Reviewer #3 (Public Review):

The spindle checkpoint ensures the accuracy of chromosome segregation by sensing unattached kinetochores during mitosis and meiosis and delays the onset of anaphase. Unattached kinetochores catalyze the conformational activation of the latent open MAD2 (O-MAD2) to the active closed MAD2 (C-MAD2). C-MAD2 is then incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C) to delay anaphase. When all kinetochores are properly unattached, the MAD2-binding protein p31comet and the ATPase TRIP13 extract C-MAD2 from the MCC, leading to MCC disassembly and the conversion of C-MAD2 back to O-MAD2. This action turns off the spindle checkpoint, resulting in APC/C activation and anaphase onset. Cells deficient in p31comet exhibit mitotic delays.

In the current study, Huang et al. have linked p31comet mutations to female infertility. Biallelic loss-of-function alleles of p31comet cause delays in the exiting metaphase of meiosis I and polar body extrusion. The p31comet mutant proteins contain C-terminal truncations and fail to bind to MAD2. Reintroducing full-length p31comet into patient oocytes can bypass the metaphase arrest. Together with a previous study that showed biallelic mutations of TRIP13 caused female infertility, this work established a critical role of the p31comet-TRIP13 module in regulating meiotic progression during oogenesis. As such, this is a significant study.

Reviewer #3 (Public Review):

In this work, Eccleston et. al. use a computational method involving the Rosetta (Flex ddG) suite to infer epistasis in binding free energy changes for combinatorial sets of mutations in the DHFR gene and the drug pyrimethamine. They use this to estimate the most likely path of stepwise mutation accumulation in the evolution of antimalarial drug resistance. The authors also infer likely pathways from different geographical regions from isolated data using a method based on mutation frequencies. They report that these results are broadly consistent with their computational predictions as well.

In contrast to machine learning approaches, the Rosetta Flex ddG method uses physical models at the atomic scale to compute various macromolecular properties. The present paper, therefore, uses atomic-scale molecular properties to make predictions at the population level. As acknowledged by the authors, their method has the limitation that chemical factors other than the free energy changes are largely ignored, as are complications arising from complex population dynamics. Nonetheless, there is reasonable agreement between their predictions and the experimental data, especially at high drug concentrations.

The authors also infer likely trajectories of mutation acquisition from isolate data from various parts of the world. The inference method is based on a simple ranking scheme of mutation frequencies. It is difficult to gauge the reliability of this method, given the complexity of infectious disease dynamics, including confounding factors introduced by varied drug treatment regimens. However, predictions from the computational method are still able to capture some of the general trends in the inferred pathways from isolates, inspiring some confidence in both approaches. The authors emphasize the importance of geographic variation in evolutionary pathways, but their computational method is limited in its ability to provide quantitative insights into the origins of such variation.

A few limitations of the work should be mentioned. It suffers from a lack of summary metrics that quantify the performance of its computational method, which is important for a clearer understanding of its accuracy. While the work is a useful indicator of the potential usefulness of the Rosetta Flex ddG method in enabling evolutionary predictions through macromolecular modeling, the method is applied to a well-studied system and the work remains limited in the novelty of the insights it generates into the dynamics of the evolution of antimalarial drug resistance.

Reviewer #3 (Public Review):

The authors of this study have examined which cation channels specifically confer to ventral tegmental area dopaminergic neurons their autonomic (spontaneous) firing properties. Having brought evidence for the key role played by NALCN and TRPC6 channels therein, the authors aimed at measuring whether these channels play some role in so-called depression-like (but see below) behaviors triggered by chronic exposure to different stressors. Following evidence for a down-regulation of TRPC6 protein expression in ventral tegmental area dopaminergic cells of stressed animals, the authors provide evidence through viral expression protocols for a causal link between such a down-regulation and so-called depression-like behaviors. The main strength of this study lies on a comprehensive bottom-up approach ranging from patch-clamp recordings to behavioral tasks. However, the interpretation of the results gathered from these behavioral tasks might also be considered one main weakness of the abovementioned approach. Thus, the authors make a confusion (widely observed in numerous publications) with regard to the use of paradigms (forced swim test, tail suspension test) initially aimed (and hence validated) at detecting the antidepressant effects of drugs and which by no means provide clues on "depression" in their subjects. Indeed, in their hands, the authors report that stress elicits changes in these tests which are opposed to those theoretically seen after antidepressant medication. However, these results do not imply that these changes reflect "depression" but rather that the individuals under scrutiny simply show different responses from those seen in nonstressed animals. These limits are even more valid in nonstressed animals injected with TRPC6 shRNAs (how can 5-min tests be compared to a complex and chronic pathological state such as depression?). With regard to anxiety, as investigated with the elevated plus-maze and the open field, the data, as reported, do not allow to check the author's interpretation as anxiety indices are either not correctly provided (e.g. absolute open arm data instead of percents of open arm visits without mention of closed arm behaviors) or subjected to possible biases (lack of distinction between central and peripheral components of the apparatus).

Reviewer #3 (Public Review):

In this study, authors used the Drosophila model to characterize molecular details underlying traumatic brain injury (TBI). The authors used the transcriptomic analysis of astrocytes collected by FACS sorting of cells derived from Drosophila heads following brain injury and identified upregulation of multiple genes, such as Pvr receptor, Jun, Fos, and MMP1. Additional studies identified that Pvr positively activates AP-1 transciption factor (TF) complex consisting of Jun and Fos, of which activation leads to the induction of MMP1. Finally, authors found that disruption of endocytosis and endocytotic trafficking facilitates Pvr signaling and subsequently leads to induction of AP-1 and MMP1.

Overall, this study provides important clues to understanding molecular mechanisms underlying TBI. The identified molecules linked to TBI in astrocytes could be potential targets for developing effective therapeutics. The obtained data from transcriptional profiling of astrocytes will be useful for future follow-up studies. The manuscript is well-organized and easy to read. However, I would like to request the authors to address the following issue to improve the quality of their study.

It is unclear why the authors did not explore the involvement of the JNK pathway in their study. While they described the potential involvement of the JNK pathway based on previous literature, they did not include any evidence on the JNK pathway in their own study.

It is important to note that the mechanism by which JNK activates AP-1 is primarily through phosphorylation, not the quantitative control of amounts, as much as I know. This raises questions about the authors' proposed hierarchical relationship between Pvr and AP-1 and the potential involvement of the JNK pathway in mediating this relationship.

Given the significance of the mechanistic link between Pvr and AP-1 in solidifying the authors' conclusion, it would have been beneficial for them to explore the involvement of the JNK pathway in their study, even if only minimally. The lack of such exploration may weaken the overall strength of their findings and the potential implications for understanding TBI.

a person who breaks a law or moral rule:

in a way that proudly refuses to obey authority 對抗地；對立地；違抗地

an act that is criminal or bad 違法行為；罪行；不端行為

a formal agreement or promise between two or more people盟約；契約；協定；承諾

the act of having sex 性交，交媾

a wheeled vehicle for living or travelling in, especially for holidays, that contains beds and cooking equipment and can be pulled by a car （尤指度假時使用，由汽車拖曳的）宿營拖車，旅行拖車

to make something appear by magic, or as if by magic 變戲法；用魔法變出；像變魔術般變出

a man who takes care of a large group of animals of the same type 放牧人

If an animal such as a dog fawns on/upon you, it is very friendly towards you and rubs itself against you.（狗等動物）向…搖尾乞憐

a feeling of great pleasure and attraction, especially because something is very beautiful 陶醉，入迷

Reviewer #3 (Public Review):

This work provides new insights into the regulation of the intracellular effector protein Calcineurin B homologous protein 3 (CHP3). The authors precisely delineate how intracellular calcium signals and myristoylation affect the binding of CHP3 to lipid membranes and the sodium/proton exchanger NHE1. Different mechanisms are known to trigger the exposure of the myristoyl-moiety in the calcium-binding protein family and CHP3 was proposed to use a "calcium-myristoyl switch", which leads to exposure of the myristoyl group due to conformational changes in the protein triggered by calcium-binding. Becker and Fuchs et al. now demonstrate that CHP3 uses a novel mechanism, in which not calcium-binding but binding to the target protein NHE1 triggers exposure of its myristoyl-group. This paper represents a detailed functional characterization of CHP3 and the maximum level of mechanistic interpretation that can be achieved without high-resolution structural information.

The conclusions of this paper are fully supported by the data.

Strengths<br /> The protein biochemistry is of an exceptionally high level, both with respect to the quality of the material and the stringency with which the authors assess and assure the protein quality. The authors purify CHP3 without any affinity tags, and thus in its most representative relevant state. Their validations indicate that complete myristoylation of CHP3 is achieved and that all protein is functional with respect to calcium binding.

The authors go to extensive lengths to convince themselves of the quality of their data and their interpretation. They use an extensive amount of replicates, including both biological and technical replicates. Assays and experimental procedures are verified using model proteins, such as Recoverin. In addition, the authors employ an extensive set of complementary approaches to assure their observations are universal.

Weaknesses<br /> A small weakness is the fact that the interpretation in terms of mechanistic insights contributed by some of the assays employed is rather limited, resulting in comparably unprecise descriptions of the state of the protein such as "affects the conformation and/or flexibility of CHP3" or the "open" and "closed" conformations. As indicated by the authors, structural studies are required to precisely detail the conformational states and delineate their mechanism of action.

The authors imply that the major form of CHP3 is the myristoylated state. However, it remains unclear whether the source of the biological material, which appears to be membrane-only, already implies a significant experimental bias that only allows (or highly favors) the identification of myristoylated CHP3. Without a calcium-signal, unmyristoylated CHP may not associate with membranes, or be less strong, resulting in its depletion upon isolation of the vesicles.

Reviewer #3 (Public Review):

The authors present a study of 13000, Salmonella Typhi genomes from across the globe. Here, they present an overview of the global genomic epidemiology of Salmonella Typhi, in the context of the evolution of antimicrobial resistance. The authors present the temporal trends in the prevalence of Salmonella Typhi genotypes in select regions/ countries as well as the prevalence and antimicrobial resistance. The authors cite travel isolates of Salmonella Typhi as a useful proxy for surveillance in high burden settings where there exists a paucity of genomic data. While the authors acknowledge the limitations of their study, there remain major concerns over sampling bias and representativeness that question the generalizability of their findings.

Based on the methods section, the authors did not make mention of adjusting their prevalence estimates for outbreak investigations. When conducting a population analysis, including outbreak samples can lead to an overestimation of the prevalence of the outbreak strain. First, outbreaks tend to be sampled more densely than isolates from routine surveillance of endemic disease, secondly in an outbreak, you are essentially sampling the same strain multiple times. This needs to be taken into consideration when estimating the prevalence of genotypes in the population. Treating outbreak investigations and routine surveillance equally in calculating prevalence can be misleading if the proportion of outbreak isolates sequenced is greater than the proportion of isolates in the surveillance area that are sequenced.

There are concerns regarding the validity of the results presented in Figures 1-3. These results require a nuanced assessment of the factors that are likely to influence genotypic diversity including type of study, duration of sampling and total number of genomes sequenced. In big Countries like Nigeria and India, where can be heterogeneity in different regions of the country and this needs to also be considered in inferring the prevalence of genotypes.

This heterogeneity in prevalence of genotypes was observed in countries with multiple laboratories. In India for example, the prevalence of lineage 4.3.1.2 ranged from 39% to 82%, in different cities/ regions. The authors have not provided sufficient context on the underlying source of this variation in prevalence. In order to understand the reason for observing these differences there needs to be a discussion around when the samples in each place/ region were conducted, how long the study was conducted, how many isolates were collected and whether this was a routine surveillance, outbreak investigation or other type of study. Similar variability is observed in Nigeria where most isolates were from Abuja (Zankli Medical Center, n=105, 2010-2013) and other sources included Ibadan (University of Ibadan, n=14, 2017-2018), and reference laboratories in England (n=15, 2015-2019) and the USA (n=10, 2016-2019). Given the small sample sizes and the fact that the time periods for sample collection varied, using this dataset to get a snapshot of the prevalence of genotypes in Nigeria can be potentially misleading.

Moreover, the authors cite that 70% of cases in Pakistan are caused by XDR. Is this based on the proportions of isolates that are XDR in this dataset? Klemm et al 2018 sequenced primarily XDR isolates, therefore that dataset is not representative of the wider population. Rasheed et al included on 27 genomes, which were isolated at hospitals. Hospitals isolates may give an overestimated XDR burden because susceptible isolates are likely to get treated successfully with antibiotics alleviating the need for hospitalization. Similarly, Yousafzai et al 2019 was an investigation into an outbreak of ceftriaxone-resistant Salmonella Typhi in Hyderabad, which is a densely sample dataset and not necessarily a representation of the wider population. Aggregating these data may lead to an accumulation of bias that gives a distorted snap shot of the diversity on genotypes. Also, it is unclear whether the number of isolates collected from each of these studies was consistent with time. Thus, changes in the prevalence may be representative of a change in the proportion of genomes that were sampled from individual studies.

One of the major recommendations from this study was that travel associated isolates can be a proxy for surveillance in high burden regions where there is paucity of data. The authors have not demonstrated a rigorous test for representativeness of the travel associated samples. The test conducted by the authors looked at how well the travel isolates correlated with the isolates from other studies conducted in the source population. However, they have not factored in potential biases associated with the studies conducted in the host countries. Also, travel is more likely to encompass a specific socio-economic demography of people who can afford to travel. This leads to underrepresentation of low income individuals and communities, especially in low-income countries. Moreover, the authors have not shown that the phylogenetic placement of the travel isolates supports the claim that they originated from that country. Conclusions drawn from travel associated isolates need to be tempered, while it can be a useful tool for early detection of potentially virulent lineages or lineages that have novel resistance mechanisms, using it to determine prevalence can be misleading.

Other minor observations include:

Introduction needs to trim significantly to be more concise. The authors can demonstrate that Salmonella typhi accumulates resistance genotypes over time and as new antibiotics are introduced resistance mutations become selected for and fixed in the population.

Figure 4 is very similar to Figure 1 of Klemm et al 2018, does not add any new insights.

Reviewer #3 (Public Review):

The authors study the spatio-temporal dynamics of gap and pair-rule pattern formation in the Tribolium embryo. Their main contributions are (1) to perform DNA accessibility profiles at multiple time points and in three domains along the A/P axis, (2) to establish a reporter gene system to examine reporter gene expression driven by candidate enhancers (including live imaging), (3) identify at least three new enhancers, and (4) provide some evidence in favor of the "Enhancer Switching" model.

This is an interesting study that marks solid progress towards an organizing principle of pattern formation. The two practical contributions of the work are impactful: (1) germband region-specific accessibility profiling provides a novel view of the epigenome, especially when combined with profiling of temporal variation. (2) the live imaging system has been powerful in Drosophila studies and this work establishes this system for Tribolium, which has certain advantages as a model.

I have two major concerns: First, the claim about differential accessibility being related to enhancer activity is not really established from the presented data, in my view. This needs to be clarified. (I do believe in the claim to some extent, but not based on presented evidence.) Second, the evidence in support of the Enhancer Switching model for runt should be accompanied by identification of and spatiotemporal profiling of the "speed regulator", if this is not established yet. In addition to these two concerns, the simulations of the Enhancer Switching model need to be described, at least in the outline, in the Methods section.

Reviewer #3 (Public Review):

This work attempts to introduce a new attribute of the receptor- efficiency, a fraction of an agonist binding energy consumed by conformational transition of the receptor from resting to active (open) states. Furthermore, the authors use an impressive set of experimental data (single channel recordings with 23 agonists and 53 mutations) to measure the efficiency for each agonist and mutant receptor. All the estimated efficiencies fall into a few groups and inside each of the efficiency groups there is a strong correlation between agonist affinity and receptor opening efficacy.

The main finding in this study is that estimated efficiencies fall into 5 groups. There is no clear description of the method how the efficiencies were allocated into different groups. Most importantly, it is not clear if the method used takes into account the uncertainty of the efficiency estimate. The study does not show any statistical metrics of the efficiency estimates as well as any other calculated variable such as dissociation equilibrium constants to resting or open states. Surely, the uncertainty of the efficiency should matter especially considering how near the efficiency group values are (eg. difference about 10% between 0.51 and 0.56 or 0.41 and 0.45).

All the tested agonists fell into groups according to the efficiency value attributed to them. It is difficult to see why some of the agonists belong to the same group. For example, it is not obvious at all why such agonists as epibatidine, decamethonium and TMP are in the same group. The question, I guess, arises if this grouping based on efficiency has any predictability value. Furthermore, if a series of mutations with the same agonist fall into different groups, the prediction power of this approach is very limited if one attempts to design a new agonist or look for a new mutation.

Reviewer #3 (Public Review):

My general assessment of the paper is that the analyses done after they find the model are exemplary and show some interesting results. However, the method they use to find the number of states (Calinski-Harabasz score instead of log-likelihood), the model they use generally (HMM), and the fact that they don't show how they find the number of states on HCP, with the Schaeffer atlas, and do not report their R^2 on a test set is a little concerning. I don't think this perse impedes their results, but it is something that they can improve. They argue that the states they find align with long-standing ideas about the functional organization of the brain and align with other research, but they can improve their selection for their model.

Strengths:

- Use multiple datasets, multiple ROIs, and multiple analyses to validate their results<br /> - Figures are convincing in the sense that patterns clearly synchronize between participants<br /> - Authors select the number of states using the optimal model fit (although this turns out to be a little more questionable due to what they quantify as 'optimal model fit')<br /> - Replication with Schaeffer atlas makes results more convincing<br /> - The analyses around the fact that the base state acts as a flexible hub are well done and well explained<br /> - Their comparison of synchrony is well-done and comparing it to resting-state, which does not have any significant synchrony among participants is obvious, but still good to compare against.<br /> - Their results with respect to similar narrative engagement being correlated with similar neural state dynamics are well done and interesting.<br /> - Their results on event boundaries are compelling and well done. However, I do not find their Chang et al. results convincing (Figure 4B), it could just be because it is a different medium that explains differences in DMN response, but to me, it seems like these are just altogether different patterns that can not 100% be explained by their method/results.<br /> - Their results that when there is no event, transition into the DMN state comes from the base state is 50% is interesting and a strong result. However, it is unclear if this is just for the sitcom or also for Chang et al.'s data.<br /> - The involvement of the base state as being highly engaged during the comedy sitcom and the movie are interesting results that warrant further study into the base state theory they pose in this work.<br /> - It is good that they make sure SM states are not just because of head motion (P 12).<br /> - Their comparison between functional gradient and neural states is good, and their results are generally well-supported, intuitive, and interesting enough to warrant further research into them. Their findings on the context-specificity of their DMN and DAN state are interesting and relate well to the antagonistic relationship in resting-state data.

Weaknesses:

- Authors should train the model on part of the data and validate on another<br /> - Comparison with just PCA/functional gradients is weak in establishing whether HMMs are good models of the timeseries. Especially given that the HMM does not explain a lot of variance in the signal (~0.5 R^2 for only 27 brain regions) for PCA. I think they don't report their own R^2 of the timeseries<br /> - Authors do not specify whether they also did cross-validation for the HCP dataset to find 4 clusters<br /> - One of their main contributions is the base state but the correlation between the base state in their Song dataset and the HCP dataset is only 0.399<br /> - Figure 1B: Parcellation is quite big but there seems to be a gradient within regions<br /> - Figure 1D: Why are the DMNs further apart between SONG and HCP than the other states<br /> - Page 5 paragraph starting at L25: Their hypothesis that functional gradients explain large variance in neural dynamics needs to be explained more, is non-trivial especially because their R^2 scores are so low (Fig 1. Supplement 8) for PCA<br /> - Generally, I do not find the PCA analysis convincing and believe they should also compare to something like ICA or a different model of dynamics. They do not explain their reasoning behind assuming an HMM, which is an extremely simplified idea of brain dynamics meaning they only change based on the previous state.<br /> - For the 25- ROI replication it seems like they again do not try multiple K values for the number of states to validate that 4 states are in fact the correct number.<br /> - Fig 2B: Colorbar goes from -0.05 to 0.05 but values are up to 0.87<br /> - P 16 L4 near-critical, authors need to be more specific in their terminology here especially since they talk about dynamic systems, where near-criticality has a specific definition. It is unclear which definition they are looking for here.<br /> - P16 L13-L17 unnecessary<br /> - I think this paper is solid, but my main issue is with using an HMM, never explaining why, not showing inference results on test data, not reporting an R^2 score for it, and not comparing it to other models. Secondly, they use the Calinski-Harabasz score to determine the number of states, but not the log-likelihood of the fit. This clearly creates a bias in what types of states you will find, namely states that are far away from each other, which likely also leads to the functional gradient and PCA results they have. Where they specifically talk about how their states are far away from each other in the functional gradient space and correlated to (orthogonal) components. It is completely unclear to me why they used this measure because it also seems to be one of many scores you could use with respect to clustering (with potentially different results), and even odd in the presence of a log-likelihood fit to the data and with the model they use (which does not perform clustering).<br /> - Grammatical error: P24 L29 rendering seems to have gone wrong

Questions:

- Comment on subject differences, it seems like they potentially found group dynamics based on stimuli, but interesting to see individual differences in large-scale dynamics, and do they believe the states they find mostly explain global linear dynamics?<br /> - P19 L40 why did the authors interpolate incorrect or no-responses for the gradCPT runs? It seems more logical to correct their results for these responses or to throw them out since interpolation can induce huge biases in these cases because the data is likely not missing at completely random.

Reviewer #3 (Public Review):

Rajan et al. used scRNAseq to identify transcription factors responsible for fine-tuning stemness gene expression in neural stem cells (neuroblasts), identifying Fruitless (fru) as a putative regulator of this process. Specifically, loss of the fru isoform C (fruc) results in increased stemness gene expression, while its overexpression leads to the opposite effect. Consistently, overexpression of fruc in a brat-null neuroblast-tumor background is sufficient to partially restore differentiation. Furthermore, by performing extensive genome-wide binding studies, the authors show that Fruc preferentially binds to cis-regulatory elements of stemness genes, with evidence that this transcription factor regulates the Notch-pathway via co-binding with Notch-target genes. The overall impact of FruC on transcription was not assessed.

Their data also shows that instead of regulating the deposition of histone marks associated with active transcription, such as H3K27ac or H3K4me3, loss of fruc results in decreased levels of the repressive mark H3K27me3, namely in the Notch locus or in Notch downstream effector genes, indicating that FruC fine-tunes the expression of their bound genes through maintenance of low-levels of repressive marks at cis-regulatory elements of its target genes. Given the extensive binding profile of FruC the effects promoted by its misexpression in neuroblasts are likely multifactorial.

In addition, the authors also show that PRC2 subunits, Caf1 and Su(z)12, the multisubunit complex responsible for catalyzing H3k27me3 deposition, (1) co-localize with Fru in Fruc-bound regions and (2) their loss partially phenocopies the previous results obtained for fruc depletion. The authors propose a model in which Fruc, via synergistic work with PRC2, is capable of fine-tuning the expression of stemness genes, in particular, Notch and Notch targets in neuroblasts by promoting low levels of transcriptionally repressive histone marks at their target cis-regulatory elements. If FruC and PRC2 functionally interact, and if the recruitment of one factor affects the binding of the other remains unknown.

The authors present an assortment of results that will be useful for those working in transcription and chromatin regulation, namely in the field of Drosophila neural stem cells (neuroblasts). Specifically, the authors provide robust single-cell RNA sequencing results and analysis that can be used by researchers interested in trying to understand the transcriptional state of neuroblasts and their progeny. Additionally, genome-wide binding studies for FruC or PRC2 subunits, together with the profiling of active/repressive histone marks, offer new insights regarding transcription factor or transcriptional repressor binding and the respective read-out in terms of histone modifications. Moreover, the authors propose an interesting model via which transcription regulation of Notch and Notch downstream effectors is rendered via fine-tuning of the transcriptional output. Hence, FruC restrains and limits the levels of its target genes within neuroblasts, avoiding the segregation of high levels of stemness-associated proteins to the progeny, which would incur in fate and differentiation defects. The model proposed here highlights how transcription regulation by histone marks is much more dynamic and layered, other than being dictated only by the mutually exclusive presence of either active or repressive marks.

Reviewer #3 (Public Review):

Previously, it has been shown the essential role of IDA peptide and HAESA receptor families in driving various cell separation processes such as abscission of flowers as a natural developmental process, of leaves as a defense mechanism when plants are under pathogenic attack or at the lateral root emergence and root tip cell sloughing. In this work, Olsson et al. show for the first time the possible role of IDA peptide in triggering plant innate immunity after the cell separation process occurred. Such an event has been previously proposed to take place in order to seal open remaining tissue after cell separation to avoid creating an entry point for opportunistic pathogens. The elegant experiments in this work demonstrate that IDA peptide is triggering the defense-associated marker genes together with immune specific responses including release of ROS and intracellular CA2+. Thus, the work highlights an intriguing direct link between endogenous cell wall remodeling and plant immunity. Moreover, the upregulation of IDA in response to abiotic and especially biotic stimuli are providing a valuable indication for potential involvement of HAE/IDA signalling in other processes than plant development.

Strengths:<br /> The various methods and different approaches chosen by the authors consolidates the additional new role for a hormone-peptide such as IDA. The involvement of IDA in triggering of the immunity complex process represents a further step in understanding what happens after cell separation occurs. The Ca2+ and ROS imaging and measurements together with using the haehsl2 and haehsl2 p35S::HAE-YFP genotypes provide a robust quantification of defense responses activation. While Ca2+ and ROS can be detected after applying the IDA treatment after the occurrence of cell separation it is adequately shown that the enzymes responsible for ROS production, RBOHD and RBOHF, are not implicated in the floral abscission.<br /> Furthermore, IDA production is triggered by biotic and abiotic factors such as flg22, a bacterial elicitor, fungi, mannitol or salt, while the mature IDA is activating the production of FRK1, MYB51 and PEP3, genes known for being part of plant defense process.

