are the systematic approach by which the authors identify the start codons. They also provide data investigating if eIF2D is needed for DPR production, building upon previous findings. A major weakness of this work includes the lack of full characterization of their models as they relate to disease, including the need to define any potential toxicity associated with DPR production and any DPR aggregation. Additionally, the study would be strengthened by the quantification and further validation of some of the data presented. Overall, the findings within the study have the potential to be important in advancing our understandings of toxic dipeptide production from the G4C2 repeat expansion in C9orf72-ALS/FTD. This has implications clinically and increases our biological interpretation of this disease.

Reviewer #1 (Public Review):

The manuscript by Prem and colleagues uses neural progenitor cells (NPCs) from individuals with different types of autism (either idiopathic or 16p11.2 deletion) to determine whether cells from these individuals show similar or varying phenotypes. An equal number of control cell lines are also used for a total of 6 autism compared to 6 control lines. The results are surprising in that the NPCs from individuals with autism have common cell biology defects (in neurite outgrowth and cell migration) yet have no overlapping genetic defects between the groups. Using proteomics, the authors also show converging biology at the level of the phospho-proteome with mTOR signaling being affected (albeit in differing directions depending on the idiopathic case). Finally, the authors use a number of pharmacological approaches to show that the various cell biology phenotypes can be rescued or induced (in controls) through manipulation of mTOR signaling. In summary, it is a remarkable result that would seem to indicate that many forms of autism somehow alter mTOR signaling, at least in the NPC stage. I have only a handful of comments about the study:

A sample size of 3 idiopathic seems underpowered relative to the many types of genetic changes that can occur in ASD. Since the authors carried out WGS, it would be useful to know what potential causative variants were found in these 3 individuals and even if not overlapping if they might expect to be in a similar biological pathway. If the authors randomly selected 3 more idiopathic cell lines from individuals with autism, would these cell lines also have altered mTOR signaling? And could a line have the same cell biology defects without a change in mTOR signaling? The authors argue that the sample size could be the reason for lack of overlap of the proteomic changes (unlike the phosphor-proteomic overlaps), which makes the overlapping cell biology findings even more remarkable. Or is the phenotyping simply too crude to know if the phenotypes truly are the same?

Reviewer #1 (Public Review):

The authors used high-throughput nanodroplet-based microfluidics to measure the effect of individual species and the joint effects of species pairs and trios on the growth of six fluorescently labeled strains (E. coli and five soil isolates). In total, over 14,000 bacterial communities composed of subsets of a library of 61 soil and leaf isolates were quantified. They found that the effects of multiple species combine non-additively and are heavily dominated by the strongest single species effect.

Single species showed large variability in the ability to use different carbon sources and survive antibiotics. Interaction assays were carried out in minimal M9 media with 0.5% [w/v] glucose for 24 hours. Authors found that single species' effects on the focal strain is positive in ~1/3 of cases, and is negative in 60% of cases. When two species were added, 3/4 of cases became negative. The similarity of phenotypes (e.g. metabolic profile or phylogeny) between the focal and affecting species did not correlate strongly with the effect on focal species. Joint effects of two or more species are not additive, but rather dominated by the strongest single effect.

The paper presents an interesting example of how complexities of communities may be reduced. The assay combines different growth aspects (lag, rate, yield), and these different aspects should ideally be untangled. The mechanism of why joint effects are dominated by the strongest effect is not known. The generality of the findings awaits further studies (e.g. what will happen if environments are changed?) However, these future directions do not diminish the value of this study.

Reviewer #1 (Public Review):

By careful analyses of single particle images, the authors could identify two distinct structural forms of PSII, a stretched and a compact one. Furthermore, they could analyze a pair of two PSII complexes facing each other on their flat stroma-facing surfaces forming a protein sandwich, which can be made of both the stretched as well as the compact PSII forms. A crucial change in the positioning of the CP29 subunit in the two PSII configurations was identified that favors energy transfer between chlorophylls either to light-harvesting complex II or to the PSII-core antenna complexes. Further aspects like water channels to the oxygen-evolving complex, identification of a Na+ ion, and post-translational modifications were discovered. Overall, the manuscript provides a deep view into the structure of PSII unraveling novel aspects of its structural flexibility and fine details. The experiments and data analysis were conducted very carefully and thoroughly. The work could spark new discussions about the importance of PSII flexibility in photosynthetic membranes.

Reviewer #1 (Public Review):

The goal of the present study was to provide the spatial, morphological, and connectional properties of molecularly defined CeA neurons. The authors provide compelling, high-quality profiling of the spatial distribution of up to 29 molecular markers in the CeA and its surrounding. In addition, by combining their EASI-FISH pipeline with retrograde labeling, the authors offer a comprehensive view of the connectional and molecular diversity within the CeA. The spatial resolution and utility of the EASI-fish technique are showcased elegantly in the present study. The authors also develop a new method to combine EASI-FISH with retrograde labeling to provide information on the anatomical connectivity of molecularly defined cells. Overall, the authors do provide a novel resource for the field. However, the scRNAseq dataset presented at the beginning of the study contains ~1300 cells, which is a low number of cells. From this, the authors report that three of their sequencing clusters (c1, c4, and c12) do not have specific molecular markers and were seemingly excluded from spatial validation. As such, throughout the study, how to interpret the relationship between the sequencing clusters and the molecular clusters may require additional experiments or analyses.

Reviewer #1 (Public Review):

This paper shows that nuclear pore complex components are required for Kras/p53 driven liver tumors in zebrafish. The authors previously found that nonsense mutation in ahctf1 disrupted nuclear pore formation and caused cell death in highly proliferative cells in vivo. In the absence of this gene, there are multiple mitotic functions involving the nuclear pore that are defective, leading to p53 dependent cell death. Heterozygous fish are viable but have reduced kras/p53 liver tumor growth, and this is associated with multiple nuclear and mitotic defects that lead to cancer cell death/lack of growth. This therapeutic window suggests targetability of this pathway in cancer. I think the data are robust, rigorous, and clearly presented. I believe this in vivo work will encourage therapeutic targeting of NPCs in cancer.

Reviewer #1 (Public Review):

The study addresses an important question - how the composition of the microbiota influences the intestinal colonization of encapsulated vs unencapsulated B. theta, an important commensal organism. To answer the question, the authors develop a refurbished WITS with extended mathematical modeling to quantify B. theta population bottlenecks during intestinal colonization in the setting of different microbiota. Interestingly, they show that the colonization defect of an acapsular mutant is dependent on the composition of the microbiota, suggesting (but not proving) that interactions between gut bacteria, rather than with host immune mechanisms, explains why the mutant has a colonization defect. However, it is fairly difficult to evaluate some of the claims because experimental details are not easy to find and the number of animals is very small. Furthermore, some of the analyses and claims are compromised because the authors do not fully explain their data; for example, leaving out the zero values in Fig. 3 and not integrating the effect of bottlenecks into the resulting model, undermines the claim that the acapsular mutant has a longer in vivo lag phase.

Limitations:

1. The experiments do not allow clear separation of effects derived from the microbiota composition and those that occur secondary to host development without a microbiota or with a different microbiota. Furthermore, the measured bottlenecks are very similar in LCM and Oligo mice, even though these microbiotas differ in complexity. Oligo-MM12 was originally developed and described to confer resistance to Salmonella colonization, suggesting that it should tighten the bottleneck. Overall, an add-back experiment demonstrating that conventionalizing germ-free mice imparts a similar bottleneck to SPF would strengthen the conclusions.

2. It is often difficult to evaluate results because important parameters are not always given. Dose is a critical variable in bottleneck experiments, but it is not clear if total dose changes in Figure 2 or just the WITS dose? Total dose as well as n0 should be depicted in all figures.

3. This is in part a methods paper but the method is not described clearly in the results, with important bits only found in a very difficult supplement. Is there a difference between colonization probability (beta) and inoculum size at which tags start to disappear? Can there be some culture-based validation of "colonization probability" as explained in the mathematics? Can the authors contrast the advantages/disadvantages of this system with other methods (e.g. sequencing-based approaches)? It seems like the numerator in the colonization probability equation has a very limited range (from 0.18-1.8), potentially limiting the sensitivity of this approach.

4. Figure 3 and the associated model is confusing and does not support the idea that a longer lag-phase contributes to the fitness defect of acapsular B.theta in competitive colonization. Figure 3B clearly indicates that in competition acapsular B. theta experiences a restrictive bottleneck; i.e., in competition, less of the initial B. theta population is contributed by the acapsular inoculum. There is no need to appeal to lag-phase defects to explain the role of the capsule in vivo. The model in Figure 3D should depict the acapsular population with less cells after the bottleneck. In fact, the data in Figure 3E-F can be explained by the tighter bottleneck experienced by the acapsular mutant resulting in a smaller acapsular founding population. This idea can be seen in the data: the acapsular mutant shedding actually dips in the first 12-hours. This cannot be discerned in Figure 3E because mice with zero shedding were excluded from the analysis, leaving the data (and conclusion) of this experiment to be extrapolated from a single mouse.

5. The conclusions from Figure 4 rely on assumptions not well-supported by the data. In the high fat diet experiment, a lower dose of WITS is required to conclude that the diet has no effect. Furthermore, the authors conclude that Salmonella restricts the B. theta population by causing inflammation, but do not demonstrate inflammation at their timepoint or disprove that the Salmonella population could cause the same effect in the absence of inflammation (through non-inflammatory direct or indirect interactions).

6. Several of the experiments rely on very few mice/groups.

Reviewer #1 (Public Review):

In this valuable study, authors Sabanayagam and colleagues used multiple ML models on longitudinal data from a cohort of Chinese, Malay and Indian participants with diabetes to identify predictors for incident DKD.

The study involves a large multi-ethnic data cohort of Asian patients with diabetes and the use of machine learning methods to predict 6-yr CKD incidence risk in patients with diabetes. The final sample size for the study cohort included almost 1365 patients and 339 features. The authors tested multiple ML methods to identify which ML method provided the best prediction accuracy based on a select set of features.

Strengths:

The study is very interesting and timely as efforts are needed to develop prognostic methods for the incidence of Chronic Kidney disease in patients with diabetes. The strength of the study is the diversity in its cohort and the impressive breadth of associated covariates ranging from demographic, lifestyle, socioeconomic, physical, laboratory, retinal imaging, genetic, and blood metabolomics profile for patients.<br /> An important factor to consider when assessing a predictive risk for the progression of a disease is to consider all possible risk components ranging from environmental, metabolic, physiological, and Social determinants of health, which the authors have done very well.

The authors also did not restrict their analysis by selecting a single algorithm upfront for their analysis which strengthens the scientific process without any bias in the outcome.