Weaknesses:<br /> Even though there is shown a clear involvement of IDA in activating the after-cell separation immune system, the use of p35S:HAE-YFP line represent a weak point in the scientific demonstration. The mentioned line is driving the HAE receptor by a constitutive promoter, capable of loading the plant with HAE protein without discriminating on a specific tissue. Since it is known that IDA family consist of more members distributed in various tissues, it is very difficult to fully differentiate the effects of HAE present ubiquitously.<br /> The co-localization of HAE/HSL2 and FLS2 receptors is a valuable point to address since in the present work, the marker lines presented do not get activated in the same cell types of the root tissues which renders the idea of nanodomains co-localization (as hypothetically written in the discussion) rather unlikely.

Reviewer #3 (Public Review):

Abstract:

The paper follows a recent study by the same team (Jaroenlak et al Plos Pathogens 2020), which documented the dramatic ejection dynamics of the polar tube (PT) in microsporidia using live-imaging and scanning electron microscopy. Although several key observations were reported in this paper (the 3D architecture of the PT within the spore, the speed and extent of the ejection process, the translocation dynamics of the nucleus during germination), the precise geometry of the PT during ejection remain inaccessible to imaging, making it difficult to physically understand the phenomenon.

This paper aims to fill this gap with an indirect "data-driven" approach. By modeling the hydrodynamic dissipation for different unfolding mechanisms identified in the literature and by comparing the predictions with experiments of ejection in media of various viscosities, authors shows that data are compatible with an eversion (caterpillar-like) mechanism but not compatible with a "jack-in-the-box" scenario. In addition, the authors observe that most germinated spores exhibit an inward bulge, which they attribute to buckling due to internal negative pressure and which they suggest may be a mean of pushing the nucleus out of the PT during the final stage of ejection.

Major strengths:

Probably the most impressive aspect of the study is the experimental analysis of the ejection dynamics (velocity, ejection length) in medium of various viscosities over 3 orders of magnitudes, which, combined with a modeling of the viscous drag of the PT tube, provides very convincing evidence that the unfolding mechanism is not a global displacement of the tube but rather an apical extension mechanism, where the motion is localized at the end of the tube. The systematic classification of the different unfolding scenarios, consistent with the previous literature, and their confrontation with data in terms of energy, pressure and velocity also constitute an original approach in microbiology where in-situ and real time geometry is often difficult to access.

Major weaknesses:

1) While the experimental part of the paper is clear, I had (and still have) a hard time understanding the modeling part. Overall, the different unfolding mechanisms should be much better explained, with much more informative sketches to justify the dissipation and pressure terms, magnifying the different areas where dissipation occurs, showing the velocity field and pressure field, etc. In particular, a key parameter of eversion models is the geometry of the lubrication layers inside and outside the spore (h_sheath, h_slip). Where do the values of h_sheath and h_slip come from? What is the physical process that selects these parameters? For clarity, the figures showing the unfolding mechanics in the different scenario should be in the main text, not in the supplemental materials.

2) The authors compute and discuss in several places "the pressure" required to ejection, but no pressure is indicated in the various sketches and no general "ejection mechanism" involving this pressure is mentioned in the paper. What is this "required pressure" and to what element does it apply? I understand that the article focuses on the dissipation required to the deployment of the PT but I find it difficult to discuss the unfolding mechanism without having any idea on the driving mechanism of the movement. How could eversion be initiated and sustained?

3) Finally, the authors do not explain how pressure, which appears to be a positive, driving quantity at the beginning of the process, can become negative to induce buckling at the end of ejection. Although the hypothesis of rapid translocation induced by buckling is interesting, a much better mechanistic description of the process is needed to support it.

Reviewer #3 (Public Review):

In this study, the authors identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66, and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to ZMYND12-variant-bearing human sperm. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma. The manuscript is informative for the clinical and basic research in the field of spermatogenesis and male infertility.

Reviewer #3 (Public Review):

The study from Grechi et al showed that emerging environmental microplastics (MPs) are present in both human and bovine follicular fluid. Moreover, based on the characterization and quantification data, authors treated bovine oocytes with environmentally relevant levels of polystyrene (PS) MPs and found that PS MPs interfered with oocyte maturation in vitro. This study is novel, particularly the first part of MP characterization and quantification, and for the first time confirms the presence of MPs in follicular fluid of humans and large farm animals. These results provide a possible mechanism by which the female infertility rate has been increasing in both humans and large farm animals. The session of exposing MPs to bovine and related oocyte health evaluation can be further improved. For example, authors examined the morphology of the oocyte zona pellucida (ZP) and degeneration and stained oocyte DNA to determine the meiotic maturation status. However, a much more comprehensive oocyte health evaluation can be performed including but not limited to the examination of oocyte spindle morphology, meiotic division, fertilization, early embryo development, mitochondria, and accumulation of ROS. These additional endpoints can provide more robust evidence to determine the impact of MPs on oocyte health. While the oocyte proteomic analysis identified altered proteins, more functional studies and causation experiments can be performed. In addition, authors exposed cumulus-oocyte-complexes (COCs) but not denuded oocytes with MPs, it is crucial to determine whether MPs accumulate in cumulus cells or oocytes or both as well as the compromised oocyte quality is caused by the direct effect of MPs or the indirect impact on somatic cumulus cells to cause a secondary effect on the oocytes.

Reviewer #3 (Public Review):

The authors collected BALF samples from lung cancer patients newly diagnosed with PCP, DI-ILD or ICI-ILD. CyTOF was performed on these samples, using two different panels (T-cell and B-cell/myeloid cell panels). Results were collected, cleaned-up, manually gated and pre-processed prior to visualisation with manifold learning approaches t-SNE (in the form of viSNE) or UMAP, and analysed by CITRUS (hierarchical clustering followed by feature selection and regression) for population identification - all using Cytobank implementation - in an attempt to identify possible biomarkers for these disease states. By comparing cell abundances from CITRUS results and qualitative inspection of a small number of marker expressions, the authors claimed to have identified an expansion of CD16+ T-cell population in PCP cases and an increase in CD57+ CD8+ T-cells, FCRL5+ B-cells and CCR2+ CCR5+ CD14+ monocytes in ICI-ILD cases.

By the authors' own admission, there is an absence of healthy donor samples and, perhaps as a result of retrospective experimental design, also an absence of pre-treatment samples. The entire analysis effectively compares three yet-established disease states with no common baseline - what really constitutes a "biomarker" in such cases? The introduction asserts that "y characterizing the cellular and molecular changes in BAL from patients with these complications, we aim to improve our understanding of their pathogenesis and identify potential therapeutic targets" (lines 82-84). Given these obvious omissions, no real "changes" have been studied in the paper. These are very limited comparisons among three, and only these three, states.

Even assuming more thorough experimental design, the data analysis is unfortunately too shallow and has not managed to explore the wealth of information that could potentially be extracted from the results. CITRUS is accessible and convenient, but also make a couple of big assumptions which could affect data analysis - 1) Is it justified to concatenate all FCS files to analyse the data in one batch / small batches? Could there be batch effects or otherwise other biological events that could confuse the algorithm? 2) With a relatively small number of samples, and after internal feature selection of CITRUS, is the regression model suitable for population identification or would it be too crude and miss out rare populations? There are plenty of other established methods that could be used instead. Have those methods been considered?

Colouring t-SNE or UMAP (e.g. Figure 6C) plots by marker expression is useful for quick identification of cell populations but it is not a quantitative analysis. In a CyTOF analysis like this, it is common to work out fold changes of marker expressions between conditions. It is inadequate to judge expression levels and infer differences simply by looking at colours.

The relatively small number of samples also mean that most results presented in the paper are not statistical significant. Whilst it is understandable that it is not always possible to collect a large number of patient samples for studies like this, having several entire major figures showing "n.s." (e.g. Figures 3A, 4B and 5C), together with limitations in the comparisons themselves and inadequate analysis, make the observations difficult to be convincing, and even less so for the single fatal PCP case where N = 1.

It would also be good scientific practice to show evidence of sample data quality control. Were individual FCS files examined? Did the staining work? Some indication of QC would also be great.

This dataset generated and studied by the authors have the potential to address the question they set out to answer and thus potentially be useful for the field. However, in the current state of presentation, more evidence and more thorough data analysis are needed to draw any conclusions, or correlations, as the authors would like to frame them.

Reviewer #3 (Public Review):

This manuscript by Fisher and colleagues documents the change in clinical activity in English general practices during the COVID-19 pandemic according to a set of indicators of clinical activity. The indicators include measures of clinical reviews (e.g. blood pressure, asthma, chronic obstructive pulmonary disease, medication, and cardiovascular risk reviews), blood tests (e.g. cholesterol, liver function, thyroid function, full blood counts, diabetes monitoring blood tests, and kidney function). All these measures saw a drop during the pandemic, to a varying degree, and some recovered afterwards but others did not.

Clinical activity was measured using SNOMED CT codes, which are standard codes used for recording clinical events in UK GP records.

Strengths:

This is a large and comprehensive study including data from 99% of general practices in England. The indicators are clinically relevant, cover a broad range of disease areas, and have been chosen in a sensible manner, involving relevant stakeholders such as GPs, pharmacists, and pathologists.

The OpenSAFELY platform has the ability to enable federated analyses to be run on raw coded data of almost all patients registered with a GP in England.

The study demonstrates the value of OpenSAFELY in being able to monitor clinical activity in general practice at a detailed level, which is essential for planning and improving health services. The statistical methodology is broadly sound.

Weaknesses:

The measures are all related to chronic physical diseases in adults, with a particular focus on cardiometabolic and respiratory conditions. There are no measures related to mental health, maternal or child health.

The description of the measures does not distinguish between different types of clinical activity e.g. lab tests, clinical measurements, or diagnoses, and all are lumped together as 'codes'. This is a peculiarity of the way that information is recorded in GP systems - many different types of clinical information (such as diagnoses and lab tests) are recorded using a SNOMED CT 'code', and only the exact code differentiates what type of information is in the record.

The codelists were broad and comprehensive, but it is unclear how necessary this is because for some measures e.g. lab tests, laboratories typically record a particular type of test using a single standardised code. Instead of using a broad set of codes in the analysis, the authors could have initially verified which codes are associated with the clinical activity being measured (e.g. a numerical value of a blood pressure measurement) in all practices, as I would expect the same single or small number of codes would be used in all practices. This would have provided a smaller and simpler final codelist.

Reviewer #3 (Public Review):

In this manuscript, Fang and colleagues found that IQGAP1 interacts with TNFAIP2, which activates Rac1 to promote drug resistance in TNBC. Furthermore, they found that ITGB4 could interact with TNFAIP2 to promote TNBC drug resistance via the TNFAIP2/IQGAP1/Rac1 axis by promoting DNA damage repair.

This work has good innovation and high potential clinical significance.

Reviewer #3 (Public Review):

The authors explore an important question concerning the underlying mechanism of representational drift, which despite intense recent interest remains obscure. The paper explores the intriguing hypothesis that drift may reflect changes in the intrinsic excitability of neurons. The authors set out to provide theoretical insight into this potential mechanism.

They construct a rate model with all-to-all recurrent connectivity, in which recurrent synapses are governed by a standard Hebbian plasticity rule. This network receives a global input, constant across all neurons, which can be varied with time. Each neuron also is driven by an "intrinsic excitability" bias term, which does vary across cells. The authors study how activity in the network evolves as this intrinsic excitability term is changed.

They find that after initial stimulation of the network, those neurons where the excitability term is set high become more strongly connected and are in turn more responsive to the input. Each day the subset of neurons with high intrinsic excitability is changed, and the network's recurrent synaptic connectivity and responsiveness gradually shift, such that the new high intrinsic excitability subset becomes both more strongly activated by the global input and also more strongly recurrently connected. These changes result in drift, reflected by a gradual decrease across time in the correlation of the neuronal population vector response to the stimulus.

The authors are able to build a classifier that decodes the "day" (i.e. which subset of neurons had high intrinsic excitability) with perfect accuracy. This is despite the fact that the excitability bias during decoding is set to 0 for all neurons, and so the decoder is really detecting those neurons with strong recurrent connectivity, and in turn strong responses to the input. The authors show that it is also possible to decode the order in which different subsets of neurons were given high intrinsic excitability on previous "days". This second result depends on the extent by which intrinsic excitability was increased: if the increase in intrinsic excitability was either too high or too low, it was not possible to read out any information about past ordering of excitability changes.

Finally, using another Hebbian learning rule, the authors show that an output neuron, whose activity is a weighted sum of the activity of all neurons in the network, is able to read out the activity of the network. What this means specifically, is that although the set of neurons most active in the network changes, the output neuron always maintains a higher firing rate than a neuron with randomly shuffled synaptic weights, because the output neuron continuously updates its weights to sample from the highly active population at any given moment. Thus, the output neuron can readout a stable memory despite drift.

Strengths:<br /> The authors are clear in their description of the network they construct and in their results. They convincingly show that when they change their "intrinsic excitability term", upon stimulation, the Hebbian synapses in their network gradually evolve, and the combined synaptic connectivity and altered excitability result in drifting patterns of activity in response to an unchanging input (Fig. 1, Fig. 2a). Furthermore, their classification analyses (Fig. 2) show that information is preserved in the network, and their readout neuron successfully tracks the active cells (Fig. 3). Finally, the observation that only a specific range of excitability bias values permits decoding of the temporal structure of the history of intrinsic excitabililty (Fig. 2f and Figure S1) is interesting, and as the authors point out, not trivial.

Weaknesses:<br /> 1) The way the network is constructed, there is no formal difference between what the authors call "input", Δ(t), and what they call "intrinsic excitability" Ɛ_i(t) (see Equation 3). These are two separate terms that are summed (Eq. 3) to define the rate dynamics of the network. The authors could have switched the names of these terms: Δ(t) could have been considered a global "intrinsic excitability term" that varied with time and Ɛ_i(t) could have been the external input received by each neuron i in the network. In that case, the paper would have considered the consequence of "slow fluctuations of external input" rather than "slow fluctuations of intrinsic excitability", but the results would have been the same. The difference is therefore semantic. The consequence is that this paper is not necessarily about "intrinsic excitability", rather it considers how a Hebbian network responds to changes in excitatory drive, regardless of whether those drives are labeled "input" or "intrinsic excitability".

2) Given how the learning rule that defines input to the readout neuron is constructed, it is trivial that this unit responds to the most active neurons in the network, more so than a neuron assigned random weights. What would happen if the network included more than one "memory"? Would it be possible to construct a readout neuron that could classify two distinct patterns? Along these lines, what if there were multiple, distinct stimuli used to drive this network, rather than the global input the authors employ here? Does the system, as constructed, have the capacity to provide two distinct patterns of activity in response to two distinct inputs?

Impact:<br /> Defining the potential role of changes in intrinsic excitability in drift is fundamental. Thus, this paper represents a potentially important contribution. Unfortunately, given the way the network employed here is constructed, it is difficult to tease apart the specific contribution of changing excitability from changing input. This limits the interpretability and applicability of the results.

Reviewer #3 (Public Review):

Swallowing is an essential daily activity for survival, and pharyngo-laryngeal sensory function is critical for safe swallowing. In Drosophila, it has been reported that the mechanical property of food (e.g. Viscosity) can modulate swallowing. However, how mechanical expansion of the pharynx or fluid content sense and control swallowing was elusive. Qin et al. showed that a group of pharyngeal mechanosensory neurons, as well as mechanosensory channels (nompC, Tmc, and Piezo), respond to these mechanical forces for regulation of swallowing in Drosophila melanogaster.

Strengths:<br /> There are many reports on the effect of chemical properties of foods on feeding in fruit flies, but only limited studies reported how physical properties of food affect feeding especially pharyngeal mechanosensory neurons. First, they found that mechanosensory mutants, including nompC, Tmc, and Piezo, showed impaired swallowing, mainly the emptying process. Next, they identified cibarium multidendritic mechanosensory neurons (md-C) are responsible for controlling swallowing by regulating motor neuron (MN) 12 and 11, which control filling and emptying, respectively.

Weaknesses:<br /> While the involvement of md-C and mechanosensory channels in controlling swallowing is convincing, it is not yet clear which stimuli activate md-C. Can it be an expansion of cibarium or food viscosity, or both? In addition, if rhythmic and coordinated contraction of muscles 11 and 12 is essential for swallowing, how can simultaneous activation of MN 11 and 12 by md-C achieve this? Finally, previous reports showed that food viscosity mainly affects the filling rather than the emptying process, which seems different from their finding.

Reviewer #3 (Public Review):

The manuscript by Zhang and colleagues attempts to combine genetically barcoded rabies viruses with spatial transcriptomics in order to genetically identify connected pairs. The major shortcoming with the application of a barcoded rabies virus, as reported by 2 groups prior, is that with the high dropout rate inherent in single cell procedures, it is difficult to definitively identify connected pairs. By combining the two methods, they are able to establish a platform for doing that, and provide insight into connectivity, as well as pros and cons of their method, which is well thought out and balanced.

Overall the manuscript is well-done, but I have a few minor considerations about tone and accuracy of statements, as well as some limitations in how experiments were done. First, the idea of using rabies to obtain broader tropism than AAVs isn't really accurate - each virus has its own set of tropisms, and it isn't clear that rabies is broader (or can be made to be broader). Second, rabies does not label all neurons that project to a target site - it labels some fraction of them. Third, the high rate of rabies virus mutation should be considered - if it is, or is not a problem in detecting barcodes with high fidelity, this should be noted. Fourth, there are a number of implicit assumptions in this manuscript, not all of which are equally backed up by data. For example, it is not clear that all rabies virus transmission is synaptic-specific; in fact, quite a few studies argue that it is not (e.g., detection of rabies transcripts in glial cells). Thus, arguments about lost-source networks and the idea that if a cell is lost from the network, that will stop synaptic transmission, is not clear. There is also the very real propensity that, the sicker a starter cell gets, the more non-specific spread of virus (e.g., via necrosis) occurs. Fifth, in the experiments performed in Figure 5, the authors used a FLEx-TVA expressed via a retrograde Cre, and followed this by injection of their rabies virus library. The issue here is that there will be many (potentially thousands) of local infection events near the injection site that TVA-mediated but are Cre-dependent (=off-target expression of TVA in the absence of Cre). This is a major confound in interpreting the labeling of these cells. They may express very low levels of TVA, but still have infection be mediated by TVA. The authors did not clearly explore how expression of TVA related to rabies virus infection of cells near the rabies injection site. A modified version of TVA, such as 66T, should have been used to mitigate this issue. Otherwise, it is impossible to determine connectivity locally. The authors do not go to great lengths to interpret the findings of these observations, so I am not sure this is a critical issue, but it should be pointed out by the authors as a caveat to their dataset. Sixth, the authors are making estimates of rabies spread by comparison to a set of experiments that was performed quite differently. In the two studies cited (Liu et al., done the standard way, and Wertz et al., tracing from a single cell), the authors were likely infecting with a rabies virus using a high multiplicity of infection, which likely yields higher rates of viral expression in these starter cells and higher levels of input labeling. However, in these experiments, the authors need to infect with a low MOI, and explicitly exclude cells with >1 barcode. Having only a single virion trigger infection of starter cells will likely reduce the #s of inputs relative to starter neurons. Thus, the stringent criteria for excluding small networks may not be entirely warranted. If the authors wish to only explore larger networks, this caveat should be explicitly noted.

Overall, if the caveats above are noted and more nuance is added to some of the interpretation and discussion of results, this would greatly help the manuscript, as readers will be looking to the authors as the authority on how to use this technology.

Reviewer #3 (Public Review):

The fundamental question that the authors address in this work is how our brain encodes when two events occur in the same context as against two events occurring in different contexts. Often in life, we encounter situations where it is difficult to alter the memory specifically associated with a place, for e.g. when we try to find our favourite brand of soap after the shopkeeper rearranges the shelves. Here the authors hypothesise that the acquisition of new memory, bound by a context /space, results in extensive remapping. Presumably, such remapping is manifested as an increased difficulty in acquiring new memories that are linked through space, especially when we have to remember both the old and the new. Using a combination of a modified behavioural task and in vivo imaging of neuronal activity, the authors test this hypothesis in mice. The spatial task requires the animal to learn two navigational rules of when to make a turn. Rule 1 requires the animal to make a turn with respect to itself (turn right), and Rule 2 requires the animal to turn with respect to the outside world (turn East). This is achieved by training the mice in two distinct contexts (mazes). Having trained the mice, they acquire the neuronal activation data and analysis through i) correlation matrices and ii) population vectors they test and show that the hypothesis is true. The manuscript is well-written and easy to follow in general. One of the important aspects of this manuscript is the clarity and detail with which the methods are described. The descriptions are unambiguous and complete in detail. This needs to be appreciated.

One of the soft spots of the study is the following: The animal learns to perform the task in two different contexts. It could also be interpreted as a change in context triggering the change in rule rather than a specific context predicting a specific rule as interpreted. I would like to know the authors' views on this. Additionally, the data is from one experiment with six mice, and the data is analyzed through different frameworks to glean information. This is both the boon and bane of the study. Independent/additional cross-validation of the overall effect would be nice to establish the observed phenomena. For e.g., the use of IEGs to identify the ensembles across the two scenarios, and/or inactivation of CA1 to show that rule change is affected or the first memory is also affected.

Reviewer #3 (Public Review):

Flagella are crucial for bacterial motility and virulence of pathogens. They represent large molecular machines that require strict hierarchical expression control of their components. So far, mainly transcriptional control mechanisms have been described to control flagella biogenesis. While several sRNAs have been reported that are environmentally controlled and regulate motility mainly via control of flagella master regulators, less is known about sRNAs that are co-regulated with flagella genes and control later steps of flagella biogenesis.

In this carefully designed and well-written study, the authors explore the role of four E. coli σ28-dependent 3' or 5' sUTR-derived sRNA in regulating flagella biogenesis. UhpU and MotR sRNAs are generated from their own σ28(FliA)-dependent promoter, while FliX and FlgO sRNAs are processed from the 3'UTRs of flagella genes under control of FliA. The authors provide an impressive amount of data and different experiments, including phenotypic analyses, genomics approaches as well as in-vitro and in-vivo target identification and validation methods, to demonstrate varied effects of three of these sRNAs (UhpU, FliX and MotR) on flagella biogenesis and motility. For example, they show different and for some sRNAs opposing phenotypes upon overexpression: While UhpU sRNA increases flagella number and motility, FliX has the opposite effect. MotR sRNA also increases the number of flagella, with minor effects on motility.

While the mechanisms and functions of the fourth sRNA, FlgO, remain elusive, the authors provide convincing experiments demonstrating that the three sRNAs directly act on different targets (identified through the analysis of previous RIL-seq datasets), with a variety of mechanisms. The authors demonstrate, UhpU sRNA, which derives from the 3´UTR of a metabolic gene, downregulates LrhA, a transcriptional repressor of the flhDC operon encoding the early genes that activate the flagellar cascade. According to their RIL-seq data analyses, UhpU has hundreds of additional potential targets, including multiple genes involved in carbon metabolism. Due to the focus on flagellar biogenesis, these are not further investigated in this study and the authors further characterize the two other flagella-associated sRNAs, FliX and MotR. Interestingly, they found that these sRNAs seem to target coding sequences rather than acting via canonical targeting of ribosome binding sites. The authors show FliX sRNA represses flagellin expression by interacting with the CDS of the fliC mRNA. Both FliX and MotR sRNA turn out to modulate the levels of ribosomal proteins of the S10 operon with opposite effects. MotR, which is expressed earlier, interacts with the leader and the CDS of rpsJ mRNA, leading to increased S10 protein levels and S10-NusB complex mediated anti-termination, promoting readthrough of long flagellar operons. FliX interacts with the CDSs of rplC, rpsQ, rpsS-rplV, repressing the production of the encoded ribosomal proteins. The authors also uncover MotR and FliX affect transcription selected representative flagellar genes, with an unknown mechanism.

Overall, this comprehensive study expands the repertoire of characterized UTR derived sRNAs and integrate new layers of post-transcriptional regulation into the highly complex flagellar regulatory cascade. Moreover, these new flagella regulators (MotR, FliX) act non-canonically, and impact protein expression of their target genes by base-pairing with the CDS of the transcripts. Their findings directly connect flagella biosynthesis and motility, highly energy consuming processes, to ribosome production (MotR and FliX) and possibly to carbon metabolism (UhpU).

Specific points to be considered:

- The authors use a crl- hyper-motile strain as WT strain for the study and sometimes also a crl+ strain is used. Can the authors comment on potential reasons why some phenotypes (e.g., UhpU and MotR effects on motility) are only detectable in the crl+ strain or vice versa? Is σS regulation important for the function of these sRNAs?

- In several experiments, a variant of MotR sRNA, MotR* that harbors a 3 nt mutation upstream of the seed sequence is used and seems to mediate stronger phenotypes (impact on flagellar number) upon overexpression compared to WT or phenotypes not retrieved for WT MotR (increased flagellin expression). It would be helpful to have some more clarification throughout the text, why this variant was used, even when OE of WT MotR already has impact on the target and how these three mutated nucleotides impact target regulation. For example, does MotR* show increased RNA stability or Hfq binding compared to MotR? Does the mutation in MotR* impact MotR structure (e.g., based on secondary structure predictions) or increase the complementarity with selected targets at potential secondary binding sites (e.g., based on target predictions)? For example, Fig. S7 shows additional regions of interaction between MotR and fliC mRNA beside the seed sequence. It is also suggested that MotR might have multiple interaction sites on rpsJ mRNA. Additional structure probing or biocomputational predictions could clarify these points.

- It is suggested that UphU impacts on motility via regulation of LrhA, which represses transcription of flhDC, and therefore the flagellar cascade. While LhrA-mediated regulation by UphU is validated based on reporter genes, the effect of UhpU OE on FlhDC levels is not directly examined (Fig. 3). Furthermore, as deletion of LrhA de-represses the flagellar cascade and UhpU was also shown to increase motility, the conclusions could be further strengthened by examining flhDC levels and/or the effect of ∆UhpU (if the sRNA part can be deleted) on motility (reduction) due to relieved down-regulation of LrhA.