The authors do go about a data-driven approach by recursively eliminating features that may not be significant in providing them with statistically significant results. With a data set of a given size, this would be a logical way to go about the analysis.<br /> The authors do accept the limitations of their study in the context of not having a validation dataset which is important to address in the scientific process.

Shortcomings:

However, the study does have a few shortcomings which, hopefully when addressed/clarified can help strengthen and streamline the analysis.

1. Statistical significance versus clinical significance:<br /> The authors seem to use recursive feature elimination to come up with a set of top features for each Ml algorithm and select features from a varied feature set. However, the authors may need to pay attention to what the features (that come up as significant) are trying to allude to. For e.g. the authors seem to have dropped the datasets with features that contain the genetic and imaging parameters: D= B+ Genetic parameters and F= B+ Imaging parameters+ Blood metabolites+ Genetic parameters.<br /> They provide reasons for the low performance of the ML models for dropping the features but do not elaborate on whether they investigated the reasons for the drop in performance.<br /> They state this in the manuscript with no citation:<br /> (line 82) "Similarly, genetic abnormalities in diabetes have also been shown to increase the risk of DKD."<br /> ... which makes it difficult to assess which of the 76 snps were associated with CKD and in which population and to what extent.

Similarly, the authors also have previously found features in imaging data have shown an association with CKD:

We and several others have previously shown that retinal microvascular changes including retinopathy, vessel narrowing, or dilation, and vessel tortuosity were associated with CKD [6, 7].

However, they also drop the dataset that includes the imaging features citing poor model performance and no investigations beyond that.

2. The authors speak about the advantage of using ML approaches to overcome shortcomings of traditional assumptions from linear models, however, in the consideration of their covariates they might also want to understand the clinical association between some of their selected features. for e.g. BMI, HbA1c, duration of diabetes, and systolic BP may somehow not be entirely independent of each other (especially in the context of influencing one another and driving diabetes) and multi-collinearity may need to be looked into.

3. The following sections seem to require citation:<br /> no citation:<br /> 59: As CKD is asymptomatic till more than 50% of kidney function decline, early detection of individuals with diabetes who are at risk of developing DKD may facilitate prevention and appropriate intervention for DKD.

Elaborate on rationale (what is challenging?) and citation needed:<br /> 62 Early identification of individuals at risk of developing CKD in type 2 diabetes is challenging. Therefore, characterization of new biomarkers is urgently needed for identifying individuals at risk of progressive decline of eGFR and timely intervention for improving outcomes in DKD.

Citation needed or rationale needs to be back:<br /> Machine learning methods using 'Big data', or multi-dimensional data may improve prediction as they have less restrictive statistical assumptions compared to traditional regression models which assume linear relationships between risk factors and the logit of the outcomes and absence of multi-collinearity among explanatory variables.

Citation:<br /> Similarly, genetic abnormalities in diabetes have also been shown to increase the risk of DKD.

Citation:<br /> 81 Similarly, genetic abnormalities in diabetes have also been shown to increase the risk of DKD.

Citation:<br /> The detailed methodology of the SEED has been published elsewhere.

Citation:<br /> Malay ethnicity has been identified to be a high-risk group for CKD by several studies conducted in Singapore.

4. The authors are attempting to rationalize the outcome of their findings rather than challenge them to improve the robustness of their analysis. In this section, it would help strengthen their analysis if they could find ways to eliminate reasons other than the one they provided or perform additional analysis that could show proof of their claim:<br /> While black ethnicity was a risk factor for CKD in the meta-analysis, in our study, we found Chinese and Malay ethnicity to be at higher risk of developing incident DKD compared to Indian ethnicity. One reason for the Indian ethnicity to be at lower risk of developing DKD could be Indian ethnicity being a high-risk group for diabetes, they may be well aware of the risk, and comply with screening, medication, etc. that could reduce their risk of developing DKD.

5. Following up on the above point, the authors have decided to use SDOH (social determinants of health) to identify prognostic risk factors for the incidence of CKD in diabetic patients without considering what the model may be trying to say regarding ethnicity vs socioeconomic status? it would be good to look at the association of SDOH metrics against ethnicity to see if the ethnic populations at higher risk for CKD could be disadvantaged due to socioeconomic factors and if so these need to be mentioned in the analysis.

6. EN vs other models: the authors claim that EN has much better results than other models in a study where the entire cohort has patients with diabetes possibly progressing towards CKD. usually, Risk models assume that disease progresses in a certain trajectory. However, multiple trajectories for the disease may exist due to heterogeneity of the disease and also non-linear relationships between features and disease outcome might influence this. This is what ML models can specifically address over traditional linear models. However, the pathophysiological progression from diabetes to CKD isn't as non-linear as assumed to be since heterogeneity in disease at that stage (~CKD stage 4) is primarily low and non-additive effects are most likely negligible, which also explains why EN and then LASSO perform so much better than the other models - This needs to be addressed by the authors in the paper.

I hope that addressing these points will help strengthen the paper and streamline it while also making the analysis and the outcomes clinically and statistically significant.

Reviewer #1 (Public Review):

Caligaris et al set out to uncover the mechanism(s) underlying AMPK/Snf1-dependent regulation of TORC1 during glucose starvation. As a first step, they map the global phosphorylation changes that occur when cells are shifted from high glucose to low glucose concentrations (to activate Snf1) both in the presence and absence of the Snf1 inhibitor 2NM-pp1. They then follow this up by carrying out an on-bead in vitro kinase assay (OBIKA) to identify direct Snf1 targets. Together these very high-quality data lead to the identification of nearly 1300 Snf1-dependent phosphorylation sites and at least 150 direct Snf1 target sites. The target proteins are involved in a variety of processes--such as transcription, ribosome biogenesis, signaling, and vesicle trafficking-and will serve as an important resource for those studying Snf1/AMPK-dependent regulation of a wide variety of cellular programs.

Among the Snf1 targets the authors identify several proteins involved in TORC1 signaling and follow up on two new connections; Snf1-dependent phosphorylation of (i) the recently identify TORC1 regulator Pib2, and (ii) the key TORC1 substrate Sch9. Using a set of rigorous experiments, they confirm that Pib2 is a direct target of Snf1, and then show that cells carrying a phosphomimic version of Pib2 rescue the TORC1 inhibition defect seen in cells with no Snf1 activity. They then carry out a very similar set of experiments with Sch9 and show that Snf1-dependent phosphorylation of Sch9 is also important for the inhibition of TORC1 signaling during glucose starvation, and furthermore that impact of the Snf1-dependent phosphorylation of Pib2 and Sch9 is additive.

This paper provides some of the first mechanistic insights into the way that glucose starvation triggers inhibition of TORC1 (particularly in yeast) and will serve as an important resource for those interested in AMPK/Snf1-dependent regulation of a variety of other pathways and processes. The paper also provides the clearest picture yet of the regulation of Pib2, an important but poorly understood TORC1 regulator in yeast and likely beyond.

This important and rigorous work is very well presented but there are two areas that could do with further discussion and clarification.

(1) The authors show that there are several classes of Snf1 targets (Fig. 3e), most notably some that are phosphorylated immediately after Snf1 activation by glucose (<5 min) and others that are only phosphorylated after 15 min. In a simple view, all direct Snf1 targets should be phosphorylated immediately after Snf1 activation. Is that the case? What is the overlap between the direct targets found using the OBIKA assay and the slow and fast responding in vivo targets? What about the phosphorylation motif, does it differ between the groups? These points are not discussed in the text except to point out that the direct Snf1 target Msn4 is among the slowly phosphorylated group.

(2) The data showing that Snf1-dependent phosphorylation of Pib2 plays a key role in triggering inhibition of TORC1 is convincing but is entirely dependent on a rescue of the TORC1 inhibition defect seen in cells where Snf1 is inhibited. That is, TORC1 is normally inactivated during glucose starvation; this does not occur when Snf1 is inhibited by 2nm-pp1 but does occur when Snf1 is inhibited in a strain carrying a phosphomimetic version of Pib2 (Pib2SESE). This indicates that Pib2 phosphorylation is sufficient to replace Snf1 signaling and inhibit TORC1 during glucose starvation. However, in a simple model, a phosphodead version of Pib2 (SASA) should have the opposite effect. That is TORC1 should remain active during glucose starvation in the Pib2SASA strain-but that is not the case (Fig. 4g). This point is not discussed in the paper; why do the authors think that TORC1 is inhibited normally in the SASA mutant inhibits TORC1 normally?

Reviewer #1 (Public Review):

Taste perception is a complicated phenomenon. There are many well-established signaling pathways for the transduction of major taste modalities, however, there are also several taste phenomena that are not well understood. Among these is the mechanism for the perception of low concentrations of sodium chloride as sweet. In the present manuscript by Atsumi et al., the authors present solid evidence that identifies the T1r (sweet /umami) taste receptors as chloride (Cl-) receptors.

The authors make use of an array of modern experimental approaches to demonstrate that T1r receptors from Medaka fish, which are formed by a heterodimer of T1r2a/T1r3 proteins, are able to bind chloride and that this binding induces a conformational change in the heteromeric receptor. The authors demonstrate binding both by solving the x-ray structure of the T1r2a/T1r3 ligand binding domain with Cl- and demonstrating a chloride-induced conformational change by measuring FRET. This conformational change leads to low concentration, chloride-specific action potential firing in nerves from neurons that contain these receptors in mice.

The authors suggest that their results solve the standing question of how and why is the low concentration of NaCl perceived as sweet. In general, the results presented here support the conclusions and represent an important advance in our understanding of the logic of taste perception.

Reviewer #1 (Public Review):

The authors present a very detailed short report on a previously undocumented behaviour where flying squirrels are believed to have created grooves in various species of nuts to aid their secure storage in the crotch or forks of twigs. The behaviour is suggested to have evolved as an adaptive strategy in this population of flying squirrels because of the challenges for nut caching in a rainforest environment.

Using detailed photographs, GPS locations, measurements and camera trap videos, the authors describe the behaviour in great depth providing a useful base for comparative and future studies. However, the weakest point of this study is that the authors did not detect any squirrels making the grooves and only monitored nuts once they were cached. Therefore more research needs to be done to ascertain who, how and where the grooves are produced in the first place.

This work will be of great interest to scholars of animal behaviour and cognition and draws attention to a novel behaviour that warrants further study in similar species.

Reviewer #1 (Public Review):

HLA genes have long been known to harbor trans-species polymorphism (TSP). This manuscript aimed to use state-of-the-art analyses and updated genotyping data to rigorously test for the presence of TSP in HLA genes, quantify the timescales associated with HLA TSP, and relate HLA disease associations to evolutionary rates. To do this, the authors chose HLA alleles across great apes, old world monkeys, and new world monkeys on which to perform phylogenetic analyses, alongside non-parametric tests that compare patterns of synonymous diversity. Finally, HLA genetic associations with the disease were correlated with evolutionary rate.

Strengths:

The manuscript is well written and neatly organized, the figures are clear, and there are many supplementary analyses that will make this paper a great resource for MHC phylogenetics at allelic resolution.

Deployment of modern methodology such as BEAST2 can also test if the hypothesis of TSP is supported while accounting for uncertainties in tree topology and evolutionary rates, necessary additions to analyses of the MHC.

Weaknesses:

Because TSP has already been convincingly demonstrated to occur in the MHC, the primary benefit of the current study is to ensure these previous observations are still supported by the wealth of genetic data that is now available and modern phylogenetic approaches. However, the benefit of using the robust BEAST2 method comes with the weakness of not using all available data. Focusing on single gene trees with only a small subset of alleles may bias results, and inclusion/exclusion criteria should be better defined.

One major point that is somewhat overlooked is the presence of multiple copy numbers for the MHC genes through classic birth and death evolution. For example, MHC-B in new world monkeys is duplicated many times (up to 10; PMID: 23715823). This duplication is naturally accompanied by gene loss and pseudogene formation. All of these things muddy the waters considerably yet are not addressed here. A good example is MHC-A, where it has been very difficult to apportion orthologs, even amongst closely related species, due to alternative or incomplete duplication/loss across the species, or region configuration polymorphism (e.g. PMID: 26371256). An example is chimpanzee Patr-AL which shares similarities with human HLA-A*02 lineage, but is a separate locus, could this show up as TSP under the current analysis?

Similarly, an alternative hypothesis for TSP is convergent gene conversion mutations: intergenic gene conversion has been repeatedly observed in HLA genes and the possibility of it occurring with the same two genes becomes more realistic over 45 million years. If the same two MHC genes recombined in humans and in an NWM, each on their own lineages, this would appear as TSP and would cause an overlap of pairwise synonymous divergence between human-human and human-NWM allele comparisons. This might be especially possible in MHC-DR and MHC-DQ genes presented in Figure 2 since both humans and NWM have multiple MHC-DRB and DQB genes (unless e.g. were genes besides HLA/MHC-DRB1 such as DRB3,4,5 included in the DRB phylogenies?). While BEAST2 may be a good way of robustly modeling and identifying TSP, and I understand these analyses cannot support many more sequences, the authors should consider adding an analysis that rules out gene conversion as an explanation for their results (especially the often repeated claim of 45 million year TSP). For example, can the authors use BLAST to ensure that the alleles that underlie 45 million years of TSP do not share close similarities to other HLA genes present in their respective human and NWM genomes? This seems like it could be fairly quickly performed for all genes, and even if it argued against TSP, it would be an interesting finding.

Finally, the authors have limited themselves to a small subset of HLA/MHC alleles and do not provide sufficient information in the methods to understand how these were chosen nor sufficient discussion surrounding how inclusion/exclusion criteria could bias results. For example, the authors say the alleles were chosen at 2-digit (i.e. 1 field) resolution, but in the phylogenies of Fig. 2, I see variable numbers of alleles chosen for each 2-digit allele family - what metric was used to decide on these alleles?

"We also collected associations between amino acids and TCR phenotypes". It is not clear either what was analyzed, or the results for this part of the analysis. This is a topic of much debate and none of the previous work has been discussed (PMID: 18304006, PMID: 29636542 as primers for this contentious subject)

MHC class I also interact with NK cell receptors, including polymorphic KIR. Through their interactions during infection control and reproduction, the two complexes co-evolve across primates, contributing to the maintenance of MHC diversity. Interaction with KIR likely has a greater impact on HLA polymorphism than interactions with TCR, yet this is not factored into any of the models, or indeed mentioned in the text.

One additional reason inclusion of the KIR binding is important relates to the point above about gene conversion, where it is established that gene conversion reproducibly swaps KIR-binding motifs among MHC class I alleles and genes. HLA-A*23, *24, and *25, *32, for example, are characterized by the acquisition of the 'Bw4' motif from HLA-B (PMID: 26284483), likely followed by positive natural selection. For exon 2 (which encodes the motif), these alleles turn up in a clade distinct from other human HLA-A (Fig 2-S1). What is the impact of the Bw4 motif on this phylogeny? Could this shuffling of motifs be interpreted as indirect TSP?

The analysis that shows the most rapidly evolving sites occur in the peptide binding domain brings little new to the field. This has been established by the Hughes and Nei (cited) and Parham, Lawlor, etc of 1988 (e.g. PMID: 3375250), and replicated multiple times across human populations and many other species.<br /> Likewise, the disease association part. It is nice to have a summary of the known associations, but there are others out there and this one is far from thorough. Here, 50% of the information about infectious diseases appears to be taken from one reference, leaving out some major bodies of work; for example identifying specific peptide binding residues or peptides that associate with HIV (PMID: 22896606) or malaria control (PMID: 1280333). It is also missing some major concepts -such as the DRB1 'shared epitope' of peptide binding residues that predispose to Rheumatoid Arthritis and protects from Parkinson's disease (35 years of work from PMID: 2446635 through PMID: 30910980). The nasopharyngeal carcinoma and EBV story (e.g. PMID: 23209447). Another huge gap here is the pregnancy syndromes -associations of specific HLA C and NK cell receptor allotypes with preeclampsia for example. There are thousands of HLA associations not considered in this section, and to do them justice would likely require an enormous amount of work.<br /> Thus - neither the idea that HLA/MHC polymorphism is focused on peptide binding nor that this binding drives resistance to infection and associations with the disease are new concepts. The previous work in these areas is inadequately acknowledged.

The paper is written in a very approachable language, which is nice to read and friendly to non-experts, but perhaps a little too much so in places. I find that the paper follows a very non-traditional format with respect to for example the results section, which seems a mixture of Introduction/methods/figure legends/discussion with no real solid result description.

Reviewer #1 (Public Review):

The paper addresses an interesting question - how genetic changes in Y. pestis have led to phenotypic divergence from Y. pseudotuberculosis - and provides strong evidence that the frameshift mutation in rcsD is involved. Overall, I found the data to be clearly presented, and most of the conclusions well supported by the data. The authors convincingly show that (i) the frameshift mutation in rcsD alters the regulation of biofilm formation, (ii) this effect depends upon expression of a small protein that corresponds to the C-terminal portion of RcsD, and (iii) the frameshift mutation in rcsD prevents loss of the pgm locus. I felt that the discussion/conclusions about what phosphorylates/dephosphorylates RcsB and how this impacts biofilm formation are overstated, as there are no experiments that directly address this question. I also felt that the authors' model for what phosphorylates/dephosphorylates RcsB in Y. pestis should be more clearly articulated, even if it is only presented as speculation. Lastly, the authors propose that full-length RcsD is made in Y. pestis and contributes to phosphorylation of RcsB, but the evidence for this is weak (faint band in Figure 2d). It may be that the N-terminal domain of RcsD is functional. I recommend either softening this conclusion or testing this hypothesis further, e.g., by introducing an in-frame stop codon early in rcsD after the frame-shift.

Reviewer #1 (Public Review):

Liu et al, use a combination of Calcium imaging, behavioral analysis, optogenetics, and synaptic connectivity analysis to investigate the neuronal mechanisms underlying the regulation of speed during crawling in Drosophila larvae. They find that the larva, although a crawling animal uses a similar strategy as limb animals to control locomotion speed, by differentially varying the different phases of the locomotion cycle. Similar to limbed animals, in which the stance phase is varied, while the swing phase remains mostly constant, in Drosophila larva where crawling is generated through waves of propagation along the body, the duration of the inter-wave phase is varied. The precise techniques in the larva allowed the authors to tackle the mechanisms underlying this type of speed regulation. In a behavioral screen, they identified one type of GABA-ergic neuron (A31c) that is activated during the inter-wave phase, synchronously across multiple segments. They found this neuron is GABA-ergic. Using EM connectomics they identified one of its post-synaptic partners: another GABA-ergic neuron: A26f that strongly innervates the transverse motor neurons that contract perpendicular to the crawling direction. A26f also shows synchronized activity across multiple segments. Activating and silencing A26f using optogenetics they determine that A26f is required and sufficient to modulate the amplitude and duration of the contraction of LT muscles. This type of implementation of speed regulation based on inhibitory neural circuit motifs could be a general neural circuit mechanism shared across species.

The paper uses a combination of cutting-edge techniques to investigate the mechanisms of speed regulation in Drosophila larvae and makes parallels in the mechanics of the Drosophila larva crawling and limbed locomotion. While the evidence is compelling and the analysis rigorous, at times the presentation and writing could be clearer.

Reviewer #1 (Public Review):

The authors present a clearly written manuscript detailing a phylogenomic analysis of SARS-CoV-2 isolates collected from nonhuman hosts. The methods and statistical analysis are appropriate and clearly stated. All claims are adequately justified. Despite the relatively broad host range of the virus (both predicted, https://doi.org/10.1073/pnas.201014611, and directly observed, https://www.aphis.usda.gov/aphis/dashboards/tableau/sars-dashboard), few cross-species transmission events can be confidently assessed from retrospective sequence analysis alone. It is highly likely that the majority of cross-species transmission events go undetected and that as sequencing resources become more widely distributed, more will be resolved.

Despite these limitations, the authors were able to resolve 3 mutations overrepresented among mink (including S:N501T which has previously been suggested to confer an adaptive benefit) and 26 among deer relative to human hosts. In contrast to the present study, most prior work assessing the mutational landscape associated with zoonosis or reverse-zoonosis has focused on a single non-human species or even a single outbreak. Such a targeted analysis is more susceptible to false-positive predictions of adaptive mutations and in addition to verifying several mutations of interest, the present study serves as a generalizable framework for assessing the statistical significance of putative host-specific adaptations.

Globally, identifying characteristics of host population structure and viral molecular features which shift the balance between the emergence of generalist vs. specialist adaptations is a principal open question in viral ecology, https://doi.org/10.15252/embr.202255393. It is yet unclear whether most mutations observed to be overrepresented among non-human hosts, in this study and elsewhere, are under host-specific positive selection; subject to host-specific restriction factor activity (a possibility raised by the authors for several mutations overrepresented among deer), or represent substitutions which may be adaptive among many diverse mammalian hosts for which the probability of fixation is primarily determined by host-level dynamics (rate of contact, etc.). Additionally, while the impact of most individual mutations may be modest and, consequently, essentially host-nonspecific, slight variations among conserved host proteins (for example, the receptor ACE2, https://doi.org/10.1038/s41598-021-92388-5) may result in different epistatic landscapes. Within the receptor binding domain, however, the effect of most substitutions appears largely insensitive to context, https://doi.org/10.1128/mbio.00135-22.

The comprehensive analysis of SARS-CoV-2 mutations observed within non-human animal hosts presented in this work serves as a generalizable framework for assessing the significance of host-specific adaptations. Such analysis is essential to predicting changes in viral ecology and mitigating the risks of future zoonoses with pandemic potential.