-This study provides many opportunities for future follow-work. Now that the four sRNAs and some of their targets and opposing effects on flagella biogenesis have been identified, it will be interesting to see how the sRNAs themselves are temporally regulated throughout the flagella biogenesis cascade and which other targets are regulated by them. Future studies could also provide insights into the mechanism and function of FlgO sRNA, which seems to act via a different mechanism than base-pairing to target RNAs, as well as the global effects of regulation of ribosomal genes via FliX and MotR.

Reviewer #3 (Public Review):

This work focuses on the important problem of how to access the highly polymorphic var gene family using short-read sequence data. The approach that was most successful, and utilized for all subsequent analyses, employed a different assembler from their prior pipeline, and impressively, more than doubles the N50 metric.

The authors then endeavor to utilize these improved assemblies to assess differential RNA expression of ex vivo and short-term cultured samples, and conclude that their results "cast doubt on the validity" of using short-term cultured parasites to infer in vivo characteristics. Readers should be aware that the various approaches to assess differential expression lack statistical clarity and appear to be contradictory. Unfortunately there is no attempt to describe the rationale for the different approaches and how they might inform one another.

It is unclear whether adjusting for life-cycle stage as reported is appropriate for the var-only expression models. The methods do not appear to describe what type of correction variable (continuous/categorical) was used in each model, and there is no discussion of the impact on var vs. core transcriptome results.

Reviewer #3 (Public Review):

In this study, the authors investigate the role of the Notch signalling regulator RBP-J on Ly6Clow monocyte biology starting with the observation that RBP-J-deficient mice have increased circulating Ly6low monocytes. Using myeloid specific conditional mouse models, the authors investigate how RBP-J deficiency effects circulating monocytes and lung interstitial macrophages.

A major strength of this study is that it describes RBP-J as a novel, critical factor regulating Ly6Clow monocyte cell frequency in the blood. The authors demonstrate that RBP-J deficiency leads to increased Ly6Clow monocytes in the blood and lung and CD16.2+ interstitial macrophages in steady state. The authors use a number of different techniques to confirm this finding including bone marrow transplantation experiments and parabiosis.

There are several critical weaknesses that need to be assessed to improve the manuscript, in summary the data presented in the current manuscript are highly descriptive and without mechanistic insight. The inclusion of more mechanistic insight would greatly improve the manuscript.

The authors begin to explore the potential mechanism underlying why Ly6Clow monocytes are increased in the absence of RBP-J - is it through increased survival, increased conversion from Ly6C+ monocytes, increased proliferation or increased egress from the bone marrow. The majority of the data they present here is negative. Whilst I applaud the authors for including negative data, I think that their exploration into how RBP-J leads to increased monocytes does not go far enough and it is critical to understand the mechanism by which RBP-J increases circulating monocytes. Low n-numbers in multiple figures mean that the claims made are not fully supported.

The current title of the paper "RBP-J regulates homeostasis and function of circulating Ly6Clo monocytes" does not fully reflect the manuscript in its current form - there is no exploration of Ly6Clow monocyte functionality in the paper as it stands.<br /> Given that targeting monocytes and macrophages in a range of inflammatory diseases is an attractive yet elusive therapeutic option, understanding the underlying biology that regulates monocyte biology are critically important. This manuscript has the potential to add to our current knowledge of how Ly6Clow monocyte biology is regulated and potentially opens novel avenues for preferentially enhancing Ly6Clow monocytes without influencing Ly6C+ monocytes. This is an attractive proposition for many inflammatory conditions however, considerably more in-depth analysis is required to understand the role of RBP-J in monocyte biology.

Reviewer #3 (Public Review):

Mizukami et al. compare the structure of the coronary arteries in multiple species of amniotes, amphibians, and fish. By selecting species from each of these taxa, the authors were able to evaluate modifications to the coronary arteries during key evolutionary transitions. In mice and quail, they show two populations of vessels that are visible on the developing heart-true coronary arteries on the ventricle and a second population of vessels on the outflow tract known as the ASV., They found that in amphibians, outflow tract vessels were present but ventricular coronary arteries were completely absent. In zebrafish (a more ancestral species) an arterial branch off the rostral section of the hypobranchial artery was shown to have similar anatomical features to outflow tract vessels found in higher organisms. These zebrafish outflow tract arteries also appeared conserved in several chondriichthyes specimens. The authors conclude that rearrangement of the outflow tract vasculature or hypobranchial arteries in fish during evolution, could be homologous to the ASV population of coronary arteries in amphibians and amniotes. These data give new insight into the evolutionary origins of the coronary vasculature.

Major Points

1. The manuscript presents important data on the coronary vascular structure of several different species. However, these data alone do not conclusively demonstrate whether the developmental origins of ASV like vessels are homologous. Therefore, care should be taken when concluding that the outflow tract vessels found in all different species are conserved features. While this is a reasonable hypothesis and should be presented, the manuscript could be improved by also discussing alternate explanations. For example, ASVs in mice originate during embryonic development, while in fish and amphibians outflow tract vessels are formed only in mature animals.

2. Figure 3 A-D: The authors state that "the ASV ran through the outflow tract, then entered the aortic root before reaching the ventricle to form a secondary orifice". Do the authors have serial sections to conclude that the vessel branching off the carotid runs the length of the aorta and is continuous with an orifice at the aortic root? The endothelial projection off the aorta in panel C could reasonably be an independent projection. For example, Chen et al., described similar looking projections in the base of the aorta that were not attached to external vessels. A whole mount approach would be the most convincing to show the attachments of the ASV vessel.

3. Figure 3E: Similar as above, how is it concluded that the orifice is continuous with the ASV and that this projection is not the coronary artery stem?

4. The discussion section could be improved by making some statements more consistent, using more precise or appropriate terminology accepted in the field, and being more cognizant of how the authors' findings fit within the history of the field. For example, when referring to coronary arteries, please clarify whether this refers to ASV/ outflow tract coronary arteries, or true ventricular coronary arteries. In addition, the first sentence of the discussion makes it seem like the origins of coronary arteries were unknown prior to this study, however, their origins have been described in multiple papers previously. The authors could revise their statement to acknowledge these previous findings.

Reviewer #3 (Public Review):

The authors develop and analyze a novel model of microbial communities that considers both space and chemical mediator dynamics explicitly, with the goal of understanding the impact of spatial structure on coexistence. The authors' primary method for assessing the impact of space is to compare numerical simulations of their spatial model to simulations of an equivalent well-mixed model. They explore how spatial structure changes coexistence over a wide range of parameter space, varying parameters such as the ratio of facilitative to inhibitory interactions and the degree of mediator diffusion. They find that spatial structure can have variable effects on richness (the number of cell types within a community), in contrast to existing intuition in the field that spatial structure increases diversity.

Overall, I think the approach that the authors have taken is sound. A very interesting aspect of this model is that the diffusion of mediators and microbes can occur at different rates. In other spatial systems, such as the classic Turing model of pattern formation, differences in diffusion timescales are the key ingredient needed for interesting spatial dynamics. However, while the authors have thoroughly characterized the impact of model parameters on ecological richness, their focus on this single metric provides a somewhat limited view of coexistence in their models. For example, richness considers neither the population composition nor the spatial patterns of coexistence emerging from the model. I also have some concerns about the implementation of the carrying capacity in the model, which in its current form may lead to non-physical outcomes in a small part of the phase space.

Reviewer #3 (Public Review):

Modi et al. developed a novel data-driven computational framework to investigate interactions between multiple brain oscillations and validated this approach in hippocampal CA1 utilizing well-studied changes in oscillations across CA1 layers. This approach provides a new way to investigate complex interactions between diverse neural oscillations during different behaviors. In contrast to standard approaches that classify LFP recordings into a few different oscillatory states which simplify patterns in the LFP, this approach maps a complex state space. The essential idea behind the method is novel and interesting with the potential to expand to other studies of other brain regions or interactions between regions. The authors provide a comprehensive analysis showing how this state space relates to traditional oscillatory states (like delta, theta, and gamma). Among the reported results, it is sometimes unclear what is a validation of their approach versus a novel scientific finding (in the context of the larger literature) and the significance of the finding. Although the overall results seem convincing, the paper is a lacking a demonstration that shows why this approach is of high physiological significance. Furthermore, more evidence showing the specific advantages of using this method in LFP data from a single CA1 layer would make this approach more readily adoptable for the community.

Major concerns:<br /> 1. My primary concern is to provide clear evidence that this approach will provide key insights of high physiological significance, especially for readers who may think the traditional approaches are advantageous (for example due to their simplicity). I think the authors' findings of distinct sleep state signatures or altered organization of the NLG3-KO mouse could serve this purpose. However, right now the physiological significance of these results is unclear. For example, do these sleep state signatures predict later behavior performance, or is altered organization related to other functional impairments in the disease model? Do neurons with distinct sleep state signatures form distinct ensembles and code for related information?<br /> 2. For cells with different mean firing rates during exploration: is that because they are putative fast-spiking interneurons and pyramidal cells? From the reported mean firing rates, I think some of these cells are interneurons. Since mean firing rates are well known to vary with cell type, this should be addressed. For example, the sleep state signatures may be distinct for different putative pyramidal cells and interneurons. This would be somewhat expected considering prior work that has shown different cell types have different oscillatory coupling characteristics. I think it would be more interesting to determine if pyramidal cells had distinct sleep state signatures and, if so, whether pyramidal cells from the same sleep state signature have similar properties like they code for similar things or commonly fire together in an ensemble. It seems the number of cells in Fig. 8 may be limited for this analysis. The authors could use the hc-11 data in addition, which was also tested in this work.<br /> 3. Example traces are needed to show how LFPs change over the state-space. Example traces should be included for key parts of the state-space in Figures 2 and 3.<br /> 4. What is the primary rationale for 200ms time bins? Is this time scale sufficient to capture the slow dynamics of delta rhythm (1-5Hz) with a maximum of 1s duration?<br /> 5. Since oscillatory frequency and power are highly associated with running speed, how does speed vary over the state space. Is the relationship between speed and state-space similar to the results of previous studies for theta (Slawinska and Kasicki, Brain Res 1998; Maurer et al, Hippocampus 2005) and gamma oscillations (Ahmed and Mehta J. Neurosci 2012; Kemere et al PLOS ONE 2013), or does it provide novel insights?<br /> 6. The separation of 9 states (Fig. 6ABC) seems arbitrary, where state 1 (bin 1) is never visited. I suggest plotting the density distribution of the data in Fig. 2A or Fig. 6A to better determine how many states are there within the state space. For example, five peaks in such a density plot might suggest five states. Alternately, clustering methods could be useful to determine how the number of states.<br /> 7. The results in Fig. 4G are very interesting and suggest more variation of sub-states during nonREM periods in sleep1 than in sleep2. What might explain this difference? Was it associated with more frequent ripple events occurring in sleep2?<br /> 8. The state transition results in Fig. 6 are confusing because they include two fundamentally different timescales: fast transitions between oscillatory states and slow dynamics of sleep states. I recommend clarifying the description in the results and the figure caption. Furthermore, how can an animal transition between the same sleep state (Fig. 6EF)? Would they both be in a single sleep state?

Reviewer #3 (Public Review):

Bohannon and colleagues demonstrate that aromatic PUFA analogues positively modulate delayed rectifier potassium channel (Iks) currents, identifying new compounds that could be useful for the treatment of long QT syndrome. The data suggest that aromatic PUFA analogues have two modulatory effects that occur by distinct mechanisms involving hydrogen bonds and ionic interactions. However, the exact determinants of these molecular interactions remain unclear.

Strengths of the study include the following:<br /> 1) By examining a large panel of aromatic PUFA analogues, the study provides a thorough understanding of the relationship between the structure of these analogues and the modulatory effect. Of note, these aromatic PUFA analogues are more efficacious than previously characterized PUFAs such as DHA and N-AT. This knowledge will be important for the design of PUFA analogues for the modulation of IKs current, which could be a strategy for the treatment of long QT syndrome.<br /> 2) By examining the effect of mutations previously shown to disrupt two mechanisms of PUFA modulation, the results suggest that aromatic PUFAs act through the same mechanisms. Furthermore, the effects of the different analogues shed light on the determinants of these binding sites such as the presence of additional hydrogen bonds and electrostatic interactions between the aromatic PUFAs and ion channels.

One limitation of the study is that the structure-activity relationships and effects of the mutations do not provide a complete molecular understanding of how the aromatic PUFA analogues are interacting with the channel. This understanding will require additional studies to examine PUFA analogue binding combined with more extensive mutagenesis. Specifically, the model in Figure 5 suggests that the effect of aromatic PUFAs on the voltage dependence of activation depends on an electrostatic interaction with R231 and a hydrogen bond interaction possibly with T224. Similarly, the effect on channel conductance depends on an electrostatic interaction with K326 through the carboxylate anion of the aromatic PUFA as well as an additional electrostatic interaction with some other part of the protein. It is unclear what residues mediate these interactions. Additionally, the authors propose that T224 is forming a hydrogen bond interaction with the hydroxyl group of NALT, but there appears to be a relatively similar effect of the T224V mutation on NAL-phe, only that the spread in the data makes this effect statistically insignificant. Therefore, the conclusion that T224 mediates NALT action by forming a hydrogen bond with the hydroxyl group (a chemical moiety that is absent in NAL-phe) is not fully supported by the data. A structural model to indicate that T224 is well-positioned to form a hydrogen bond with NALT when it is also interacting with R231 would strengthen this model.

Reviewer #3 (Public Review):

In animals, several recent studies have revealed a substantial role for non-replicative mutagenic processes such as DNA damage and repair rather than replicative error as was previously believed. Much less is known about how mutation operates in plants, with only a handful of studies devoted to the topic. Authors Satake et al. aimed to address this gap in our understanding by comparing the rates and patterns of somatic mutation in a pair of tropical tree species, slow-growing Shorea lavis and fast-growing S. leprosula. They find that the yearly somatic mutation rates in the two species is highly similar despite their difference in growth rates. The authors further find that the mutation spectrum is enriched for signatures of spontaneous mutation and that a model of mutation arising from different sources is consistent with a large input of mutation from sources uncorrelated with cell division. The authors conclude that somatic mutation rates in these plants appears to be dictated by time, not cell division numbers, a finding that is in line with other eukaryotes studied so far.

In general, this work shows careful consideration and study design, and the multiple lines of evidence presented provide good support for the authors' conclusions. In particular, they use a sound approach to identify rare somatic mutations in the sampled trees including biological replicates, multiple SNP-callers and thresholds, and without presumption of a branching pattern. By applying these methods consistently across both species, the authors provide confidence in the comparative mutation rate results. Further steps could be taken to ensure the validity of the results; however, these issues are relatively minor and should minimally impact the overall findings.

Some of the identified somatic mutations (primarily those in individual F1) appear to require two mutation events-one on each chromosome-to be generated and should be either removed or accounted for. Also, while the authors provide estimates of their false positive rate at different filtering thresholds, an assessment of the false negative rate is absent and would help assure readers that the differing number of somatic mutations found is not due to differences in statistical power.

The authors compare the mutation rate per meter of growth, demonstrating that the rate is higher in slow-growing S. laevis: a key piece of evidence in favor of the authors' conclusion that somatic mutations track absolute time rather than cell division. To estimate the mutation rate per unit distance, they regress the per base-pair rate of mutations found between all pairwise branch tips against the physical distance separating the tips (Fig. 2a). While a regression approach is appropriate, the narrowness of the confidence interval is overstated as the points are not statistically independent: internal branches are represented multiple times. (For example, all pairwise comparisons involving a cambium sample will include the mutations arising along the lower trunk.) Regressing rates and lengths of distinct branches might be more appropriate. Judging from the data presented, however, the point estimates seem unlikely to change much.

The most obvious drawback of this study is the low sample size with only two individuals of each species sequenced. To eliminate lingering doubts, it would be helpful to include a more in-depth discussion about stray factors that might affect the authors' conclusions. For example: Could an error in estimation of the trees' ages affect the yearly mutation rate comparisons? If mutations are replicatively driven, could the 30% species difference in the number of cell divisions per meter be sufficient to explain the results?

This work deepens our understanding of how mutation operates at the cellular level by adding plants to the list of eukaryotes in which many mutations appear to derive from non-replicative sources. Given these results, it is intriguing to consider whether there is a fundamental mechanism linking mutation across distantly related species. Plants, generally, present a unique opportunity in the study of mutation as the germline is not sequestered, as it is in animals, and thus the forces of both mutation and selection acting throughout an individual plant's life could in principle affect the mutations transmitted to seed. The authors touch on this aspect, finding no evidence for a reduction in non-synonymous somatic mutations relative to the background rate, but more work-both experimental and observational-is needed to understand the dynamics of mutation and cell-competition within an individual plant. Overall, these results open the door to several intriguing questions in plant mutation. For example, is somatic mutation age-dependent in other species, and do other tropical plants harbor a high mutation rate relative to temperate genera? Any future inquiries on this topic would benefit from modeling their approach for identifying somatic mutations on the methods laid out here.

Reviewer #3 (Public Review):

Ding et al. address the experimental question of whether the microbially derived I3A can exert pro-metabolic effects in an experimental model of diet induced obesity/hepatic steatosis. This was based on previous findings by the authors that high fat diet alters levels of I3A, and that I3A can exert anti-steatotic and anti-inflammatory effects in vitro. The data are robust and the authors provide a plethora of omics-based platforms including proteomics and metabolomics under a variety of treatment paradigms. By performing these studies in vivo in mouse liver tissue, these atlases of proteomic and metabolomic datasets would be of interest to the field of metabolism for future analysis. However, there are several weaknesses identified within this manuscript. Primarily, weaknesses in the interpretation and organization of presented data overshadow the robust data presented and make it difficult for the reader to draw any new biological conclusions. Specifically, this manuscript in its current form is primarily of descriptive nature and does not distill any of the complex datasets presented into digestable conclusions that shed new insight into regulation of hepatic metabolism and inflammation by I3A. In essence, this manuscript in its current form is an in vivo extension to the author's previous in vitro assessment of I3A on liver function. Finally, there is a flaw in the model presented (Supplemental Fig. 9) with regards to the authors linking the anti-inflammatory effects of I3A with the metabolic effects. In fact, the authors present data (Fig. 1&2) that show the opposite of this interpretation in which inflammation is uncoupled from the metabolic effects of I3A in the low dose treatment group. While the authors achieved their main goal of addressing the metabolic effects of I3A in vivo, the organization and interpretation of the data presented in its current form is likely to result in a modest impact on the field.

Reviewer #3 (Public Review):

The manuscript entitled, "Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in ESRD" demonstrates that indoxyl sulfate (IS) induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. The authors conducted well-designed experiments to show that the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Overall, this is a very interesting study that highlights that IS mediated trained immunity may have deleterious outcomes in augmented immune responses to the secondary insult in ESRD. Key findings would help to understand accelerated inflammation in CKD or RSRD.

Reviewer #3 (Public Review):

The study focuses on in vivo and in vitro cellular responses intranasal instillation of glycoforms and mutants of SARS-CoV2 spike trimer or spike bearing VLP. Collectively, the experiments suggest that SARS-CoV2 spike has pro-inflammatory roles through increase M1 macrophage associated cytokines and induction of neutrophil netosis, a proinflammatory cell death mechanisms. These effects seem largely independent of hACE2 interaction and partly depend upon interactions with scavenger receptors on macrophages and neutrophils. A strength of the study is that a number sophisticated methods are used, including intravital microscopy in the cramaster and liver as well as acute lung slice models, to look at uptake of the spike proteins and immune cell dynamics. The weakness is that some of the reagents maybe contaminated with uncharacterized glycoforms and some important controls, such as control spike protein and control VLP are unevenly applied or not included. Given the breadth of the studies, it would be ideal for the authors to prioritise strengthening the most important in vivo results in the best animal models with the strongest controls to be able to realise the full impact of the results.

Reviewer #3 (Public Review):

The manuscript entitled "SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state" by Sebastian-Perez et al. adopted the iPOTD method to compare the chromatin-bound proteome in ESCs and 2C-like cells generated by Dux overexpression. The authors identified 397 chromatin-bound proteins enriched only in ESC and 2C- cells, among which they further investigated TOPBP1 due to its potential role in controlling chromocenter reorganization. SMARCD1, a known interacting protein of TOPBP1, was also investigated in parallel. The authors observed increased size and decreased number of H3K9me3-heterochromatin foci in Dux-induced 2C+ cells. Interestingly, depletion of TOPBP1 or SMARCD1 also led to increased size and decreased number of H3K9me3 foci. However, depletion of these proteins did not affect entry into or exit from the 2C-like state. Nevertheless, the authors showed that both TOPBP1 and SMARCD1 are required for early embryonic development.

Although this manuscript provides new insights into the features of 2C-like cells regarding H3K9me3-heterochromatin reorganization, it remains largely descriptive at this stage. It does not provide new insights into the following important aspects: 1) how SMARCD1 associates with H3K9me3 and contributes to heterochromatin maintenance, 2) how TOPBP1 regulates the expression of SMARCD1 and facilitates its localization in heterochromatin foci, 3) whether the remodelling of chromocenter is causally related to the mutual transitions between ESCs and 2C-like cells. Furthermore, some results are over-interpreted. Additional experiments and analyses are needed to increase the strength of mechanistic insights and to support all claims in the manuscript.

Reviewer #3 (Public Review):

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease with progressive and irreversible deterioration of respiratory functions that lacks curative therapies. The authors investigated a new therapeutic approach to treat idiopathic pulmonary fibrosis by targeting P2RX7/IL-18/IFNG axis.

The current data are mainly based on P2RX7 activator HEI3090 and genetic experiments are lacking to support the primary claim that activation P2RX7/IL-18/IFNG axis is beneficial for IPF.

- Parenteral systemic administration of IFN-γ failed in clinical trials (INSPIRE; NCT00075998). However, this study used i.p. administration of P2RX7 activator HEI3090 to activate P2RX7/IL-18/IFNG axis.

- Activation of P2RX7 NLRP3 inflammasome triggers cell death and the current experiments do not explore IL-18 as a potential therapy that would avoid harmful cell death as a consequence of P2RX7/NLRP3 inflammasome activation.

- Reciprocal bone marrow chimera model is needed to demonstrate the requirement of a hematopoietic compartment for HEI3090's antifibrotic effect.

- There is no evidence to show whether P2RX7 interferes with bleomycin during the generation of the IPF model. Independent IPF models would validate the therapeutic effect of P2RX7.

https://docdrop.org/pdf/Larson---1998---Schenkerian-analysis-of-modern-jazz-Question-p5tbw.pdf/

Schenkerian Analysis of Modern Jazz: Questions about Method Larson, S 1998

Reviewer #3 (Public Review):

"Continuous, long-term crawling behavior characterized by a robotic transport system" by Yu et al. presents their new robotic device to track, reposition, and feed Drosophila larvae as they crawl on an arena. By using a water droplet (or if necessary, suction) to transport larvae from the edge of the arena to the middle, long behavior trajectories can be recorded without losing larvae from the arena or camera field of view. The picker robot is also able to dispense small amounts of apple juice at precise locations to keep larvae alive for extended periods although the food was not sufficient to trigger molting and the development to the next instar stage.

The approach is interesting, but the authors could provide more details on why the approach is necessary for non-expert readers. For example, what are the advantages of using the robot picker compared to simply confining larvae in a closed arena? It's not obvious (to me) that being picked back to the center of the arena is a smaller perturbation compared to running into a chamber wall and changing direction.

The first paragraph of the introduction emphasizes the multiple time scales that are relevant for behavior from rapid stimulus response up to developmental times. This is to set the context of the authors' contribution but I'm not sure it's a fair representation of the state of the art. For example, the authors state that high-bandwidth measurement over long times is prohibitive and cite three Drosophila papers, but there are home-cage monitoring systems that allow continuous recording of mouse behavior over long times with high resolution. At the other end of the spectrum, there have been some long-term behaviour experiments done on worm behaviour with reasonably high time resolution (e.g Stern et al. 10.1016/j.cell.2017.10.041).

The authors train a neural network to segment and track the larvae, however, little information is given on the training process and I don't think it would be possible to reproduce the model based on the description. More details of the network, hyperparameters, and training data would be required to evaluate it.

The authors also state several times that larval identity is maintained throughout the recording, but this isn't quantified. It's not clear whether identity is maintained across collisions of two or more animals by the tracking algorithm or whether these collisions simply don't happen in their data because density is low.

The environment is nominally isotropic, but once larvae have been crawling on the surface for hours, including periodic feeding, there will likely be multiple gradients the larvae may sense. This may not be observable in the data, but should perhaps be mentioned in the text.

The authors show that the picking action results in a small but detectable increase in speed. The degree of perturbation overall depends on the picking frequency so some quantification of the inter-pick time interval would help to interpret whether this perturbation is relevant for a particular experiment. Is there a difference in excitation when larvae are picked successfully on the first try compared to when multiple tries or suction are required?

From the reconstructed trajectory in Figure 4, this interval looks very long compared to speed increase after picking. When reconstructing the trajectory, how are the segments joined? Is it simply by resetting the xy position or also updating rotating to match the previous direction of travel? (I'm guessing the larva can rotate during transport?)

The authors present a simple model in Figure 6 to illustrate the differences between individuals that can be hidden when looking at population distributions. However, the differences they show in the simulation don't seem relevant to the differences they observe in the experiments. Specifically, Fig. 6A and B show a contrast between individuals with similar mean speeds compared to individuals with different (but still unimodal) mean speeds. In contrast, the experimental data in Fig. D shows individual distributions that are quite similar but that are bimodal. So, there is indeed a difference between the individual distributions that is obscured in the population distribution, but is there evidence of larval personality types (line 444)? Similarly, the sentence beginning line 381 doesn't seem right either.