Reviewer #1 (Public Review):

The basic problem the authors are addressing is the step of assigning cell types. They assume a segmented (divided into cells) EM volume, then try to divide the cells into similar looking cell types, based on shape and internal structure. This step has previously been entirely manual and is quite time consuming, even in well known model animals (on the fly hemibrain, this took several months of effort by a team of experts, and was the long step in getting to publication).

Their main technical advance is using machine learning to calculate a feature vector for each cell that describes the shape and ultrastructure. Importantly, this vector is shown to capture what humans consider cell types, as clusters in this vector space match both human cell typing and gene expression driven cell typing. Also important, both the finding of the vectors, and the clustering, can be done without human definition of any cell types (they are unsupervised) which is very helpful as sample sizes of EM volumes are increasing much faster than the human effort needed to try to classify them.

The next advance is considering the vectors from the adjoining cells to classify groups of cells into tissues. On one hand, there are many fewer tissues than cell types, and division into tissues is already done for many model organisms. On the other hand, sometimes organs are not divided by anatomical features, in which case the methods proposed here may be particularly useful. This will also be very helpful when looking at entirely unknown creatures. It would be good to test this on a case where division into organs is thought to be well known.

The authors demonstrate that the proposed techniques work well on a model organism, Platynereis dumerilii, matching or exceeding results based on human cell typing, and typing by gene expression, and uncovering previously unknown cell types and organs.

The main limitation, in my mind, is that the methods were only verified on one species. This is understandable, as EM volumes are still time consuming and expensive to acquire, but does not answer how generally applicable the methods are.

Reviewer #1 (Public Review):

This is an important and timely manuscript describing the use of anti-sense oligos (ASO) to re-activate the silent paternal allele of Ube3a in juvenile and adult Ube3a deficient mice. The main finding presented here is that rescue at these postnatal stages appears to rescue EEG power and sleep problems, if not balance/ataxia and anxiety.

Previous studies have shown that through genetic manipulation, Ube3a can be turned on developmentally in a Ube3a deficient background at embryonic stages with complete rescue of "critical" phenotypes (ataxia, anxiety, repetitive behavior and epilepsy). The current study suggests that at least EEG power and sleep rhythms can be rescued at the juvenile and, to a lesser extent, adult stages. However, these previous studies did not examine EEG or sleep disturbance in any detail and may have missed these clinically relevant phenotypes.<br /> Clearly, the current manuscript addresses the issue of rescue postnatally, which is more likely in a clinical setting as Angelman therapeutics are developed. The impact of the EEG rescue is yet to be determined, as the authors did not do these studies in a 129sv background, it is impossible to tell if they could rescue the seizure phenotype in Ube3a deficient mice (c57BL/6 mice are resistant to seizure). The finding that the ASOs could rescue defects in REM sleep may be the most important results in the current study as it stands. Children with AS suffer from sleep problems, as do their caregivers, since REM sleep is disrupted, and they have difficulty sleeping through the night. In sum, I think the authors make a strong case that at least some clinically relevant phenotypes can be rescued using ASO approaches postnatally in Ube3a deficient mice. The need for more information on which isoforms of Ube3a are rescued and whether cognitive defects are rescued as well by late ASO therapeutics is a critical weakness of the current study as it stands.

Reviewer #1 (Public Review):

The authors provide a simple and clear way to understand an aspect of the implicit bias of a neural population code linking it with well-known machine learning methods and concepts such as kernel regression, sample complexity and efficiency.

Although the mathematical results the authors employ are not novel, the way they apply them to the problem of neural coding is novel and interesting to a broad audience.<br /> In particular, the computational neuroscience community can benefit from this work being it is one of the few dealing with the impact of the model implicit bias in explaining real data.

Reviewer #1 (Public Review):

Rensen et al investigated the mechanisms involved in FGF21-mediated improvement in nonalcoholic steatohepatitis (NASH) and fibrosis using APOE*3-Leiden.CETP mice with AAV-8 induced overexpression of FGF21. They find that FGF21 overexpression, in the presence of an obesogenic diet, reduced hepatic lipid influx and accumulation, reduced macrophage activation and monocyte recruitment, decreased lipid- and scar-associated macrophages, and limited activation of hepatic stellate cells. Together these biological effects led to decreased steatohepatitis and fibrosis. These data delve into molecular changes underlying FGF21 phenotypic effects and add to the body of knowledge supporting the potential pharmacologic application of FGF21 in NASH.

The conclusions of the paper are mostly well supported by data, but there are some generalizations and details that need to be further clarified or supported.

Strength:

The AAV8 expression is a powerful tool for in vivo study of potential underlying mechanisms in a disease model. Applying this method to studying FGF21 is timely and innovative given the dire need for pharmacotherapies for NASH, as well as the need to understand the potential mechanisms through which these pharmacotherapies might exert their effects.

Weakness:

The proposed endocrine and paracrine signaling, terms that are introduced for the first time in the discussion section, need more support. There is no information in the introduction section introducing this idea. More importantly, the results section does not include mechanistic data on cell binding by FGF21 in an endocrine or autocrine fashion to exert its effects as posited in the manuscript.

Reviewer #1 (Public Review):

This paper aims to test whether a series of light activated ion channels (GtCCRT4, KnChR) and enzymes that regulate second messengers (BeGC1, bPac, OaPac) can be used to manipulate cells in the zebrafish.

Among the strengths of the paper are the use of several independent methods to test whether the tools are functional - e.g. electrophysiology of mammalian cells for GtCCR4, calcium and cAMP imaging in zebrafish cells in vivo, behaviour tests (tail movement) and monitoring of heart beat. Multiple transgenic lines were established, to select for lines with optimal expression levels. Experiments are carried out in two cell types - reticulospinal neurons in the hindbrain and cardiomyocytes.

The authors have largely achieved their aim of determining whether the rhodopsins can be used in zebrafish. They demonstrate that the cation channel KnChR is particularly sensitive in triggering depolarization of the reticulospinal neurons, as indicated by tail movement. They show that the photoactivatable adenylyl cyclase bPAC and cation channels have an effect on heartbeat. Two other photoactivatable enzymes OaPAC and BeGC1 have no effect on heartbeat, although it is not evident whether this is due to lack of effect on cAMP and cGMP levels.

The abstract sets out to investigate the role of second messengers, emphasizing the need for specificity. However, KnChR is not specific for Na+. As noted by Tashiro et al, the channel can also conduct H+, Ca2+ and Mg2+. The knowledge gap that is being addressed by the manuscript thus needs to be reframed. The concluding statement of the abstract, that the tools tested here can be used to investigate second messengers, is not accurate given the broad conductance of KnChr.

The tools described here have been tested previously in other species, either in cultured mammalian cells (GtCCR4, KnChR, OaPAC) or in vivo (bPAC and BeGC1). The current work thus does not introduce novel tools, but provides evidence that some of these tools can be used in zebrafish. Overall, the lines characterized here will be of use to scientists using zebrafish as the experimental system in a variety of areas.

Reviewer #1 (Public Review):

This paper investigates whether bistable rhodopsins can be used to manipulate GPCR signalling in zebrafish. As a first step, the authors compared the performance of bistable rhodopsins fused with a flag tag or with a fluorescent protein tag (TagCFP). Constructs were compared by expressing in HEK cells followed by calcium imaging with aequorin or cAMP monitoring with GloSensor. This showed that the protein with a smaller flag tag performed better. Then, a series of transgenic zebrafish lines were made, in which tagged rhodopsins were expressed in reticulospinal neurons or cardiomyocytes.

The data indicate that bistable rhodopsin can be used to manipulate Gq and Gi/o signalling in zebrafish. The Gq-coupled SpiRh1 was effective in manipulating reticulospinal neurons, as indicated by analysis of tail movements and calcium imaging of the neurons. Gi/o signalling could be manipulated by Opn3 from mosquitoes, TMT from pufferfish, and parapinopsin from lamprey, as shown by their effects on the heartbeat. Lamprey parapinopsin has the interesting property that it can be turned on and off by different wavelengths of light, and this was used to stop and restart the heart. Finally, the authors show that the cardiac effects are mediated by an inward-rectifier K+ channel, through the use of pharmacological inhibitors.

A strength of this paper is the testing of a range of bistable rhodopsins, with a total of 10 proteins tested. This provides a good resource for future experiments. A weakness is the failure to show that some experiments involved repeated sampling of the same animal. Figure 3 gives the impression that there are 48 independent datapoints. However, there are 8 animals, with 6 datapoints coming from each. Similarly, Figure 4 shows the data from 6 trials of 4 animals, not 24 independent animals. Repeated sampling should be reflected in the data presentation, and in the statistical analysis. Was there an effect of trial number, which is suggested in Figure 6?

Delta F/F refers to relative change, which should be (F-F0)/F0. This should be zero when t = 0. The values in Figure 3E, and 3F are ~ 1 when t = 0, however. Are these figures showing F/F0?

The authors' conclusions that the bistable rhodopsins are useful tools in the zebrafish system appear largely justified. This is consistent with findings from other organisms, including mouse (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097317/, https://www.sciencedirect.com/science/article/pii/S0896627321001616). The tools here are likely to find broad use by scientists who use the zebrafish as the experimental system for a variety of different areas.

Reviewer #1 (Public Review):

In this work, the authors set out to understand quantitatively how, in co-existing populations of bacteria and phages, immune diversity emerges and relates to the level of immunity. The work achieves this goal through a number of numerical and theoretical analyses.<br /> The strength of the work relies in the insight gained via a solvable model, which allows the authors to: discover law-like dependencies between average number of clones and other parameters of the dynamics (e.g. mutation rates); to identify an observable, the average immunity, as a main driver of the coupled phage/bacteria dynamics that can also be measured in real data; to assess the impact of cross-reactivity between CRISPR in bacteria and its targets on phages.

Claims and conclusions are justified by the simulations and the data presented. The paper is also a useful resource for all readers interested in this type of models, since it proposes an extensive documentation of the field linking the results to other theoretical work and experimental findings. (For this reason, however, the article can be found a bit demanding to fully navigate through).

To avoid compromising the analytical insight, the model does not include a series of other factors that are likely to impact the dynamics observed in the experiments (spatial structure, niche partitioning), however all these limitations are appropriately mentioned. The agreement to the experimental data, while extremely valuable, remains qualitative, so additional work in the future could be devoted to devising some parameter fitting that allows for a more quantitative fitting to the data.

The work has the potential to have a broader impact, both by suggesting targets of measurement in experimental settings and by providing insights that could be shared by other types of immune systems, for instance, vertebrate adaptive immunity.

Reviewer #1 (Public Review):

The authors sought to demonstrate the specific two-stage maturation process that results in the development of cardiomyocyte crests. The authors also sought to demonstrate the importance of EphrinB1 in CM crest development and cardiac function, with disruption of EphrinB1 resulting in diastolic dysfunction and subsequently systolic dysfunction.