Reviewer #3 (Public Review):

Bushy cells are one of the principal neurons in the cochlear nucleus that provide temporal information to higher auditory nuclei to compare sound signals from both ears. One prominent feature in the auditory processing of bushy cells is that they show enhanced temporal responses compared to the auditory nerve (AN) inputs, thus providing a better temporal representation of the acoustic signals. Another feature of the AN-bushy cell circuit is that AN fibers form large synapses termed "endbulbs" around the soma of bushy cells. Scientists have proposed that the temporal enhancement can be due to the coincidence detection of subthreshold convergent AN inputs, or a first-spike latency-based detection of convergent supra-threshold inputs. However, testing these hypotheses requires knowledge of the detailed anatomical arrangement of the AN inputs onto bushy cells. This paper provides a first look at the 3-D organizations of the pre- and postsynaptic structures of mouse bushy cells at a nanoscale resolution. Furthermore, the authors create a morphology-constrained biophysical model to examine how these structures may affect synaptic integration and auditory processing.

The main finding of the paper is that the authors found two input motifs in the AN-bushy cell circuit: one with all small, subthreshold endbulb inputs (all < 180 um2), and the other with 1-2 large, suprathreshold endbulb inputs (> 180 um2) plus other smaller endbulb inputs. Using modeling, the authors argue that the former group correlates with a physiological phenotype of "coincidence detection", and the latter correlates with a phenotype termed "mixed-mode detection". "Coincidence detection" cells require the coincident firing of many subthreshold presynaptic inputs to evoke a postsynaptic spike; "mixed-mode" cells can either have postsynaptic spikes evoked by the largest input(s) alone, or by the coincident firing of small (plus large) inputs. Interestingly, the authors found that even though the large inputs alone can trigger spikes in the "mixed mode" cells, smaller inputs can further enhance the temporal precision of the spikes. The structural data are of very high quality and clearly show the endbulb inputs comprise various sizes. Whether these inputs are really supra/sub-threshold remains to be explored physiologically, but nevertheless, the model provides a hypothesis for the functional roles of the endbulb of different sizes.

In addition to the finding of "two convergent motifs', the authors also report a first complete map of synaptic inputs to a single bushy cell, and structures that have not been observed before, such as synapses at axon-hillock and axon initial segment, dendritic "hub", "braided" dendrites, non-innervated dendrites, etc. These data, like the previous "two input motifs" observation, are also of very high quality and can be useful resources for the ultrastructural study of the bushy cells.

Strengths:<br /> The strengths of this paper are that the authors obtained unprecedented high-resolution 3-D images of the AN-bushy cell circuit, and they implemented a biophysical model to simulate the neural processing of AN inputs based on these structural data. The 3-D reconstruction of the pre- (input organization) and post- (dendrites and axons) synaptic structures of bushy cells are of high quality, as exemplified by the high-resolution figures and animations. The biophysical modeling, although lacking comparison with in vivo physiological data due to the chosen species (mice), is also solid and well documented. The combination of high-resolution imaging and structure-based modeling, together with the detailed documentation, provides rich information for not only auditory scientists but non-auditory scientists who want to use similar techniques to explore neural circuits.

Weaknesses:<br /> Despite the high quality of the data, the paper is marred by the species they chose: there are very few published in vivo single-unit results from mouse bushy cells, so it is hard to evaluate how well the model predictions fit the real-world data, and how the structural findings address the "fundamental questions" in physiology. If we look at data from other animals such as cats and gerbils, it is true that high-frequency (globular) bushy cells show envelope phase locking, but compared to ANs they are at best only moderately enhanced (gerbils: Frisina et al. 1990: Fig 7 and 10; cats: Joris and Yin 1998 Fig 4); the most prominent enhancement is actually to the temporal fine structures of low-frequency bushy cells (cells tuned to < 1 kHz), which mice lack. Furthermore, the temporal modulation transfer function (tMTF, i.e. the vector strengths vs modulation frequency plots in Fig 7O of the paper) of (globular) bushy cells are mostly low-pass filtered, with a cutoff frequency close to 1 kHz, and the highest vector strength rarely surpasses 0.9 (cats: Rhode 1994 Fig 9, 16, Rhode 2008 Fig 8G, Joris and Yin 1998 Fig 7; and there's one report from mice: Kopp-Scheinpflug et al 2003 Fig 8). Thus, the band-pass tMTFs tuned to 100-200 Hz with vector strengths > 0.9 or 0.95 in this paper (Fig 7O, Fig 8M) do not really match known physiology (in non-mouse species). Again, we know very little about in vivo physiology of mouse (globular) bushy cells and there is of course a possibility that responses in mice may be closer to the predictions of this paper. No rationale (e.g. use of molecular tools or in vitro physiology) is given why the authors focus on the mouse. It seems that the analyses provided here could as well have done on a species with good low-frequency hearing, which may have provided a much more interesting case for understanding the spectacular temporal transformation performed by bushy cells.

Reviewer #3 (Public Review):

In this paper, Ichinose et al. examine mechanisms that contribute to building inhibitory synapses through differential protein release from microtubules. They find that tenurin-2 plays a role in this process in cultured hippocampal neurons via EB1 using a variety of genetic and imaging methods. Overall, the experiments are generally designed well, but it is unclear whether their findings offer a significant advance. The experimental logic flow and rational difficult for readers to follow in the manuscript's current form.

Strengths:<br /> 1) The experiments are generally well designed overall, and appropriate to the questions posed.<br /> 2) Several experimental methods are combined to validate key results.<br /> 3) Use of cutting-edge technologies (i.e. STORM imaging) to help answer key questions in the paper.

Weakness:<br /> 1) Simplifying the text and story line would go a long way to ensure the study results are more effectively communicated. Additional specific suggestions are provided in the recommendations for the authors.<br /> 2) The introduction overall would benefit from simplification so that the reader is given only the information they need to know to understand the question at hand.<br /> 3) MT dynamics are important for paper results, but the background in the paper does not appropriately introduce this topic.<br /> 4) It is a bit unclear from the abstract and introduction how the findings of this paper have significantly advanced the field or taught something fundamentally new about how inhibitory synapses are regulated.<br /> 5) Figure 1 - Line 109, it is obscure why "it was found appropriate" to divide the data into three clusters. This section would better justified by starting with cellular functions and then basing the clusters on these functions.<br /> 6) The proteomic screen and candidate selection is not well justified and the logic steps for arriving at TEN2 are a bit weak. Again, less is more here.<br /> 7) Fig. 2 - The authors should consider whether EB1 overexpression would have functional consequences that alter the results and colocalization.<br /> 8) Fig. 3 - Is immobilization of COS cells using HA tag antibodies a relevant system for study of these questions?<br /> 9) Fig. 4 - The authors should confirm post-synaptic localization in vivo (brain).<br /> 10) Figure 4D-E - The way the STORM results are presented is confusing. The authors state is shows that TEN2 is postsynaptic but before this say that the Abs are the same size as the synaptic cleft so that the results cannot be considered conclusive. This issue should be resolved.<br /> 11) Figure 5 -The authors should examine the levels of gephyrin relative to the levels of knockdown given the knockdown variability.<br /> 12) Functional validation of a reduction in inhibition following TEN2 manipulation would elevate the paper.<br /> 13) Figure 6E - The expression levels of TEN2TM and TEN2NL are important to the outcome of these experiments. How did the authors ensure that the levels of two proteins were the same to begin with?

Reviewer #3 (Public Review):

In this study, the authors examined the expression of GPR110 in a HFD-fed mouse model and validated their findings in human samples. They then performed both gain- and loss-of-function studies on the cellular and systemic metabolic effects of manipulating the levels of GPR110. They further demonstrated that SCD-1 was a downstream effector of GPR110, and the effects of GPR110 could be mediated by SCD-1. This study provides a novel target in NAFLD. Overall the data and analyses well performed and convincing. As the GPR110-SCD1-lipid metabolic phenotype axis is a central theme of the study, I would suggest that the authors further discuss the connection between GPR110 and SCD1, especially the persistent upregulation of SCD1 at late stage of HFD-fed mice (obese mouse model) when GPR110 is very low, for example, whether another regulator plays a more relevant role at this time point.

Reviewer #3 (Public Review):

The manuscript by Shin et al, "Aerobic exercise reverses aging-induced depth-dependent decline in cerebral microcirculation", addresses fundamental questions on the mechanism by which aerobic exercise can reverse several age-related dysfunctions in the cerebral vasculature. This work is solid as they use a wide range of complementary in vivo imaging modalities including two-photon fluorescent imaging, optical coherence tomography, and measurements of PO2 as well as behavioral tests. The experiments specifically examined region-specific differences in the young and aged vasculature and the response to aerobic exercise in superficial cortical areas and importantly in deeper white matter areas. This is a solid contribution because it provides additional understanding of age-related changes in the white matter microcirculation, a brain region where our understanding is incomplete. This work effectively sets the stage to further examine aging-related white matter degeneration, how aerobic exercise ameliorates the vascular decline in aging, and will potentially lead to novel interventions targeting the white matter.

• definition
  • the three inevitables

• the third inevitable

  • bad things will happen

• comment

  • progress traps are the right framework to describe the AI problem

Reviewer #3 (Public Review):

In the work presented in "A label-free method to track individuals and lineages of budding cells", Pietsch et al. use multiple machine learning approaches to identify, delineate, and track yeast cells in microscopy images.

I commend the authors for putting a lot of work into this manuscript and coming up with many new ideas to solve their problem of interest. However, throughout the manuscript, I felt that this manuscript does not work well as a 'methods' paper. Maybe it should have been a paper about the biology, which I find very interesting. My main reason for finding this manuscript not well-suited for a methods paper is that their approach as well as the goal are so specific that it may not be readily adopted by others. I would like to list the number of limitations and particularities of their set-up to support this conclusion:

- The whole problem of small cells not being in focus in a single plane is to a large part due to the high ceiling of the authors' microfluidic chips (6 um according to Crane et al.). Other microfluidic chips have much lower ceilings, keeping cells essentially in 2D. If Pietsch et al. used a lower ceiling, small cells would presumably not be out of focus so frequently nor appear to overlap with other cells, and the usual single z-stack approach would suffice. (Another configuration in which cells appear to overlap is in wells, e.g., 96-well plates, which are similarly not ideal for imaging.) Thus, for the problem of interest to Pietsch et al., I would have used a different chip first and then seen what remains of the identification, segmentation, and tracking problem.

- The method requires a number of z-stacks (although I read somewhere how many z-stacks the method needs, I now cannot find that information any more, which highlights a general problem with the presentation, which I will get to below). This means that the already large amount of data that needs to be acquired with regular 2D images now is multiplied by "n" for each z-stack. More importantly, initially, z-stacks have to be individually labeled for training the neural network. That is n times what other segmentation methods require. So, one would presumably only invest this amount of work if one really cared about the tiniest buds because that is, from what I understand, the main selling point of the method. But how many labs do care about this question and going about it in the exact same way as Pietsch et al.? For example, to just find the exact time a bud appears, most people could just extrapolate the size of a new bud in time to zero or simply use a fluorescent budneck marker. Somebody would have to want to measure the growth rates of the smallest buds without fluorescent labels, which the authors do in this present work. But unless someone wants to repeat this exact measurement, say, with other mutants, I do not see who else would invest a large amount of time and resources for this. Other quantities such as fluorescent protein levels cannot be measured with this approach anyway, i.e., by going through z-stacks with a widefield microscope. One would presumably have to use a confocal microscope.

- Could the problem have been simplified by taking z-stacks but analyzing each as a regular 2D image with existing segmentation methods? If a new bud is detected in any of the z-stacks, it is counted as a new cell. This would allow one to use existing 2D training sets and methods and only add a few images of one's own, whether taken in a single z-stack or not. It would only involve tweaking or augmenting existing methods slightly.

- While a 3D image needs to be fed to the neural network, ultimately, all measurements in this manuscript are 2D measurements, e.g., all growth rates are in units of um^2/h. (Somewhat unexpectedly, the authors use a Myo1-GFP construct to identify the budded phase of cells in Fig. 4, i.e., exactly what this method was designed to avoid.) Thus, the effort of going to 3D is only to make the identification of buds more accurate. So, we are not really dealing with a method that goes from 2D to 3D and reconstructs, for example, the shape of cells in 3D. So, while z-stacks go in, it is not 3D annotations that come out.

- The authors may argue that they want to use their high-ceiling chips because they want to follow aging cells. Or, they may argue that indeed, this method is going to be used more widely because people want to study the growth rates of tiny buds in various mutants. However, then the limitation of their method to convex shapes or shapes that can be represented in cylindrical coordinates is a problem since old cells and many mutants can have strange shapes. In this way, the authors have gone a step back methodologically for reasons that I do not understand.

- Given that the method is tailored to detecting small buds, I also do not understand why the authors do not use a higher magnification objective, e.g., a 100x objective instead of 60x? Maybe the problem becomes much easier that way?

- It is unclear how well the tracking method generalizes for other configurations. Here, the tracking problem is somewhat special because there are only a few cell in and around the traps and frequently cells are washed away. For a method paper, the tracking method would need to be compared and contrasted with others for different kinds of experiments. Since tracking is in the title of the manuscript, it is presumably an important selling point of the manuscript.

- The same applies to the segmentation problem. The traps in the authors' microfluidic chips only keep a small number of cells, avoiding problems that emerge when many cells of similar sizes abut.

Reviewer #3 (Public Review):

Lee et al. identify the Stranded at second (Sas) cell surface protein as an extracellular vesicle (EV) component in Drosophila. They first show that different isoforms of Sas exhibit differential tissue distribution in vivo, with the EV-enriched full-length Sas isoform exhibiting distribution at distant sites away from its cells of origin. They show that Sas is present in EVs purified from Drosophila S2 cells, as assessed using exosome isolation kits and via immuno-electron microscopy. Their data suggest that Sas-bearing EVs preferentially target cells expressing Ptp10D, a receptor tyrosine phosphatase that is a known binding partner of Sas, both in the context of S2 cells and imaginal discs engineered to overexpress Ptp10D and the endocytosis regulatory protein Numb. Through immunoprecipitation (IP) of Sas from S2 cell EVs, as well as validation co-IPs and peptide binding assays, the authors found that Sas can interact with the dArc1 protein (i.e. the orthologue of mammalian Arc, which has the ability to form capsid-like structures) via a conserved protein motif of Sas. Finally, they show that Sas increases the transfer of dArc1 protein and mRNA from Sas-expressing cells to Ptp10D-enriched tissues in vivo. The authors conclude that Sas facilitates the delivery of dArc1 capsids that carry dArc1 mRNA to recipient cells that express Ptp10D.

General Strengths: The in vivo and in vitro data conveying the selective targeting of the full-length Sas isoform to EVs, and the impact on the delivery of dArc1 to distant Ptp10D-expressing cells, are generally strong and supportive of the proposed model. The authors also show convincing data confirming the interaction of Sas with dArc1 by IP-MS and binding assays.

General Weakness: It is not clear if the major biological function of the endogenous Sas-Ptp10D interaction is mediated via EVs. The inclusion of additional data evaluating dArc1 mRNA EV-mediated transfer to the trachea in Sas and/or Ptp10D null mutant flies would strongly enhance the paper and support the role of these proteins in tissue-specific EV targeting in vivo. Moreover, throughout the paper, there are several controls and quantifications missing that would be required in order to strengthen the general conclusions and proposed regulatory model. For instance, it is not clear to what extent Sas and dArc1 proteins are co-enriched within purified EV specimens. Immuno-EM studies or nanoparticle analysis strategies should be implemented to address this aspect. Several of the IF- and FISH-based labeling experiments lacked controls. Also, there are few if any quantifications provided as to the number of tissue specimens that were examined in the various assays as a basis for making specific conclusions.

Reviewer #3 (Public Review):

In this manuscript, Yang et al. claimed the creation of a single-cell atlas of the human anterior cruciate ligament (ACL) using scRNA-seq, spRNA-seq, and transcriptomic profiling. Upon analysis of about 25K cells from healthy and degenerated human ACL, the authors reported the existence of fibroblasts, endothelial cells, pericytes, and immune cells in healthy ACL. Their ratios altered in the degenerative ACL, featuring an increase in fibroblasts and immune cells, as demonstrated by the UMAP. Further characterization revealed the presence of subclusters in each of the four major types of cells. The evolution trajectory, spatial transcriptome, and signaling pathways that may contribute to biphasic ACL degeneration were also explored. These data are valuable, to some extent, in improving the current knowledge regarding ACL cellular heterogeneity, homeostasis, and ligamental degeneration. However, the abovementioned findings are purely derived from computational modeling; the authors haven't validated any of them experimentally in vitro and in vivo, particularly regarding whether there are multiple fibroblast subclusters in the ACL with distinct biology. The spatial transcriptomic analysis is also superficial, and few novel insights were generated. The reported work seems like a window show of fancy technologies rather than a hypothesis-driven investigation. Some figures were not clearly labeled, and figure legends were too brief to follow up the studies. Therefore, the significance of this work and its value as a cell atlas of ACL are compromised.

Reviewer #3 (Public Review):

Macrophages play an important role during heart regeneration. This has been shown in the mouse and zebrafish for example by treating the animals with clodronate liposomes to eliminate phagocytic cells.<br /> The manuscript follows up on a previous observation by the authors performing these experiments in the zebrafish (Lai et al eLife 2017). When comparing regenerative vs non-regenerative teleosts zebrafish resp Medaka they found that macrophages and neutrophils were the cell types more differentially responding in these two species to a cardiac injury.

Here the authors anaylse in extenso neutrophil and macrophage populations using single-cell RNA-seq at different stages of regeneration. They perform FAC sorting of the two populations using specific reporter lines. They also assess the change in these populations upon clodronate treatment. They find that clodronate treatment affects the gene expression profiles of different subsets of macrophages and neutrophils as well as their abundance.

They also show that chlodronate treatment performed several days before cryoinjury depleted macrophages from the heart but after injury overall macrophage number recovers. However, heart regeneration does not. Cardiomyocyte is the only parameter that is not affected, but vasculogenesis and scar resolution is impaired.

The authors conclude that (1) there are different subsets of macrophages and neutrophils, (2) that they interact with each other during regeneration through specific ligand and receptor pairs, and (3) that a cardiac resident population rather than a circulating macrophage population is important for heart regeneration.

The transcriptomic characterization of the two immune cell populations is very exhaustive and rigorous. No functional validation of subpopulation marker genes was performed, but the data as it stands will already be of great value to the community. The figure quality is outstanding.

Reviewer #3 (Public Review):

The authors examined mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. They investigated endothelial cell morphology, migration speed, cell shape, cytoskeletal and focal adhesion maturation in human derived ECFC. To substantiate their in vitro data set, they imaged intersegmental vessel development in zebrafish embryos treated with various inhibitors of translation and acto-myosin remodelling . They conclude that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates endothelial cell motility. Mechanistically, YAP and TAZ induced transcriptional suppression of myosin II activity to maintain dynamic cytoskeletal equilibria. Such transcriptional feedback loop may be necessary for persistent endothelial cell migration and vascular morphogenesis. The authors addressed an interesting aspect of vascular development and I have some comments and suggestions that are listed below.

Comments:

The authors used ECFC - endothelial colony forming cells (circulating endothelial cells that activate in response to vascular injury).

Q: did the authors characterize these cells and made sure that they are truly endothelial cells - for example examine specific endothelial markers, arterial-venous identity markers & Notch signalling status, overall morphology etc prior to the start of the experiment. How were ECFC isolated from human individuals, are these "healthy" volunteers - any underlying CVD risk factors, cells from one patient or from pooled samples, what injury where these humans exposed to trigger the release of the ECPFs into the circulation, etc. The materials & methods on ECFC should be expanded.

The authors suggest that loss of YAP/TAZ phenocopies actinomycin-D inhibition - "both transcription inhibition and YAP/TAZ depletion impaired polarization, and induced robust ventral stress fiber formation and peripheral focal adhesion maturation". However, the cell size of actinomycin-D treated cells (Fig. 1B, top right panel), differs from the endothelial cell size upon siYAP/TAZ (Fig. 1E, top right panel) - and vinculin staining seems more pronounced in actinomyocin-D treated cells (B, bottom right) when compared to siYAP/TAZ group. Cell shape is defined by acto-myosin tension.

Q: besides Fraction of focal adhesion >1um; focal adhesion number did the authors measure additional parameters related to cytoskeleton remodelling / focal adhesions that can substantiate their statement on similarity between loss of YAP/TAZ and actinomycin-D treatment. Would it be possible to make a more specific genetic intervention (besides YAP/TAZ) interfering with the focal adhesion pathway as opposed to the broad spectrum inhibitor actinomyocin-D.<br /> Q: does the actinomycin-D treatment affect responsiveness to Vegf? induce apoptosis or reduce survival of the ECFC?<br /> Q: Which mechanism links ECM stiffness with endothelial surface area in the authors scenario. In zebrafish, activity of endothelial guanine exchange factor Trio specifically at endothelial cell junctions (Klems, Nat Comms, 2020) and endoglin in response to hemodynamic factors (Siekmann, Nat Cell Biol 2017) have been show to control EC shape/surface area - do these factors play a role in the scenario proposed by the authors.<br /> Q: the authors report that EC migrate faster on stiff substrate, and concomitantly these cells have a larger surface area. What is the physiological rationale behind these observations. Did the authors observe such behaviors in their zebrafish ISV model? How do these observation integrate with the tip - stalk cell shuffling model (Jakobsson&Gerhardt, Nat Cell Biol, 2011) and Notch activity in developing ISVs.

The authors examined the formation of arterial intersegmental vessels in the trunk of developing zebrafish embryos in vivo. They used a variety of pharmacological inhibitors of transcription and acto-myosin remodelling and linked the observed morphological changes in ISV morphogenesis with changes in endothelial cell motility.<br /> Q: reduced formation and dorsal extension of ISVs may have several reasons, including reduced EC migration and proliferation. The Tg(fli1a:EGFP) reporter however is not the most suitable line to monitor migration of individual endothelial cells. Can the authors repeat the experiments in Tg(fli1a:nEGFP); Tg(kdrl:HRAS-mCherry) double transgenics to visualise movement-migration of the individual endothelial cells and EC proliferation events, in the different treatment regimes.<br /> ISV formation is furthermore affected by Notch signalling status and a series of (repulsive) guidance cues.<br /> Q: Does de novo blockade of gene expression with Actinomycin D affect Notch signalling status, expression of PlexinD - sFlt1, netrin1 or arterial-venous identify genes.

Remark: the authors may want to consider using the Tg(fli1:LIFEACT-GFP) reporter for in vivo imaging of actin remodelling events.

Remark: the authors report "As with broad transcription inhibition, in situ depletion of YAP and TAZ by RNAi arrested cell motility, illustrated here by live-migration sparklines over 10 hours: siControl: , siYAP/TAZ: (25 μm scale-bar: -)". Can the authors make a separate figure panel for this, how many cells were measured?<br /> Remark: in the wash-out experiments, exposure to the inhibitors is not the same in the different scenarios - could it be that the longer exposure time induces "toxic" side effect that cannot be "washed out" when compared to the short treatment regimes?

Reviewer #3 (Public Review):

This study focuses on defining the specific importance of HSP90 isoforms in stress-resistance. Specifically, addressing the importance of the two HSP90 isoforms alpha and beta in adapting cells to chronic stress. Noting that chronic stresses of different types can induce increases of cellular size, the authors investigated the role of HSP90a/b in this process. Intriguingly, they found that KO of either of these isoforms did not influence chronic stress-dependent increases of cell size. However, they did find that HSF1 plays an important role in this process through undefined mechanisms. The authors go on to show that this increase in cell size appears to be correlated with enhanced protein synthesis during conditions of stress, which allow cells to maintain protein density in the enlarged cell. Intriguingly, this correlation is disrupted in HSP90a/b KO cells, where cell size increases, but there is a deficiency in recovery of protein synthesis following the initial insult. This appears to involve sustained ISR signaling that does not resolve in HSP90-deficient cells. Using a number of different compounds that increase (e.g., CDKi) or inhibit (e.g., rapamycin) cell size changes, the authors demonstrate that protection against chronic stress correlates with cell size and protein density, linking cell expansion to stress resistance.

Overall, this is an observational study that heavily relies on correlation to define a proposed stress responsive signaling mechanism termed the 'rewiring stress response' to explain the coordinated increase in cell size and protein translation in protection against chronic stress.

Due to the reliance on correlation, there remains many questions unanswered related to this work. For example. What is the specific role for HSP90a/b in regulating protein translation during chronic stress through the ISR or related pathways? The authors indicate that the induction of the eIF2a phosphatase GADD34 is not impacted in HSP90-deficient cells, so what role does HSP90 have in this process. Is HSP90 required for proper folding of GADD34? Would you see similar effects in protein translation recovery if other ISR activators are used in HSP90-deficient cells? Addressing this central unanswered question that would significantly enhance the current study. While the authors are undoubtedly pursuing this in subsequent studies, it is difficult to fully gauge the impact of this work without more clarity on that point specifically.

Along the same lines, another critical unanswered question is 'Are similar effects observed in non-dividing cells?' Does chronic stress lead to increases of size and regulation of protein translation in primary cell models that are not undergoing division.

Ultimately, this is an interesting study that does a good job of establishing correlations between increases in cell size and protein translation, but does not get to the really intriguing questions related to this coordination. As this study is extended through either revisions to this manuscript or subsequent papers, the importance of this rewiring stress response in the context of cellular stress and pathologic conditions (e.g., age-associated disease) will become increasing apparent.

Reviewer #3 (Public Review):

In this study, Wang et al extend on their previous finding of a novel quality control pathway, the MAGIC pathway. This pathway allows misfolded cytosolic proteins to become imported into mitochondria and there they are degraded by the LON protease. Using a screen, they identify Snf1 as a player that regulates MAGIC. Snf1 inhibits mitochondrial protein import via the transcription factor Hap4 via an unknown pathway. This allows cells to adapt to metabolic changes, upon high glucose levels, misfolded proteins an become imported and degraded, while during low glucose growth conditions, import of these proteins is prevented, and instead import of mitochondrial proteins is preferred.

This is a nice and well-structured manuscript reporting on important findings about a regulatory mechanism of a quality control pathway. The findings are obtained by a combination of mostly fluorescent protein-based assays. Findings from these assays support the claims well.

While this study convincingly describes the mechanisms of a mitochondria-associated import pathway using mainly model substrates, my major concern is that the physiological relevance of this pathway remains unclear: what are endogenous substrates of the pathway, to which extend are they imported and degraded, i.e. how much does MAGIC contribute to overall misfolded protein removal (none of the experiments reports quantitative "flux" information). Lastly, it remains unclear by which mechanism Snf1 impacts on MAGIC or whether it is "only" about being outcompeted by mitochondrial precursors.