Strengths of the work included studies in two species (rat and mouse), cardiac-specific KO models, and careful considerations of myocardial structure-function.

Reviewer #1 (Public Review):

The manuscript by Gomolka et al. aims to characterize the impact of genetic AQP4 deletion in mice on brain‐water morphometry and transport. The results suggest that the markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic fluid export, leading to stagnation of ISF and enlargement of the interstitial space. The interstitial fluid stagnation will in turn reduce CSF influx and give rise to an overall reduction in glymphatic transport.

The design of the study and the technical quality of the work looks convincing and results can be of general interest. The manuscript is well-written and the authors used correct statistical approaches to analyse their findings. The methods are appropriate, described in good detail (for most parts), and properly conducted. The claims are supported by the experimental data.

Reviewer #1 (Public Review):

In their paper "Efficacy and safety of endocrine therapy (ET) after mastectomy in patients with hormone receptor positive breast ductal carcinoma in situ: retrospective cohort study", Nan Niu et al describe the outcome of patients with DCIS treated by unilateral mastectomy with (n=791) and without (n=216) adjuvant endocrine therapy. This is a retrospective multicentre study with follow-up for at least 5 years.

Whilst this approach (ET post-mastectomy for DCIS) is rarely prescribed in the Western world (as the authors note) because the risk of recurrence of DCIS is very low (as shown in this series), some consider this appropriate for reducing the risk of contralateral breast cancer. This series of patients with unilateral mastectomy for DCIS thus provides more globally applicable information regarding the recommendation for adjuvant ET in the context of a potential reduction in contralateral cancer risk following a diagnosis of DCIS.

791 of the 1007 eligible patients received ET. The disease-free survival of patients in both groups was excellent, with no difference between those with and without ET. There was no difference in overall tumour recurrence, albeit with small numbers in both arms; 4 cases had invasive local recurrence (a comment confirming no radiotherapy had been received would be relevant), 3 had contralateral breast cancer and 12 had distant metastasis in the ET group while 4 cases with distant metastases were recorded in the non-ET group. Thus overall recurrence was low in both groups and the DFS was 98.36% vs. 99.07% between the ET and non-ET groups.

Conversely, adverse events (including fracture and endometrial cancer, but largely musculoskeletal) were seen in 37% of patients receiving ET. The grade of the adverse events is not reported so it is not possible to determine if these are mild or severe.

Strengths<br /> This is a large and novel series of cases of DCIS, with detailed clinicopathological information.

Weaknesses<br /> The authors themselves note that this study is retrospective and with limited follow-up. The number of cases with recurrence is small, as would be expected. It would be important to note, as I presume, that none had radiotherapy.

They have reported that they have included all hormone receptor positive cases. The definition of 'hormone receptor positive" has not been described in further detail, i.e. whether oestrogen and/or progesterone receptor and scoring system and cut-offs applied. It is presumed, therefore, that cases with any degree of receptor positivity are included. It is not apparent therefore whether there were any differences between those with low ER expression vs those with strong uniform reactivity.

All grades of DCIS were included. However, the % of cases (in both arms) with high-grade DCIS is surprisingly low (29% vs 24%); this is markedly different from what one sees, for example in the UK, where approx. 60% of all cases are of high histological grade. Conversely, the % of microinvasion reported is relatively high, particularly when considering the grade of the DCIS; it is well-recognised that microinvasion is much more common in association with high-grade DCIS. However, the Kaplan-Meier curve for disease-free survival of those with microinvasive carcinoma (Fig 2; D) is interesting and appears to show some separation (not significant presumably because of the small numbers not receiving ET).

Overall this is an interesting and thought-provoking manuscript highlighting the excellent outcome of patients with a wide range of DCIS lesions treated with unilateral mastectomy (whether they are in receipt of ET or not) and the high proportion of adverse events in those in receipt of adjuvant endocrine therapy.

Reviewer #1 (Public Review):

Understanding the evolution of broadly neutralizing influenza antibodies is key to developing a more universal vaccine. In this study, Phillips et al. performed a comprehensive analysis of the evolutionary pathway of CH65, which is an H1-specific broadly neutralizing antibody. The authors generated a combinatorial mutant library with 2^16 members that contained all possible evolutionary intermediates between the unmutated common ancestor (UCA) and CH65, less two mutations that did not affect binding. The binding affinity of each member in the library was measured against HAs from MA90 and SI06, which were isolated 16 years apart, as well as MA90 with a UCA escape mutation G189E. The binding affinity was measured using a high-throughput approach that combined yeast display and Tite-Seq, with careful experimental validation. The results showed that epistasis between mutations within the heavy chain and also across heavy and light chains plays an important role in CH65 to evolve breadth. Although this study highly resembles a previous study by the authors that focused on another broadly neutralizing influenza antibody called CR9114 (Phillips et al., eLife 2021), there are several key differences. Firstly, CR9114 is a HA stem-directed antibody, whereas CH65 binds to the receptor-binding site of HA. Secondly, their previous study only studied the mutations in the heavy chain, whereas the present study looked at mutations in both heavy and light chains. Lastly, the present study provided a structural mechanism of epistasis by solving crystal structures. Such investigation of structural mechanisms was absent in their previous study. Overall, the data quality in this study is very high. In addition, the results have important implications for vaccine development.

Reviewer #1 (Public Review):

The article "Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function" explored the functional roles of MTIF3 during adipocyte differentiation. In persons living with obesity, genetic variation at the MTIF3 locus associates with body mass index and responses to weight loss interventions. MTIF3 regulates mitochondrial protein expression and gene knockouts cause cardiomyopathy in mice. This paper provides insight into the impacts of MTIF3 knockout on adipocyte differentiation and the expression effects of the eQTL on MTIF3 levels. The authors implement a CRISPR/Cas9 gene editing approach coupled with an in vitro platform to detect influences of MTIF3 on adipocyte glucose metabolism and gene expression. This method may serve as a platform to explore knockouts in human cell lines, so it may allow the discovery of new gene x environment influences on in vitro outcomes related to differentiation, growth, and metabolism.

The conclusions of this paper are mostly well supported by data, but some experimental conditions and data analysis needs to be clarified and extended.<br /> 1) The authors use CRISPR/Cas9 to generate the rs1885988 variant in the human white adipocyte cell line and performed a comprehensive validation analysis of gene editing (Figure 1). qPCR analysis showed reduced MTIF3 expression during human adipocyte differentiation (Figure 1E, F). To expand the importance of the rs1885988 variant, the authors should have provided target gene measurements to verify the canonical differentiation profile (e.g., FABP4, ADIPOQ) and help readers understand the overall impact of gene editing at the MTIF3 locus.<br /> 2) The direct mechanistic influences of MTIF3 on adipocyte function remain unclear. MTIF3 regulates the translation initiation of mitochondrial protein synthesis. Western blots of OXPHOS proteins do not per se underscore supercomplex formation, which is also a process mediated by MTIF3. Blue native gel electrophoresis may prove a better method to establish the effects of MTIF3 loss-of-function on supercomplex formation.<br /> 3) Based on the findings, the authors argue that MTIF3 knockout alters the function of adipocytes. However, many of the experiments show fairly small effect sizes (Figure 5A, Figure 6A). How does the MTIF3 knockout explicitly perform functions related to body weight regulation? Gene editing in vivo would have helped to substantiate the authors' conclusions.<br /> 4) In several instances, the authors refer to 'feeding' cells with glucose (line 206, line 171). Feeding experiments often imply complex nutrient interventions in animal models and people, which cannot be easily recapitulated in cell culture. The in vitro experiments simply alter levels of glucose and more precise language would state the specific challenges accurately.

Reviewer #1 (Public Review):

This is thorough, quantitative microbial ecology research on one of the most important problems of species coexistence in infection biology. The intermediate disturbance hypothesis is supported once again, and they show unsurprisingly that nutrition matters for their ratio of coexistence, but more specifically as a novel function of the ratio of metabolic fueling to reproductive rate, which the authors term absolute growth. I like this study for its care and completeness even though the results are fairly intuitive to those in the field of cystic fibrosis microbial ecology.

Reviewer #1 (Public Review):

This manuscript provides evidence of a new pathway of endometrial decidualization through COX2-PGI2-PPARδ through fibroblast activation in response to embryo- derive TNFα, conserved in both mice and humans. This is an interesting finding that sheds light on the understanding of the decidua contribution to pregnancy.

The major inconvenience is how the authors perform the experiments and how they expose their findings. The work needs to be completely rewritten, starting with a clear abstract, with an interconnected introduction, using a defined objective, explaining which organisms/tissues/cells are used in each experiment, the sample size and replicates should be specified in each experiment, and ending with a strong conclusion.

Reviewer #1 (Public Review):

IRF8 is a key transcription factor in the differentiation of hematopoietic cell lineages including dendritic cell (DC) and monocyte/macrophage lineages. The promoter and enhancer regions of Irf8 have been a focus of intense research in recent times. In the submitted study Xu H. et. Al., have first time reported a lncRNA transcribed specifically in the pDC subtype from +32Kb which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. Authors have employed modern-day tools for an in-depth understanding of the role of lncIrf8, its promoter region, and crosstalk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. In the attempt to decipher the crosstalk between the promoter regions of IRF8 and lncIRF8 by employing various in vitro artificial systems, the study falls short of identifying the real significance of the lncIRF8 which is specifically expressed in pDC subtype.

Reviewer #1 (Public Review):

Zhao et al., first show that learning that one odour is paired with a shock and another one is not (a differential associative learning), is impeded by another differential associative learning happening less than 20 min before, the proactive interference. However, the effect of differential associative learning on a previous one (retroactive interference) lasts for at least 60 min (even 1.5h in previous studies, Shuai et al., 2010; Cervantes-Sandoval et al., 2016; Gai et al., 2016). Consistent with previous studies (Shuai et al., 2010), they demonstrate that retroactive interference is dependent on the expression of the small G protein Rac1 which is involved in actin cytoskeleton dynamics in the mushroom body (MB), the insect learning and memory center (Heisenberg et al., 2003). While a reduced expression of Rac1 in the MB only at the adult stage induces less retroactive interference effect, adult expression of a constitutively active Rac1 protein increases retroactive interference. Interestingly, this Rac1 expression manipulation did not alter proactive interference. By interacting with the MAPK pathway, Corkskrew, a tyrosine phosphatase SHP2, regulates the spacing effect during repeated associative learning (Pagani et al., 2009). Given this function, the authors then investigated the function Corkskrew and found that proactive but not retroactive interference involves Corkskrew. They found that reducing adult expression of Corkskrew enhances and prolongs proactive interferences while overexpression of Corkskrew in MB γ neurons reduces proactive interference. The authors finally showed that Corskrew regulates proactive interference upstream of Raf and MAPK in the MB. Overall this work demonstrates with solid evidence that proactive and retroactive interference have different temporal dynamics and molecular bases, as summarized in Figure 4F. A more complete cellular and molecular model of their findings could help the reader to understand how proactive and retroactive interference works.