Reviewer #3 (Public Review):

This study performs in vivo recordings of neurons in the mouse superior colliculus and their afferents from the retina, retinal ganglion cells (RGCs). Building on a preparation they previously published, this study adds the use of optogenetic identification of inhibitory neurons (aka optotagging) to compare RGC connectivity to excitatory and inhibitory neurons in SC. Using this approach, the authors characterize connection probability, strength, and response correlation between RGCs and their target neurons in SC, finding several differences from what is observed in the retina-thalamus-visual cortex pathway. As such, this may be a useful dataset for efforts to understand retinocollicular connectivity and computations.

Reviewer #3 (Public Review):

This work provides a new approach to simultaneously control elbow and wrist degrees of freedom using movement based inputs, and demonstrate performance in a virtual reality environment. The work is also demonstrated using a proof-of-concept physical system. This control algorithm is in contrast to prior approaches which electrophysiological signals, such as EMG, which do have limitations as described by the authors. In this work, the movements of proximal joints (eg shoulder), which generally remain under voluntary control after limb amputation, are used as input to neural networks to predict limb orientation. The results are tested by several participants within a virtual environment, and preliminary demonstrated using a physical device, albeit without it being physically attached to the user.

Strengths:<br /> Overall, the work has several interesting aspects. Perhaps the most interesting aspect of the work is that the approach worked well without requiring user calibration, meaning that users could use pre-trained networks to complete the tasks as requested. This could provide important benefits, and if successfully incorporated into a physical prosthesis allow the user to focus on completing functional tasks immediately. The work was also tested with a reasonable number of subjects, including those with limb-loss. Even with the limitations (see below) the approach could be used to help complete meaningful functional activities of daily living that require semi-consistent movements, such as feeding and grooming.

Weaknesses:<br /> While interesting, the work does have several limitations. In this reviewer's opinion, main limitations are: the number of 'movements' or tasks that would be required to train a controller that generalized across more tasks and limb-postures. The authors did a nice job spanning the workspace, but the unconstrained nature of reaches could make restoring additional activities problematic. This remains to be tested.

The weight of a device attached to a user will impact the shoulder movements that can be reliably generated. Testing with a physical prosthesis will need to ensure that the full desired workspace can be obtained when the limb is attached, and if not, then a procedure to scale inputs will need to be refined.

The reliance on target position is a complicating factor in deploying this technology. It would be interesting to see what performance may be achieved by simply using the input target positions to the controller and exclude the joint angles from the tracking devices (eg train with the target positions as input to the network to predict the desired angles).

Treating the humeral rotation degree of freedom is tricky, but for some subjects, such as those with OI, this would not be as large of an issue. Otherwise, the device would be constructed that allowed this movement.

Overall, this is an interesting preliminary study with some interesting aspects. Care must be taken to systematically evaluate the method to ensure clinical impact.

Reviewer #3 (Public Review):

Drougard et al. explore microglial detection of a switch to high-fat diet and a subsequent metabolic response that benefits memory. The findings are both surprising and novel in the context of acute high-fat intake, with convincing evidence of increased CSF palmitate after 3 days of HFD. While the authors demonstrate compelling signs of microglial activation in multiple brain regions and unique metabolite release in tracing studies, they should address the following areas prior to acceptance of this manuscript.

Major Points:<br /> 1. It appears that the authors perform key metabolic assays in vitro/ex vivo using primary microglia from either neonatal or adult mice, which should be more clearly delineated especially for the 13C-palmitate tracing. In the case of experiments using primary microglia derived from mixed glial cultures stimulated with M-CSF, this system relies on neonatal mice. This is understandable given the greater potential yield from neonatal mice, but the metabolic state and energetic demands of neonatal and adult microglia differ as their functional roles change across the lifespan. The authors should either show that the metabolic pathways they implicate in neonatal microglia are also representative of adult microglia or perform additional experiments using microglia pooled from adult mice, especially because they link metabolites derived from neonatal microglia (presumably not under the effects of acute HFD) to improved performance in behavioral assays that utilize adult mice.

2. The authors demonstrate that 3 days of HFD increases circulating palmitate by CSF metabolomics and that microglia can readily metabolize palmitate, but the causal link between palmitate metabolism specifically by microglia and improved performance in behavioral paradigms remains unclear. A previous body of research, alluded to by the authors, suggests that astrocyte shuttling of lactate to neurons improves long-term and spatial memory. The authors should account for palmitate that also could be derived from astrocyte secretion into CSF, and the relative contribution compared to microglia-derived palmitate. Specifically, although microglia can metabolize the palmitate in circulation, there is no direct evidence that the palmitate from the HFD is directly shuttled to microglia and not, for example, to astrocytes (which also express CX3CR1). Thus, the Barnes Maze results could be attributed to multiple cell types. Furthermore, the evidence provided in Figure 5J is insufficient to claim a microglia-dependent mechanism without showing data from mice on HFD with and without microglia depletion (analogous to the third and fourth bars in panel K).

3. Given the emphasis on improved cognitive function, there is minimal discussion of the actual behavioral outcomes in both the results and discussion sections. The data that HFD-treated animals outperform controls should be presented in more detail both in the figure and in the text. For example, data from all days/trials of the Barnes Maze should be shown, including the day(s) HFD mice outperform controls. Furthermore, the authors should either cite additional literature or provide experimental evidence supporting the notion that microglia release of TCA-associated substrates into the extracellular milieu after HFD specifically benefits neuronal function cellularly or regionally in the brain, which could translate to improved performance in classical behavioral paradigms. The single reference included is a bit obscure, given the study found that increased lactate enhances fear memory which is a neural circuit not studied in the current manuscript. Are there no additional studies on more relevant metabolites (e.g., itaconate, succinate)?

Minor Points:<br /> 1. In Figure 5J the latency to find the hole was noticeably higher (mean around 150s) than the latency in panel K (mean around 100s for controls, and 60s for Drp1MGWT on HFD). This suggests high variability between experiments using this modified version of the Barnes Maze, despite the authors' assertion that a "standard" Barnes Maze was employed and the results were reproducible at multiple institutions. Why do Drp1MGWT mice on control diet find the escape hole significantly faster than WT mice on control diet in panel J? Given the emphasis on cognitive improvement following acute HFD as a novel finding, the authors should explain this discrepancy.

2. The authors highlight in the graphical abstract and again in Figure 4A the formation of lipid droplets following palmitate exposure as evidence of that microglia can process fatty acids. They later suggest that a lack of substantial induction of lipid droplet accumulation suggests that microglia are metabolically wired to release carbon substrates to neighboring cells. Clarification as to the role of lipid droplet formation/accumulation in explaining the results would eliminate any possible confusion.

3. In many bar graphs showing relatively modest effects, it would be helpful to use symbols to also show the distribution of sample and animal replicates (especially behavioral paradigms).

Reviewer #3 (Public Review):

In this manuscript by Douglas et al., the authors used a functional genomics approach to understand how Staphylococcus aureus survives in the bloodstream to cause bacteraemia. They identified seven novel genes that affect serum survival. The study focused on tcaA, a gene associated with resistance to the antibiotic teicoplanin and is activated when exposed to serum and plays a role in producing a critical virulence factor called wall teichoic acids (WTA) in the cell envelope. This protein affects the bacteria's sensitivity to cell wall attacking agents, human defense fatty acids, and antibiotics, as well as autolytic activity and lysostaphin sensitivity. The data in this study suggested that TcaA play a role in the ligation or retention of WTA within the cell wall. However, more work is needed to clarify that part. Interestingly, despite making the bacteria more vulnerable to serum killing, tcaA contributes to S. aureus virulence by altering the cell wall architecture, as demonstrated by the wild type strain outcompeting the tcaA mutant in a Mouse Co-infection model. The study raises an important point that TcaA in S. aureus may represent a system balancing two scenarios: it makes the bacteria more susceptible to serum killing, potentially limiting bacteraemia and providing long-term benefits between hosts; however, once established in the bloodstream, the bacteria survive and thrive, causing successful bacteraemia, as per the short-sighted evolution of virulence hypothesis. This duality highlights the complex interplay between within-host and between-host fitness in bacterial evolution. I strongly suggest creating a graphical abstract to illustrate the complex relationship between within-host and between-host fitness scenarios involving TcaA. Having this visual representation in the discussion will enhance comprehension and provide a concise summary of the complex system for the reader.

In this manuscript, the authors achieved their aims, and the results support their conclusions. This work will be important for understanding this complex system and for developing novel therapeutics and vaccines for S. aureus.

Reviewer #3 (Public Review):

The regulation of transporters in many physiological systems is poorly known. Here, Forster and colleagues describe how activity of an inorganic carbon transporter, SbtA, in the bacterial carbon concentrating mechanism is regulated by the PII protein SbtB. Although there is now significant structural knowledge of the system and many potential SbtB-regulating small molecule effectors are known, Forster and colleagues clarify, how the adenylate charge in the cell, rather than any single metabolite, is the important regulatory effector. This is critical for the endogenous function, as the cyanobacterial host undergoes dramatic changes in adenylate charge over the course of a diurnal cycle and this result explains how the channel is regulated to efficiently function in CO2 assimilation. The manuscript is generally clear and the data generally supportive of the conclusions as written. However, there are several instances where additional clarification and/or experiments are needed to confirm the major findings of the paper.

Reviewer #3 (Public Review):

In this manuscript, the authors use behavior, calcium imaging, and circuit modulation (DREADDs etc) to assess dopamine regulation of prefrontal cortical circuits in the mouse. The authors have previously established that activation of dopamine inputs to prefrontal cortex during adolescence can drive increases in mPFC DA bouton number and enhanced mPFC activity in WT mice. Here the authors use two mouse models - one with a reporter replacing the Arc gene, and another with knockout of the schizophrenia-associated gene Disc1, both of which are thought to have reduced prefrontal cortical activity. First they trained mice on a Y-maze and showed impaired performance in the Arc knockout. Then they demonstrated selective disruption of neuronal firing with calcium imaging at the time of the decision in the task. The Arc mice were found to have reduced dopamine bouton density, and adolescent activation of the DA neurons corrected this as well as the PFC firing and the behavior. Similar data were shown in the Disc1 KO. The data are well controlled and the authors use a number of leading edge methods.

Reviewer #3 (Public Review):

As a follow up from a manuscript previously published (Ruby et al. 2018), the authors use basic survival analysis methods to estimate hazard rates on an extended dataset of naked mole rats. They conclude that naked mole rats do not show the common exponential increase in mortality that has been typified in most mammals.

In fact, this species has attracted great interest due to their extreme longevity, and the physiological mechanisms that have been associated with slower aging. As the authors show, this species shows unprecedented longevity, particularly considering their body size and phylogenetic location.

However, the data available and the methods used cannot support the conclusion of an absence of increase in mortality for adults. As the authors show, the survivorship curves, calculated using Kaplan-Meier estimators, do not reach below values of 0.5. In short, nothing can be said about hazard rates after the age of median life expectancy. What the authors show is that, up to a certain age (when at least 50% of the individuals are still alive), the hazard rate is relatively constant. Beyond that age, the authors cannot draw any conclusions.

In addition, here is a summary of the methodological limitations I could find based on their limited description: 1) their survivorships do not go below 0.5 and thus cannot make any statements about actuarial senescence; 2) ignoring this last, to test whether the hazards follow a Gompertz mortality it would be more appropriate to use maximum likelihood and test alternative models (e.g., exponential, Siler), and not visually as they show in fig 1; 3) they seem to be confusing left-censoring with left-truncation; 4) given the left-truncation, they should be using product limit estimators and not Kaplan-Meier estimators (which they might, but it's not possible to know based on the limited description of the methods); 5) their treatment of the effects of colony size, breeding status, and body weight should be at least by means of a proportional hazards, not a simple visual inspection on arbitrary age intervals.

In light of these limitations, I would rank the significance of the study as not more than useful, and the strength of evidence inadequate. Still, and as I've stated above, this species is of great interest for ageing research, and the extensive work that the authors have done maintaining this captive colony is to be commended.

Reviewer #3 (Public Review):

Fission yeast is an important model organism and studies on fission yeast have provided many key insights into the understanding of genes and biological pathways. However, even in such a well-studied model organism, there are still many genes without known functions.

In this work, the authors took advantage of the availability of genome-wide fission yeast deletion mutants to systematically analyze the mutant phenotypes under 131 different conditions. This effort generated a genotype-phenotype dataset larger than the currently curated genotype-phenotype dataset, which is derived from studies over many decades by hundreds of fission yeast laboratories. The authors used the dataset to construct gene clusters that provide functional clues for many genes without previously known functions, including ones conserved in humans. This rich resource will surely be highly useful to the fission yeast community and beyond.

In addition, the authors also used machine learning to generate functional predictions of fission yeast genes and yield novel understandings, which are validated by experimental analysis of new ageing-related genes.

Overall, this study provides unprecedented and highly valuable resources for understanding fission yeast gene functions.

Reviewer #3 (Public Review):

The authors present a pipeline for generating strain-specific genome-scale metabolic models for bacteria using Klebsiella spp. as the demonstrative data. The proposed improvement of performance and accuracy in this process holds great value. However, the demonstrated evidence, justification, and validation methods require further discussion.

Apart from the claim to quickly and accurately produce strain-specific models, the manuscript highlights the need to create pan-metabolic models from manually curated models, which are relatively time-consuming and can only be done with well-established organisms. Therefore, claims to speed up the process are redundant.

The justification and evaluation of the generated models are inadequate and one-dimensional. The authors only focus on statistics such as the number of reactions and genes in the models, which does not accurately depict the completeness of the model.

Furthermore, the authors solely compare their results with the performance of the previously published CraveMe packages, and the results do not clearly demonstrate the superior performance of the Bactabolize tool that they developed.

The authors have not provided evidence or discussion on the accuracy of any metabolic fluxes, which are considered to be crucial for reconstructing metabolic models. Additionally, the authors have not mentioned the importance of non-growth associated maintenance and the criticality of biomass composition analysis, both of which significantly determine the fluxes in the system.

Overall, the work holds potential for direct application in certain specific aims and fields. However, the cryptic details and critical points of the justification regarding the completeness of the models require further discussion. A detailed discussion on the importance of manually curated models and the potential future direction of incorporating machine learning into the process would significantly enhance the quality of the manuscript.

Reviewer #3 (Public Review):

In their manuscript, Schneider et al. aim to develop voyAGEr, a web-based tool that enables the exploration of gene expression changes over age in a tissue- and sex-specific manner. The authors achieved this goal by calculating the significance of gene expression alterations within a sliding window, using their unique algorithm, Shifting Age Range Pipeline for Linear Modelling (ShARP-LM), as well as tissue-level summaries that calculated the significance of the proportion of differentially expressed genes by the windows and calculated enrichments of pathways for showing biological relevance. Furthermore, the authors examined the enrichment of cell types, pathways, and diseases by defining the co-expressed gene modules in four selected tissues. The voyAGEr was developed as a discovery tool, providing researchers with easy access to the vast amount of transcriptome data from the GTEx project. Overall, the research design is unique and well-performed, with interesting results that provide useful resources for the field of human genetics of aging. I have a few questions and comments, which I hope the authors can address.

1. In the gene-centric analyses section of the result, to improve this manuscript and database, linear regression tests accounting for the entire range of age should be added. The authors' algorithm, ShARP-LM, tests locally within a 16-year window which makes it has lower power than the linear regression test with the whole ages. I suspect that the power reduction is strongly affected in the younger age range since a larger number of GTEx donors are enriched in old age. By adding the results from the lm tests, readers would gain more insight and evidence into how significantly their interest genes change with age.<br /> 2. In line with the ShARP-LM test results, it is not clear which criterion was used to define the significant genes and the following enrichment analyses. I assume that the criterion is P < 0.05, but it should be clearly noted. Additionally, the authors should apply adjusted p-values for multiple-test correction. The ideal criterion is an adjusted P < 0.05. However, if none or only a handful of genes were found to be significant, the authors could relax the criteria, such as using a regular P < 0.01 or 0.05.<br /> 3. In the gene-centric analyses section, authors should provide a full list of donor conditions and a summary table of conditions as supplementary.<br /> 4. The tissue-specific assessment section has poor sub-titles. Every title has to contain information.<br /> 5. I have an issue understanding the meaning of NES from GSEA in the tissue-specific assessment section. The authors performed GSEA for the DEGs against the background genes ordered by t-statistics (from positive to negative) calculated from the linear model. I understand the p-value was two-tailed, which means that both positive and negative NES are meaningful as they represent up-regulated expression direction (positive coefficient) and down-regulated expression direction (negative coefficient) with age, respectively, within a window. However, in the GSEA section of Methods, authors were not fully elaborate on this directionality but stated, "The NES for each pathway was used in subsequent analyses as a metric of its over- or down-representation in the Peak". The authors should clearly elaborate on how to interpret the NES from their results.<br /> 6. In the Modules of co-expressed genes section, the authors did not explain how or why they selected the four tissues: brain, skeletal muscle, heart (left ventricle), and whole blood. This should be elaborated on.<br /> 7. In the modules of the co-expressed genes section, the authors did not provide an explanation of the "diseases-manual" sub-tab of the "Pathway" tab of the voyAGEr tool. It would be helpful for readers to understand how the candidate disease list was prepared and what the results represent.

Reviewer #3 (Public Review):

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

The gene at the center of this study (PBANKA_0902300) was identified in an earlier genetic screen by Russell et al. as being a female specific gene with essential role in transmission and named Fd2 (for female-defective 2). Since this name entered the literature first and is equally descriptive, the Fd2 name should be used instead of PFG to maintain clarity and avoid unnecessary confusion.

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

The gene at the center of this study (PBANKA_0902300) was identified in an earlier genetic screen by Russell et al. as being a female specific gene with essential role in transmission and named Fd2 (for female-defective 2). Since this name entered the literature first and is equally descriptive, the Fd2 name should be used instead of PFG to maintain clarity and avoid unnecessary confusion.

Reviewer #3 (Public Review):

In this study, the authors developed and tested a novel framework for extracting muscle synergies. The approach aims at removing some limitations and constrains typical of previous approaches used in the field. In particular, the authors propose a mathematical formulation that removes constrains of linearity and couple the synergies to their motor outcome, supporting the concept of functional synergies and distinguishing the task-related performance related to each synergy. While some concepts behind this work were already introduced in recent work in the field, the methodology provided here encapsulates all these features in an original formulation providing a step forward with respect to the currently available algorithms. The authors also successfully demonstrated the applicability of their method to previously available datasets of multi-joint movements.

Preliminary results positively support the scientific soundness of the presented approach and its potential. The added values of the method should be documented more in future work to understand how the presented formulation relates to previous approaches and what novel insights can be achieved in practical scenarios and confirm/exploit the potential of the theoretical findings.

Strengths:

This work proposes a novel framework that addresses physiologically non-verified hypothesis of standard muscle synergy methods: it removes restrictive model assumptions (e.g. linearity, same mixing coefficients) and the reliance on variance-accounted-for (VAF) metrics.

The method is solid and achieves the prescribed objectives at a computational level and in preliminary laboratory data.

A toolbox is available for testing the methods on a larger scale.

The paper is well written and shows a high level of innovation, original content and analysis

Weaknesses:

Task performance variables could be specified in more quantitative definition in future work (e.g.: articular angles rather than a generic starting point- end point).

The paper does not show a comparison with previous approaches (e.g.: NMF) or recently developed approaches (such as MMF).

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

In this work, the effort of the authors aimed at developing the field is clear. It is fundamental to develop novel frameworks for synergy extraction and use them to make them more interpretable and applicable to real scenarios, as well as more adherent to recent findings achieved in motor control and neuroscience that are not reflected in the standard models. At the same time, muscle synergies are being used more and more in research but their impact in practical scenarios is still limited, probably because synergies have rarely been analyzed in a functional context. This paper shows a very in-depth analysis and a novel framework to interpret data that links to the task space from a functional perspective. I also found that the results on the datasets are very well commented but could expand more to show why using this framework is advantageous.

There are some key points for discussion that follow from this paper which can be described more, maybe in future work, and that might contribute to major developments in the field, including:

The understanding of how the separation between relevant (redundant and synergistic) and irrelevant synergies impact on synergy analysis in practical works;

Interpreting how different synergistic organizations described in this work allows to better describe data from real scenarios (e.g.: motor recovery of patients after neurological diseases);

Discussing in detail how the presented findings compare with standard algorithms such as NMF to determine the added value provided with this approach;

Describe how redundant synergies reflect real neural organization and - if their "existence" is confirmed - how they contribute to redesign the concept of muscle synergies and of modular/synergistic control in general.

Reviewer #3 (Public Review):

Yuan et al., set out to examine the role of functional and structural interaction between Slac and NaVs on the Slack sensitivity to quinidine. Through pharmacological and genetic means they identify NaV1.6 as the privileged NaV isoform in sensitizing Slac to quinidine. Through biochemical assays, they then determine that the C-terminus of Slack physically interacts with the N- and C-termini of NaV1.6. Using the information gleaned from the in vitro experiments the authors then show that virally-mediated transduction of Slack's C-terminus lessens the extent of SlackG269S-induced seizures. These data uncover a previously unrecognized interaction between a sodium and a potassium channel, which contributes to the latter's sensitivity to quinidine.

The conclusions of this paper are mostly well supported by data, but some aspects of functional and structural studies in vivo as well as physically interaction need to be clarified and extended.

1) Immunolabeling of the hippocampus CA1 suggests sodium channels as well as Slac colocalization with AnkG (Fig 3A). Proximity ligation assay for NaV1.6 and Slac or a super-resolution microscopy approach would be needed to increase confidence in the presented colocalization results. Furthermore, coimmunoprecipitation studies on the membrane fraction would bolster the functional relevance of NaV1.6-Slac interaction on the cell surface.

2) Although hippocampal slices from Scn8a+/- were used for studies in Fig. S8, it is not clear whether Scn8a-/- or Scn8a+/- tissue was used in other studies (Fig 1J & 1K). It will be important to clarify whether genetic manipulation of NaV1.6 expression (Fig. 1K) has an impact on sodium-activated potassium current, level of surface Slac expression, or that of NaV1.6 near Slac.

3) Did the epilepsy-related Slac mutations have an impact on NaV1.6-mediated sodium current?

4) Showing the impact of quinidine on persistent sodium current in neurons and on NaV1.6-expressing cells would further increase confidence in the role of persistent sodium current on sensitivity of Slac to quinidine.

Reviewer #3 (Public Review):

This study from the Flores group aims at understanding neuronal circuit changes during adolescence which is an ill-defined, transitional period involving dramatic changes in behavior and anatomy. They focus on DA innervation of the prefrontal cortex, and their interaction with the guidance cue Netrin-1. They propose DA axons in the PFC increase in the postnatal period, and their density is reduced in a Netrin 1 knockdown, suggesting that Netrin abets the development of this mesocortical pathway. In such mice impulsivity gauged by a go-no go task is reduced. They then provide some evidence that Unc5c is developmentally regulated in DA axons. Finally they use an interesting hamster model, to study the effect of light hours on mesocortical innervation, and make some interesting observations about the timing of innervation and Unc5c expression, and the fact that females housed in winter day length conditions display an accelerated innervation of the prefrontal cortex. While this work is novel and on an interesting, understudied topic, several aspects need to be further consolidated, to make it more persuasive.

Main comments<br /> 1. Fig 1 A and B don't appear to be the same section level.<br /> 2. Fig 1C. It is not clear that these axons are crossing from the shell of the NAC.<br /> 3. Fig 1. Measuring width of the bundle is an odd way to measure DA axon numbers. First the width could be changing during adult for various reasons including change in brain size. Second, I wouldn't consider these axons in a traditional bundle. Third, could DA axon counts be provided, rather than these proxy measures.<br /> 4. TH in the cortex could also be of noradrenergic origin. This needs to be ruled out to score DA axons<br /> 5. Netrin staining should be provided with NeuN + DAPI; its not clear these are all cell bodies. An in situ of Netrin would help as well.<br /> 6. The Netrin knockdown needs validation. How strong was the knockdown etc?<br /> 7. If the conclusion that knocking down Netrin in cortex decreases DA innervation of the IL, how can that be reconciled with Netrin-Unc repulsion.<br /> 8. The behavioral phenotype in Fig 1 is interesting, but its not clear if its related to DA axons/signaling. IN general, no evidence in this paper is provided for the role of DA in the adolescent behaviors described.<br /> 9. Fig2 - boxes should be drawn on the NAc diagram to indicate sampled regions. Some quantification of Unc5c would be useful. Also, some validation of the Unc5c antibody would be nice.<br /> 10. "In adolescence, dopamine neurons begin to express the repulsive Netrin-1 receptor UNC5C, and reduction in UNC5C expression appears to cause growth of mesolimbic dopamine axons to the prefrontal cortex".....This is confusing. Figure 2 shows a developmental increase in UNc5c not a decrease. So when is the "reduction in Unc5c expression" occurring?<br /> 11. In Fig 3, a statistical comparison should be made between summer male and winter male, to justify the conclusions that the winter males have delayed DA innervation.<br /> 12. Should axon length also be measured here (Fig 3)? It is not clear why the authors have switched to varicosity density. Also, a box should be drawn in the NAC cartoon to indicate the region that was sampled.<br /> 13. In Fig 3, Unc5c should be quantified to bolster the interesting finding that Unc5c expression dynamics are different between summer and winter hamsters. Unc5c mRNA experiments would also be important to see if similar changes are observed at the transcript level.<br /> 14. Fig 4. The peak in exploratory behavior in winter females is counterintuitive and needs to be better discussed. IN general, the light dark behavior seems quite variable.

Reviewer #3 (Public Review):

This study investigated cognitive mechanisms underlying approach-avoidance behavior using a novel reinforcement learning task and computational modelling. Participants could select a risky "conflict" option (latent, fluctuating probabilities of monetary reward and/or unpleasant sound [punishment]) or a safe option (separate, generally lower probability of reward). Overall, participant choices were skewed towards more rewarded options, but were also repelled by increasing probability of punishment. Individual patterns of behavior were well-captured by a reinforcement learning model that included parameters for reward and punishment sensitivity, and learning rates for reward and punishment. This is a nice replication of existing findings suggesting reward and punishment have opposing effects on behavior through dissociated sensitivity to reward versus punishment.

Interestingly, avoidance of the conflict option was predicted by self-reported task-induced anxiety. This effect of anxiety was mediated by the difference in modelled sensitivity to reward versus punishment (relative sensitivity). Importantly, when a subset of participants were retested over 1 week later, most behavioral tendencies and model parameters were recapitulated, suggesting the task may capture stable traits relevant to approach-avoidance decision-making.

However, interpretation of these findings are severely undermined by the fact that the aversiveness of the auditory punisher was largely determined by participants, with the far-reaching impacts of this not being accounted for in any of the analyses. The manipulation check to confirm participants did not mute their sound is highly commendable, but the thresholding of punisher volume to "loud but comfortable" at the outset of the task leaves substantial scope for variability in the punisher delivered to participants. Indeed, participants' ratings of the unpleasantness of the punishment was moderate and highly variable (M = 31.7 out of 50, SD = 12.8 [distribution unreported]). Despite having this rating, it is not incorporated into analyses. It is possible that the key finding of relationships between task-induced anxiety, reward-punishment sensitivity and avoidance are driven by differences in the punisher experienced; a louder punisher is more unpleasant, driving greater task-induced anxiety, model-derived punishment sensitivity, and avoidance (and vice versa). This issue can also explain the counterintuitive findings from re-tested participants; lower/negatively correlated task-induced anxiety and punishment-related cognitive parameters may have been due to participants adjusting their sound settings to make the task less aversive (retest punisher rating not reported). It can therefore be argued that the task may not actually capture meaningful cognitive/motivational traits and their effects on decision-making, but instead spurious differences in punisher intensity.

This undercuts the proposed significance of this task as a translational tool for understanding anxiety and avoidance. More information about ratings of punisher unpleasantness and its relationship to task behavior, anxiety and cognitive parameters would be valuable for interpreting findings. It would also be of interest whether the same results were observed if the aversiveness of the punisher was titrated prior to the task.

Although the procedure and findings reported here remain valuable to the field, claims of novelty including its translational potential are perhaps overstated. This study complements and sits within a much broader literature that investigates roles for aversion and cognitive traits in approach-avoidance decisions. This includes numerous studies that apply reinforcement learning models to behavior in two-choice tasks with latent probabilities of reward and punishment (e.g., see doi: 10.1001/jamapsychiatry.2022.0051), as well as other translationally-relevant paradigms (e.g., doi: 10.3389/fpsyg.2014.00203, 10.7554/eLife.69594, etc).

Reviewer #3 (Public Review):

Eichler et al. set out to map the locations of the mechanosensory bristles on the fly head, examine the axonal morphology of the bristle mechanosensory neurons (BMNs) that innervate them, and match these to electron microscopy reconstructions of the same BMNs in a previously published EM volume of the female adult fly brain. They used BMN synaptic connectivity information to create clusters of BMNs that they show occupy different regions of the subesophageal zone brain region and use optogenetic activation of subsets of BMNs to support the claim that the morphological projections and connectivity of defined groups of BMNs are consistent with the parallel model for behavioral sequence generation.

The authors have beautifully cataloged the mechanosensory bristles and the projection paths and patterns of the corresponding BMN axons in the brain using detailed and painstaking methods. The result is a neuroanatomy resource that will be an important community resource. To match BMNs reconstructed in an electron microscopy volume of the adult fly brain, the authors matched clustered reconstructed BMNs with light-level BMN classes using a variety of methods, but evidence for matching is only summarized and not demonstrated in a way that allows the reader to evaluate the strength of the evidence. The authors then switch from morphology-based categorization to non-BMN connectivity as a clustering method, which they claim demonstrates that BMNs form a somatotopic map in the brain. This map is not easily appreciated, and although contralateral projections in some populations are clear, the distinct projection zones that are mentioned by the authors are not readily apparent. Because of the extensive morphological overlap between connectivity-based clusters, it is not clear that small projection differences at the projection level are what determines the post-synaptic connectivity of a given BMN cluster or their functional role during behavior. The claim the somatotopic organization of BMN projections is preserved among their postsynaptic partners to form parallel sensory pathways is not supported by the result that different connectivity clusters still have high cosine similarity in a number of cases (i.e. Clusters 1 and 3, or Clusters 1 and 2). Finally, the authors use tools that were generated during the light-level characterization of BMN projections to show that specifically activating BMNs that innervate different areas of the head triggers different grooming behaviors. In one case, activation of a single population of sensory bristles (lnOm) triggers two different behaviors, both eye and dorsal head grooming. This result does not seem consistent with the parallel model, which suggests that these behaviors should be mutually exclusive and rely on parallel downstream circuitry.

This work will have a positive impact on the field by contributing a complete accounting of the mechanosensory bristles of the fruit fly head, describing the brain projection patterns of the BMNs that innervate them, and linking them to BMN sensory projections in an electron microscopy volume of the adult fly brain. It will also have a positive impact on the field by providing genetic tools to help functionally subdivide the contributions of different BMN populations to circuit computations and behavior. This contribution will pave the way for further mechanistic study of central circuits that subserve grooming circuits.

Reviewer #3 (Public Review):

The six-transmembrane epithelial antigen of the prostate (STEAP) family comprises four members in metazoans. STEAP1 was identified as integral membrane protein highly upregulated on the plasma membrane of prostate cancer cells (PMID: 10588738), and it later became evident that other STEAP proteins are also over expressed in cancers, making STEAPs potential therapeutic targets (PMID: 22804687). Functionally, STEAP2-4 are ferric and cupric reductases that are important for maintaining cellular metal uptake (PMIDs: 16227996, 16609065). The cellular function of STEAP1 remains unknown, as it cannot function as an independent metalloreductase. In the last years, structural and functional data have revealed that STEAPs form trimeric assemblies and that they transport electrons from intracellular NADPH, through membrane bound FAD and heme cofactors, to extracellular metal ions (PMIDs: 23733181, 26205815, 30337524). In addition, numerous studies (including a previous study from the senior authors) have provided strong implications for a potential metalloreductase function of STEAP1 (PMIDs: 27792302, 32409586).

This new study by Chen et al. aims to further characterize the previously established electron transport chain in STEAPs in high molecular detail through a variety of assays. This is a well-performed, highly specialized study that provides some useful extra insights into the established mechanism of electron transport in STEAP proteins. The authors first perform a detailed spectroscopic analysis of Fe3+-NTA reduction by STEAP2 and STEAP1, confirming that both purified proteins are capable of reducing metal ions. A cryo-EM structure of STEAP2 is also presented. It is then established that STEAP1 can receive electrons from cytochrome b5 reductase, and the authors provide experimental evidence that the flavin in STEAP proteins becomes diffusible.

The specific aims of the study are clear, but it is not always obvious why certain experiments are performed only on STEAP2, on STEAP1, or on both isoforms. A better justification of the performed experiments through connecting paragraphs and proper referencing of the literature would improve readability of the manuscript. Experimentally, the conclusions are appropriate and mostly consistent with the experimental data, although one important finding can benefit from further clarification. Namely, the observation that STEAP1 can form an electron transfer chain with cytochrome b5 reductase in vitro is an exciting finding, but its physiological relevance remains to be validated. The metalloreductase activity of STEAP1 in vitro has been described previously by the authors and by others (PMIDs: 27792302, 32409586). However, when over expressed in HEK cells, STEAP1 by itself does not display metal ion reductase activity (PMID: 16609065), and it was also found that STEAP1 over expression does not impact iron uptake and reduction in Ewing's sarcoma (cancer) cells (PMID: 22080479). Therefore, the physiological relevance of metal ion reduction by STEAP1 remains controversial. The current work establishes an electron transfer chain between STEAP1 and cytochrome b5 reductase in vitro with purified proteins. However, the conformation of this metalloreductase activity of the STEAP1-cytochrome b5 complex will be required in a cell line to prove that the two proteins indeed form a physiological relevant complex and that the results are not just an in vitro artefact from using high concentrations of purified proteins.

The work will be interesting for scientists working within the STEAP field. However, some of the presented data are redundant with previous findings, moderating the study's impact. For instance, the new structural insights into STEAP2 are limited because the structure is virtually identical to the published structures of STEAP4 and STEAP1 (PMIDs: 30337524, 32409586), which is not surprising because of the high sequence similarity between the STEAP isoforms. Moreover, the authors provide experimental evidence to prove the previous hypothesis (PMID: 30337524) that the flavin in STEAP proteins becomes diffusible, but the molecular arrangement of a STEAP protein, in which the flavin interacts with NADPH, remains unknown. Based on the manuscript title, I would also expect the in-depth characterization of STEAP1/STEAP2 hetero trimers (first identified by the authors), but this is only briefly mentioned. When taken together, this study by Chen et al. strengthens and supports previously published biochemical and structural data on STEAP proteins, without revealing many prominent conceptual advances.

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Reviewer #3 (Public Review):

The authors present the mechanism, validation, and modular application of LOVtag, a light-responsive protein degradation tag that is processed by the native degradosome of Escherichia coli. Upon exposure to blue light, the c-terminal alpha helix unfolds, essentially marking the protein for degradation. The authors demonstrate the engineered tag is modular across multiple complex regulatory systems, which shows its potential widespread use throughout the synthetic biology field. The step-by-step rational design of identifying the protein that was most dark-stabilized as well as most light-responsive for degradation, was useful in terms of understanding the key components of this system. The most compelling data shows that the engineered LOVTag can be fused to multiple proteins and achieve light-based degradation, without affecting the original function of the fused protein; however, results are not benchmarked against similar degradation tagging and optogenetic control constructs. Creating fusion proteins that do not alter either of the original functions, is often difficult to achieve, and the novelty of this should be expanded upon to drive further impact.

Reviewer #3 (Public Review):

The authors show that ILC2s seem to be important during pregnancy to achieve an optimal fetal growth. This is an important finding to the field and provides ILC2s with new roles distinct from parasite protection and allergy inducers. However, the fetal weight restriction phenotype does not seem very striking. Moreover, the mechanism by which ILC2s promote homeostasis in pregnancy are not well shown. The data shown in Figure 3 is overall a bit confusing and does not lead the reader to the conclusions stated in the text. Figure 4 conclusions are not very informative. The authors also show that ILC2s are protective to fetal loss during LPS infection. Again, the means by which ILC2s could be doing so are not well presented and the supporting data not fully convincing. Throughout the manuscript, the authors present quantitative data as fold change, expressing data in fold change is not as clear as showing actual numbers of cells for each group. Moreover, they should show the flow cytometry plots and gating strategy for all their FACS analysis.

Reviewer #3 (Public Review):

The manuscript by Guss et al. characterizes an extracellular matrix protein, Perlecan (trol), in maintaining axon and synapse stability in motor neurons through its function in maintaining the neural lamella's integrity in Drosophila. Using a combination of immunostaining and protein labeling with fluorescent tags, the authors find that perlecan localizes to the neural lamella. When perlecan is deleted, the authors identify a synapse retraction phenotype as the subsequent result of axon damage. They further suggest that this axon instability is the result of loss of perlecan causing a disruption in the neural lamella, due to the mislocalization of neural lamella protein, Collagen IV (Vkg). Moreover, they find that perlecan acts independently of previously characterized interactions with the wnt signaling and Wallerian degradation pathways, however important controls for these negative results are lacking.

The manuscript offers an interesting and important role for perlecan in motor neuron axon maintenance. However, the experiments attempting to elucidate the mechanism of action of this protein require further validation and clarification.

Reviewer #3 (Public Review):

This study documents the dynamics of Merkel cells and their axonal afferents during the hair growth cycle. Methodologically, the study is impressive-using two transgenic lines and repeated 2-photon imaging allowed the researchers to monitor Merkel cells and afferent axons over the course of weeks. These exciting tools and methods will enable future studies of these cell interactions. The manuscript is well written, the figures are clear and appealing, the statistical analyses are rigorous and appropriate, and potentially confounding issues (e.g., damage caused by 2-photon imaging or hair removal) were thoughtfully considered and controlled for. The clear and rigorously analyzed findings make the conclusions well justified. The impact of this study could be enhanced with further experiments that provide more functional characterization of boutons and kylikes, and that characterize axonal dynamics in Atoh mutants lacking Merkel cells.

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Reviewer #3 (Public Review):

In this manuscript, the authors characterized the clonal composition in the medaka pallium and found the dorsal pallium region to be a compartment constituting repeatedly identifiable clonal units. By performing ATAC-seq on the clonal units as well as RNA-seq on different subregions of the pallium, the dorsal pallium was further identified as a unique region with open chromatin regions with regulatory elements enriched for synapse-related genes. Further experimental and bioinformatic evidence supports the region's putative function of synapse generation, with similar TF-binding motifs to homologous brain regions in human. Although the "uniqueness" of the dorsal pallium might be a coincidence of the timing of clonal tracing, the conclusions in the manuscript are largely supported by experimental evidence. The study showcases an elegant model of how anatomical, molecular, and functional diversity arises in a previously under-characterized brain region. The enriched genes in Dd2 are an interesting candidate for future investigation, and the function of the dorsal pallium of teleost fish is of general interest for studies of brain evolution.

Reviewer #3 (Public Review):

This is a highly interesting paper that comprehensively investigates the electrophysiological properties of granule cells in the dentate gyrus at different developmental stages. Using state-of-the-art in vitro electrophysiological techniques, the authors record granule cell responses to fluctuating current injections to study how they encode stimuli. The authors find that while immature granule cells produce less reliable stimulus responses and worse stimulus representations than mature cells (8wks and older), cell populations containing neurons of mixed ages improve overall stimulus reconstruction. These data suggest that the cellular diversity contributed by immature granule cells could be beneficial for transmitting distinct properties of stimuli with rich temporal structure, potentially improving the cellular process of pattern separation.<br /> Major strengths of the paper lie in the precise age determination of immature neurons in Ascl1-CreERT2-Tom mice, recordings of immature neurons, which are rare in in vivo and in vitro studies, precise control over cell-intrinsic properties by blocking excitatory and inhibitory inputs in vitro, and characterization of encoding properties using a spike response model (SRM).

The conclusions drawn are supported by the data, and the results are likely of great interest to a specialist community of hippocampal electrophysiologists.

Reviewer #3 (Public Review):

The manuscript by JY Toshima et al. is an excellent and important study that demonstrates very clearly the existence of an endosomal compartment in yeast, distinct from the trans-Golgi network, to which endocytic vesicles fuse upon internalization. They show that this compartment is enriched in the SNARE protein Tlg2, a yeast homologue of syntaxin, and is segregated from the Golgi-localized Sec7-containing compartment, indicating that the organization of the endocytic system in yeast is similar to that of animal cells. Furthermore, they demonstrate the trafficking machinery required for maturation of this compartment, and that it is also a station on the pathway back to the plasma membrane. Because there have been conflicting reports in the literature as to the existence of an endosomal compartment in yeast distinct from the trans-Golgi network, this paper is of great importance for the cell biology community.

Major strengths of this study are the cutting-edge imaging technology used, and the careful, quantitative analyses carried out. The authors use a super-resolution live cell imaging approach that allows them to discriminate to a high resolution different compartments and membrane domains of highly dynamic yeast organelles, and to follow an internalizing cargo over time. With their manuscript, they have provided a full set of movies, along with quantifications, to support their conclusions.

The authors use fluorescent-protein-labelled endocytic cargo (alpha-factor) and florescent-protein-labelled compartment markers, assaying them in high resolution and super-resolution live cell imaging microscopy systems. In this way, they are able to follow trafficking of cargo through compartments in real time. The authors first demonstrate that the alpha-factor cargo substantially colocalized with the SNARE protein Tlg2, a marker of early endosomes, but very little with Sec7. They also show that Tlg2 marks a sub-compartment distinct from the Sec7 compartment, but adjacent to it. Furthermore, they demonstrate using super-resolution microscopy and triple color 4D imaging that endocytosed alpha-factor cargo structures make contact with the Tlg2 compartment, adjacent to the Sec7 compartment, then disappear, supporting the conclusion that endocytic vesicles first fuse with the Tlg2 compartment. Next the authors show that alpha factor is transported from the Tlg2 compartment to the Vps21 compartment, a process that requires the GGA adaptors Gga1 and Gga2. Finally, the authors show that recycling of the endocytic R-SNARE Snc1 also occurs by passage through the Tlg2 compartment.

The use of mutants that affect different stages of endosomal trafficking is a strength of the manuscript, as it allows elucidation of the mechanism of transport through successive compartments. Importantly, using a gga1-delta gga2-delta mutant, the authors demonstrate convincingly that the GGA adaptors Gga1 and Gga2 are required for alpha factor transport from the Tlg2 compartment to the Vps21 compartment.

Throughout this study, the authors use fluorescent protein-labelled cargo and compartment markers (EGFP, mCherry, iRFP), but don't explicitly state to what extent these fusion proteins are functional compared to the endogenous proteins. They could cite previous publications or their results describing the functionality of the fusion proteins used.

Reviewer #3 (Public Review):

One key finding of this work is the identification of Xanthomonas oryzae pv. oryzae (Xoo) strains in Africa, based on their genomes sequence and their TALE repertoires, have high similarity with Asian strains. Asian Xoo strains typically overcome NLR-mediated recognition of TALEs in rice by so-called iTALEs. Moreover, some Asian strains contain a TALE resembling PthXo1, a TALE protein that was shown to overcome xa5 resistance.

The authors now found that some of the newly identified African strains have iTALEs and PthXo1-like TALEs. Such newly evolved African strains were found to be fully virulent on the African rice elite variety Komboka, which is resistant to a broad panel of African Xoo strains.

Previous studies have shown that TALEs bind to effector binding elements (EBEs) present in promoters of rice SWEET genes to promote disease. Work from the lab of the authors and other labs has shown that TALEs can no longer promote the disease if matching EBEs are changed or deleted by CRISPR or TALEN-mediated mutagenesis. In fact, pioneering work by Bing Yang, one of the authors of this article published about ten years ago a Nature Biotechnology article where he showed that rice plants with mutated EBEs are resistant to Xoo. Recently, a combined effort of the Yang and Frommer labs resulted in two further Nature Biotechnology publications (2019), in which they described along with other useful tools rice lines where multiple EBEs were mutagenized in parallel and that provide broad spectrum resistance.

The work under review describes now CRISPR mutagenesis of an African elite cultivar resulting in a line that mediates resistance to Asian and newly evolved African strains.

Overall, the work is technically sound. Yet, the approach that has been described - mutagenesis of multiple EBEs - has been used before and is a routine procedure for labs that are focused on such undertakings. While such approaches do not provide new insights for fundamental research, they nevertheless are certainly important and useful in translational research, as demonstrated here.

Reviewer #3 (Public Review):

Huff et.al further characterise the anatomy and function of a population of excitatory medullary neurons, the Post-inspiratory Complex (PiCo), which they first described in 2016 as the origin of the laryngeal adduction that occurs in the post-inspiratory phase of quiet breathing. They propose an additional role for the glutamatergic and cholinergic PiCo interneurons in coordinating swallowing and protective airway reflexes with breathing, a critical function of the central respiratory apparatus, the neural mechanics of which have remained enigmatic. Using single allelic and intersectional allelic recombinase transgenic approaches, Huff et al. selectively excited choline acetyltransferase (ChAT) and vesicular glutamate transporter-2 (VGluT2) expressing neurons in the intermediate reticular nucleus of anesthetised mice using an optogenetic approach, evoking a stereotyped swallowing motor pattern (indistinguishable from a water-induced swallow) during the early phase of the breathing cycle (within the first 10% of the cycle) or tonic laryngeal adduction (which tracked tetanically with stimulus length) during the later phase of the breathing cycle (after 70% of the cycle).

They further refine the anatomical demarcation of the PiCo using a combination of ChAT immunohistochemistry and an intersectional transgenic strategy by which fluorescent reporter expression (tdTomato) is regulated by a combinatorial flippase and cre recombinase-dependent cassette in triple allelic mice (Vglut2-ires2-FLPO; ChAT-ires-cre; Ai65).

Lastly, they demonstrate that the PiCo is anatomically positioned to influence the induction of swallowing through a series of neuroanatomical experiments in which the retrograde tracer Cholera Toxin B (CTB) was transported from the proposed location of the putative swallowing pattern generator within the caudal nucleus of the solitary tract (NTS) to glutamatergic ChAT neurons located within the PiCo.

Methods and Results<br /> The experimental approach is appropriate and at the cutting edge for the field: advanced neuroscience techniques for neuronal stimulation (virally driven opsin expression within a genetically intersecting subset of neurons) applied within a sophisticated in vivo preparation in the anaesthetized mouse with electrophysiological recordings from functionally discrete respiratory and swallowing muscles. This approach permits selective stimulation of target cell types and simultaneous assessment of gain-of-function on multiple respiratory and swallowing outputs. This intersectional method ensures PiCo activation occurs in isolation from surrounding glutamatergic IRt interneurons, which serve a diverse range of homeostatic and locomotor functions, and immediately adjacent cholinergic laryngeal motor neurons within the nucleus ambiguous (seen by some as a limitation of the original study that first described the PiCo and its roll in post-I rhythm generation Anderson et al., 2016 Nature 536, 76-80). These experiments are technically demanding and have been expertly performed.

The supplemental tracing experiments are of a lower standard. CTB is a robust retrograde tracer with some inherent limitations, paramount of which is the inadvertent labelling of neurons whose axons pass through the site of tracer deposition, commonly leading to false positives. In the context of labelling promiscuity by CTB, the small number of PiCo neurons labelled from the NTS (maybe 5 or 6 at most in an optical plane that features 20 or more PiCo neurons) is a concern. Even assuming that only a small subset of PiCo neurons makes this connection with the presumed swallowing CPG within the cNTS, interpretation is not helped by the low contrast of the tracer labelling (relative to the background) and the poor quality of the image itself. The connection the authors are trying to demonstrate between PiCo and the cNTS could be solidified using anterograde tracing data the authors should already have at hand (i.e. EYFP labelling driven by the con-fon AAV vectors from PiCo neurons (shown in Fig5), which should robustly label any projections to the cNTS).

The retrograde labelling from laryngeal muscles seems unnecessary: the laryngeal motor pool is well established (within the nAmb and ventral medulla), and it would be unprecedented for a population of glutamatergic neurons to form direct connections with muscles (beyond the sensory pool).

The authors support their claim that PiCo neurons gate laryngeal activity with breathing through the demonstration that selective activation of glutamatergic and cholinergic PiCo neurons is sufficient to drive oral/pharyngeal/laryngeal motor responses under anaesthesia and that such responses are qualitatively shaped by the phase of the respiratory cycle within which stimulation occurs. Optical stimulation within the first 10% of the respiratory cycle was sufficient to evoke a complete, stereotyped swallow that reset the breathing cycle, while stimuli within the later 70% of the cycle, evoked discharge of the laryngeal muscles in a stimulus length-dependent manner. Induced swallows were qualitatively indistinguishable from naturalistic swallow induced by the introduction of water into the oral cavity. The authors note that a detailed interpretation of induced laryngeal activity is probably beyond the technical limits of their recordings, but they speculate that this activity may represent the laryngeal adductor reflex. This seems like a reasonable conclusion.

The authors propose a model whereby the PiCo impinges upon the swallowing CPG (itself a poorly resolved structure) to explain their physiological data. The anatomical data presented in this study (retrograde transport of CTB from cNTS to PiCo) are insufficient to support this claim. As suggested above, complementary, high-quality, anterograde tracing data demonstrating connectivity between these structures as well as other brain regions would help to support this claim and broaden the impact of the study.

This study proposes that the PiCo in addition to serving as the site of generation of the post-I rhythm also gates swallowing and respiration. The scope of the study is small, and limited to the subfields of swallowing and respiratory neuroscience, however, this is an important basic biological question within these fields. The basic biological mechanisms that link these two behaviors, breathing and swallowing, are elusive and are critical in understanding how the brain achieves robust regulation of motor patterning of the aerodigestive tract, a mechanism that prevents aspiration of food and drink during ingestion. This study pushes the PiCo as a key candidate and supports this claim with solid functional data. A more comprehensive study demonstrating the necessity of the PiCo for swallow/breathing coordination through loss of function experiments (inhibitory optogenetics applied in the same transgenic context) along with robust connectivity data would solidify this claim.

Reviewer #3 (Public Review):

The relevance of Y90 phosphorylation as a regulatory mechanism is shown by the comparison of Src kinase activity, transforming potential, cell invasiveness, and lateral diffusion in membranes. Mechanistically, Y90E mutation affects the opening of the structure, estimated from FRET experiments, and the phosphorylation status of the three main downstream signaling pathways.

The effect of the Y90E mutation is very clear, although its description as "phosphomimicking" is, in my opinion, not accurate. Glutamic acid has a negative charge but is significantly different from phosphotyrosine. Maybe other polar mutants (lysine, glutamine...) would have a similar destabilizing effect on hydrophobic interactions. Erpel 1995 showed some effects of the Y90A mutants.

The effect of tyrosine phosphorylation on the SH3 domain of proteins having the conserved ALYDY motif supports the proposed role, although the evidence for in vivo Y90 phosphorylation in c-Src is scarce. The possible autophosphorylation of Y90 is suggested but the evidence is not very strong and does not rule out other kinases, especially some downstream of Src itself -as already suggested by the authors.

The authors suggest that the perturbation of Y90 reduces the interaction with the connector domain. This is a reasonable explanation, supported by the opening of the structure, but additional effects may exist: The SH3 hydrophobic region including Y90 is also the binding site for the myristoyl group (Le Roux et al. iScience, 12, 194-203) and mutations in the SH3 RT loop significantly affected lipid binding. This could contribute to the observed reduced diffusion in the lipid bilayer.