The strength of this manuscript relies on the clear bidirectional effect of opposite genetic manipulations (over- and decreased gene expression) on proactive or retroactive interference analysed at the behavioural level.

A weakness of this work is that the authors did not pursue the last part of their investigation (on the Raf/MAPK) in the MB neurons but in the overall MB. In addition, Corkskrew seems to regulate the duration of proactive interference but they have not tested such a thing in the downstream Raf/MAPK. Another weakness of this manuscript relies on the absence of some background genetic controls that the field usually use to conclude the effect of a genetic tool (e.g the UAS tools). More explanation in the text is also needed to understand a bit more about the tools used and the brain structure targeted to tackle the cellular and molecular bases of proactive and retroactive interference.

Reviewer #1 (Public Review):

Slusarczyk et al present a very well written manuscript focused on understanding the mechanisms underlying aging of erythrophagocytic macrophages in the spleen (RPM) and its relationship to iron loading with age. The manuscript is diffuse with a broad swath of data elements. Importantly, the manuscript demonstrates that RPM erythrophagocytic capacity is diminished with age, restored in iron restricted diet fed aged mice. In addition, the mechanism for declining RPM erythrophagocytic capacity appears to be ferroptosis-mediated, insensitive to heme as it is to iron, and occur independently of ROS generation. These are compelling findings. However, some of the data relies on conjecture for conclusion and a clear causal association is not clear. The main conclusion of the manuscript points to the accumulation of unavailable insoluble forms of iron as both causing and resulting from decreased RPM erythrophagocytic capacity. In addition, the finding that IR diet leads to increased TF saturation in aged mice is surprising. Furthermore, whether the finding in RPMs is intrinsic or related to RBC-related changes with aging is not addressed. Finally, these findings in a single strain and only female mice is intriguing but warrants tempered conclusions.

Major points:<br /> 1) The main concern is that there is no clear explanation of why iron increases during aging although the authors appear to be saying that iron accumulation is both the cause of and a consequence of decreased RPM erythrophagocytic capacity. This requires more clarification of the main hypothesis on Page 4, line 17-18.<br /> 2) It is unclear if RPMs are in limited supply. Based on the introduction (page 4, line 13-15), they have limited self-renewal capacity and blood monocytes only partially replenished. Fig 4D suggests that there is a decrease in RPMs from aged mice. The %RPM from CD45+ compartment suggests that there may just be relatively more neutrophils or fewer monocytes recruited. There is not enough clarity on the meaning of this data point point.<br /> 3) Anemia of aging is a complex and poorly understood mechanistically. In general, it is considered similar to anemia of chronic inflammation with increased Epo, mild drop in Hb, and erythroid expansion, similar to ineffective erythropoiesis / low Epo responsiveness. It is not surprising that IR diet did not impact this mild anemia. However, was the MCV or MCH altered in aged and IR aged mice?<br /> 4) Page 6, line 23 onward: the conclusion is that KC compensate for the decreased function of RPM in the spleen, based on the expansion of KC fraction in the liver. Is there evidence that KCs are engaged in more erythrophagocytosis in aged mice? Furthermore, iron accumulation in the liver with age does not demonstrate specifically enhanced erythrophagocytosis of KC. Please clarify why liver iron accumulation would not be simply a consequence of increased parenchymal iron similar to increased splenic iron with age, independent of erythrophagocytic activity in resident macrophages in either organ.<br /> 5) Unclear whether the effect on RPMs is intrinsic or extrinsic. Would be helpful to evaluate aged iRPMs using young RBC vs. young iRPMs using old RBCs.<br /> 6) Discussion of aggregates in the spleen of aged mice (Fig 2G-2K and Fig 3) is very descriptive and non-specific. For example, if the iron-rich aggregates are hemosiderin, a hemosiderin-specific stain would be helpful. This data specifically is correlatory and difficult to extract value from.<br /> 7) The aging phenotype in RPMs appears to be initiated sometime after 2 months of age. However, there is some reversal of the phenotype with increasing age, e.g. Fig 4B with decreased lipid peroxidation in 9 month old relative to 6 month old RPMs. What does this mean? Why is there a partial spontaneous normalization?<br /> 8) Does the aging phenotype in RPMs respond to ferristatin? It appears that NAC, which is a glutathione generator and can reverse ferroptosis, does not reverse the decreased RPM erythrophagocytic capacity observed with age yet the authors still propose that ferroptosis is involved. A response to ferristatin is a standard and acceptable approach to evaluating ferroptosis.<br /> 9) The possible central role for HO-1 in the pathophysiology of decreased RPM erythrophagocytic capacity with age is interesting. However, it is not clear how the authors arrived at this hypothesis and would be useful to evaluate in the least whether RBCs in young vs. aged mice have more hemoglobin as these changes may be primary drivers of how much HO-1 is needed during erythrophagocytosis.

Reviewer #1 (Public Review):

The study by Xie et al., investigates whether the entorhinal-DG/CA3 pathway is involved in working memory maintenance. The main findings include a correlation between stimulus and neural similarities that was specific for cued stimulus and entorhinal-DG/CA3 locations. The authors observed similar results (cuing and region specificity) using inverted encoding modeling approach. Finally, they also showed that trials in which participants made a smaller error showed a better reconstruction fidelity on the cued side (compared to un-cued). This effect was absent for larger-error trials.

The study challenges a widely held traditional view that working memory and episodic memory have largely independent neural implementations with the MTL being critical for episodic memory but not for working memory. The study adds to a large body of evidence showing involvement of the hippocampus across a range of different working memory tasks and stimuli. Nevertheless, it still remains unclear what functions may hippocampus play in working memory.

Reviewer #1 (Public Review):

In this study, they demonstrate that neonatal mice produce more CD43- B cell-derived IL-10 following anti-BCR stimulation than adult mice. This is due to autocrine mechanisms whereby anti-BCR stimulation leads to pSTAT5 upregulation and production of IL-6 which then enhances IL-10 production via pSTAT3. These are interesting results for the regulatory B cell field, demonstrating that signaling is different in adult vs neonatal B cells and in particular for researchers studying the mechanisms underpinning the enhanced susceptibility to infection. The authors in the main achieved their aim and the results support their conclusions. However, considering that other studies have previously addressed the mechanisms contributing to enhanced IL-10 production in neonates, in the manuscript, there are some experimental decisions and data presentation decisions that at times need more explanation. An important additional comment is that the introduction/discussion is at times insufficiently referenced to put the data in context for non-experts in this field and that numbers in general are low for an in vitro study.

Reviewer #1 (Public Review):

In this study, the protein composition of exocytotic sites in dopaminergic neurons is investigated. While extensive data are available for both glutamatergic and GABA-ergic synapses, it is far less clear which of the known proteins (particularly proteins localized to the active zone) are also required for dopamine release, and whether proteins are involved that are not found in "classical" synapses. The approach used here uses proximity ligation to tag proteins close to synaptic release sites by using three presynaptic proteins (ELKS, RIM, and the beta4-subunit of the voltage-gated calcium channel) as "baits". Fusion proteins containing BirA were selectively expressed in striatal dopaminergic neurons, followed by in-vivo biotin labelling, isolation of biotinylated proteins and proteomics, using proteins labelled after expression of a soluble BirA-construct in dopaminergic neurons as reference. As controls, the same experiments were performed in KO-mouse lines in which the presynaptic scaffolding protein RIM or the calcium sensor synaptotagmin 1 were selectively deleted in dopaminergic neurons. To control for specificity, the proteomes were compared with those obtained by expressing a soluble BirA construct. The authors found selective enrichments of synaptic and other proteins that were disrupted in RIM but not Syt1 KO animals, with some overlap between the different baits, thus providing a novel and useful dataset to better understand the composition of dopaminergic release sites.

Technically, the work is clearly state-of-the-art, cutting-edge, and of high quality, and I have no suggestions for experimental improvements. On the other hand, the data also show the limitations of the approach, and I suggest that the authors discuss these limitations in more detail. The problem is that there is very likely to be a lot of non-specific noise (for multiple reasons) and thus the enriched proteins certainly represent candidates for the interactome in the presynaptic network, but without further corroboration it cannot be claimed that as a whole they all belong to the proteome of the release site.

Reviewer #1 (Public Review):

This is an exciting paper describing the development of a robust differentiation of the common marmoset induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells and subsequently into spermatogonia-like cells when combined with testis somatic cells. The work is of high quality, but some experimental details and protocols are missing which are necessary for a new protocol development - for example, reconstitution methods and protocols are missing completely in the manuscript and additional details in various aspects of the differentiation and cell maintenance are missing. Despite this, the work is valuable and would be of interest to the germ cell and in vitro gametogenesis communities. The data suggest that marmosets are very similar to humans and macaques, and indeed previously established protocols for PGCLC induction and likely previously published testis reconstitution methods/differentiation were employed here to generate the spermatogonia-like cells.

Reviewer #1 (Public Review):

Sayin et al. sought to determine if bacterial drug resistance has impact on drug efficacy. They focused on gemcitabine, a drug used for pancreatic cancer that is metabolized by E. coli. Using an innovative combination of genetic screens, experimental evolution, and cancer cell co-cultures to reveal that E. coli can evolve resistance to gemcitabine through loss-of-function mutations in nupC, with potential downstream consequences for drug efficacy.

Major strengths include:<br /> • Paired use of genetic screens and experimental evolution<br /> • The spheroid model is a creative approach to modeling the tumor microbiome that I hadn't seen before<br /> • Rigorous microbiology, including accounting for mutation rate in both selective and non-selective conditions<br /> • Timely research question

Major weaknesses of the methods and results include the following:

1. Limited scope of the current work. Just a single drug-bacterial pair is evaluated and there are no experiments with microbial communities, animal models, or attempts to test the translational relevance of these findings using human microbiome datasets.

2. No direct validation of the primary genetic screen. The authors use a very strict cutoff (16-fold-change) without any rationale for why this was necessary. More importantly, a secondary screen is necessary to evaluate the reproducibility of the results, either by testing each KO in isolation or by testing a subset of the library again.

3. Some methodological concerns about the spheroid system. As I understood it, these cells are growing aerobically, which may not be the best model for the microbiome. Furthermore, bacterial auxotrophs are used and only added for 4 hours, which will really limit their impact. It also was unclear if the spheroids are truly sterile. Finally, the data lacks statistical analysis, making it unclear which KOs are meaningful. Delta-cdd looks clearly distinct by eye, but the other two genes are more subtle.