Reviewer #3 (Public Review):

By using chemogenetic manipulations of direct pathway neurons in the dorsomedial part of the striatum (DMS) of anesthetized mice combined with fMRI, Markicevic et al explore changes in BOLD dynamics at local (striatum) and macro-scale (brain-wide) levels. The article is appropriately written, and the main findings are well organized and presented in 7 figures. Figures 1 and 2 schematize the techniques and document the motor effects of chemogenetic manipulations. Figures 3-7 describe neural changes induced by these manipulations. The main strength of this work is the level of specificity of the chemogenetic manipulations, which combined with brain-wide functional exploration, provide a very useful map of the consequences of activating a specific striatal subpopulation. In my opinion, the main weakness of this work is that the results are under-discussed and not appropriately contextualized in the current views of the functions of the basal ganglia. My main concerns are exposed in the following lines:

1. In the first finding the authors show that D1 activation/inactivation produces reliable changes in the infected region (DMS), but most importantly, also produced changes in adjacent areas, suggesting intra-striatal communication. The way the data is presented and discussed appears to be confirmatory of what has been previously described with electrophysiological recordings. In my opinion, the most important part of this section would be to fully describe the differences between activation and inactivation groups. Is interesting that opposite manipulations of D1 receptors produced very similar maps of discrimination (Fig. 3). Therefore, it would be necessary to discuss the meaning of obtaining similar classification accuracy indices with opposite manipulations. Perhaps, the use of SVM classifiers can be complemented with other analytical techniques to further disentangle the consequences of manipulating intrastriatal D1 receptors.

2. The second finding (Fig. 4) indicates that thalamic regions forming "closed loops" with the striatum were more affected by chemogenetic manipulations. We knew from anatomical studies that the BG are part of anatomically segregated cortico-BG-thalamic loops. Therefore, it would be expected that these anatomical boundaries would somehow limit functional connectivity maps. Here again, I consider that the manuscript would be improved with further analysis or discussion. For example, it would be interesting to perform further analysis relating the previous section (local striatal connectivity) with this one. In this section, several thalamic nuclei presented higher levels of classification accuracy, but in the previous section, the authors showed that DMS manipulation also produced the same effects in different intrastriatal regions. Therefore, it is not possible to know if the thalamic effects are related to the manipulation of D1 in the DMS or its adjacent regions.

3. In the third finding (Fig. 5) the authors show that the most "sensitive" cortical regions to the manipulations were classified as "unimodal". This is an interesting result; however, it would be necessary to at least provide further discussion on its potential meaning. It is important to consider that the cortical regions with significant changes, for example, primary sensorimotor cortices, mainly target the dorsolateral, not the dorsomedial striatum. In this context, would it be possible to establish a new analysis to characterize potential correlations between cortical regions and striatal subregions?

4. The fourth finding (Figure 6) is that thalamic but not cortical regions presented low-frequency fluctuations. What is the meaning of an increase in slow fluctuations? Why did D1 activation (and not inactivation) induced this effect? Are striatal sub-regions also presenting these slow fluctuations?

5. In the last finding (Figure 7), the authors explored potential changes in functional connectivity (FC) between the striatum and cortical and subcortical regions. Contrary to the results obtained with the SVM-based analytical tool, FC analysis revealed that D1 activation and inactivation produced opposite results, while D1 activation decreased FC in several cortical and subcortical regions, D1 inactivation increased it. While this set of data is clearly described, the implications of these relationships could be further discussed. For example, how do the authors explain that FC with SSp was not significantly changed with this analytical method, but was one of the most affected regions with the Balanced Classification Accuracy method?

6. Finally, there is no section in the discussion where the behavioral effects observed in figure 2 are contextualized in the massive set of BOLD results presented in the following sections.

Reviewer #3 (Public Review):

The manuscript by Bravo-Plaza et al. identifies and characterizes new mutations (E6K and G540S) in the Uso1 globular head domain that suppress the loss of function mutations in Rab1. Further experiments show that the combined E6K/G540S mutant restores apparent Golgi-localization of Uso1 in Rab1 deficient cells, that this mutant preferentially co-purifies with ER/Golgi SNARE proteins, that monomeric E6K/G540S globular head-domain binds more avidly to purified Bos1 SNARE protein than wild type head-domain, and that overexpression of E6K/G540S or wild type head-domain alone is sufficient for viability. Based on these findings the authors propose that long-distance tethering by Uso1 is dispensable and that the head domain provides an essential function to directly regulate ER/Golgi SNARE-dependent membrane fusion.

Strengths of the study are that an unbiased screen was used to identify new Rab1 suppresser mutations that land in the Uso1 globular head domain. Characterization of these suppressor mutants reveals that SNARE binding activity of Uso1 resides in the head domain and that elevated expression of the Uso1 head domain is sufficient for viability. Imaging experiments document the localization and dynamics of Uso1 on Golgi compartments and biochemical studies show the properties and binding activity of Uso1 domain mutants. These are new findings and the conclusion that monomeric globular head-domain interacts with specific SNAREs to maintain viability is justified.

Weaknesses are that it is well documented that both Rab1 and Uso1 activity can be bypassed by activation of ER/Golgi SNARE machinery either by overexpression of SNARE proteins or by the single copy SLY1-20 allele. Therefore, it was not surprising that tethering by the Uso1 coiled-coil domain is dispensable. The proposal that the E6K mutation in the head domain of Uso1 promotes membrane targeting was not well supported by experimental evidence. And while the AlphaFold modeling of Uso1 with the ER/Golgi fusion machinery was intriguing, the proposed molecular models remain speculative until further tested.

Reviewer #3 (Public Review):

The strongest aspects of this study are the structural analysis of the 90 residue KER domain. This is an important advance, discovering a founding member of a novel class of DNA binding motifs, termed a SAH-DBD (single alpha helix-DNA binding domain). Interestingly, they define a subregion of KER (termed "middle-A", residues 155-204 of Cac1) that has nearly the same DNA binding affinity and confers similar in vivo phenotypes as the full KER domain.

This study also shows that the biological role of KER partially overlaps compensatory factors in vivo, both within the same Cac1 protein subunit (e.g. the WHD domain) and also with other proteins acting in parallel (e.g. Rtt106). That is, the presence of either WHD or Rtt106 renders the drug-resistance and silencing assays employed here insensitive to loss of the KER domain.

However, the drug resistance and gene silencing phenotypes are inherently indirect measures of the most important claim of this work, that KER is a molecular ruler for DNA for the purpose of ensuring sufficiently large templates deposition of histone H3/H4 cargoes. Therefore, this study would be of greater impact if the authors more directly tested this measurement idea in assays that directly assess histone deposition. There are multiple options. Since the authors have in hand recombinant wild-type and mutant CAF-1 complexes, one could examine the number and/or spacing of nucleosomes formed during in vitro deposition reactions. Complementary in vivo experiments using the authors' existing mutant strains could be based on the finding that CAF-1 is particularly important for histone deposition onto nascent Okazaki fragments during DNA replication (Smith and Whitehouse, 2012; pmid: 22419157), and that the spacing pattern of nucleosomes on this DNA is greatly perturbed in cac1-delete cells.

Reviewer #3 (Public Review):

This study describes a descending circuit that can modulate pain perception in the drosophila larvae. While descending inhibition is a major component of mammalian pain perception, it is not known if a similar circuit design exists in fruit flies. Overall the authors use clean logic to establish a role for DSK and its receptor in regulating nociception. The following concerns still stand:

1) It's not completely clear why the authors are staining animals with an FLRFa antibody. Can the authors stain WT and DSK KO animals with a DSK antibody? Also, can the authors show in supplemental what antigen the FLRFa antibody was raised against, and what part of that peptide sequence is retained in the DSK sequence? This overall seems like a weakness in the study that could be improved on in some way by using DSK-specific tools.

2) What is the phenotype of DSK-Gal4 x UAS-TET animals? They should be hyper-reactive. If it's lethal maybe try an inducible approach.

3) Figure 9. This was not totally clear, but I think the authors were evaluating spontaneous (i.e. TRPA1-driven) rolling at 35C. The critical question is "Does activating DSK-expressing neurons suppress acute heat nociception?" and this hasn't really been addressed. The inclusion of PPK Gal4 + DSK Gal4 in the same animal clouds the overall conclusions the reader can draw. The essential experiment is to express UAS-dTRPA1 in DSK-Gal4 or GORO-Gal4 cells, heat the animals to ~29C, and then test latency to a thermal heat probe (over a range of sub and noxious temperatures). Basically, prove the model in Figure 10 showing ectopic activation or inhibition for each major step, then test heat probe responses.

4) It would also then be interesting to see how strong the descending inhibition circuit is in the context of UV burn. If this is a real descending circuit, it should presumably be able to override sensitization after injury.

Reviewer #3 (Public Review):

Lauterbur et al. present an expansion of the whole-genome evolution simulation software "stdpopsim", which includes new features of the simulator itself, and 15 new species in their catalog of demographic models and genetic parameters (which previously had 6 species). The list of new species includes mostly animals (12), but also one species of plant, one of algae, and one of bacteria. While only five of the new animal species (and none of the other organisms) have a demographic model described in the catalog, those species showcase a variety of demographic models (e.g. extreme inbreeding of cattle). The authors describe in detail how to go about gathering genetic and demographic parameters from the literature, which is helpful for others aiming to add new species and demographic models to the stdpopsim catalog. This part of the paper is the most widely relevant not only for stdpopsim users but for any researcher performing population genomics simulations. This work is a concrete contribution towards increasing the number of users of population genomic simulations and improving reproducibility in research that uses this type of simulations.

Reviewer #3 (Public Review):

Dominici et al studied the effects of the type I PRMT inhibitor MS023 on skeletal muscle stem cells (MuSCs) and on muscle strength in dystrophin-deficient mdx mice. The authors observed an enhanced proliferative capacity of cultured MuSCs with an increase of Pax7+/MyoD- cells. The observations are more or less in line with previous studies of the same group, describing reduced differentiation but enhanced proliferation of MuSCs after genetic inactivation of Prmt1. scRNA-seq identified different subpopulations of MuSCs, showing a shift to increased Pax7 expression and elevated oxidative phosphorylation and glycolysis after treatment with MS023. Treatment of MuSC with MS023 during expansion in vitro enhanced engraftment of MuSCs and treatment of dystrophic mdx mice increased muscle strength.

Overall, the manuscript provides new insights into the beneficial effects of the type I PRMT inhibitor MS023 for skeletal muscle regeneration. The description of the MS023-induced transcriptional and metabolic changes in MuSC is interesting and the effects on MuSC transplantation and muscle strength are stunning. However, I have the following comments and concerns:

* Control experiments with the TP-064 inhibitor (previously shown to be specific for CARM1/PRMT4) were not done for the transplantation and muscle strength experiments, which is a clear shortcoming in my view. Since MS023 is a non-selective inhibitor of type I PRMTs with comparable IC50 values for PRMT1 and PRMT4 (CARM1), and lower IC50 values for PRMT6 and PRMT8, it is still not clear whether the enhanced transplantation efficiency and the increased muscle strength is indeed only caused by inhibition of PRMT1. The authors justify their statements by pointing out that gene expression of Prmt1 is highest among the type I PRMTs in MuSCs, which is a rather poor argument, as seen by the strong effects caused by the inactivation of PRMT4.

* Clustering of the M1-M5 subpopulations. I expressed my concern about the separation of the subclusters, which appear more or less in the same cloud. The authors answered that each cluster has some genes, which are only expressed in the respective cluster. I do not doubt this observation but apparently, the transcriptional differences are minor, otherwise one would have seen a much better separation of the subpopulations.

* The authors have not done additional experiments but simply toned-down the statements about the relevance of the proposed "metabolic reprogramming" of MuSC by the type I PRMT inhibitor MS023, which was a major conclusion in the original submission. Again, the changes in the expression of metabolically relevant genes upon MS023 treatment are interesting and should be analyzed in respect to causality. It is not a solution to more or less disabandon the original hypothesis by changing the wording.

* I specifically asked the authors to check whether the dramatic six-fold increase of MuSC engraftment after MS023 treatment really goes along with the incorporation of transplanted MuSC into the MuSC niche, raising concerns that a huge share of the transplanted cells may linger around in the interstitium. It should be very easy to identify and quantify transplanted MuSC outside and inside the basal lamina. Instead of doing the requested experiment, the authors argue about suppression of endogenous MuSC competition by irradiation, at the same time admitting that several GFP-negative fibers have formed.

* I expressed my doubts that a 3-day treatment with MS023 is sufficient to dramatically enhance muscle function in mdx mice via "improvement" of the MuSC population, as reported by the authors, even 30 days after administration of MS023. It seems much more likely that MS023 exerts additional effects that are responsible for the dramatic improvement of muscle function in mdx mice. I maintain my view that this needs to be interrogated more carefully since the improvement of muscle function of dystrophic mice is a central point of the study. It has to be made clear whether this is really due to "improved" functions of MuSC. Many other processes might be involved or responsible for the effect (e.g. impact on inflammation?).

Reviewer #3 (Public Review):

The authors report that the secretion of endosome-derived exosomes is enhanced by a calcium-dependent response to damage to the plasma membrane of cells. The authors present convincing evidence that in response to the influx of calcium that follows damage to the plasma membrane annexin A6 is recruited to multivesicular bodies (MVBs) and likely serves to tether the MVBs to the plasma membrane causing a concomitant release of exosomes. Although it is not directly addressed in the Discussion, I am left with the impression that the authors are hinting that exosome secretion is more a byproduct of plasma membrane repair rather than a means of intercellular communication. In other words, the cell needs the membrane material from the MVB to patch and repair holes in the plasma membrane and exosome ejection from the cell is a secondary (perhaps even irrelevant) consequence. Obviously, these two possibilities are not mutually exclusive. The authors are encouraged to speculate about which possibility they favor and how their findings might change our understanding of the cell biology of exosome secretion.

Reviewer #3 (Public Review):

In this manuscript, Li et al. examine how the expression of the chemokine receptor CCR4 impacts the movement of thymocytes within the thymus. It is currently known that the chemokine receptor CCR7 is important for developing thymocytes to migrate from the cortical region into the medullary region and CCR7 expression is therefore often used to define medullary localization. This is important because key developmental outcomes, like enforcing tolerance to self-antigens amongst others, occur in the medullary environment. The authors demonstrate that the chemokine receptor CCR4 is induced on thymocytes prior to expression of CCR7 and thymocytes exhibit responsiveness to CCR4 ligands earlier in development. Using elegant live confocal microscopy experiments, the authors demonstrate that CCR4 expression is important for the entry and accumulation of specific thymocyte subsets while CCR7 expression is needed for the accumulation of more mature thymocyte subsets. The use of cells deficient in both CCR4 and CCR7 and competitive migration/accumulation experiments provide strong support for this conclusion. The elimination of CCR4 expression results in decreases in apoptosis of thymocyte subsets that have been signalled through their antigen receptor and are responsive to CCR4 ligands. As expected, more mature thymocyte subsets show decreased apoptosis when CCR7 is absent. Distinct antigen-presenting cells in the thymus express CCR4 ligands supporting a model where CCR4 expressing thymocytes can interact with thymic antigen-presenting cells for induction of apoptosis. The absence of CCR4 results in an increase in peripheral T cells that can respond to self-antigens presented by LPS-activated antigen-presenting cells providing further support for the model. Collectively, the manuscript convincingly demonstrates a previously unappreciated role for CCR4 in directing a subset of thymocytes to the medulla.

Strengths:

Relevant model systems and elegant experimental techniques are used throughout the manuscript. The experiments are extensively replicated resulting in robust and convincing data sets. These findings represent an important conceptual advance in our understanding of the processes and cellular regulation of T cell development in the thymus.

Weaknesses:

Evidence demonstrating a direct interaction between CCR4 expressing thymocytes and CCR4-ligand expressing antigen-presenting cells is lacking. Furthermore, increased self-reactivity in the absence of CCR4 is measured using mature peripheral CD4 T cells, but altered self-reactivity of thymocytes is not evaluated similarly.

Reviewer #3 (Public Review):

The authors put together a rigorous study to model the impact of HPV vaccine programme disruptions on cervical cancer incidence and meeting WHO elimination goals in a low-income country - using India as an example. The study explores possible scenarios by varying HPV vaccination strategies for 10-year-old children between a) increasing vaccine coverage in a girls-only vaccination programme and b) vaccinating boys in addition to girls (i.e a gender-neutral vaccination programme).

The main strength of this study is the strength of the modelling methodology in helping to make predictions and in contingency planning. The study methodology is rigorous and uses models that have been validated in other settings. The study employs a high level of detail in calibrating and adapting the model to the Indian context despite poor data availability. The detailed methodology allows future studies to employ the model and techniques with locally-contextualised parameters to study the potential impact of HPV vaccine programme disruptions in other countries.

The work in this field can begin to help lower-income countries explore varying HPV vaccination strategies to reduce cervical cancer incidence, keeping in mind the potential for future supply chains or other related disruptions. However, the scenarios could be better sculpted to model potentially realistic scenarios to guide policymakers to make decisions in situations with limited vaccine supplies - in other words comparing scenario alternatives based on a fixed number of vaccines being available. Using comparative alternatives will help policymakers grapple with the decisions that need to be made regarding planning national HPV vaccination programmes. The results could afford to provide readers with a clearer measure of vaccine strategy 'resilience'.

In all, the authors are able to successfully explore the potential impact of varying HPV vaccination strategies on cervical cancer cases prevented in the context of vaccine disruptions, and make valid conclusions. The results produced are rich in information and are worthy of deeper discussion.

Reviewer #3 (Public Review):

Using the zebrafish model system, this manuscript assessed the roles of Rif1 protein in replication timing control and transcription during early development, and successfully demonstrated the differential impact of Rif1 protein in replication timing control and transcription. Moreover, the comprehensive assessments of the impacts of mutating Rif1 on animal development (including animal survival and sexual development) were assessed. Although there are works that examined Rif1's implications in replication timing and transcription separately, this work is unique in assessing all these points at once.

The strength of this manuscript is the genomic analyses of replication timing and transcription being combined in a single model system. Consequently, this manuscript clearly demonstrates the differential impact of Rif1 in these processes during zebrafish development.

The weakness of this manuscript is, as the authors comment in the Discussion, analyses of replication timing and transcription were performed using bulk embryos. There is a possibility that tissue-specific changes could have been masked. Tissue-specific or single-cell analysis in the future will fill the gap in the knowledge.

Some of the findings presented in this manuscript are consistent with previous findings using different models such as Drosophila and mice, whereas other findings do not necessarily agree. I hope further studies will reveal more clearly what is common in these systems, and what is different.

Also, the suggestion that the Rif1 protein may be implicated in a function similar to Fanconi-Anemia genes/proteins is very intriguing.

Overall, the data presented in this manuscript sufficiently justify the authors' claims. Moreover, this manuscript provides interesting insights into Rif1's function, as well as how development could be controlled.

Reviewer #3 (Public Review):

Mullen et al present an important study describing how DHODH inhibition enhances efficacy of immune checkpoint blockade by increasing cell surface expression of MHC I in cancer cells. DHODH inhibitors have been used in the clinic for many years to treat patients with rheumatoid arthritis and there has been a growing interest in repurposing these inhibitors as anti-cancer drugs. In this manuscript, the Singh group build on their previous work defining combinatorial strategies with DHODH inhibitors to improve efficacy. The authors identify an increase in expression of genes involved in the antigen presentation pathway and MHC I after BQ treatment and they narrow the mechanism to be strictly pyrimidine and CDK9/P-TEFb dependent. The authors rationalize that increased MHC I expression induced by DHODH inhibition might favor efficacy of dual immune checkpoint blockade. This combinatorial treatment prolonged survival in an immunocompetent B16F10 melanoma model.

Previous studies have shown that DHODH inhibitors can increase expression of innate immunity-related genes but the role of DHODH and pyrimidine nucleotides in antigen presentation has not been previously reported. A strength of the manuscript is the use of multiple controls across a panel of cell lines to exclude off-target effects and to confirm that effects are exclusively dependent on pyrimidine depletion. Overall, the authors do a thorough characterization of the mechanism that mediates MHC I upregulation using multiple strategies. Furthermore, the in vivo studies provide solid evidence for combining DHODH inhibitors with immune checkpoint blockade.

However, despite the use of multiple cell lines, most experiments are only performed in one cell line, and it is hard to understand why particular gene sets, cell lines or time points are selected for each experiment. It would be beneficial to standardize experimental conditions and confirm the most relevant findings in multiple cell lines. The differential in vivo survival depending on dosing schedule is interesting. However, this section could be strengthened with a more thorough evaluation of the tumors at endpoint.

Overall, this is an interesting manuscript proposing a mechanistic link between pyrimidine depletion and MHC I expression and a novel therapeutic strategy combining DHODH inhibitors with dual checkpoint blockade. These results might be relevant for the clinical development of DHODH inhibitors in the treatment of solid tumors, a setting where these inhibitors have not shown optimal efficacy yet.

Reviewer #3 (Public Review):

Summary:

In their study the authors analyze the localization of multiple organelles and subcellular structure of blood stage malaria parasites with unprecedented detail. They use a 3D super-resolution imaging technique that has gained popularity in the protozoan field, ultrastructure expansion microscopy. Building on markers and labels established in the field they generate an appealing collection of images for all stages of the intraerythrocytic developmental stages of asexual blood stage parasites with some focus on nuclear division and cell segmentation stages.

Strengths:

The authors generated an impressive amount of imaging data that presents the most comprehensive analysis of ultrastructural organization of the parasite cell so far. This atlas can serve as a reference for researchers studying the cell biology of the intraerythrocytic development cycle. The authors achieve a nice catalogue of the reorganization of well-established markers, which together with the improved resolution allows them to highlight some novel observations and consolidate previous findings. They e.g. improve our understanding of organization, duplication and constitutive tethering of the malaria parasite centrosome to the plasma membrane. Further they provide some interesting observations on rhoptry biogenesis, cytostome morphology, and organelle fission during segmentation.

Weaknesses:

While the comprehensiveness of the study is its strength the authors do not present any novel markers, stainings, or imaging protocols. There is no fundamentally new mechanistic insight derived from this study although some earlier findings are consolidated by the higher spatial resolution.

In the following I want to comment on some major points.

Most importantly, in order to justify the authors claim to provide an "Atlas", I want to strongly suggest they share their raw 3D-imaging data (at least of the main figures) in a data repository. This would allow the readers to browse their structure of interest in 3D and significantly improve the impact of their study in the malaria cell biology field.

The organization of the manuscript can be improved. Aside some obvious modifications as citing the figures in the correct order (see also further comments and recommendations), I would maybe suggest one subsection and one figure per analyzed cellular structure/organelle (i.e. 13 sections). This would in my opinion improve readability and facilitate "browsing the atlas".

Considering the importance of reliability of the U-ExM protocol for this study the authors should provide some validation for the isotropic expansion of the sample e.g. by measuring one well defined cellular structure.

In the absence of time-resolved data and more in-depth mechanistic analysis the authors must down tone some of their conclusions specifically around mitochondrial membrane potential, supellicular microtubule depolymerization, and kinetics of the basal complex. More detailed suggestions for improvement are provided as further comments.

In conclusion the authors provide an exciting cell biological reference framework and new working hypotheses about the function of some subcellular structures, which are still largely enigmatic in the malaria parasite, and can be investigated in the future.

Reviewer #3 (Public Review):

The manuscript by Yang et al. investigated in mice how hypobaric hypoxia can modify the RBC clearance function of the spleen, a concept that is of interest. Via interpretation of their data, the authors proposed a model that hypoxia causes an increase in cellular iron levels, possibly in RPMs, leading to ferroptosis, and downregulates their erythrophagocytic capacity. However, most of the data is generated on total splenocytes/total spleen, and the conclusions are not always supported by the presented data. The model of the authors could be questioned by the paper by Youssef et al. (which the authors cite, but in an unclear context) that the ferroptosis in RPMs could be mediated by augmented erythrophagocytosis. As such, the loss of RPMs in vivo which is indeed clear in the histological section shown (and is a strong and interesting finding) can be not directly caused by hypoxia, but by enhanced RBC clearance. Such a possibility should be taken into account.

Major points:

1) The authors present data from total splenocytes and then relate the obtained data to RPMs, which are quantitatively a minor population in the spleen. Eg, labile iron is increased in the splenocytes upon HH, but the manuscript does not show that this occurs in the red pulp or RPMs. They also measure gene/protein expression changes in the total spleen and connect them to changes in macrophages, as indicated in the model Figure (Fig. 7). HO-1 and levels of Ferritin (L and H) can be attributed to the drop in RPMs in the spleen. Are any of these changes preserved cell-intrinsically in cultured macrophages? This should be shown to support the model (relates also to lines 487-88, where the authors again speculate that hypoxia decreases HO-1 which was not demonstrated). In the current stage, for example, we do not know if the labile iron increase in cultured cells and in the spleen in vivo upon hypoxia is the same phenomenon, and why labile iron is increased. To improve the manuscript, the authors should study specifically RPMs.

2) The paper uses flow cytometry, but how this method was applied is suboptimal: there are no gating strategies, no indication if single events were determined, and how cell viability was assessed, which are the parent populations when % of cells is shown on the graphs. How RBCs in the spleen could be analyzed without dedicated cell surface markers? A drop in splenic RPMs is presented as the key finding of the manuscript but Fig. 3M shows gating (suboptimal) for monocytes, not RPMs. RPMs are typically F4/80-high, CD11-low (again no gating strategy is shown for RPMs). Also, the authors used single-cell RNAseq to detect a drop in splenic macrophages upon HH, but they do not indicate in Fig. A-C which cluster of cells relates to macrophages. Cell clusters are not identified in these panels, hence the data is not interpretable).

3) The authors draw conclusions that are not supported by the data, some examples:

a) they cannot exclude eg the compensatory involvement of the liver in the RBCs clearance (the differences between HH sham and HH splenectomy is mild in Fig. 2 E, F and G)

b) splenomegaly is typically caused by increased extramedullary erythropoiesis, not RBC retention. Why do the authors support the second possibility? Related to this, why do the authors conclude that data in Fig. 4 G,H support the model of RBC retention? A significant drop in splenic RBCs (poorly gated) was observed at 7 days, between NN and HH groups, which could actually indicate increased RBC clearance capacity = less retention.

c) lines 452-54: there is no data for decreased phagocytosis in vivo, especially in the context of erythrophagocytosis. This should be done with stressed RBCs transfusion assays, very good examples, like from Youssef et al. or Threul et al. are available in the literature.

d) Line 475 - ferritinophagy was not shown in response to hypoxia by the manuscript, especially that NCOA4 is decreased, at least in the total spleen.