Despite these concerns, this paper is a valuable addition to the growing literature on interactions between cancer chemotherapy and the microbiome, which will definitely inspire follow-up work in complex microbial communities, animal models, and human cohorts.

Reviewer #1 (Public Review):

Monfared et al. construct a three-dimensional phase-field model of cell layers and use it to examine cellular extrusion by independently tuning cell-substrate and cell-cell adhesion. In line with earlier studies (in some of which some of the authors were involved), they find that extrusion is linked to topological defects in cellular arrangement and relieving stress.<br /> The authors claim that their development of the three-dimensional phase field model is crucial for understanding cell extrusion (which I agree with the authors is inherently three-dimensional). However, I don't think the data they currently present clearly demonstrate that the three-dimensional model adds significantly more to our understanding of extrusion events than earlier two-dimensional models.

In the end, I think that the more important achievement of this work -- and one that is likely to be more influential -- is developing a three-dimensional phase field model for cell monolayers rather than any specific result regarding extrusion.

Reviewer #1 (Public Review):

Junctophilin is mostly known as a structural anchor to keep excitation-contraction (E-C) proteins in place for healthy contractile function of skeletal muscle. Here the authors provide a new interesting role in skeletal muscle for Junctophilin (44 kD segment, JPh44), where it translocates to the nuclei and influences gene transcription. Also, the authors have shown that Calpain 1 can digest junctophilin to generate the 44 kDa segment. The field of skeletal muscle generally knows little about how E-C coupling proteins have dual role and influence gene regulation that subsequently may alter the muscle function and metabolism. This part of the manuscript is solid, informative, and novel. The authors use advanced imaging and genetic manipulations of junctophilin etc to support their hypothesis. The authors then also aim to link this mechanism to hyperglycemia in individuals susceptible for malignant hyperthermia as they have elevated levels of the 44kDa segment. However, the power of the analyses are low and the included data comparisons complicates the possibility to interpret the results and its relevance. Nevertheless, the data supporting the novel dual role of junctophilin would likely be appreciated and gain attention to the muscle field.

Reviewer #1 (Public Review):

Xin Gao et al. have performed mouse and human studies on the role of Tfh17 cells in the maintenance and function of central memory (Tcm). The authors conclude that antigen-specific Tfh17 cells outcompete Tfh1 or Tfh2 cells for persistence in the memory phase. Overall, the manuscript is well written and addresses an important issue in the field of Tfh biology. However, further investigation is warranted to understand how the CCR6 expressing Tfhcm contributes to the recall of humoral responses.

The strength of this manuscript is the experimental system in the mouse model, indicating that the adoptive transfer of the in vitro induced Tfh17-like cells induced higher antibody responses and more GC responses than those received Tfh1 or Tfh2 cells. Another strength is the analysis of multiple human cohorts indicating that cTfh17 cells are superior in memory maintenance for HBV, influenza virus, and tetanus toxin vaccines.

The weakness of this manuscript is not clear enough about how the CCR6 expressing Tfhcm contributes to the humoral responses. CCR6 controls mainly the localization of T cells into the inflammatory site but not into the GC site. Therefore, I could not understand the advantage of cTfh17 cells for memory maintenance in vaccination.

Reviewer #1 (Public Review):

This paper provides biochemical and structural evidence for how two different phage proteins inhibit the RecBCD system. The paper provides interesting new insights into the battle that takes place between bacteria and phages and shows how convergent evolution has led to two different phages inhibiting RecBCD in two manners.

Reviewer #1 (Public Review):

This study developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxis-lineage (ScxLin) cells in young mice (DTR). The authors found the depletion reduced cell numbers to similar baselines as aged tendons, indicating that a minimum cell number threshold exists to maintain tendon. This cell loss coincided with disrupted ECM organization and reduced mechanical properties. The DTR and aged tendons had similar protein composition with the main difference compared to young healthy tendons being a loss of high turnover ECM proteins. Via scRNA-seq, DTR and aged tendon had fewer biosynthetic cells, correlating with loss of certain ECM proteins. Interestingly, the remaining cells in the DTR model differed from aged tendons. While somewhat artificial, this depletion model system is an interesting way to investigate mechanisms that lead to reduced ECM turnover and matrix degeneration, and may have inform the mechanisms by which aging affects the maintenance of dense connective tissues.

Reviewer #1 (Public Review):

Zhang et al. have submitted a manuscript demonstrating that STAT3 regulates RNA polymerase III transcription in human tumor cell lines. They present several lines of evidence for this proposal. They show that short hairpin (sh)RNAs that repress STAT3 inhibit Pol III transcription and limit proliferation in HepG2, HuH-7, and 293T cells. Accordingly, overexpression of STAT3 enhances Pol III transcription and increases proliferation in the same cell lines. STAT3-dependent EdU incorporation into synthesized DNA confirmed the proliferation results. The Pol-III transcription inhibitor ML-60218 reversed the positive proliferation effects of STAT3 overexpression. In a mouse xenograft model, overexpression of STAT3 enhanced tumor growth of HepG2 cells, whereas suppression of STAT3 inhibited its growth. Consistent with these results, overexpression of STAT3 enhanced colony formation of HepG2 cells in soft agar, whereas STAT3 suppression inhibited it. ChIP data suggest that STAT3 shRNAs reduce the presence of TBP, BRF1, TFIIIC subunits, and POLR3A at Pol III genes regulated by gene-internal promoters. However, STAT3 does not bind to 5S, 7SL, U6, and tRNA Met genes. In addition, STAT3 does not affect the expression of various Pol III transcription factors. RNA-seq in STAT3-shRNA-expressing HepG2 cells and in shRNA-expressing control cells revealed upregulation of 356 and downregulation of 590 Pol II-transcribed genes. None of the Pol III transcription factors were affected. Among the genes whose expression was enhanced by silencing STAT3 was TP73. Accordingly, overexpression of STAT3 decreased mRNA expression of TP73. To show that TP73 acts downstream of STAT3, the authors demonstrated that HepG2 cells expressing both STAT3 shRNAs and TP73 shRNAs did not exhibit decreased Pol III transcription or proliferation. Consistent with these results, TP73 shRNAs enhance Pol III transcription in HepG2 cells, and overexpression of endogenous TP73 represses Pol III transcription. This inhibition of TP73 is caused by the disruption of TFIIIB assembly. Consequently, TP73shRNAs increase the presence of Pol III factors at Pol III genes without affecting their expression. However, co-IP with alphaTP73 antibodies detected TBP, TFIIIC2, and TFIIICC3 but not BRF1, and vice versa. Moreover, shTP73 enhanced the co-IP of TBP with antiBRF1 antibodies. To discover molecular mechanisms explaining how TP73 expression is indirectly regulated by STAT3, the authors identified miR-106a-5p as a potential regulator. In agreement with a regulatory role, miR-106a-5p mimics reduce TP73 expression and enhance Pol III transcription. Finally, Zhang et al. show that STAT3 binds to the miR-106a-5p promoter and activates miR-106a-5p promoter transcription in a luciferase assay.

Overall, the data presented in this manuscript is clean and convincing and clearly supports the proposed model.

Reviewer #1 (Public Review):

LIS1 is a key dynein regulator and mutations in LIS1 cause the human brain developmental disease lissencephaly. The authors have previously reported a 3.1Å structure of yeast dynein bound to Pac1 (budding yeast LIS1) (Gillies et al., 2022, Elife). However, mutations they designed using the yeast dynein-PAC1 structure had mild effects on human dynein activation in vitro. Here they reported cryo-EM structures of human dynein-LIS1 complexes. While LIS1 and Pac1 bind to roughly the same sites (ring and stalk) of the dynein motor domains at the level of the 2D class averages, their current 3D cryo-EM structures of human dynein bound to one and two human LIS1 beta-propeller domains (4.0 Å and 4.1 Å resolution respectively) have revealed interesting similarities and differences in the interaction sites. In addition, they have provided the locations of missense mutations of LIS1 and dynein that cause lissencephaly and other human brain developmental or neurodegenerative disorders in the context of the human dynein-LIS1 structure. Overall, this first detailed structural analysis on human dynein-LIS1 interaction is well presented and will be important to the dynein field as well as people interested in lissencephaly and/or other neurodevelopmental disorders.

Methods are convincing. I do think it is important to point out that the dynein motor domain rather than full length dynein was used in this study. A relative weakness is the lack of functional analyses on the involved amino acids in the dynein-LIS1 and LIS1-LIS1 interaction interfaces. This is in contrast to the Gillies et al., 2022 paper, in which multiple functional assays were presented. However, knowing that functional assays are much more difficult to perform in human cells than in budding yeast, functional tests can be done in the future after this structural work is published.

Reviewer #1 (Public Review):

This paper provides de novo assembly of full-length 18S and 28S rRNA sequences from 33 mosquito species for whom no genome sequence exists. This is a very useful approach and dataset and provides a new tool by which wild-caught mosquitoes can be species-identified. Additionally, the existence of rRNA reference sequences will allow more effective depletion of these hyperabundant species of RNA prior to investing in RNA-seq of other cellular RNAs from a given sample. It is interesting how phylogenetic trees constructed using 28s rRNA compare to the more standard mitochondrial cytochrome c oxidase I gene. The availability of these data will be very useful for field entomologists and the method by which the rRNAs were obtained may be broadly useful for scientists contemplating a similar approach in less-studied species of medical or biological importance.

Reviewer #1 (Public Review):

The author's findings are as follows:<br /> (A) S.pombe Rlc1 is highly phosphorylated at Ser35 during the mitotic phase under respiratory metabolism.<br /> (B) This phosphorylation promotes the assembly and contraction of the contractile actomyosin ring (CAR).<br /> (C) This mechanism sustains CAR assembly and contraction under the respiratory metabolism, generating ROS. The ROS activates SAPK, which inhibits F-actin formation by reducing For3 expression.

They are important findings explaining the robustness of proliferative and regenerative activity of the eukaryotic cells.<br /> The data presented in the paper support the author's model.

Although there are controversial reports on Rlc1 phosphorylation, whether it activates or inactivates type II myosin in S.pombe, this paper does not terminate the debate. Inhibitory phosphorylation on Rlc1 was reported by Mohan Balasubramanian laboratory and Susan Lowey laboratory before, as the authors referred to in this paper. In contrast, the author's model showed that Rlc1 is phosphorylated to facilitate cell division. Since the molecular mechanism of the CAR assembly and contraction is still not defined well yet, the research field should welcome this study to facilitate the discussion in the future.