4) In a few cases, the authors show only representative dot plots or histograms, without quantification for n>1. In Fig. 4B the authors write about a significant decrease (although with n=1 no statistics could be applied here; of note, it is not clear what kind of samples were analyzed here). Another example is Fig. 6I. In this case, it is even more important as the data are conflicting the cited article and the new one: PMCID: PMC9908853 which shows that hypoxia stimulates efferocytosis. Sometimes the manuscript claim that some changes are observed, although they are not visible in representative figures (eg for M1 and M2 macrophages in Fig. 3M)

5) There are several unclear issues in methodology:

- what is the purity of primary RPMs in the culture? RPMs are quantitatively poorly represented in splenocyte single-cell suspensions. This reviewer is quite skeptical that the processing of splenocytes from approx 1 mm3 of tissue was sufficient to establish primary RPM cultures. The authors should prove that the cultured cells were indeed RPMs, not monocyte-derived macrophages or other splenic macrophage subtypes.<br /> - (around line 183) In the description of flow cytometry, there are several missing issues. In 1) it is unclear which type of samples were analyzed. In 2) it is not clear how splenocyte cell suspension was prepared.<br /> - In line 192: what does it mean: 'This step can be omitted from cell samples'?<br /> - 'TO method' is not commonly used anymore and hence it was unclear to this Reviewer. Reticulocytes should be analyzed with proper gating, using cell surface markers.<br /> - The description of 'phagocytosis of E. coli and RBCs' in the Methods section is unclear and incomplete. The Results section suggests that for the biotinylated RBCs, phagocytosis? or retention? Of RBCs was quantified in vivo, upon transfusion. However, the Methods section suggests either in vitro/ex vivo approach. It is vague what was indeed performed and how in detail. If RBC transfusion was done, this should be properly described. Of note, biotinylation of RBCs is typically done in vivo only, being a first step in RBC lifespan assay. The such assay is missing in the manuscript. Also, it is not clear if the detection of biotinylated RBCs was performed in permeablized cells (this would be required).

Reviewer #3 (Public Review):

The mechanistically diverse SLC26 transporters play a variety of physiological roles. The current manuscript establishes the SLC26A6 subtype as electroneutral chloride/bicarbonate exchanges and reports its high-resolution structure with chloride bound.

The claims in this manuscript are all well-supported by the data. Strengths include the comprehensive functional analysis of SLC26A6 in reconstituted liposome vesicles. The authors employ an array of assays, including chloride sensors, a newly developed fluorescent probe for bicarbonate, and assays to detect the electrogenicity of anion exchange. With this assortment of assays, the authors are able to establish the anion selectivity and stoichiometry of SLC26A6. Another strength of the manuscript is the functional comparison with SLC26A9, which permits fast, passive chloride transport, in order to benchmark the SLC26A6 activity. The structural analysis, including the assignment of the chloride binding site, is also convincing. The structural details and the chloride binding site are well-conserved among SLC26s. Finally, the authors present an interesting discussion comparing the structures of SLC26A5, SLC26A6, and SLC26A9, and how the details of the chloride binding site might influence the mechanistic distinctions between these similar transporters.

Reviewer #3 (Public Review):

Dux (or DUX4 in human) is a master transcription factor regulating early embryonic gene activation and has garnered much attention also for its involvement in reprogramming pluripotent embryonic stem cells to totipotent "2C-like" cells. The presented work starts with the recognition that DUX contains five conserved c. 100-amino acid carboxy-terminal repeats (called C1-C5) in the murine protein but not in that of other mammals (e.g. human DUX4). Using state-of-the-art techniques and cell models (BioID, Cut&Tag; rescue experiments and functional reporter assays in ESCs), the authors dissect the activity of each repeat, concluding that repeats C3 and C5 possess the strongest transactivation potential in synergy with a short C-terminal 14 AA acidic motif. In agreement with these findings, the authors find that full-length and active (C3) repeat containing Dux leads to increased chromatin accessibility and active histone mark (H3K9Ac) signals at genomic Dux binding sites. A further significant conclusion of this mutational analysis is the proposal that the weakly activating repeats C2 and C4 may function as attenuators of C3+C5-driven activity.

By next pulling down and identifying proteins bound to Dux (or its repeat-deleted derivatives) using BioID-LC/MS/MS, the authors find a significant number of interactors, notably chromatin remodellers (SMARCC1), a histone chaperone (CHAF1A/p150) and transcription factors previously (ZSCAN4D) implicated in embryonic gene activation.

The experiments are of high quality, with appropriate controls, thus providing a rich compendium of Dux interactors for future study. Indeed, a number of these (SMARCC1, SMCHD1, ZSCAN4) make biological sense, both for embryonic genome activation and for FSHD (SMCHD1).

A critical question raised by this study, however, concerns the function of the Dux repeats, apparently unique to mice. While it is possible, as the authors propose, that the weak activating C1, C2 C4 repeats may exert an attenuating function on activation (and thus may have been selected for under an "adaptationist" paradigm), it is also possible that they are simply the result of Jacobian evolutionary bricolage (tinkering) that happens to work in mice. The finding that Dux itself is not essential, in fact appears to be redundant (or cooperates with) the OBOX4 factor, in addition to the absence of these repeats in the DUX protein of all other mammals (as pointed out by the authors), might indeed argue for the second, perhaps less attractive possibility.

In summary, while the present work provides a valuable resource for future study of Dux and its interactors, it fails, however, to tell a compelling story that could link the obtained data together.

Reviewer #3 (Public Review):

Summary. This study sought to clarify the connection between inositol pyrophosphates (PP-IPs) and their regulation of phosphate homeostasis in the yeast Saccharomyces cerevisiae to answer the question of whether any of the PP-IPs (1-IP7, 5-IP7, and IP8) or only particular PP-IPs are involved in regulation. PP-IPs bind to SPX domains in proteins to affect their activity, and there are several key proteins in the PHO pathway that have an SPX domain, including Pho81. The authors use the latest methodology, capillary electrophoresis and mass spectrometry (CE-MS), to examine the cytosolic concentrations of PP-IPs in wild-type and strains carrying mutations in the enzymes that metabolize these compounds in rich medium and during a phosphate starvation time-course for the wild-type.

Major strengths and weaknesses. The authors have strong premises for performing these experiments: clarifying the regulatory molecule(s) in yeast and providing a unifying mechanism across eukaryotes. They use the latest methodologies and a variety of approaches including genetics, biochemistry, cell biology and protein structure to examine phosphate regulation. Their experiments are rigorous and well controlled, and the story is clearly told. The consideration of physiological levels of PP-IPs throughout the study was critical to the interpretation of the data and the strength of the manuscript.

There were a few places in which a deeper discussion of the data was warranted: not discussed was an explanation for the decrease in the levels of all of the PP-IPs upon phosphate starvation, nor of the phosphate regulation of two target genes of Pho4 when Pho4 is constitutively nuclear.

Appraisal. The authors achieved their goal of determining the mechanistic details for phosphate regulation, revising the prior model with new insights. Additionally, they provided strong support for the idea that IP8 regulates phosphate metabolism across eukaryotes - including animals and plants in addition to fungi.

Impact. This study is likely to have a broad impact because it addresses prior findings that are inconsistent with current understanding, and they provide good reasoning as to how older methods were inadequate.

Reviewer #3 (Public Review):

The study by Thommen et al. sought to identify the native role of the Plasmodium falciparum FKBP35 protein, which has been identified as a potential drug target due to the antiplasmodial activity of the immunosuppressant FK506. This compound has multiple binding proteins in many organisms; however, only one FKBP exists in P. falciparum (FKBP35). Using genetically-modified parasites and mass spectrometry-based cellular thermal shift assays (CETSA), the authors suggest that this protein is in involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is separate from its activity on the FKBP35 protein. The authors first created a conditional knockdown using the destruction domain/shield system, which demonstrated no change in asexual blood stage parasites. A conditional knockout was then generated using the DiCre system. FKBP35KO parasites survived the first generation but died in the second generation. The authors called this "a delayed death phenotype", although it was not secondary to drug treatment, so this may be a misnomer. This slow death was unrelated to apicoplast dysfunction, as demonstrated by lack of alterations in sensitivity to apicoplast inhibitors. Quantitative proteomics on the FKBP35KO vs FKBP35WT parasites demonstrated enrichment of proteins involved in pre-ribosome development and the nucleolus. Interestingly, the KO parasites were not more susceptible to cycloheximide, a translation inhibitor, in the first generation (G1), suggesting that mature ribosomes still exist at this point. The SunSET technique, which incorporates puromycin into nascent peptide chains, also showed that in G1 the FKBP35KO parasites were still able to synthesize proteins. But in the second generation (G2), there was a significant decrease in protein synthesis. Transcriptomics were also performed at multiple time points. The effects of knockout of FKBP35 were transcriptionally silent in G1, and the parasites then slowed their cell cycles as compared to the FKBP35WT parasites.

The authors next sought to evaluate whether killing by FK506 was dependent upon the inhibition of PfKBP35. Interestingly, both FKBP35KO and FKBP35WT parasites were equally susceptible to FK506. This suggested that the antiplasmodial activity of FK506 was related to activity targeting essential functions in the parasite separate from binding to FKBP35. To identify these potential targets, the authors used MS-CETSA on lysates to test for thermal stabilization of proteins after exposure to drug, which suggests drug-protein interactions. As expected, FK506 bound FKBP35 at low nM concentrations. However, given that the parasite IC50 of this compound is in the uM range, the authors searched for proteins stabilized at these concentrations as putative secondary targets. Using live cell MS-CETSA, FK506 bound FKBP35 at low nM concentrations; however, in these experiments over 50 ribosomal proteins were stabilized by the drug at higher concentrations. Of note, there was also an increase in soluble ribosomal factors in the absence of denaturing conditions. The authors suggested that the drug itself led to these smaller factors disengaging from a larger ribosomal complex, leading to an increase in soluble factors. Ultimately, the authors conclude that the native function of FKBP35 is involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is not related to the binding of FKBP35, but instead results from inhibition of essential functions of secondary targets.

Strengths:

This study has many strengths. It addresses an important gap in parasite biology and drug development, by addressing the native role of the potential antiplasmodial drug target FKBP35 and whether the compound FK506 works through inhibition of that putative target. The knockout data provide compelling evidence that the KBP35 protein is essential for asexual parasite growth after one growth cycle. Analysis of the FKBP35KO line also provides evidence that the effects of FK506 are likely not solely due to inhibition of that protein, but instead must have secondary targets whose function is essential. These data are important in the field of drug development as they may guide development away from structure-based FK506 analogs that bind more specifically to the FKBP35 protein.

Weaknesses:

There are also a few notable weaknesses in the evidence that call into question the conclusion in the article title that FKBP35 is definitely involved in ribosomal homeostasis. While the proteomics supports alterations in ribosome biogenesis factors, it is unclear whether this is a direct role of the loss of the FKBP35 protein or is more related to non-specific downstream effects of knocking down the protein. The CETSA data clearly demonstrate that FK506 binds PfKB35 at low nM concentrations, which is different than the IC50 noted in the parasite; however, the evidence that the proteins stabilized by uM concentrations of drug are actual targets is not completely convincing. Especially, given the high uM amounts of drug required to stabilize these proteins. This section of the manuscript would benefit from validation of a least one or two of the putative candidates noted in the text. In the live cell CETSA, it is noted that >50 ribosomal components are stabilized in drug treated but not lysate controls. Similarly, the authors suggest that the -soluble fraction of ribosomal components increases in drug-exposed parasites even at 37{degree sign}C and suggests that this is likely from smaller ribosomal proteins disengaging from larger ribosomal complexes. While the evidence is convincing that this protein may play a role in ribosome homeostasis in some capacity, it is not sure that the title of the paper "FKBP secures ribosome homeostasis" holds true given the lack of mechanistic data. A minor weakness, but one that should nonetheless be addressed, is the use of the term "delayed death phenotype" with regards to the knockout parasite killing. This term is most frequently used in a very specific setting of apicoplast drugs that inhibit apicoplast ribosomes, so the term is misleading. It is also possible that the parasites are able to go through a normal cycle because of the kinetics of the knockout and that the time needed for protein clearance in the parasite to a level that is lethal.

Overall, the authors set out to identify the native role of FKB35 in the P. falciparum parasites and to identify whether this is, in fact, the target of FK506. The data clearly demonstrate that FKBP35 is essential for parasite growth and provide evidence that alterations in its levels have proteomic but not transcriptional changes. However, the conclusion that FKBP35 actually stabilizes ribosomal complexes remains intermediate. The data are also very compelling that FK506 has secondary targets in the parasite aside from FKBP35; however, the high uM concentrations of the drug needed to attain results and the lack of biological validation of the CETSA hits makes it difficult to know whether any of these are actually the target of the compound or instead are nonspecific downstream consequences of treatment.

Reviewer #3 (Public Review):

The authors investigated the mechanism of transport of the GLUT5 sugar porter using enhanced sampling molecular dynamics simulations and biochemical analysis.<br /> The results suggest a possible general mechanism by which binding to a transported substrate stabilizes an occluded intermediate conformation between outward and inward-facing states of the alternating access conformational change of the protein, thereby enabling transport.

The authors also identified key elements of this transition, associated with residues involved in sugar binding, and through elegant biochemical experiments demonstrated how mutations of the latter affect the protein function, including mutations of gating residues that can recover the function of inactive mutants.<br /> The general computational methodology used by authors is appropriate for addressing these questions and compared to other techniques has the advantage of bringing forth an unbiased molecular description of the transport process. The results are overall qualitatively in line with the proposed conclusions.

A major weakness of this work is that, in contrast to previous studies with the same type of methodology, the authors do not report error analysis or careful statistical assessment of the computational results. Therefore, it is not clear whether the latter is solid or if they support the proposed conclusions. The computational data might generally benefit from an improved methodological design, such as including more degrees of freedom (or collective variables) in the description of the minimum free energy pathway, e.g. the salt-bridges.

Another weakness is that some of the details of the computational analysis are not reported, therefore other investigators would not know how to reproduce the results.

Once these issues are addressed, this work could potentially provide important insights into the mechanism of transport of sugar porters, which as suggested by other recent studies might also apply to other types of membrane transporters.

Reviewer #3 (Public Review):

In this work, Elbahnsi and colleagues use enhanced sampling MD simulation, to recapitulate step by step, the electromechanical coupling between VSD and the pore in HCN1 channels. Building on the available cryoEM structures of HCN1 with the VSD in resting and active state, the authors characterize by MD a subset of interactions that seemingly stabilize the open channel. This subset is, in turn, used in enhanced-sampling simulations to guide channel opening.<br /> The main findings are that S4 movement induces a rearrangement of the hydrophobic interaction at the level of S1- S4- and S5 interfaces. Occupancy of lipids seems therefore state-dependent and highlights their regulatory role in HCN gating.

The approach is rather innovative, and it apparently allows the reconstruction of the whole mechanism of gating, pushing the predictive power of MD simulation well beyond its actual temporal limitations. At the same time, the initial choice of interactions is crucial for this approach, because the result cannot differ from the inputs. And reading the paper it does not emerge clearly how the correctness of the reconstructed gating pathway can be verified, if not by functional validation.

Here are my comments on the main interactions that were used to feed the final MD simulation:

1. W281-N300: this interaction, previously identified and studied in SpH channels (Ramentol et al, 2020; Wu et al, 2021), has been elegantly confirmed in this paper. Its inclusion in the initial subset seems appropriate.<br /> In the other two cases, the choice of interactions requires further explanations and experimental validation.

2. D290 and K412: the validation of this interaction shown in Figure 3 and suppl Figure 1 is missing a control, i.e., the effect of the addition of Cd++ on the wt channel. Please add.

3. Modelling the open state of HCN1 pore (page 18), is done on the structure of the distantly related hERG rather than on the available open pore structure of HCN4. This choice is justified as follows by the authors:

a) "Available structures in the CNBD channel family for which representative structures have been solved in closed and open states".<br /> b) "The structural mechanism of pore gating (i.e. the ⍺ to 𝜋 helix occurring at the glycine657 hinge in hERG) observed in rEAG/hERG may be a conserved gating transition in the CNBD family of channels"<br /> I encourage the authors to consider the following:

a) The structure of hERG channel is not available in the closed/open configuration, indeed the comparison must be done with the closed configuration of the related channel rEAG. On the contrary, HCN4 is available in the closed/open configurations. Moreover, one of the open pore structures shows S4-S5-S6 in a very similar conformation to the lock open mutant (F186C/S264C) of HCN1 (Saponaro et al, 2021). With an available HCN4 open structure, forcing HCN1 to the open pore structure of hERG channel (which opens in depolarization and is not regulated by cAMP) seems not necessary.

To my knowledge, hERG is the only channel of the CNBD family for which the transition ⍺ to 𝜋 helix reported by the Authors, occurs in S6. It is not reported for other CNBD family members, in particular for the CNG channels mentioned by the Authors (Zheng et al., 2020; Xue et al., 2021, 2022). Task 4 (Zheng et al) does not show it. Its pore opens by a right-handed twist of S6 at glycine 399, a conserved glycine in all CNG. Human CNGA1 too, opens the pore by a rotational movement of S6 hinged at the equivalent glycine (glycine 385) (Xue et al, 2021). And the same occurs in the non-symmetrical channel CNGA1/B1 (Xue te al, 2022). So, it seems that CNG channels do not show the ⍺ to 𝜋 helix transition in the open pore. Moreover, hERG excluded, all other members of the CNBD family, CNG, EAG, and HCN4 included, do not bend at the hinge glycine 657 of hERG, but at another glycine (gly 648 in hERG numbering) located upstream. Further, their opening is due to a rotation of S6 associated with an outward movement, rather than to the lifting of the lower part of S6, as in hERG.

4- V390-I302: this interaction is predicted to stabilize the open pore configuration and was included in the subset. The contact between V390 on S6 and I302 on S5 is observed in the homology model discussed above when the S6 is twisted at the glycine hinge, rotating the preceding residue (V390) out of its pore-lining position and is.<br /> Again, I can only disagree with this hypothesis because it has been experimentally demonstrated (Cheng et al, J Pharmacol Exp Ther. 2007 Sep;322(3):931-9) that the side chain of Valine390 is inside the cavity of the open pore of HCN1 channels as it controls the affinity for the pore blocker ZD7288.

In conclusion, modelling the open state pore of HCN1 on hERG rather than on that of HCN4 seems not justified based on accumulated evidence in the published literature. Therefore, the choice of the authors to use it as the open pore model of HCN1 channels needs to be experimentally validated. One possibility is to mutate the glycine hinge, gly391 in HCN1, into an Alanine in order to remove the flexible hinge. If this mutation alters pore gating, it will support the choice of the Authors.

Reviewer #3 (Public Review):

Parab et al. investigate the requirement of specific Vegf ligands during the embryonic development of new blood vessels in different brain regions. The authors implement their previously published experimental paradigm (Parab et al 2021 eLife) combined with new transgenic and mutant zebrafish lines to show that vegf ligands (vegfaa, vegfab, vegfc, and vegfd) are required in various combinations to drive angiogenesis in distinct brain regions. Specifically, they show that individual loss of different vegf ligands causes either undetectable or partial effects in angiogenesis, while combined loss of vegf ligands results in severe defects in brain region-specific angiogenesis. As different blood vessel types (i.e. arteries, veins, lymphatics) require specific angiogenic cues, this study provides interesting new data on how the combination of these signals drives brain region-specific vascular development.

While the conclusions of the paper are generally well supported by the data, the authors overstate some of their findings, particularly with respect to the development of fenestrated capillaries. In this study, the authors use the zebrafish transgenic reporter line, plvap:EGFP, as an indicator of fenestrations. However, the authors do not provide any evidence of fenestrations of the blood vessels of the choroid plexuses or the cranial vessels used for quantification (Figures 1, 3, and 4). While expression of Plvap protein is often used as a marker for non-blood brain barrier endothelial cells, as Plvap is the major component of the diaphragms of fenestrated capillaries, plvap:EGFP expression alone does not indicate fenestrations. This is an important point because previous work has demonstrated that targeted deletion of Plvap does not cause a loss of fenestrations, but instead a loss of the diaphragms associated with fenestrations (Stan et al 2012 Dev Cell; Gordon et al 2019 Development). Similarly, Plvap expression alone does not necessarily indicate fenestrations as an expression of Plvap is not sufficient for fenestration formation. In fact, Plvap has initially been expressed in brain endothelial cells during initial angiogenesis to the brain without evidence of fenestrations, and subsequently, Plvap expression disappears during the maturation of the BBB. Thus, to conclude that specific vegf ligands are required for the development of fenestrated capillaries, transmission electron microscopy (TEM) should be used on the capillaries examined in this study or the language describing the results should be modified accordingly. Conversely, the authors did show TEM for the choriocapillaris (Figure 5A-C) but did not show plvap:EGFP expression in these vessels.

Additionally, the authors' usage of the phrase "development of fenestrated vessels" suggests that the study was examining signals that regulate the formation of fenestrations and not angiogenesis of vessels that may become fenestrated as demonstrated here. Therefore, as Plvap expression does not necessarily equate fenestrations (and vice-versa), the title and some of the major claims of the study are somewhat overstated.

Reviewer #3 (Public Review):

The study presents a systematic analysis of how a range of dystroglycan mutations alter CCK/CB1 axonal targeting and inhibition in hippocampal CA1 and impact seizure susceptibility. The study follows up on prior literature identifying a role for dystroglycan in CCK/CB1 synapse formation. The careful assay includes comparison of 5 distinct dystroglycan mutation types known to be associated with varying degrees of muscular dystrophy phenotypes: a forebrain specific Dag1 knockout in excitatory neurons at 10.5, a forebrain specific knockout of the glycosyltransferase enzyme in excitatory neurons, mice with deletion of the intracellular domain of beta-Dag1 and 2 lines with missense mutations with milder phenotypes. They show that forebrain glutamatergic deletion of Dag1 or glycosyltransferase alters cortical lamination while lamination is preserved in mice with deletion of the intracellular domain or missense mutation. The study extends prior works by identifying that forebrain deletion of Dag1 or glycosyltransferase in excitatory neurons impairs CCK/CB1 and not PV axonal targeting and CB1 basket formation around CA1 pyramidal cells. Mice with deletion of the intracellular domain or missense mutation show limited reductions in CCK/CB1 fibers in CA1. Carbachol enhancement of CA1 IPSCs was reduced both in forebrain knockouts. Interestingly, carbachol enhancement of CA1 IPSCs was reduced when the intracellular domain of beta-Dag1was deleted, but not I the missense mutations, suggesting a role of the intracellular domain in synapse maintenance. All lines except the missense mutations , showed increased susceptibility to chemically induced behavioral seizures. Together, the study, is carefully designed, well controlled and systematic. The results advance prior findings of the role for dystroglycans in CCK/CB1 innervations of PCs by demonstrating effects of more selective cellular deletions and site specific mutations in extracellular and intracellular domains. The interesting finding that deletion of intracellular domain reduces both CB1 terminals in CA1 and carbachol modulation of IPSCs warrants further analysis. Lack of EEG evaluation of seizure latency is a limitation.

Specific comments<br /> 1. Whether CCK/CB1 cell numbers in the CA1 are differentially affected in the transgenic mice is not clarified.<br /> 2. Whether basal synaptic inhibition is altered by the changes in CCK innervation is not examined.

Reviewer #3 (Public Review):

With a soft-spoken, matter-of-fact attitude and almost unwittingly, this brilliant study chisels away one of the pillars of hippocampal neuroscience: the special role(s) ascribed to theta oscillations. These oscillations are salient during specific behaviors in rodents but are often taken to be part of the intimate endowment of the hippocampus across all mammalian species, and to be a fundamental ingredient of its computations. The gradual anticipation or precession of the spikes of a cell as it traverses its place field, relative to the theta phase, is seen as enabling the prediction of the future - the short-term future position of the animal at least, possibly the future in a wider cognitive sense as well, in particular with humans. The present study shows that, under suitable conditions, place cell population activity "sweeps" to encode future positions, and sometimes past ones as well, even in the absence of theta, as a result of the interplay between firing rate adaptation and precise place coding in the afferent inputs, which tracks the real position of the animal. The core strength of the paper is the clarity afforded by the simple, elegant model. It allows the derivation (in a certain limit) of an analytical formula for the frequency of the sweeps, as a function of the various model parameters, such as the time constants for neuronal integration and for firing rate adaptation. The sweep frequency turns out to be inversely proportional to their geometric average. The authors note that, if theta oscillations are added to the model, they can entrain the sweeps, which thus may superficially appear to have been generated by the oscillations.

The main weakness of the study is the other side of the simplicity coin. In its simple and neat formulation, the model envisages stereotyped single unit behavior regulated by a few parameters, like the two time constants above, or the "adaptation strength", the "width of the field" or the "input strength", which are all assumed to be constant across cells. In reality, not only assigning homogeneous values to those parameters seems implausible, but also describing e.g. adaptation with the simple equation included in the model may be an oversimplification. Therefore, it remains important to understand to what extent the mechanism envisaged in the model is robust to variability in the parameters or to eg less carefully tuned afferent inputs.

The weak adaptation regime, when firing rate adaptation effectively moves the position encoded by population activity slightly ahead of the animal, is not novel - I discussed it, among others, in trying to understand the significance of the CA3-CA1 differentiation (2004). What is novel here, as far as I know, is the strong adaptation regime, when the adaptation strength m is at least larger than the ratio of time constants. Then population activity literally runs away, ahead of the animal, and oscillations set in, independent of any oscillatory inputs. Can this really occur in physiological conditions? A careful comparison with available experimental measures would greatly strengthen the significance of this study.