Reviewer #1 (Public Review):

Starrett, Gabriel et al. investigated 43 bladder cancers (primary tumors), 5 metastases and 14 normal tissues from 43 solid organ transplant recipients of 5 Transplant Cancer Match Study participating registries (US) for the presence of viral genetic signatures, their host genome integration and possible contribution in carcinogenesis. They isolated DNA and RNA from FFPE tissues to perform state of the art whole genome and transcriptome sequencing. They find that 20 of the primary tumors, 3 of the metastases and 7 of the normal tissues harbor viral signatures with BKPyV and JCPyV being the most prevalent viruses detected. The bulk of the experiments focuses on the 9 BKPyV-positive primary tumors. They report that several of the BKPyV-positive tumors show host genome integration of BKPyV with associated focal amplifications of adjacent host chromosome regions, with chromosome 1 being the most prevalent. Furthermore, BKPyV-positive tumors show a distinct transcriptomic signature with gene expression changes related to DNA damage responses, cell cycle progression, angiogenesis, chromatin organization, mitotic spindle assembly, chromosome condensation/separation and neuronal differentiation. The authors only touch the features of other virus-positive tumors, e.g. those with JCPyV and HPV signals, without offering further detail or thought. The overall mutation signature analysis reveals no clear correlation between presence of viral sequences and tumor mutation burden suggesting that many different, virus-unrelated, factors possibly contribute to bladder cancer genesis and progression. Most striking are cases potentially linked to aristolochic acid, ABOBUCK3 and SBS5. Thus, while the approach is state-of-the-art, the causality of viral signatures and oncogenesis and vice versa remains unsolved.

Strengths:<br /> 1) The study assesses 43 primary tumors, 5 metastases and 14 normal tissues from 43 solid organ transplants of different kinds (24x kidney, 4x liver, 14x heart and/or lung, 1x pancreas) rather than just focusing on a particular organ.

2) The study makes use of whole genome sequencing and transcriptomics and the assayed material is extracted from FFPE tissue, which shows a high level of practical, technical and computational skills and expertise.

Weaknesses:<br /> 1) There have been multiple inconsistencies in sample number and figure references throughout the publication. Is it 19 or 20 cases that have viral sequences detected? A comprehensive checker board table showing all cases, the available tissue samples and respective analyses would be in order.

2) The overall low coverage of the whole genome sequencing, which the authors mention, and the relatively big variation in coverage in both datasets (WGS, transcriptomics) are major limitations of the study. Possibly, this was done to increase specificity, but sorting out and discarding reads may also be problematic. Please comment.

Reviewer #1 (Public Review):

In the manuscript Malagon et al. investigate the nano-organization of asynchronous release at glutamatergic synapses. The authors conduct near-TIRF imaging to probe the localization of synchronous and asynchronous release sites at a single synapses using vGlut-pHluorin. Recent work in the field of synaptic neurobiology has focused on investigating how different modes of neurotransmission are organized in the presynaptic bouton, however, discrepancy remains on the sub-synaptic localization of asynchronous release sites and whether these are independent from synchronous release locations. While a variety of techniques including flash-freeze EM and super resolution microscopy have been employed, the use of live imaging by Malagon et al. provides further insight at the single synapse level.

With an impressive resolution of 27nm in live synapses the authors are able delineate synchronous and asynchronous release events within the same active zone. Furthermore, beyond the pure localization of release sites, how the vGlut-pHluorin fluorescent signal decays following fusion provides insight into distinct endocytic mechanisms. The authors delineate two populations of asynchronous events - one located within the active zone center and one ectopically outside this map (as defined by synchronous release sites). Synchronous and asynchronous demonstrate similar kinetics for the ultra-fast component of endocytosis with major differences in the fast component, which is calcium dependent for synchronous release. The authors demonstrate a consistent pattern in the localization and kinetics of release across multiple types of experiments with both EGTA and Sr2+ manipulation.

Inclusion of further analyses on already acquired data would greatly strengthen the paper, such as if single synapses preference one type of release over another. While this paper reconciles differences in the field major questions still remain; what is the mechanism for calcium independent and calcium dependent endocytosis and how does this differ between synchronous and asynchronous release. This paper sets the stage for further work probing what presynaptic machinery drives the segregation of release, what proteins mediate the differences in exocytosis-endocytosis coupling, and how the nano-organization of asynchronous release sites imparts autonomous roles for asynchronous release.

Reviewer #1 (Public Review):

In this manuscript, May et al use H2B overexpression driven by Keratin14 Cre-mediated excision of a loxP-stop cassette to quantify bulk chromatin dynamics in the live epidermis. They observe heterogeneity of H2B distribution within the basal stem cell layer and a change in distribution when the stem cells delaminate into the suprabasal layers. They further show that these chromatin rearrangements precede cell fate commitment, as detected by adding another Cre-mediated transgene on top (tetO-Cre mediated Keratin10 reporter). Finally, they generate an MST stem-loop transgene for the keratin 10 transcript and observe transcriptional bursting.

The manuscript uses elegant in vivo imaging approaches to describe a set of observations that are logically based on a panel of studies that have used genetic approaches to dissect the role of heterochromatin and histone/DNA modifications in epidermal state transitions (Aarenstrup et al., 2008; Driskell et al., 2012; Eckert et al., 2011; Ezhkova et al., 2011; Ezhkova et al., 2009; Fessing et al., 2011; Indra et al., 2005; Kashiwagi et al., 2007; Lien et al., 2011; Luis et al., 2011; Mejetta et al., 2011; Sen et al., 2010). In addition, the MST stem-loop analysis is a nice technical advance, confirming transcriptional bursting as a general phenomenon of how transcription is regulated in cells (see work from Daniel Larsson, Jonathan Chubb, Arjun Raj, and others). The value of the study in my view is recapitulating these known phenomena in a live tissue setting with high-quality imaging and careful quantification. Overall the analyses appear thorough, although the overall changes appear relatively minor, which is perhaps to be expected from imaging bulk H2B distribution as a proxy for chromatin states.

There is one major technical concern that might impact the interpretation of the data. The authors combine Cre lines for their key conclusions (Krt10 reporter and SRF KO) and analyze single cells that thus express very high levels of Cre. Knowing that Cre will target non-loxP sites and is genotoxic, it is possible that the effect of chromatin is due to high levels of Cre expression in single cells rather than specific effects due to cell state transitions. I would encourage the authors to carefully quantify the dose-dependent effects of the Cre protein (independent of the LoxP sites) on chromatin organization. Along these lines, is the phenotype of the SRF KO similar in the presence of two Cre alleles versus just one?

The second issue is the conclusion of "chromatin spinning". Concluding that chromatin is spinning would in my view require that the authors demonstrate that the nuclear envelope is not moving or is moving less than the chromatin. To support this conclusion the authors should do double imaging for example with LINC complex proteins, an ER/outer nuclear membrane marker, or equivalent.

Reviewer #1 (Public Review):

The shift from outcrossing to selfing is one of the most prevalent evolutionary events in flowering plants. The ecological and genetic backgrounds of these transitions have been of major interest for decades, and one of the key questions was the dating of this transition. Timing of pseudogenization of the self-incompatibility (SI) genes has been used as a proxy for this transition because loss-of-function mutations of SI genes are often responsible for the evolution of predominant selfing. However, SI genes are identified only in a limited number of taxa, and in some cases, the evolution of selfing is not necessarily associated with loss of SI. Therefore, an independent time estimate of the evolution of selfing by genome-wide polymorphism data has been considered important in this field.<br /> <br /> This study provides two statistical methods: SMC-based and ABC-based methods. Both methods intend to detect the genome-wide signatures of the outcrossing-to-selfing transition that alters the ratio of population recombination rate and mutation rate. Authors validated these methods by using the simulated data, confirming that both methods can generally infer the timing of the outcrossing-to-selfing transition jointly with population size changes, although its precision depends on several population history settings.  <br /> <br /> This study would be an important contribution to the field of mating system evolution. By applying the proposed methods to many other selfing organisms, we may be able to see a general picture of the timescale of the outcrossing-to-selfing transition combined with population size dynamics. At the same time, this is one of the extensions of the SMC method, which has already been well utilized for various inferences, including population size and recombination rate heterogeneity.  <br /> <br /> I do not find a major weakness in the methodologies of this study, but I have a few comments on their applications to the data of Arabidopsis thaliana. It is important that these estimates largely depend on what input data is used, especially the mutation rate and recombination rate. While the authors claim that their estimate is older than Bechsgaard's estimate (<413 kyrs), these two studies used different mutation rates: the authors used Ossowski's mutation rate, and Bechsgaard used Koch's mutation rate (Koch et al. MBE 2010). To compare these two estimates, it is important to use the same mutation rate. Shimizu & Tsuchimatsu (2015; Ann Rev Eco Evo Syst) in detail discussed this point and showed that Bechsgaard's estimate becomes <1.48 myrs when Ossowski's mutation rate was used (see Figure 4). Then it happens to overlap with the estimate of this study.<br /> <br /> I am also concerned about the genomic regions of Arabidopsis thaliana used for this study. Authors chose specific five regions based on homogeneity of recombination rates and diversity, but how does the estimated change when randomly chosen genomic regions are used? If it is important to choose "preferable" regions according to the homogeneity of recombination rates and diversity, it may be useful to describe how these regions should be chosen for future applications of this method to other organisms.

Reviewer #1 (Public Review):

Landshammer et al. characterized the role of LNCSOX17, a previously not annotated lncRNA, in the regulation of human endoderm differentiation, further reinforcing the importance of lncRNAs in the regulation of human stem cells differentiation and embryonic development. LNCSOX17 is a unique lncRNA as it does not regulate neighboring SOX17 gene within the TAD.

Employing different loss-of-function methods (i.e. CRISPR-Cas9 MECP2, CRISPR-pAS), the authors manage to untangle the complexity of the LNCSOX17 locus, showing that it contains a distal enhancer of SOX17, a transcription factor crucial for the determination of endodermal cell fate, and, on the other hand, it operates as RNA transcript to guarantee endodermal cell differentiation.<br /> Although lncRNA LNCSOX17 does not regulate SOX17 levels and chromatin occupancy, the authors show that its loss leads to the impairment of definitive endodermal differentiation, in line with the downregulation of endoderm-related genes and markers (eg CXCR4). These data fit well with the LNCSOX17 expression profile, which indeed appears to be restricted to early human definitive endoderm. The combination of multiple genomic techniques to manipulate the LNCSOX17 locus, together with the evidence of a clear phenotype upon loss of this lncRNA, constitutes the strength of the paper.

The mechanism of how LNCSOX17 regulates endoderm differentiation is not clear and should be strengthened. The reader had a feeling that the identification of the LNCSOX17 molecular mechanism in definitive endoderm differentiation was not the focus of the work, but at the same time, it was also clear that the authors put a lot of effort to address this biological question by employing several-omics approaches (i.e. RNA pulldown, RNA-seq, CRISPR, HI-C).

Overall the conclusions are supported by the data but some methods used in this manuscript (eg RIP, Pulldown) should be strengthen with alternative tools. The manuscript is easy to read and the figures are nicely represented.